高良姜素调节SIRT1/AMPK/mTOR信号通路对脂多糖诱导的大鼠关节软骨细胞自噬和凋亡的影响  

Effects of Galangin on LPS-induced Autophagy and Apoptosis of Rat Articular Chondrocytes by Regulating the SIRT1/AMPK/mTOR Signaling Pathway

作　　者：杨瑜 吴洁 高晓 YANG Yu;WU Jie;GAO Xiao(Clinical Laboratory,Xinxiang Central Hospital,Xinxiang 453000 Henan,China;Rheumatology and Immunology Department,Xinxiang Central Hospital,Xinxiang 453000 Henan,China)

机构地区：[1]新乡中心医院检验科,河南新乡453000 [2]新乡中心医院风湿免疫科,河南新乡453000

出　　处：《中药新药与临床药理》2024年第9期1299-1305,共7页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：河南省医学科技攻关计划(联合共建)项目(LHGJ20230888)。

摘　　要：目的探讨高良姜素调节沉默信息调节因子1(SIRT1)/增强腺苷酸活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对脂多糖(LPS)诱导的大鼠关节软骨细胞自噬和凋亡的影响。方法将大鼠关节软骨细胞分为对照组,模型组(1.0μg·mL^(-1)LPS),高良姜素低(1.0μg·mL^(-1)LPS+10μmol·L^(-1)高良姜素)、中(1.0μg·mL^(-1)LPS+20μmol·L^(-1)高良姜素)、高(1.0μg·mL^(-1)LPS+40μmol·L^(-1)高良姜素)剂量组,高良姜素高剂量+EX527(SIRT1抑制剂)组(1.0μg·mL^(-1)LPS+40μmol·L^(-1)高良姜素+40μmol·L^(-1)EX527)。用流式细胞术检测关节软骨细胞凋亡;丹酰尸胺(MDC)染色观察关节软骨细胞自噬;酶联免疫吸附法检测细胞中白细胞介素1β(IL^(-1)β)、肿瘤坏死因子α(TNF-α)、IL-6水平;Western Blot法检测苄氯素(Beclin)、微管相关蛋白1轻链3(LC3)Ⅱ/Ⅰ、B淋巴细胞瘤-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、SIRT1、AMPK、磷酸化AMPK(p-AMPK)、mTOR、p-mTOR表达情况。结果与对照组比较,模型组MDC阳性细胞比例,Beclin、LC3Ⅱ/Ⅰ、Bcl-2、SIRT1蛋白表达及p-AMPK/AMPK比值均降低(P<0.05);细胞凋亡率、IL^(-1)β、TNF-α、IL-6水平、Bax蛋白表达、p-mTOR/mTOR比值均升高(P<0.05)。与模型组比,高良姜素低、中、高剂量组的MDC阳性细胞比例及Beclin、LC3Ⅱ/Ⅰ、Bcl-2、SIRT1蛋白表达及p-AMPK/AMPK比值均增加(P<0.05);细胞凋亡率、IL^(-1)β、TNF-α、IL-6水平、Bax蛋白表达及p-mTOR/mTOR比值均降低(P<0.05)。与高良姜素高剂量组比较,高良姜素高剂量+EX527组的MDC阳性细胞比例及Beclin、LC3Ⅱ/Ⅰ、Bcl-2、SIRT1蛋白表达以及p-AMPK/AMPK比值明显降低(P<0.05),细胞凋亡率IL^(-1)β、TNF-α、IL-6水平、Bax蛋白表达、p-mTOR/mTOR比值明显升高(P<0.05)。结论高良姜素能促进LPS诱导的关节软骨细胞自噬,抑制细胞凋亡,可能是通过激活SIRT1/AMPK/mTOR信号通路发挥作用。Objective To investigate the effect of galangin on lipopolysaccharide(LPS)-induced autophagy and apoptosis of rat articular chondrocytes by regulating the silent information regulator 1(SIRT1)/adenosine 5’-monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)signaling pathway.Methods Rat articular chondrocytes were grouped into control group,model group(1.0μg·mL^(-1)LPS),low concentration galangin group(1.0μg·mL^(-1)LPS+10μmol·L^(-1)galangin),medium concentration galangin group(1.0μg·mL^(-1)LPS+20μmol·L^(-1)galangin),high concentration galangin group(1.0μg·mL^(-1)LPS+40μmol·L^(-1)galangin),high concentration galangin+EX527(SIRT1 inhibitor)group(1.0μg·mL^(-1)LPS+40μmol·L^(-1)galangin+40μmol·L^(-1)EX527).Apoptosis of articular chondrocyte was detected by flow cytometry.Monodansylcadaverine(MDC)staining was applied to observe autophagy of articular chondrocytes.Enzyme linked immunosorbent assay was applied to detect the levels of interleukin(IL)-1β,tumor necrosis factorα(TNF-α),and IL-6 in cells.Western Blot was applied to detect the expression of Beclin,microtubule-associated protein 1 light chain 3(LC3)Ⅱ/Ⅰ,B lymphoblastoma-2-associated X protein(Bax),B lymphoblastoma-2(Bcl-2),SIRT1,AMPK,phosphorylated(p)-AMPK,mTOR,and p-mTOR.Results Compared with the control group,the proportion of MDC positive cells,protein expression of Beclin,LC3Ⅱ/Ⅰ,Bcl-2 and SIRT1,as well as p-AMPK/AMPK ratio in the model group decreased(P<0.05),while the apoptosis rate,levels of IL^(-1)β,TNF-αand IL-6,Bax protein expression,and p-mTOR/mTOR ratio increased(P<0.05).Compared with the model group,the proportion of MDC positive cells,protein expression of Beclin,LC3Ⅱ/Ⅰ,Bcl-2 and SIRT1,as well as p-AMPK/AMPK ratio in the low-,medium-,and high-concentration galangin groups all increased(P<0.05),but the apoptosis rate,levels of IL^(-1)β,TNF-αand IL-6,Bax protein expression,and p-mTOR/mTOR ratio decreased(P<0.05).Compared with the high concentration galangin group,the proportion of MD

关 键 词：高良姜素 SIRT1/AMPK/mTOR信号通路 脂多糖 大鼠关节软骨细胞 自噬 凋亡 

分 类 号：R285.5[医药卫生—中药学]