archives of oral biology 59 (2014) 258–267
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Long-term curcumin treatment antagonizes masseter muscle alterations induced by chronic unpredictable mild stress in rats Min Cui 1, Qiang Li 1, Min Zhang *, Ya-Juan Zhao, Fei Huang, Yong-Jin Chen * Department of General Dentistry & Emergency, School of Stomatology, Fourth Military Medical University, Xi’an 710032, Shaanxi, China
article info
abstract
Article history:
Objective: To investigate the correlation between psychological stress and masseter muscle
Received 27 February 2013
(MM) alterations, and explore the therapeutic agents for restoring the impaired masticatory
Received in revised form
muscle.
9 November 2013
Design: We established a chronic unpredictable mild stress (CUMS) animal model and
Accepted 2 December 2013
observed the changes of ultrastructure, redox homeostasis and energy metabolism in
Keywords:
Results: The depressive-like behavior in stressed rats was confirmed by the evidences of
MM in rats with and without curcumin treatment. Curcumin
altered behaviors in sucrose preference test and open field test; while these phenomena
Masseter muscle
were eased by curcumin. Except for the pathological changes in ultrastructure, decreased
Metabolic disorder
SOD, GSH-Px, CAT, Na+-K+ATPase, and Ca2+-Mg2+ATPase activities as well as increased MDA
Oxidative stress
and LD content and LDH activity were also observed in MM in stressed rats. However,
CUMS
curcumin was capable of reversing CUMS-induced MM disorder by improving the activities of the examined anti-oxidant enzymes and energy metabolism enzymes. Additionally, the increased MDA content, LD content, and LDH activity in stressed rats were reduced by curcumin. Conclusion: All the findings indicate the adverse effects of CUMS on MM function in rats, and raise the possibility of developing curcumin as a potential therapeutic agent for psychological stress-induced masseter dysfunction. # 2013 Elsevier Ltd. All rights reserved.
Introduction Temporomandibular disorders (TMD) is a heterogeneous array of pathological changes that affects the temporomandibular joint (TMJ), the jaw muscles, or both.1 The etiology of this disease is considered to be multifactorial, which involved both psychological and physiological components, including
mental stress, occlusion disorder, trauma, and autoimmune diseases. In recent years, the role of psychosocial factors in TMD has been intensively investigated, and the findings suggested that it was associated with abnormal physiology of masticatory muscle, and considered an important risk indicator for the development of TMD.2,3 Psychological stress is ubiquitous and virtually all diseases, are affected by this phenomenon.4 Anxiety and
* Corresponding authors at: Department of General Dentistry and Emergency, School of Stomatology, Fourth Military Medical University, Changle West Road 145, Xi’an, Shaanxi Province, 710032, China. Tel.: +86 29 84776488; fax: +86 29 84776488. E-mail addresses: [email protected] (M. Zhang), [email protected] (Y.-J. Chen). 1 These authors contributed equally to the work. 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.12.001
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depression are the most common stress-related psychological disorders. In our previous studies, we found that anxiety caused by psychological stress induces oxidative damage and up-regulates the expression of HSP70 in the masseter muscle (MM) of rats; these molecular changes are associated with behaviors that resemble anxiety, which induced by a communication box.5 However, the communication box only tended to result in anxious state in rats,5 while depression is also a commonly seen emotional disorder. And the single stressor of communication box has a potential to cause adaption of animals more easily over time, consequently weakens the effect on animal model. Therefore, in this study, we used a CUMS procedure to produce depressive stated in rats. Multiple stressors in this kind of procedure could also prevent habituation, which can occur rapidly if a single stressor is presented repeatedly.6 The MM is the largest and strongest muscle among the masticatory muscles in rats,7 and is very sensitive at perceiving the changing state of the stomatognathic system, for it contains abundant proprioceptors, such as muscle spindles.8 However, it is unknown whether the depression caused by psychological stress will affect the structure and function of the MM. Anti-depressants improve the clinical symptoms of depression by enhancing or adjusting the functions of monoamine neurotransmitters or receptors. The commonly used chemical anti-depressants, such as tricyclic anti-depressants (TCAs), monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs), atypical anti-depressant drugs, and other compounds, have been reported to produce several unwelcome reactions, including orthostatic hypotension, blurred vision, dry mouth, sinus tachycardia, various gastrointestinal reactions, hepatotoxicity, and even suicidal intentions.9 Therefore, a novel anti-depressant with improved safety and fewer side effects is needed. Curcumin, a Curcuma longa extract, which has been used to effectively manage depression-related disorders in China,10 successfully piqued our interest. Actually, the anti-depressant effects of curcumin has been proved in various animal models of depression, such as forced swim, tail suspension, unpredictable mild stress, olfactory bulbectomy, and chronic fatigue model of depression.11 In addition, curcumin has great potential in stimulating muscle regeneration after traumatic injury, including masseter, and tibialis anterior muscles,12 and the protective effects of curcumin against ischemia-reperfusion injury in rat skeletal muscle have been revealed, due to its great antioxidant ability.13 Moreover, the usage of curcumin has not been reported to cause the aforementioned side effects that can be produced by commonly used chemical anti-depressants to our knowledge. Thus, the present study sought to characterize the ultrastructure, oxidative level, and energy metabolism in the MMs of rats with depressive-like behavior that had been subjected to psychological stress induced by the chronic unpredictable mild stress (CUMS) procedure. Furthermore, curcumin was administered as an anti-depressant to assess whether it plays a protective role in this process. We hypothesized that CUMS caused alterations in the MM that are fully or partially reversible through the administration of curcumin.
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Methods and materials An animal model of CUMS14 The rats were subjected to 7 various and repeated unpredictable stressors during the administration of a CUMS procedure that lastedfor 12 weeks; the duration of this procedure were determined by preliminary experiment. The stressors were as follows: (i) damp sawdust for 12 h, (ii) food deprivation for 12 h, (iii) water deprivation for 12 h, (iv) inversion of the lightdark cycle, (v) immersing in 4 8C cold water for 5 min, (vi) immersing in 45 8C hot water for 5 min (The temperature was determined by Yang’s research.15 To avoid physically stimulating the MM of rats, we put the rats in a cage with 8 cm depth of preheated or cooled water. The rats’ head was above water from beginning to the end.), and (vii) 1 h of restraint.16 (For this last stressor, the rats were placed in a restraining device that was composed of inflexible wire mesh; during the stress procedure, the rats were not allowed to move freely, but their bodies were not constricted.) Over the course of each week of the CUMS procedure, one of the 7 stressors was applied each day, started at 9:00 AM on a random schedule. The same stressor was never applied for 2 consecutive days.
