Arch Dermatol Res DOI 10.1007/s00403-014-1521-3
ORIGINAL PAPER
Oxidative stress and substance P mediate psychological stress-induced autophagy and delay of hair growth in mice Lei Wang • Ling-Ling Guo • Lin-Hui Wang • Guo-Xing Zhang • Jing Shang • Koji Murao • Deng-Feng Chen • Xiang-Hua Fan • Wen-Qing Fu
Received: 5 October 2013 / Revised: 2 October 2014 / Accepted: 30 October 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Neuropeptide substance P (SP) and reactive oxygen species (ROS) have been demonstrated to play an important role in psychological stress-induced alteration of hair cycle, the underlying mechanism remains unknown. The present study aims to investigate possible contribution of SP and ROS in chronic restraint stress (CRS, a chronic psychological stress model) induced abnormal of hair cycle and induction of autophagy. Mouse CRS model was applied for 18 days with or without treatment antioxidant Tempol (a free radical scavenger) or SP receptor (NK1) antagonist (RP67580). After CRS procedure, hair growth cycle, oxidative stress markers and skin tissue autophagy levels were analyzed by ELISA or western blot. Our results revealed that CRS reduced body weight gain, distance of movement and times of standing, affected hair cycle by prolonging the telogen stage and delaying subsequent anagen and catagen stage. In addition, CRS resulted in
L. Wang, L. - L. Guo, L.- H. Wang these authors contributed equally to this work. L. Wang W.-Q. Fu (&) Department of Clinical Psychology, Medical College of Soochow University, 199 Ren-Ai Road, Dushu Lake Campus, Suzhou Industrial Park, Suzhou 215123, People’s Republic of China e-mail: [email protected] L.-L. Guo Department of Pathology, Medical College of Soochow University, 199 Ren-Ai Road, Dushu Lake Campus, Suzhou Industrial Park, Suzhou 215123, People’s Republic of China L.-H. Wang G.-X. Zhang (&) Department of Physiology, Medical College of Soochow University, 199 Ren-Ai Road, Dushu Lake Campus, Suzhou Industrial Park, Suzhou 215123, People’s Republic of China e-mail: [email protected]
increase of lipid peroxidation levels and reduction of the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and increase of autophagy markers (microtubule-associated proteins, light chain 3-II, LC3-II, and Beclin-1) in mice skin. Treatment with Tempol restored GSH-Px activity, and significantly reduced increases of lipid peroxidation levels and LC3-II and Beclin-1 expressions, as well as normalized hair cycle. In addition; RP67580 also restored SOD and GSH-Px activities, and markedly reduced increases of lipid peroxidation levels and LC3-II and Beclin-1 expressions, and normalized hair cycle. Our study provides the first strong evidence for SP and ROS play a role not only in alteration of hair cycle but also in induction of autophagy in psychological stress model, suggesting autophagy may contribute to psychological stress-induced abnormal of hair cycle. Keywords Chronic restraint stress Hair cycle Reactive oxygen species Substance P Autophagy
J. Shang New Drug Screening Center, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China K. Murao Department of Clinical Laboratory, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe Miki-cho, Kita-gun, Kagawa 761-0793, Japan D.-F. Chen Department of Surgery, Harrison International Peace Hospital, 2 Ren-Ming Road, Hengsui 053000, People’s Republic of China X.-H. Fan Department of Dermatology, the Second Affiliated Hospital of Nangtong University, 6 Hai-Er Road, Nantong 226001, People’s Republic of China
123
Arch Dermatol Res
Introduction Psychological stress has been reported to be highly related to the development of skin diseases [9, 15, 16, 36]. Previous studies showed that psychological stress affected hair cycle mediated by neurohormone, neurotransmitter and cytokines [33, 46]. Arck et al. [6] have found that when the mice were irritated by the noise for 24 h, hair growth was inhibited by induction of premature catagen development via neuropeptide substance P (SP)-dependent pathways. Studies of the foot shock model also demonstrated that the telogen has been prolonged and the anagen has been deferred [4, 22]. However, neither of the 24-h noise nor the foot shock is sufficient to imitate the state of human being’s chronic stress and to prove the existence of somatic stress element. Chronic restrained stress (CRS) can efficiently simulate the long-term stress situation of human kind [13, 20, 43]. It could evoke depression-like behavioral changes accompanied by working memory and learning deficits. Using this model, it may provide better understanding of abnormal of hair growth in psychological stress condition. Clinical studies have shown that SP-mass cell pathway is involved in various pathogenesis of skin disease. Arck et al. reported SP is the central neurotransmitter of brainskin axis [5–7, 45, 48]. SP is one kind of the neuropeptides related to stress. It is synthesized in the neurons of dorsal root ganglion and released by the sensory nerve ending of skin. It can induce activation of mast cells and then release the pro-inflammatory factor resulting in neurogenic scytitis [7, 23, 45, 48]. SP has three main receptors, which are named as NK1, NK2 and NK3 receptor. Of them, NK1 receptor has the highest affinity to SP and is widely distributed [3, 21, 30]. In the previous studies, pseudo-peptide, RP 67580, was reported as the higher potency blocker of NK1 because of its highest affinity on NK1 [10, 24]. Under normal circumstances, oxidation-antioxidant system is stable and can maintain the metabolic balance of free radicals. The reactive oxygen species (ROS) are controlled at lower levels [39]. But under internal and external stimulation, ROS generation may increase in rapid speed and cause the disorders of oxidation and antioxidant balance, which cause oxidative damage of biofilm and macromolecular material. Studies from Akar et al. and Naziroglu et al. proved that oxidative stress plays an important role in the process of hair loss [1, 32]. In addition, CRS resulted in the oxidative stress injury of the rat’s stomach, liver, kidney, heart, brain and viscera [38, 50]. Furthermore, our recent study also demonstrated that ROS play a role in regulation of psychological stress-induced SP-Mast cell pathway and hair growth cycle [29]. 4-Hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol) is a membrane-permeable superoxide dismutase (SOD) mimetic that possesses potent antioxidant activity. Our
123
previous studies have demonstrated its strong effects on ROS scavenge [51, 52]. Autophagy played an important role in maintaining cellular homeostasis, differentiation and tissue remodeling. Paradoxically, the role of autophagy in cell survival is still controversial. Nutrient depletion induced autophagy provides amino acids for synthesis of essential proteins, thus prolonging cell survival [25]. It was also reported that autophagy could counteract cell apoptosis [37]. Other studies, however, have shown that autophagy served as an alternative form of cell death [11, 14]. ROS have been reported to be essential for induction of autophagy [41, 49]. Similarly, role of ROS-induced autophagy is still controversial, it may depend on the degree of autophagy, in which mild induction of autophagy by ROS may have cytoprotective effects, but massive induction of autophagy by ROS may cause excessive self-digestion of cell components and lead to death [19]. Whether CRS will induce autophagy and how it is regulated in skin are still unknown. Based on the above reports, the present study will apply antioxidant and SP receptor (NK1) antagonist in the experimental CRS mice model, observing hair growth cycle, detecting autophagy protein markers (LC3-II, Beclin-1), exploring the mechanisms in CRS-induced abnormal of hair growth.
Method Animals Six to eight weeks old healthy male C57BL/6 mice were used (weight 20 ± 2 g), provided by center of experimental animal, Soochow University. All mice were acclimated under the following conditions for one week: room temperature 21–24 °C; humidity 50–60 % and a 12-h light: dark cycle (lights on at 6:00 a.m. and off at 18:00 p.m.). During this period, food and water are provided ad libitum. The mice were randomly divided into the following groups: (1) Control group (10 mice); (2) CRS group (10 mice): no drug injection and application of CRS; (3) Tempol group (10 mice): application of CRS concomitant with 200 mg/kg/day of intraperitoneal injection of Tempol for 18 days; (4) NK1 blocker, RP67580 group (10 mice): application of CRS concomitant with 200 mg/mouse of intraperitoneal injection of RP67580 for 18 days (every other day). The present study was designed according to our previous report [29]. Induction of anagen and application of CRS Method for induction of anagen was as described previously [6]. On the first day of the experiment, wax/rosin mixture (1:1 on weight) was applied to the dorsal skin
Arch Dermatol Res
(from neck to tail) of mice, after it dried and then peelingoff the wax/rosin mixture, removed all hair shafts and immediately induced a highly synchronized hair growth, as evidenced by the homogeneously pink skin color in the back, indicating all hair follicles in telogen [26, 34]. CRS starts on day 1 after depilation for 6 h daily (10:00 a.m.16:00 p.m.) while food and water were prohibited. Period of CRS was lasted for 9 or 18 days and the control mice were kept in their original cage, but food and water were not provided during the CRS period. All mice were taken pictures with a digital camera (Panasonic, Japan) on the 9th and 18th day, respectively.
