Mechanism of electroacupuncture at acupoints of the lung meridian through PKA/PKC regulation of TRPV1 in chronic cough after lung surgery in guinea pigs

Introduction

With the recent progress in imaging technology and the popularization of low-dose spiral computed tomography, an increasing number of patients have been clinically identified with pulmonary nodules, some of which have been identified as early non-small cell lung cancer after surgery (1,2). However, some patients develop acute or chronic cough symptoms after lung surgery, with a deleterious effect on their quality of life (3-5).

Cough is a common symptom of respiratory disease and the most important respiratory defense reflex. The related afferent nerves affecting the airway reflex mainly include myelinated Aδ fibers, which are sensitive to various mechanical stimuli, and unmyelinated C fibers, which are sensitive to various types of chemical stimuli (6,7). The C-fiber nerve endings of the lung contain TRPV1, and various physical and chemical stimuli can activate TRPV1-induced cough (8-10). Our previous study found that among patients undergoing lung surgery, those diagnosed with postoperative acute or chronic cough had significantly higher levels of serum prostaglandin E2 (PGE2) and bradykinin (BK; the upstream protein of TRPV1) (11). Therefore, we believe that PGE2 and BK increase respiratory inflammation and trigger an increase in cough by activating the TRPV1 pathway. Other studies have reported that PGE2 and BK cause conscious animal and human coughs when inhaled in aerosol form, and patients taking angiotensin converting enzyme inhibitors also develop excessive coughing due to reduced decomposition of BK (12-14). Therefore, we believe that intervention in the TRPV1 pathway and reduction in airway inflammation can prevent and reduce chronic cough after lung surgery. Revealing the pathogenesis of postoperative cough symptoms and its effective treatment has important theoretical implications and practical clinical significance.

Acupuncture is an important part of Traditional Chinese Medicine (15,16). It has been reported that acupuncture can reduce the postoperative inflammatory response in patients undergoing lung surgery, thus reducing postoperative pulmonary complications such as cough (17,18). In addition, clinical studies have confirmed that acupuncture can achieve good results in the treatment of cough, asthma, chronic obstructive pulmonary disease (COPD) and other lung diseases (19-21). However, the mechanism of acupuncture in the treatment of chronic cough caused by pulmonary surgery is unknown. Therefore, in the present study, a model of chronic cough after lung operation in guinea pigs was established, and electroacupuncture at acupoints of the lung meridian, the protein kinase A (PKA) inhibitor H89 and the protein kinase C (PKC) inhibitor Go6983 were used to confirm that acupuncture of the lung meridian regulates TRPV1 through PKA/PKC. PKA/PKC could activate TRPV1 to induce cough. Changes in cough frequency and lung histopathology were observed in different groups of guinea pigs with chronic cough after lung surgery. The airway inflammation indexes of the chronic cough model after lung surgery were evaluated, and the protein expression levels of PGE2, BK, TRPV1, p-PKA, p-PKC and p-TRPV1 were measured. The TRPV1, SP, CGRP and NK1R mRNA levels in the lung tissue of guinea pigs were measured by real-time fluorescence quantitative polymerase chain reaction (PCR). We present the following article in accordance with the ARRIVE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-409/rc).

