TRPV1 Agonist Capsaicin Attenuates Lung Ischemia-Reperfusion ...

Journal of Surgical Research 173, 153–160 (2012) doi:10.1016/j.jss.2010.08.053

TRPV1 Agonist Capsaicin Attenuates Lung Ischemia-Reperfusion Injury in Rabbits Maohua Wang, M.D.,*,† Peng Ji, M.D.,* Rurong Wang, M.D., Ph.D.,*,1 Lifang Zhao, M.D.,* and Zhengyuan Xia, M.D., Ph.D.‡ *Department of Anesthesiology, and Laboratory of Anesthesia and Intensive Care Medicine, West China Hospital of Sichuan University, Chengdu, Sichuan, China; †Department of Anesthesiology, Affiliated Hospital of Luzhou Medical College, Luzhou, China; and ‡Department of Anesthesiology and Research Centre for Heart, Brain, Hormone, and Healthy Aging, The University of Hong Kong, Hong Kong, China Originally submitted June 7, 2010; accepted for publication August 27, 2010

Background. Capsaicin, a transient receptor potential vanilloid type1 (TRPV1) agonist, was found to protect against myocardial and renal ischemiareperfusion (IR) injury. This study was carried out to investigate the role of capsaicin in lung IR injury in vivo. Methods. Forty male New Zealand rabbits were randomized into four groups (10 per group) as follows: sham group (sham thoracotomy), IR group (occlusion of the left pulmonary hilus for 1 h followed by reperfusion for 3 h), CAP (capsaicin) group (a bolus injection of CAP 5 min before ischemia), CPZ (capsazepine) group (a bolus injection of the TRPV1 antagonist CPZ 5 min before ischemia). Blood and lung tissue samples were obtained for blood gas and biochemical analyses, wet/ dry weight ratio measurements, and histologic evaluation. Protein levels and neutrophils in the bronchoalveolar lavage fluid (BALF) were also measured. Results. Pretreatment with capsaicin improved gas exchange function, decreased lung wet/dry ratio and protein levels and neutrophil counts in BALF, decreased lung malondialdehyde levels and myeloperoxidase activities, increased superoxide dismutase activities, along with an elevation of calcitonin generelated peptide (CGRP) level (P < 0.05 versus IR group). Capsaicin also attenuated IR-induced pathological lesions. By contrast, capsazepine exacerbated gas exchange abnormality, increased pulmonary microvascular permeability, oxidative stress, neutrophils infiltration, and also revealed a decreased CGRP level (P < 0.05 versus IR group).

1 To whom correspondence and reprint requests should be addressed at Department of Anesthesiology, West China Hospital, Sichuan University, no. 37 Wai Nan Guo Xue Xiang, Chengdu, Sichuan, China. E-mail: [email protected].

Conclusion. Results from the present study show that capsaicin confers protection against lung IR injury. These protective effects seem to be closely related to the inhibition of inflammation and oxidative stress via the activation of TRPV1 and the release of CGRP. Ó 2012 Elsevier Inc. All rights reserved. Key Words: capsaicin; lung; ischemia-reperfusion; TRPV1.

INTRODUCTION

Blood flow to the ischemic lung induces greater injury than the original ischemia itself, causing what is known as lung ischemia-reperfusion (IR) injury [1], thus resulting in severe complications after lung transplant and cardiopulmonary bypass [2]. Over the past two decades, the role of neutrophils, free radicals, and other inflammatory mediators in IR injury has been extensively investigated [3]. Because of the involvement of multiple factors in lung IR injury, it has been difficult to achieve an obvious pulmonary protection by targeting any one single factor. Recent studies have shown that stimulating vagus nerve generated a protective effect in myocardial IR injury [4, 5]. The protective role of sensory neurons in renal and liver IR injury have also been documented [6–8]. However, lung IR injury, due to its distinct inflammatory response, is different in many ways from the IR in other organs. Moreover, the exact role of sensory neurons in lung IR injury has not yet been clearly understood. Approximately 75% of the afferent nerves, which are conducted primarily by branches of the vagus nerves, arising from the lungs and airways, are pulmonary sensory nerves [9, 10]. Transient receptor potential vanilloid

