柴胡皂甙d对特发性肺纤维化肺泡上皮细胞自噬作用机制研究  

Mechanism of Saikosaponin d on Autophagy of Alveolar Epithelial Cells in Idiopathic Pulmonary Fibrosis

作　　者：尚璐璐 张军[2] 黄彬 丁慢玲 吴妍 郑金旭[1] SHANG Lu-lu;ZHANG Jun;HUANG Bin;DING Man-ling;WU Yan;ZHENG Jin-xu(Department of Respiratory and Critical Care Medicine,Affiliated Hospital of Jiangsu University,Jiangsu 212001;Department of Respiratory and Critical Care Medicine,Affiliated Aoyang Hospital of Jiangsu University,Jiangsu 215600)

机构地区：[1]江苏大学附属医院呼吸与危重症医学科,江苏212001 [2]江苏大学附属澳洋医院呼吸与危重症医学科,江苏215600

出　　处：《中国中西医结合杂志》2022年第8期996-1001,共6页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：镇江市重大(社会发展)项目(No.SH2018048);苏州市社会发展(民生医疗)项目(No.SYSD2020010)。

摘　　要：目的研究柴胡皂甙d(SSd)对特发性肺纤维化(IPF)中Ⅱ型肺泡上皮细胞(AT-Ⅱ)自噬的调控作用及其对纤维化程度的干预作用。方法转化生长因子β1(TGF-β1,5 ng/mL)诱导A549细胞48 h建立肺纤维化细胞模型,分别联合SSd低、中、高浓度(2.5、5、10μg/mL)、雷帕霉素(100 ng/mL)处理细胞。CCK8法检测各组细胞存活率,Western Blot法检测E-钙黏蛋白(E-cadherin)、Ⅰ型胶原蛋白(Col-Ⅰ)、α-平滑肌肌动蛋白(α-SMA)、Beclin1、微管相关蛋白1轻链3B-Ⅱ(LC3B-Ⅱ)/LC3B-Ⅰ、p62、哺乳动物雷帕霉素靶蛋白(mTOR)通路活化蛋白表达水平,单丹磺酰尸胺(MDC)染色法检测细胞内自噬泡形成。结果与正常组比较,模型组细胞存活率下降(P<0.05),E-cadherin、Beclin1、LC3B-Ⅱ/LC3B-Ⅰ蛋白表达水平亦降低(P<0.05,P<0.01),Col-Ⅰ、α-SMA、p62、mTOR通路活性蛋白表达水平升高(P<0.05,P<0.01),胞质内荧光标记的自噬泡较少。与模型组比较,SSd高浓度组细胞存活率升高(P<0.05),SSd组及雷帕霉素组E-cadherin、Beclin1、LC3B-Ⅱ/LC3B-Ⅰ蛋白表达水平升高(P<0.05,P<0.01),Col-Ⅰ、α-SMA、p62蛋白表达水平降低(P<0.05,P<0.01),SSd高浓度组及雷帕霉素组p-mTOR/mTOR、p-AKT1/AKT1、p-S6K1/S6K1表达水平降低(P<0.05,P<0.01),SSd高浓度组及雷帕霉素组细胞质内荧光标记的自噬泡增多。与SSd低、中低浓度组比较,SSd高浓度组及雷帕霉素组Ecadherin、LC3B-Ⅱ/LC3B-Ⅰ表达水平升高(P<0.05,P<0.01),Col-Ⅰ、α-SMA、p62表达水平降低(P<0.05,P<0.01)。结论SSd可改善TGF-β1诱导的ATⅡ纤维化改变,可能通过抑制mTOR通路激活诱导细胞自噬发挥作用。Objective To study the regulatory effect of Saikosaponin d(SSd)on autophagy of typeⅡalveolar epithelial cells(AT-Ⅱ)in idiopathic pulmonary fibrosis(IPF),and to explore its intervention of the severity of fibrosis.Methods A cell model of pulmonary fibrosis was built with A549 cells induced by transforming growth factorβ1(TGF-β1,5 ng/mL)for 48 h.Combined treatments with SSd of different concentrations(2.5,5,and 10μg/mL)and rapamycin(100 ng/mL)were given to the cells respectively.The cell viability in different groups were determined by CCK8.The expression level of E-cadherin,Col-Ⅰ,α-SMA,Beclin1,microtubule associated protein 1 light chain 3B-Ⅱ(LC3B-Ⅱ)/LC3B-Ⅰ,p62 and activated protein in mammalian targetof rapamycin(mTOR)pathway were detected by Western Blot.The formation of autophagic vacuoles in cell was detected by monodansylcadavrine.Results Compared with the normal group,the cell viability decreased(P<0.05),the protein expression levels of E-cadherin,Beclin1 and LC3B-Ⅱ/LC3B-Ⅰdecreased(P<0.05,P<0.01),the expression levels of Col-Ⅰ,α-SMA,p62 and active protein in mTOR pathway increased in the model group(P<0.05,P<0.01),and fewer fluorescein-labelled autophagic vacuoles in cytoplasm.Compared with the model group,the cell viability increased in high concentration SSd group(P<0.05).The expression of E-cadherin,Beclin1 and LC3B-Ⅱ/LC3B-Ⅰprotein increased(P<0.05,P<0.01),the expression of Col-Ⅰ,α-SMA and p62 protein decreased in SSd groups and rapamycin group(P<0.05,P<0.01).The expression of p-mTOR/mTOR,p-AKT1/AKT1,p-S6K1/S6K1 decreased in high concentration SSd group and rapamycin group(P<0.05,P<0.01).There were more fluorescently labeled autophagic vesicles in the cytoplasm of high concentration SSd group and rapamycin group.Compared with low and media concentration SSd group,the expression of E-cadherin,LC3B-Ⅱ/LC3B-Ⅰincreased in the high concentration SSd group and rapamycin group(P<0.05,P<0.01),the expression of Col-Ⅰ,α-SMA,p62 decreased(P<0.05,P<0.01).Conclusion SSd could improv

关 键 词：特发性肺纤维化 柴胡皂甙D 哺乳动物雷帕霉素靶蛋白 巨自噬 

分 类 号：R285[医药卫生—中药学]