Experimental Lung Research, 41, 12–20, 2015 Copyright © 2015 Informa Healthcare USA, Inc. ISSN: 0190-2148 print / 1521-0499 online DOI: 10.3109/01902148.2014.959140
ORIGINAL ARTICLE
Substance P protects against hyperoxic-induced lung injury in neonatal rats Bo Huang,1 Qing Li,1 Shuhong Xu,1 Mingyang Tian,1 Xinghui Zhen,1 Yunxia Bi,1 and Feng Xu2 1
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Pediatric Intensive Care Unit, The First People’s Hospital of Zunyi, Zunyi, China Pediatric Intensive Care Unit, Children’s Hospital, Chongqing Medical University, Chongqing, China A B STRACT The aim of the study was to investigate the effects of substance P (SP) in hyperoxia-induced lung injury in newborn rats. Thirty-two rat pups were randomly divided into four groups: normoxia/saline, normoxia/SP, hyperoxia/saline and hyperoxia/SP. In a separate set of experiments, the neonatal rat pups were exposed to 21% or >95% O2 for 14 days with or without intraperitoneal administration of SP. On day 14, the animals were sacrificed and the lungs were processed for histology and biochemical analysis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used for the detection of apoptosis. Antioxidant capacity was assessed by glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), oxidative stress was assessed by determining the extent of formation of malondialdehyde (MDA), activities of NADPH oxidase activity, and formation of reactive oxygen species (ROS). The activity of phospho-p38 (p-p38) and -ERK1/2 (p-ERK1/2) proteins and expression of NF-E2-related factor 2 (NRF2) were detected by Western blot, and the expression of p-p38 was detected by immunofluorescence analysis. Compared with the hyperoxia treatment, the lung damage was significantly ameliorated following the SP treatment. Furthermore, the lungs from the pups exposed to hyperoxia TUNEL-positive nuclei increased markedly and decreased significantly after SP treatment. The levels of MDA decreased and that of GSH-Px and SOD increased following the SP treatment. The SP treatment significantly suppressed the activity of NADPH oxidase and reduced ROS production. SP stimulation may result in blocking p38 MAPK and ERK signaling pathways, and the activities of p-p38 and p-ERK, and expression of NRF2 decreased following the SP treatment. These findings indicate that SP can ameliorate hyperoxic lung injury through decreasing cell apoptosis, elevating antioxidant activities, and attenuating oxidative stress. KEYWORDS apoptosis, hyperoxic lung injury, oxidative stress, Substance P
on cell structures, such as membranes, mitochondria, and nuclei to injure pulmonary cells through enzyme oxidation, which includes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) and lipid peroxidation [4,5]. Then both necrosis and apoptosis events emerge [6]. Developing therapeutic strategies based on the molecular pathogenesis of hyperoxia-induced neonatal lung injury is critical. The mitogen-activated protein kinases (MAPKs) play an important role in hyperoxia-induced cell injury [7]. The MAPK families are critical signal transduction mediators that can be stimulated by some growth factors [8], which consist of at least three major subfamilies: (1) C-Jun N-terminal kinases (JNK), (2) extracellular signal-regulated kinase (ERK), and (3) the p38 MAPK subfamily [9]. The p38 MAPK is mostly associated with cellular responses to stress [7,10]. The activation of p38
INTRODUCTION With improvements in neonatal care in general, premature infants with immature lungs frequently require respiratory care, such as hyperoxia. Supplement of very high concentrations of oxygen is required to maintain sufficient blood oxygenation in the patients, which can lead to the development of chronic lung disease in infancy, also known as bronchopulmonary dysplasia (BPD) [1,2]. In recent years, reactive oxygen species (ROS) generated during hyperoxic insult have been generally accepted to play a critical role in lung injury [3]. ROS can direct detrimental effects Received 12 December 2013; accepted 25 August 2014 Address correspondence to Dr. Bo Huang, PhD, Pediatric Intensive Care Unit, The First People’s Hospital of Zunyi, 149 Dalian Road, Zunyi 563003, Guizhou, China. E-mail: [email protected]
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Substance P Protects Against Hyperoxic Lung Injury
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MAPK has been shown to be associated with the hyperoxia-induced cell damage. The substance P (SP) was first found to be a neuropeptide of the tachykinin family that is involved in the regulation of some different biological processes [11–13]. It is distributed widely in the airway endothelial cell layer, bronchus smooth muscle, bronchus ganglion, trachea, pulmonary vessel, and surrounding glands. There is ample evidence supporting that SP might serve as an antagonist of apoptotic processes of impaired cells [14,15], and the protective effects of SP may be relevant in various neurodegenerative disorders [16]. Interestingly, in vitro studies have demonstrated that SP had a protective effect on the type II alveolar epithelial cell (AECIIs) and decreases AECIIs apoptosis [17]. These results suggest that SP is a possible therapeutical benefit in hyperoxia-induced neonatal lung injury. We hypothesize that SP could prevent hyperoxic lung injury during the newborn period, and that this protective effect is mediated by blocking hyperoxia-induced activation of p38 MAPK pathway. This study demonstrated that SP ameliorates hyperoxic lung injury by modulating the MAPK pathway and the expression of NRF2. The findings suggest a potentially novel and easily applicable solution to hyperoxic lung injury, and provide new insight into our scientific knowledge of SP.
a day and the other groups, i.e., control and hyperoxia groups, received equal volumes of saline. SP was purchased from Sigma company (Sigma-Aldrich, St. Louis, MO). Experimental treatments were started immediately after birth and continued throughout postnatal day 14. All animal procedures in this study were in accordance with the guidelines established by the Association for Assessment and Accreditation of Laboratory Animal Care. The protocols were approved by the committee on the ethics of animal experiments of the First People’s Hospital of Zunyi (Protocol number IACUC-F201304-113).