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type 1 (TRPV1) channels are widely expressed in these sensory nerve fibers as well as in the cell bodies of pulmonary neurons in the jugular and nodose ganglia [11]. TRPV1 can be activated by a variety of stimuli, such as mechanical stimuli or chemical irritants [12]. Capsaicin is the pungent ingredient in hot pepper and is a potent selective stimulant of the TRPV1 channel. It has been most commonly used in the studies of the physiologic properties and functions of sensory neurons with C and Ad fibers [13]. When TRPV1 is activated, neuropeptides, including substance P (SP) and calcitonin gene-related peptide (CGRP), are released from capsaicin-sensitive sensory nerves [12, 14]. Moreover, capsaicin, which activates TRPV1, has been shown to have a protective effect against myocardial, hepatic, and renal IR injuries in rats [6, 15–17]. However, the role of capsaicin in lung IR injury is unclear. In this study, we have demonstrated that capsaicin plays an important role in ameliorating IR-induced lung injury, inflammation, and oxidative stress in rabbits. This study also evaluated the possible involvement of TRPV1 and CGRP in the above mentioned effects. MATERIALS AND METHODS Animal Preparation The experimental protocols described in this study were approved by the Institutional Animal Care and Use Committee of Sichuan University. All experiments were conducted in adult male New Zealand rabbits (2.0–3.0 kg). They were fasted for 8 h before surgery with free access to water. Before anesthesia induction, both venous and arterial lines were established for drug and fluid administrations and arterial blood pressure monitoring. Anesthesia was induced with a bolus injection of pentobarbital (35 mg/kg, i.v.) and maintained by continuous infusion of pentobarbital (0.3 mg/kg/min). An endotracheal tube was introduced and positive pressure ventilation was provided by a DH-150 rodent ventilator (Zhejiang University Medical Instrument, Zhejiang, China) with an inspiratory oxygen fraction (FiO2) of 50% at a rate of 30 breaths/min and a tidal volume of 15 mL/kg. A left anterolateral thoracotomy in the fifth intercostal space was also performed. Before inducing lung ischemia, 250 U/kg heparin was administered intravenously, and 15 min stabilization was provided for all animals. The ischemic injury was induced by clamping the left pulmonary hilus with a non-crushing microvascular clamp, including the left main bronchus, vein, and artery for 1 h. Reperfusion was achieved by removing the clamp and reventilating and reperfusing the left lung for 3 h. At the end of the experiment, rabbits were euthanized by intravenous injection of overdose pentobarbital (50 mg/kg) after collecting the arterial blood sample. And left lung tissues were harvested for further examination.

Chemical Preparation Stock solutions of capsaicin (Sigma-Aldrich, St. Louis, MO), capsazepine (Sigma-Aldrich) were prepared and stored at 4
C. The desired drug concentrations were prepared daily. The solution with 1% Tween 80, 1% ethanol, and 98% saline was used for vehicle injection.

Experimental Protocol After 15 min stabilization, animals subjected to 1 h of left pulmonary helium occlusion and 3 h of reperfusion were randomized into

three groups (n ¼ 10, respectively): (1) vehicle injection 5 min before ischemia (IR group), (2) bolus injection of capsaicin 50 mg/kg 5 min before ischemia (CAP group), (3) bolus injection of capsazepine 50 mg/kg 5 min before ischemia (CPZ group). Another 10 rabbits were used as sham group, which underwent the same surgical procedures except that the left pulmonary helium was not occluded (n ¼ 10). The variables of HR and mean arterial pressure (MAP) were recorded throughout the experiment. The doses of capsaicin and capsazepine used in the present study were based on previous experiments and preliminary study [15]. In pilot studies, capsaicin doses of 20 mg/kg and 50 mg/kg were tested. Fifty mg/kg of capsaicin was shown to have more significant inhibitory effect on IR-induced changes in vascular permeability.

Blood Gas Analyses Before exsanguination, 1 mL of arterial blood was obtained from an ear artery and gas analysis was performed (ABL3; Radiometer, Copenhagen, Denmark). Arterial-alveolar oxygen pressure gradient (A-aDO2) was also calculated with the following formula: A-aDO2 ¼ fraction inspired O2 3(Patm – PH2O) – (arterial PCO2/0.8) – arterial PO2 [18], where Patm ¼ 760 mmHg and PH2O ¼ 47 mmHg.

