目的探讨JAZF1过表达对棕榈酸诱导肝细胞促炎因子肿瘤坏死因子(TNF)α、单核细胞趋化蛋白-1(MCP-1)及白细胞介素(IL)-8表达的影响。方法采用棕榈酸作用于肝细胞以构建肝细胞脂肪变性模型,并行油红O染色;将Ad—JAZF1转染入脂肪变性的肝细胞模型中,用RT-pCR法检测转染前后肝细胞JAZF1、TNFα、MCP-1及IL-8mRNA表达,Westem blot法检测JAZF1蛋白表达水平,酶联免疫吸附法检测上述促炎因子的分泌情况。对数据的比较,组内采用配对t检验;组问采用方差分析,进一步两两比较采用LSD法。结果与对照组相比,不同浓度(0、0.125、0.25、0.5、1mmol/L)棕榈酸诱导肝细胞促炎因子TNFα、MCP-1及IL-8mRNA的表达,其相对表达量随着浓度升高呈逐渐升高趋势(P〈0.01),但在0.5mmol/L和1mmol/L浓度组间,差异没有统计学意义。以0.25mmol/L棕榈酸作用于肝细胞0、6、12、24、48h,促炎因子TNFα、MCP-1及IL-8mRNA的相对表达水平随时间延长逐渐升高,在12h达高峰,随后逐渐下降,呈时间依赖性,且各时间组组间差异均有统计学意义(FTNFα=26.51,FMCP-1=57.20,FIL-8=353.85,P值均〈0.01)。与Ad-GFP空载体组相比,Ad—JAZFl转染组肝细胞JAZF1 mRNA和蛋白质的相对表达水平明显增高。JAZF1过表达可以抑制肝细胞中由棕榈酸诱导的TNFα、MCP-1及IL-8mRNA的表达,使其相对表达量分别降低89.69%、77.68%及83.21%护〈0.01),同时TNFα、MCP-1及IL-8相对分泌量也分别减少37%、37%及41%(P〈0.01)。结论棕榈酸能促进肝细胞促炎因子mRNA的表达;JAZF1基因过表达能够抑制棕榈酸诱导的肝细胞促炎因子的表达及释放。
Objective To investigate the effects of JAZF1 overexpression on the pro-inflammatory cytokines in hepatic steatosis. Methods The model of hepatic steatosis was established by incubating hepatocytes with palmitic acid (PA) at 0, 0.125, 0.25, 0.5 and 1 mM dose and for 0, 6, 12, 24 and 48 hours, after which recombinant adenovirus expressing JAZF 1 (Ad-JAZF 1) was introduced to up-regulate expression. Triglyceride level was measured by GOD. Cell viability was detected by CCK-8. The mRNA and protein expression of TNF-α, MCP-1, IL-8 and JAZF1 was examined by RT-PCR, ELISA, and western blotting. Results The PA-treated hepatocytes showed dose-dependent significant increases in TNF-α, MCP-1 and IL-8 mRNA expression for doses up to 0.25 mM; there were no significant increases for the highest doses of 0.5 and 1 raM. The 0.25 mM PA-treated hepatocytes showed time-dependent significant increases in TNF-α, MCP-1 and IL-8 mRNA expressions (FTNF-α = 26.51, FMCP-1 = 57.20, FIL-8 = 353.85, P 〈 0.01), with the maximum level reached at 12 h and followed by a gradual decrease with longer treatment times. JAZF1 mRNA and protein expression was markedly increased in hepatocytes infected with Ad-JAZF1 (P 〈 0.01). However, the AP-treated hepatocytes with JAZF1 overexpression showed down-regulation of TNF-α, MCP- 1 and IL-8 mRNA expression (decreased by 89.69%, 77.68%, and 83.21%, respectively) and secretion (37%, 37% and 41%, respectively, P 〈 0.01). Conclusion Stimulation ofhepatocytes by the PA fatty acid in vitro promotes mRNA expression of TNF-α, MCP-1 and IL-8, but overexpression of JAZF1 inhibits the PA- induced expression and secretion of these factors.