cycle of control mice just spontaneously enters the catagen transformation [26, 31], mice were killed; plasma and skin tissues were sampled according to above mentioned method. Measurement of body weight and plasma corticosterone concentration All mice were monitored body weight on day 0, 4, 9, 14, 18 after depilation. Plasma corticosterone concentration was determined by mouse corticosterone RIA kit following manufacturer’s instruction (Beyotime, China). Protein concentration measurement
Reagents RP67580 (Santa Cruz biotechnology,CA, USA), a selective antagonist of the tachykinin NK1 receptor, was dissolved in ethanol and then diluted with PBS to make a final concentration of 1 mg/ml. 4-Hydroxy-2, 2, 6, 6-tetramethyl piperidinoxyl, Tempol (Sigma, MO, USA), is a stable membranepermeable superoxide dismutase (SOD) mimetic that exhibits potent antioxidant activity against superoxide as well as hydroxyl radicals [42]. In the present study, Tempol was injected intraperitoneally to against oxidative stress in skin at a dose of 200 mg/kg/day according to previous report [18]. Open-field test Two time points were chosen to evaluate motor function and state anxiety of the mice. One was on day 9; the other was on day18. The mice were placed into the central grid, recording the mice’s distance of movement and times of standing in 3 min. Distance of movement reflects the ability of autonomous activity in mice; times of standing reflect exploratory behavior in mice. The reduction of distance of movement and times of standing shows that restraint stress results in a state of depression in mice. Tissue and plasma preparation Two time points were chosen to indicate different hair cycle stage. One, on day 9 after depilation when hair cycle of control mice was in the late anagen [26, 31], blood samples were collected in the evening after 9 days CRS under anesthesia by eyeball removal method, then centrifuged at 3,000 rpm for 15 min to obtain plasma, then stored at -80 °C immediately. Skin specimens from back skin (the region of neck) were harvested about 2 9 5 cm, which is the region of neck, which is an essential section for the quantitative histomorphology of the hair cycle [6], was fixed in 4 % paraformaldehyde, then paraffin-wax embedded. The remaining skin specimens are cryopreserved at -80 °C immediately. Second, on day 18 after depilation when hair
The skin tissues were homogenized in a solution containing 0.15 mol/L KCl and 0.02 mol/L Tris–HCl (pH 7.4), then used BCA Protein Assay Kit (Beyotime, China) to determine protein concentration. Finally, the skin tissue homogenates were adjusted at a concentration of 1 mg/ml for further analysis. Measurement of lipid peroxidation levels in skin The method for measurement of thiobarbituric acid reactive substances (TBARS) levels has been described previously [17]. Briefly, 100 lL sample solution was mixed with 15 % trichloroacetic acid and 0.375 % thiobarbituric acid. Butylated hydroxytoluene (0.01 %) was added to the assay mixture to prevent autoxidation of the sample, and the mixture was heated at 100 °C for 15 min. After cooling, the mixture was centrifuged at 14,000 rpm for 5 min, and the absorbance of the organic phase was measured at 535 nm. The amount of thiobarbituric acid reactive substances (TBARS) was determined by the malondialdehyde standard curve and expressed as lmol/L in 100 lg protein. Measurement of SOD and GSH-Px activities in skin SOD activity was detected according to previous report [35]. In brief, xanthine–xanthine oxidase complex produces superoxide radicals, which react with water-soluble tetrazolium (WST-1). GSH-Px activity using H2O2 as a substrate was assayed by an adaptation of the method reported by Lawrence and Burk [27]. Using 96-well plate, SOD activity was measured at 450 nm and GSH-Px activity was measured at 412 nm by Micro-plate reader, and both of the data were presented as U/100 lg protein. Assessment of hair cycle Mouse skin color changes according to hair growth cycle, which becomes darker and darker from anagen to catagen, then gradually become lighter from catagen to telogen [31].
123
Arch Dermatol Res
The grayscale (0–255) of specific area in photograph (the region of neck) was analyzed by Image J software and presented as ratios (grayscale/255) on day 9 after depilation when the mice were shaved. HE stain was used to quantify the stage of the hair follicles using a published classification technique based on the morphology of the dermal papilla and sebaceous glands [26]. To identify accurately, all hair follicles were scored which was assigned to the telogen, anagen and catagen stages according to its morphological and histological difference (anagen I–VI; catagen I–VIII; telogen) as described in previous reports [31, 38]. In brief, after identifying the stage of each hair follicles according to corresponding special characteristic of each stage, every stage of anagen or catagen is assigned a factor in ascending numerical order (anagen: anagen I = factor 1, anagen II = factor 2, anagen III = factor 3, etc.; for catagen: catagen I = factor 1, catagen II = factor 2, catagen III = factor 3, etc.). The number of HF in each specific stage is multiplied by the corresponding factor. The results of each sum were totaled and divided by the total number of HF counted [31]. Western blotting for LC3-II and Beclin-1 expression Expressions of LC3-II, Beclin-1 in the mice skin were measured by western blotting, as previously described [51]. Briefly, equal amount of protein from each homogenate was resolved by 10 % SDS–PAGE. Proteins were transferred to PVDF membranes (Hybond TM-ECL; Amersham Pharmacia Biotech, Inc.). The membranes were blocked for 2 h at room temperature with 5 % skimmed milk in PBS and 0.1 % Tween 20. The blots were incubated overnight with 1:1,000 diluted primary antibodies: anti-LC3-II and anti-Beclin-1 (Abcam, Inc.) followed by incubation for 1 h with a secondary antibody (HRP-conjugated anti-rabbit IgG; 1:2,000). Immunoreactive bands were visualized using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech) and analyzed by NIH image software. Data were normalized by GAPDH. Statistical analysis All data were presented as the mean ± S.E.M. Statistical significance between two groups was tested using one way ANOVA followed by the Post hoc test. P values \0.05 were considered statistically significant.
Results
Fig. 1 Tempol and RP67580 have no effect on CRS-induced body weight gain loss. a A representative picture of each group on day 9 and day 18 after CRS. b Body weight gains were monitored in each group on day 0, 4, 9, 14, 19. CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580. Data are presented as mean ± SEM, n = 9 in each group, *p \ 0.05 compared with control group
significantly on day 4, 9, 14 and 18 compared with control group. Neither of Tempol and RP67580 affected CRSinduced reduction of body weight gain (Fig. 1). Through the open-field test we recorded the distance of movement and the times of standing in each group. Distance of movement reflects the ability of autonomic activities, and times of standing reflect the exploratory behavior in mice. CRS markedly reduced the distance of movement and number of standing, suggesting CRS resulted in a state of depression in mice. Neither Tempol nor RP67580 had any effects on CRS-induced reduction of the distance of movement and number of standing, indicating both of Tempol nor could RP67580 not ameliorate CRS-induced depression (Fig. 2a, b).
Assessment of body weight gain and behavior The plasma levels of corticosterone Body weight was monitored on day 0, 4, 9, 14 and 18. Body weight gain was obtained by calculation. Our results showed that CRS inhibited mice body weight gain
123
The primary glucocorticoid, corticosterone, was named as stress hormone to monitor the state of stress. Plasma
Arch Dermatol Res Fig. 2 Tempol and RP67580 do not affect CRS-induced depressive behavior. a Times of standing are shown in each group on day 9 and 19. b distance of movement in each group is shown on day 9 and 19. CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580. Data were presented as mean ± SEM, n = 9 in each group, *p \ 0.05 compared with control group
Table 1 Levels of plasma corticosterone in different experiment groups after 9-day and 18-day CRS Group
The levels of lipid peroxide and the activities of SOD and GSH-Px in skin tissue
Levels of plasma corticosterone (ng/L) 9-day
18-day
Control
38.52 ± 5.03
37.91 ± 4.65
CRS
48.15 ± 3.87*
47.29 ± 3.50*
CRS ? T CRS ? N
46.73 ± 4.12* 46.22 ± 3.64*
49.85 ± 6.86* 48.32 ± 5.11*
The plasma corticosterone concentrations of mice on day 9 and 18 were measured Data are presented as mean ± SEM, n = 9 in each group CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580 * p \ 0.05 compared with control group
concentrations of corticosterone on day 9 and day 18 after depilation both were significantly increased in CRS group compared with control group (Table 1), confirming that CRS successfully induced psychological stress. Both of Tempol and RP67580 does not reduce CRS-induced increase of corticosterone concentration in the plasma (Table 1).
Thiobarbituric acid reactive substances (TBARS) are an ideal index to reflect the levels of lipid peroxidation. On day 9 and day 18 after depilation, TBARS levels in CRS group were significantly increased compared with control group. As a strong antioxidant, Tempol reasonable reduces CRS-induced increase of TBARB levels. RP67580 also normalized CRS-induced increase of TBARB levels (Table 2). Activities of SOD and GSH-Px in CRS group were markedly decreased than control group in skin. These results indicated that CRS-induced increase of oxidative stress might be through reduction of antioxidant enzyme activity. Tempol could increase CRS-induced reduction of GSH-Px activity but did not affect CRS-induced reduction of SOD activity. RP67580 also normalized CRS-induced reduction of SOD and GSH-Px activities (Tables 3, 4). These results suggested that CRS-induced alterations were mediated by SP-NK1-mast cell-ROS pathway, which were consistent with our previous report [29].
123
Arch Dermatol Res Table 2 Levels of skin TBARS in different experiment groups after 9-day and 18-day CRS Group
TBARS (lmmol/L/100 lg protein) 9-day
Control CRS CRS ? T CRS ? N
18-day
6.66 ± 2.27
7.09 ± 2.28
12.29 ± 3.08*
14.40 ± 5.12*
6.97 ± 3.01 7.43 ± 1.88
6.07 ± 4.13 7.47 ± 2.40
The Levels of skin TBARS on day 9 and 18 were measured Data are presented as mean ± SEM, n = 9 in each group CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580 * p \ 0.05 compared with control group p \ 0.05 compared with CRS group
Table 3 Activities of skin GSH-Px in different experiment groups after 9-day and 18-day CRS Group
Control
GSH-Px (U/100 lg protein) 9-day
18-day
422.22 ± 57.29
403.53 ± 82.09
CRS
239.58 ± 53.48*
259.22 ± 58.40*
CRS ? T
353.16 ± 66.63
394.97 ± 77.12
CRS ? N
377.59 ± 65.24
384.82 ± 47.76
were examined in mice subjected to CRS compared to control mice. On day 9 after depilation (late anagen), the skin color of neck region in control mice was darker than the stressed mice, and corresponding skin gray-scale ratio was significantly lower than CRS mice (Fig. 3). Morphological study shown most hair follicles of control mice enter anagen V/VI, and the majority of hair follicles in CRS mice was in anagen II/III. On day 18 after depilation (catagen), skin color of neck region in control mice was lighter than the CRS group, and corresponding skin grayscale ratio was significantly higher than CRS mice (Fig. 3). Meanwhile, most of hair follicles of control mice entered catagen V/VI; however, it was still in anagen VI in CRS mice. On day 9 and day 18, scores of hair cycle in control mice were both significantly higher than in CRS mice (Fig. 4). These results suggest CRS inhibited hair growth by prolonging the telogen stage and delaying subsequent onset of anagen and catagen stage. Both of Tempol and RP67580 significantly ameliorated CRS-induced alterations in skin gray scale and hair follicle score both on day 9 and day 18 (Figs. 3, 4), suggesting that ROS and SP are involved in CRS-induced delay of hair growth, which were also consistent with our previous observation [29]. Levels of autophagy marker, LC3-II and Beclin-1 proteins expression
The activities of skin GSH-Px on day 9 and 19 were measured Data are presented as mean ± SEM, n = 9 in each group CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580 * p \ 0.05 compared with control group p \ 0.05 compared with CRS group Table 4 Activities of skin SOD in different experiment groups after 9-day and 18-day CRS Group
Control
SOD (U/100 lg protein)) 9-day
18-day
11.22 ± 1.95
11.50 ± 2.26
CRS
8.38 ± 1.80*
7.52 ± 2.56*
CRS ? T CRS ? N
7.69 ± 1.32* 9.34 ± 0.85
6.70 ± 2.44* 9.58 ± 0.95
The activities of skin GSH-Px on day 9 and 19 were measured Data are presented as mean ± SEM, n = 9 in each group CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580 * p \ 0.05 compared with control group p \ 0.05 compared with CRS group
CRS induces delay of hair growth To investigate whether CRS affects the hair growth in vivo, the onset and termination of anagen stage after depilation
123
Expressions of autophagy protein levels were calculated by the ratio of LC3-II and Beclin-1 to GAPDH. Our results shown that CRS increased this ratio both on day 9 and day 18, indicating that CRS increased tissue autophagy levels, which may play a role in hair growth (Figs. 5, 6). Both of Tempol and RP67580 markedly reduced CRSinduced increase of LC3-II and Beclin-1 expressions, suggesting that CRS-induced increase of autophagy is mediated by ROS and SP signal pathways (Figs. 5, 6).
