The ATP-P2X signaling pathway mediates the effect of electroacupuncture on excessive bladder detrusor muscle contraction in a rat model of neurogenic bladder

The ATP-P2X signaling pathway mediates the effect of electroacupuncture on excessive bladder detrusor muscle contraction in a rat model of neurogenic bladder | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The ATP-P2X signaling pathway mediates the effect of electroacupuncture on excessive bladder detrusor muscle contraction in a rat model of neurogenic bladder Qiong Liu, Ming Xu, Li-Fen Zhan, Qi-Rui Qu, Li-Ya Tang, Yue Zhuo, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4229154/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Neurogenic bladder (NB) is a type of neurological bladder dysfunction characterized by increased detrusor muscle contraction. Adenosine triphosphate (ATP)-P2X receptor signaling plays a role in muscle contraction. This study aimed to explore whether ATP-P2X signaling is involved in the mechanism through which electroacupuncture (EA) affects excessive detrusor muscle contraction in NB. Methods: Forty rats were divided into CON, NB, SHAM, PPADS (a P2X1/2 receptor antagonist), and EA groups. The NB model was induced using the modified Hassan Shaker spinal cord transection method. After one week of EA treatment, urodynamic tests were used to assess bladder function, hematoxylin and eosin (H&E) staining was used to evaluate morphological changes, enzyme-linked immunosorbent assays (ELISAs) were used to measure ATP concentrations, and Western blotting was used to analyze the protein levels of P2X 1 , P2X 2 , phosphorylated myosin light chain kinase (p-MLCK), and phosphorylated myosin light chain (p-MLC). Results: NB treatment led to morphological abnormalities, impaired urodynamics, increased ATP/P2X 1 /P2X 2 /p-MLC levels ( P <0.01), and decreased p-MLCK protein levels ( P <0.01). Both EA and the P2X 1/2 receptor antagonist reversed these changes induced by NB dysfunction ( P <0.05). Conclusion: The findings suggest that the ATP-P2X signaling pathway is involved in the therapeutic effect of EA on excessive detrusor contraction in a rat model of NB. Health sciences/Diseases Health sciences/Medical research Health sciences/Urology neurogenic bladder spinal cord injury electroacupuncture ATP-P2X signaling p-MLCK p-MLC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Neurogenic bladder (NB) a neurological condition characterized by urinary incontinence and retention, is a serious complication following spinal cord injury 1 , 2 . Injury to the micturition reflex arc of the central nervous system leads to uninhibited contraction of the detrusor reflex. Increased internal pressure and decreased compliance and volume are considered the main neurogenic causes in patients with spinal cord injury 3 , 4 . Therefore, inhibiting the internal pressure of detrusor contraction is an effective intervention strategy for improving urine storage capacity. In fact, drugs that inhibit noninhibitory contraction of the detrusor can be used to clinically treat neurogenic bladder and often improve urination efficiency and function 5 . Detrusor contraction is calcium-dependent and positively correlated with calcium concentration 6 . The concentration of Ca 2+ in the detrusor is regulated by adenosine triphosphate (ATP) 7 . ATP, a neurotransmitter in the purine system, plays a crucial role in detrusor contraction 7 , 8 . Previous studies have revealed that ATP can induce calcium influx by acting on P2X 1 and P2X 2 (gated ion channel receptors) on smooth muscle cells 9 , 10 . Ca 2+ ions combine with calmodulin (CaM) to form a Ca 2+ -CaM complex that, in turn, binds with myosin light chain kinase (MLCK) in the cytoplasm. This activation of MLCK leads to detrusor contraction through downstream phosphorylation of the myosin light chain (MLC) 4 , 11 . It is increasingly evident that acupuncture is an effective intervention for treating neurogenic diseases 12 , 13 . Previous studies have shown that acupuncture has a regulatory effect on the ATP-P2X signaling pathway 14 , 15 . Our previous research demonstrated that acupuncture can inhibit detrusor muscle contraction by activating MLCK to phosphorylate MLC 4 . However, whether acupuncture inhibits detrusor muscle contraction through modulation of the ATP-P2X-p-MLCK/p-MLC pathway remains unclear. Therefore, the current study sought to investigate the role of the ATP-P2X pathway in the mechanism by which acupuncture inhibits detrusor contraction in rats. Methods Animals The experimental animals were obtained from the Experimental Animal Center of Hunan University of Traditional Chinese Medicine. Eight-week-old female Sprague Dawley rats weighing between 190 and 230 g were acclimatized to room temperature (22–24°C) with a 12-hour light/dark cycle and provided food and water ad libitum. This study was approved by the Animal Ethics Committee of Hunan University of Traditional Chinese Medicine (LLBH-202201090002). All experimental procedures were conducted following the guidelines of the National Institutes of Health for the Care and Use of Experimental Animals. Forty-eight rats were randomly assigned to the CON, NB, SHAM, PPADS (P2X1/2 antagonist), and EA groups. The study is reported in accordance with ARRIVE guidelines ( https://arriveguidelines.org ). The methodology utilized in this study was based on the Hassan Shaker spinal cord transection method supplemented by an improved technique developed by researchers. Rats in the NB model group underwent a 24-hour fast prior to surgery and were given an intraperitoneal injection of penicillin sodium (200,000 U) to prevent infection. The surgical procedure involved spinal cord transection at the T8 level under anesthesia with isoflurane. Postoperatively, the rats received antibiotics, rehydration, and ongoing care, such as cage cleaning, manual urination, and dressing changes. In the SHAM group, only the spinal canal was exposed without severing the spinal cord. A follow-up period of 14 days was included to confirm the stability of the NB model and ensure that the animals had recovered from the spinal shock phase. EA administration In this study, BL32 (Ciliao), CV3 (Zhongji), and SP6 (Sanyinjiao) were chosen as the electroacupuncture points 4 , following the standardized reference GB12346-90. The measurement method used was similar to that used for human acupoint bone degree measurements. Rats in the EA group were treated with electroacupuncture for 7 days using 0.18 mm × 13 mm stainless steel needles (Huatuo brand, Jiangsu, China). The needles were obliquely inserted 5 mm into the CV3 acupoints, 5 mm into the SP6 acupoints, and 15 mm into the