Methods

Experimental materials

A guinea pig PGE2 enzyme-linked immunosorbent assay (ELISA) kit and BK ELISA kit were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China). The TRPV1 ELISA kit was purchased from Cloud-Clone Corp. (formerly Uscn Life Science Inc., Wuhan, China). TRIzol was purchased from Ambion Inc. (Austin, TX, USA). HiScript Reverse Transcriptase (RNase H), 5 × HiScript Buffer and SYBR Green Master Mix were purchased from Vazyme (Nanjing, China). DNTP, Taq Plus DNA Polymerase and DL2000 DNA Marker were purchased from the TIANGEN Company (Beijing, China). Random Primer (N6) was purchased from Takara Bio USA, Inc. (San Jose, CA, USA). Concentrated normal goat serum (closed), fluorescent labeled sheep anti-rabbit IgG and horseradish peroxidase (HRP)-labeled sheep anti-rabbit secondary antibody were purchased from China Wuhan Boshi Bioengineering Co., Ltd. (Wuhan, China). DAPI, phosphatase inhibitor, PMSF, RIPA lysate and BCA protein concentration determination kits were purchased from Beijing Biyuntian Company (Beijing, China). The anti-fluorescence quenching tablet was purchased from SouthernBiotech (Birmingham, AL, USA). P-TRPV1 (rabbit), p-PKA (rabbit) and p-PKC (rabbit) were purchased from Affinity Company (San Francisco, CA, USA). The protein marker (10–250 kD) was purchased from Fermentas (Waltham, MA, USA). PVDF film (0.45 µm) was purchased from Millipore (Burlington, MA, USA). Rabbit multi-antibody GAPDH (37 KD) was purchased from Hangzhou Xianzhi Shengwu Co., Ltd., China (Hangzhou, China). p-PKC (rabbit) (77 kD), p-PKA (rabbit) (41 kD) and P-TRPV1 (rabbit) (95 kD) were purchased from Affinity Company, USA. ECL substrate liquid was purchased from Thermo Scientific, USAs. Hematoxylin was purchased from Sigma, Germany. Water-soluble eosin Y, xylene, embedded paraffin and neutral gum were purchased from China National Pharmaceutical Group Corporation. Capsaicin was purchased from Beijing Solarbio Biotechnology Co., Ltd. (Beijing, China).

Experimental animal model

The study animals comprised 40 healthy adult male Dunkin-Hartley guinea pigs aged 5–7 weeks and weighing 300–350 g (Experimental Animal Center of Anhui University of Chinese Medicine; License No. Wan SCXK2017-001). Excluding those that died or were injured during feeding, finally 30 guinea pigs were included in the study. The guinea pigs were housed in a specific pathogen-free environment with a temperature of 20–26 ℃, daily temperature fluctuation ≤4 ℃ and a humidity of 40–70%. The light/dark cycle was 12 h and the animals had free access to food and water. A protocol was prepared before the study without registration. Animal experiments were performed under a project license (No. AHUCM-mouse-2022059) granted by the ethics board of Anhui University of Chinese Medicine, in compliance with institutional guidelines for the care and use of animals.

Grouping of experimental animals

After 1 week of acclimation, all guinea pigs were randomly divided into 5 groups (n=6): Sham Group (Sham), Model Group (Model), Electroacupuncture + Model Group (EA + M), H89 + Model Group (H89 + M) and Go6983 + Model Group (Go6983 + M). We followed Zhu et al. in the establishment of the animal experimental model (22).

According to the results of our previous study, referring to the route of human meridians, combined with the acupuncture and moxibustion acupoint positioning standard of Chinese Veterinary Acupuncture and moxibustion on guinea pigs, based on the literature (23). Two Φ 0.30×25 mm filigree needles were used and connected to an SDZ-IV type electronic acupuncture apparatus with the following stimulation parameters: the current (IP-P) was 1 mA, the frequency was 2 Hz, and electroacupuncture was performed continuously for 30 min. The stimulation parameters were the same for each group. Acupuncture intervention lasted for 1 week. The guinea pigs in the inhibitor groups were given intraperitoneal injections of H89 and Go6983 (injection doses of 20 mg/kg and 22 µg/kg, respectively) for the 1 week of intervention. The experimental schedule is shown in Figure 1.

Figure 1 Experimental timeline.

Experimental protocol

Monitoring of the number of coughs in guinea pigs

The whole body plethysmography system (WBP, Shanghai TOW Intelligent Technology Co., Ltd., Shanghai, China) was used to determine the number of coughs. The guinea pigs in each group were placed in the transparent chamber of the WBP and allowed to move freely for 6 min while the number of coughs was recorded. The guinea pigs were considered to be coughing based on characteristic body positions, such as opening their mouths, extending their front feet and necks forward (24,25). Cough counts were recorded and analyzed by two trained observers.

Histological evaluation

Lung tissue was sectioned (4 µm), deparaffinized, and dehydrated in decreasing concentrations of ethanol, followed by rinsing with distilled water and staining with hematoxylin and eosin (H&E). Microscopic images of stained sections were obtained at ×100 magnification.

ELISA

The levels of PGE2, BK, and TRPV1 protein expression in bronchoalveolar BALF supernatant and blood were determined using ELISA kits according to the manufacturers’ instructions.