Discussion In the present study, we demonstrated that CRS-induced psychological stress, altered hair growth cycle, which was mediated by SP induced increase of ROS. In addition, CRS also induced skin autophagy levels, which was also regulated by SP and ROS signal pathway, suggesting the possible involvement of autophagy in CRS-induced delay of hair growth. Under stress state, the body will activate the autonomic nervous system to react quickly to cope with the stress stimulus. Meanwhile, it also activates the Hypothalamic– Pituitary–Adrenal axis (HPA) to facilitate the secretion of hormone, thus causing series of stress responses. CRS can influence the behaviors of mice, which has been confirmed
Arch Dermatol Res Fig. 3 Tempol and RP67580 suppress CRS-induced delay of hair growth cycle. The hair shafts of murine skin were shaven by animal clippers beforehand. a The representative area of the mouse dorsal skin is shown in each group on day 9 and 19. b The corresponding skin color grayscale ratio on day 9 was shown on the left and day 19 on the right. CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580. Data were presented as mean ± SEM, n = 9 in each group, *p \ 0.05 compared with control group, p \ 0.05 compare with CRS group
by the decreases of movement distance and standing times in the opening test, reflecting the anxious and the depressive behaviors in mice which have been demonstrated to be highly related with feeding behaviors [2, 13]. In the present study, we also observed that body weight gain was reduced in CRS model supporting CRS could affect feeding behaviors. All of these psychological changes were not related by increase of ROS and secretion of SP, which has been demonstrated by application of Tempol and RP67580 in the present study. It should be noted that cell death may also contribute to body weight gain loss. We found CRSinduced skin tissue to increase apoptosis in our paralleled experiments, although Tempol could reduce CRS-induced skin apoptosis, but not affect body weight gain, suggesting cell death may play minor role in CRS-induced body weight gain loss (Data not shown). Although compared with the activities of the autonomic nervous system, HPA axis responds relatively slower, its activity plays a major role in the prolonged stress. Thus, the corticosterone levels in the blood reflect the chronic stress state of the body. Our results shown the plasma corticosterone levels of mice in CRS group were significantly higher than that of in the Control group both on day 9 and
18, indicating that mice were under chronic stress state throughout whole experimental period. Long-term high levels of corticosterone may affect the immune system resulting in detrimental effects for the health [2]. Our results clearly demonstrated high relationship between corticosterone levels and hair growth conditions. Neither Tempol nor RP67580 affected the CRS-induced increase of corticosterone levels, suggesting corticorsterone release was the up-stream signal substance. The present time points (0, 4, 9, 14, and 18 days) were chosen according to the murine hair growth cycle, which was synchronized under telogen stage on day 0 after depilation, from late anagen entered catagen stage on day 9 after depilation, on day 18 after depilation when hair cycle of control mice just spontaneously enters the catagen transformation [26, 31]. Therefore, the present designed observing period could provide the effects of CRS on the whole hair growth cycle. Lots of research results shown the close relationship between lipid peroxidation malondialdehyde (MDA) and stress [38, 44, 50]. Silva et al. [44] found that after 72 h of sleep deprivation, hippocampus MDA in rat increased significantly compared with the control group. Sahin and Zafir also reported the increase of oxidative stress in the
123
Arch Dermatol Res Fig. 4 Tempol and RP67580 ameliorate CRS-induced delay of hair follicle growth on the hair cycle stage. a A representative area of each group on day 9 and day 18 after depilation with the majority of hair follicles. Original magnification was 9100. b The results of hair follicles score for day 9 were shown on the left and the data for day 18 on the right. The Y axis depicted histometric score assessed hair follicles stages. For each mouse a minimum of 10 individual visual fields were assigned to define hair follicles score. CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580. Green arrows point to the typical follicles in each group. Data were presented as mean ± SEM, n = 9 in each group, *p \ 0.05 compared with control group, p \ 0.05 compared with CRS group
Fig. 5 Tempol and RP67580 inhibit CRS-induced increase in LC3-II expression. a The representative blots of LC3-II and GAPDH are shown in each group. b Densitometric analysis of the ratio of LC3-II to GAPDH. CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580. Data were presented as mean ± SEM, n = 6 in each group, *p \ 0.05 compared with control group, p \ 0.05 compared with CRS group
123
Arch Dermatol Res Fig. 6 Tempol and RP67580 inhibit CRS-induced increase in Beclin-1 expression. a The representative blots of Beclin-1 and GAPDH are shown in each group. b Densitometric analysis of the ratio of Beclin-1 to GAPDH. CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580. Data were presented as mean ± SEM, n = 6 in each group, *p \ 0.05 compared with control group, p \ 0.05 compared with CRS group
brain, liver, stomach, heart, kidney tissue and the other organs of rat after 15 and 21 days of CRS [38, 50]. In skin diseases, oxidative stress also plays an important role. Schallreuter et al. [40] detected sustained accumulation of hydrogen peroxide in the epidermis of patients with vitiligo; Akar and Naziroglu reported, respectively, the increased activity of GSH-Px and SOD in the scalp tissue of patients with alopecia areata and the decreased activity of GSH-Px in plasma [1, 32]. In the present, we also observed the increase of oxidative stress in skin after CRS, which was consistent with previous study. In addition, our results also demonstrated that oxidative stress plays a pivotal role in CRS-induced delay of hair growth, because Tempol treatment could normalize CRS-induced abnormal of hair cycle. Previous studies have demonstrated SP induced neurogenic inflammation and mast cell degranulation which were involved in skin disorders, such as atopic dermatitis and psoriasis [5, 6, 23, 45, 48]. Recently, it was reported that ROS were second messengers of neurokinin signaling in peripheral sensory neurons [28]. Our results show that oxidative stress and antioxidant system could be regulated by administration of NK1 receptor blocker, confirming previous conception about SP-ROS signal pathway in CRS
model. The main target of SP is mast cells, which has been proven by previous reports [5, 6, 23, 45, 48]. In the present study, we did not show the possible source of ROS, it could be speculated that activation of mast cell would be the main cause of increase of ROS generation. Specific inhibition of mast cell activation or application of genetically mast cell-deficient mice such as W/Wv and SI/SId mice will provide better information for this issue [8, 47]. Furthermore, disodium cromoglycate is known to be a mast cell stabilizer, which may also provide information of the role of mast cell in the present research model [12]. Most of researchers focused on the tissue apoptosis in response to stress-induced injury. Recently, autophagy has emerged as a powerful mediator in response to cellular injury. Increase of oxidative stress and ROS has been reported to be important stimulation of autophagy [41, 49]. Since CRS has been demonstrated to increase oxidative stress in skin, it is reasonable that increased oxidative stress will induce autophagy. Our present data clearly demonstrated that CRS increased skin autophagy levels by elevation of LC3-II and Beclin-1 expressions through SP-ROS signal pathway. Although the roles of autophagy in the development of various kinds of diseases are still controversial, our results shown the highly relationship between
123
Arch Dermatol Res
the hair growth cycle and autophagy levels, indicating autophagy might play a role in CRS-induced delay of hair growth. Recently, clinical studies revealed that the activities of GSH-Px and SOD were increased in scalp of patients with alopecia areata [1]. Relatively, the activity of GSH-Px in plasma of the patients with alopecia was significantly decreased [32]. These clinical evidences indicated that antioxidant enzyme system played a critical role in the occurrence and development of alopecia. Our study now reports the first experimental evidence that oxidative stress is mediated by SP signal pathway in the abnormal of hair cycle as well as its role in regulation of cell autophagy. The present data may provide a better understanding and strategic application in clinical with psychological stressinduced hair growth disorders.
11.
12.
13.
14.
15. Acknowledgments This work is supported by the National Nature Science Foundation of China (81270316), the Research Program of Soochow University (Q413400111). Conflict of interest
16.
No declare. 17.
References 1. Akar A, Arca E, Erbil H, Akay C, Sayal A, Gur AR (2002) Antioxidant enzymes and lipid peroxidation in the scalp of patients with alopecia areata. J Dermatol Sci 29:85–90 2. Anisman H, Merali Z (2002) Cytokines, stress, and depressive illness. Brain Behav Immun 16:513–524 3. Ansel JC, Armstrong CA, Song I, Quinlan KL, Olerud JE, Caughman SW, Bunnett NW (1997) Interactions of the skin and nervous system. J Investig Dermatol Symp Proc 2:23–26 4. Aoki E, Shibasaki T, Kawana S (2003) Intermittent foot shock stress prolongs the telogen stage in the hair cycle of mice. Exp Dermatol 12:371–377 5. Arck PC, Handjiski B, Hagen E, Joachim R, Klapp BF, Paus R (2001) Indications for a ‘brain-hair follicle axis (BHA)’: inhibition of keratinocyte proliferation and up-regulation of keratinocyte apoptosis in telogen hair follicles by stress and substance P. FASEB J 15:2536–2538. doi:10.1096/fj.00-0699fje 6. Arck PC, Handjiski B, Peters EM, Peter AS, Hagen E, Fischer A, Klapp BF, Paus R (2003) Stress inhibits hair growth in mice by induction of premature catagen development and deleterious perifollicular inflammatory events via neuropeptide substance P-dependent pathways. Am J Pathol 162:803–814. doi:10.1016/ S0002-9440(10)63877-1 7. Arck PC, Slominski A, Theoharides TC, Peters EM, Paus R (2006) Neuroimmunology of stress: skin takes center stage. J Invest Dermatol 126:1697–1704. doi:10.1038/sj.jid.5700104 8. Askenase PW, Van Loveren H, Kraeuter-Kops S, Ron Y, Meade R, Theoharides TC, Nordlund JJ, Scovern H, Gerhson MD, Ptak W (1983) Defective elicitation of delayed-type hypersensitivity in W/Wv and SI/SId mast cell-deficient mice. J Immunol 131:2687–2694 9. Bauer ME, Jeckel CM, Luz C (2009) The role of stress factors during aging of the immune system. Ann NY Acad Sci 1153:139–152. doi:10.1111/j.1749-6632.2008.03966.x 10. Beaujouan JC, Heuillet E, Petitet F, Saffroy M, Torrens Y, Glowinski J (1993) Higher potency of RP 67580, in the mouse
123
18.
