清肝降脂方对脂肪变性LO2细胞HO-1、Egr-1基因及蛋白表达的影响

Effect of Qinggan Jiangzhi Decoction on Expressions of HO-1,Egr-1 Gene and Protein in Steatosis LO2 Cells in vitro

目的:通过建立游离脂肪酸体外诱导的LO2细胞非酒精脂肪肝模型,探讨不同剂量的清肝降脂方对肝脏脂肪变LO2细胞HO-1、Egr-1基因及蛋白表达水平的影响。方法:建立游离脂肪酸诱导脂肪变性LO2肝细胞体外模型。人肝LO2细胞采用含10%胎牛血清的RPMI 1640培养基培养,待细胞均匀分布,随机分为6组:正常对照组、模型组、清肝降脂方高剂量组、中剂量组、低剂量组、水林佳对照组,每组6瓶。除正常对照组外,其余5组细胞均以0.5 mmol/L的游离脂肪酸诱导LO2非酒精脂肪肝细胞模型,诱导24 h后,各组随机抽取1瓶进行油红O染色,观察LO2细胞的脂肪化程度。确定模型诱导成功后,正常对照组和模型组分别加入10%正常大鼠血清,低、中、高剂量清肝降脂方药物血清组及水林佳药物血清组添加10%含相对应药物的大鼠血清,培养24h后,收集细胞;采用RT-PCR及Western blot法检测血红素氧化酶-1(HO-1)、早期生长反应因子-1(Egr-1)的基因及蛋白表达水平。结果:与正常对照组比较,模型对照组LO2细胞HO-1、Egr-1 mRNA和蛋白表达有所增加;与模型对照组比较,自拟清肝降脂方三剂量组和对照药物组HO-1 mRNA及蛋白表达有所增高,Egr-1mRNA和蛋白表达明显降低;与对照药物组比较,清肝降脂方高剂量组HO-1 mRNA表达有所增高,Egr-1 mRNA表达有所降低。结论:Egr-1高表达可能会导致肝损伤机制之一;HO-1高表达可能是保护肝损伤机制之一;两者可能都是通过调节TNF-α表达来调节NAFLD细胞的损伤进展。清肝降脂方可明显改善LO2细胞Egr-1、HO-1 mRNA及蛋白的表达,可能为其治疗机制、临床诊断和判断进展参考指标。

Objective： By establishing the nonalcoholic fatty liver model of free fatty acid induced LO2 cells in vitro,we investigated the expression levels of HO-1,Egr-1 gene and protein in steatosis LO2 cells under the effection of the various doses of Qinggan Jiangzhi Decoction. Method： Establish the nonalcoholic fatty liver model of free fatty acid induced LO2 cells in vitro. Cultivate human liver LO2 cells with RPMI 1640 culture-medium which obtains 10% fetal bovine serum,and then divide them into 6 groups when the cells spread evenly： normal contrast group,model group,three groups（ g1,g2,g3） with high,middle and low dose of Qinggan Jiangzhi Decoction and contrast group with Shuilinjia,and each group had 6 bottles. Except normal contrast group,induce LO2 non alcoholic fatty liver cell model with 0. 5 mmol/L free fatty acid in the other 5 groups and then pick one bottle randomly and operate red oil O staining after being induced for 24 h. Observe the level of steatosis of LO2 cells. After confirming the success of inducing model,add 10% normal rat serum correspondingly into normal contrast group and model group,and add 10% rat serum with corresponding drug into g1,g2 and g3 and Shuijialin group. Collect cells after culturing for 24 h. Investigate the expression levels of HO-1,Egr-1gene and protein by RT-PCR and Western blot. Results： Compared with normal contrast group,the expression levels of HO-1,Egr-1 mRNA and protein in model group increased. Compared with model group,in three groups（ g1,g2,g3）and contrast drug group,the expression levels of HO-1 mRNA and protein increased and the expression levels of Egr-1 mRNA and protein decline obviously. Compared with contrast drug group,in high dose of Qinggan Jiangzhi Decoction group,the expression level of HO-1 mRNA increased and the expression level of Egr-1 mRNA and protein declined.Conclusion： The high expression of Egr-1 may lead to liver impairment and the high expression of HO-1 may be one of the protecting liver mechanisms. The two may regulate the process of NAFLD cells＇ impairment by regulating the expression of TNF-α. Qinggan Jiangzhi Decoction can dramatically improve the expression levels of HO-1 and Egr-1 gene and protein in LO2 cells and provide the reference index for the treatment mechanism,clinical diagnosis and estimating process.