基于PACAP-cAMP-PKA信号通路探讨J型针刀调节神经源性膀胱逼尿肌的效应机制

Study on the Effect of J-type Needle-knife Treatment on Neurogenic Bladder Detrusor Based on PACAP-cAMP-PKA S

目的:探讨J型针刀通过PACAP-cAMP-PKA信号通路改善脊髓损伤后神经源性膀胱大鼠逼尿肌的作用机制。方法:36只SD雄性大鼠随机分为6组:空白组、假手术组、模型组、J型针刀治疗组、治疗+Bupivacaine抑制剂组、治疗+H-89抑制剂组,各6只。采用脊髓横断法制作神经源性膀胱模型。空白组为正常SD大鼠;假手术组为切开相关部位组织,无脊髓切断;其余4组予以手术脊髓切断造模。J型针刀治疗组造模后第19天予J型针刀针刺大鼠次髎穴,2 d 1次,共治疗7次。干预结束后各组行HE染色观察膀胱逼尿肌组织形态变化,Elisa检测膀胱逼尿肌组织中c AMP、PKA含量,免疫组化检测膀胱逼尿肌组织中PACAP38蛋白表达,Western blot检测膀胱逼尿肌组织中PKA、p-MLC蛋白表达。结果:HE染色结果显示,与空白组相比,模型组中膀胱逼尿肌组织严重坏死,可见大量炎症细胞浸润;与模型组相比,J型针刀治疗组、治疗+Bupivacaine抑制剂组及治疗+H-89抑制剂组均有不同程度坏死组织修复情况。免疫组化检测PACAP38蛋白表达在模型组中表达量最低,针刀治疗之后,PACAP38表达量均不同程度上调;与J型针刀治疗组相比,治疗+Bupivacaine抑制剂组及治疗+H-89抑制剂组PACAP38蛋白表达均不同程度下调(P<0.05),但高于模型组(P<0.05)。Elisa检测结果显示,cAMP、PKA表达量模型组显著下调(P<0.05),J型针刀治疗组与模型组比显著上调(P<0.05),治疗+Bupivacaine抑制剂组及治疗+H-89抑制剂组比无显著差异(P>0.05)。WB结果显示:与空白组相比,模型组PKA、p-MLC蛋白表达上调;针刀治疗组表达量下调,其中PKA蛋白表达有显著性(P<0.05),p-MLC蛋白表达无显著性(P>0.05)。与J型针刀治疗组比,PKA蛋白表达量在治疗+Bupivacaine抑制剂组及治疗+H-89抑制剂组中无显著差异(P>0.05),p-MLC蛋白表达量显著下降(P<0.05)。结论:J型针刀针刺次髎穴可改善脊髓损伤后神经源性膀胱大鼠膀胱功能,其机制与J型针刀上调膀胱逼尿肌中PACAP38、cAMP、PKA表达,激活PACAP-cAMP-PKA信号通路,从而促进逼尿肌舒张有关。

Objective:To investigate the effect of J-type needle-knife on detrusor PACAP-cAMP-PKA signaling pathway in rats with neurogenic bladder after spinal cord injury.Methods:36 SD rats(male)were randomly divided into 6 groups with 6 rats in each group:blank group,sham operation group,model group,J-type acupotomy group,treatment+Bupivacaine inhibitor group,treatment+H-89 inhibitor group.Neurogenic bladder model was established by spinal cord transection.The blank group was normal SD rats.Sham operation group(relevant tissues were cut open without spinal cord amputation);The other 4 groups were treated with surgical spinal cord amputation.On the 19th day after modeling,the J-type acupotomy group was injected with J-type acupotomy at secondary scalene point of rats,once every 2 days,for a total of 7 treatments.After the intervention,HE staining was performed in each group to observe the morphological changes of the detrusor tissue of the bladder,cAMP and PKA contents in the detrusor tissue of the bladder were detected by Elisa,PACAP38 protein expression in the detrusor tissue of the bladder was detected by immunohistochemistry,and PKA and p-MLC protein expression in the detrusor tissue of the bladder was detected by Western blot.Results:HE staining showed that,compared with the blank group,the detrusor tissue in the model group was severely necrotic and a large number of inflammatory cells could be seen.Compared with the model group,the J-type acupotomy group,the treatment+Bupivacaine inhibitor group and the treatment+H-89 inhibitor group had different degrees of necrotic tissue repair.Immunohistochemical test showed the lowest expression of PACAP38 protein in the model group.After acupotomy,the expression of PACAP38 was up-regulated to varying degrees.Compared with the J-type acupotomy group,PACAP38 protein expression in treatment+Bupivacaine inhibitor group and treatment+H-89 inhibitor group was down-regulated in different degrees(P<0.05),but higher than that in model group(P<0.05).Elisa results showed that the expression levels of cAMP and PKA were significantly down-regulated in the model group(P<0.05),and significantly up-regulated in the J-type acupotomy group compared with the model group(P<0.05).There was no significant difference in the ratio of treatment+Bupivacaine inhibitor group and treatment+H-89 inhibitor group(P<0.05).WB results showed that compared with blank group,the expressions of PKA and p-MLC proteins in model group were up-regulated.In the acupotomy group,the expression of PKA protein was significantly decreased(P<0.05),but P-mlC protein was not significantly decreased(P>0.05).Compared with J-type acupotomy group,PKA protein expression level was not significantly different in treatment+Bupivacaine inhibitor group and treatment+H-89 inhibitor group(P>0.05),while P-mlC protein expression level was significantly decreased(P<0.05).Conclusions: J-type needle-knife acupuncture at Ciliu point can improve the bladder function of neuro-induced bladder rats after spinal cord injury,and its mechanism is related to up-regulating the expression of PACAP38,cAMP and PKA in the detrusor muscle of bladder,activating the PACAP-cAMP-PKA signaling pathway,so as to promote the relaxation of detrusor muscle.