过氧化物酶体增殖活化受体-α激活对HepG2细胞脂肪变性及血红素加氧酶-1表达的影响

Effects of PPAR-alpha activation on oleic acid-induced steatosis and expression of heme oxygenase-1 in HepG2 eeDs

目的研究过氧化物酶体增殖活化受体-α（PPAR-α）激活对油酸谬莹导的HepG2细胞脂肪变性及血红素加氧酶-1（HO-1）表达的影响。方法以油酸（OA）诱导人肝癌HepG2细胞脂肪变性为模型组，采用不同浓度的PPAR-α激动剂非诺贝特（FF）处理HepG2细胞24h，油红O染色观察细胞内脂滴数量，甘油-3-磷酸氧化酶法检测细胞内甘油三酯（TG）含量，采用实时荧光定量PCR检测各组HepG2细胞PPAR-α、HO-1mRNA水平，采用免疫细胞化学法检测PPAR-α与HO-1蛋白表达。采用SPSS13．0软件，采用单因素方差分析及Peatrmn直线相关进行相关分析。结果（1）模型组HepG2细胞内TG含量为（379．98土23．19）mg／g，对照组为（185．03±12．68）mg／g，模型组HepG2细胞内TG含量明显增加，t=24．385，P〈0．01。PPAR-α的mRNA及蛋白相对表达水平模型组分别为0．42±0．38和0．47±0．14，对照组分别为1．00±0．00和1．85±0．12，模型组明显降低，f=0．583和1．382，P值均〈O．01。HO-1的mRNA及蛋白相对表达水平模型组分别为0．36土0．66和0．26士0．10，对照组分别为1．00±0．00和1．22士0．12，模型组明显降低，f=0．637和f=0．967，P值均〈0．01；（2）FF浓度在5、10、50umol／L时，HepG2细胞内TG值分别为（294．00±19．80）mg／g、（250．33土9．96）mg／g和（196．99士9．14）mg／g，值分别为10．747、16．200和22．873，，=148．555 P值均〈0．01}PPAR-α的mRNA相对表达量分别为0．55士0．65，0．85士0．61，1．31土0．36，t值分别为0．137、0．430和0．893，F=177．637，P值均〈O．01lPPAR-α蛋白累积光密度值分别为0．82士0．11、1．31士0．16和1．75±0．13，t值分别为0．352，0．840，1．280，F=120．764，P值均〈0．01,HO-1的mRNA相对表达量分别为0．62土0．05、0．84±0．07和1．304-0．11，t值分别为0．257，0．480，0．937，F=74．768，P值均〈0．01；HO-1蛋白累积光密度值分别为0．44士0．08、0．81±0．08和1．20士0．10，f值分别为0．180，0．553，0．943，F=119．903，P值均〈0．01。结论PPAR-α的激活可以抑制HepG2细胞脂肪变陛，HO-1可能是其重要下游因子。

Objective To investigate the effects of peroxisome proliferator activated receptor-alpha （PPAR-ct） activation on oleic acid （OA）-indueed steatosis and hepatic expression of heme oxygenase-1 （HO-1） using an in vitro cell model system. Methods A steatotic human hepatocyte in vitro model system was established by treating HepG2 cells with 0.2 mmol/L of oleic acid for 24 hours. The steatotic cells were then divided into four groups for an additional 24 hours of trealment with 0.2 mmol/L of oleic acid alone （modelcontrol group） or with 5, 10 or 50 pmol/L of fenofibrate （FF, a selective PPAR-a agonist; experimental groups）. Untreated HepG2 cells served as non-steatotic controls. Effect of PPAR-a activation on fat accumulation was detected by Oil Red O staining and on intracellular triglyceride （TG） levels by enzymatic assay, mRNA and protein expression of PPAR-a and HO-1 were quantified by real-time PCR and immunocytochemistry, respectively. One-way ANOVA and the LSD t- test were used for between-group comparisons, and correlation analysis was performed with the Pearson＇s correlation coefficient. Results The steatosis model control cells showed significantly increased TG deposition （379.98 ± 23.19 mg/g protein, vs. non-steatotic conlrols F = 148.56, P 〈 0.01）, significantly decreased mRNA and protein expression of PPAR-a （0.42 ± 0.38,F = 177.64, P 〈 0.01 and 0.47 ± 0.14, F = 120.76, P 〈 0.01 ） and HO- 1 （0.36 ± 0.66,F = 74.77,P 〈 0.01 and 0.26 ± 0.10,F = 119.90, P 〈 0.01 ）. FF （5, 10 and 50 Bmol/L） inhibited the steatosis induced by OA in a concentration-dependent manner （294.00 ± 19.80, 250.33 ± 9.96, and 196.99 ± 9.14, F = 148.56, P 〈 0.01） and increased the mRNA and protein expression of PPAR-a （0.55 ± 0.65, 0.85 ± 0.61, and 1.31 ± 0.36, F = 177.64, P 〈 0.01; 0.82 ± 0.11, 1.31 ± 0.16, and 1.75 ± 0.13, F = 120.76, P 〈 0.01 ） and HO- 1 （0.62 ± 0.05, 0.84 ± 0.07, and 1.30 ± 0.11, F = 74.77, P 〈 0.01; 0.44 ± 0.08, 0.81 ± 0.08, 1.20±0.10,F= 119.90,P〈0.01）. Conclusion Activation of PPAR-a prevents OA-induced steatosis in HepG2 cells, and HO-1 may function as a downstream effector of this mechanism.