Experimental groups and drug administration A total of 48 male Sprague–Dawley rats (8 weeks old, provided by the Laboratory Animal Center of the Fourth Military Medical University, Xi’an, China) with a mean weight of 230 10 g were housed in groups of 4 in a temperature-controlled room (24 1 8C) with a 12 h light-dark cycle (light on from 08:00 to 20:00 h). Twice each week, the cages were cleaned, and new bedding was provided. The animals were acclimated to the laboratory conditions for 1 week prior to the experiment, with food and water available ad libitum. The animals were randomly divided into the following 6 groups, with 8 rats in each group: a blank control group, a group of rats with CUMS, groups of rats pretreated with curcumin (10, 40, and 80 mg/kg of curcumin) and then treated with CUMS, and a group of rats pretreated with fluoxetine and then treat with CUMS. Curcumin (Sigma–Aldrich Co. St. Louis, MO, USA) was dissolved in 1 ml of peanut oil and administered to the rats once daily by gavage. The doses of curcumin were determined based on the report of Sivalingam et al., with minor modification.17 Fluoxetine (5 mg/kg, Eli Lilly, Indianapolis, IN, USA) was dissolved in saline solution and administered to the rats by gavage. The dose of fluoxetine was determined based on the report of Bonilla-Jaime et al.18 The rats in control group received 1 ml of peanut oil but did not receive CUMS. All the drugs were administered to the rats 0.5 h before they experienced CUMS. This study was performed in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the National Institutes of Health, and was approved by the committee on the ethics of animal research of the Fourth Military Medical University (Xi’an, China). All of the surgeries were performed under anesthesia, and every effort was made to minimize the animals’ suffering.
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Behavioral test
group were calculated as: the number of sample with microstructural changes/the number of total samples 100%.
Sucrose preference test The sucrose preference test was based on the method of Willner with minor modification.19 The test was performed right after 12 weeks of CUMS stress. Prior to this test, animals were separated in 1 cage each, with their food and water being deprived for 24 h. Then each rat was given both 1 bottle of water and 1 bottle of 1% sucrose. Fluid consumption in 24 h was recorded by reweighing preweighed bottles of test solution. Bottles were counterbalanced across left and right sides of the cages throughout the experiment, varying for each animal. The percentage preference for sucrose was calculated as Sucrose preference (%) = sucrose consumption (ml)/total fluid consumption (ml) 100%.
Muscle oxidative stress measurements Muscle samples were minced and homogenized with physiological saline into 10% homogenates and were centrifuged at 3000 rpm for 10 min at 4 8C. The supernatants were transferred into new tubes and kept at 80 8C until they were used in experiments.24 The protein content, superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity, catalase (CAT) activity, and malondialdehyde (MDA) content were determined using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Muscle energy metabolism measurements Open field test An open-field chamber (RD1412-OF, Shanghai Mobile Datum Information Technology Co., Shanghai, China) consisted of a 100 cm 100 cm 80 cm Plexiglas box was used in this test. This chamber was placed in a temperature-controlled room and was illuminated by one fluorescent light suspended over the chamber. A digital video camera was used to record the activities of rats. Each rat was monitored for 15 min. After each test, the maze was cleaned with 20% alcohol to eliminate the odor and other traces of the previously tested rat. The total distance moved, the velocity, and the time spent in the center were recorded and calculated.20
Histological observation The rats were anesthetized with intraperitoneal injections of 1% pentobarbital sodium (30 mg/kg) and euthanatized by cervical dislocation.21 For histological observation, the muscle sample was cut into size of 5 mm3 and fixed in 40 g/L paraformaldehyde for 24 h. The sections of masseter muscle were stained with hematoxylin and eosin (H&E). Sections were incubated in Harris’ hematoxylin (0.75% w/v) for 12 min, then immersed in acid alcohol for 30 s and in Scott’s tap water for 2 min, and finally stained with 1% (w/v) aqueous eosin for 5 min. The sections were washed with running tap water before and after each solution, dehydrated in serial alcohol, and mounted with gum.22
The Na+-K+ATPase, Ca2+-Mg2+ATPase, and lactate dehydrogenase (LDH) activities and the lactic acid (LD) content were determined using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Statistical analysis Statistical analyses were performed using SPSS 16.0 software package (SPSS Inc., USA). All data acquisition and analyses were performed blindly. The normal distributions of experimental measures were test by Q–Q plot. Uniformity of variance was determined by a Bartlett’s test. When uniform variance was found, significant differences for each measure were determined by one-way analysis of variance (ANOVA) tests and post hoc test. The chi-square test was used to compare the percentage of microstructurally changed MMs. The Bonferroni correction was applied for adjusting p value. Statistical significance was set at p < 0.05/15 (in sucrose test, open field test, and ultrastructure comparison), p < 0.05/6 (in muscle oxidative stress measurements and energy metabolism measurements). Additionally, a student t test was performed between SL and DL in Muscle oxidative stress measurements and energy metabolism measurements, and statistical significance was set at p < 0.05. Data are expressed as the means standard deviation (SD).
Muscle ultrastructure observation
Results
Eight MM samples in each subgroup were investigated by transmission electron microscopy as following. After the rats were executed, the superficial layer (SL) and deep layer (DL) of the MM were quickly excised from both the left and right side, freed of fascia and other connective tissue. The middle bellies of left SL and DL of masseter muscle were cut into 1 mm3 pieces and fixed with glutaraldehyde solution; these samples were then embedded in Epon812, sectioned using an LKB-V ultramicrotome (LKB, Bromma, Sweden), and stained with uranyl acetate and lead citrate. The ultrastructures of the muscles were then observed by transmission electron microscopy (JEM-100SX, JEOL Company, Japan).23 The occurrence of microstructural changes was counted in each group. The percentages of microstructural changed muscles in each
The evaluation of behavioral test Sucrose preference test As shown in Fig. 1, the sucrose preference of stressed rats was significantly decreased ( p < 0.003, Fig. 1), which was partially reversed after the administration of curcumin (40 and 80 ml/ kg) and fluoxetine ( p < 0.003, Fig. 1).
Open field test The open-field test was designed to evaluate the stress state of experimental animals in a new environment. As shown in Fig. 2, CUMS resulted in decreases in the velocity ( p < 0.003, Fig. 2B) and the distance moved ( p < 0.003, Fig. 2C) of experimental rats. After the administration of curcumin, the
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above indices were gradually reversed in a dose-dependent manner (Fig. 2B and C). Moreover, the distance and velocity of the rats treated with 80 mg/kg curcumin approached normal levels ( p < 0.003, Fig. 2B and C). With respect to the time that was spent in the center of the apparatus, the duration of this time was longer in the CUMS rats than in the control animals ( p < 0.003, Fig. 2A) and significantly decreased in 80 mg/kg treated rats( p < 0.003, Fig. 2A). The fluoxetine exhibited similar effect of decreased duration of this time as curcumin does, but no significance was found ( p > 0.003, Fig. 2A).
Histological observation The cross-section views of masseter muscle with magnification of 400 are shown in Fig. 3. The SL and DL of masseter muscle in each group exhibited a normal histological appearance, with the presence of orderly arranged fibers with peripheral nuclei and evenly stained sarcoplasm. No signs of inflammation or injury were observed in CUMS group or any other groups. Fig. 1 – Sucrose Preference of rats. All of the data were shown as the mean WSD. Each bar indicates the standard deviation. *p < 0.003.
Ultrastructure Fig. 4 illustrates the ultrastructures of the SL and DL of masseter in the control group, the CUMS group, the curcumin (10, 40, and 80 mg/kg) treated groups, and the fluoxetine
Fig. 2 – Time spent in center A, velocity B, and the distance moved C of rats in the open-field test. All of the data are shown as the mean WSD. *p < 0.003.
Fig. 3 – The hematoxylin and eosin (H&E) staining cross-section for histological observation (400T) A1 SL of control group; A2 DL of control group; B1 SL of CUMS group; B2 DL of CUMS group; C1 SL of curcumin (10 mg/kg) group; C2 DL of curcumin (10 mg/kg) group; D1 SL of curcumin (40 mg/kg) group; D2 DL of curcumin (40 mg/kg) group; E1 SL of curcumin (80 mg/kg) group; E2 DL of curcumin (80 mg/kg) group; F1 SL of fluoxetine group; F2 DL of fluoxetine group. Scale bar = 100 mm.