METHODS AND MATERIALS
Measurement of SP Levels
Animals and Hyperoxia Exposure Protocol First-time pregnant Sprague-Dawley rats were kept in humidity- and temperature-controlled rooms (12hour dark–light cycle) and fed a standard rat chow ad libitum. Pups delivered spontaneously naturally were pooled, randomized, and returned to the nursing dams. Nursing mothers were rotated between hyperoxia- and normoxia-groups every 24 hours to prevent oxygen toxicity. Rats were exposed continuously to either normoxia (21% O2 ) or hyperoxia (>95% O2 ) at a flow rate of 3 L/minute in a chamber (90 × 60 × 40 cm) flow-through system. Thirty-two rat pups were divided into four groups (N = 8 per group). Two groups of pups were kept in room air (normoxia group) as controls and the second group of pups was treated with SP. The third group of pups was exposed to hyperoxia (hyperoxia group) and the fourth group was exposed to hyperoxia and treated with SP (95% O2 , hyperoxia/SP group). Pups in the hyperoxia/SP and normoxia/SP groups were injected intraperitoneally (i.p.) with SP at a dose of 1 × 10−6 mol/L/kg body weight once C
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Assessment of Lung Function and Injury and Tissue Collection The pups were killed on day 14 under anesthesia by i.p. injection of pentobarbital sodium (200 mg/kg). The thorax was opened and the lungs were harvested, lavaged, or inflation-fixed. The lungs were then perfused through the main pulmonary artery with phosphate-buffered saline prior to tissue collection and immediately snap-frozen in liquid nitrogen and stored at –70◦ C until use for biochemical investigations except in samples collected to assess the wetto-dry (W/D) weight ratio. W/D ratio was measured as described previously [18].
The lung samples were thawed, weighed, and homogenized at a ratio of 1:9 (w/v) in 1 mL ice-cold SP assay buffer for 20 seconds. The homogenate was then centrifuged at 4000× g for 20 minutes at 4◦ C. The levels of SP in the supernatant were measured using a rat SP ELISA kit according to the supplier’s assay instructions [19]. The concentration of SP was expressed in pg/mg.
Histopathological Analysis For histological evaluation, the pulmonary circulation was first perfused with heparinized saline at a constant hydrostatic pressure of 20 cm water until the left atrial effluent was clear of blood. Formalin was then instilled through the pulmonary artery catheter at a pressure of 30 cm water. The left lung was then inflated through the tracheal catheter using 10% formalin at a pressure of 25 cm water until the pleural surface became smooth. The lung was stored in 10% formalin and processed for light microscopy. Tissues were embedded in paraffin, and sectioned and stained with hematoxylin and eosin respectively. Lung injury
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was blindly scored according to previously described criteria [18].
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Biochemical Analysis Activities of GSH-Px and SOD, MDA content, and total ROS production of lung tissues were measured using cell-free supernatants of right lung homogenates by spectrophotometric analyses. The GSH-Px activity was measured using the dithiodinitrobenzoic acid method as described previously [20]. The tissue SOD activity was assayed using a method involving spectrophotometric detection of formazan production at 550 nm by inhibition of nitroblue tetrazolium reduction as described previously [21]. The tissue MDA content was determined with the thiobarbituric acid reaction of MDA with thiobarbituric acid at 95◦ C according to the method described previously [22]. The total ROS production of lung tissues was determined by the DCFDA fluorescence method [23].
TUNEL Assay Lung tissue was analyzed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Staining was performed on paraffinembedded sections by using the in situ cell death detection kit (Roche, Indianapolis, IN, USA). TUNEL-positivity was detected for labeled cells with yellow-brown particle deposition in nuclei. Ten fields were randomly selected in each section under a light microscope (×400). The apoptotic index was determined by the percentage of labeled cells.
Western Blot Analysis Western blot analysis was performed on 30 μg of lung homogenates, which were resolved by SDS-PAGE for the proteins as described previously [24]. The following primary antibodies were used: phospho-p38 (p-p38), p38 MAPK, phospho-ERK1/2 (p-ERK1/2), ERK1/2, NF-E2-related factor 2 (NRF2), fibrillarin, and Beta-actin (Santa Cruz Biotechnology, Santa Cruz, CA) at a concentration of 1:1000 dilution overnight at 4◦ C. The blots were then incubated with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody at a concentration of 1:1000 for 1 hour. Immunodetection was enhanced by chemiluminescence using ECL Plus. Images were captured on Image pro plus 6.0 software. The total proteins were used for analysis of phospho-p38 MAPK, p38 MAPK, phospho-
ERK1/2, ERK1/2, and Beta-actin; the separated nucleus proteins were used for the analysis of NRF2 and fibrillarin.
Immunofluorescent Analysis The method was previously described for lung tissues [25]. For immunofluorescent analysis, anti-phosphop38 (Santa Cruz Biotechnology, Santa Cruz, CA) was used.
NADPH Oxidase Activity Lucigenin-enhanced chemiluminescence was used to measure NADPH oxidase activity in lungs according to the method described previously [26].
Statistical Analysis Statistical differences between the groups were evaluated by two-way ANOVA followed by Bonferroni ttest. A level of P < .05 was considered to represent statistical significance. Experimental group mean values and standard errors (SEs) were determined using descriptive statistics, and values are means ± SE. Statistical analysis was performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA).
RESULTS SP Reduced Hyperoxic Lung Injury and Lung Edema Induced by Hyperoxia To investigate the effects of SP on hyperoxic lung injury, the rats of hyperoxia/SP and normoxia/SP groups were injected SP for 14 days. The SP level of lungs increased significantly in hyperoxia/SP (6690 ± 440 pg/mg) and normoxia/SP (6695 ± 563 pg/mg) groups compared with hyperoxia/saline (1244 ± 188 pg/mg) and normoxia/saline (1034 ± 125 pg/mg) groups (Figure 1A). To evaluate the extent of oxygen-induced lung damage, the W/D weight ratio was measured at the same time in all four groups after 14 days. The W/D ratio of the rat lung tissue increased significantly in rats exposed to hyperoxic conditions (hyperoxia: F[1,20] = 15.4, P < .01, Figure 1B). Compared with the lungs from hyperoxia/saline rats, SP ameliorated hyperoxia-induced lung edema as indicated by a significant decrease in W/D ratio (SP: F[1,20] = 6.3, P < .05; hyperoxia×SP: F[1,20] = 3.2, P < .05, Figure 1B). The effects of SP treatment on the histopathologic changes in rat lungs are shown in Figure 1C. Experimental Lung Research
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FIGURE 1. Measurement of SP level, W/D ratio, and histopathology analysis in rats. (A) SP level of lungs measured by ELISA after 14 days of normoxia or hyperoxia. N = 6 for each group, # P < .01, ∗ P < .05. (B) W/D ratio of lungs after 14 days of normoxia or hyperoxia. N = 6 for each group, # P < .01, ∗ P < .05. (C) Representative images of hematoxylin-eosin staining of lung. Top panels: Lungs exposed to normoxic conditions with 21% O2 . Bottom panels: Lungs exposed to hyperoxic conditions with 95% O2 . 400× magnification. (D) The lung injury scores of hematoxylin-eosin staining lungs. The score of lung injury was assessed by (1) infiltration or aggregation of neutrophils in airspace, the alveolar wall, or the vessel wall, (2) thickness of the alveolar wall, and (3) alveolar congestion, and each item was graded on a four-point scale. The score of each component ranged from 0 to 3, and higher scores indicated more severe damage. A total score was calculated as the sum of three components. At least eight fields were chosen randomly from each section. 400× magnification. N = 6 for each group, # P < .01, ∗ P < .05.