BL32 acupoints toward the affected area. The electroacupuncture device was programmed with a density wave of 10 Hz/50 Hz and a current intensity of 1.5 mA. Ventral CV3 was paired with SP6, while dorsal left and right BL32 were grouped together; each pair underwent electroacupuncture for 20 minutes. The PPADS group received an intraperitoneal injection of a P2X receptor antagonist (1.5 mg/kg). Both electroacupuncture and intraperitoneal injections began on Day 15 and ended on Day 22. Throughout this period, the management of the other groups remained consistent, except for the exclusion of the electroacupuncture intervention. Urodynamic testing Following one week of EA intervention, all rats were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg) and placed in a supine position. The bladder was emptied, and an MP-150 manometer was connected to an F3 catheter microinjection pump for future use. A 1 cm incision was made above the pubic symphysis along the anterior midline of the abdomen of the rats, and the F3 catheter was carefully inserted at the fornix and secured in place. After a 30-minute stabilization period, a 0.9% sodium chloride solution was infused at a rate of 6.0 ml/h. The computer system recorded parameters, including maximum bladder pressure (MBP), maximal bladder capacity (MBC), abdominal leak-point pressure (ALPP), and the duration of perfusion, for each rat. Sample collection and processing After terminal anesthesia, the rat bladder detrusor muscle was isolated and divided into two parts. One part was fixed in 4% paraformaldehyde solution, and the other was stored at -80°C. Hematoxylin and eosin staining The samples were fixed in 4% paraformaldehyde for 48 hours, heated at 60°C for 1–2 hours, placed in xylene for 10 minutes, dehydrated in an ethanol gradient, and embedded in wax. Continuous sections of approximately 4 µm thickness were prepared, and two samples from each were observed under an HE staining microscope (×400; BA210T, Motic). Measurement of ATP concentrations in bladder tissue Bladder detrusor samples were collected in EDTA and centrifuged at 3000 × g for 15 minutes at 4°C. The supernatant was then stored at -80°C. The ATP concentration in the bladder tissues was measured using an enzyme-linked immunosorbent assay (ELISA) kit (JM-11430R2, JINGMEI BIOTECHNOLOGY, China) according to the manufacturer’s instructions. Western blotting Bladder samples were homogenized in lysis buffer and centrifuged at 12000 × g for 15 minutes at 4°C. The supernatants were collected, and protein concentrations were determined by a bicinchoninic acid (BCA) assay. Equal amounts of protein (50–100 µg) from each sample were loaded onto 8% polyacrylamide gels and subjected to sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE). After separation, the proteins were electroblotted onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 hour and incubated with the following primary antibodies at 4°C overnight: rabbit anti-P2X 1 (1:1000; bs-21358R, Bioss), rabbit anti-P2X 2 (1:1000; bs-23813R, Bioss), rabbit anti-(p)-MLCK (1:1000; AF8387, Affinity), rabbit anti-p-MLC (1:1000; AF8618, Affinity), and rabbit anti-β-actin (1:3000; AF7018, Affinity). After washing three times with TBST, the membranes were incubated with secondary goat anti-rabbit HRP-conjugated AffiniPure (1:3000; SA00001-2, Proteintech). The blots were washed again three times in TBST, and the immunoreactive bands were detected using enhanced chemiluminescence (ECL) reagents. Statistical analysis All the data are presented as the mean ± standard error of the mean (SEM). Statistical differences were evaluated using one-way analysis of variance (ANOVA) followed by Fisher’s post hoc test for least significant difference (LSD). A P value < 0.05 was considered to indicate statistical significance. Results Effect of EA on the urodynamics of the rats in each group As shown in Fig. 2 A, compared with that in the CON group, the time at which the leak point appeared in the NB group was delayed, and the abdominal leak point pressure increased. Compared with those in the NB, SHAM, PPADS, and EA groups, the leakage point appeared earlier, and the abdominal leak-point pressure was relatively lower. As shown in Fig. 2 B, compared with those in the CON group, the MBP and MBC in the NB group were greater ( P < 0.01), the perfusion time was greater ( P < 0.01), and the ALPP was greater ( P < 0.01). Compared with those in the NB group, the MBP ( P < 0.01), MBC ( P < 0.01, P < 0.05), perfusion time ( P < 0.05), and ALPP ( P 0.05). The effect of EA on the morphological changes in bladder detrusor muscle tissue in each group The transitional epithelium in the bladder tissue of rats in the CON group (Fig. 3 A) and the SHAM group (Fig. 3 B) was tightly and neatly arranged, with an intact lamina propria and no signs of inflammatory infiltration or tissue edema. The muscle fiber bundles in the detrusor layer were neatly arranged and structurally dense, with abundant elastic fibers in the muscle space and no evidence of proliferation or fibrosis. In the NB group (Fig. 3 C), the arrangement of the transitional epithelium in the bladder tissue was disrupted, the structure of the lamina propria was loose and damaged, and there were signs of tissue edema and hemorrhagic changes. The arrangement of muscle fibers was disordered, accompanied by local fibrous connective tissue proliferation. Compared with the NB group, the PPADS (Fig. 3 D) and EA (Fig. 3 E) groups exhibited relatively regular and complete epithelial structures, reduced damage to the lamina propria, and improved edema and hemorrhagic changes. They also exhibited a relatively regular arrangement of muscle fibers, clear boundaries, and minimal fibrous tissue proliferation. EA indirectly inhibits bladder detrusor muscle contraction by regulating ATP levels in bladder tissue As shown in Fig. 4 , compared with that in the CON group, the ATP level in the NB group significantly increased ( P < 0.01), while compared with that in the NB group, the ATP levels in the SHAM, PPADS, and EA groups significantly decreased ( P < 0.01). EA directly inhibits bladder detrusor muscle contraction by regulating the release of purinergic synaptic ATP signals Compared with those in the CON group, the expression of P2X 1 , P2X 2 , and p-MLC2 in the detrusor muscle of the NB group was significantly greater ( P < 0.01). Compared with those in the NB group, the expression of P2X 1 , P2X 2 , and p-MLC2 in the detrusor muscle of the SHAM, PPADS, and EA groups was significantly lower ( P < 0.01). Compared with that in the CON group, the expression of p-MLCK in the detrusor muscle of the NB group was significantly lower ( P < 