基于NGF-p38MAPK信号通路探讨盆痛灵方逆转EMs疼痛的分子机制

Pain is a main symptom of Endometriosis, It is seriously and a tough problem for clinical treatment.the pathogenesis is also complex. NGF regulate p38MAPK signaling pathway of DRG,TRPV1 expression increased, It may participate in the pain of EMs. We form PenTongLing compound by Activate blood, clear heat and wet. Previous clinical study showed that treating EMs pain with PenTongLing compound has a significant effect, animal experiments showed using this compound by enema can significantly increase the pain domain in mice, reduce the level of NGF. Based on the above two points, We put forward the hypothesis " PenTongLing may through contraversy p38MAPK signaling pathway, affect TRPV1 expression, inhibition the pain of Endometriosis ". we will measure the pain domain of EMs rat model，the expression of related signaling molecules and transduction protein like NGF、p38、TRPV1 will detected by immunohistological labeling and molecular biology, transmission electron microscopy will be used to asses the EMs lesions ultrastructure. At the same time, treat the DRG cell invitro with PenTongLing medicated serum at different times，to explore the process of NGF-p38MAPK-TRPV1 signal pathway in EMs pain,and the role of PenTongLing compound in this signal pathway,in order to provide hard scientific evidence for Chinese medicine prevent and treat pain associated with Endometriosis.

子宫内膜异位症（EMs）相关疼痛临床治疗困难，发病机制复杂，NGF调控DRG内p38MAPK信号通路，使TRPV1表达增强可能参与EMs疼痛的发病。本课题组前期以活血化瘀，清热利湿法自拟盆痛灵灌肠，临床对EMs疼痛患者效果明显，并可显著提高小鼠痛阈，降低NGF水平。基于以上两点，我们提出“盆痛灵方可能通过NGF-p38MAPK信号通路，影响TRPV1表达，抑制内异症疼痛”的假说。拟通过建立SD大鼠EMs模型,鞘内注射NGF、p38MAPK抑制剂；不同剂量盆痛灵灌肠，观察模型大鼠行为学及内异灶结构变化；另盆痛灵含药血清干预离体DRG细胞，Western blot、PCR法检测NGF/p-p38MAPK/TRPV1等基因及蛋白的表达，研究NGF-p38MAPK-TRPV1信号通路在EMs疼痛中的作用，探讨盆痛灵方通过该通路逆转EMs疼痛的分子机制，为中医药治疗EMs疼痛提供新的、确切的科学依据

子宫内膜异位症（EMs）相关疼痛临床治疗困难，发病机制复杂，前期研究表明盆痛灵临床取得较好疗效。课题组提出“盆痛灵方可能通过NGF-p38MAPK信号通路调控TRPV1表达，逆转EMs疼痛”的假说，研究通过体内实验，构建SD大鼠Ems模型，鞘内注射NGF诱导剂，模型大鼠痛阈明显下降，同时腹腔液及血清中NGF、TNF-ɑ、IL-1β的水平升高，内异灶及背根神经节内NGF及其受体TrkA，p75NTR以及p38MAPK、TRPV1的表达增加，与正常组比较差异有统计学意义（P＜0.05）；鞘内注射NGF抑制剂、p38MAPK抑制剂可以明显提高大鼠痛阈，腹腔液及血清中NGF、TNF-ɑ、IL-1β的水平下降，内异灶及DRG内NGF及其受体TrkA，p75NTR以及p38MAPK、TRPV1的表达下调，差异有统计学意义（P＜0.05）；通过不同剂量盆痛灵方灌肠治疗，各组均能改善大鼠的疼痛行为学，能缩小内异灶体积，降低大鼠血清及腹腔液内NGF及炎症因子TNF-ɑ、IL-1β的水平；下调内异灶、DRG内NGF及其受体trkA、p75NTR、p-p38、TRPV1的基因及蛋白表达量，差异具有统计学意义（P＜0.05）；不同剂量盆痛灵方以高剂量组镇痛效果最优；体外实验采用盆痛灵中药提取液干预培养DRG细胞，不同时间干预以24小时干预时间为最佳；同时，不同浓度的盆痛灵方干预DRG细胞后，细胞内NGF、trkA、p75NTR、p38、TRPV1的基因及蛋白表达均明显下降，差异具有统计学意义（p<0.001）。通过体内、体外研究结果表明：NGF通过调控p38MAPK信号通路，使TRPV1表达增强参与EMs疼痛的发病。盆痛灵方的镇痛机理可能是干预了NGF与其受体结合激活p38通路的过程，通过抑制p38激活，调控下游信号分子TRPV1的表达，降低内异灶周围腹腔液及血清中炎症因子TNF-ɑ、IL-1β的表达，从而逆转EMs疼痛，盆痛灵从多环节、多靶点起到镇痛作用，为中医药治疗EMs疼痛提供新的、确切的科学依据。