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[摘要]
目的 从抑制早期生长反应因子-1(Egr-1)介导的白细胞介素8(IL-8)表达的角度探讨强肝胶囊对大鼠非酒精性脂肪肝的改善作用。方法 Wistar大鼠随机分为对照组、模型组和强肝胶囊组,每组各10只。模型组和强肝胶囊组喂食高脂饲料,建立高脂血症非酒精性脂肪肝模型,造模同时强肝胶囊组大鼠ig强肝胶囊内容物0.53 mg/g,给药体积为1 mL/100 g体质量,模型组ig等量生理盐水。每周给药6 d,持续8周。治疗结束取动物肝脏,氧化酶法测定肝组织中三酰甘油(TG)、总胆固醇(TC)水平;实时荧光定量PCR(RT-qPCR)法检测Egr-1和IL-8 mRNA表达;Western blotting法测定Egr-1蛋白表达;ELISA法测定肝组织中IL-8水平。将大鼠肝脏HepG2细胞分别进行0.25 mmol/L油酸处理、转染Egr-1 siRNAs并油酸处理、转染pcDNA3.1-Egr-1阳性表达质粒并油酸处理后,RT-qPCR法检测细胞中Egr-1和IL-8 mRNA表达,WB法检测Egr-1蛋白表达,ELISA法检测培养液上清中IL-8水平。结果 经过8周治疗,强肝胶囊组TG和、TC水平明显下降(P<0.01);Egr-1 mRNA、Egr-1蛋白表达和IL-8 mRNA、IL-8水平均明显下降(P<0.01)。Egr-1 siRNA#1、siRNA#2能明显降低Egr-1表达水平,采用siRNA干扰Egr-1基因,并用油酸处理HepG2细胞后,细胞内和培养液上清中IL-8水平较对照细胞显著降低(P<0.01)。转染Egr-1阳性表达质粒导致Egr-1表达量上升(P<0.01),IL-8表达也同步升高(P<0.01)。结论 Egr-1可正向调节IL-8表达。强肝胶囊通过抑制Egr-1介导的IL-8表达,达到改善非酒精性脂肪肝的作用。
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[Abstract]
Objective To discuss the improvement of Qianggan Capsules on non-alcoholic fatty liver in rats based on regulation of Egr-1 on IL-8 expression. Methods Wistar rats were randomly divided into the control, model, and Qianggan Capsules groups, and each group had 10 rats. Rats in model and Qianggan Capsules groups were fed high-fat diet to establish hyperlipidemic nonalcoholic fatty liver model. At the same time, the rats in Qianggan Capsules group were ig administered with Qianggan Capsules 0.53 mg/g, and the volume was 1 mL/100 g body weight. And rats in model group were given equivalent saline. Rats were treated 6 d weekly for 8 weeks. After treatment, liver tissues were separated. The levels of three glycerol oxidase (TG) and total cholesterol (TC) level in liver tissue were measured by oxidase method. Egr-1 and IL-8 expression of mRNA were detected by real-time fluorescence quantitative PCR (RT-qPCR) method, Egr-1 protein expression was detected by Western blotting (WB) method, and IL-8 levels were detected by ELISA method. The HepG2 cells were treated with 0.25 mmol/L oleic acid, transfected with Egr-1 siRNAs and then treated with oleic acid, or transfected with pcDNA3.1-Egr-1 and then treated with oleic acid. The expression of Egr-1 and IL-8 mRNA in the cells was detected by RT-qPCR method, the expression of Egr-1 protein was detected by Western blotting method, and IL-8 level in the supernatant of the culture liquid was detected by ELISA method. Results After treatment for 8 weeks, the levels of TG and TC were significantly decreased in Qianggan Capsules group (P < 0.01), and the expression of Egr-1 mRNA and Egr-1 protein, IL-8 mRNA and IL-8 levels were significantly decreased (P < 0.01). Egr-1 siRNA#1 and siRNA#2 could significantly decrease the levels of Egr-1 expression. After siRNA interference with Egr-1 gene and HepG2 treated with oleic acid, IL-8 levels in cells and culture supernatants were significantly lower than those in control cells (P < 0.01). The expression of Egr-1 was increased by transfection of Egr-1 positive expression plasmid (P < 0.01), and IL-8 expression also increased (P < 0.01). Conclusion Egr-1 can positively regulate the expression of IL-8. Qianggan Capsules can improve the effect of nonalcoholic fatty liver by inhibiting the expression of Egr-1 mediated IL-8.
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[基金项目]
天津市卫生和计划生育委员会中医中西医结合科研课题(2015107)