摘要
目的以游离脂肪酸(FFA)诱导的HepG2细胞脂质堆积为细胞模型,探讨牛蒡子苷改善肝脏脂质蓄积的作用机制。方法MTT法检测牛蒡子苷对HepG2细胞活性的影响;设立正常对照组,模型组,阳性药洛伐他汀组(10µmol·L^(-1)),雷帕霉素组(100 nmol·L^(-1)),牛蒡子苷低、中、高剂量组(10、20、30µmol·L^(-1)),自噬抑制剂氯喹组(10µmol·L^(-1));采用油红O染色检测细胞内脂滴形成情况;试剂盒检测细胞中甘油三酯(TG)、总胆固醇(TC)水平;激光共聚焦显微镜观察荧光标记的微管相关蛋白轻链3(LC3)与脂滴以及溶酶体标志物的共定位;蛋白免疫印迹法(Western Blot)检测LC3、溶酶体关联膜蛋白2(LAMP2)、泛素结合蛋白p62(p62/SQSTM1)、磷脂酰肌醇3激酶(PI3K)、磷酸化(p)-PI3K、蛋白激酶B(Akt)、p-Akt、哺乳动物雷帕霉素靶蛋白(mTOR)、p-mTOR蛋白表达情况;实时荧光定量PCR(RT-qPCR)法检测自噬相关蛋白5(ATG5)、自噬相关蛋白7(ATG7)、自噬相关蛋白12(ATG12)、LAMP2及脂肪甘油三酯脂肪酶(ATGL)和激素敏感性脂肪酶(HSL)的mRNA表达情况。结果MTT结果显示牛蒡子苷在0~30µmol·L^(-1)范围内无明显的细胞毒性;与正常对照组比较,模型组脂滴增加(P<0.001),TC、TG水平升高(P<0.001),LC3-Ⅱ、LAMP2蛋白表达降低(P<0.05),p62/SQSTM1蛋白表达升高(但差异无统计学意义,P>0.05),ATG5、ATG7、ATG12、LAMP2的mRNA水平降低(P<0.01,P<0.001),PI3K/Akt/mTOR通路相关蛋白表达量升高(P<0.01,P<0.001);与模型组比较,牛蒡子苷高剂量组明显改善游离脂肪酸诱导的HepG2细胞的脂质蓄积(P<0.001),TC、TG水平下降(P<0.001),LC3-Ⅱ、LAMP2的蛋白表达水平明显升高(P<0.001),p62/SQSTM1蛋白表达水平降低(P<0.001),ATG5、ATG7、ATG12、LAMP2的mRNA水平呈上升趋势(P<0.001),PI3K/Akt/mTOR通路相关蛋白表达量呈下降趋势(P<0.001);共定位结果发现牛蒡子苷干预促进了LC3的表达,并增加了LC3与脂滴和溶酶体标志物的共定位;而ATGL及HSL等脂解酶的mRNA水平无明显变化(P>0.05);使用自噬抑制剂氯喹后,牛蒡子苷对游离脂肪酸诱导的HepG2细胞中TC、TG的下调作用被逆转(P<0.001)。结论牛蒡子苷通过抑制PI3K/Akt/mTOR信号通路调控肝细胞脂噬改善肝脏脂质蓄积。
Objective To investigate the mechanism by which arctiin ameliorates hepatic lipid accumulation,using a cellular model of lipid accumulation induced by free fatty acids(FFA)in HepG2 cells.Methods The effect of arctiin on HepG2 cell viability was assessed by MTT assay.Cells were divided into the following groups:normal control group,model group,positive control lovastatin group(10µmol·L^(-1)),rapamycin group(100 nmol·L^(-1)),arctiin low-,medium-,and high-dose groups(10,20,30µmol·L^(-1)),and the autophagy inhibitor chloroquine group(10µmol·L^(-1)).Oil Red O staining was used to detect intracellular lipid droplet formation.Triglyceride(TG)and total cholesterol(TC)levels were measured using commercial kits.Colocalization of fluorescently labeled microtubule-associated protein 1 light chain 3(LC3)with lipid droplets and lysosomal markers was observed by confocal laser scanning microscopy.Protein expression levels of LC3,lysosome-associated membrane protein 2(LAMP2),Ubiquitin-binding protein p62(p62/SQSTM1),phosphatidylinositol 3-kinase(PI3K),p-PI3K,protein kinase B(Akt),p-Akt,mammalian target of rapamycin(mTOR),and p-mTOR were detected by Western Blot.mRNA expression of autophagy-related protein 5(ATG5),ATG7,ATG12,LAMP2,adipose triglyceride lipase(ATGL),and hormone-sensitive lipase(HSL)was measured Real-time fluorescent quantitative PCR(RT-qPCR).Results MTT results indicated no significant cytotoxicity of arctiin within the concentration range of 0-30µmol·L^(-1).Compared with the normal control group,the model group showed increased lipid droplets(P<0.001),elevated TC and TG levels(P<0.001),decreased protein expression of LC3-Ⅱand LAMP2(P<0.05),increased p62/SQSTM1 protein expression(the differences were not statistically significant,P>0.05),reduced mRNA levels of ATG5,ATG7,ATG12,and LAMP2(P<0.01,P<0.001),and upregulated expression of proteins related to the PI3K/Akt/mTOR pathway(P<0.01,P<0.001).Compared with the model group,the high-dose arctiin group significantly ameliorated FFA-induced lipid accumulation in HepG2 cells(P<0.001),reduced TC and TG levels(P<0.001),markedly increased protein expression of LC3-II and LAMP2(P<0.001),decreased p62/SQSTM1 protein expression(P<0.001),elevated mRNA levels of ATG5,ATG7,ATG12,and LAMP2(P<0.001),and downregulated expression of PI3K/Akt/mTOR pathway-related proteins(P<0.001).Colocalization analysis revealed that arctiin intervention promoted LC3 expression and enhanced its colocalization with both lipid droplets and lysosomal markers.Furthermore,the mRNA levels of lipolytic enzymes such as ATGL and HSL showed no significant changes.The downregulatory effects of arctiin on TC and TG in FFA-induced HepG2 cells were reversed upon co-treatment with the autophagy inhibitor chloroquine(P<0.001).Conclusion Arctiin ameliorates hepatic lipid accumulation by regulating lipophagy in hepatocytes through inhibition of the PI3K/Akt/mTOR signaling pathway.
作者
申炎炎
张效威
张振强
徐江雁
高改
谢治深
SHEN Yanyan;ZHANG Xiaowei;ZHANG Zhenqiang;XU Jiangyan;GAO Gai;XIE Zhishen(Collaborative Innovation Center of Prevention and Treatment of Major Diseases by Chinese and Western Medicine,Henan Province,Henan University of Chinese Medicine,Zhengzhou 450046 Henan,China;Collaborative Innovation Center of Research and Development on the Whole Industry Chain of Yu-Yao,Henan Province,Henan University of Chinese Medicine,Zhengzhou 450046 Henan,China;Academy of Chinese Medicine Sciences,Henan University of Chinese Medicine,Zhengzhou 450046 Henan,China)
出处
《中药新药与临床药理》
北大核心
2026年第2期235-244,共10页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
国家自然科学基金项目(82174267,82004019)
河南省中医药科学研究专项课题(2023ZYZD15,2024ZY1027)。