19.
20.
21.
22.
23.
24.
25. 26.
27.
and the rat compared with other nonpeptide and peptide tachykinin NK1 antagonists. Br J Pharmacol 108:793–800 Canu N, Tufi R, Serafino AL, Amadoro G, Ciotti MT, Calissano P (2005) Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells. J Neurochem 92:1228–1242. doi:10.1111/j.1471-4159.2004. 02956.x Chen L, Schrementi ME, Ranzer MJ, Wilgus TA, Dipietro LA (2014) Blockade of mast cell activation reduces cutaneous scar formation. PLoS One 9:e85226. doi:10.1371/journal.pone.0085226 Chiba S, Numakawa T, Ninomiya M, Richards MC, Wakabayashi C, Kunugi H (2012) Chronic restraint stress causes anxietyand depression-like behaviors, downregulates glucocorticoid receptor expression, and attenuates glutamate release induced by brain-derived neurotrophic factor in the prefrontal cortex. Prog Neuropsychopharmacol Biol Psychiatry 39:112–119. doi:10. 1016/j.pnpbp.2012.05.018 Codogno P, Meijer AJ (2005) Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ 12(Suppl 2):1509–1518. doi:10.1038/sj.cdd.4401751 Dhabhar FS (2013) Psychological stress and immunoprotection versus immunopathology in the skin. Clin Dermatol 31:18–30. doi:10.1016/j.clindermatol.2011.11.003 Gu HF, Tang CK, Yang YZ (2012) Psychological stress, immune response, and atherosclerosis. Atherosclerosis 223:69–77. doi:10. 1016/j.atherosclerosis.2012.01.021 Gutteridge JM (1995) Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin Chem 41:1819–1828 Hahn SM, Tochner Z, Krishna CM, Glass J, Wilson L, Samuni A, Sprague M, Venzon D, Glatstein E, Mitchell JB et al (1992) Tempol, a stable free radical, is a novel murine radiation protector. Cancer Res 52:1750–1753 Huang J, Lam GY, Brumell JH (2011) Autophagy signaling through reactive oxygen species. Antioxid Redox Signal 14:2215–2231. doi:10.1089/ars.2010.3554 Hutchinson KM, McLaughlin KJ, Wright RL, Bryce Ortiz J, Anouti DP, Mika A, Diamond DM, Conrad CD (2012) Environmental enrichment protects against the effects of chronic stress on cognitive and morphological measures of hippocampal integrity. Neurobiol Learn Mem 97:250–260. doi:10.1016/j.nlm. 2012.01.003 Joachim RA, Sagach V, Quarcoo D, Dinh QT, Arck PC, Klapp BF (2004) Neurokinin-1 receptor mediates stress-exacerbated allergic airway inflammation and airway hyperresponsiveness in mice. Psychosom Med 66:564–571. doi:10.1097/01.psy. 0000132878.08780.93 Katayama M, Aoki E, Suzuki H, Kawana S (2007) Foot shock stress prolongs the telogen stage of the spontaneous hair cycle in a non-depilated mouse model. Exp Dermatol 16:553–560. doi:10. 1111/j.1600-0625.2007.00558.x Kawana S, Liang Z, Nagano M, Suzuki H (2006) Role of substance P in stress-derived degranulation of dermal mast cells in mice. J Dermatol Sci 42:47–54. doi:10.1016/j.jdermsci.2005.12. 004 Kennedy PG, Rodgers J, Jennings FW, Murray M, Leeman SE, Burke JM (1997) A substance P antagonist, RP-67,580, ameliorates a mouse meningoencephalitic response to Trypanosoma brucei brucei. Proc Natl Acad Sci USA 94:4167–4170 Klionsky DJ, Emr SD (2000) Autophagy as a regulated pathway of cellular degradation. Science 290:1717–1721 Krause K, Foitzik K (2006) Biology of the hair follicle: the basics. Semin Cutan Med Surg 25:2–10. doi:10.1016/j.sder.2006. 01.002 Lawrence RA, Burk RF (1976) Glutathione peroxidase activity in selenium-deficient rat liver. Biochem Biophys Res Commun 71:952–958
Arch Dermatol Res 28. Linley JE, Ooi L, Pettinger L, Kirton H, Boyle JP, Peers C, Gamper N (2012) Reactive oxygen species are second messengers of neurokinin signaling in peripheral sensory neurons. Proc Natl Acad Sci USA 109:E1578–E1586. doi:10.1073/pnas. 1201544109 29. Liu N, Wang LH, Guo LL, Wang GQ, Zhou XP, Jiang Y, Shang J, Murao K, Chen JW, Fu WQ, Zhang GX (2013) Chronic restraint stress inhibits hair growth via substance P mediated by reactive oxygen species in mice. PLoS One 8:e61574. doi:10. 1371/journal.pone.0061574 30. Maggi CA, Patacchini R, Rovero P, Giachetti A (1993) Tachykinin receptors and tachykinin receptor antagonists. J Auton Pharmacol 13:23–93 31. Muller-Rover S, Handjiski B, van der Veen C, Eichmuller S, Foitzik K, McKay IA, Stenn KS, Paus R (2001) A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J Invest Dermatol 117:3–15. doi:10. 1046/j.0022-202x.2001.01377.x 32. Naziroglu M, Kokcam I (2000) Antioxidants and lipid peroxidation status in the blood of patients with alopecia. Cell Biochem Funct 18:169–173. doi:10.1002/1099-0844(200009)18:3\169: AID-CBF870[3.0.CO;2-T 33. Paus R, Botchkarev VA, Botchkareva NV, Mecklenburg L, Luger T, Slominski A (1999) The skin POMC system (SPS). Leads and lessons from the hair follicle. Ann NY Acad Sci 885:350–363 34. Paus R, Cotsarelis G (1999) The biology of hair follicles. N Engl J Med 341:491–497. doi:10.1056/NEJM199908123410706 35. Peskin AV, Winterbourn CC (2000) A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1). Clin Chim Acta 293:157–166 36. Peters EM (2012) Psychological support of skin cancer patients. Br J Dermatol 167:105–110. doi:10.1111/j.1365-2133.2012.11094.x 37. Ravikumar B, Berger Z, Vacher C, O’Kane CJ, Rubinsztein DC (2006) Rapamycin pre-treatment protects against apoptosis. Hum Mol Genet 15:1209–1216. doi:10.1093/hmg/ddl036 38. Sahin E, Gu¨mu¨s¸ lu¨ S (2007) Immobilization stress in rat tissues: alterations in protein oxidation, lipid peroxidation and antioxidant defense system. Comp Biochem Physiol C: Toxicol Pharmacol 144:342–347. doi:10.1016/j.cbpc.2006.10.009 39. Scandalios JG (2005) Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses. Braz J Med Biol Res 38:995–1014 (S0100-879X2005000700003) 40. Schallreuter KU, Moore J, Wood JM, Beazley WD, Gaze DC, Tobin DJ, Marshall HS, Panske A, Panzig E, Hibberts NA (1999) In vivo and in vitro evidence for hydrogen peroxide (H2O2) accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase. J Investig Dermatol Symp Proc 4:91–96 41. Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, Elazar Z (2007) Reactive oxygen species are essential for autophagy and
42.
43.
44.
45.
46. 47.
48.
49.
50.
51.
52.