0.01). Compared with that in the NB group, the expression of p-MLCK in the detrusor muscle of the SHAM, PPADS, and EA groups was significantly greater ( P < 0.01) (Fig. 5 ). Discussion In this study, we demonstrated that electroacupuncture can inhibit detrusor contraction and improve the function of rats with spinal cord injuries and revealed the important role of the ATP-P2X-p-MLCK/p-MLC pathway in mediating this effect. In numerous clinical studies, surface electroacupuncture has been demonstrated to enhance urine storage capacity and urination efficiency in patients with neurogenic disorders following spinal cord injury 16 , 17 . Animal studies have further supported these findings by showing that electroacupuncture can increase bladder volume and decrease internal pressure in neurogenic rats 4 , 18 . Our study aligns with previous research, revealing that electroacupuncture effectively suppresses detrusor muscle contraction in neurogenic rats, leading to increased internal pressure and improved function. Detrusor contraction is a fundamental pathological feature of neurogenic bladder following spinal cord injury. Clinical treatment of neurogenic bladder diseases often involves inhibiting detrusor contraction 19 , 20 。 Amend et al. demonstrated that antimuscarinic drugs can effectively increase bladder volume and detrusor compliance 19 while Peyronnet et al. also supported this finding 20 . Li et al. utilized sacral nerve stimulation to modulate detrusor contraction and enhance function through nerve discharge 21 . These studies collectively highlight the effectiveness of inhibiting detrusor contraction in treating neurogenic bladder diseases. Our research indicated that acupuncture can effectively inhibit detrusor contraction and improve function. Importantly, acupuncture stands out for its high safety profile, minimal side effects, and cost-effectiveness compared to other therapies. Numerous studies have demonstrated the substantial involvement of the purinergic ATP system in detrusor contraction 8 , 22 . This system is responsible for regulating the detrusor muscle by activating P2X 1 and P2X 2 receptors 23 . P2X 1 and P2X 2 , known as ATP-gated nonselective cation channels, directly influence the ion channel state by altering the protein configuration, bypassing the need for rapid transduction through membranes and intracellular signaling 24 . Activation of the P2X 1 and P2X 2 receptors leads to the opening of cation channels, causing an influx of extracellular Ca 2+ ions. Ca 2+ ions combined with calmodulin (CaM) form a complex that, in turn, binds with myosin light chain kinase (MLCK) in the cytoplasm. This interaction activates MLCK, triggering detrusor contraction through the phosphorylation of myosin light chain (MLC). These findings highlight the crucial role of the ATP-P2X-p-MLCK/p-MLC pathway in regulating detrusor contraction (Fig. 6 A). The levels of ATP and its receptors P2X 1 and P2X 2 were found to be significantly higher in the model group than in the control group, with the exception of urodynamic indices, indicating their involvement in detrusor contraction. Conversely, the EA group exhibited significantly lower levels of ATP and its receptors P2X 1 and P2X 2 than the model group, suggesting that the ATP-P2X pathway may play a crucial role in the EA-induced inhibition of detrusor contraction. Furthermore, in the P2X inhibitor group, there was a significant reduction in ATP receptor expression, providing further evidence for the importance of the ATP-P2X pathway in the EA-mediated inhibition of detrusor contraction. The results of our study demonstrated alterations in the phosphorylation levels of MLCK and MLC downstream of ATP in NB model rats, leading to increased expression of p-MLCK and p-MLC. These findings suggest that the ATP-P2X-p-MLCK/p-MLC pathway plays a crucial role in detrusor contraction. Following EA intervention, the changes in ATP-P2X-p-MLCK/p-MLC ratio and detrusor contraction were abrogated. Overall, our results indicate that EA effectively enhances detrusor relaxation by activating the ATP-P2X-p-MLCK/p-MLC pathway (Fig. 6 B). In this study, EA was administered at CV3, SP6, and BL32, as they are commonly used traditional acupuncture points for treating NB 4 . The tissues at CV3 are innervated by the T12-L1 segments of the spinal cord, while SP6 is innervated by the S2 segment. BL32 is situated in the second posterior sacral foramen, below the branch of the second sacral nerve. Acupuncture at the SP6 and BL32 can stimulate the second sacral nerve and regulate the S2-S4 parasympathetic center. Overall, acupuncture at these three sites together has been shown to improve excessive contraction of the bladder detrusor muscle in rats, possibly through modulation of the sympathetic and/or parasympathetic nerves innervating the bladder detrusor. 18 , 25 , 26 Conclusion This study is the first to demonstrate that acupuncture can inhibit detrusor contraction by modulating the ATP-P2X pathway in the treatment of neurogenic spinal cord injury. These findings offer a scientific rationale for the clinical application of acupuncture and moxibustion in managing detrusor contraction and neurogenic bladder. Given the important role of ATPase in ATP breakdown, future research elucidating the role of ATPase in electroacupuncture-mediated modulation of detrusor contraction will enhance our understanding of the mechanisms underlying the effects of acupuncture and moxibustion in treating detrusor contraction and neurogenic bladder. Declarations Data acailability Data is provided within the manuscript or supplementary information files. Contributions Study design: KA, XL. Project administration: LQ, KA. Experimental implementation: QL, MX and LFZ. Data analysis: LYT, YZ, SFD and QRQ. Paper writing: QL, XL and KA. Paper review and editing: HZ. Experimental support: KA QL and LX. All authors read and approved the final version of the manuscript accepted for publication. Acknowledgments We would like to thank all the participants in this study. Funding This study was supported by the National Natural Science Foundation of China (82205255), Natural Science Foundation of Hunan Province (2022JJ40301,2022JJ40312) and Undergraduate Research Innovation Fund of Hunan University of Traditional Chinese Medicine(2023BKS093). ORCID iD Qiong Liu iD https://orcid.org/0000-0002-5634-1555 Declaration of confliction interests The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article. References Truzzi, J. C., de Almeida, F. G., Sacomani, C. A., Reis, J. & Rocha, F. E. T. Neurogenic bladder - concepts and treatment recommendations. International braz j urol: official journal of the Brazilian Society of Urology 48, 220–243. http://doi.org/10.1590/s1677-5538.Ibju.2021.0098 (2022). Lee, J. W., Lee, S. S., Yang, S. H. & Choe, H. S. Assessment of Bacterial Communities Within the Biofilm of Bladder Calculi in the Neurogenic Bladder Rat Model Following Spinal Cord Injury. 