和厚朴酚对脂变性状态下HepG2细胞脂合成的影响

The effects of honokiol on lipid synthesis in HepG2 cells with steatosis

本文采用油红O染色法检测细胞脂滴含量,荧光光度法检测细胞的葡萄糖摄取量,实时荧光定量PCR及蛋白印迹法检测目标基因的表达量,研究了和厚朴酚(honokiol,HN)对正常、TO901317(TO)和油酸(oleic acid,OA)处理的Hep G2细胞脂合成、葡萄糖摄取及细胞色素P450酶(cytochrome P450 proteins,CYP)家族的CYP2E1、CYP4A蛋白表达的影响。结果显示:(1)TO和OA处理能明显提高细胞中脂滴数量,以及固醇调节元件结合蛋白1c(sterol regulatory element binding protein-1,SREBP-1c)和脂肪营养蛋白3(patatin-like phospholipase domain-containing 3,PNPLA3)的m RNA和蛋白表达水平;经HN(10、20、40μmol·L^(-1))处理细胞24 h后,肝细胞中脂滴明显减少,SREBP-1c和PNPLA3 m RNA及蛋白表达量降低。(2)OA处理能明显抑制细胞的糖摄取;经HN处理24 h后,肝细胞对荧光葡萄糖[2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose,2-NBDG]的吸收呈剂量依赖性增加。(3)正常及TO、OA处理的Hep G2细胞经HN处理24 h后,胞内CYP2E1的蛋白表达量较对照组明显降低,但HN给药仅抑制了经OA处理的Hep G2细胞中CYP4A的蛋白表达量。上述结果表明,HN可通过抑制脂变性状态下肝细胞内SREBP-1c和PNPLA3的表达而降低肝细胞的脂堆积,通过下调肝细胞内CYP2E1、CYP4A的蛋白表达及促进肝细胞的糖摄取,改善肝细胞的脂质过氧化和胰岛素抵抗。

In this study,the effects of honokiol（HN）treatment for 24 h on lipid synthesis was examined in Hep G2 cells.The parameters include intracellular lipid droplet and the expression of SREBP-1c and PNPLA3,glucose uptake,and oxidative stress including the expression of CYP2E1 and CYP4A in normal,TO901317（TO）-and oleic acid（OA）-treated Hep G2 cells.The lipid droplets were detected by oil red O staining.The glucose uptake was measured by fluorescence spectrophotometry using[2-（N-（7-nitrobenz-2-oxa-1,3-diazol-4-yl）amino）-2-deoxyglucose,2-NBDG]as probe.The expression levels of target genes were detected by quantitative PCR and Western blot.The results showed that：（1）TO（5μmol·L^-1）and OA（0.5 mmol·L^-1）treatment increased the levels of intracellular lipid accumulation and the m RNA and protein expression of SREBP-1c and PNPLA3.After HN（10,20,40μmol·L^-1）treatment for 24 h,the lipid accumulation and the expression of SREBP-1c and PNPLA3 were all decreased in the tested cells.（2）OA treatment significantly suppressed glucose uptake,while HN treatment dose-dependently increased the glucose uptake in OA-treated cells.（3）Compared with control group,CYP2E1 protein level significantly decreased in the three tested cells,and CYP4A protein level significantly decreased only in OA-treated cells following HN treatment.The above results suggest that HN may attenuate lipid accumulation by suppressing the expression of SREBP-1c and PNPLA3,and reduce lipid peroxidation and insulin resistance by down-regulation of the protein levels of CYP2E1 and CYP4A in Hep G2 cells with steatosis.