The lungs of the rats exposed to hyperoxia showed marked severe edema and cellular infiltration in the interstitial area and the associated thickening of the alveolar septum. In the presence of SP, both edema and inflammatory cell infiltration were reduced despite exposure to hyperoxia; there was no significant histopathologic difference between normoxia/saline and normoxia/SP groups (Figure 1C). In addition, the lung injury scores in hyperoxia/SP rats were significantly lower than those of hyperoxia/saline rats (hyperoxia: F[1,20] = 24.2, P < .01; SP: F[1,20] = 7.8, P < .05; hyperoxia×SP: F[1,20] = 2.4, P < .05, Figure 1D). C
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GSH-Px and SOD Activities, MDA Content Measurements in Lung Tissues Biochemical analyses revealed that the rats to hyperoxia significantly decreased tissue GSH-Px and SOD activities, whereas it increased tissue MDA content, which are shown in Table 1. By contrast, the GSHPx and SOD activities were significantly higher in the hyperoxia/SP group as compared with the hyperoxia/saline group (40.4 ± 3.1 vs. 27.9 ± 1.6 U/mg, P < .05, and 250 ± 41 vs. 142 ± 25 U/mg, P < .05 respectively), indicating the efficacy of SP on decreasing oxidative stress. The MDA content (4.65 ±
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TABLE 1. Results Showing GSH-Px and SOD Activities, and MDA Content in Lung Tissue of Rats
Effect of SP Treatment on Hyperoxia-Induced Oxidative Stress
Biochemical analyses
When compared with the normoxia/saline group, oxygen exposure resulted in the upregulated activities of NADPH oxidase (hyperoxia: F[1,20] = 23.1, P < .01, Figure 3A) and enhanced formation of ROS (hyperoxia: F[1,20] = 31.4, Figure 3B). Treatment with SP significantly suppressed NADPH oxidase activity (SP: F[1,20] = 12.5, P < .05; hyperoxia×SP: F[1,20] = 5.2, P < .05, Figure 3A) and reduced ROS production (SP: F[1,20] = 19.1, P < .05; hyperoxia×SP: F[1,20] = 4.5, P < .05, Figure 3B).
Normoxia/saline Normoxia/SP Hyperoxia/saline Hyperoxia/SP
GSH-Px (U/mg)
SOD (U/mg)
MDA (nmol/mg)
57.3 ± 1.91 53.2 ± 3.1 27.9 ± 1.6# 40.4 ± 3.1∗
355 ± 15 358 ± 35 142 ± 25# 250 ± 41∗
3.21 ± 0.32 3.64 ± 0.43 8.77 ± 0.15# 4.65 ± 0.42∗
P < .05 significantly compared with normoxia/saline group. P < .05 significantly compared with hyperoxia/saline group.
#
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∗
0.42 vs. 8.77 ± 0.15 nmol/mg, P < .05) was significantly lower in the hyperoxia/SP group as compared with that in the hyperoxia/saline group, suggesting an SP effect on decreasing lipid peroxidation (Table 1). There was no significant difference in GSH-Px, SOD, and MDA levels in normoxia/saline and normoxia/SP groups.
Detection of Apoptotic Cell Death As shown in Figure 2, the number of TUNELpositive cells was increased in rats exposed to hyperoxic conditions after 14 days (hyperoxia: F[1,20] = 15.2, P < .01). The SP treatment comparably decreased the percentage of TUNEL-positive cells relative to the hyperoxia/saline group (SP: F[1,20] = 4.9, P < .05; hyperoxia×SP: F[1,20] = 3.3, P < .05). The normoxia/SP group showed no significant increasing number of TUNEL-positive cells compared with hyperoxia/SP group.
Effect of SP on Hyperoxia-Induced Activation of MAP Kinases and NRF2 in Nucleus To probe into the mechanism of SP action, we focused on p38 MAPK and ERK. The rats were exposed to hyperoxia for 14 days, which resulted in significant increase in p-p38 and pERK1/2 activation, expressed as the ratio of phosphorylated/total p38 protein levels and phosphorylated/total ERK1/2 protein levels; SP treatment blocked p-p38 and pERK1/2 activation (Figure 4A). In accord with these observed effects of SP on p-p38 and p-ERK1/2, under the same experimental conditions, there was an increase in staining for p-p38, which was blocked by SP (hyperoxia: F[1,12] = 31.2, P < .01; SP: F[1,12] = 11.4, P < .05; hyperoxia×SP: F[1,12] = 2.3, P < .05, Figures 4C and D). We also found the increased expression of NRF2 in nucleus when lungs were exposed to hyperoxia, and treatment with SP
FIGURE 2. Detection of apoptotic cell death by TUNEL staining in all four groups after 14 days of oxygen exposure. (A)
Representative images of TUNEL staining of lung sections. Upper panels: Lungs exposed to normoxic conditions with 21% O2 . Lower panels: Lungs exposed to hyperoxic conditions with 95% O2 . 400× magnification. (B) Shown in a number of TUNEL-positive cells. N = 6 for each group, # P < .01, ∗ P < .05.
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FIGURE 3. Effect of SP treatment on hyperoxia-induced oxidative stress. Rats were exposed to hyperoxia for 14 days. N = 5 for
each group, # P < .01, ∗ P < .05. (A) The lungs were removed for assessment of activities of NADPH oxidase. (B) Total ROS accumulation was measured after 14 days of normoxia or hyperoxia with or without SP treatment.
significantly downregulated NRF2 expression (Figure 3B).