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Go it alone no more - P2X7 joins the society of heteromeric ATP-gated receptor channels. Molecular Pharmacology 72, 1402–1405. http://doi.org/10.1124/mol.107.042077 (2007). Ai, K. et al. Effect of electroacupuncture on urodynamics of neurogenic bladder and PACAP/cAMP/PKA signaling pathway in detrusor tissue of rats after suprasacral spinal cord injury. Zhen ci yan jiu 46, 728–734. http://doi.org/10.13702/j.1000-0607.200880 (2021). He, Y. et al. Acupoint selection rules of acupuncture and moxibustion in treating neurogenic bladder based on data mining. Zhen ci yan jiu 49, 198–207. http://doi.org/10.13702/j.1000-0607.20230018 (2024). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4229154","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":292712773,"identity":"0941f07f-21d6-4325-8d7b-75e91c2a9bf7","order_by":0,"name":"Qiong Liu","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qiong","middleName":"","lastName":"Liu","suffix":""},{"id":292712778,"identity":"7b39d848-00cf-4f8d-ae37-d2aad238e83e","order_by":1,"name":"Ming Xu","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Xu","suffix":""},{"id":292712779,"identity":"ff01c9d0-97c9-4b8a-9691-b9e4bd9691b5","order_by":2,"name":"Li-Fen Zhan","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Li-Fen","middleName":"","lastName":"Zhan","suffix":""},{"id":292712780,"identity":"1cbeba78-deaf-4b6b-8c48-2b9f153c8527","order_by":3,"name":"Qi-Rui Qu","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qi-Rui","middleName":"","lastName":"Qu","suffix":""},{"id":292712781,"identity":"d470f7e3-b3c8-4f17-b503-0d02285f046c","order_by":4,"name":"Li-Ya Tang","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Li-Ya","middleName":"","lastName":"Tang","suffix":""},{"id":292712782,"identity":"a2a7392e-2980-496a-853a-5e54b3f548a8","order_by":5,"name":"Yue Zhuo","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Zhuo","suffix":""},{"id":292712783,"identity":"775abfc2-db72-42d9-8800-f593de12fbe7","order_by":6,"name":"Shi-Feng Deng","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shi-Feng","middleName":"","lastName":"Deng","suffix":""},{"id":292712784,"identity":"a41c2b15-3a00-41d9-b71a-e149e42c25c1","order_by":7,"name":"Hong Zhang","email":"","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Zhang","suffix":""},{"id":292712785,"identity":"aa5309a1-de1d-419a-836e-4e3b17a58acf","order_by":8,"name":"Xia Liu","email":"","orcid":"","institution":"Chongqing Three Gorges Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Liu","suffix":""},{"id":292712786,"identity":"527db405-c935-4ec9-af0b-7afa865583cd","order_by":9,"name":"Kun Ai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYDACZjYgUfGPh59ELWcOyEk2EK8HqIWx5YCxwQFiNfC3s6VJFzbcSdx8PHkDw4+KbYS1SBxmOyY9c8ezxG1nnhUw9py5TYQ1h9nbpHnPMCduu5FjwMzYRoQWebCWNubEzTOI1WIAchhv22FjAwlitRgeZku25jmTJicB9MtBovwid/6Y4W2eChse/vbkjQ9+VBDjfQRIID5qEFpI1TEKRsEoGAUjBAAAve47y49dIWIAAAAASUVORK5CYII=","orcid":"","institution":"Hunan University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Kun","middleName":"","lastName":"Ai","suffix":""}],"badges":[],"createdAt":"2024-04-07 02:14:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4229154/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4229154/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54966282,"identity":"3fc38506-f86a-4add-9b3e-9f2e91e45f20","added_by":"auto","created_at":"2024-04-19 09:53:46","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1559180,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental schedule and design. Forty rats were acclimatized for one week and then randomly divided into the CON group and the NB group. The NB group was used for model replication, and the replication results were then randomly divided into the SHAM group, the PPADS group, and the EA group. After 14 days of successful model replication, interventions were conducted. After one week of intervention, all groups underwent urodynamic testing, and samples were collected after the testing had been completed (created with BioRender.com).\u003c/p\u003e","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4229154/v1/fa951b10bf96e70d284d40e4.jpeg"},{"id":54967147,"identity":"35697116-83c2-46f8-886c-228d6316ae9e","added_by":"auto","created_at":"2024-04-19 10:01:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":619583,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in urodynamics of rats in each group. A: Urodynamic curve. B(a): Maximal bladder capacity. B(b): Perfusion time. B(c): Maximum bladder pressure. B(d): Abdominal leak-point pressure. Values are expressed as mean ± SEM, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. CON group; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. NB group.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4229154/v1/0ff8017b04d380e68d9bbc37.jpg"},{"id":54966285,"identity":"b0c67951-7583-454f-96a9-98f2f5fa6cb0","added_by":"auto","created_at":"2024-04-19 09:53:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1508972,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological changes of bladder tissue in different groups under light microscopy after hematoxylin and eosin stain (×400). A: CON group. B: NB group. C: SHAM group. D: PPADS group. E: EA group.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4229154/v1/5c2fffffbfedb6311cc65e9d.jpg"},{"id":54966283,"identity":"13a60646-b5e1-47a1-9889-351a2ba4e79b","added_by":"auto","created_at":"2024-04-19 09:53:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":312426,"visible":true,"origin":"","legend":"\u003cp\u003eATP content in bladder tissue of rats in each group. Values are expressed as mean ± SEM. \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. CON group. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. NB group.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4229154/v1/61682133a333add3731ffe1e.jpg"},{"id":54967149,"identity":"ace56c89-b343-49ff-b1ad-7d818ed8a6a7","added_by":"auto","created_at":"2024-04-19 10:01:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":574314,"visible":true,"origin":"","legend":"\u003cp\u003eProtein content of P2X\u003csub\u003e1\u003c/sub\u003e, P2X\u003csub\u003e2\u003c/sub\u003e, p-MLCK, and p-MLC in the detrusor muscle of rats in each group. A: Quantification of P2X\u003csub\u003e1\u003c/sub\u003e expression in detrusor tissue, and bands of P2X\u003csub\u003e1\u003c/sub\u003e expression in detrusor tissue. B: Quantification of P2X\u003csub\u003e2 \u003c/sub\u003eexpression, and bands of P2X\u003csub\u003e2\u003c/sub\u003e expression in detrusor tissue. C: Quantification of p-MLCK expression in detrusor tissue, and bands of p-MLCK expression in detrusor tissue. D: Quantification of p-MLC2 expression in detrusor tissue, and bands of p-MLC2 expression in detrusor tissue. Values are expressed as mean ± SEM. \u0026nbsp;\u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs CON group. