基于miR-221介导的细胞自噬探讨补肾温阳化瘀方治疗肾阳虚血瘀型子宫内膜异位症的作用机制

Mechanism of Bushen Wenyang Huayu Formula in treating endometriosis of kidney yang deficiency and blood stasis based on miR-221 mediated autophagy

目的:探讨补肾温阳化瘀方治疗大鼠肾阳虚血瘀型子宫内膜异位症(EMs)的疗效及可能的作用机理。方法:将100只实验大鼠随机分为假手术组、模型组、中药高剂量组、中药低剂量组和西药组,每组20只,肾阳虚血瘀组EMs造模成功后分别给予补肾温阳化瘀方高、低剂量及孕三烯酮灌胃,假手术组和模型组纯净水灌胃,连续3周。Western blot检测Beclin-1、P62蛋白表达;RT-PCR检测miR-221及Beclin-1、P62 mRNA水平。结果:与假手术组比较,模型组在位内膜的Beclin-1蛋白及mRNA水平显著升高(P<0.01,P<0.05),P62蛋白及mRNA水平显著下降(P<0.01,P<0.05),miR-221水平显著升高(P<0.01)。与模型组比较,中药高、低剂量组在位内膜及异位灶的Beclin-1蛋白及mRNA水平显著下降(P<0.05),P62蛋白及mRNA水平显著升高(P<0.01,P<0.05),miR-221水平显著下降(P<0.01,P<0.05)。miR-221与Beclin-1 mRNA呈现正相关,与P62 mRNA呈现负相关,相关系数(r)分别为0.515、-0.796(P<0.05,P<0.01)。结论:补肾温阳化瘀方能够通过调节自噬基因Beclin-1、P62活性从而抑制细胞自噬水平,其机制可能与在转录水平下调miR-221有关。

Objective:To investigate the therapeutic effect and possible mechanism of Bushen Wenyang Huayu Formula on endometriosis of kidney yang deficiency and blood stasis in rats.Methods:One hundred experimental rats were randomly divided into sham operation group,model group,high-dose group,low-dose group and Western medicine group,20 rats in each group.After successful modeling of endometriosis of kidney yang deficiency and blood stasis,high and low doses of Bushen Wenyang Huayu Formula and gestrinone were given by gavage,and pure water was given by gavage in sham operation and model group for 3 weeks.The protein expressions of Beclin-1 and P62 were detected by Western blot.The expressions of miR~221,Beclin-1 mRNA and P62 mRNA were detected by RT-PCR.Results:Compared with sham operation group,the levels of Beclin-1 protein and mRNA in eutopic endometrium in the model group increased significantly(P<0.01,P<0.05),the levels of P62 protein and mRNA decreased significantly(P<0.01,P<0.05),and the level of miR-221 increased significantly(P<0.01).Compared with model group,the levels of Beclin-1 protein and mRNA decreased(P<0.05),P62 protein and mRNA increased(P<0.01,P<0.05)and miR-221 decreased(P<0.01,P<0.05)in eutopic endometrium and ectopic lesions in high and low dose groups with statistical significance(P<0.01,P<0.05).MiR-221 was positively correlated with Beclin-1 mRNA and negatively correlated with P62 mRNA.The correlation coefficients(r)were 0.515 and-0.796,respectively(P<0.05,P<0.01).Conclusion:Bushen Wenyang Huayu Formula can inhibit the level of autophagy by regulating the activities of autophagy genes Beclin-1 and P62.Its mechanism may be related to down-regulate miR-221 at the transcriptional level.