加味盆痛灵方对子宫内膜异位症模型大鼠TRPV1/ERK1/2/p-CREB通路的影响

Effect of modified Pentongling Formula on TRPV1/ERK1/2/p-CREB pathway in endometriosis model rats

目的:基于瞬时受体电位香草酸亚型1/细胞外信号调节激酶1/2/磷酸化环磷酸腺苷反应元件结合蛋白(TRPV1/ERK1/2/p-CREB)信号通路探讨加味盆痛灵方对子宫内膜异位症(EMs)疼痛模型大鼠的镇痛机制。方法:采用自体内膜移植法建立SD大鼠EMs疼痛模型。造模成功的大鼠采用完全随机设计法分为模型组、TRPV1抑制剂组(CPZ组)、加味盆痛灵方组(JWPTL组)和TRPV1抑制剂+加味盆痛灵方组(CPZ+JWPTL组),每组8只;另设假手术组(n=8)。JWPTL组和CPZ+JWPTL组大鼠自造模成功后第1天起给予7.77 g/kg加味盆痛灵方灌肠,1次/d,其余大鼠灌肠给予等体积0.9%NaCl溶液,连续干预28 d。CPZ组和CPZ+JWPTL组自造模成功后第21天起鞘内注射辣椒平(CPZ),10μL/d,1次/d;其余大鼠鞘内注射等体积助溶剂10%二甲基亚砜(DMSO),连续干预7 d。采用“Up&Down”法测量大鼠的50%机械缩足反应阈值(PMWT),Western blot和RT-qPCR法检测脊髓组织中TRPV1、ERK1/2、p-CREB蛋白和mRNA的表达水平,ELISA法观察即刻早期基因c-fos蛋白的表达情况。结果:给药前,与假手术组比较,各造模组大鼠50%PMWT均明显降低(P<0.05);给药28 d后,CPZ组、JWPTL组和CPZ+JWPTL组50%PMWT较模型组显著升高(P<0.05),CPZ+JWPTL组50%PMWT较CPZ组显著升高(P<0.05)。模型组TRPV1、ERK1/2、CREB蛋白和mRNA的表达水平较假手术组显著升高(P<0.01,P<0.001),模型组c-fos蛋白表达水平较假手术组亦显著升高(P<0.001);与模型组比较,TRPV1抑制剂CPZ和加味盆痛灵方可不同程度下调TRPV1、ERK1/2、CREB蛋白和mRNA表达水平(P<0.05,P<0.01),且CPZ+JWPTL组效果最优。与模型组比较,JWPTL组和CPZ+JWPTL组c-fos蛋白表达水平明显降低(P<0.001)。相关性分析显示,TRPV1、ERK1/2、CREB蛋白和mRNA表达水平及c-fos蛋白表达水平均与50%PMWT呈负相关。结论:加味盆痛灵方可能通过干预TRPV1/ERK1/2/p-CREB通路及c-fos的表达缓解EMs疼痛。

Objective:To explore the analgesic mechanism of modified Pengtongling Formula on endometriosis(EMs)pain rats based on the transient receptor potential vanillic acid subtype 1/extracellular signal regulated kinase 1/2/phosphorylated cyclic adenosine monophosphate responsive element binding protein(TRPV1/ERK1/2/p-CREB)signaling pathway.Method:A rat EMs pain model was established by autologous endometrial transplantation.The successfully modeled rats were divided into model group,TRPV1 inhibitor group(CPZ group),modified Pentongling Formula group(JWPTL group),and TRPV1 inhibitor+modified Pentongling Formula group(CPZ+JWPTL group)by a completely randomized design method,with 8 rats in each group;another sham surgery group was set up(n=8).The rats in JWPTL group and CPZ+JWPTL group were given 7.77 g/kg modified Pentongling Formula enema once a day from the first day after successful modeling,and the other rats were given equal volume 0.9%NaCl solution for continuous intervention for 28 d.CPZ group and CPZ+JWPTL group received intrathecal injection of capsazepine(CPZ),10μL/d,once a day from the 21st day after successful modeling;The other rats were injected intrathecally with 10%dimethyl sulfoxide(DMSO),which was treated continuously for 7 d.The"Up&Down"method was used to measure the 50%paw mechanical withdrawal threshold(PMWT)of rats.Western blot and RT-qPCR methods were used to detect the expression levels of TRPV1,ERK1/2,p-CREB proteins and mRNA in spinal cord tissues.ELISA method was used to observe the expression of the immediate early gene c-fos protein.Results:Before administration,compared with the sham surgery group,the 50%PMWT of rats in each modeling group was significantly reduced(P<0.05).After 28 d of administration,compared with the model group,the 50%PMWT of the CPZ group,JWPTL group,and CPZ+JWPTL group was significantly increase(P<0.05);Compared with the CPZ group,the 50%PMWT of the CPZ+JWPTL group was significantly increase(P<0.05).Compared with the sham surgery group,the protein and mRNA expression levels of TRPV1,ERK1/2 and CREB in the model group was significantly increased(P<0.01,P<0.001),and the expression level of c-fos protein in the model group was also significantly increased(P<0.001);Compared with the model group,TRPV1 inhibitors CPZ and modified Pentongling Formula could down-regulate the protein and mRNA expression levels of TRPV1,ERK1/2 and CREB in different degrees(P<0.05,P<0.01),and CPZ+JWPTL group has the best effect.Compared with the model group,the expression level of c-fos protein was significantly decreased.The JWPTL group and CPZ+JWPTL group(P<0.001).Correlation analysis showed that the expression levels of TRPV1,ERK1/2,CREB protein and mRNA as well as c-fos protein were negatively correlated with 50%PMWT.Conclusion:Modified Pengtongling Formula may alleviate EMs pain by intervening in the TRPV1/ERK1/2/p-CREB pathway and the expression of c-fos.