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. NB group.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4229154/v1/9d20937f5c4f6e3027a1ccbe.jpg"},{"id":54967148,"identity":"dda187cd-cc67-4c2b-9024-5dd7596bce8b","added_by":"auto","created_at":"2024-04-19 10:01:46","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":180464,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism diagram of electroacupuncture promoting detrusor muscle relaxation by inhibiting the activation of the ATP-P2X-p-MLCK/p-MLC pathway.(created with Figdraw.com).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4229154/v1/63db089ca474d27565c92834.jpg"},{"id":55504814,"identity":"3bc0f8ac-9b2d-44c7-b43b-36fcf0e685b2","added_by":"auto","created_at":"2024-04-29 11:18:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":897060,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4229154/v1/d945c620-0bd2-43b0-b4cd-d7ea1ce7cfb9.pdf"},{"id":54967146,"identity":"33b61d2a-336e-4d82-a7d7-0ffc5b3e12b6","added_by":"auto","created_at":"2024-04-19 10:01:46","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":181171,"visible":true,"origin":"","legend":"","description":"","filename":"ConfirmationofPublicationandLicensingRights.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4229154/v1/16b5271bdd300ff565ca1720.pdf"},{"id":54966288,"identity":"82bae487-f103-4bc0-8d64-46642b641451","added_by":"auto","created_at":"2024-04-19 09:53:46","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":129073,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarydataOriginalblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4229154/v1/b22f3b694b1437d569d499e5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The ATP-P2X signaling pathway mediates the effect of electroacupuncture on excessive bladder detrusor muscle contraction in a rat model of neurogenic bladder","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNeurogenic bladder (NB) a neurological condition characterized by urinary incontinence and retention, is a serious complication following spinal cord injury\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Injury to the micturition reflex arc of the central nervous system leads to uninhibited contraction of the detrusor reflex. Increased internal pressure and decreased compliance and volume are considered the main neurogenic causes in patients with spinal cord injury\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Therefore, inhibiting the internal pressure of detrusor contraction is an effective intervention strategy for improving urine storage capacity. In fact, drugs that inhibit noninhibitory contraction of the detrusor can be used to clinically treat neurogenic bladder and often improve urination efficiency and function\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDetrusor contraction is calcium-dependent and positively correlated with calcium concentration\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The concentration of Ca\u003csup\u003e2+\u003c/sup\u003e in the detrusor is regulated by adenosine triphosphate (ATP)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. ATP, a neurotransmitter in the purine system, plays a crucial role in detrusor contraction\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Previous studies have revealed that ATP can induce calcium influx by acting on P2X\u003csub\u003e1\u003c/sub\u003e and P2X\u003csub\u003e2\u003c/sub\u003e (gated ion channel receptors) on smooth muscle cells\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Ca\u003csup\u003e2+\u003c/sup\u003e ions combine with calmodulin (CaM) to form a Ca\u003csup\u003e2+\u003c/sup\u003e-CaM complex that, in turn, binds with myosin light chain kinase (MLCK) in the cytoplasm. This activation of MLCK leads to detrusor contraction through downstream phosphorylation of the myosin light chain (MLC)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is increasingly evident that acupuncture is an effective intervention for treating neurogenic diseases\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that acupuncture has a regulatory effect on the ATP-P2X signaling pathway\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Our previous research demonstrated that acupuncture can inhibit detrusor muscle contraction by activating MLCK to phosphorylate MLC\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, whether acupuncture inhibits detrusor muscle contraction through modulation of the ATP-P2X-p-MLCK/p-MLC pathway remains unclear. Therefore, the current study sought to investigate the role of the ATP-P2X pathway in the mechanism by which acupuncture inhibits detrusor contraction in rats.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eThe experimental animals were obtained from the Experimental Animal Center of Hunan University of Traditional Chinese Medicine. Eight-week-old female Sprague Dawley rats weighing between 190 and 230 g were acclimatized to room temperature (22\u0026ndash;24\u0026deg;C) with a 12-hour light/dark cycle and provided food and water ad libitum. This study was approved by the Animal Ethics Committee of Hunan University of Traditional Chinese Medicine (LLBH-202201090002). All experimental procedures were conducted following the guidelines of the National Institutes of Health for the Care and Use of Experimental Animals. Forty-eight rats were randomly assigned to the CON, NB, SHAM, PPADS (P2X1/2 antagonist), and EA groups. The study is reported in accordance with ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe methodology utilized in this study was based on the Hassan Shaker spinal cord transection method supplemented by an improved technique developed by researchers. Rats in the NB model group underwent a 24-hour fast prior to surgery and were given an intraperitoneal injection of penicillin sodium (200,000 U) to prevent infection. The surgical procedure involved spinal cord transection at the T8 level under anesthesia with isoflurane. Postoperatively, the rats received antibiotics, rehydration, and ongoing care, such as cage cleaning, manual urination, and dressing changes. In the SHAM group, only the spinal canal was exposed without severing the spinal cord. A follow-up period of 14 days was included to confirm the stability of the NB model and ensure that the animals had recovered from the spinal shock phase.