摘要
目的观察姜黄素对油酸诱导的大鼠脂肪变性肝细胞模型过氧化物酶体增殖物活化受体-α(PPAR-α)启动子区的去甲基化作用。方法将大鼠正常肝细胞株(BRL)分为4组:正常组以同体积的DMSO处理96 h;模型组、姜黄素组、5-氮杂-2'-脱氧胞苷(5-Aza-CdR)组均采用15μg/mL油酸处理48 h,建立脂肪变性肝细胞模型,然后,模型组再用油酸诱导48 h,姜黄素组用10μmol/L姜黄素+油酸干预48 h,5-Aza-CdR组用5.0μmol/L 5-Aza-CdR+油酸干预48 h。检测各组细胞上清液ALT、AST、TG、TC及细胞内TG、TC含量,用油红O染色镜下观察细胞内脂肪变程度,RT-PCR检测PPAR-αmRNA表达,焦磷酸测序法分析PPAR-α启动子区甲基化水平。结果模型组上清液ALT、AST、TG、TC及细胞内TG、TC均较正常组显著升高(P<0.05,P<0.01),PPAR-αmRNA相对表达水平显著下降(P<0.01),-378、-375、-373 CpG位点的甲基化率显著升高(P<0.05,P<0.01)。姜黄素组上清液ALT、AST、TG、TC及细胞内TG、TC均较模型组显著降低(P<0.05,P<0.01),油红O染色细胞内脂滴减少甚至消失,PPAR-αmRNA表达水平显著升高(P<0.01),-378、-375、-373CpG位点的甲基化率显著降低(P<0.05,P<0.01)。与模型组相比,5-Aza-CdR组PPAR-αmRNA表达显著升高(P<0.01),但仍低于正常组(P<0.01),-378、-375、-373 CpG的甲基化率显著降低(P<0.05,P<0.01),但与姜黄素组相比,两者的去甲基化作用无明显差异(P>0.05)。-378 CpG的甲基化率与PPAR-αmRNA表达水平呈负相关(r=-0.663,P=0.026)。结论姜黄素可能通过降低PPAR-α启动子区CpG甲基化率,促进PPAR-αmRNA表达,改善非酒精性脂肪性肝病大鼠肝细胞模型的脂肪变性。
Objective To investigate the cureumin - induced demethylation of the promoter of peroxisome prolife- rator - activated receptor α （ PPAR - α） in the hepatocyte steatosis models induced by oleic acid. Methods The normal rat hepatocytes （BRL） were divided into 4 groups ： normal group （ DMSO for 96 hours）, non - alcoholic fatty liver disease （NAFLD） group, eurcumin group, and 5 -Aza- CdR group. In NAFLD, cureumin and 5 -Aza- CdR groups, the cells were cultivated with oleic acid （ 15 μg/mL） for 48 hours, and subsequently oleie acid alone, plus curcumin （ 10. 0 μmol/ L） , or plus 5 - Aza - CdR （5.0 p, mol/L） were given to the subjects in NAFLD, curcumin or 5 - Aza - CdR group, re- spectively, for the next 48 hours. Biochemical assays were used to assess the ALT, AST, TG, TC levels in supernatant and TG, TC contents in cells. Oil red O staining was used to observe the fatty droplets in cells. The mRNA expression level of PPAR - α was measured by real time reverse transcription - polymerase chain reaction. The DNA methylation level was analyzed by pyro -sequencing. Results Compared with the normal group, significant increase was observed in su- pernatant ALT, TG, TC levels and cellular TG, TC contents in the NAFLD group （P 〈 0. 05, P 〈 0. 01 ）, so was the down - regulation of PPAR - α mRNA （P 〈 0. 01 ）. However, the DNA methylation of CpG - 378, - 375, - 373 was significant higher in NAFLD group （P 〈0. 05, P 〈0. 01 ）. Compared with NAFLD group, significant reduction in the su- pernatant ALT, TG, TC levels and cellular TG, TC contents were observed in curcumin group; so was the reduction of cellular fatty droplets through Oil red O staining. Besides, the expression of PPAR - α mRNA in curcumin group was sig- nificantly up - regulated than that of NAFLD group （P 〈 0. 01 ）. In addition, the DNA methylation of CpG - 378, - 375, - 373 in curcumin group was significantly lower than that of NAFLD group （ P 〈 0.05, P 〈 0. 01 ）. Compared with NAFLD group, the expression of PPAR - α mRNA of 5 - Aza - CdR group was significantly up - regulated （ P 〈 0. 01 ）, though it was still lower than that of normal group ; and the DNA methylation of CpG - 378, - 375, - 373 were also sig- nificantly reduced （P 〈 0. 05, P 〈 0. 01 ）, however, there was no significant difference between 5 - Aza - CdR group and normal group. A significantly negative correlation was revealed between the PPAR - c~ mRNA expression and DNA methyl- ation of CpG - 378 （ r = - 0. 663, P = 0. 026）. Conclusion Curcumin increases the expression of PPAR -α mRNA through the demethylation on CpG promoters, thereby ameliorating NAFLD hepatocyte steatosis.
出处
《广东医学》
CAS
CSCD
北大核心
2014年第2期175-179,共5页
Guangdong Medical Journal
基金
广州市医药卫生科技重点项目(编号:2009-ZDi-03)