Western blotting

Total protein of the lung tissue was extracted according to the instructions of RIPA lysis buffer, and the BCA method was used to prepare protein samples. The prepared protein samples and MAKER were added to sample wells with a micropipette, and the total protein content of each sample was 40 µg. After adding the sample, electrophoresis was run at a constant current of 80 V until the bromophenol blue indicator became a line at the junction of the concentrating and separating gels, and a constant current of 120 V brought bromophenol blue to the bottom of the sample. The target protein was transferred to a PVDF membrane and blocked with 5% nonfat milk powder for 2 h. After washing, the PVDF membranes were immersed in primary antibody and incubated overnight at 4 ℃. The next day, the corresponding HRP-labeled secondary antibody was diluted with blocking solution at 1:50,000, and the PVDF membrane was immersed in the secondary antibody solution and incubated at 37 ℃ for 2 h on a shaker. The enhanced solution in the electrochemoluminescent reagent was mixed with stabilized peroxidase solution at a ratio of 1:1, the working solution was dropped onto the PVDF membrane, the membrane was allowed to react for a few minutes, and the fluorescence band appeared. The film was then cleaned, dried, scanned, and then analyzed for greyscale values using IPP (Image Pro-Plus).

Real-time fluorescence quantitative PCR

A small amount lung tissue was frozen with liquid nitrogen and stored in a 1.5-mL Eppendorf tube. TRIzol reagent (1 mL) was added to extract RNA. OD260, OD280 and OD260/OD280 were measured by microspectrophotometer to calculate the purity and concentration of RNA. cDNA was synthesized according to the instructions of the reverse transcription kit. The related mRNA was amplified by PCR, and the total reaction volume was 20 µL. The primer sequence is given in Table S1. The reaction conditions were as follows: predenaturation at 95 ℃ and annealing at 60 ℃ for 30 s, repeated for 40 cycles. The relative gene expression was calculated by the 2^−ΔΔCt method.

Statistical analysis

All the data in the experiment are expressed as the mean ± standard deviation (mean ± SD). The fluorescence area of each group was analyzed by ImageJ software. Statistical analysis was carried out with GraphPad Prism 6 software. One-way ANOVA was used to compare the mean and multiple groups, the Bonferroni test was used to compare groups, and Spearman linear correlation analysis was used to test the correlation. R squared was recorded as R². P<0.05 indicated that the difference was statistically significant.

Results

Monitoring cough in experimental groups

The cough frequency (times/6 min) was 2.50±0.54 in the Sham Group, 15.67±1.63 in the Model Group, 7.50±2.88 in the EA + M Group, 9.17±1.17 in the H89 + M Group and 9.50±1.05 in the Go6983 + M Group. There was a significant difference between the Model Group and the EA + M, H89 + M and Go6983 + M groups (P<0.05, Figure 2). The specific P values are given in Table S2.

Figure 2 Changes of cough in each group after intervention. ^&, Sham Group compared with the other groups, P<0.05. ^#, Model Group compared with the other groups, P<0.05. EA, electroacupuncture; M, Model.

Pathological results in experimental group

Lung tissue was collected from guinea pigs in each group (Figure 3) and compared. In the Sham Group, the lung tissue appeared normal whereas in the EA + M, H89 + M and Go6983 + M groups the lung epithelial cells were swollen and degenerated to varying degrees, accompanied by varying degrees of inflammatory cell infiltration. The Model Group had more severe pathological lung damage, and extensive inflammatory cell infiltration. Compared with the H89 + M and Go6983 + M groups, the EA + M Group showed alleviated lung damage.

Figure 3 Pathological changes of guinea pig lung tissue (hematoxylin and eosin staining). (A) Sham Group, (B) Model Group, (C) EA + Model Group, (D) H89 + Model Group, (E) Go6983 + Model Group. Arrows represent swelling, degeneration or inflammatory cell infiltration of lung epithelial cells. A, electroacupuncture; M, Model.

ELISA detection of changes in PGE2, BK, and TRPV1 protein expression and correlation analysis in the BALF and blood of guinea pigs in each group

The levels of PGE2, BK and TRPV1 protein in the BALF from the Sham Group were significantly different from those in the Model Group, and there were significant differences between the Model Group and the EA + M, H89 + M and Go6983 + M groups (P<0.05, Figure 4). There were no significant differences among the EA + M, H89 + M and Go6983 + M groups (P>0.05, Figure 4; specific P values are given in Table S3). Chronic cough after pneumonectomy in the guinea pigs positively correlated with the expression of PGE2, BK and TRPV1 in BALF (Figure S1).