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEA administration\u003c/h2\u003e \u003cp\u003eIn this study, BL32 (Ciliao), CV3 (Zhongji), and SP6 (Sanyinjiao) were chosen as the electroacupuncture points\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, following the standardized reference GB12346-90. The measurement method used was similar to that used for human acupoint bone degree measurements. Rats in the EA group were treated with electroacupuncture for 7 days using 0.18 mm \u0026times; 13 mm stainless steel needles (Huatuo brand, Jiangsu, China). The needles were obliquely inserted 5 mm into the CV3 acupoints, 5 mm into the SP6 acupoints, and 15 mm into the BL32 acupoints toward the affected area. The electroacupuncture device was programmed with a density wave of 10 Hz/50 Hz and a current intensity of 1.5 mA. Ventral CV3 was paired with SP6, while dorsal left and right BL32 were grouped together; each pair underwent electroacupuncture for 20 minutes. The PPADS group received an intraperitoneal injection of a P2X receptor antagonist (1.5 mg/kg). Both electroacupuncture and intraperitoneal injections began on Day 15 and ended on Day 22. Throughout this period, the management of the other groups remained consistent, except for the exclusion of the electroacupuncture intervention.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eUrodynamic testing\u003c/h2\u003e \u003cp\u003eFollowing one week of EA intervention, all rats were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg) and placed in a supine position. The bladder was emptied, and an MP-150 manometer was connected to an F3 catheter microinjection pump for future use. A 1 cm incision was made above the pubic symphysis along the anterior midline of the abdomen of the rats, and the F3 catheter was carefully inserted at the fornix and secured in place. After a 30-minute stabilization period, a 0.9% sodium chloride solution was infused at a rate of 6.0 ml/h. The computer system recorded parameters, including maximum bladder pressure (MBP), maximal bladder capacity (MBC), abdominal leak-point pressure (ALPP), and the duration of perfusion, for each rat.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSample collection and processing\u003c/h2\u003e \u003cp\u003eAfter terminal anesthesia, the rat bladder detrusor muscle was isolated and divided into two parts. One part was fixed in 4% paraformaldehyde solution, and the other was stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin and eosin staining\u003c/h2\u003e \u003cp\u003eThe samples were fixed in 4% paraformaldehyde for 48 hours, heated at 60\u0026deg;C for 1\u0026ndash;2 hours, placed in xylene for 10 minutes, dehydrated in an ethanol gradient, and embedded in wax. Continuous sections of approximately 4 \u0026micro;m thickness were prepared, and two samples from each were observed under an HE staining microscope (\u0026times;400; BA210T, Motic).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of ATP concentrations in bladder tissue\u003c/h2\u003e \u003cp\u003eBladder detrusor samples were collected in EDTA and centrifuged at 3000 \u0026times; g for 15 minutes at 4\u0026deg;C. The supernatant was then stored at -80\u0026deg;C. The ATP concentration in the bladder tissues was measured using an enzyme-linked immunosorbent assay (ELISA) kit (JM-11430R2, JINGMEI BIOTECHNOLOGY, China) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eBladder samples were homogenized in lysis buffer and centrifuged at 12000 \u0026times; g for 15 minutes at 4\u0026deg;C. The supernatants were collected, and protein concentrations were determined by a bicinchoninic acid (BCA) assay. Equal amounts of protein (50\u0026ndash;100 \u0026micro;g) from each sample were loaded onto 8% polyacrylamide gels and subjected to sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE). After separation, the proteins were electroblotted onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 hour and incubated with the following primary antibodies at 4\u0026deg;C overnight: rabbit anti-P2X\u003csub\u003e1\u003c/sub\u003e (1:1000; bs-21358R, Bioss), rabbit anti-P2X\u003csub\u003e2\u003c/sub\u003e (1:1000; bs-23813R, Bioss), rabbit anti-(p)-MLCK (1:1000; AF8387, Affinity), rabbit anti-p-MLC (1:1000; AF8618, Affinity), and rabbit anti-β-actin (1:3000; AF7018, Affinity). After washing three times with TBST, the membranes were incubated with secondary goat anti-rabbit HRP-conjugated AffiniPure (1:3000; SA00001-2, Proteintech). The blots were washed again three times in TBST, and the immunoreactive bands were detected using enhanced chemiluminescence (ECL) reagents.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical differences were evaluated using one-way analysis of variance (ANOVA) followed by Fisher\u0026rsquo;s post hoc test for least significant difference (LSD). A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffect of EA on the urodynamics of the rats in each group\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, compared with that in the CON group, the time at which the leak point appeared in the NB group was delayed, and the abdominal leak point pressure increased. Compared with those in the NB, SHAM, PPADS, and EA groups, the leakage point appeared earlier, and the abdominal leak-point pressure was relatively lower.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, compared with those in the CON group, the MBP and MBC in the NB group were greater (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), the perfusion time was greater (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the ALPP was greater (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with those in the NB group, the MBP (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), MBC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), perfusion time (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and ALPP (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the PPADS and EA groups were lower. There was no significant difference in MBP, MBC, perfusion time, or ALPP between the SHAM group and the CON group (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe effect of EA on the morphological changes in bladder detrusor muscle tissue in each group\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe transitional epithelium in the bladder tissue of rats in the CON group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and the SHAM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) was