安胃汤含药血清对胃黏膜肠上皮化生细胞内质网应激-自噬通路的影响  

Effects of Anwei Decoction-Containing Serum on Endoplasmic Reticulum Stress-Autophagy Pathway in Gastric Intestinal Metaplasia Cells

作　　者：吴德坤[1] 唐友明[1] 郑景辉[1] 覃树辉 莫少丹 黄敏燕 吕明艳 吴鸿运 梁柳观 胡鑫 WU Dekun;TANG Youming;ZHENG Jinghui;QIN Shuhui;MO Shaodan;HUANG Minyan;LYU Mingyan;WU Hongyun;LIANG Liuguan;HU Xin(Ruikang Hospital Affiliated to Guangxi University of Traditional Chinese Medicine,Nanning 530011 Guangxi,China;Guangxi University of Traditional Chinese Medicine,Nanning 530200 Guangxi,China)

机构地区：[1]广西中医药大学附属瑞康医院,广西南宁530011 [2]广西中医药大学,广西南宁530200

出　　处：《中药新药与临床药理》2023年第10期1377-1386,共10页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(81860843);广西壮族自治区中医药管理局科研项目(20210718)。

摘　　要：目的观察安胃汤(黄连、制半夏、白芍等)含药血清对胃黏膜肠上皮化生(GIM)细胞的影响,并基于内质网应激(ERS)-自噬通路探讨其作用机制。方法将SD大鼠随机分为空白组及安胃汤低、中、高剂量组(5.4、10.7、21.4 g·kg^(-1)·d^(-1)),每组5只;每日1次,连续灌胃给药7 d,制备空白血清及安胃汤含药血清。采用150μmol·L^(-1)鹅去氧胆酸(CDCA)干预24 h,诱导人胃黏膜上皮细胞(GES-1),复制GIM细胞模型。将细胞分为正常对照组(未经CDCA干预的GES-1细胞)、模型组及安胃汤低、中、高剂量组,以10%空白血清及安胃汤低、中、高剂量含药血清干预24 h。采用Western Blot法检测肠上皮细胞的特异因子MUC2、VILLIN、CDX2蛋白表达情况;MTT法检测细胞增殖活力;流式细胞术检测细胞周期及细胞凋亡;免疫荧光法检测细胞中ATF6蛋白表达水平;RT-qPCR法检测细胞中GRP78、LC3-Ⅱ、Beclin1 mRNA表达水平;Western Blot法检测细胞中GRP78、LC3-Ⅱ、LC3-Ⅰ及Beclin1蛋白表达水平。结果与正常对照组(0μmol·L^(-1))比较,CDCA 50、100、150、200μmol·L^(-1)浓度组GES-1细胞的MUC2、VILLIN、CDX2蛋白表达均显著上调(P<0.01);模型组细胞的增殖水平显著升高(P<0.01);细胞在G2/M期的细胞比例显著升高(P<0.01),在G0/G1期和S期的细胞比例明显降低(P<0.05,P<0.01);细胞的凋亡率显著降低(P<0.01);ATF6蛋白表达显著下调(P<0.01);GRP78、LC3-Ⅱ、Beclin1 mRNA表达显著下调(P<0.01);GRP78、Beclin1、LC3-Ⅱ/LC3-Ⅰ蛋白表达显著下调(P<0.01)。与模型组比较,安胃汤含药血清低、中、高剂量组GIM细胞的增殖水平均显著降低(P<0.01);细胞在G2/M期的细胞比例显著降低(P<0.01),在G0/G1期和S期的细胞比例显著升高(P<0.01);细胞的凋亡率显著升高(P<0.01);ATF6蛋白表达显著上调(P<0.01);GRP78、LC3-Ⅱ、Beclin1 mRNA表达均明显上调(P<0.05,P<0.01);GRP78、Beclin1、LC3-Ⅱ/LC3-Ⅰ蛋白表达显著上调(P<0.01)。结论安胃汤含药�Objective To observe the effects of Anwei Decoction-containing serum on gastric intestinal metaplasia(GIM)cells,and to explore the mechanism of action based on the endoplasmic reticulum stress(ERS)-autophagy pathway.Methods SD rats were randomly divided into a blank group and a low-,medium-and highdose group of Anwei Decoction(5.4,10.7 and 21.4 g·kg^(-1)·d^(-1)),5 rats in each group;all the groups were administered by gavage once a day for 7 days.Human gastric mucosal epithelial cells(GES-1)were induced by chenodeoxycholic acid(CDCA)(150μmol·L^(-1))for 24 hours to replicate GIM cell model.The cells were divided into normal control group(GES-1 cells without CDCA intervention),model group and Anwei Decoction low-,medium-and high-dose groups.10%blank serum and low-,medium-and high-dose drug-containing serum of Anwei Decoction were intervened for 24 hours.The protein expressions of the specific factors MUC2,VILLIN,and CDX2 in the gastrointestinal epithelial cells was detected by Western Blot;the proliferative viability of the cells was detected by MTT;and the proliferative activity of the cells was detected by flow cytometric assay;flow cytometry was used to detect cell cycle and apoptosis;immunofluorescence was used to detect protein expression of ATF6 in cells;RT-qPCR was used to detect mRNA expression levels of GRP78,LC3-Ⅱ,and Beclin1 in cells;and Western Blot was used to detect protein expression levels of GRP78,LC3-Ⅱ,LC3-Ⅰand Beclin1 in cells.Results Compared with the normal control group(0μmol·L-1),the protein expression of MUC2,VILLIN,and CDX2 in GES-1 cells in the CDCA 50,100,150,and 200μmol·L^(-1)concentration groups were significantly upregulated(P<0.01);the proliferation level of the cells in the model group was significantly increased(P<0.01);the proportion of cells in the G2/M phase of cells was significantly increased(P<0.01),and the proportion of cells in G0/G1 phase and S phase was significantly reduced(P<0.05,P<0.01);apoptosis rate of cells was significantly reduced(P<0.01);ATF6 protein

关 键 词：安胃汤 胃黏膜肠上皮化生 内质网应激 自噬 增殖 凋亡 大鼠 人胃黏膜上皮细胞 

分 类 号：R285.5[医药卫生—中药学]