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Fig. 4 – The ultrastructures of the masseter muscle observed by transmission electron microscopy (20,000T). A1 SL of control group; A2 DL of control group; B1 SL of CUMS group; B2 DL of CUMS group; C1 SL of curcumin (10 mg/kg) group; C2 DL of curcumin (10 mg/kg) group; D1 SL of curcumin (40 mg/kg) group; D2 DL of curcumin (40 mg/kg) group; E1 SL of curcumin (80 mg/kg) group; E2 DL of curcumin (80 mg/kg) group; F1 SL of fluoxetine group; F2 DL of fluoxetine group. Mitochondria with normal ultrastructures are marked by white arrows. Swollen mitochondria with loss of the cristae, reductions in the matrix density and vacuolar changes are marked by black arrows. G1 Percentage of microstructurally changed SL of MM. G2 Percentage of microstructurally changed DL of MM. *p < 0.003.
treated group. The ultrastructures of the masseter myocytes (SL and DL) of the rats in the control and curcumin (80 mg/kg) treated groups demonstrated evenly distributed muscular nuclei under the sarcolemma around muscle fibers, and no signs of hyperplasia, swelling, or pyknosis were observed. The myofibrils and myotomes of the CUMS rats were normal and resembled to those of the rats in the other 5 groups. The light I bands and dark A bands of the myotomes in the 6 groups of rats were intact and located in the correct positions. Dramatic vacuolar changes in the mitochondria appeared in the masseter myocytes of the CUMS rats (7 out of 8 samples of SL and 7 out of 8 samples of DL, marked by black arrows, Fig. 4B1 and B2, respectively). Swollen mitochondria with a loss of the cristae and a reduction in the matrix density were found in the MM in the 10 mg/kg curcumin group (6 out of 8 samples of SL and 6 out of 8 samples of DL, marked by black arrows, Fig. 4C1 and C2, respectively), the 40 mg/kg curcumin group (5 out of 8 samples of SL, and 6 out of 8 samples of DL, marked by black arrows, Fig. 4D1 and D2, respectively), and the fluoxetine treated rats (6 out of 8 samples of SL, and 6 out of
8 samples of DL, marked by black arrows, Fig. 4F1 and F2, respectively). Only a few slightly loss of cristae and a reduction in the matrix density were found in SL of masseter muscle of the 80 mg/kg curcumin group (2 out of 8 samples, marked by black arrows, Fig. 4E1). In contrast, there were nearly no swollen/abnormal mitochondria or vacuolar changes being observed in control group and in the DL of masseter muscle of the 80 mg/kg curcumin group (the normal mitochondria was marked by white arrows, Fig. 4A1, A2, and E2, respectively). The occurrence of degenerated mitochondria was counted in each group. The percentages of microstructural changes for mitochondria degeneration in each group were calculated as: the number of sample with mitochondria degeneration/the number of total samples 100%. The chi-square test showed significant difference of microstructurally changed SL of masseter muscle between the CUMS group and control group (Fig. 4G1, p < 0.003). The significant differences were also noted for DL of masseter muscle between the CUMS group and the control group, as well as the CUMS group and 80 mg/kg curcumin group (Fig. 4G2, p < 0.003).
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Fig. 5 – The SOD activity A, GSH-Px activity B, CAT activity C, and MDA content D of rats. All of the data are shown as the mean WSD. *p < 0.008.
All of the results indicated the successful establishment of the CUMS animal model as a rodent stress state, and 80 mg/kg was selected as the effective dosage for curcumin for the next experiment, which evaluated its protective effects against CUMS.
The activities of SOD, GSH-Px, and CAT and the MDA content As shown in Fig. 5, the activities of SOD, GSH-Px, and CAT in the SL and DL were significantly lower in the CUMS rats than in the control rats ( p < 0.008, Fig. 5A–C), whereas curcumin treatment (80 mg/kg) reversed the deficit in total anti-oxidant capacity ( p < 0.008, Fig. 5A–C). In the masseters of the fluoxetine group, the SOD, GSH-Px, and CAT activity exhibited improvements, but no significance was found ( p > 0.008, Fig. 5A–C). With respect to the MDA content, the CUMS treatment caused an increase in the masseter compared with the control group ( p < 0.008, Fig. 5D). However, this value was reduced following the administration of curcumin (80 mg/kg) ( p < 0.008, Fig. 5D), but not fluoxetine ( p >0.008, Fig. 5D). No significant difference of SOD, GSH-Px, CAT activities, and MDA content were found between the SL and DL of masseter muscle ( p > 0.05).
The activities of Na+-K+ATPase, Ca2+-Mg2+ATPase, and LDH and the LD content The energy metabolism enzymatic activities in the masseter were remarkably altered in the rats that received CUMS.
Specifically, the Na+-K+ATPase and Ca2+-Mg2+ATPase activities significantly decreased ( p < 0.008, Fig. 6A and B), whereas the LDH activity and LD content significantly increased ( p < 0.008, Fig. 6C and D) in the CUMS rats compared with the control rats. However, curcumin (80 mg/kg) treatment restored normal enzymatic activities ( p < 0.008, Fig. 6A–C). Compared with the CUMS group, the masseters of the fluoxetine treatment group demonstrated a similar trend as curcumin group (80 mg/kg), but with no significance ( p > 0.008, Fig. 6A–C). The LD content in the masseters of the CUMS rats was elevated compared with the control animals, and a clear decrease in this parameter was observed in the curcumin (80 mg/kg), ( p < 0.008, Fig. 6D) group, but not in fluoxetine group ( p > 0.008). No significant difference of these indices was found between the SL and DL of masseter muscle ( p > 0.05), except the activity of Ca2+-Mg2+ATPase is higher in SL than DL of MM in control group ( p < 0.05,Fig. 6B).
Discussion Depression is a common human psychological stress state. In recent years, the therapeutic potential of curcumin for many types of depression-related diseases has attracted researchers’ attention.25–27 However, there is still lack of animal studies to investigate the adverse effects of depression on the MM. In addition, it has not yet been determined whether curcumin exerts protective effects in the jaw muscle. To our knowledge,
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Fig. 6 – The Na+-K+ATPase activity A, Ca2+-Mg2+ATPase activity B, LDH activity C, and LD content D of rats. All of the data are shown as the mean WSD. *p < 0.008.
