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灵芝酸C2调控S6K/SREBPs信号通路对肝细胞脂代谢的影响及机制研究
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| 篇名： | 灵芝酸C2调控S6K/SREBPs信号通路对肝细胞脂代谢的影响及机制研究 |
| TITLE： | Study on the Effect and Mechanism of Ganoderic Acid C 2 on Lipid Metabolism of Hepatocytes by Regulating S6K/SREBPs Signaling Pathway |
| 摘要： | 目的：研究灵芝酸C2（GAC2）的体外降脂作用，并基于核糖体S6蛋白激酶（S6K）/固醇调节元件结合蛋白（SREBPs）信号通路探讨其可能机制。方法：以人肝细胞HL-7702为对象，采用MTT法考察低、中、高剂量（5、10、20μmol/L，下同）GAC2作用后细胞的相对活力；以洛伐他汀为阳性对照，采用酶联免疫吸附测定法检测低、中、高剂量GAC2作用后细胞中胆固醇（TC）、三酰甘油（TG）的含量，并采用尼罗红染色法观察细胞内的脂质堆积情况；转染SREBPs报告基因质粒后，以25-羟基胆固醇（25-HC）为阳性对照，采用荧光素酶报告基因法检测低、中、高剂量GAC2作用后细胞中SREBPs荧光素酶的相对活性；以25-HC为阳性对照，采用实时荧光定量聚合酶链式反应法检测中、高剂量GAC2作用后细胞中SREBPs及其下游基因mRNA的表达情况；以SREBPs抑制剂（25-HC）、S6K抑制剂（雷帕霉素）为参照，采用Westernblotting法检测细胞中SREBP-1、SREBP-2的表达情况[以成熟SREBPs（n-SREBPs）表示]以及磷酸化S6K与S6K的相对表达量比值（p-S6K/S6K比值）；利用AutoDock4.0等软件对S6K与GAC2进行分子对接。结果：低、中、高剂量GAC2对细胞相对活力无显著影响（P＞0.05）。与空白对照组比较，洛伐他汀组和GAC2高剂量组细胞中TC含量以及洛伐他汀组和GAC2中、高剂量组细胞中TG含量均显著降低（P＜0.05或P＜0.01），各给药组细胞中的脂滴均有所减少。与空白对照组比较，25-HC组和GAC2低、中、高剂量组细胞中的SREBPs荧光素酶相对活性均显著降低；25-HC组和GAC2中、高剂量组细胞中HMGCS1、MVK、SCD、HMGCR基因mRNA，25-HC组细胞中DHCR7基因mRNA，GAC2高剂量组细胞中SREBP-2基因mRNA，25-HC组和GAC2高剂量组细胞中DHCR24、MSMO2基因mRNA的相对表达量均显著降低；25-HC组和GAC2低、中、高剂量组细胞中SREBP-1蛋白的相对表达量，25-HC组和GAC2高剂量组n-SREBP-2蛋白的相对表达量以及霉帕霉素组和GAC2各剂量组p-S6K/S6K比值均显著降低（P＜0.05或P＜0.01）。分子对接结果显示，GAC2可通过氢键与S6K的氨基酸残基Arg335、Arg330、Ala332结合。结论：GAC2可降低HL-7702细胞的脂质水平，其调控作用可能与抑制S6K/SREBPs信号通路的表达有关。 |
| ABSTRACT： | OBJECTIVE：To stu dy in vitro lipid-lowering effect of ganoderic acid C 2（GAC2），and to investigate its potential mechanism on the basis of S 6K/SREBPs signaling pathway. METHODS ：Using human liver cells HL- 7702 as objects ，MTT assay was used to test relative cell viability after treated with low ，medium and high doses （5，10，20 μmol/L，hereinafter）of GAC 2. Using lovastatin as positive control ，ELISA method was used to detect the contents of TC and TG in cells after treated with low ， medium and high doses of GAC 2. Nile red staining was used to observe the accumulation of lipids in cells. After transfected SREBPs report gene plasmid ，using 25-HC as positive control ，relative viability of SREBPs luciferase in cells were determined by luciferase assay after treated with low ，medium and high doses of GAC 2. Using 25-HC as positive control ，real-time fluorescent quantitative PCR was used to measure the mRNA expression of SREBPs and their downstream genes in cells after treated with medium and high doses of GAC 2. Using SREBPs inhibitor （25-HC）and S 6K inhibitor （rapamycin）as control ，Western blotting assay was adopted to determine the expression of SREBP- 1 and SREBP- 2（in the case of n-SREBPs ），relative expression ratio of phosphorylated S 6K to S 6K（p-S6K/S6K ratio ）. AutoDock 4.0 and other softwares were used for molecular docking of S 6K and GAC2. RESULTS ：There was no significant effect of low ， 0.05）. Compared with blank control group ，the content of TC qq.com in lovastatin group and GAC 2 high-dose group as well as thecontent of TG in lovastatin group ， GAC2 medium- and 床应用。电话：0371-65962746。E-mail：whui3697@126.com high-dose groups were decreased significantly （P＜0.05 or P＜ 0.01）；the number of lipid droplets in the cells of all medication groups decreased. Compared with blank control group ，relative viability of SREBPs luciferase in 25-HC group ，GAC2 low-，medium- and high-dose groups were decreased significantly ；mRNA expression of HMGCS1，MVK，SCD，HMGCR gene in 25-HC group and GAC 2 medium-，high-dose groups ，mRNA expression of DHCR7 gene in 25-HC group ，mRNA expression of SREBP-2 gene in GAC- 2 high-dose group as well as mRNA expression of DHCR24 and MSMO2 gene in 25-HC group and GAC 2 high-dose group were all decreased significantly ；relative protein expression of n-SREBP- 1 in 25-HC group ，GAC2 low-，medium- and high-dose groups ，relative protein expression of n-SREBP- 2 in 25-HC group and GAC 2 high-dose group as well as p-S 6K/S6K ratio in rapamycin group and GAC 2 groups were decreased significantly （P＜0.05 or P＜0.01）. The molecular docking results showed that GAC 2 could bound to amino acid residues Arg 335，Arg330 and Ala332 of S 6K via hydrogen bond. CONCLUSIONS ：GAC2 can reduce the lipid level of HL- 7702 cells，which may be associated with inhibiting the expression of S 6K/SREBPs signaling pathway. |
| 期刊： | 2020年第31卷第19期 |
| 作者： | 蒋亚丽，袁永，董帅，高改，赵建平，王辉 |
| AUTHORS： | JIANG Yali，YUAN Yong，DONG Shuai，GAO Gai，ZHAO Jianping ，WANG Hui |
| 关键字： | 灵芝酸C2；核糖体S6蛋白激酶/固醇调节元件结合蛋白信号通路；脂代谢；HL-7702细胞 |
| KEYWORDS： | Ganoderic acid C 2；S6K/SREBPs signaling pathway ；Lipid metabolism ；HL-7702 cells |
| 阅读数： | 304 次 |
| 本月下载数： | 5 次 |
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