tightly and neatly arranged, with an intact lamina propria and no signs of inflammatory infiltration or tissue edema. The muscle fiber bundles in the detrusor layer were neatly arranged and structurally dense, with abundant elastic fibers in the muscle space and no evidence of proliferation or fibrosis. In the NB group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), the arrangement of the transitional epithelium in the bladder tissue was disrupted, the structure of the lamina propria was loose and damaged, and there were signs of tissue edema and hemorrhagic changes. The arrangement of muscle fibers was disordered, accompanied by local fibrous connective tissue proliferation. Compared with the NB group, the PPADS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and EA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) groups exhibited relatively regular and complete epithelial structures, reduced damage to the lamina propria, and improved edema and hemorrhagic changes. They also exhibited a relatively regular arrangement of muscle fibers, clear boundaries, and minimal fibrous tissue proliferation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEA indirectly inhibits bladder detrusor muscle contraction by regulating ATP levels in bladder tissue\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, compared with that in the CON group, the ATP level in the NB group significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while compared with that in the NB group, the ATP levels in the SHAM, PPADS, and EA groups significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003cp\u003e \u003cb\u003eEA directly inhibits bladder detrusor muscle contraction by regulating the release of purinergic synaptic ATP signals\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCompared with those in the CON group, the expression of P2X\u003csub\u003e1\u003c/sub\u003e, P2X\u003csub\u003e2\u003c/sub\u003e, and p-MLC2 in the detrusor muscle of the NB group was significantly greater (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with those in the NB group, the expression of P2X\u003csub\u003e1\u003c/sub\u003e, P2X\u003csub\u003e2\u003c/sub\u003e, and p-MLC2 in the detrusor muscle of the SHAM, PPADS, and EA groups was significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with that in the CON group, the expression of p-MLCK in the detrusor muscle of the NB group was significantly lower (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with that in the NB group, the expression of p-MLCK in the detrusor muscle of the SHAM, PPADS, and EA groups was significantly greater (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated that electroacupuncture can inhibit detrusor contraction and improve the function of rats with spinal cord injuries and revealed the important role of the ATP-P2X-p-MLCK/p-MLC pathway in mediating this effect.\u003c/p\u003e \u003cp\u003eIn numerous clinical studies, surface electroacupuncture has been demonstrated to enhance urine storage capacity and urination efficiency in patients with neurogenic disorders following spinal cord injury\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Animal studies have further supported these findings by showing that electroacupuncture can increase bladder volume and decrease internal pressure in neurogenic rats\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Our study aligns with previous research, revealing that electroacupuncture effectively suppresses detrusor muscle contraction in neurogenic rats, leading to increased internal pressure and improved function.\u003c/p\u003e \u003cp\u003eDetrusor contraction is a fundamental pathological feature of neurogenic bladder following spinal cord injury. Clinical treatment of neurogenic bladder diseases often involves inhibiting detrusor contraction\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003csub\u003e。\u003c/sub\u003e Amend et al. demonstrated that antimuscarinic drugs can effectively increase bladder volume and detrusor compliance\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e while Peyronnet et al. also supported this finding\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Li et al. utilized sacral nerve stimulation to modulate detrusor contraction and enhance function through nerve discharge\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. These studies collectively highlight the effectiveness of inhibiting detrusor contraction in treating neurogenic bladder diseases. Our research indicated that acupuncture can effectively inhibit detrusor contraction and improve function. Importantly, acupuncture stands out for its high safety profile, minimal side effects, and cost-effectiveness compared to other therapies.\u003c/p\u003e \u003cp\u003eNumerous studies have demonstrated the substantial involvement of the purinergic ATP system in detrusor contraction\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. This system is responsible for regulating the detrusor muscle by activating P2X\u003csub\u003e1\u003c/sub\u003e and P2X\u003csub\u003e2\u003c/sub\u003e receptors\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. P2X\u003csub\u003e1\u003c/sub\u003e and P2X\u003csub\u003e2\u003c/sub\u003e, known as ATP-gated nonselective cation channels, directly influence the ion channel state by altering the protein configuration, bypassing the need for rapid transduction through membranes and intracellular signaling\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Activation of the P2X\u003csub\u003e1\u003c/sub\u003e and P2X\u003csub\u003e2\u003c/sub\u003e receptors leads to the opening of cation channels, causing an influx of extracellular Ca\u003csup\u003e2+\u003c/sup\u003e ions. Ca\u003csup\u003e2+\u003c/sup\u003e ions combined with calmodulin (CaM) form a complex that, in turn, binds with myosin light chain kinase (MLCK) in the cytoplasm. This interaction activates MLCK, triggering detrusor contraction through the phosphorylation of myosin light chain (MLC). These findings highlight the crucial role of the ATP-P2X-p-MLCK/p-MLC pathway in regulating detrusor contraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe levels of ATP and its receptors P2X\u003csub\u003e1\u003c/sub\u003e and P2X\u003csub\u003e2\u003c/sub\u003e were found to be significantly higher in the model group than in the control group, with the exception of urodynamic indices, indicating their involvement in detrusor contraction. Conversely, the EA group exhibited significantly lower levels of ATP and its receptors P2X\u003csub\u003e1\u003c/sub\u003e and P2X\u003csub\u003e2\u003c/sub\u003e than the model group, suggesting that the ATP-P2X pathway may play a crucial role in the EA-induced inhibition of detrusor contraction. Furthermore, in the P2X inhibitor group, there was a significant reduction in ATP receptor expression, providing further evidence for the importance of the ATP-P2X pathway in the EA-mediated inhibition of detrusor contraction.