Figure 4 Bronchoalveolar lavage fluid and blood analysis of PGE2, BK and TRPV1 protein expression changes in each group of guinea pigs. ^&, Sham Group compared with the other groups, P<0.05; ^#, Model Group compared with the other groups, P<0.05. EA, electroacupuncture; M, Model; PGE2, prostaglandin E2; BK, bradykinin; TRPV1, transient receptor potential vanilloid-1.

Quantitative analysis of p-PKA, p-PKC and p-TRPV1 protein expressions and correlation in lung tissue of guinea pigs by Western blotting

Compared with the Sham Group, the protein expression of p-PKA in the lung tissue of the chronic cough Model Group after lung surgery was significantly increased on the 15th day of modeling (P<0.05); compared with the Model Group, the expression of p-PKA protein in the lung tissue of the EA + M Group and the H89 + M Group was significantly decreased (P<0.05); compared with the EA + M Group, the expression of p-PKA protein in the H89 + M Group was significantly decreased (P<0.05), as shown in Figure 5 (Table S4 for P values.) According to p-PKA protein expression in lung tissue, chronic cough after lung resection in guinea pigs positively correlated with PKA (R²=0.2828, P<0.05, Figure 5).

Figure 5 Western blot quantitative analysis of p-PKA, p-PKC and p-TRPV1 in guinea pig lung. ^&, Sham Group compared with the other groups, P<0.05. ^#, Model Group compared with the other groups, P<0.05. *, EA + M Group compared with the other groups, P<0.05. ^Δ, H89 + M Group compared with the other groups, P<0.05. EA, electroacupuncture; M, Model; PKA, protein kinase A; PKC, protein kinase C; TRPV, transient receptor potential vanilloid.

Compared with the Sham Group, the protein expression of p-PKC in the lung tissue of the Model Group on the 15th day of modeling was significantly increased (P<0.05). Compared with the EA + M Group, the p-PKC protein expression of the Go6983 + M Group decreased significantly (P<0.05), as shown in Figure 5 (specific P values are given in Table S4). According to p-PKC protein expression in lung tissue, chronic cough after lung resection in guinea pigs positively correlated with p-PKC (R²=0.2038, P<0.05, Figure 5).

Compared with that in the Sham Group, the protein expression of p-TRPV1 in the lung tissue of the chronic cough Model Group after lung surgery was significantly increased on the 15th day of modeling (P<0.05). The expression of p-TRPV1 protein in the lung tissue of the H89 + M Group and the Go6983 + M Group was significantly decreased (P<0.05), as shown in Figure 5, and the specific P values are shown in Table S4. According to p-TRPV1 protein expression in lung tissue, chronic cough after lung resection in guinea pigs positively correlated with p-TRPV1, R²=0.5760, P<0.05, Figure 5.

Measurement of TRPV1, SP, CGRP and NK1R mRNA in lung tissue of guinea pigs by real-time fluorescence quantitative PCR and correlation analysis

Real-time quantitative PCR was used to measure the TRPV1 mRNA content in guinea pig lung tissue. Compared with the Sham, EA + M, H89 + M and Go6983 + M groups, the Model Group showed a significant difference (P<0.05). Compared with the H89 + M Group, the EA + M Group had no statistical significance (P>0.05). Compared with the Go6983 + M Group, the EA + M Group showed a significant difference (P<0.05, Figure 6; specific P value is shown in Table S5). The level of TRPV1 mRNA in lung tissue positively correlated with chronic cough after pneumonectomy in guinea pigs (R²=0.3023, P<0.05, Figure 6).

Figure 6 Real-time fluorescence quantitative PCR analysis of TRPV1, SP, CGRP and NK1R mRNA in guinea pig lungs. ^&, Sham Group compared with the other groups, P<0.05. ^#, Model Group compared with the other groups, P<0.05. *, EA + M Group compared with the other groups, P<0.05. EA, electroacupuncture; M, Model; PCR, polymerase chain reaction; SP, substance P; NK1R, neurokinin-1R; CGRP, calcitonin gene-related peptide; TRPV1, transient receptor potential vanilloid-1.

