乌丹丸对寒凝血瘀子宫内膜异位大鼠PI3K/Akt/mTOR自噬信号轴及相关分子表达的影响

Effects of Wudan Pills on the expression of PI3K/Akt/mTOR autophagy signaling pathway and related molecules in endometriosis rats with cold congelation and blood stasis

目的探讨乌丹丸对寒凝血瘀型子宫内膜异位症(endometriosis,EMs)大鼠自噬信号轴磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB又称Akt)/雷帕霉素靶分子(mammalian target of rapamycin,mTOR)及相关分子表达的影响。方法随机将49只雌性SD大鼠分为假手术组、模型组、乌丹丸高、中、低剂量组、散结镇痛胶囊组、地诺孕素组,每组7只,采用冰水浴联合自体子宫内膜移植法建立寒凝血瘀EMs大鼠模型。假手术组及模型组每天给予10 mL/kg生理盐水灌胃,乌丹丸高、中、低剂量组分别给予2.4 g/kg、1.2 g/kg、0.6 g/kg灌胃,散结镇痛胶囊组每天给予0.48 g/kg灌胃,地诺孕素组每天给予0.2 mg/kg灌胃,各组持续用药28日。应用蛋白免疫印迹法和免疫组化法检测异位内膜组织自噬信号轴PI3K、p-Akt、p-mTOR蛋白以及相关自噬分子LC3、Beclin-1蛋白表达。结果与假手术组比较,模型组大鼠异位内膜组织PI3K/Akt/mTOR蛋白表达上调,Beclin-1、LC3蛋白表达下调(P<0.01)。中药干预后,乌丹丸高、中剂量组、散结镇痛胶囊组和地诺孕素组异位内膜上PI3K/Akt/mTOR蛋白表达降低,而Beclin-1、LC3蛋白表达升高,与模型组比较差异有统计学意义(P<0.01,P<0.05)。结论PI3K/Akt/mTOR信号通路参与子宫内膜异位症发生,乌丹丸可能通过下调PI3K/Akt/mTOR通路蛋白的表达,激活自噬相关蛋白LC3、Beclin-1表达,促进细胞自噬,治疗子宫内膜异位症。

Objective To investigate the effect of Wudan pill on phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)/target of rapamycin(mTOR)autophagy signaling pathway and related molecules in rats with endometriosis(EMs)of cold congelation and blood stasis.Methods Forty-nine female SD rats were randomly divided into the sham operation group,model group,high-dose,medium-dose and low-dose Wudan pill groups,Sanjie Zhentong capsule group,and dienogest group,seven in each group.The cold coagulation and blood stasis EMs rat model was established by ice water bath combined with autologous endometrium transplantation,and were given Wudan Pill high-dose,medium-dose and low-dose(2.4 g/kg,1.2 g/kg,0.6 g/kg),the sham operation group and the model group were given normal saline at the rate of 10 mL/kg per day,the Sanjie Zhentong capsule group was given at the rate of 0.48 g/kg per day,and the dienogest group was given at the rate of 0.2 mg/kg per day.Each group was treated for 28 days.The method of western blot and immunohistochemistry were used to detect the expression of autophagy signal axis PI3K,p-Akt,p-mTOR proteins and related autophagy molecules LC3 and beclin-1 proteins in ectopic endometrial tissues.Results Compared with the sham operation group,the expression of PI3K/Akt/mTOR protein in the model group was up-regulated,and the expressions of Beclin-1 and LC3 protein were down-regulated(P<0.01).After TCM intervention,the expression of PI3K/Akt/mTOR protein in the ectopic endometrium of the high dose groups,the medium dose groups,the Sanjie Zhentong capsule group and the dienogest group were decreased,while the expressions of Beclin-1 and LC3 protein were increased,and the difference was statistically significant compared with the model group(P<0.01,P<0.05).Conclusion PI3K/Akt/mTOR signaling pathway is involved in the occurrence of endometriosis.Wudan pill may promote autophagy and treat endometriosis by down-regulating the expression of PI3K/Akt/mTOR pathway proteins,activating the expression of autophagy related proteins LC3 and Beclin-1.