specifically regulate the activity of Atg4. EMBO J 26:1749–1760. doi:10.1038/sj.emboj.7601623 Schnackenberg CG, Wilcox CS (1999) Two-week administration of tempol attenuates both hypertension and renal excretion of 8-Iso prostaglandin f2alpha. Hypertension 33:424–428 Seo JS, Park JY, Choi J, Kim TK, Shin JH, Lee JK, Han PL (2012) NADPH oxidase mediates depressive behavior induced by chronic stress in mice. J Neurosci 32:9690–9699. doi:10.1523/ JNEUROSCI.0794-12.2012 Silva RH, Abilio VC, Takatsu AL, Kameda SR, Grassl C, Chehin AB, Medrano WA, Calzavara MB, Registro S, Andersen ML, Machado RB, Carvalho RC, Ribeiro Rde A, Tufik S, Frussa-Filho R (2004) Role of hippocampal oxidative stress in memory deficits induced by sleep deprivation in mice. Neuropharmacology 46:895–903. doi:10.1016/j.neuropharm.2003.11.032 Singh LK, Pang X, Alexacos N, Letourneau R, Theoharides TC (1999) Acute immobilization stress triggers skin mast cell degranulation via corticotropin releasing hormone, neurotensin, and substance P: a link to neurogenic skin disorders. Brain Behav Immun 13:225–239. doi:10.1006/brbi.1998.0541 Spencer LV, Callen JP (1987) Hair loss in systemic disease. Dermatol Clin 5:565–570 Taguchi Y, Tsuyama K, Watanabe T, Wada H, Kitamura Y (1982) Increase in histidine decarboxylase activity in skin of genetically mast-cell-deficient W/Wv mice after application of phorbol 12-myristate 13-acetate: evidence for the presence of histamine-producing cells without basophilic granules. Proc Natl Acad Sci USA 79:6837–6841 Theoharides TC, Donelan JM, Papadopoulou N, Cao J, Kempuraj D, Conti P (2004) Mast cells as targets of corticotropin-releasing factor and related peptides. Trends Pharmacol Sci 25:563–568. doi:10.1016/j.tips.2004.09.007 Wu HH, Hsiao TY, Chien CT, Lai MK (2009) Ischemic conditioning by short periods of reperfusion attenuates renal ischemia/ reperfusion induced apoptosis and autophagy in the rat. J Biomed Sci 16:19. doi:10.1186/1423-0127-16-19 Zafir A, Banu N (2009) Induction of oxidative stress by restraint stress and corticosterone treatments in rats. Indian J Biochem Biophys 46:53–58 Zhang GX, Kimura S, Murao K, Shimizu J, Matsuyoshi H, Takaki M (2009) Role of neuronal NO synthase in regulating vascular superoxide levels and mitogen-activated protein kinase phosphorylation. Cardiovasc Res 81:389–399. doi:10.1093/cvr/ cvn304 Zhang GX, Kimura S, Nishiyama A, Shokoji T, Rahman M, Abe Y (2004) ROS during the acute phase of Ang II hypertension participates in cardiovascular MAPK activation but not vasoconstriction. Hypertension 43:117–124. doi:10.1161/01.HYP. 0000105110.12667.F8
123
ORIGINAL PAPER
Oxidative stress and substance P mediate psychological stress-induced autophagy and delay of hair growth in mice Lei Wang • Ling-Ling Guo • Lin-Hui Wang • Guo-Xing Zhang • Jing Shang • Koji Murao • Deng-Feng Chen • Xiang-Hua Fan • Wen-Qing Fu
Received: 5 October 2013 / Revised: 2 October 2014 / Accepted: 30 October 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Neuropeptide substance P (SP) and reactive oxygen species (ROS) have been demonstrated to play an important role in psychological stress-induced alteration of hair cycle, the underlying mechanism remains unknown. The present study aims to investigate possible contribution of SP and ROS in chronic restraint stress (CRS, a chronic psychological stress model) induced abnormal of hair cycle and induction of autophagy. Mouse CRS model was applied for 18 days with or without treatment antioxidant Tempol (a free radical scavenger) or SP receptor (NK1) antagonist (RP67580). After CRS procedure, hair growth cycle, oxidative stress markers and skin tissue autophagy levels were analyzed by ELISA or western blot. Our results revealed that CRS reduced body weight gain, distance of movement and times of standing, affected hair cycle by prolonging the telogen stage and delaying subsequent anagen and catagen stage. In addition, CRS resulted in
L. Wang, L. - L. Guo, L.- H. Wang these authors contributed equally to this work. L. Wang W.-Q. Fu (&) Department of Clinical Psychology, Medical College of Soochow University, 199 Ren-Ai Road, Dushu Lake Campus, Suzhou Industrial Park, Suzhou 215123, People’s Republic of China e-mail: [email protected] L.-L. Guo Department of Pathology, Medical College of Soochow University, 199 Ren-Ai Road, Dushu Lake Campus, Suzhou Industrial Park, Suzhou 215123, People’s Republic of China L.-H. Wang G.-X. Zhang (&) Department of Physiology, Medical College of Soochow University, 199 Ren-Ai Road, Dushu Lake Campus, Suzhou Industrial Park, Suzhou 215123, People’s Republic of China e-mail: [email protected]
increase of lipid peroxidation levels and reduction of the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and increase of autophagy markers (microtubule-associated proteins, light chain 3-II, LC3-II, and Beclin-1) in mice skin. Treatment with Tempol restored GSH-Px activity, and significantly reduced increases of lipid peroxidation levels and LC3-II and Beclin-1 expressions, as well as normalized hair cycle. In addition; RP67580 also restored SOD and GSH-Px activities, and markedly reduced increases of lipid peroxidation levels and LC3-II and Beclin-1 expressions, and normalized hair cycle. Our study provides the first strong evidence for SP and ROS play a role not only in alteration of hair cycle but also in induction of autophagy in psychological stress model, suggesting autophagy may contribute to psychological stress-induced abnormal of hair cycle. Keywords Chronic restraint stress Hair cycle Reactive oxygen species Substance P Autophagy
J. Shang New Drug Screening Center, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China K. Murao Department of Clinical Laboratory, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe Miki-cho, Kita-gun, Kagawa 761-0793, Japan D.-F. Chen Department of Surgery, Harrison International Peace Hospital, 2 Ren-Ming Road, Hengsui 053000, People’s Republic of China X.-H. Fan Department of Dermatology, the Second Affiliated Hospital of Nangtong University, 6 Hai-Er Road, Nantong 226001, People’s Republic of China
123
Arch Dermatol Res
Introduction Psychological stress has been reported to be highly related to the development of skin diseases [9, 15, 16, 36]. Previous studies showed that psychological stress affected hair cycle mediated by neurohormone, neurotransmitter and cytokines [33, 46]. Arck et al. [6] have found that when the mice were irritated by the noise for 24 h, hair growth was inhibited by induction of premature catagen development via neuropeptide substance P (SP)-dependent pathways. Studies of the foot shock model also demonstrated that the telogen has been prolonged and the anagen has been deferred [4, 22]. However, neither of the 24-h noise nor the foot shock is sufficient to imitate the state of human being’s chronic stress and to prove the existence of somatic stress element. Chronic restrained stress (CRS) can efficiently simulate the long-term stress situation of human kind [13, 20, 43]. It could evoke depression-like behavioral changes accompanied by working memory and learning deficits. Using this model, it may provide better understanding of abnormal of hair growth in psychological stress condition. Clinical studies have shown that SP-mass cell pathway is involved in various pathogenesis of skin disease. Arck et al. reported SP is the central neurotransmitter of brainskin axis [5–7, 45, 48]. SP is one kind of the neuropeptides related to stress. It is synthesized in the neurons of dorsal root ganglion and released by the sensory nerve ending of skin. It can induce activation of mast cells and then release the pro-inflammatory factor resulting in neurogenic scytitis [7, 23, 45, 48]. SP has three main receptors, which are named as NK1, NK2 and NK3 receptor. Of them, NK1 receptor has the highest affinity to SP and is widely distributed [3, 21, 30]. In the previous studies, pseudo-peptide, RP 67580, was reported as the higher potency blocker of NK1 because of its highest affinity on NK1 [10, 24]. Under normal circumstances, oxidation-antioxidant system is stable and can maintain the metabolic balance of free radicals. The reactive oxygen species (ROS) are controlled at lower levels [39]. But under internal and external stimulation, ROS generation may increase in rapid speed and cause the disorders of oxidation and antioxidant balance, which cause oxidative damage of biofilm and macromolecular material. Studies from Akar et al. and Naziroglu et al. proved that oxidative stress plays an important role in the process of hair loss [1, 32]. In addition, CRS resulted in the oxidative stress injury of the rat’s stomach, liver, kidney, heart, brain and viscera [38, 50]. Furthermore, our recent study also demonstrated that ROS play a role in regulation of psychological stress-induced SP-Mast cell pathway and hair growth cycle [29]. 4-Hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol) is a membrane-permeable superoxide dismutase (SOD) mimetic that possesses potent antioxidant activity. Our
123
previous studies have demonstrated its strong effects on ROS scavenge [51, 52]. Autophagy played an important role in maintaining cellular homeostasis, differentiation and tissue remodeling. Paradoxically, the role of autophagy in cell survival is still controversial. Nutrient depletion induced autophagy provides amino acids for synthesis of essential proteins, thus prolonging cell survival [25]. It was also reported that autophagy could counteract cell apoptosis [37]. Other studies, however, have shown that autophagy served as an alternative form of cell death [11, 14]. ROS have been reported to be essential for induction of autophagy [41, 49]. Similarly, role of ROS-induced autophagy is still controversial, it may depend on the degree of autophagy, in which mild induction of autophagy by ROS may have cytoprotective effects, but massive induction of autophagy by ROS may cause excessive self-digestion of cell components and lead to death [19]. Whether CRS will induce autophagy and how it is regulated in skin are still unknown. Based on the above reports, the present study will apply antioxidant and SP receptor (NK1) antagonist in the experimental CRS mice model, observing hair growth cycle, detecting autophagy protein markers (LC3-II, Beclin-1), exploring the mechanisms in CRS-induced abnormal of hair growth.
Method Animals Six to eight weeks old healthy male C57BL/6 mice were used (weight 20 ± 2 g), provided by center of experimental animal, Soochow University. All mice were acclimated under the following conditions for one week: room temperature 21–24 °C; humidity 50–60 % and a 12-h light: dark cycle (lights on at 6:00 a.m. and off at 18:00 p.m.). During this period, food and water are provided ad libitum. The mice were randomly divided into the following groups: (1) Control group (10 mice); (2) CRS group (10 mice): no drug injection and application of CRS; (3) Tempol group (10 mice): application of CRS concomitant with 200 mg/kg/day of intraperitoneal injection of Tempol for 18 days; (4) NK1 blocker, RP67580 group (10 mice): application of CRS concomitant with 200 mg/mouse of intraperitoneal injection of RP67580 for 18 days (every other day). The present study was designed according to our previous report [29]. Induction of anagen and application of CRS Method for induction of anagen was as described previously [6]. On the first day of the experiment, wax/rosin mixture (1:1 on weight) was applied to the dorsal skin
Arch Dermatol Res
(from neck to tail) of mice, after it dried and then peelingoff the wax/rosin mixture, removed all hair shafts and immediately induced a highly synchronized hair growth, as evidenced by the homogeneously pink skin color in the back, indicating all hair follicles in telogen [26, 34]. CRS starts on day 1 after depilation for 6 h daily (10:00 a.m.16:00 p.m.) while food and water were prohibited. Period of CRS was lasted for 9 or 18 days and the control mice were kept in their original cage, but food and water were not provided during the CRS period. All mice were taken pictures with a digital camera (Panasonic, Japan) on the 9th and 18th day, respectively.
cycle of control mice just spontaneously enters the catagen transformation [26, 31], mice were killed; plasma and skin tissues were sampled according to above mentioned method. Measurement of body weight and plasma corticosterone concentration All mice were monitored body weight on day 0, 4, 9, 14, 18 after depilation. Plasma corticosterone concentration was determined by mouse corticosterone RIA kit following manufacturer’s instruction (Beyotime, China). Protein concentration measurement
Reagents RP67580 (Santa Cruz biotechnology,CA, USA), a selective antagonist of the tachykinin NK1 receptor, was dissolved in ethanol and then diluted with PBS to make a final concentration of 1 mg/ml. 4-Hydroxy-2, 2, 6, 6-tetramethyl piperidinoxyl, Tempol (Sigma, MO, USA), is a stable membranepermeable superoxide dismutase (SOD) mimetic that exhibits potent antioxidant activity against superoxide as well as hydroxyl radicals [42]. In the present study, Tempol was injected intraperitoneally to against oxidative stress in skin at a dose of 200 mg/kg/day according to previous report [18]. Open-field test Two time points were chosen to evaluate motor function and state anxiety of the mice. One was on day 9; the other was on day18. The mice were placed into the central grid, recording the mice’s distance of movement and times of standing in 3 min. Distance of movement reflects the ability of autonomous activity in mice; times of standing reflect exploratory behavior in mice. The reduction of distance of movement and times of standing shows that restraint stress results in a state of depression in mice. Tissue and plasma preparation Two time points were chosen to indicate different hair cycle stage. One, on day 9 after depilation when hair cycle of control mice was in the late anagen [26, 31], blood samples were collected in the evening after 9 days CRS under anesthesia by eyeball removal method, then centrifuged at 3,000 rpm for 15 min to obtain plasma, then stored at -80 °C immediately. Skin specimens from back skin (the region of neck) were harvested about 2 9 5 cm, which is the region of neck, which is an essential section for the quantitative histomorphology of the hair cycle [6], was fixed in 4 % paraformaldehyde, then paraffin-wax embedded. The remaining skin specimens are cryopreserved at -80 °C immediately. Second, on day 18 after depilation when hair
The skin tissues were homogenized in a solution containing 0.15 mol/L KCl and 0.02 mol/L Tris–HCl (pH 7.4), then used BCA Protein Assay Kit (Beyotime, China) to determine protein concentration. Finally, the skin tissue homogenates were adjusted at a concentration of 1 mg/ml for further analysis. Measurement of lipid peroxidation levels in skin The method for measurement of thiobarbituric acid reactive substances (TBARS) levels has been described previously [17]. Briefly, 100 lL sample solution was mixed with 15 % trichloroacetic acid and 0.375 % thiobarbituric acid. Butylated hydroxytoluene (0.01 %) was added to the assay mixture to prevent autoxidation of the sample, and the mixture was heated at 100 °C for 15 min. After cooling, the mixture was centrifuged at 14,000 rpm for 5 min, and the absorbance of the organic phase was measured at 535 nm. The amount of thiobarbituric acid reactive substances (TBARS) was determined by the malondialdehyde standard curve and expressed as lmol/L in 100 lg protein. Measurement of SOD and GSH-Px activities in skin SOD activity was detected according to previous report [35]. In brief, xanthine–xanthine oxidase complex produces superoxide radicals, which react with water-soluble tetrazolium (WST-1). GSH-Px activity using H2O2 as a substrate was assayed by an adaptation of the method reported by Lawrence and Burk [27]. Using 96-well plate, SOD activity was measured at 450 nm and GSH-Px activity was measured at 412 nm by Micro-plate reader, and both of the data were presented as U/100 lg protein. Assessment of hair cycle Mouse skin color changes according to hair growth cycle, which becomes darker and darker from anagen to catagen, then gradually become lighter from catagen to telogen [31].
