WNT1诱导信号通路蛋白2对肝细胞脂质代谢的影响及其作用机制

The effects of WNT1 inducible signaling pathway protein 2 on hepatocyte lipid metabolism and associated mechanisms

目的探讨不同浓度人重组WNT1诱导信号通路蛋白2(WISP2)对人肝癌细胞(HepG2)脂质代谢的影响及相关作用机制。方法分别用不同浓度(0、0.4、1和2 μg/L)人重组WISP2作用于HepG2细胞48 h, Cell-Titer发光法测定细胞活力, 酶法检测各组HepG2细胞内三酰甘油(TG)及总胆固醇(TC)含量, 同时应用实时荧光定量聚合酶链式反应(RT-qPCR)及免疫印迹法(western blot)检测HepG2细胞内脂质合成、分解、转运相关基因mRNA和蛋白表达水平。结果与对照组比较, 各浓度人重组WISP2处理组均未降低HepG2细胞的活性;人重组WISP2处理上调HepG2细胞TG、TC含量, 0.4、1、2 μg/L人重组WISP2处理组TG的含量分别为未处理组的1.254±0.039、1.216±0.028、1.174±0.014倍(F=6.791, P=0.006), TC含量分别为未处理组的1.264±0.057、1.394±0.101、1.392±0.077倍(F=7.045, P=0.005)。进一步研究发现, 与对照组比较, 加入人重组WISP2明显增加HepG2细胞脂质合成蛋白甾醇调节元件结合蛋白1(SREBP1)、羟甲基戊二酸单酰辅酶A还原酶(HMGCR)、乙酰辅酶A羧化酶(ACC)及二酰基甘油酰基转移酶(DGAT2)mRNA表达(均P<0.05), 显著上调SREBP1、ACC及脂肪酸合成酶(FAS)蛋白水平的表达(均P<0.05);但对低密度脂蛋白受体(LDLR)、载脂蛋白B(ApoB)、载脂蛋白E(ApoE)这类脂质转运蛋白及关键的脂质分解蛋白--脂肪三酰甘油脂肪酶(ATGL)的表达无明显影响。结论 rh-WISP2能显著增加肝细胞脂质含量, 促进脂质合成增加可能是其作用的关键机制。

Objective To inves tigate the effects of various concentrations of recombinant human WISP2 protein(WISP2)on lipid metabolism in HepG2 cells.Methods HepG2 cells were treated with different concentrations(0,0.4.1 and 2μug/L)of recombinant human WISP2 for 48 hours.Cell viability was detected by Cell-Titer,and enzymatic hydrolysis methods were used to measure intracellular triacylglycerol(TG)and total cholesterol(TC)levels.The mRNA expression was detected by quantitative real-time reverse transcription PCR(RT-qPCR)and protein expression in HepG2 cells was detected by western blot.Results Compared with the control group,the WISP2 groups treated with various concentration did not significantly reduce the viability of HepG2 cells.TG and TC in HepG2 cells were significantly increased by recombinant human W ISP2 treatment(all P<0.05).The concentrations of TG in the0.4.1 and 2 ug/L recombinant human WISP2 treated groups were(1.254±0.039,1.216±0.028 and 1.174±0.014)times the concentration in the untreated group.respectively(F=6.791,P=0.006).The concentration of TC in the untreated group was(1.264±0.057,1.394±0.101 and 1.392±0.077),respectively,times the concentration in each of the treated groups(F=7.045,P=0.005).Further experiments found that the mRNA expression of sterol regulatory element binding protein 1(SREBP1),3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGCR),acetyl-CoA carboxylase(ACC),type 2 diacylglycerol acyltransferase(DGAT2)and the protein expression of SREBPI,ACC and fatty acid synthase(FAS)were significantly increased in the recombinant human WISP2-treated groups.compared with the control group(all P<0.05).However,the expression of lipid transporters such as the low-density lipoprotein receptor(LDLR),ApoB and ApoE and adipose triglyceride lipase(ATGL).a key lipolysis protein,was not significantly affected.Conclusions Human recombinant WISP2 protein increases lipid levels in hepatocytes and the key underlying mechanisms may be through promoting lipid synthesis.