this study is the first to report the capacity of curcumin to reverse the pathological changes in the ultrastructures and biochemistry of the masticatory muscles of rats that have been exposed to CUMS. CUMS is widely used in experiments and investigations at present because it provides a realistic model of the stresses of daily life. Besides, the prolonged time course of the model was suitable for determining the effects of chronic drug treatments.6 Sucrose preference and the open-field test results indicated that the CUMS procedure successfully induced depressive-like behaviors in rats. Similarly to the classical anti-depressant fluoxetine, curcumin is capable of alleviating the depressive state of experimental rats, as evidenced by the fact that curcumin significantly restored the altered behavior parameters of CUMS treated rats. These results are similar to the findings of Wang.28 Furthermore, the data revealed the superior anti-depressant effect of a dose of 80 mg/kg curcumin compared with doses of 10 or 40 mg/kg curcumin, raising the possibility that curcumin acts in a dose-dependent manner in treating depression. However, the upper limit of the drug dose in relieving depressed mood is unclear from these data;
therefore, this parameter should be evaluated in future studies. The transmission electron microscopy indicated large numbers of severe vacuolar pathological changes in the mitochondria of both SL and DL of the masseters of the CUMS rats. These ultrastructural observations are consistent with the possibility that the mitochondria from the muscles of stressed rats may exhibit altered permeability characteristics.23 We also found swollen mitochondria with loss of the cristae and reductions in the matrix density in the MMs of 10, 40 mg/kg curcumin treat rats and the fluoxetine treated rats. By contrast, mitochondrial abnormalities were significantly reduced in the 80 mg/kg curcumin treated rats. These observations indicated that lesions of mitochondria are likely secondary to the CUMS procedure and 80 mg/kg curcumin is more effective than fluoxetine at protecting mitochondria from damage caused by stress. That was probably because, other than curcumin, fluoxetine might have a directly negative influence on mitochondria, for it was reported that the in vitro administration of fluoxetine resulted in disruption of mitochondrial function.29
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Oxidative stress has been implicated in many psychiatric disorders, including major depressive disorder.30 In the current study, the SOD, GSH-Px, and CAT activities and the MDA content, which are the major indices of oxidative stress, were examined in the masseter. The results, which demonstrated decreased anti-oxidase activities and increased MDA contents in stressed animals, indicated that CUMS can disturb the balance between oxidant formation and anti-oxidant defenses in the masseter. Stress can activate the sympathetic nervous system (SNS) and the hypothalamus-pituitary-adrenal axis (HPA). These systems respond to chronic maladaptive stimulation by producing catecholamine (SNS) and by subsequently activating the immune response and inflammatory response system (HPA); these responses induce hypoxia and ischemia, and increase the levels of proinflammatory cytokines that interact with inflammatory cells, respectively. These processes eventually lead to the generation of reactive oxygen species (ROS), resulting in oxidative stress in the muscle tissue.31 The microstructural changes that were observed could be caused by the excessive accumulation of ROS, given that these molecules can damage the cell membrane, disrupt the structures of protein molecules, and alter the functions of mitochondrial transport.32 In our experiment, we found that curcumin have positive effects on maintaining redox homeostasis in the MM. This finding is unsurprising because of the powerful antioxidant effect of curcumin, which possesses a natural phenolic hydroxyl structure that can directly capture and eliminate free radicals, thereby protecting tissues against oxidative stress injuries.33–35 The activities of Na+-K+ATPase and Ca2+-Mg2+ATPase were significantly reduced in the CUMS group. A reduction in ATPase activity is a sign of a damaged cell membrane and is primarily caused by hypoxia, acid metabolites, and free radicals.36,37 The low activities of Na+-K+ATPase and Ca2+Mg2+ATPase result in high concentrations of Na+ and Ca2+ inside the cells, which causes the swelling and damage. This result further supports our ultrastructural observations. LDH activity is typically viewed as a sign of anaerobic oxidation in skeletal muscle. LD is a product of glycolysis; this molecule is an important product of muscle activity and is recognized as the fatigue-causing compound. In the present study, the increased LDH activity and LD content in the MMs of stressed rats indicated the enhancement of anaerobic metabolism in muscle, which is consistent with the clinical phenomena of MM fatigue, pain and other symptoms of depressive disorder.2,3 The administration of curcumin (80 mg/kg) reversed the lower activities of Na+-K+ATPase and Ca2+-Mg2+ATPase and decreased LDH activities and LD levels. This result is in agreement with the findings of a previous report.38,39 We know that the masseter muscle is a very important and powerful muscle in the human mastication system. This organ can be anatomically divided into the superficial and the deep portion, with both their functions 40 and myofiber constituents being different.41 Therefore, we separately focused on the superficial and deep portions of the MM in the study of rats that were exposed to CUMS and/or receiving antidepressants. Hardly any differences between the SL and DL of masseter after CUMS procedure and anti-depressant treatment with curcumin and fluoxetine were observed after biomechanical analysis, indicating that the superficial part and
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the deep part of masseter muscle displayed similar redox and energy metabolism responses to the CUMS. In the current study, we used 2 types of anti-depressants, curcumin, and fluoxetine, as the antagonists to stress. The antidepressant effects of both of these substances are related to the central monoaminergic neurotransmitter system. According to the behavioral test results, fluoxetine did the same way with the high-dose curcumin (80 mg), which showed significant effects against behavioral changes by mental stress. However, the micrographs of mitochondria of the fluoxetine group still generally exhibited swollen mitochondria with loss of the cristae and reduction in the matrix density, which indicated that the anti-stress effect of the fluoxetine for muscular mitochondria was far less obvious compared with that of the high-dose curcumin. Whereas, we also noticed that there were rarely dramatic vacuolar degenerations of mitochondria in fluoxetine treated group, and also less mitochondria degenerations compared with the samples from the CUMS group. This phenomenon was generally in accordance with the trends of biochemical assays, which convinced that the anti-depressant effect of fluoxetine could partly neutralize the adverse impact of depression induced by stress, which helped to maintain the muscular metabolism and homeostasis to some extent. Compared with fluoxetine, our results for curcumin showed a potential therapeutic benefit, not only as a behavioral antidepressant but also in restoring the abnormal ultrastructural features and biochemical indices that were caused by CUMS. Other studies have demonstrated the protective effects of curcumin against skeletal muscle ischemia-reperfusion injury and muscle atrophy,13,42 indicating that curcumin favors the recovery of muscle dysfunction. Additionally, curcumin is also well known as an anti-oxidant agent.43 The oxidation resisting character of curcumin may as well contribute to the protective effect of curcumin against muscle disorder. Moreover, curcumin is a very safe anti-depressant. 44,45 The side effects that have been reported for fluoxetine, such as hypomania,46 congenital cardiovascular defects and suicidal ideation in children,47,48 have not been observed for curcumin. A close relation has been found in depressive disorder with a decreased activity of main peripheral anti-oxidant defences, including SOD, GSH-Px, CAT and the increased level of MDA.49,50 While an ameliorated depressive state after treatment with anti-depressant is confirmed to be related to the reforming of redox homeostasis in vivo, therefore, the protective role of curcumin played against oxidative stress and energy metabolism disorder in CUMS rats may be ascribed to the anti-depressant effect of curcumin in the present study.51,10,11 In addition, another potential cause that curcumin could scavenge free radicals directly and maintain homeostasis in tissue, should be taken into consideration. Since previous studies have demonstrated that curcumin could enhance the activities of oxidant enzymes,52,53 and energy metabolic enzymes, which are very sensitive for free radical reaction,54 the improved redox homeostasis and energy metabolism in stressed rats after the administration of curcumin are also considered to be the results of the antioxidant effect of curcumin. However, such speculations still need to be confirmed in future studies. In conclusion, the CUMS procedure resulted in pathological ultrastructural changes, oxidative damage, and energy
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metabolism disorders in the MMs of rats, providing evidence for the pathogenesis of masticatory muscle dysfunction after psychological stress. We found that curcumin was capable of restoring CUMS-induced deficits in the MM. In this case, the muscle alterations induced by the CUMS procedure can be reversed by the anti-depressant and anti-oxidant curcumin in a dose-dependent manner. Our study provided a potential preventive treatment for depression patients who are at risk of developing MM disorders.
