The effect of electroacupuncture on the protein expression levels of Cyt-C, Caspase-3 and Caspase-9 in the bladder tissue of rats with neurogenic bladder after suprasacral spinal cord injury

Objective To observe the effect of electroacupuncture on the morphological change of the bladder tissue and the protein expression levels of cytochrome C, Caspase-3 and Caspase-9 in the bladder tissue of rats with neurogenic bladder after suprasacral spinal cord injury, and to preliminarily explore its partial mechanism of action. Methods Forty-eight female SD rats were randomly divided into blank group（group B）, sham operation group（group S）, model group（group M） and electroacupuncture group（group EA） according to random number table method with 12 rats in each group. Neurogenic bladder models after suprasacral spinal cord injury were established by adopting modified spinal cord transection method. Electroacupuncture intervention was conducted at ＂Cìliáo（次髎 BL 32）＂＂Zhōngjí（中极 CV 3）＂ ＂Sānyīnjiāo（三阴交 SP 6）＂ and ＂Dàzhuī（大椎 GV 14）＂ on the 19 th day after modeling. After treatment for 7 consecutive days, the rats were sacrificed, and the bladder tissues were removed rapidly for microscopic observation of morphological change after HE stain and for determination of the protein expression levels of cytochrome C, Caspase-3 and Caspase-9 via Western blot method. Results① Under light microscope after HE stain, injury in varying degrees in muscle fiber arrangement and the shape and size of cells, etc. of bladder detrusor of rats in group M and group EA was found, and the degree of injury of detrusor in group M was severer than that in group EA;② comparison of the protein expression levels of cytochrome C, Pro-Caspase-9 and activated Caspase-9 between group M as well as group EA and group B as well as group S： the protein expression levels of cytochrome C, Pro-Caspase-9 and activated Caspase-9 in bladder tissue of rats in group M and group EA obviously increased（ P〈0.01, P〈0.05）; comparison between group EA and group M： the protein expression levels of cytochrome C, Pro-Caspase-9 and activated Caspase-9 in bladder tissue of rats in group EA obviously reduced（ P〈0.05）;③ comparison of the protein expression level of Caspase-3 between group M as well as group EA and group B as well as group S： the protein expression level of Pro-Caspase-3（35 KD） in group M and group EA reduced, and the protein expression level of activated Caspase-3（17 KD/19 KD） obviously increased（ P〈0.01, P〈0.05）; comparison between group EA andgroup M： the protein expression level of Pro-Caspase-3（35 KD） in group EA obviously increased, and the protein expression level of activated Caspase-3（17 KD/19 KD） obviously reduced（ P〈0.05）. Conclusion suprasacral spinal cord injury can increase the protein expression levels of cytochrome C, activated Caspase-3 and Caspase-9 in bladder tissues of rats, and electroacupuncture at BL 32, CV 3, SP 6 and GV 14 can down-regulate the protein expression levels of cytochrome C, activated Caspase-3 and Caspase-9 in bladder tissues of rats after suprasacral spinal cord injury and protect bladder tissue, which may be one of the mechanisms that electroacupuncture protects the involved bladder detrusor after suprasacral spinal cord injury.