Lung Tissue Edema The left lungs were harvested for measurement of lung wet/dry weight ratio, which is an estimation of lung tissue edema. Approximately 500 mg of the lung tissue was taken from the upper left quadrant of the left lower lobe. The wet/dry ratio was calculated after weighing the freshly harvested organ and heating it to 80
C in an oven over a 72 h period until the weight of the remaining residue was found to be constant.

Myeloperoxidase Activity and Oxidative Stress Status Assays At 3 h after reperfusion, the upper right quadrant of the left lower lobe was used to determine myeloperoxidase activity and to perform an oxidative stress assay. The lung tissues were frozen immediately after being harvested and stored at –80
C until analyzed. Tissue myeloperoxidase (MPO) activity was measured to assess the extent of polymorphonuclear leukocyte accumulation in the lungs. Lung tissues were homogenized and 2 mL of the homogenate was used to measure MPO activity without centrifugation. The remainder of the homogenate was centrifuged at 1,500 3 g at 4
C for 15 min. The supernatant was used to determine the activities of superoxide dismutase (SOD) and the content of malondialdehyde (MDA). All these measurements were performed using assay kits (Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions.

Cell Counting and Protein Level Determination in Bronchoalveolar Lavage Fluid At 3 h after reperfusion, lungs were lavaged as described with ligation of the right main bronchus and left lower lobe [19]. After this, a tracheotomy was performed using a 16-gauge intravenous cannula. Bronchoalveolar lavage was also performed in the upper lobe of left lung by gently instilling and aspirating 4
C 5 mL saline through the intratracheal tube three times. The amount of bronchoalveolar lavage fluid retrieved was more than 2.5 mL in each rabbit. Total cells were then counted using a hemocytometer. Cells in 1 mL BALF were differentiated as basophilic granulocyte, acidophilic granulocyte, or neutrophils utilizing cytocentrifuge preparations stained with Wright’s stain. The remaining lavage fluid was cold-centrifuged at 1500 3 g for 20 min and frozen at –80
C. It was then used to measure protein concentration by the method of Lowry et al. [20].

WANG ET AL.: CAPSAICIN REDUCES LUNG ISCHEMIA-REPERFUSION INJURY

Radioimmunoassay for CGRP At the end of the experiment, lung tissues were taken from the lower left quadrant of the left lower lobe. The CGRP levels in tissue were determined with a CGRP radioimmunoassay (RIA) kit. (Dongya Immunity Technology Institution, Beijing, China) Lungs were homogenized in RIA buffer, and the homogenates were centrifuged at 1500 3 g at 4
C for 15 min. The supernatant was separated and used for RIA reactions in duplicate.

Histologic Analysis Lung tissue blocks of approximately 0.5 cm3 were taken from the lower right quadrant of the left lower lobe. After processing and staining with hematoxylin and eosin, lung samples were graded by a pathologist who was blinded to research groups. The pathologic features of the lungs were scored based on alveolar edema, interstitial edema, hemorrhage, and inflammatory infiltration. The scoring system used in this work was adapted and modified from a previous study [21]. In our scoring system, values from 0 to 3 represent the severity of the pathologic feature as follows: 0 ¼ normal appearance, 1 ¼ slight damage in those above four features, 2 ¼ intermediate damage, and 3 ¼ severe damage. A further part of our system was used to describe the extent of involvement in each field of view, as follows: 0 ¼ lack of involvement of the above four features, 1 ¼ up to 25%, 2 ¼ 25%–50%, 3 ¼ 50%–75%, and 4 > 75%. For each pathologic feature evaluated, the severity and extent of involvement values were multiplied, leading to values in the range of 0–12.

Statistical Analysis Data were expressed as means 6 SD. Differences were examined by one-way ANOVA followed by LSD test. The histological analyses between groups were assessed by Kruskal-Wallis Test. P < 0.05 was considered statistically significant. All analyses were carried out using SPSS ver. 10.0 (SPSS Inc., Chicago, IL).

