NF-κB信号途径在小鼠狼疮性肾炎发病中的可能作用

Effect of NF-κB signal pathway in murine lupus nephritis

目的:探讨NF-κB信号途径在小鼠狼疮性肾炎发病中的可能作用。方法:选取16周龄的雄性BXSB小鼠(狼疮性肾炎模型组)和同周龄C57BL/6小鼠(正常对照组)作为研究对象,透射电镜和PAS染色观察肾组织的超微结构形态改变;RT-PCR技术检测小鼠全血中HMGB1mRNA的表达变化。采用ELISA方法检测血清中HMGB1蛋白浓度;免疫组织化学检测肾组织中HMGB1和PCNA蛋白的表达变化;Western blot和流式细胞术检测肾组织中RAGE、p-NF-κB和IκB蛋白的表达。结果:16周时,与正常的C57BL/6小鼠相比,BXSB小鼠血清中BUN水平及尿中微球白蛋白水平明显升高;与正常的C57BL/6小鼠相比,BXSB小鼠全血中HMGB1mRNA水平和血清中HMGB1蛋白浓度明显升高;16周时,与正常的C57BL/6小鼠相比,BXSB基底膜明显增厚,部分足突融合,内皮细胞下可见团块状电子致密物沉积;与正常的C57BL/6小鼠相比,BXSB小鼠肾组织的肾小球中可见较多的PCNA阳性表达,肾小管上皮细胞核内也可见少量的表达;BXSB小鼠肾组织中HMGB1蛋白表达升高,HMGB1蛋白尤其在细胞增生明显而肥大的肾小球呈高表达,主要位于细胞浆和细胞外;而在C57BL/6小鼠肾脏组织中以小管细胞核表达为主;与对照组相比,BXSB小鼠肾组织p-NF-κB和RAGE蛋白表达明显升高;而IκB蛋白表达明显降低;HMGB1蛋白与p-NF-κB蛋白表达呈显著正相关(r=0.833,P=0.000);p-NF-κB蛋白与RAGE蛋白表达呈显著正相关(r=0.621,P=0.018);HMGB1蛋白与RAGE蛋白表达呈显著正相关(r=0.848,P=0.000);p-NF-κB蛋白与IκB蛋白表达呈显著负相关(r=-0.759,P=0.002)。结论:HMGB1在小鼠狼疮性肾炎中的致炎作用可能部分通过结合其受体RAGE,激活NF-κB信号途径,促进肾小球固有细胞的增生,从而导致增生性肾小球肾炎形成而实现的。

Objective： To investigate the expression and mechanism of NF-κB signal pathway in murine lupus nephritis. Methods： The BXSB mice as well as C57BL/6 of 16 weeks were used. Transmission electron microscope and PAS were used to detect the pathological change of renal tissue. RT-PCR and ELISA were used to detect the expression of HMGBI mRNA and protein. The expression of HMGB1, p- NF-κB, RAGE, IκB and PCNA protein was detected by immunohistochemical stain, FCM and Western blot. Results：The level of BUN in serum and Micro-albumin in urine of BXSB mice was higher than that in C57BL/6 mice.The expression of HMGB1 mRNA and HMGB1 protein level in peripheral blood increased significantly in BXSB group. Compared with those in control group, electron microscopy and PAS revealed the thickness of glomerular basement membrane（ GBM）, fusion of foot processes partly of epithelial dell and subepithelial electron-dense deposits in the renal tissue of BXSBA mice. Compared with that of control group, expression of PCNA was higher in glomeruli of BXSB mouse. HMGB1 protein overexpression localized in cytoplasm and extracellular milieu, especially in proliferative glomeruli in BXSB group, while the HMGB1 protein primarily cofined to the nuclear of tubule in control group. In BXSB group, the expression of p-NF-κB and RAGE increased, while the expression of IκB decreased. There were positive correlation between the expression of HMGB 1, RAGE and p- NF-κB protein （ r = 0.833, 0.621,0. 848, P 〈 0.01 ）,while the expression of p-NF-κB protein negatively correlated with that of IκB. Conclusion： HMGBI could activate NF-κB through combining with its receptor-RAGE, induce the fonn of proliferative glomerulonephritis by promoting the proliferation of inherent cell of glomeruli, which may play an important role in the murine lupus nephritis.