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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 observe the demethylation of curcumin on the promoter region of peroxisome proliferator activated receptor-α (PPAR-α) induced by oleic acid in rat hepatic steatosis model. Methods Rat normal liver cell line (BRL) was divided into four groups: normal group treated with DMSO for 96 h; the model group, curcumin group, 5-Aza- CdR groups were treated with oleic acid at 15μg / mL for 48 hours to establish a model of steatosis; then induced by oleic acid for 48 hours in model group, curcumin treated with 10μmol / L curcumin + oleic acid for 48 hours, 5 -Aza-CdR group with 5.0μmol / L 5-Aza-CdR + oleic acid for 48 h. The levels of ALT, AST, TG, TC and the content of intracellular TG and TC in each group were measured. The degree of fatty degeneration was observed with oil red O staining. The expression of PPAR-αmRNA was detected by RT-PCR and pyrosequencing PPAR-α promoter methylation level. Results The levels of ALT, AST, TG, TC and intracellular TG and TC in the model group were significantly higher than those in the normal group (P <0.05, P <0.01), while the relative expression levels of PPAR-α mRNA were significantly decreased The methylation rates of -378, -375 and -373 CpG sites were significantly increased (P <0.05, P <0.01). The levels of ALT, AST, TG, TC in the curcumin group and the contents of TG and TC in the curcumin group were significantly lower than those in the model group (P <0.05, P <0.01). The lipid droplets in the oil red O staining decreased or even disappeared. (P <0.01). The methylation rates of - 378, -375 and -373CpG sites were significantly decreased (P <0.05, P <0.01). Compared with the model group, the expression of PPAR-αmRNA in 5-Aza-CdR group was significantly increased (P <0.01), but still lower than that in normal group (P <0.01), methylation of -378, -375 and -373 CpG (P <0.05, P <0.01). However, there was no significant difference in the demethylation between curcumin and curcumin (P> 0.05). The methylation rate of -378 CpG was negatively correlated with the expression of PPAR-αmRNA (r = -0.663, P = 0.026). Conclusion Curcumin may improve steatosis of hepatocellular model in nonalcoholic fatty liver disease rats by decreasing the CpG methylation rate in PPAR-α promoter and promoting PPAR-α mRNA expression.