RESULTS Blood Gas Exchange

PaO2, PaCO2, and A-aDO2 are shown in Table 1. IR in the lung was shown to reduce PaO2 and elevate A-aDO2 (P < 0.05 versus sham). Capsaicin pretreatment increased PaO2 and decreased A-aDO2 compared with the IR group (P < 0.05). CPZ treatment resulted in the lowest PaO2 and the highest A-aDO2 among the four groups, reflecting impaired gas exchange efficiency (P < 0.05 versus other groups). There was no significant statistical difference between the groups in response to

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the hemodynamic parameters in the pre-ischemic and post-ischemic periods (data not shown). Oxidative Stress

IR was found to increase the oxidative reaction. As shown in Fig. 1A, the lung MDA content, an index of lipid peroxidation, was significantly increased in the IR group that was reduced by capsaicin pretreatment (P < 0.05). Furthermore, capsaicin caused a significant increase in the lung tissue SOD activity, compared with the groups of CPZ and IR (P < 0.05, Fig. 1B). In contrast, CPZ increased MDA level (P < 0.05, compared with IR group). Neutrophil Activation

As shown in Fig. 2, IR elevated PMN% and total cell number in BALF and lung MPO level, compared with sham group (P < 0.05). CAP reduced the MPO level, PMN%, and total cell number (P < 0.05 versus IR group). In contrast, CPZ resulted in the highest level of MPO, PMN%, and total cell number (P < 0.05 versus other groups). The change of MPO level in lung tissue was in parallel with that of PMN% in BALF. Pulmonary Vascular Permeability

Rabbits in the IR group had a higher protein level in BALF and higher lung wet/dry weight ratio compared with the sham group (P < 0.05). Capsaicin decreased the protein level in BALF compared with the IR group, while capsazepine did not increase the protein level and wet/dry ratio (Fig. 3A, B). Lung Tissue CGRP Level

As shown in Fig. 4, both IR and capsaicin elevated lung tissue CGRP level (P < 0.05 versus sham). Rabbits in the CAP group had the highest CGRP level among four groups (P < 0.05 versus all other groups). The CGRP level in CPZ group was lower than that of CAP group or IR group (P < 0.05). Histology

TABLE 1 Comparisons of the Blood Gases between Four Groups Groups Sham IR CAP CPZ

PaO2 (mmHg)

PaCO2 (mmHg)

A-aDO2 (mmHg)

266.33 6 16.39 121.30 6 38.18* 196.88 6 47.23*,y 105.50 6 26.55*,y

34.29 6 3.58 37.54 6 7.61 34.64 6 3.02 36.78 6 6.65

47.31 6 15.28 188.28 6 35.88* 116.33 6 46.29*,y 205.03 6 22.66*

IR ¼ ischemia-reperfusion; CAP ¼ capsaicin; CPZ ¼ capsazepine. P < 0.05 compared with Sham group. y P < 0.05 compared with IR group. *

Results of histologic evaluation are summarized in Table 2. The scores of intra-alveolar edema, interstitial edema, and inflammatory infiltration in IR group were higher than that in the sham group (P < 0.05). Compared with the IR group, capsaicin pretreatment decreased the scores of interstitial edema and inflammatory infiltration, while CPZ increased the scores of intra-alveolar edema. Figure 5B shows the typical histologic graphs in rabbit lung tissues of four groups. Obvious intra-alveolar and interstitial edema,

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FIG. 1. Comparing the malondialdehyde (MDA) content (A) and superoxide dismutase (SOD) activity (B) between four groups in the rabbit lung. *P < 0.05 compared with sham group. yP < 0.05 compared with IR group.

neutrophils infiltration, and hemorrhage were found in the IR and CPZ groups. However, such pathologic changes were less obviously seen in the CAP and sham groups. DISCUSSION

The major findings of the present study are that pretreatment with the TRPV1 agonist capsaicin reduced IR-induced pulmonary inflammation and oxidative stress, attenuated IR-mediated impairment of blood gas exchanges, along with elevated pulmonary CGRP levels in rabbits. In contrast, pretreatment with a TRPV1 antagonist exacerbated the injuries with decreased pulmonary CGRP levels. Evidences of capsaicin mediated attenuation of lung IR were as follows: (1) although PaO2 was above 100 mmHg in the four groups due to 50% oxygen inhalation, IR and CPZ decreased the PaO2 and increased A-aDO2, whereas capsaicin

increased the PaO2 and reduced the A-aDO2. This indicates that capsaicin improved IR-mediated impairment of oxygen exchange; (2) capsaicin reduced pulmonary microvascular permeability reflected by decreased protein level in BALF. In addition, capsaicin pretreatment ameliorated the interstitial edema and neutrophil infiltration seen histopathologically. Neutrophil infiltration and inflammation have been suggested to be major factors that lead to lung IR injury [1]. MPO is an enzyme secreted by activated leukocytes, and its activity has been used as a sensitive index of tissue PMN sequestration and activation. The present study demonstrated that IR increased pulmonary MPO activity and neutrophil infiltration in BALF. Capsaicin pretreatment reduced the level of MPO in lung tissue and PMN% in BALF. In contrast, CPZ pretreatment elevated neutrophil activation status with a high PMN% in BALF. Therefore, current results indicate that capsaicin modifies IR-induced lung inflammation. A previous