DISCUSSION For the first time, the current study demonstrated the protective actions of SP on the hyperoxic lung injury in vivo. This study demonstrated significant improvement in W/D, less lung injury as measured by histopathology analysis, increased antioxidant enzyme activities (SOD, GSH-Px), and decreased oxidative stress marker (MDA) when hyperoxic neonatal rats were treated with SP. Our data demonstrated that SP downregulated phosphorylated p38, and decreased cell apoptosis in hyperoxic lung injury, supporting the idea that SP attenuates apoptosis by decreasing p38 activation. Substance P is crucial for proliferation, migration, and differentiation of impaired cells [27,28]. Recent studies have demonstrated that SP treatment decreases the apoptosis of AECII, and attenuates the morphological alteration of AECII at different time points after hyperoxia exposure [17]. In this study, SP was shown to be able to markedly decrease TUNELpositive nuclei in hyperoxia-exposed lung, suggesting that SP could partially reverse apoptosis increasing effects on hyperoxia-exposed lung. Oxygen causes tissue injury through the formation of destructive radicals, such as hydroxyl radicals, which and highly reactive, and through the peroxidation of membrane lipids [29,30]. Prolonged hyperoxia exposure will cause oxidative stress, which is decisive for hyperoxic lung injury, and antioxidant therapies are thought to improve lung morphol C
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ogy [31]. Moreover, the activity of SOD and GSHPx, which are intracellular enzymatic antioxidant defense, makes premature infants highly susceptible to oxidative injury [32]. A previous study has found that hyperoxia could induce ROS formation in pulmonary capillary endothelial cells in situ, and it might be relevant to the initiation of hyperoxia-induced lung injury [32]. Therefore we observed a significant increase in lung MDA level in the hyperoxia group, suggesting increased lipid peroxidation. MDA, the last product of lipid breakdown caused by oxidative stress, is considered to be a good indicator of free radical-induced lipid peroxidation [33]. The results showed that on day 14, lung tissues of neonatal rats with SP treatment had high MDA levels and low activity of SOD and GSH-Px compared with the hyperoxia group. The sources of intracellular ROS during oxygen exposure remain unclear but may involve increased mitochondrial generation and activation of NADPH oxidases. The pathological changes in hyperoxia-injured lungs coincide with the injury or death of pulmonary capillary endothelial cells and alveolar epithelial cells [34]. In this work, we also found that treatment with SP suppressed the upregulation of activity of NADPH oxidase, which was reported as a major producer of ROS in lungs of rodents exposed to hyperoxia [35]. Our results demonstrated the protective role of SP in hyperoxic lung injury through its inhibitory action on oxygen-derived free radicals. MAPKs, including ERK, JNK, and p38, are phosphorylated in response to a series of specific pathways through which different extracellular signals can be conducted from the cell surface into the nucleus [36]. Studies in several different cellular systems showed that MAPK could be activated by ROS. The
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FIGURE 4. SP inhibits hyperoxia-induced phosphorylated p38 MAPK, phosphorylated pERK, and NRF2 expression in nucleus.
(A) Western blots for phosphorylated and total p38 MAPK and ERK1/2 on protein extracts from the lungs taken after 14 days of normoxia or hyperoxia with or without treatment of SP. (B) Western blots for proteins expression of NRF2 and fibrillarin in nucleus from the lungs taken after 14 days of normoxia or hyperoxia with or without SP treatment. (C) Representative images of immunofluorescent staining for phosphorylated p38 in lungs after 14 days of normoxia or hyperoxia with or without SP treatment. (D) Shown in a number of phospho-p38 positive cells. N = 4 for each group, # P < .01, ∗ P < .05.
p38 protein has been shown to be a inducer of cell death following exposure of cells to a wide variety of stimuli. The activation of p38 has also been shown to be associated with hyperoxia-induced epithelial damage [7], and another study further confirmed that hyperoxia activated p38 signaling pathways in type II cells [4]. To emphasize the effect of SP on phosphorylation of p38 MAPK and ERK1/2, we found that hyperoxia exposure significantly induced the activity of p-p38 and pERK1/2 in hyperoxic lung injury. Interestingly, treatment with additional SP effectively downregulated the activity of p-p38 and pERK1/2. Meanwhile, SP decreased apoptosis of lung tissues. Furthermore, NRF2 is a key transcription factor that regulates antioxidant genes as an adaptive response to oxidative stress or pharmocological stimuli, and its overexpression confers cellular protection against hyperoxia [37]. It is likely that NRF2 acts as one
of the important downstream effectors of PI3K pathway to regulate the expression of genes that contribute to cell survival following hyperoxic insult. We found increased expression of NRF2 in nucleus, and treatment with SP significantly downregulated NRF2 expression when lungs were exposed to hyperoxia. These data together are summarized in Figure S1. In conclusion, this study demonstrated, for the first time, that SP ameliorates hyperoxic lung injury by modulating the MAPK pathway. The findings suggest a potentially novel and easily applicable solution to hyperoxic lung injury, and provide new insight into our scientific knowledge of SP. Although further investigation is required, SP is expected to be an innovative therapeutic tool for unmet medical needs that currently cause considerable health burdens, in particular for critically ill patients with lung diseases. Experimental Lung Research
Substance P Protects Against Hyperoxic Lung Injury [7]
[8] [9]
[10]
[11]
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[15] FIGURE S1. Schematic summary of the protection of SP on
hyperoxic-induced lung injury in neonatal rats. SP suppresses the activity of NADPH oxidase and reduced SOD production on hyperoxic-induced lung injury, which blocks the activities of p-p38 and p-ERK, leading to the expression of decreased NRF2. In addition, SP ameliorates hyperoxia lung injury through decreasing cell apoptosis.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. This work was supported by the project of the Science and Technology Foundation of Guizhou Province (No. J[2011]2341), Department of Health Science and Technology Foundation of Guizhou Province (No. qzwkj2011-1-092), and Science and Technology Bureau Foundation of Zunyi City (No. [2011]22).