Real-time quantitative PCR was used to detect the SP (Substance P) mRNA content in guinea pig lung tissue. Compared with the Sham Group, EA + M Group, H89 + M Group and Go6983 + M Group, there was a significant difference in the Model Group (P<0.05). Comparing the EA + M, H89 + M and Go6983 + M groups, there was no statistical significance (P>0.05) (Figure 6; specific P value is shown in Table S5). Regarding the level of SP mRNA in lung tissue, chronic cough after pneumonectomy in guinea pigs positively correlated with SP (R²=0.5344, P<0.05, Figure 6).

Real-time quantitative PCR was used to measure CGRP (calcitonin gene-related peptide) mRNA content in guinea pig lung tissue. Compared with the Sham, EA + M, H89 + M and Go6983 + M groups, there was a significant difference in the Model Group (P<0.05). Compared with the H89 + M Group, the EA + M Group had no significant difference (P>0.05). Compared with the H89 + M and Go6983 + M groups, the EA + M Group showed a significant difference (P<0.05, Figure 6; specific P value is shown in Table S5). Based on the level of CGRP mRNA in lung tissue, chronic cough after pneumonectomy in guinea pigs positively correlated with CGRP (R²=0.3339, P<0.05, Figure 6).

Real-time quantitative PCR was used to measure NK1R mRNA content in guinea pig lung tissue. Compared with the Sham, EA + M, H89 + M and Go6983 + M groups, there was a significant difference in the Model Group (P<0.05). Comparing the EA + M, H89 + M and Go6983 + M groups, there was no significant difference (P>0.05, Figure 6; specific P value is shown in Table S5). Based on the level of NK1R mRNA in lung tissue, chronic cough positively correlated with NK1R after pneumonectomy in guinea pigs (R²=0.4549, P<0.05, Figure 6).

Discussion

Lung surgery can induce cough and if present for more than 8 weeks it is defined as chronic. Cough is not only a physiological response to airway stimulation but is also related to the lymph node dissection area, surgical site, surgical method, and protective effect on healthy lungs during surgery (26-28). Cough is also one of the most common symptoms of respiratory diseases. Although it is a protective reflex, long-term coughing seriously affects quality of life.

Airway inflammation an important pathological mechanism of cough and is divided into infectious, allergic and neurogenic (29,30). The different types of inflammation may occur simultaneously. Various physical and chemical factors will stimulate the respiratory system after lung surgery, such as local pleurisy and pleural effusion, chronic irritation caused by surgical scars and foreign bodies, and postoperative airway irritation.

Acupuncture is an integral part of Traditional Chinese Medicine and can enhance the functioning of the patient’s immune system, thereby reducing phlegm and inflammation (31). According to a number of clinical reports, acupuncture and moxibustion have achieved good results in the treatment of cough, asthma, COPD and other lung diseases (19-21). For patients with poor drug treatment effect, you can try acupuncture treatment. Acupuncture treatment makes cough treatment methods diversified. In the present study using an animal model of postoperative cough, we found that the number of coughs within 6 min in the EA + M, H89 + M and Go6983 + M groups was significantly lower than that in the Model Group. In addition, the results of pathological investigations showed that the pulmonary epithelial cells in the EA + M, H89 + M and Go6983 + M groups were swollen and variously degenerated, accompanied by different degrees of inflammatory cell infiltration. In the Model Group, the lung pathological damage was more serious, with many infiltrated inflammatory cells. Compared with the H89 + M and Go6983 + M groups, the pathological lung damage in the EA + M Group was alleviated. Electroacupuncture at acupoints of the lung meridian and application of the inhibitors H89 and Go6983 alleviated the cough symptoms of these guinea pigs after pneumonectomy.

In our previous study (11), we found that the postoperative serum PGE2, BK and TRPV1 levels in 60 patients who underwent lung surgery were significantly higher than before surgery. Of them, 22 developed chronic cough, and 25 were diagnosed with non-chronic cough. The serum levels of PGE2, BK and TRPV1 in the chronic cough group were significantly higher than those in the non-chronic cough group at 8 weeks after surgery. As endogenous inflammatory mediators, PGE2 and BK activate the TRPV1 pathway by activating G protein-coupled receptors, increasing airway inflammation and triggering cough. Grace et al. found that the TRPV1 blockade in the guinea pig cough model inhibited the cough response of PGE2 and BK, and that the TRPV1 pathway was a key regulator of the cough response (32). In the present study, by comparing the results of BALF and blood ELISA to analyze the changes in the expression of PGE2 and BK in the guinea pigs in each group, we found that the expression of PGE2 and BK in the chronic cough Model Group was significantly increased after lung surgery. The expression levels of PGE2 and BK proteins in the acupuncture group were significantly decreased. Therefore, it can be inferred that electroacupuncture intervention can inhibit TRPV1 by reducing the expression of PGE2 and BK.