FIG. 2. Comparisons of myeloperoxidase (MPO) activities in lung tissue and the percentage of neutrophils and the total cell number in BALF in four groups. *P < 0.05 compared with sham group. yP < 0.05 compared with IR group.

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FIG. 3. Protein levels in BALF (A) and lung wet/dry weight ratio (B) in rabbits of four groups. *P < 0.05 compared with sham group. yP < 0.05 compared with IR group.

study reported that capsaicin pretreatment reduced the proinflammatory cytokines (TNF-a, IL-6) and promoted the anti-inflammatory cytokine IL-10 in a rat model of sepsis [22]. Direct measurements of cytokine levels were not performed in the current study. However, based on previous studies, it seems reasonable to postulate that capsaicin may reduce pulmonary inflammation by decreasing the production of proinflammatory cytokines or by prompting anti-inflammatory cytokines following lung IR injury. This hypothesis needs to be further investigated. Oxidative stress is an important factor that leads to lung IR injury [1]. Oxygen free radicals are generated from activated macrophages or from lung tissue after pulmonary IR [23]. MDA is liberated as an end product from hydroxyperoxide destruction and is a good indicator of lipid peroxidation. SOD is a major endogenous antioxidative enzyme, reflecting the antioxidative capability. In the present study, intravenous administration of capsaicin before IR reduced lung MDA level and elevated SOD activity. By contrast, capsazepine treatment reduced lung tissue activity of antioxidant SOD and increased MDA level. In accordance with our study, a

previous study demonstrated that systemic administration of capsaicin before inducing sepsis decreased the lipid peroxidation in various tissues in rats [22]. Similarly, in the current study capsaicin pretreatment before ischemia reduced lipid peroxidation and increased antioxidant effects in the rabbit model of lung IR. It is well known that capsaicin is a potent selective stimulus for the TRPV1 channel, and capsazepine is a competitive antagonist of capsaicin [24–26]. In our previous study, we found that administration of capsaicin before IR significantly improved the cardiac function and limited myocardial infarct size in a rat model of heart IR [16].Pretreatment with capsazepine prior to capsaicin significantly eliminated capsaicininduced cardio-protective effects [16]. Furthermore, numerous investigations have demonstrated that effects of capsaicin on respiration and IR liver and kidney were produced by activating TRPV1 [6, 17, 27]. In the present study, pretreatment with capsaicin reduced IR-induced pulmonary inflammation and oxidative stress, attenuated lung dysfunction, whereas the opposite effects were found in the capsazepine pretreatment group. A potential limitation of the current study is that the direct measurement of TRPV1 activity was not performed. Also, this study was not designed to observe the effect of co-administration of capsaicin and capsazepine on lung IR injury. However, based on aforementioned findings that administration of capsazepine prior to capsaicin cancelled the protective effect of capsaicin

TABLE 2 Scores of Histologic Evaluations in Four Groups Groups

FIG. 4. Comparing calcitonin gene-related peptide (CGRP) levels in lung tissue. *P < 0.05 compared with sham group. yP < 0.05 compared with IR group.

Sham IR CAP CPZ

Intra-alveolar edema

Interstitial edema

Hemorrhage

Inflammatory infiltration

0.6 6 0.5 2.2 6 0.6* 1.5 6 0.5* 2.9 6 0.9*,y

1.1 6 0.9 3.7 6 0.9* 1.8 6 0.9*,y 4.3 6 0.9*

0.9 6 0.8 3.1 6 1.0* 2.8 61.1* 2.7 6 0.9*

1.5 6 0.9 4.4 6 1.2* 2.5 6 1.1*,y 5.4 6 0.9*

P < 0.05 compared with Sham group. P < 0.05 compared with IR group.

