基于PI3K/Akt信号通路探讨加味盆痛灵方对子宫内膜异位症模型大鼠的镇痛作用机制

Exploring mechanism of modified Pentongling Formula on analgesic in endometriosis model rats based on PI3K/Akt signaling pathway

目的:基于磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路探讨加味盆痛灵方对子宫内膜异位症(EMs)模型大鼠的镇痛作用机制。方法:采用自体子宫内膜移植法建立EMs大鼠模型,用“Up&Down”法筛选有疼痛行为的EMs大鼠,随机分为模型组及加味盆痛灵低、中、高剂量组(n=8);另设假手术组8只。假手术组、模型组大鼠给予0.9%NaCl溶液灌肠,加味盆痛灵各组给予不同剂量的加味盆痛灵方灌肠,连续灌肠4周并观察大鼠50%机械缩足反应阈值(50%PMWT)的变化。HE染色观察大鼠子宫内膜组织形态学变化,免疫组化法测定内异灶PI3K和Akt蛋白表达水平,ELISA法检测腹腔液雌二醇(E_(2))、血清脑源性神经营养因子(BDNF)的浓度,RT-qPCR法检测内异灶PI3K和Akt mRNA表达水平。结果:①各造模组大鼠造模后50%PMWT均明显降低(P<0.05)。给药4周后,与模型组比较,加味盆痛灵各组50%PMWT均明显升高(P<0.001)。②HE染色结果显示,模型组内膜组织上皮细胞呈扁平状,腺体明显减少甚至消失,血管丰富,间质有炎性细胞浸润,各剂量加味盆痛灵方均可不同程度改善内膜组织病理形态,以高剂量效果最佳。③与假手术组比较,模型组E_(2)、BDNF表达上调(P<0.01,P<0.001);与模型组比较,加味盆痛灵各组E_(2)、BDNF表达下调(P<0.05,P<0.001);④与假手术组比较,模型组PI3K、Akt mRNA及蛋白表达上调(P<0.05,P<0.01);与模型组比较,加味盆痛灵各剂量组Akt蛋白表达下调(P<0.05,P<0.01),加味盆痛灵高剂量组Akt mRNA表达下调(P<0.05),加味盆痛灵中、高剂量组PI3K mRNA及蛋白表达下调(P<0.05,P<0.01)。(5)相关性分析显示,E_(2)、BDNF表达和PI3K、Akt mRNA及蛋白表达均与50%PMWT呈负相关。结论:加味盆痛灵方可能通过抑制E_(2)调控PI3K/Akt信号通路缓解EMs疼痛。

Objective: To explore the mechanism of modified Pentongling Formula on analgesic in endometriosis(EMs) model rats based on phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt) signaling pathway. Methods: The rat model of EMs was established by autologous endometrial transplantation. The EMs rats with pain behavior were chosen by the “Up & Down” method and randomly divided into model group, modified Pentongling low, medium and high dose group(n=8);and the sham group was set with 8 rats. The sham group and model group were given 0.9% NaCl solution enema, and each modified Pentongling group was given different doses of modified Pentongling Formula enema. After continuous enema for 4 weeks, the changes of 50% paw mechanical withdrawal threshold(50% PMWT) in rats were observed. HE staining was used to observe the pathological changes of the endometrium in rats, immunohistochemistry was used to detect the protein expression levels of PI3K and Akt in the ectopic lesion, ELISA was used to detect the concentrations of estradiol(E_(2)) in the peritoneal fluid and brain-derived neurotrophic factor(BDNF) in the serum, and RT-qPCR was used to detect the mRNA expression levels of PI3K and Akt in the ectopic lesion. Results:① After modeling, the 50% PMWT of rats in each modeling group significantly decreased(P<0.05). Compared with the model group, the 50% PMWT of the modified Pentongling groups significantly increased after 4weeks administration(P<0.001).② HE staining showed that the epithelial cells were flat, the glands significantly decreased or even disappeared, blood vessels were abundant, and there was inflammatory cell infiltration in the stroma in the endometrial tissue of the model group. Each dose of modified Pentongling could improve the pathological morphology of endometrial tissue to different degrees, and the high dose had the best effect.③Compared with the sham group, the expression of E_(2), BDNF was up-regulated in the model group(P<0.01, P<0.001). Compared with the model group, E_(2)and BDNF expression was down-regulated in modified Pentongling each group(P<0.05, P<0.001).④Compared with the sham group, PI3K and Akt mRNA and protein expressions in the model group was up-regulated(P<0.05, P<0.01). Compared with the model group, the Akt protein expression was down-regulated in modified Pentongling each dose group(P<0.05, P<0.01), the Akt mRNA expression was down-regulated in modified Pentongling high dose group(P<0.05), and the PI3K mRNA and protein expressions were down-regulated in modified Pentongling medium and high dose groups(P<0.05, P<0.01).(5)Correlation analysis showed that the expression of E_(2), BDNF and the mRNA and protein expression of PI3K and Akt were negatively correlated with 50%PMWT. Conclusion: Modified Pentongling Formula may relieve EMs pain by inhibiting E_(2)to regulate PI3K/Akt signaling pathway.