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
- 上海中医药大学学报 2024年38卷第3期 页码：50-56
- 作者机构：
1.上海中医药大学龙华临床医学院（上海 200032）
2.上海中医药大学附属龙华医院妇科（上海 200032）
- 作者简介：
刘晓庆，女，在读硕士生，主要从事子宫内膜异位症的基础研究
付金荣，教授、主任医师，博士生导师；E-mail：fujinrong2006@sina.com
- 基金信息：国家自然科学基金资助项目(81674009)
DOI：10.16306/j.1008-861x.2024.03.008
中图分类号：纸质出版日期：2024-05-25，
收稿日期：2023-05-18，
修回日期：2024-03-28，
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刘晓庆,余思云,夏玉等.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[J].上海中医药大学学报,2024,38(03):50-56.
LIU Xiaoqing,YU Siyun,XIA Yu,et al.Effect of modified Pentongling Formula on TRPV1/ERK1/2/p⁃CREB pathway in endometriosis model rats[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2024,38(03):50-56.
刘晓庆,余思云,夏玉等.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[J].上海中医药大学学报,2024,38(03):50-56. DOI： 10.16306/j.1008-861x.2024.03.008.
LIU Xiaoqing,YU Siyun,XIA Yu,et al.Effect of modified Pentongling Formula on TRPV1/ERK1/2/p⁃CREB pathway in endometriosis model rats[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2024,38(03):50-56. DOI： 10.16306/j.1008-861x.2024.03.008.
摘要
目的
2
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
2
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），10 μL/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
2
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
2
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。
Abstract
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
2
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）， 10 μL/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
2
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
2
Modified Pengtongling Formula may alleviate EMs pain by intervening in the TRPV1/ERK1/2/p-CREB pathway and the expression of c-fos.
关键词
加味盆痛灵方子宫内膜异位症疼痛TRPV1/ERK1/2/p-CREB通路c-fos
Keywords
modified Pentongling FormulaendometriosispainTRPV1/ERK1/2/p-CREB pathwayc-fos
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