Acknowledgment This work was financially supported by the National Nature Science Foundation of China(No. (81070851, (31170888, (81371188).
references
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Available online at www.sciencedirect.com
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Long-term curcumin treatment antagonizes masseter muscle alterations induced by chronic unpredictable mild stress in rats Min Cui 1, Qiang Li 1, Min Zhang *, Ya-Juan Zhao, Fei Huang, Yong-Jin Chen * Department of General Dentistry & Emergency, School of Stomatology, Fourth Military Medical University, Xi’an 710032, Shaanxi, China
article info
abstract
Article history:
Objective: To investigate the correlation between psychological stress and masseter muscle
Received 27 February 2013
(MM) alterations, and explore the therapeutic agents for restoring the impaired masticatory
Received in revised form
muscle.
9 November 2013
Design: We established a chronic unpredictable mild stress (CUMS) animal model and
Accepted 2 December 2013
observed the changes of ultrastructure, redox homeostasis and energy metabolism in
Keywords:
Results: The depressive-like behavior in stressed rats was confirmed by the evidences of
MM in rats with and without curcumin treatment. Curcumin
altered behaviors in sucrose preference test and open field test; while these phenomena
Masseter muscle
were eased by curcumin. Except for the pathological changes in ultrastructure, decreased
Metabolic disorder
SOD, GSH-Px, CAT, Na+-K+ATPase, and Ca2+-Mg2+ATPase activities as well as increased MDA
Oxidative stress
and LD content and LDH activity were also observed in MM in stressed rats. However,
CUMS
curcumin was capable of reversing CUMS-induced MM disorder by improving the activities of the examined anti-oxidant enzymes and energy metabolism enzymes. Additionally, the increased MDA content, LD content, and LDH activity in stressed rats were reduced by curcumin. Conclusion: All the findings indicate the adverse effects of CUMS on MM function in rats, and raise the possibility of developing curcumin as a potential therapeutic agent for psychological stress-induced masseter dysfunction. # 2013 Elsevier Ltd. All rights reserved.
Introduction Temporomandibular disorders (TMD) is a heterogeneous array of pathological changes that affects the temporomandibular joint (TMJ), the jaw muscles, or both.1 The etiology of this disease is considered to be multifactorial, which involved both psychological and physiological components, including
mental stress, occlusion disorder, trauma, and autoimmune diseases. In recent years, the role of psychosocial factors in TMD has been intensively investigated, and the findings suggested that it was associated with abnormal physiology of masticatory muscle, and considered an important risk indicator for the development of TMD.2,3 Psychological stress is ubiquitous and virtually all diseases, are affected by this phenomenon.4 Anxiety and
* Corresponding authors at: Department of General Dentistry and Emergency, School of Stomatology, Fourth Military Medical University, Changle West Road 145, Xi’an, Shaanxi Province, 710032, China. Tel.: +86 29 84776488; fax: +86 29 84776488. E-mail addresses: [email protected] (M. Zhang), [email protected] (Y.-J. Chen). 1 These authors contributed equally to the work. 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.12.001
archives of oral biology 59 (2014) 258–267
depression are the most common stress-related psychological disorders. In our previous studies, we found that anxiety caused by psychological stress induces oxidative damage and up-regulates the expression of HSP70 in the masseter muscle (MM) of rats; these molecular changes are associated with behaviors that resemble anxiety, which induced by a communication box.5 However, the communication box only tended to result in anxious state in rats,5 while depression is also a commonly seen emotional disorder. And the single stressor of communication box has a potential to cause adaption of animals more easily over time, consequently weakens the effect on animal model. Therefore, in this study, we used a CUMS procedure to produce depressive stated in rats. Multiple stressors in this kind of procedure could also prevent habituation, which can occur rapidly if a single stressor is presented repeatedly.6 The MM is the largest and strongest muscle among the masticatory muscles in rats,7 and is very sensitive at perceiving the changing state of the stomatognathic system, for it contains abundant proprioceptors, such as muscle spindles.8 However, it is unknown whether the depression caused by psychological stress will affect the structure and function of the MM. Anti-depressants improve the clinical symptoms of depression by enhancing or adjusting the functions of monoamine neurotransmitters or receptors. The commonly used chemical anti-depressants, such as tricyclic anti-depressants (TCAs), monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs), atypical anti-depressant drugs, and other compounds, have been reported to produce several unwelcome reactions, including orthostatic hypotension, blurred vision, dry mouth, sinus tachycardia, various gastrointestinal reactions, hepatotoxicity, and even suicidal intentions.9 Therefore, a novel anti-depressant with improved safety and fewer side effects is needed. Curcumin, a Curcuma longa extract, which has been used to effectively manage depression-related disorders in China,10 successfully piqued our interest. Actually, the anti-depressant effects of curcumin has been proved in various animal models of depression, such as forced swim, tail suspension, unpredictable mild stress, olfactory bulbectomy, and chronic fatigue model of depression.11 In addition, curcumin has great potential in stimulating muscle regeneration after traumatic injury, including masseter, and tibialis anterior muscles,12 and the protective effects of curcumin against ischemia-reperfusion injury in rat skeletal muscle have been revealed, due to its great antioxidant ability.13 Moreover, the usage of curcumin has not been reported to cause the aforementioned side effects that can be produced by commonly used chemical anti-depressants to our knowledge. Thus, the present study sought to characterize the ultrastructure, oxidative level, and energy metabolism in the MMs of rats with depressive-like behavior that had been subjected to psychological stress induced by the chronic unpredictable mild stress (CUMS) procedure. Furthermore, curcumin was administered as an anti-depressant to assess whether it plays a protective role in this process. We hypothesized that CUMS caused alterations in the MM that are fully or partially reversible through the administration of curcumin.
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Methods and materials An animal model of CUMS14 The rats were subjected to 7 various and repeated unpredictable stressors during the administration of a CUMS procedure that lastedfor 12 weeks; the duration of this procedure were determined by preliminary experiment. The stressors were as follows: (i) damp sawdust for 12 h, (ii) food deprivation for 12 h, (iii) water deprivation for 12 h, (iv) inversion of the lightdark cycle, (v) immersing in 4 8C cold water for 5 min, (vi) immersing in 45 8C hot water for 5 min (The temperature was determined by Yang’s research.15 To avoid physically stimulating the MM of rats, we put the rats in a cage with 8 cm depth of preheated or cooled water. The rats’ head was above water from beginning to the end.), and (vii) 1 h of restraint.16 (For this last stressor, the rats were placed in a restraining device that was composed of inflexible wire mesh; during the stress procedure, the rats were not allowed to move freely, but their bodies were not constricted.) Over the course of each week of the CUMS procedure, one of the 7 stressors was applied each day, started at 9:00 AM on a random schedule. The same stressor was never applied for 2 consecutive days.