123
Arch Dermatol Res
The grayscale (0–255) of specific area in photograph (the region of neck) was analyzed by Image J software and presented as ratios (grayscale/255) on day 9 after depilation when the mice were shaved. HE stain was used to quantify the stage of the hair follicles using a published classification technique based on the morphology of the dermal papilla and sebaceous glands [26]. To identify accurately, all hair follicles were scored which was assigned to the telogen, anagen and catagen stages according to its morphological and histological difference (anagen I–VI; catagen I–VIII; telogen) as described in previous reports [31, 38]. In brief, after identifying the stage of each hair follicles according to corresponding special characteristic of each stage, every stage of anagen or catagen is assigned a factor in ascending numerical order (anagen: anagen I = factor 1, anagen II = factor 2, anagen III = factor 3, etc.; for catagen: catagen I = factor 1, catagen II = factor 2, catagen III = factor 3, etc.). The number of HF in each specific stage is multiplied by the corresponding factor. The results of each sum were totaled and divided by the total number of HF counted [31]. Western blotting for LC3-II and Beclin-1 expression Expressions of LC3-II, Beclin-1 in the mice skin were measured by western blotting, as previously described [51]. Briefly, equal amount of protein from each homogenate was resolved by 10 % SDS–PAGE. Proteins were transferred to PVDF membranes (Hybond TM-ECL; Amersham Pharmacia Biotech, Inc.). The membranes were blocked for 2 h at room temperature with 5 % skimmed milk in PBS and 0.1 % Tween 20. The blots were incubated overnight with 1:1,000 diluted primary antibodies: anti-LC3-II and anti-Beclin-1 (Abcam, Inc.) followed by incubation for 1 h with a secondary antibody (HRP-conjugated anti-rabbit IgG; 1:2,000). Immunoreactive bands were visualized using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech) and analyzed by NIH image software. Data were normalized by GAPDH. Statistical analysis All data were presented as the mean ± S.E.M. Statistical significance between two groups was tested using one way ANOVA followed by the Post hoc test. P values \0.05 were considered statistically significant.
Results
Fig. 1 Tempol and RP67580 have no effect on CRS-induced body weight gain loss. a A representative picture of each group on day 9 and day 18 after CRS. b Body weight gains were monitored in each group on day 0, 4, 9, 14, 19. CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580. Data are presented as mean ± SEM, n = 9 in each group, *p \ 0.05 compared with control group
significantly on day 4, 9, 14 and 18 compared with control group. Neither of Tempol and RP67580 affected CRSinduced reduction of body weight gain (Fig. 1). Through the open-field test we recorded the distance of movement and the times of standing in each group. Distance of movement reflects the ability of autonomic activities, and times of standing reflect the exploratory behavior in mice. CRS markedly reduced the distance of movement and number of standing, suggesting CRS resulted in a state of depression in mice. Neither Tempol nor RP67580 had any effects on CRS-induced reduction of the distance of movement and number of standing, indicating both of Tempol nor could RP67580 not ameliorate CRS-induced depression (Fig. 2a, b).
Assessment of body weight gain and behavior The plasma levels of corticosterone Body weight was monitored on day 0, 4, 9, 14 and 18. Body weight gain was obtained by calculation. Our results showed that CRS inhibited mice body weight gain
123
The primary glucocorticoid, corticosterone, was named as stress hormone to monitor the state of stress. Plasma
Arch Dermatol Res Fig. 2 Tempol and RP67580 do not affect CRS-induced depressive behavior. a Times of standing are shown in each group on day 9 and 19. b distance of movement in each group is shown on day 9 and 19. CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580. Data were presented as mean ± SEM, n = 9 in each group, *p \ 0.05 compared with control group
Table 1 Levels of plasma corticosterone in different experiment groups after 9-day and 18-day CRS Group
The levels of lipid peroxide and the activities of SOD and GSH-Px in skin tissue
Levels of plasma corticosterone (ng/L) 9-day
18-day
Control
38.52 ± 5.03
37.91 ± 4.65
CRS
48.15 ± 3.87*
47.29 ± 3.50*
CRS ? T CRS ? N
46.73 ± 4.12* 46.22 ± 3.64*
49.85 ± 6.86* 48.32 ± 5.11*
The plasma corticosterone concentrations of mice on day 9 and 18 were measured Data are presented as mean ± SEM, n = 9 in each group CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580 * p \ 0.05 compared with control group
concentrations of corticosterone on day 9 and day 18 after depilation both were significantly increased in CRS group compared with control group (Table 1), confirming that CRS successfully induced psychological stress. Both of Tempol and RP67580 does not reduce CRS-induced increase of corticosterone concentration in the plasma (Table 1).
Thiobarbituric acid reactive substances (TBARS) are an ideal index to reflect the levels of lipid peroxidation. On day 9 and day 18 after depilation, TBARS levels in CRS group were significantly increased compared with control group. As a strong antioxidant, Tempol reasonable reduces CRS-induced increase of TBARB levels. RP67580 also normalized CRS-induced increase of TBARB levels (Table 2). Activities of SOD and GSH-Px in CRS group were markedly decreased than control group in skin. These results indicated that CRS-induced increase of oxidative stress might be through reduction of antioxidant enzyme activity. Tempol could increase CRS-induced reduction of GSH-Px activity but did not affect CRS-induced reduction of SOD activity. RP67580 also normalized CRS-induced reduction of SOD and GSH-Px activities (Tables 3, 4). These results suggested that CRS-induced alterations were mediated by SP-NK1-mast cell-ROS pathway, which were consistent with our previous report [29].
123
Arch Dermatol Res Table 2 Levels of skin TBARS in different experiment groups after 9-day and 18-day CRS Group
TBARS (lmmol/L/100 lg protein) 9-day
Control CRS CRS ? T CRS ? N
18-day
6.66 ± 2.27
7.09 ± 2.28
12.29 ± 3.08*
14.40 ± 5.12*
6.97 ± 3.01 7.43 ± 1.88
6.07 ± 4.13 7.47 ± 2.40
The Levels of skin TBARS on day 9 and 18 were measured Data are presented as mean ± SEM, n = 9 in each group CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580 * p \ 0.05 compared with control group p \ 0.05 compared with CRS group
Table 3 Activities of skin GSH-Px in different experiment groups after 9-day and 18-day CRS Group
Control
GSH-Px (U/100 lg protein) 9-day
18-day
422.22 ± 57.29
403.53 ± 82.09
CRS
239.58 ± 53.48*
259.22 ± 58.40*
CRS ? T
353.16 ± 66.63
394.97 ± 77.12
CRS ? N
377.59 ± 65.24
384.82 ± 47.76
were examined in mice subjected to CRS compared to control mice. On day 9 after depilation (late anagen), the skin color of neck region in control mice was darker than the stressed mice, and corresponding skin gray-scale ratio was significantly lower than CRS mice (Fig. 3). Morphological study shown most hair follicles of control mice enter anagen V/VI, and the majority of hair follicles in CRS mice was in anagen II/III. On day 18 after depilation (catagen), skin color of neck region in control mice was lighter than the CRS group, and corresponding skin grayscale ratio was significantly higher than CRS mice (Fig. 3). Meanwhile, most of hair follicles of control mice entered catagen V/VI; however, it was still in anagen VI in CRS mice. On day 9 and day 18, scores of hair cycle in control mice were both significantly higher than in CRS mice (Fig. 4). These results suggest CRS inhibited hair growth by prolonging the telogen stage and delaying subsequent onset of anagen and catagen stage. Both of Tempol and RP67580 significantly ameliorated CRS-induced alterations in skin gray scale and hair follicle score both on day 9 and day 18 (Figs. 3, 4), suggesting that ROS and SP are involved in CRS-induced delay of hair growth, which were also consistent with our previous observation [29]. Levels of autophagy marker, LC3-II and Beclin-1 proteins expression
The activities of skin GSH-Px on day 9 and 19 were measured Data are presented as mean ± SEM, n = 9 in each group CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580 * p \ 0.05 compared with control group p \ 0.05 compared with CRS group Table 4 Activities of skin SOD in different experiment groups after 9-day and 18-day CRS Group
Control
SOD (U/100 lg protein)) 9-day
18-day
11.22 ± 1.95
11.50 ± 2.26
CRS
8.38 ± 1.80*
7.52 ± 2.56*
CRS ? T CRS ? N
7.69 ± 1.32* 9.34 ± 0.85
6.70 ± 2.44* 9.58 ± 0.95
The activities of skin GSH-Px on day 9 and 19 were measured Data are presented as mean ± SEM, n = 9 in each group CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580 * p \ 0.05 compared with control group p \ 0.05 compared with CRS group
CRS induces delay of hair growth To investigate whether CRS affects the hair growth in vivo, the onset and termination of anagen stage after depilation
123
Expressions of autophagy protein levels were calculated by the ratio of LC3-II and Beclin-1 to GAPDH. Our results shown that CRS increased this ratio both on day 9 and day 18, indicating that CRS increased tissue autophagy levels, which may play a role in hair growth (Figs. 5, 6). Both of Tempol and RP67580 markedly reduced CRSinduced increase of LC3-II and Beclin-1 expressions, suggesting that CRS-induced increase of autophagy is mediated by ROS and SP signal pathways (Figs. 5, 6).
