摘要
目的:探讨Wnt1/β-连环蛋白(β-catenin)信号通路在补阳还五汤(BYHWT)含药血清干预人肺动脉内皮细胞(HPAECs)内皮间质转化(EndMT)过程中的作用及其相关机制。方法:常法煎煮BYHWT,分别以53.36 g·kg^(-1)·d^(-1) BYHWT和等体积生理盐水灌胃新西兰兔制备含药血清和空白血清。体外培养HPAECs,采用转化生长因子-β1(TGF-β1)诱导建立EndMT模型。设立空白组(10%空白血清)、模型组(TGF-β1+10%空白血清)、BYHWT低剂量组(TGF-β1+2.5%含药血清+7.5%空白血清)、BYHWT中剂量组(TGF-β1+5%含药血清+5%空白血清)和BYHWT高剂量组(TGF-β1+10%含药血清)5个组,采用蛋白免疫印迹法(Western blot)检测Wnt1、β-catenin和糖原合成激酶-3β(GSK-3β)蛋白的表达。为进一步明确Wnt1/β-catenin信号通路在BYHWT含药血清干预EndMT过程中的机制,构建β-catenin过表达质粒转染至HPAECs,经聚合酶链式反应确认β-catenin过表达后,采用前期研究效果最优的10%含药血清进行干预,另设空白组(10%空白血清)、模型组(TGF-β1+10%空白血清)、BYHWT组(TGF-β1+10%含药血清)、BYHWT+过表达质粒组(TGF-β1+10%含药血清+空白质粒)和BYHWT+β-catenin过表达质粒组(TGF-β1+10%含药血清+β-catenin)5个组。采用噻唑蓝(MTT)比色法检测细胞增殖能力,划痕实验和Transwell法共同检测细胞迁移能力,免疫荧光法检测血小板内皮细胞黏附分子(CD31)、血管内皮钙黏蛋白(VE-cadherin)、成纤维细胞特异性蛋白1(FSP1)和α-平滑肌肌动蛋白(α-SMA)的表达。结果:与空白组比较,模型组Wnt1、β-catenin蛋白表达均显著升高(P<0.01),GSK-3β蛋白表达显著降低(P<0.01)。与模型组比较,BYHWT高剂量组Wnt1、β-catenin蛋白表达均显著降低(P<0.01),GSK-3β蛋白表达显著升高(P<0.01);BYHWT中剂量组β-catenin蛋白表达显著降低(P<0.01),GSK-3β蛋白表达显著升高(P<0.01);而BYHWT低剂量组差异无统计学意义。与空白组比较,模型组细胞增殖和迁移能�
Objective:To investigate the role of the Wnt1/β-catenin signaling pathway in the intervention of medicated serum of Buyang Huanwutang(BYHWT) in endothelial-to-mesenchymal transition(EndMT) of human pulmonary artery endothelial cells(HPAECs) as well as its related mechanisms.Method:Medicated serum of BYHWT was prepared by gavage to New Zealand rabbits with a dosage of 53.36 g·kg^(-1)·d^(-1)after decocting the medicine as usual.In addition,the same volume of normal saline was used to prepare blank serum.The HPAECs were cultured in vitro,and then induced by the transforming growth factor-β1(TGF-β1) to establish the EndMT model.Five groups were established:blank group(10% blank serum),model group(TGF-β1+10% blank serum),low-dose BYHWT group(TGF-β1+2.5% medicated serum+7.5% blank serum),medium-dose BYHWT group(TGF-β1+5% medicated serum+5% blank serum) and high-dose BYHWT group(TGF-β1+10% medicated serum).Through Western blot,the expressions of Wnt1,β-catenin,and glycogen synthase kinase-3β(GSK-3β) were detected.In order to further clarify the mechanism of the Wnt1/β-catenin signaling pathway in the intervention of the medicated serum of BYHWT in inhibiting EndMT,the overexpression of β-catenin was confirmed by polymerase chain reaction after plasmid of overexpression β-catenin was constructed and transfected into the HPAECs.The HPAECs were intervened by 10% medicated serum with the optimal effect in previous studies.Then,they were divided into another five groups:the blank group(10% blank serum),the model group(TGF-β1+10% blank serum),the BYHWT group(TGF-β1+10%medicated serum),the BYHWT+overexpression plasmid control group(TGF-β1+10% medicated serum+blank plasmid) and the BYHWT+β-catenin overexpression plasmid group(TGF-β1+10% medicated serum+β-catenin).Apart from that,cell proliferation ability was detected by the methyl thiazolyl tetrazolium(MTT) method and cell migration ability by scratch assay and Transwell assay together.Immunofluorescence was adopted to detect the expressions of platelet en
作者
杨其芬
渠景连
赵惠亮
郭永胜
陈隆
YANG Qifen;QU Jinglian;ZHAO Huiliang;GUO Yongsheng;CHEN Long(Guizhou University of Traditional Chinese Medicine,Guiyang 550025,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第7期49-58,共10页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81660789,82160892)。