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of oxidative stress. Nutr Metab Cardiovasc Dis. 2005;15:316– 328. Parinandi NL, Kleinberg MA, Usatyuk PV, Cummings RJ, Pennathur A, Cardounel AJ, Zweier JL, Garcia JG, Natarajan V: Hyperoxia-induced NAD (P) H oxidase activation and regulation by MAP kinases in human lung endothelial cells. Am J Physiol Lung Cell Mol Physiol. 2003;284:L26– L38. Carnesecchi S, Deffert C, Pagano A, Garrido-Urbani S, M´etrailler-Ruchonnet I, Sch¨appi M, Donati Y, Matthay MA, Krause K-H, Argiroffo CB: NADPH oxidase-1 plays a crucial role in hyperoxia-induced acute lung injury in mice. Am J Respir Crit Care Med. 2009;180:972. Maulik D, Ashraf QM, Mishra OP, Delivoria-Papadopoulos M: Activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) during hypoxia in cerebral cortical nuclei of guinea pig fetus at term: role of nitric oxide. Neurosci Lett. 2008;439:94–99. Papaiahgari S, Kleeberger SR, Cho H-Y, Kalvakolanu DV, Reddy SP: NADPH oxidase and ERK signaling regulates hyperoxia-induced Nrf2-ARE transcriptional response in pulmonary epithelial cells. J Bio Chem. 2004;279:42302– 42312.
Experimental Lung Research
ORIGINAL ARTICLE
Substance P protects against hyperoxic-induced lung injury in neonatal rats Bo Huang,1 Qing Li,1 Shuhong Xu,1 Mingyang Tian,1 Xinghui Zhen,1 Yunxia Bi,1 and Feng Xu2 1
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Pediatric Intensive Care Unit, The First People’s Hospital of Zunyi, Zunyi, China Pediatric Intensive Care Unit, Children’s Hospital, Chongqing Medical University, Chongqing, China A B STRACT The aim of the study was to investigate the effects of substance P (SP) in hyperoxia-induced lung injury in newborn rats. Thirty-two rat pups were randomly divided into four groups: normoxia/saline, normoxia/SP, hyperoxia/saline and hyperoxia/SP. In a separate set of experiments, the neonatal rat pups were exposed to 21% or >95% O2 for 14 days with or without intraperitoneal administration of SP. On day 14, the animals were sacrificed and the lungs were processed for histology and biochemical analysis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used for the detection of apoptosis. Antioxidant capacity was assessed by glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), oxidative stress was assessed by determining the extent of formation of malondialdehyde (MDA), activities of NADPH oxidase activity, and formation of reactive oxygen species (ROS). The activity of phospho-p38 (p-p38) and -ERK1/2 (p-ERK1/2) proteins and expression of NF-E2-related factor 2 (NRF2) were detected by Western blot, and the expression of p-p38 was detected by immunofluorescence analysis. Compared with the hyperoxia treatment, the lung damage was significantly ameliorated following the SP treatment. Furthermore, the lungs from the pups exposed to hyperoxia TUNEL-positive nuclei increased markedly and decreased significantly after SP treatment. The levels of MDA decreased and that of GSH-Px and SOD increased following the SP treatment. The SP treatment significantly suppressed the activity of NADPH oxidase and reduced ROS production. SP stimulation may result in blocking p38 MAPK and ERK signaling pathways, and the activities of p-p38 and p-ERK, and expression of NRF2 decreased following the SP treatment. These findings indicate that SP can ameliorate hyperoxic lung injury through decreasing cell apoptosis, elevating antioxidant activities, and attenuating oxidative stress. KEYWORDS apoptosis, hyperoxic lung injury, oxidative stress, Substance P
on cell structures, such as membranes, mitochondria, and nuclei to injure pulmonary cells through enzyme oxidation, which includes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) and lipid peroxidation [4,5]. Then both necrosis and apoptosis events emerge [6]. Developing therapeutic strategies based on the molecular pathogenesis of hyperoxia-induced neonatal lung injury is critical. The mitogen-activated protein kinases (MAPKs) play an important role in hyperoxia-induced cell injury [7]. The MAPK families are critical signal transduction mediators that can be stimulated by some growth factors [8], which consist of at least three major subfamilies: (1) C-Jun N-terminal kinases (JNK), (2) extracellular signal-regulated kinase (ERK), and (3) the p38 MAPK subfamily [9]. The p38 MAPK is mostly associated with cellular responses to stress [7,10]. The activation of p38
INTRODUCTION With improvements in neonatal care in general, premature infants with immature lungs frequently require respiratory care, such as hyperoxia. Supplement of very high concentrations of oxygen is required to maintain sufficient blood oxygenation in the patients, which can lead to the development of chronic lung disease in infancy, also known as bronchopulmonary dysplasia (BPD) [1,2]. In recent years, reactive oxygen species (ROS) generated during hyperoxic insult have been generally accepted to play a critical role in lung injury [3]. ROS can direct detrimental effects Received 12 December 2013; accepted 25 August 2014 Address correspondence to Dr. Bo Huang, PhD, Pediatric Intensive Care Unit, The First People’s Hospital of Zunyi, 149 Dalian Road, Zunyi 563003, Guizhou, China. E-mail: [email protected]
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Substance P Protects Against Hyperoxic Lung Injury
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MAPK has been shown to be associated with the hyperoxia-induced cell damage. The substance P (SP) was first found to be a neuropeptide of the tachykinin family that is involved in the regulation of some different biological processes [11–13]. It is distributed widely in the airway endothelial cell layer, bronchus smooth muscle, bronchus ganglion, trachea, pulmonary vessel, and surrounding glands. There is ample evidence supporting that SP might serve as an antagonist of apoptotic processes of impaired cells [14,15], and the protective effects of SP may be relevant in various neurodegenerative disorders [16]. Interestingly, in vitro studies have demonstrated that SP had a protective effect on the type II alveolar epithelial cell (AECIIs) and decreases AECIIs apoptosis [17]. These results suggest that SP is a possible therapeutical benefit in hyperoxia-induced neonatal lung injury. We hypothesize that SP could prevent hyperoxic lung injury during the newborn period, and that this protective effect is mediated by blocking hyperoxia-induced activation of p38 MAPK pathway. This study demonstrated that SP ameliorates hyperoxic lung injury by modulating the MAPK pathway and the expression of NRF2. The findings suggest a potentially novel and easily applicable solution to hyperoxic lung injury, and provide new insight into our scientific knowledge of SP.
a day and the other groups, i.e., control and hyperoxia groups, received equal volumes of saline. SP was purchased from Sigma company (Sigma-Aldrich, St. Louis, MO). Experimental treatments were started immediately after birth and continued throughout postnatal day 14. All animal procedures in this study were in accordance with the guidelines established by the Association for Assessment and Accreditation of Laboratory Animal Care. The protocols were approved by the committee on the ethics of animal experiments of the First People’s Hospital of Zunyi (Protocol number IACUC-F201304-113).
