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目的观察大鼠肾小球系膜细胞中内脏脂肪素(Visfatin)对肾素血管紧张素系统(RAS)mRNA表达的影响及探讨其机制。方法分别构建Visfatin表达载体质粒及Visfatin RNAi表达载体质粒,将细胞分为8组:正常糖对照组、正常糖+NF-κB特异性抑制剂吡咯烷二硫基甲酸酯(PDTC)组、过表达Visfatin组、过表达Visfatin+PDTC组、空白过表达载体组、空白过表达载体+PDTC组、Visfatin RNAi组及空白沉默载体组,用Realtime-PCR方法检测血管紧张素原(AGT)、血管紧张素转化酶(ACE)、血管紧张素Ⅱ1型受体(AT1R)、血管紧张素Ⅱ2型受体(AT2R)等RAS相关基因表达,使用电泳迁移率变动分析法(EMSA)检测NF-κB活性,比较各组间基因表达及NF-κB活性的差异;以及通过观察PDTC处理对照组、过表达Visfatin组、空白过表达载体组前后上述指标的变化。结果细胞过表达Visfatin后,NF-κB活性、Visfatin、AGT、AT1R mRNA表达量显著升高,分别为正常糖对照组的1.52、11.42、2.85、1.25倍(P均<0.01);转染RNAi质粒pSIREN-Visfatin/sh RNA后,NF-κB活性、Visfatin、AGT、AT1R mRNA表达量显著降低,分别为正常糖对照组的10.90%、26.83%、30.00%、73.47%(P均<0.01)。经PDTC处理的各组NF-κB活性、AGT mRNA表达量与相应的未经PDTC处理组相比显著降低(P<0.01),正常糖+PDTC干预组NF-κB活性、AGT mRNA表达量是正常糖对照组的21.60%、34.59%;过表达Visfatin+PDTC干预组的NF-κB活性、AGT mRNA表达量是过表达Visfatin组的37.96%、19.72%;空白过表达载体+PDTC干预组NF-κB活性、AGT mRNA表达量是空白表达载体组的52.53%、33.08%,差异均具有统计学意义(P<0.01)。结论在大鼠肾小球系膜细胞中,Visfatin可通过NF-κB引起RAS相关基因表达的变化。
Objective To investigate the effect of visfatin on the expression of renin-angiotensin system (RAS) mRNA in rat mesangial cells and to explore its mechanism. Methods The plasmids of Visfatin expression vector and Visfatin RNAi expression vector were constructed and divided into 8 groups: normal control group, normal glucose + PDTC group, a specific inhibitor of NF-κB Visfatin group, overexpression Visfatin + PDTC group, blank overexpression vector group, blank overexpression vector + PDTC group, Visfatin RNAi group and blank silencing vector group. Realtime-PCR was used to detect angiotensinogen (AGT), vascular tone (ACE), angiotensin Ⅱ type 1 receptor (AT1R) and angiotensin Ⅱ type 2 receptor (AT2R) were detected by flow cytometry. The activity of NF-κB was detected by electrophoretic mobility shift assay (EMSA) The differences of gene expression and NF-κB activity among different groups were compared. The changes of these indexes were observed before and after PDTC-treated control group, Visfatin-overexpression group, and blank overexpression vector group. Results After overexpression of Visfatin, the expression of NF-κB, Visfatin, AGT and AT1R were significantly increased, which were 1.52,11.42,2.85 and 1.25 times higher than those in normal control group (all P <0.01) The expression of NF-κB, Visfatin, AGT and AT1R mRNA were significantly decreased in pSIREN-Visfatin / sh RNA group, which were 10.90%, 26.83%, 30.00% and 73.47% of those in normal control group respectively (all P <0.01). The PDTC-treated NF-κB activity and AGT mRNA expression levels were significantly lower than those without PDTC treatment (P <0.01). NF-κB activity and AGT mRNA expression were normal in normal glucose + PDTC intervention groups 21.60% and 34.59% of the control group, respectively. The overexpression of Visfatin + PDTC in the intervention group, the NF-κB activity, AGT mRNA expression was 37.96%, 19.72% The activity of AGT mRNA was 52.53% and 33.08% of the blank vector group, respectively, with significant difference (P <0.01). Conclusion Visfatin can induce the expression of RAS related gene in rat glomerular mesangial cells through NF-κB.