Discussion In the present study, we demonstrated that CRS-induced psychological stress, altered hair growth cycle, which was mediated by SP induced increase of ROS. In addition, CRS also induced skin autophagy levels, which was also regulated by SP and ROS signal pathway, suggesting the possible involvement of autophagy in CRS-induced delay of hair growth. Under stress state, the body will activate the autonomic nervous system to react quickly to cope with the stress stimulus. Meanwhile, it also activates the Hypothalamic– Pituitary–Adrenal axis (HPA) to facilitate the secretion of hormone, thus causing series of stress responses. CRS can influence the behaviors of mice, which has been confirmed
Arch Dermatol Res Fig. 3 Tempol and RP67580 suppress CRS-induced delay of hair growth cycle. The hair shafts of murine skin were shaven by animal clippers beforehand. a The representative area of the mouse dorsal skin is shown in each group on day 9 and 19. b The corresponding skin color grayscale ratio on day 9 was shown on the left and day 19 on the right. CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580. Data were presented as mean ± SEM, n = 9 in each group, *p \ 0.05 compared with control group, p \ 0.05 compare with CRS group
by the decreases of movement distance and standing times in the opening test, reflecting the anxious and the depressive behaviors in mice which have been demonstrated to be highly related with feeding behaviors [2, 13]. In the present study, we also observed that body weight gain was reduced in CRS model supporting CRS could affect feeding behaviors. All of these psychological changes were not related by increase of ROS and secretion of SP, which has been demonstrated by application of Tempol and RP67580 in the present study. It should be noted that cell death may also contribute to body weight gain loss. We found CRSinduced skin tissue to increase apoptosis in our paralleled experiments, although Tempol could reduce CRS-induced skin apoptosis, but not affect body weight gain, suggesting cell death may play minor role in CRS-induced body weight gain loss (Data not shown). Although compared with the activities of the autonomic nervous system, HPA axis responds relatively slower, its activity plays a major role in the prolonged stress. Thus, the corticosterone levels in the blood reflect the chronic stress state of the body. Our results shown the plasma corticosterone levels of mice in CRS group were significantly higher than that of in the Control group both on day 9 and
18, indicating that mice were under chronic stress state throughout whole experimental period. Long-term high levels of corticosterone may affect the immune system resulting in detrimental effects for the health [2]. Our results clearly demonstrated high relationship between corticosterone levels and hair growth conditions. Neither Tempol nor RP67580 affected the CRS-induced increase of corticosterone levels, suggesting corticorsterone release was the up-stream signal substance. The present time points (0, 4, 9, 14, and 18 days) were chosen according to the murine hair growth cycle, which was synchronized under telogen stage on day 0 after depilation, from late anagen entered catagen stage on day 9 after depilation, on day 18 after depilation when hair cycle of control mice just spontaneously enters the catagen transformation [26, 31]. Therefore, the present designed observing period could provide the effects of CRS on the whole hair growth cycle. Lots of research results shown the close relationship between lipid peroxidation malondialdehyde (MDA) and stress [38, 44, 50]. Silva et al. [44] found that after 72 h of sleep deprivation, hippocampus MDA in rat increased significantly compared with the control group. Sahin and Zafir also reported the increase of oxidative stress in the
123
Arch Dermatol Res Fig. 4 Tempol and RP67580 ameliorate CRS-induced delay of hair follicle growth on the hair cycle stage. a A representative area of each group on day 9 and day 18 after depilation with the majority of hair follicles. Original magnification was 9100. b The results of hair follicles score for day 9 were shown on the left and the data for day 18 on the right. The Y axis depicted histometric score assessed hair follicles stages. For each mouse a minimum of 10 individual visual fields were assigned to define hair follicles score. CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580. Green arrows point to the typical follicles in each group. Data were presented as mean ± SEM, n = 9 in each group, *p \ 0.05 compared with control group, p \ 0.05 compared with CRS group
Fig. 5 Tempol and RP67580 inhibit CRS-induced increase in LC3-II expression. a The representative blots of LC3-II and GAPDH are shown in each group. b Densitometric analysis of the ratio of LC3-II to GAPDH. CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580. Data were presented as mean ± SEM, n = 6 in each group, *p \ 0.05 compared with control group, p \ 0.05 compared with CRS group
123
Arch Dermatol Res Fig. 6 Tempol and RP67580 inhibit CRS-induced increase in Beclin-1 expression. a The representative blots of Beclin-1 and GAPDH are shown in each group. b Densitometric analysis of the ratio of Beclin-1 to GAPDH. CRS chronic restrained stress, T Tempol and N SP receptor antagonist, RP67580. Data were presented as mean ± SEM, n = 6 in each group, *p \ 0.05 compared with control group, p \ 0.05 compared with CRS group
brain, liver, stomach, heart, kidney tissue and the other organs of rat after 15 and 21 days of CRS [38, 50]. In skin diseases, oxidative stress also plays an important role. Schallreuter et al. [40] detected sustained accumulation of hydrogen peroxide in the epidermis of patients with vitiligo; Akar and Naziroglu reported, respectively, the increased activity of GSH-Px and SOD in the scalp tissue of patients with alopecia areata and the decreased activity of GSH-Px in plasma [1, 32]. In the present, we also observed the increase of oxidative stress in skin after CRS, which was consistent with previous study. In addition, our results also demonstrated that oxidative stress plays a pivotal role in CRS-induced delay of hair growth, because Tempol treatment could normalize CRS-induced abnormal of hair cycle. Previous studies have demonstrated SP induced neurogenic inflammation and mast cell degranulation which were involved in skin disorders, such as atopic dermatitis and psoriasis [5, 6, 23, 45, 48]. Recently, it was reported that ROS were second messengers of neurokinin signaling in peripheral sensory neurons [28]. Our results show that oxidative stress and antioxidant system could be regulated by administration of NK1 receptor blocker, confirming previous conception about SP-ROS signal pathway in CRS
model. The main target of SP is mast cells, which has been proven by previous reports [5, 6, 23, 45, 48]. In the present study, we did not show the possible source of ROS, it could be speculated that activation of mast cell would be the main cause of increase of ROS generation. Specific inhibition of mast cell activation or application of genetically mast cell-deficient mice such as W/Wv and SI/SId mice will provide better information for this issue [8, 47]. Furthermore, disodium cromoglycate is known to be a mast cell stabilizer, which may also provide information of the role of mast cell in the present research model [12]. Most of researchers focused on the tissue apoptosis in response to stress-induced injury. Recently, autophagy has emerged as a powerful mediator in response to cellular injury. Increase of oxidative stress and ROS has been reported to be important stimulation of autophagy [41, 49]. Since CRS has been demonstrated to increase oxidative stress in skin, it is reasonable that increased oxidative stress will induce autophagy. Our present data clearly demonstrated that CRS increased skin autophagy levels by elevation of LC3-II and Beclin-1 expressions through SP-ROS signal pathway. Although the roles of autophagy in the development of various kinds of diseases are still controversial, our results shown the highly relationship between
123
Arch Dermatol Res
the hair growth cycle and autophagy levels, indicating autophagy might play a role in CRS-induced delay of hair growth. Recently, clinical studies revealed that the activities of GSH-Px and SOD were increased in scalp of patients with alopecia areata [1]. Relatively, the activity of GSH-Px in plasma of the patients with alopecia was significantly decreased [32]. These clinical evidences indicated that antioxidant enzyme system played a critical role in the occurrence and development of alopecia. Our study now reports the first experimental evidence that oxidative stress is mediated by SP signal pathway in the abnormal of hair cycle as well as its role in regulation of cell autophagy. The present data may provide a better understanding and strategic application in clinical with psychological stressinduced hair growth disorders.
11.
12.
13.
14.
15. Acknowledgments This work is supported by the National Nature Science Foundation of China (81270316), the Research Program of Soochow University (Q413400111). Conflict of interest
16.
No declare. 17.
References 1. Akar A, Arca E, Erbil H, Akay C, Sayal A, Gur AR (2002) Antioxidant enzymes and lipid peroxidation in the scalp of patients with alopecia areata. J Dermatol Sci 29:85–90 2. Anisman H, Merali Z (2002) Cytokines, stress, and depressive illness. Brain Behav Immun 16:513–524 3. Ansel JC, Armstrong CA, Song I, Quinlan KL, Olerud JE, Caughman SW, Bunnett NW (1997) Interactions of the skin and nervous system. J Investig Dermatol Symp Proc 2:23–26 4. Aoki E, Shibasaki T, Kawana S (2003) Intermittent foot shock stress prolongs the telogen stage in the hair cycle of mice. Exp Dermatol 12:371–377 5. Arck PC, Handjiski B, Hagen E, Joachim R, Klapp BF, Paus R (2001) Indications for a ‘brain-hair follicle axis (BHA)’: inhibition of keratinocyte proliferation and up-regulation of keratinocyte apoptosis in telogen hair follicles by stress and substance P. FASEB J 15:2536–2538. doi:10.1096/fj.00-0699fje 6. Arck PC, Handjiski B, Peters EM, Peter AS, Hagen E, Fischer A, Klapp BF, Paus R (2003) Stress inhibits hair growth in mice by induction of premature catagen development and deleterious perifollicular inflammatory events via neuropeptide substance P-dependent pathways. Am J Pathol 162:803–814. doi:10.1016/ S0002-9440(10)63877-1 7. Arck PC, Slominski A, Theoharides TC, Peters EM, Paus R (2006) Neuroimmunology of stress: skin takes center stage. J Invest Dermatol 126:1697–1704. doi:10.1038/sj.jid.5700104 8. Askenase PW, Van Loveren H, Kraeuter-Kops S, Ron Y, Meade R, Theoharides TC, Nordlund JJ, Scovern H, Gerhson MD, Ptak W (1983) Defective elicitation of delayed-type hypersensitivity in W/Wv and SI/SId mast cell-deficient mice. J Immunol 131:2687–2694 9. Bauer ME, Jeckel CM, Luz C (2009) The role of stress factors during aging of the immune system. Ann NY Acad Sci 1153:139–152. doi:10.1111/j.1749-6632.2008.03966.x 10. Beaujouan JC, Heuillet E, Petitet F, Saffroy M, Torrens Y, Glowinski J (1993) Higher potency of RP 67580, in the mouse
123
18.