基于PACAP-cAMP信号通路研究电针治疗骶上脊髓损伤后逼尿肌反射亢进型膀胱的效应机制

Objective:To elucidate the underlying mechanism and effect of electroacupuncture(EA)on the neurogenic bladder following suprasacral spinal cord injury(ssCI).A rat model of detrusor hyperreflexia after SsCI was established to examine the urodynamics,detrusor muscle tissue morphology,the protein and mRNA expression levels of pituitary adenylate cyclase activating peptide(PACAP)and its receptor PAC1R,and cyclic adenosine monophosphate(cAMP)content in the detrusor muscle with a focus on the PACAPcAMP signaling pathway.Method:A total of 72 female SD rats were randomized into control group and sham operation group(n=12 per group)by using a random number table.The remaining 48 rats were established into the model of detrusor hyperreflexia after SsCI.After successful modeling,these rats were randomly assigned to model,EA,and EA+PACAP6-38 groups(n=12 per group).The unsuccessful modeled rats were used for exploratory observation.For the rats in EA group,"Ciliao(BL32)""Zhongji(CV3)",and"Sanyinjiao(SP6)"were needled and stimulated by EA.The PACAP receptor antagonist PACAP6-38 was administered intraperitoneally in the EA+PACAP6-38 group before EA,and EA was applied for seven consecutive days.After treatment,the urodynamics of the rats were analyzed,and hematoxylin and eosin staining was used to examine rat bladder detrusor tissue morphology.The expressions of PACAP-38 and PAC1R were detected by immunohistochemistry and Western blot.The mRNA expression levels of PACAP-38 and PAC1R were examined by RT-qPCR,while cAMP content was detected by ELISA.Results:(1)Compared with sham operation group,it was exhibited disarray in the transitional epithelium cells of the bladder in the modeled rats.The intercellular space was significantly widened,accompanied by inflammatory cell infiltration and noticeable tissue edema.Both the bladder initial pressure and leak point pressure of the rats were higher(P<0.01),whereas the maximum cystometric capacity and bladder compliance were lower(P<0.01).The protein and mRNA expression levels of PACAP-38 and PAC1R in the detrusor muscle,together with the cAMP content,were lower(P<0.05).(2)Compared with the model rats,the EA group showed reduced inflammatory response in the detrusor muscle tissue,with decreased monocyte infiltration and less severe tissue edema.The bladder smooth muscle cells exhibited increased integrity,and there was decreased cellular tissue edema,inflammatory cell infiltration,and fibroplasia.The bladder initial pressure and leak point pressure were lower(P<0.05),while the maximum cystometric capacity and bladder compliance were higher(P<0.01).The protein and mRNA expression levels of PACAP-38 and PAC1R in the detrusor muscle,along with the cAMP content,were higher(P<0.05).(3)Compared to the EA group,the EA+PACAP6-38 group showed a less organized arrangement of muscle fibers in the detrusor muscle tissue,larger intercellular space,monocyte infltration,and considerable tissue edema.The changes in bladder initial pressure and leak point pressure were not significant(P>0.05),while the maximum cystometric capacity and bladder compliance were lower(P<0.05).The changes in the protein and mRNA expressions of PACAP-38 within the detrusor muscle were not signifcant(P>0.05),whereas the protein and mRNA expressions of PAC1R were reduced(P<0.05),and the cAMP content within the detrusor muscle was lower(P<0.05).Conclusion:EA can ameliorate the uninhibited contractile condition of the detrusor muscle in the bladder following SSCI.By mediating the PACAP-cAMP signaling pathway,it reduces the pathological damage to the detrusor muscle,thereby improving bladder function.

Effect of Electroacupuncture on urodynamics, PACAP-38 and PAC1R of spinal cord in rats with neurogenic bladder

Objective： To investigate the effect and mechanism of electroacupuncture（EA） in the intervention of neurogenic bladder（NB） caused by suprasacral spinal cord injury（SCI）.Methods： The total 60 female Sprague-Dawley（SD） rats were divided into blank group, sham operation group by adopting random number table method, with 12 rats in each group. The remaining rats were made into models through operation, and 24 successful model rats were randomly divided into model group and EA group, with 12 rats in each group. Since the 19 th day after modeling, EA was conducted at ＂Cìliào＂（次髎BL 32）, ＂Zhōngjí＂（中极CV 3）,＂Sānyīnjiāo＂（三阴交SP 6） and ＂Dàzhuī＂（大椎GV 14） in EA group, remaining the needle for 20 min every time. The intervention was performed for once a day, and7 consecutive days were needed. The rest of the three groups were only tied up for the same time. Urodynamics test was conducted after the intervention. The contents of PACAP-38 and PAC1 R of the spinal cord of the injury site were detected by Western blot.Results： Compared with the blank group. there were no significant differences in the bladder capacity,leak point pressure, bladder compliance and PACAP-38 and PAC1 R contents in spinal cord in sham operation group; compared with the sham operation group, the maximum bladder capacity and the bladder compliance in the model group were lower（both P〈0.05）, the leak point pressure was higher（P〈0.05）,the contents of PACAP-38 and PAC1 R were lower（both P〈0.05）; compared with the model group, the maximum bladder capacity and the bladder compliance in the EA group were higher（both P〈0.05）, the leak point pressure was lower（P〈0.05）, the contents of PACAP-38 and PAC1 R were higher（both P〈0.05）.Conclusion： EA at BL 32, CV 3, SP 6 and GV 14 can improve the bladder function of NB rats of suprasacral SCI, and its mechanism may be related to the increase expression of PACAP-38 and PAC1 R protein in the injured spinal cord tissue.