METHODS AND MATERIALS
Measurement of SP Levels
Animals and Hyperoxia Exposure Protocol First-time pregnant Sprague-Dawley rats were kept in humidity- and temperature-controlled rooms (12hour dark–light cycle) and fed a standard rat chow ad libitum. Pups delivered spontaneously naturally were pooled, randomized, and returned to the nursing dams. Nursing mothers were rotated between hyperoxia- and normoxia-groups every 24 hours to prevent oxygen toxicity. Rats were exposed continuously to either normoxia (21% O2 ) or hyperoxia (>95% O2 ) at a flow rate of 3 L/minute in a chamber (90 × 60 × 40 cm) flow-through system. Thirty-two rat pups were divided into four groups (N = 8 per group). Two groups of pups were kept in room air (normoxia group) as controls and the second group of pups was treated with SP. The third group of pups was exposed to hyperoxia (hyperoxia group) and the fourth group was exposed to hyperoxia and treated with SP (95% O2 , hyperoxia/SP group). Pups in the hyperoxia/SP and normoxia/SP groups were injected intraperitoneally (i.p.) with SP at a dose of 1 × 10−6 mol/L/kg body weight once C
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Assessment of Lung Function and Injury and Tissue Collection The pups were killed on day 14 under anesthesia by i.p. injection of pentobarbital sodium (200 mg/kg). The thorax was opened and the lungs were harvested, lavaged, or inflation-fixed. The lungs were then perfused through the main pulmonary artery with phosphate-buffered saline prior to tissue collection and immediately snap-frozen in liquid nitrogen and stored at –70◦ C until use for biochemical investigations except in samples collected to assess the wetto-dry (W/D) weight ratio. W/D ratio was measured as described previously [18].
The lung samples were thawed, weighed, and homogenized at a ratio of 1:9 (w/v) in 1 mL ice-cold SP assay buffer for 20 seconds. The homogenate was then centrifuged at 4000× g for 20 minutes at 4◦ C. The levels of SP in the supernatant were measured using a rat SP ELISA kit according to the supplier’s assay instructions [19]. The concentration of SP was expressed in pg/mg.
Histopathological Analysis For histological evaluation, the pulmonary circulation was first perfused with heparinized saline at a constant hydrostatic pressure of 20 cm water until the left atrial effluent was clear of blood. Formalin was then instilled through the pulmonary artery catheter at a pressure of 30 cm water. The left lung was then inflated through the tracheal catheter using 10% formalin at a pressure of 25 cm water until the pleural surface became smooth. The lung was stored in 10% formalin and processed for light microscopy. Tissues were embedded in paraffin, and sectioned and stained with hematoxylin and eosin respectively. Lung injury
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was blindly scored according to previously described criteria [18].
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Biochemical Analysis Activities of GSH-Px and SOD, MDA content, and total ROS production of lung tissues were measured using cell-free supernatants of right lung homogenates by spectrophotometric analyses. The GSH-Px activity was measured using the dithiodinitrobenzoic acid method as described previously [20]. The tissue SOD activity was assayed using a method involving spectrophotometric detection of formazan production at 550 nm by inhibition of nitroblue tetrazolium reduction as described previously [21]. The tissue MDA content was determined with the thiobarbituric acid reaction of MDA with thiobarbituric acid at 95◦ C according to the method described previously [22]. The total ROS production of lung tissues was determined by the DCFDA fluorescence method [23].
TUNEL Assay Lung tissue was analyzed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Staining was performed on paraffinembedded sections by using the in situ cell death detection kit (Roche, Indianapolis, IN, USA). TUNEL-positivity was detected for labeled cells with yellow-brown particle deposition in nuclei. Ten fields were randomly selected in each section under a light microscope (×400). The apoptotic index was determined by the percentage of labeled cells.
Western Blot Analysis Western blot analysis was performed on 30 μg of lung homogenates, which were resolved by SDS-PAGE for the proteins as described previously [24]. The following primary antibodies were used: phospho-p38 (p-p38), p38 MAPK, phospho-ERK1/2 (p-ERK1/2), ERK1/2, NF-E2-related factor 2 (NRF2), fibrillarin, and Beta-actin (Santa Cruz Biotechnology, Santa Cruz, CA) at a concentration of 1:1000 dilution overnight at 4◦ C. The blots were then incubated with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody at a concentration of 1:1000 for 1 hour. Immunodetection was enhanced by chemiluminescence using ECL Plus. Images were captured on Image pro plus 6.0 software. The total proteins were used for analysis of phospho-p38 MAPK, p38 MAPK, phospho-
ERK1/2, ERK1/2, and Beta-actin; the separated nucleus proteins were used for the analysis of NRF2 and fibrillarin.
Immunofluorescent Analysis The method was previously described for lung tissues [25]. For immunofluorescent analysis, anti-phosphop38 (Santa Cruz Biotechnology, Santa Cruz, CA) was used.
NADPH Oxidase Activity Lucigenin-enhanced chemiluminescence was used to measure NADPH oxidase activity in lungs according to the method described previously [26].
Statistical Analysis Statistical differences between the groups were evaluated by two-way ANOVA followed by Bonferroni ttest. A level of P < .05 was considered to represent statistical significance. Experimental group mean values and standard errors (SEs) were determined using descriptive statistics, and values are means ± SE. Statistical analysis was performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA).