H89 is a commonly used inhibitor that can selectively inhibit a variety of protein kinases (including PKA, PKC and PKD, where PKA and PKD are involved in the control of Golgi morphology), but it has a stronger selective inhibitory effect on PKA (33,34). The inhibitor Go6983 is a classic PKC inhibitor. Ma et al. showed that PGE2 agonist-induced TRPV1 activation can be inhibited by PKA inhibitors (35). In addition, we found that the protein levels of p-PKA, p-PKC and p-TRPV1 in the chronic cough Model Group increased significantly after lung surgery. The protein levels of p-PKA, p-PKC and p-TRPV 1 in the electroacupuncture group was significantly reduced compared with the Model Group after electroacupuncture treatment. The protein content of p-PKA in guinea pigs treated with the inhibitor H89 was significantly lower than in the Model Group, and the protein content of p-PKC in guinea pigs treated with the inhibitor Go6983 was significantly lower than that in the Model Group. Therefore, we speculate that acupuncture can treat chronic cough by reducing PGE2 and BK through intervening in the PKA and PKC pathways to activate the TRPV1 gene to secrete TRPV1 protein.

Activation of TRPV1 ion channels leads to calcium influx into nerve terminals (33). The current concept is that the TRPV1 receptor acts as a "switch" for coughing. An airborne irritant that enters the lungs activates sensory nerves after contact with receptors, leading to a series of responses that manifest as coughing. In addition, studies have confirmed that TRPV1 can affect the release of neuropeptide substances (SP, CGRP and NK1R, etc.) (36,37). Our study found that the expression of SP, CGRP, and NK1R mRNA in the chronic cough Model Group was significantly increased after lung surgery, and the expression of SP, CGRP, and NK1R mRNA was significantly decreased after electroacupuncture at acupoints of the lung meridian and application of the inhibitors H89 and Go6983. These findings suggest that TRPV1 can interfere with the expression of SP, CGRP, NK1R and other neuropeptide proteins to cause chronic cough after lung surgery.

Conclusions

Electroacupuncture of the lung meridian reduced the number of coughs and the infiltration of inflammatory cells in a guinea pig model of chronic cough after lung surgery. In addition, electroacupuncture in the lung meridian inhibited the expression and secretion of SP, CGRP and NKA and reduced the infiltration of inflammatory cells by reducing the expression of the p-PKA and p-PKC-activated TRPV1 gene by interfering with PEG2 and BK (Figure 7). These findings provide new insights into the treatment of postoperative chronic cough.

Figure 7 Hypothesis diagram. PGE2, prostaglandin-E2; CGRP, calcitonin gene-related peptide; NKA, neurokinin A; SP, substance P; TRPV1, transient receptor potential vanilloid 1; PKA, cyclic-AMp dependent protein kinase A; PKC, cyclic-AMp dependent protein kinase C; EA, electroacupuncture; BK, bradykinin capsaicin.

Acknowledgments

Funding: This work was supported by grants from the Opening Project of Zhejiang Provincial Preponderant and Characteristic Subject of Key University (Chinese Traditional Medicine), Zhejiang Chinese Medical University (No. ZYXZD2019004); the National Natural Science Foundation of China (No. 81973643), the Key Projects of Natural Science Research in Anhui Universities in 2021 (No. KJ2021A0557); the Fundamental Research Funds for the Central Universities (No. WK9110000021); Key research and development projects in Anhui Province (No. 202004j07020017); Anhui Health Research Project in 2022 (No. AHWJ2022b053); Exploration project of Xin’an Physicians’ Thoughts on Treating Tumor and Its Syndromes (No. 2022AH050415).

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-409/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-409/dss

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Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-23-409/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Animal experiments were performed under a project license (No. AHUCM-mouse-2022059) granted by ethics board of Anhui University of Chinese Medicine, in compliance with institutional guidelines for the care and use of animals.

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