文献类型：期刊文献

中文题名：加味少腹逐瘀汤调控EGFR/PI3K/AKT信号通路诱导子宫内膜细胞自噬的作用机制

英文题名：The mechanism of modified Shaofu Zhuyu Decoction regulating the EGFR/PI3K/AKT signaling pathway to induce autophagy in endometrial cells

作者：杨亚玲[1];王婉润[2];张作良[2];王家兴[2];林祥羽[2];武权生[2]

第一作者：杨亚玲

机构：[1]甘肃中医药大学附属医院,兰州730000;[2]甘肃中医药大学

第一机构：甘肃中医药大学第二附属医院

年份：2024

卷号：47

期号：9

起止页码：1191

中文期刊名：北京中医药大学学报

外文期刊名：Journal of Beijing University of Traditional Chinese Medicine

收录：CSTPCD;;北大核心:【北大核心2023】；CSCD:【CSCD2023_2024】；

基金：国家自然科学基金项目(No.82260949);甘肃省自然科学基金项目(No.23JRRA1199);甘肃省中医药科研项目(No.GZKP-2022-22);兰州市科技计划项目(No.2021-1-97);甘肃中医药大学附属医院2021院内一般项目(No.gzfy-2021-03)。

语种：中文

中文关键词：子宫内膜异位症;加味少腹逐瘀汤;表皮生长因子受体(EGFR)/磷酸肌醇3-激酶(PI3K)/蛋白激酶B(AKT)信号通路;自噬;炎症;大鼠

外文关键词：endometriosis;modified Shaofu Zhuyu Decoction;epidermal growth factor receptor(EGFR)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway;autophagy;inflammation;rats