Experimental groups and drug administration A total of 48 male Sprague–Dawley rats (8 weeks old, provided by the Laboratory Animal Center of the Fourth Military Medical University, Xi’an, China) with a mean weight of 230 10 g were housed in groups of 4 in a temperature-controlled room (24 1 8C) with a 12 h light-dark cycle (light on from 08:00 to 20:00 h). Twice each week, the cages were cleaned, and new bedding was provided. The animals were acclimated to the laboratory conditions for 1 week prior to the experiment, with food and water available ad libitum. The animals were randomly divided into the following 6 groups, with 8 rats in each group: a blank control group, a group of rats with CUMS, groups of rats pretreated with curcumin (10, 40, and 80 mg/kg of curcumin) and then treated with CUMS, and a group of rats pretreated with fluoxetine and then treat with CUMS. Curcumin (Sigma–Aldrich Co. St. Louis, MO, USA) was dissolved in 1 ml of peanut oil and administered to the rats once daily by gavage. The doses of curcumin were determined based on the report of Sivalingam et al., with minor modification.17 Fluoxetine (5 mg/kg, Eli Lilly, Indianapolis, IN, USA) was dissolved in saline solution and administered to the rats by gavage. The dose of fluoxetine was determined based on the report of Bonilla-Jaime et al.18 The rats in control group received 1 ml of peanut oil but did not receive CUMS. All the drugs were administered to the rats 0.5 h before they experienced CUMS. This study was performed in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the National Institutes of Health, and was approved by the committee on the ethics of animal research of the Fourth Military Medical University (Xi’an, China). All of the surgeries were performed under anesthesia, and every effort was made to minimize the animals’ suffering.
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Behavioral test
group were calculated as: the number of sample with microstructural changes/the number of total samples 100%.
Sucrose preference test The sucrose preference test was based on the method of Willner with minor modification.19 The test was performed right after 12 weeks of CUMS stress. Prior to this test, animals were separated in 1 cage each, with their food and water being deprived for 24 h. Then each rat was given both 1 bottle of water and 1 bottle of 1% sucrose. Fluid consumption in 24 h was recorded by reweighing preweighed bottles of test solution. Bottles were counterbalanced across left and right sides of the cages throughout the experiment, varying for each animal. The percentage preference for sucrose was calculated as Sucrose preference (%) = sucrose consumption (ml)/total fluid consumption (ml) 100%.
Muscle oxidative stress measurements Muscle samples were minced and homogenized with physiological saline into 10% homogenates and were centrifuged at 3000 rpm for 10 min at 4 8C. The supernatants were transferred into new tubes and kept at 80 8C until they were used in experiments.24 The protein content, superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity, catalase (CAT) activity, and malondialdehyde (MDA) content were determined using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Muscle energy metabolism measurements Open field test An open-field chamber (RD1412-OF, Shanghai Mobile Datum Information Technology Co., Shanghai, China) consisted of a 100 cm 100 cm 80 cm Plexiglas box was used in this test. This chamber was placed in a temperature-controlled room and was illuminated by one fluorescent light suspended over the chamber. A digital video camera was used to record the activities of rats. Each rat was monitored for 15 min. After each test, the maze was cleaned with 20% alcohol to eliminate the odor and other traces of the previously tested rat. The total distance moved, the velocity, and the time spent in the center were recorded and calculated.20
Histological observation The rats were anesthetized with intraperitoneal injections of 1% pentobarbital sodium (30 mg/kg) and euthanatized by cervical dislocation.21 For histological observation, the muscle sample was cut into size of 5 mm3 and fixed in 40 g/L paraformaldehyde for 24 h. The sections of masseter muscle were stained with hematoxylin and eosin (H&E). Sections were incubated in Harris’ hematoxylin (0.75% w/v) for 12 min, then immersed in acid alcohol for 30 s and in Scott’s tap water for 2 min, and finally stained with 1% (w/v) aqueous eosin for 5 min. The sections were washed with running tap water before and after each solution, dehydrated in serial alcohol, and mounted with gum.22
The Na+-K+ATPase, Ca2+-Mg2+ATPase, and lactate dehydrogenase (LDH) activities and the lactic acid (LD) content were determined using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Statistical analysis Statistical analyses were performed using SPSS 16.0 software package (SPSS Inc., USA). All data acquisition and analyses were performed blindly. The normal distributions of experimental measures were test by Q–Q plot. Uniformity of variance was determined by a Bartlett’s test. When uniform variance was found, significant differences for each measure were determined by one-way analysis of variance (ANOVA) tests and post hoc test. The chi-square test was used to compare the percentage of microstructurally changed MMs. The Bonferroni correction was applied for adjusting p value. Statistical significance was set at p < 0.05/15 (in sucrose test, open field test, and ultrastructure comparison), p < 0.05/6 (in muscle oxidative stress measurements and energy metabolism measurements). Additionally, a student t test was performed between SL and DL in Muscle oxidative stress measurements and energy metabolism measurements, and statistical significance was set at p < 0.05. Data are expressed as the means standard deviation (SD).
Muscle ultrastructure observation
Results
Eight MM samples in each subgroup were investigated by transmission electron microscopy as following. After the rats were executed, the superficial layer (SL) and deep layer (DL) of the MM were quickly excised from both the left and right side, freed of fascia and other connective tissue. The middle bellies of left SL and DL of masseter muscle were cut into 1 mm3 pieces and fixed with glutaraldehyde solution; these samples were then embedded in Epon812, sectioned using an LKB-V ultramicrotome (LKB, Bromma, Sweden), and stained with uranyl acetate and lead citrate. The ultrastructures of the muscles were then observed by transmission electron microscopy (JEM-100SX, JEOL Company, Japan).23 The occurrence of microstructural changes was counted in each group. The percentages of microstructural changed muscles in each
The evaluation of behavioral test Sucrose preference test As shown in Fig. 1, the sucrose preference of stressed rats was significantly decreased ( p < 0.003, Fig. 1), which was partially reversed after the administration of curcumin (40 and 80 ml/ kg) and fluoxetine ( p < 0.003, Fig. 1).
Open field test The open-field test was designed to evaluate the stress state of experimental animals in a new environment. As shown in Fig. 2, CUMS resulted in decreases in the velocity ( p < 0.003, Fig. 2B) and the distance moved ( p < 0.003, Fig. 2C) of experimental rats. After the administration of curcumin, the
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above indices were gradually reversed in a dose-dependent manner (Fig. 2B and C). Moreover, the distance and velocity of the rats treated with 80 mg/kg curcumin approached normal levels ( p < 0.003, Fig. 2B and C). With respect to the time that was spent in the center of the apparatus, the duration of this time was longer in the CUMS rats than in the control animals ( p < 0.003, Fig. 2A) and significantly decreased in 80 mg/kg treated rats( p < 0.003, Fig. 2A). The fluoxetine exhibited similar effect of decreased duration of this time as curcumin does, but no significance was found ( p > 0.003, Fig. 2A).
Histological observation The cross-section views of masseter muscle with magnification of 400 are shown in Fig. 3. The SL and DL of masseter muscle in each group exhibited a normal histological appearance, with the presence of orderly arranged fibers with peripheral nuclei and evenly stained sarcoplasm. No signs of inflammation or injury were observed in CUMS group or any other groups. Fig. 1 – Sucrose Preference of rats. All of the data were shown as the mean WSD. Each bar indicates the standard deviation. *p < 0.003.
Ultrastructure Fig. 4 illustrates the ultrastructures of the SL and DL of masseter in the control group, the CUMS group, the curcumin (10, 40, and 80 mg/kg) treated groups, and the fluoxetine
Fig. 2 – Time spent in center A, velocity B, and the distance moved C of rats in the open-field test. All of the data are shown as the mean WSD. *p < 0.003.
Fig. 3 – The hematoxylin and eosin (H&E) staining cross-section for histological observation (400T) A1 SL of control group; A2 DL of control group; B1 SL of CUMS group; B2 DL of CUMS group; C1 SL of curcumin (10 mg/kg) group; C2 DL of curcumin (10 mg/kg) group; D1 SL of curcumin (40 mg/kg) group; D2 DL of curcumin (40 mg/kg) group; E1 SL of curcumin (80 mg/kg) group; E2 DL of curcumin (80 mg/kg) group; F1 SL of fluoxetine group; F2 DL of fluoxetine group. Scale bar = 100 mm.