19.
20.
21.
22.
23.
24.
25. 26.
27.
and the rat compared with other nonpeptide and peptide tachykinin NK1 antagonists. Br J Pharmacol 108:793–800 Canu N, Tufi R, Serafino AL, Amadoro G, Ciotti MT, Calissano P (2005) Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells. J Neurochem 92:1228–1242. doi:10.1111/j.1471-4159.2004. 02956.x Chen L, Schrementi ME, Ranzer MJ, Wilgus TA, Dipietro LA (2014) Blockade of mast cell activation reduces cutaneous scar formation. PLoS One 9:e85226. doi:10.1371/journal.pone.0085226 Chiba S, Numakawa T, Ninomiya M, Richards MC, Wakabayashi C, Kunugi H (2012) Chronic restraint stress causes anxietyand depression-like behaviors, downregulates glucocorticoid receptor expression, and attenuates glutamate release induced by brain-derived neurotrophic factor in the prefrontal cortex. Prog Neuropsychopharmacol Biol Psychiatry 39:112–119. doi:10. 1016/j.pnpbp.2012.05.018 Codogno P, Meijer AJ (2005) Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ 12(Suppl 2):1509–1518. doi:10.1038/sj.cdd.4401751 Dhabhar FS (2013) Psychological stress and immunoprotection versus immunopathology in the skin. Clin Dermatol 31:18–30. doi:10.1016/j.clindermatol.2011.11.003 Gu HF, Tang CK, Yang YZ (2012) Psychological stress, immune response, and atherosclerosis. Atherosclerosis 223:69–77. doi:10. 1016/j.atherosclerosis.2012.01.021 Gutteridge JM (1995) Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin Chem 41:1819–1828 Hahn SM, Tochner Z, Krishna CM, Glass J, Wilson L, Samuni A, Sprague M, Venzon D, Glatstein E, Mitchell JB et al (1992) Tempol, a stable free radical, is a novel murine radiation protector. Cancer Res 52:1750–1753 Huang J, Lam GY, Brumell JH (2011) Autophagy signaling through reactive oxygen species. Antioxid Redox Signal 14:2215–2231. doi:10.1089/ars.2010.3554 Hutchinson KM, McLaughlin KJ, Wright RL, Bryce Ortiz J, Anouti DP, Mika A, Diamond DM, Conrad CD (2012) Environmental enrichment protects against the effects of chronic stress on cognitive and morphological measures of hippocampal integrity. Neurobiol Learn Mem 97:250–260. doi:10.1016/j.nlm. 2012.01.003 Joachim RA, Sagach V, Quarcoo D, Dinh QT, Arck PC, Klapp BF (2004) Neurokinin-1 receptor mediates stress-exacerbated allergic airway inflammation and airway hyperresponsiveness in mice. Psychosom Med 66:564–571. doi:10.1097/01.psy. 0000132878.08780.93 Katayama M, Aoki E, Suzuki H, Kawana S (2007) Foot shock stress prolongs the telogen stage of the spontaneous hair cycle in a non-depilated mouse model. Exp Dermatol 16:553–560. doi:10. 1111/j.1600-0625.2007.00558.x Kawana S, Liang Z, Nagano M, Suzuki H (2006) Role of substance P in stress-derived degranulation of dermal mast cells in mice. J Dermatol Sci 42:47–54. doi:10.1016/j.jdermsci.2005.12. 004 Kennedy PG, Rodgers J, Jennings FW, Murray M, Leeman SE, Burke JM (1997) A substance P antagonist, RP-67,580, ameliorates a mouse meningoencephalitic response to Trypanosoma brucei brucei. Proc Natl Acad Sci USA 94:4167–4170 Klionsky DJ, Emr SD (2000) Autophagy as a regulated pathway of cellular degradation. Science 290:1717–1721 Krause K, Foitzik K (2006) Biology of the hair follicle: the basics. Semin Cutan Med Surg 25:2–10. doi:10.1016/j.sder.2006. 01.002 Lawrence RA, Burk RF (1976) Glutathione peroxidase activity in selenium-deficient rat liver. Biochem Biophys Res Commun 71:952–958
Arch Dermatol Res 28. Linley JE, Ooi L, Pettinger L, Kirton H, Boyle JP, Peers C, Gamper N (2012) Reactive oxygen species are second messengers of neurokinin signaling in peripheral sensory neurons. Proc Natl Acad Sci USA 109:E1578–E1586. doi:10.1073/pnas. 1201544109 29. Liu N, Wang LH, Guo LL, Wang GQ, Zhou XP, Jiang Y, Shang J, Murao K, Chen JW, Fu WQ, Zhang GX (2013) Chronic restraint stress inhibits hair growth via substance P mediated by reactive oxygen species in mice. PLoS One 8:e61574. doi:10. 1371/journal.pone.0061574 30. Maggi CA, Patacchini R, Rovero P, Giachetti A (1993) Tachykinin receptors and tachykinin receptor antagonists. J Auton Pharmacol 13:23–93 31. Muller-Rover S, Handjiski B, van der Veen C, Eichmuller S, Foitzik K, McKay IA, Stenn KS, Paus R (2001) A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J Invest Dermatol 117:3–15. doi:10. 1046/j.0022-202x.2001.01377.x 32. Naziroglu M, Kokcam I (2000) Antioxidants and lipid peroxidation status in the blood of patients with alopecia. Cell Biochem Funct 18:169–173. doi:10.1002/1099-0844(200009)18:3\169: AID-CBF870[3.0.CO;2-T 33. Paus R, Botchkarev VA, Botchkareva NV, Mecklenburg L, Luger T, Slominski A (1999) The skin POMC system (SPS). Leads and lessons from the hair follicle. Ann NY Acad Sci 885:350–363 34. Paus R, Cotsarelis G (1999) The biology of hair follicles. N Engl J Med 341:491–497. doi:10.1056/NEJM199908123410706 35. Peskin AV, Winterbourn CC (2000) A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1). Clin Chim Acta 293:157–166 36. Peters EM (2012) Psychological support of skin cancer patients. Br J Dermatol 167:105–110. doi:10.1111/j.1365-2133.2012.11094.x 37. Ravikumar B, Berger Z, Vacher C, O’Kane CJ, Rubinsztein DC (2006) Rapamycin pre-treatment protects against apoptosis. Hum Mol Genet 15:1209–1216. doi:10.1093/hmg/ddl036 38. Sahin E, Gu¨mu¨s¸ lu¨ S (2007) Immobilization stress in rat tissues: alterations in protein oxidation, lipid peroxidation and antioxidant defense system. Comp Biochem Physiol C: Toxicol Pharmacol 144:342–347. doi:10.1016/j.cbpc.2006.10.009 39. Scandalios JG (2005) Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses. Braz J Med Biol Res 38:995–1014 (S0100-879X2005000700003) 40. Schallreuter KU, Moore J, Wood JM, Beazley WD, Gaze DC, Tobin DJ, Marshall HS, Panske A, Panzig E, Hibberts NA (1999) In vivo and in vitro evidence for hydrogen peroxide (H2O2) accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase. J Investig Dermatol Symp Proc 4:91–96 41. Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, Elazar Z (2007) Reactive oxygen species are essential for autophagy and
42.
43.
44.
45.
46. 47.
48.
49.
50.
51.
52.
specifically regulate the activity of Atg4. EMBO J 26:1749–1760. doi:10.1038/sj.emboj.7601623 Schnackenberg CG, Wilcox CS (1999) Two-week administration of tempol attenuates both hypertension and renal excretion of 8-Iso prostaglandin f2alpha. Hypertension 33:424–428 Seo JS, Park JY, Choi J, Kim TK, Shin JH, Lee JK, Han PL (2012) NADPH oxidase mediates depressive behavior induced by chronic stress in mice. J Neurosci 32:9690–9699. doi:10.1523/ JNEUROSCI.0794-12.2012 Silva RH, Abilio VC, Takatsu AL, Kameda SR, Grassl C, Chehin AB, Medrano WA, Calzavara MB, Registro S, Andersen ML, Machado RB, Carvalho RC, Ribeiro Rde A, Tufik S, Frussa-Filho R (2004) Role of hippocampal oxidative stress in memory deficits induced by sleep deprivation in mice. Neuropharmacology 46:895–903. doi:10.1016/j.neuropharm.2003.11.032 Singh LK, Pang X, Alexacos N, Letourneau R, Theoharides TC (1999) Acute immobilization stress triggers skin mast cell degranulation via corticotropin releasing hormone, neurotensin, and substance P: a link to neurogenic skin disorders. Brain Behav Immun 13:225–239. doi:10.1006/brbi.1998.0541 Spencer LV, Callen JP (1987) Hair loss in systemic disease. Dermatol Clin 5:565–570 Taguchi Y, Tsuyama K, Watanabe T, Wada H, Kitamura Y (1982) Increase in histidine decarboxylase activity in skin of genetically mast-cell-deficient W/Wv mice after application of phorbol 12-myristate 13-acetate: evidence for the presence of histamine-producing cells without basophilic granules. Proc Natl Acad Sci USA 79:6837–6841 Theoharides TC, Donelan JM, Papadopoulou N, Cao J, Kempuraj D, Conti P (2004) Mast cells as targets of corticotropin-releasing factor and related peptides. Trends Pharmacol Sci 25:563–568. doi:10.1016/j.tips.2004.09.007 Wu HH, Hsiao TY, Chien CT, Lai MK (2009) Ischemic conditioning by short periods of reperfusion attenuates renal ischemia/ reperfusion induced apoptosis and autophagy in the rat. J Biomed Sci 16:19. doi:10.1186/1423-0127-16-19 Zafir A, Banu N (2009) Induction of oxidative stress by restraint stress and corticosterone treatments in rats. Indian J Biochem Biophys 46:53–58 Zhang GX, Kimura S, Murao K, Shimizu J, Matsuyoshi H, Takaki M (2009) Role of neuronal NO synthase in regulating vascular superoxide levels and mitogen-activated protein kinase phosphorylation. Cardiovasc Res 81:389–399. doi:10.1093/cvr/ cvn304 Zhang GX, Kimura S, Nishiyama A, Shokoji T, Rahman M, Abe Y (2004) ROS during the acute phase of Ang II hypertension participates in cardiovascular MAPK activation but not vasoconstriction. Hypertension 43:117–124. doi:10.1161/01.HYP. 0000105110.12667.F8
123