* y

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FIG. 5. Typical histological graphs (HEx200) taken from a rabbit lung tissue of four groups (upper left, Sham; upper right, IR, ischemiareperfusion; lower left, CAP, capsaicin; lower right, CPZ, capsazepine). (Color version of figure is available online.)

against myocardial IR injury [16] and that capsaicin is widely accepted as a TRPV1 agonist, it is reasonable to assume that capsaicin reduced lung IR-induced inflammation and oxidative stress by activating TRPV1. CGRP is primarily located in small sensory C fibers in most tissues within lining epithelia and around small blood vessels [28]. On activation of TRPV1, CGRP is released from sensory nerve terminal. In this study, pretreatment with a small dose of capsaicin caused the largest release of CGRP in lung tissue because capsaicin is a potent selective agonist of TRPV1. It was reported that ischemia-induced low pH and inflammatory mediators such as adenosine and bradykinin could activate TRPV1 and release CGRP [29]. Therefore, the CGRP level in the IR groups of rabbits is higher than those in the CPZ and the sham groups. Among the three IR groups, capsaicin treated rabbits had the highest CGRP levels, accompanied by ameliorated lung histologic lesions, improved lung blood gas exchange, and less inflammation and oxidative stress. CPZ treated rabbits had lower CGRP levels with obvious lung histologic damage, impaired lung function, and severe inflammation and oxidative stress. These data suggest that activation of TRPV1 induced CGRP release, which

mitigated the rabbits’ lung IR injury. On the other hand, the depression of TRPV1 activation would exacerbate the lung injury in the circumstance of IR. These results further support our assertion that capsaicin reduced IR-induced inflammation and oxidative stress possibly by activating TRPV1. In supporting of our findings, Mizutani et al. reported that activation of sensory neurons reduced IR-induced acute renal injury via CGRP receptor [7]. CGRP has also been shown to inhibit type 1 cytokines (e.g., IL-12 and interferon) and enhance the production of IL-10 [30, 31]. In addition, in H9c2 cardiomyocytes and cultured smooth muscle cells, CGRP protected cells from H2O2 induced oxidative stress and reduced apoptosis [32, 33]. These data suggest that capsaicin mitigated lung IR injuries possibly via activations of TRPV1-induced CGRP releases. However, we cannot exclude the possibility that capsaicin may also directly act upon macrophage/ neutrophils, which merits further study. Limitations of This Study

It is known that activation of TRPV1 can release various neuropeptides, including CGRP and SP [12]. CGRP may not be the only path by which capsaicin plays

WANG ET AL.: CAPSAICIN REDUCES LUNG ISCHEMIA-REPERFUSION INJURY

a protective role in this study. The pulmonary SP content released by capsaicin or IR and its protective effect in lung IR injury also merits study. Although TRPV1 primarily expresses in sensory neurons and nerve fibers, it also expresses in non-neuronal cells such as epithelial cells, endothelial cells, and macrophages. Since capsaicin was intravenously injected, the activation of TRPV1 in these non-neuronal cells is quite plausible. However, the current study did not attempt to differentiate the roles of TRPV1 channels in neuronal and non-neuronal cells in lung IR injury. Though there is literature reporting significant neutrophils activation and inflammatory factors release after 3 or 4 h lung reperfusion [34–36], the 3 h time point is only an early stage for inflammatory cells accumulation. To observe the delayed protective effect of capsaicin to lung IR injury, it is necessary to follow-up for longer periods of time (such as 24 h and/or 48 h of post-ischemic reperfusion) to observe the inflammatory response. Another limitation is that we did not measure TRPV1 activity in this study. Therefore, further studies using TRPV1 depletion animals are required to confirm the role of TRPV1 in mediating pulmonary protective effect of IR injury. CONCLUSION

Pretreatment with TRPV1 agonist capsaicin increased pulmonary CGRP level and attenuated lung IR injury through relieving oxidative stress and reducing inflammation in the rabbits. Conversely, pretreatment with TRPV1 antagonist capsazepine reduced CGRP level and exacerbated the injury. The present study suggests that capsaicin plays a protective role in IR-induced lung injury, possibly by activating TRPV1 and releasing CGRP, thereby relieving oxidative stress and inflammation in the rabbits. This study might shed light on the pulmonary protection in lung transplantation and cardiopulmonary bypass circumstance from a neurogenic point. ACKNOWLEDGMENTS The author thanks Nanfu Luo, Daqing Liao, and Chaoran Wu for their technical support. This work was supported by National Natural Science Foundation of China (30672260 and 81170077).

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