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Fig. 4 – The ultrastructures of the masseter muscle observed by transmission electron microscopy (20,000T). A1 SL of control group; A2 DL of control group; B1 SL of CUMS group; B2 DL of CUMS group; C1 SL of curcumin (10 mg/kg) group; C2 DL of curcumin (10 mg/kg) group; D1 SL of curcumin (40 mg/kg) group; D2 DL of curcumin (40 mg/kg) group; E1 SL of curcumin (80 mg/kg) group; E2 DL of curcumin (80 mg/kg) group; F1 SL of fluoxetine group; F2 DL of fluoxetine group. Mitochondria with normal ultrastructures are marked by white arrows. Swollen mitochondria with loss of the cristae, reductions in the matrix density and vacuolar changes are marked by black arrows. G1 Percentage of microstructurally changed SL of MM. G2 Percentage of microstructurally changed DL of MM. *p < 0.003.
treated group. The ultrastructures of the masseter myocytes (SL and DL) of the rats in the control and curcumin (80 mg/kg) treated groups demonstrated evenly distributed muscular nuclei under the sarcolemma around muscle fibers, and no signs of hyperplasia, swelling, or pyknosis were observed. The myofibrils and myotomes of the CUMS rats were normal and resembled to those of the rats in the other 5 groups. The light I bands and dark A bands of the myotomes in the 6 groups of rats were intact and located in the correct positions. Dramatic vacuolar changes in the mitochondria appeared in the masseter myocytes of the CUMS rats (7 out of 8 samples of SL and 7 out of 8 samples of DL, marked by black arrows, Fig. 4B1 and B2, respectively). Swollen mitochondria with a loss of the cristae and a reduction in the matrix density were found in the MM in the 10 mg/kg curcumin group (6 out of 8 samples of SL and 6 out of 8 samples of DL, marked by black arrows, Fig. 4C1 and C2, respectively), the 40 mg/kg curcumin group (5 out of 8 samples of SL, and 6 out of 8 samples of DL, marked by black arrows, Fig. 4D1 and D2, respectively), and the fluoxetine treated rats (6 out of 8 samples of SL, and 6 out of
8 samples of DL, marked by black arrows, Fig. 4F1 and F2, respectively). Only a few slightly loss of cristae and a reduction in the matrix density were found in SL of masseter muscle of the 80 mg/kg curcumin group (2 out of 8 samples, marked by black arrows, Fig. 4E1). In contrast, there were nearly no swollen/abnormal mitochondria or vacuolar changes being observed in control group and in the DL of masseter muscle of the 80 mg/kg curcumin group (the normal mitochondria was marked by white arrows, Fig. 4A1, A2, and E2, respectively). The occurrence of degenerated mitochondria was counted in each group. The percentages of microstructural changes for mitochondria degeneration in each group were calculated as: the number of sample with mitochondria degeneration/the number of total samples 100%. The chi-square test showed significant difference of microstructurally changed SL of masseter muscle between the CUMS group and control group (Fig. 4G1, p < 0.003). The significant differences were also noted for DL of masseter muscle between the CUMS group and the control group, as well as the CUMS group and 80 mg/kg curcumin group (Fig. 4G2, p < 0.003).
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Fig. 5 – The SOD activity A, GSH-Px activity B, CAT activity C, and MDA content D of rats. All of the data are shown as the mean WSD. *p < 0.008.
All of the results indicated the successful establishment of the CUMS animal model as a rodent stress state, and 80 mg/kg was selected as the effective dosage for curcumin for the next experiment, which evaluated its protective effects against CUMS.
The activities of SOD, GSH-Px, and CAT and the MDA content As shown in Fig. 5, the activities of SOD, GSH-Px, and CAT in the SL and DL were significantly lower in the CUMS rats than in the control rats ( p < 0.008, Fig. 5A–C), whereas curcumin treatment (80 mg/kg) reversed the deficit in total anti-oxidant capacity ( p < 0.008, Fig. 5A–C). In the masseters of the fluoxetine group, the SOD, GSH-Px, and CAT activity exhibited improvements, but no significance was found ( p > 0.008, Fig. 5A–C). With respect to the MDA content, the CUMS treatment caused an increase in the masseter compared with the control group ( p < 0.008, Fig. 5D). However, this value was reduced following the administration of curcumin (80 mg/kg) ( p < 0.008, Fig. 5D), but not fluoxetine ( p >0.008, Fig. 5D). No significant difference of SOD, GSH-Px, CAT activities, and MDA content were found between the SL and DL of masseter muscle ( p > 0.05).
The activities of Na+-K+ATPase, Ca2+-Mg2+ATPase, and LDH and the LD content The energy metabolism enzymatic activities in the masseter were remarkably altered in the rats that received CUMS.
Specifically, the Na+-K+ATPase and Ca2+-Mg2+ATPase activities significantly decreased ( p < 0.008, Fig. 6A and B), whereas the LDH activity and LD content significantly increased ( p < 0.008, Fig. 6C and D) in the CUMS rats compared with the control rats. However, curcumin (80 mg/kg) treatment restored normal enzymatic activities ( p < 0.008, Fig. 6A–C). Compared with the CUMS group, the masseters of the fluoxetine treatment group demonstrated a similar trend as curcumin group (80 mg/kg), but with no significance ( p > 0.008, Fig. 6A–C). The LD content in the masseters of the CUMS rats was elevated compared with the control animals, and a clear decrease in this parameter was observed in the curcumin (80 mg/kg), ( p < 0.008, Fig. 6D) group, but not in fluoxetine group ( p > 0.008). No significant difference of these indices was found between the SL and DL of masseter muscle ( p > 0.05), except the activity of Ca2+-Mg2+ATPase is higher in SL than DL of MM in control group ( p < 0.05,Fig. 6B).
Discussion Depression is a common human psychological stress state. In recent years, the therapeutic potential of curcumin for many types of depression-related diseases has attracted researchers’ attention.25–27 However, there is still lack of animal studies to investigate the adverse effects of depression on the MM. In addition, it has not yet been determined whether curcumin exerts protective effects in the jaw muscle. To our knowledge,
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Fig. 6 – The Na+-K+ATPase activity A, Ca2+-Mg2+ATPase activity B, LDH activity C, and LD content D of rats. All of the data are shown as the mean WSD. *p < 0.008.