\u003c/p\u003e \u003cp\u003eThe results of our study demonstrated alterations in the phosphorylation levels of MLCK and MLC downstream of ATP in NB model rats, leading to increased expression of p-MLCK and p-MLC. These findings suggest that the ATP-P2X-p-MLCK/p-MLC pathway plays a crucial role in detrusor contraction. Following EA intervention, the changes in ATP-P2X-p-MLCK/p-MLC ratio and detrusor contraction were abrogated. Overall, our results indicate that EA effectively enhances detrusor relaxation by activating the ATP-P2X-p-MLCK/p-MLC pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn this study, EA was administered at CV3, SP6, and BL32, as they are commonly used traditional acupuncture points for treating NB\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The tissues at CV3 are innervated by the T12-L1 segments of the spinal cord, while SP6 is innervated by the S2 segment. BL32 is situated in the second posterior sacral foramen, below the branch of the second sacral nerve. Acupuncture at the SP6 and BL32 can stimulate the second sacral nerve and regulate the S2-S4 parasympathetic center. Overall, acupuncture at these three sites together has been shown to improve excessive contraction of the bladder detrusor muscle in rats, possibly through modulation of the sympathetic and/or parasympathetic nerves innervating the bladder detrusor.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study is the first to demonstrate that acupuncture can inhibit detrusor contraction by modulating the ATP-P2X pathway in the treatment of neurogenic spinal cord injury. These findings offer a scientific rationale for the clinical application of acupuncture and moxibustion in managing detrusor contraction and neurogenic bladder. Given the important role of ATPase in ATP breakdown, future research elucidating the role of ATPase in electroacupuncture-mediated modulation of detrusor contraction will enhance our understanding of the mechanisms underlying the effects of acupuncture and moxibustion in treating detrusor contraction and neurogenic bladder.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData acailability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy design: KA, XL. Project administration: LQ, KA. Experimental implementation: QL, MX and LFZ. Data analysis: LYT, YZ, SFD and QRQ. Paper writing: QL, XL and KA. Paper review and editing: HZ. Experimental support: KA QL and LX. All authors read and approved the final version of the manuscript accepted for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all the participants in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (82205255), Natural Science Foundation of Hunan Province (2022JJ40301,2022JJ40312) and Undergraduate Research Innovation Fund of Hunan University of Traditional Chinese Medicine(2023BKS093).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID iD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQiong Liu iD https://orcid.org/0000-0002-5634-1555\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of confliction interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTruzzi, J. 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Zhen ci yan jiu 49, 198\u0026ndash;207. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.13702/j.1000-0607.20230018\u003c/span\u003e\u003cspan address=\"10.13702/j.1000-0607.20230018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"neurogenic bladder, spinal cord injury, electroacupuncture, ATP-P2X signaling, p-MLCK, p-MLC","lastPublishedDoi":"10.21203/rs.3.rs-4229154/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4229154/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eNeurogenic bladder (NB) is a type of neurological bladder dysfunction characterized by increased detrusor muscle contraction. Adenosine triphosphate (ATP)-P2X receptor signaling plays a role in muscle contraction. This study aimed to explore whether ATP-P2X signaling is involved in the mechanism through which electroacupuncture (EA) affects excessive detrusor muscle contraction in NB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eForty rats were divided into CON, NB, SHAM, PPADS (a P2X1/2 receptor antagonist), and EA groups. The NB model was induced using the modified Hassan Shaker spinal cord transection method. After one week of EA treatment, urodynamic tests were used to assess bladder function, hematoxylin and eosin (H\u0026amp;E) staining was used to evaluate morphological changes, enzyme-linked immunosorbent assays (ELISAs) were used to measure ATP concentrations, and Western blotting was used to analyze the protein levels of P2X\u003csub\u003e1\u003c/sub\u003e, P2X\u003csub\u003e2\u003c/sub\u003e, phosphorylated myosin light chain kinase (p-MLCK), and phosphorylated myosin light chain (p-MLC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eNB treatment led to morphological abnormalities, impaired urodynamics, increased ATP/P2X\u003csub\u003e1\u003c/sub\u003e/P2X\u003csub\u003e2\u003c/sub\u003e/p-MLC levels (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01), and decreased p-MLCK protein levels (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). Both EA and the P2X\u003csub\u003e1/2\u003c/sub\u003e receptor antagonist reversed these changes induced by NB dysfunction (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThe findings suggest that the ATP-P2X signaling pathway is involved in the therapeutic effect of EA on excessive detrusor contraction in a rat model of NB.\u003c/p\u003e","manuscriptTitle":"The ATP-P2X signaling pathway mediates the effect of electroacupuncture on excessive bladder detrusor muscle contraction in a rat model of neurogenic bladder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-19 09:53:41","doi":"10.21203/rs.3.rs-4229154/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d677a510-2c17-4b09-95f7-a054a15ebe75","owner":[],"postedDate":"April 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":30840599,"name":"Health sciences/Diseases"},{"id":30840600,"name":"Health sciences/Medical research"},{"id":30840601,"name":"Health sciences/Urology"}],"tags":[],"updatedAt":"2024-04-29T10:09:06+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-19 09:53:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4229154","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4229154","identity":"rs-4229154","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}