RESULTS SP Reduced Hyperoxic Lung Injury and Lung Edema Induced by Hyperoxia To investigate the effects of SP on hyperoxic lung injury, the rats of hyperoxia/SP and normoxia/SP groups were injected SP for 14 days. The SP level of lungs increased significantly in hyperoxia/SP (6690 ± 440 pg/mg) and normoxia/SP (6695 ± 563 pg/mg) groups compared with hyperoxia/saline (1244 ± 188 pg/mg) and normoxia/saline (1034 ± 125 pg/mg) groups (Figure 1A). To evaluate the extent of oxygen-induced lung damage, the W/D weight ratio was measured at the same time in all four groups after 14 days. The W/D ratio of the rat lung tissue increased significantly in rats exposed to hyperoxic conditions (hyperoxia: F[1,20] = 15.4, P < .01, Figure 1B). Compared with the lungs from hyperoxia/saline rats, SP ameliorated hyperoxia-induced lung edema as indicated by a significant decrease in W/D ratio (SP: F[1,20] = 6.3, P < .05; hyperoxia×SP: F[1,20] = 3.2, P < .05, Figure 1B). The effects of SP treatment on the histopathologic changes in rat lungs are shown in Figure 1C. Experimental Lung Research
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FIGURE 1. Measurement of SP level, W/D ratio, and histopathology analysis in rats. (A) SP level of lungs measured by ELISA after 14 days of normoxia or hyperoxia. N = 6 for each group, # P < .01, ∗ P < .05. (B) W/D ratio of lungs after 14 days of normoxia or hyperoxia. N = 6 for each group, # P < .01, ∗ P < .05. (C) Representative images of hematoxylin-eosin staining of lung. Top panels: Lungs exposed to normoxic conditions with 21% O2 . Bottom panels: Lungs exposed to hyperoxic conditions with 95% O2 . 400× magnification. (D) The lung injury scores of hematoxylin-eosin staining lungs. The score of lung injury was assessed by (1) infiltration or aggregation of neutrophils in airspace, the alveolar wall, or the vessel wall, (2) thickness of the alveolar wall, and (3) alveolar congestion, and each item was graded on a four-point scale. The score of each component ranged from 0 to 3, and higher scores indicated more severe damage. A total score was calculated as the sum of three components. At least eight fields were chosen randomly from each section. 400× magnification. N = 6 for each group, # P < .01, ∗ P < .05.
The lungs of the rats exposed to hyperoxia showed marked severe edema and cellular infiltration in the interstitial area and the associated thickening of the alveolar septum. In the presence of SP, both edema and inflammatory cell infiltration were reduced despite exposure to hyperoxia; there was no significant histopathologic difference between normoxia/saline and normoxia/SP groups (Figure 1C). In addition, the lung injury scores in hyperoxia/SP rats were significantly lower than those of hyperoxia/saline rats (hyperoxia: F[1,20] = 24.2, P < .01; SP: F[1,20] = 7.8, P < .05; hyperoxia×SP: F[1,20] = 2.4, P < .05, Figure 1D). C
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GSH-Px and SOD Activities, MDA Content Measurements in Lung Tissues Biochemical analyses revealed that the rats to hyperoxia significantly decreased tissue GSH-Px and SOD activities, whereas it increased tissue MDA content, which are shown in Table 1. By contrast, the GSHPx and SOD activities were significantly higher in the hyperoxia/SP group as compared with the hyperoxia/saline group (40.4 ± 3.1 vs. 27.9 ± 1.6 U/mg, P < .05, and 250 ± 41 vs. 142 ± 25 U/mg, P < .05 respectively), indicating the efficacy of SP on decreasing oxidative stress. The MDA content (4.65 ±
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TABLE 1. Results Showing GSH-Px and SOD Activities, and MDA Content in Lung Tissue of Rats
Effect of SP Treatment on Hyperoxia-Induced Oxidative Stress
Biochemical analyses
When compared with the normoxia/saline group, oxygen exposure resulted in the upregulated activities of NADPH oxidase (hyperoxia: F[1,20] = 23.1, P < .01, Figure 3A) and enhanced formation of ROS (hyperoxia: F[1,20] = 31.4, Figure 3B). Treatment with SP significantly suppressed NADPH oxidase activity (SP: F[1,20] = 12.5, P < .05; hyperoxia×SP: F[1,20] = 5.2, P < .05, Figure 3A) and reduced ROS production (SP: F[1,20] = 19.1, P < .05; hyperoxia×SP: F[1,20] = 4.5, P < .05, Figure 3B).
Normoxia/saline Normoxia/SP Hyperoxia/saline Hyperoxia/SP
GSH-Px (U/mg)
SOD (U/mg)
MDA (nmol/mg)
57.3 ± 1.91 53.2 ± 3.1 27.9 ± 1.6# 40.4 ± 3.1∗
355 ± 15 358 ± 35 142 ± 25# 250 ± 41∗
3.21 ± 0.32 3.64 ± 0.43 8.77 ± 0.15# 4.65 ± 0.42∗
P < .05 significantly compared with normoxia/saline group. P < .05 significantly compared with hyperoxia/saline group.
#
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∗
0.42 vs. 8.77 ± 0.15 nmol/mg, P < .05) was significantly lower in the hyperoxia/SP group as compared with that in the hyperoxia/saline group, suggesting an SP effect on decreasing lipid peroxidation (Table 1). There was no significant difference in GSH-Px, SOD, and MDA levels in normoxia/saline and normoxia/SP groups.
Detection of Apoptotic Cell Death As shown in Figure 2, the number of TUNELpositive cells was increased in rats exposed to hyperoxic conditions after 14 days (hyperoxia: F[1,20] = 15.2, P < .01). The SP treatment comparably decreased the percentage of TUNEL-positive cells relative to the hyperoxia/saline group (SP: F[1,20] = 4.9, P < .05; hyperoxia×SP: F[1,20] = 3.3, P < .05). The normoxia/SP group showed no significant increasing number of TUNEL-positive cells compared with hyperoxia/SP group.
Effect of SP on Hyperoxia-Induced Activation of MAP Kinases and NRF2 in Nucleus To probe into the mechanism of SP action, we focused on p38 MAPK and ERK. The rats were exposed to hyperoxia for 14 days, which resulted in significant increase in p-p38 and pERK1/2 activation, expressed as the ratio of phosphorylated/total p38 protein levels and phosphorylated/total ERK1/2 protein levels; SP treatment blocked p-p38 and pERK1/2 activation (Figure 4A). In accord with these observed effects of SP on p-p38 and p-ERK1/2, under the same experimental conditions, there was an increase in staining for p-p38, which was blocked by SP (hyperoxia: F[1,12] = 31.2, P < .01; SP: F[1,12] = 11.4, P < .05; hyperoxia×SP: F[1,12] = 2.3, P < .05, Figures 4C and D). We also found the increased expression of NRF2 in nucleus when lungs were exposed to hyperoxia, and treatment with SP
FIGURE 2. Detection of apoptotic cell death by TUNEL staining in all four groups after 14 days of oxygen exposure. (A)
Representative images of TUNEL staining of lung sections. Upper panels: Lungs exposed to normoxic conditions with 21% O2 . Lower panels: Lungs exposed to hyperoxic conditions with 95% O2 . 400× magnification. (B) Shown in a number of TUNEL-positive cells. N = 6 for each group, # P < .01, ∗ P < .05.