this study is the first to report the capacity of curcumin to reverse the pathological changes in the ultrastructures and biochemistry of the masticatory muscles of rats that have been exposed to CUMS. CUMS is widely used in experiments and investigations at present because it provides a realistic model of the stresses of daily life. Besides, the prolonged time course of the model was suitable for determining the effects of chronic drug treatments.6 Sucrose preference and the open-field test results indicated that the CUMS procedure successfully induced depressive-like behaviors in rats. Similarly to the classical anti-depressant fluoxetine, curcumin is capable of alleviating the depressive state of experimental rats, as evidenced by the fact that curcumin significantly restored the altered behavior parameters of CUMS treated rats. These results are similar to the findings of Wang.28 Furthermore, the data revealed the superior anti-depressant effect of a dose of 80 mg/kg curcumin compared with doses of 10 or 40 mg/kg curcumin, raising the possibility that curcumin acts in a dose-dependent manner in treating depression. However, the upper limit of the drug dose in relieving depressed mood is unclear from these data;
therefore, this parameter should be evaluated in future studies. The transmission electron microscopy indicated large numbers of severe vacuolar pathological changes in the mitochondria of both SL and DL of the masseters of the CUMS rats. These ultrastructural observations are consistent with the possibility that the mitochondria from the muscles of stressed rats may exhibit altered permeability characteristics.23 We also found swollen mitochondria with loss of the cristae and reductions in the matrix density in the MMs of 10, 40 mg/kg curcumin treat rats and the fluoxetine treated rats. By contrast, mitochondrial abnormalities were significantly reduced in the 80 mg/kg curcumin treated rats. These observations indicated that lesions of mitochondria are likely secondary to the CUMS procedure and 80 mg/kg curcumin is more effective than fluoxetine at protecting mitochondria from damage caused by stress. That was probably because, other than curcumin, fluoxetine might have a directly negative influence on mitochondria, for it was reported that the in vitro administration of fluoxetine resulted in disruption of mitochondrial function.29
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Oxidative stress has been implicated in many psychiatric disorders, including major depressive disorder.30 In the current study, the SOD, GSH-Px, and CAT activities and the MDA content, which are the major indices of oxidative stress, were examined in the masseter. The results, which demonstrated decreased anti-oxidase activities and increased MDA contents in stressed animals, indicated that CUMS can disturb the balance between oxidant formation and anti-oxidant defenses in the masseter. Stress can activate the sympathetic nervous system (SNS) and the hypothalamus-pituitary-adrenal axis (HPA). These systems respond to chronic maladaptive stimulation by producing catecholamine (SNS) and by subsequently activating the immune response and inflammatory response system (HPA); these responses induce hypoxia and ischemia, and increase the levels of proinflammatory cytokines that interact with inflammatory cells, respectively. These processes eventually lead to the generation of reactive oxygen species (ROS), resulting in oxidative stress in the muscle tissue.31 The microstructural changes that were observed could be caused by the excessive accumulation of ROS, given that these molecules can damage the cell membrane, disrupt the structures of protein molecules, and alter the functions of mitochondrial transport.32 In our experiment, we found that curcumin have positive effects on maintaining redox homeostasis in the MM. This finding is unsurprising because of the powerful antioxidant effect of curcumin, which possesses a natural phenolic hydroxyl structure that can directly capture and eliminate free radicals, thereby protecting tissues against oxidative stress injuries.33–35 The activities of Na+-K+ATPase and Ca2+-Mg2+ATPase were significantly reduced in the CUMS group. A reduction in ATPase activity is a sign of a damaged cell membrane and is primarily caused by hypoxia, acid metabolites, and free radicals.36,37 The low activities of Na+-K+ATPase and Ca2+Mg2+ATPase result in high concentrations of Na+ and Ca2+ inside the cells, which causes the swelling and damage. This result further supports our ultrastructural observations. LDH activity is typically viewed as a sign of anaerobic oxidation in skeletal muscle. LD is a product of glycolysis; this molecule is an important product of muscle activity and is recognized as the fatigue-causing compound. In the present study, the increased LDH activity and LD content in the MMs of stressed rats indicated the enhancement of anaerobic metabolism in muscle, which is consistent with the clinical phenomena of MM fatigue, pain and other symptoms of depressive disorder.2,3 The administration of curcumin (80 mg/kg) reversed the lower activities of Na+-K+ATPase and Ca2+-Mg2+ATPase and decreased LDH activities and LD levels. This result is in agreement with the findings of a previous report.38,39 We know that the masseter muscle is a very important and powerful muscle in the human mastication system. This organ can be anatomically divided into the superficial and the deep portion, with both their functions 40 and myofiber constituents being different.41 Therefore, we separately focused on the superficial and deep portions of the MM in the study of rats that were exposed to CUMS and/or receiving antidepressants. Hardly any differences between the SL and DL of masseter after CUMS procedure and anti-depressant treatment with curcumin and fluoxetine were observed after biomechanical analysis, indicating that the superficial part and
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the deep part of masseter muscle displayed similar redox and energy metabolism responses to the CUMS. In the current study, we used 2 types of anti-depressants, curcumin, and fluoxetine, as the antagonists to stress. The antidepressant effects of both of these substances are related to the central monoaminergic neurotransmitter system. According to the behavioral test results, fluoxetine did the same way with the high-dose curcumin (80 mg), which showed significant effects against behavioral changes by mental stress. However, the micrographs of mitochondria of the fluoxetine group still generally exhibited swollen mitochondria with loss of the cristae and reduction in the matrix density, which indicated that the anti-stress effect of the fluoxetine for muscular mitochondria was far less obvious compared with that of the high-dose curcumin. Whereas, we also noticed that there were rarely dramatic vacuolar degenerations of mitochondria in fluoxetine treated group, and also less mitochondria degenerations compared with the samples from the CUMS group. This phenomenon was generally in accordance with the trends of biochemical assays, which convinced that the anti-depressant effect of fluoxetine could partly neutralize the adverse impact of depression induced by stress, which helped to maintain the muscular metabolism and homeostasis to some extent. Compared with fluoxetine, our results for curcumin showed a potential therapeutic benefit, not only as a behavioral antidepressant but also in restoring the abnormal ultrastructural features and biochemical indices that were caused by CUMS. Other studies have demonstrated the protective effects of curcumin against skeletal muscle ischemia-reperfusion injury and muscle atrophy,13,42 indicating that curcumin favors the recovery of muscle dysfunction. Additionally, curcumin is also well known as an anti-oxidant agent.43 The oxidation resisting character of curcumin may as well contribute to the protective effect of curcumin against muscle disorder. Moreover, curcumin is a very safe anti-depressant. 44,45 The side effects that have been reported for fluoxetine, such as hypomania,46 congenital cardiovascular defects and suicidal ideation in children,47,48 have not been observed for curcumin. A close relation has been found in depressive disorder with a decreased activity of main peripheral anti-oxidant defences, including SOD, GSH-Px, CAT and the increased level of MDA.49,50 While an ameliorated depressive state after treatment with anti-depressant is confirmed to be related to the reforming of redox homeostasis in vivo, therefore, the protective role of curcumin played against oxidative stress and energy metabolism disorder in CUMS rats may be ascribed to the anti-depressant effect of curcumin in the present study.51,10,11 In addition, another potential cause that curcumin could scavenge free radicals directly and maintain homeostasis in tissue, should be taken into consideration. Since previous studies have demonstrated that curcumin could enhance the activities of oxidant enzymes,52,53 and energy metabolic enzymes, which are very sensitive for free radical reaction,54 the improved redox homeostasis and energy metabolism in stressed rats after the administration of curcumin are also considered to be the results of the antioxidant effect of curcumin. However, such speculations still need to be confirmed in future studies. In conclusion, the CUMS procedure resulted in pathological ultrastructural changes, oxidative damage, and energy
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metabolism disorders in the MMs of rats, providing evidence for the pathogenesis of masticatory muscle dysfunction after psychological stress. We found that curcumin was capable of restoring CUMS-induced deficits in the MM. In this case, the muscle alterations induced by the CUMS procedure can be reversed by the anti-depressant and anti-oxidant curcumin in a dose-dependent manner. Our study provided a potential preventive treatment for depression patients who are at risk of developing MM disorders.
Acknowledgment This work was financially supported by the National Nature Science Foundation of China(No. (81070851, (31170888, (81371188).
references
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