摘要：目的从表皮生长因子受体(EGFR)/磷酸肌醇3-激酶(PI3K)/蛋白激酶B(AKT)信号通路介导异位子宫内膜组织细胞自噬角度探讨加味少腹逐瘀汤治疗子宫内膜异位症的潜在分子机制。方法72只雌性SD大鼠按照随机数字表法分为空白组、模型组、孕三烯酮组及加味少腹逐瘀汤高、中、低剂量组,每组12只。采用自体移植法构建子宫内膜异位症模型。孕三烯酮组大鼠灌胃孕三烯酮混悬液0.25 mg/(kg·d),加味少腹逐瘀汤高、中、低剂量组大鼠分别灌胃加味少腹逐瘀汤水煎剂30、15、7.5 g/(kg·d),空白组及模型组大鼠灌胃等量生理盐水,连续4周。给药结束后,每只大鼠给予缩宫素2 U诱发宫缩,观察大鼠疼痛情况;记录大鼠异位子宫内膜组织的质量和体积;苏木素-伊红(HE)染色法观察各组大鼠子宫内膜组织病理变化;透射电镜观察各组大鼠子宫内膜组织超微结构;酶联免疫吸附测定法检测各组大鼠血清中雌二醇(E2)、孕酮(P)、白细胞介素-1β(IL-β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、转化生长因子-β(TGF-β)、表皮生长因子(EGF)、EGFR含量;免疫荧光法检测各组大鼠子宫内膜组织中重组人自噬效应蛋白Beclin 1(Beclin-1)、微管相关蛋白1轻链3(LC3B)平均荧光强度;蛋白质印迹法检测各组大鼠子宫内膜组织中EGFR、PI3K、磷酸化磷酸肌醇3-激酶(p-PI3K)、AKT、磷酸化蛋白激酶B(p-AKT)的蛋白表达水平;实时荧光PCR法检测EGFR、PI3K、AKT mRNA表达水平。结果与空白组比较,模型组大鼠出现典型扭体反应,腹壁可见异位子宫内膜组织,HE结果显示,异位内膜组织增厚,间质增生,腺体扩张;超微结构仅见自噬小体,部分视野未见明显自噬结构;大鼠血清E2、IL-6、IL-1β、TNF-α、TGF-β、EGF、EGFR含量升高,P含量降低;自噬相关蛋白Beclin-1、LC3B平均荧光强度降低;EGFR、PI3K、AKT、p-PI3K、p-AKT蛋白表达升高;EGFR、PI3K、AKT mRNA表达升高(均P<0.05)。与模型组比较,孕三烯酮组及加味少腹逐瘀汤高、中剂量组大鼠扭体次数减少、扭体反应潜伏时间延长;异位子宫内膜组织体积及质量减小;HE结果显示,病理损伤程度减轻;超微结构不同程度出现溶酶体、自噬溶酶体、自噬小体结构;血清E2、IL-6、IL-1β、TNF-α、TGF-β、EGF、EGFR含量降低,P含量升高;自噬相关蛋白Beclin-1、LC3B平均荧光强度升高;EGFR、PI3K、AKT、p-PI3K、p-AKT蛋白表达降低;EGFR、PI3K、AKT mRNA表达降低(均P<0.05)。结论加味少腹逐瘀汤治疗子宫内膜异位症的机制可能与诱导EGFR/PI3K/AKT信号通路介导的异位子宫内膜组织自噬、减轻炎性反应有关。
Objective To investigate the potential molecular mechanism of modified Shaofu Zhuyu Decoction in treating endometriosis from the perspective of autophagy in ectopic endometrial tissue cells mediated by the epidermal growth factor receptor(EGFR)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway.Methods Seventy-two female SD rats were divided into the blank group,the model group,the gestrinone group and the modified Shaofu Zhuyu Decoction high-,medium-,and low-dose groups according to the random number table method,12 rats in each group.Construction of an endometriosis model used auto-transplantation method.The rats in the gestrinone group were gavaged with 0.25 mg/(kg·d)of gestrinone suspension,the rats in the modified Shaofu Zhuyu Decoction high-,medium-,and low-dose groups were gavaged with 30,15,and 7.5 g/(kg·d)of modified Shaofu Zhuyu Decoction,respectively,and the rats in the blank and model groups were gavaged with an equal amount of physiological saline,respectively.4 weeks of continuous treatment.At the end of drug administration,2 U of oxytocin was given to each rat to induce contractions,and the pain of the rats was observed;the weight and volume of ectopic endometrial tissue of the rats were recorded.Hematoxylineosin(HE)staining method was used to observe the pathological changes of rat endometrial tissue in each group;transmission electron microscope was used to observe the ultrastructure of rat endometrial tissue.Enzyme-linked immunosorbent assay was used to detect the serum levels of oestradiol(E2),progesterone(P),interleukin-1β(IL-β),interleukin-6(IL-6),tumour necrosis factor-α(TNF-α),transforming growth factor-β(TGF-β),epidermal growth factor(EGF),and EGFR in the rat serum of each group.Mean fluorescence intensity of autophagy-associated protein yeast Atg6 homologue(Beclin-1)and microtubule-associated protein 1 light chain 3(LC3B)in endometrial tissues of rat endometrium in each group by immunofluorescence assay.Protein expression levels of EGFR,PI3K,phosphorylated phosphatidylinositol 3-kinase(p-PI3K),AKT,and phosphorylated protein kinase B(p-AKT)in endometrial tissues of rats in each group were examined by protein blotting.Real-time fluorescence PCR was used to detect EGFR,PI3K,and AKT mRNA expression levels.Results Compared with the blank group,the rats in the model group showed typical torsion reaction,ectopic endometrial tissue was visible in the abdominal wall,and the HE result showed thickening of the ectopic endometrial tissue,mesenchymal hyperplasia,and glandular dilatation;only autophagic vesicles were seen in the ultrastructure,and no obvious autophagic structures were seen in some of the fields of view;Serum E2,IL-6,IL-1β,TNF-α,TGF-β,EGF,and EGFR levels were elevated and P levels were decreased in rats;autophagy-related proteins Beclin-1 and LC3B decreased in average fluorescence intensity;protein expressions of EGFR,PI3K,AKT,p-PI3K,p-AKT was elevated;and mRNA expressions of EGFR,PI3K,AKT was elevated(P<0.05).Compared with the model group,the gestrinone group and modified Shaofu Zhuyu Decoction high-,medium-dose groups showed a decrease in the number of torsion and a prolongation of the latency time of the torsion response,a decrease in the volume and mass of ectopic endometrial tissues,a decrease in the degree of pathological damage as shown by HE,and the appearance of lysosomes,autophagolysosomes and autophagic vesicles in different degrees in the ultrastructure;Serum E2,IL-6,IL-1β,TNF-α,TGF-β,EGF,EGFR levels were decreased,and P levels were increased;the mean fluorescence intensity of autophagy-related proteins Beclin-1 and LC3B was increased;the protein expression of EGFR,PI3K,AKT,p-PI3K,and p-AKT was decreased;and the mRNA expression of EGFR,PI3K,and AKT was decreased(P<0.05).Conclusion The mechanism of modified modified Shaofu Zhuyu Decoction for the treatment of endometriosis may be related to the induction of autophagy of ectopic endometrial tissues mediated by the EGFR/PI3K/AKT signaling pathway and the attenuation of inflammatory responses.