Experimental Lung Research
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FIGURE 3. Effect of SP treatment on hyperoxia-induced oxidative stress. Rats were exposed to hyperoxia for 14 days. N = 5 for
each group, # P < .01, ∗ P < .05. (A) The lungs were removed for assessment of activities of NADPH oxidase. (B) Total ROS accumulation was measured after 14 days of normoxia or hyperoxia with or without SP treatment.
significantly downregulated NRF2 expression (Figure 3B).
DISCUSSION For the first time, the current study demonstrated the protective actions of SP on the hyperoxic lung injury in vivo. This study demonstrated significant improvement in W/D, less lung injury as measured by histopathology analysis, increased antioxidant enzyme activities (SOD, GSH-Px), and decreased oxidative stress marker (MDA) when hyperoxic neonatal rats were treated with SP. Our data demonstrated that SP downregulated phosphorylated p38, and decreased cell apoptosis in hyperoxic lung injury, supporting the idea that SP attenuates apoptosis by decreasing p38 activation. Substance P is crucial for proliferation, migration, and differentiation of impaired cells [27,28]. Recent studies have demonstrated that SP treatment decreases the apoptosis of AECII, and attenuates the morphological alteration of AECII at different time points after hyperoxia exposure [17]. In this study, SP was shown to be able to markedly decrease TUNELpositive nuclei in hyperoxia-exposed lung, suggesting that SP could partially reverse apoptosis increasing effects on hyperoxia-exposed lung. Oxygen causes tissue injury through the formation of destructive radicals, such as hydroxyl radicals, which and highly reactive, and through the peroxidation of membrane lipids [29,30]. Prolonged hyperoxia exposure will cause oxidative stress, which is decisive for hyperoxic lung injury, and antioxidant therapies are thought to improve lung morphol C
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ogy [31]. Moreover, the activity of SOD and GSHPx, which are intracellular enzymatic antioxidant defense, makes premature infants highly susceptible to oxidative injury [32]. A previous study has found that hyperoxia could induce ROS formation in pulmonary capillary endothelial cells in situ, and it might be relevant to the initiation of hyperoxia-induced lung injury [32]. Therefore we observed a significant increase in lung MDA level in the hyperoxia group, suggesting increased lipid peroxidation. MDA, the last product of lipid breakdown caused by oxidative stress, is considered to be a good indicator of free radical-induced lipid peroxidation [33]. The results showed that on day 14, lung tissues of neonatal rats with SP treatment had high MDA levels and low activity of SOD and GSH-Px compared with the hyperoxia group. The sources of intracellular ROS during oxygen exposure remain unclear but may involve increased mitochondrial generation and activation of NADPH oxidases. The pathological changes in hyperoxia-injured lungs coincide with the injury or death of pulmonary capillary endothelial cells and alveolar epithelial cells [34]. In this work, we also found that treatment with SP suppressed the upregulation of activity of NADPH oxidase, which was reported as a major producer of ROS in lungs of rodents exposed to hyperoxia [35]. Our results demonstrated the protective role of SP in hyperoxic lung injury through its inhibitory action on oxygen-derived free radicals. MAPKs, including ERK, JNK, and p38, are phosphorylated in response to a series of specific pathways through which different extracellular signals can be conducted from the cell surface into the nucleus [36]. Studies in several different cellular systems showed that MAPK could be activated by ROS. The
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FIGURE 4. SP inhibits hyperoxia-induced phosphorylated p38 MAPK, phosphorylated pERK, and NRF2 expression in nucleus.
(A) Western blots for phosphorylated and total p38 MAPK and ERK1/2 on protein extracts from the lungs taken after 14 days of normoxia or hyperoxia with or without treatment of SP. (B) Western blots for proteins expression of NRF2 and fibrillarin in nucleus from the lungs taken after 14 days of normoxia or hyperoxia with or without SP treatment. (C) Representative images of immunofluorescent staining for phosphorylated p38 in lungs after 14 days of normoxia or hyperoxia with or without SP treatment. (D) Shown in a number of phospho-p38 positive cells. N = 4 for each group, # P < .01, ∗ P < .05.
p38 protein has been shown to be a inducer of cell death following exposure of cells to a wide variety of stimuli. The activation of p38 has also been shown to be associated with hyperoxia-induced epithelial damage [7], and another study further confirmed that hyperoxia activated p38 signaling pathways in type II cells [4]. To emphasize the effect of SP on phosphorylation of p38 MAPK and ERK1/2, we found that hyperoxia exposure significantly induced the activity of p-p38 and pERK1/2 in hyperoxic lung injury. Interestingly, treatment with additional SP effectively downregulated the activity of p-p38 and pERK1/2. Meanwhile, SP decreased apoptosis of lung tissues. Furthermore, NRF2 is a key transcription factor that regulates antioxidant genes as an adaptive response to oxidative stress or pharmocological stimuli, and its overexpression confers cellular protection against hyperoxia [37]. It is likely that NRF2 acts as one
of the important downstream effectors of PI3K pathway to regulate the expression of genes that contribute to cell survival following hyperoxic insult. We found increased expression of NRF2 in nucleus, and treatment with SP significantly downregulated NRF2 expression when lungs were exposed to hyperoxia. These data together are summarized in Figure S1. In conclusion, this study demonstrated, for the first time, that SP ameliorates hyperoxic lung injury by modulating the MAPK pathway. The findings suggest a potentially novel and easily applicable solution to hyperoxic lung injury, and provide new insight into our scientific knowledge of SP. Although further investigation is required, SP is expected to be an innovative therapeutic tool for unmet medical needs that currently cause considerable health burdens, in particular for critically ill patients with lung diseases. Experimental Lung Research
Substance P Protects Against Hyperoxic Lung Injury [7]
[8] [9]
[10]
[11]
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[12] [13] [14]
[15] FIGURE S1. Schematic summary of the protection of SP on
hyperoxic-induced lung injury in neonatal rats. SP suppresses the activity of NADPH oxidase and reduced SOD production on hyperoxic-induced lung injury, which blocks the activities of p-p38 and p-ERK, leading to the expression of decreased NRF2. In addition, SP ameliorates hyperoxia lung injury through decreasing cell apoptosis.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. This work was supported by the project of the Science and Technology Foundation of Guizhou Province (No. J[2011]2341), Department of Health Science and Technology Foundation of Guizhou Province (No. qzwkj2011-1-092), and Science and Technology Bureau Foundation of Zunyi City (No. [2011]22).
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[17]
[18]
[19]
[20] [21] [22]
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Experimental Lung Research
