NF-κB介导大鼠肾小球系膜细胞Visfatin对促纤维化因子和细胞外基质表达的影响

Visfatin-induced expression of profibrogenic cytokines and extracellular matrix in rat mesangial cell through the NF-κB pathway

目的观察大鼠肾小球系膜细胞中内脏脂肪素(Visfatin)对促纤维化因子和细胞外基质(ECM)表达的影响并探讨核因子-κB(NF-κB)在此过程中的作用。方法分别构建Visfatin表达质粒及Visfatin RNAi质粒,将细胞分为八组:A组:正常糖对照组;B组:正常糖+NF-κB特异性抑制剂吡咯烷二硫基甲酸酯(PDTC)组;C组:过表达Visfatin组;D组:过表达Visfatin+PDTC组;E组:空白表达载体组;F组:空白表达载体+PDTC组;G组:Visfatin RNAi组;H组:空白沉默载体组。比较过表达或沉默Visfatin前后促纤维因子及细胞外基质基因及蛋白表达的变化。用Realtime-PCR方法检测转录生长因子β(TGF-β)、结缔组织生长因子(CTGF)、纤维蛋白溶解酶原激活物抑制剂(PAI-1)、纤维连接蛋白(FN)、I型胶原(Col I)、IV胶原(Col IV)等基因表达的变化,用Western Blot检测Visfatin、CTGF、FN等蛋白的表达,用电泳迁移率变动分析法(EMSA)检测NF-κB活性;以及通过观察PDTC处理对照组、过表达Visfatin组、空白表达载体组前后上述指标的变化。结果大鼠肾系膜细胞过表达Visfatin后,NF-κB活性、Visfatin、TGF-β、CTGF、PAI-1、FN、Col I、Col IV的m RNA表达量及Visfatin、CTGF、FN蛋白表达量显著升高(P<0.05)。下调Visfatin表达后,NF-κB活性、Visfatin、TGF-β、CTGF、PAI-1、FN、Col I、Col IV的m RNA表达量及Visfatin、CTGF、FN蛋白表达量均显著降低(P<0.05)。PDTC处理的各组,NF-κB活性、TGF-β、CTGF、PAI-1、FN、Col I、Col IV表达量与对应的未经PDTC处理组相比显著降低(P<0.05)。结论在大鼠肾小球系膜细胞中,Visfatin可通过NF-κB通路诱导促纤维化因子和细胞外基质的表达。

Objective To investigate whether visfatin induced the expression of profibrogenic eytokines and extracellular matrix via NF-κB pathway in rat mesangial cell. Methods The rat mesangial cells were transfected with the plasmids by usingLipofectamine method to overexpress or silence visfatin, or treated with pyrrolidinedithiocarbamate （PDTC）. The mRNA expression levels of visfatin, transforming growth factor-β （TGF-β）, connective tissue growth factor （CTGF）, plasminogen activator inhibitor-1 （PAI-1）, fibronection（FN）, collagen type I（Col I）, collagen typeIV （Col IV） were measured byrealtime PCR. The protein expression levels of visfatin, CTGF, FN were measured by Western Blot.The NF- κB activity was detected by electrophoretic mobility shift assay. Results The NF-κB activity, and the mRNA levels of visfatin, TGF-β, CTGF, PAI-1, FN, Col I, Col IV and the protein levels of visfatin, CTGF, FN in the overexpression of visfatin group were significantly higher than those in the control group. The opposite effect was observed in the visfatin siRNA group.When the control group, overexpression of visfatin group, or the group transfected with blank expression plasmids were treated with PDTC, the NF-κB activity, and the mRNA levels of TGF-β, CTGF, PAI-1, FN, Col I, Col IV and the protein levels of CTGF, FN were significantly reduced compared with the control group. Conclusion Visfatin upregulated the expression of profibrogenic cytokines and ECM probably via the NF-κB pathway.