基于核转录因子-κB信号通路探讨清热止血方联合雷公藤多苷治疗紫癜性肾炎大鼠的作用机制

Mechanism of Qingre Zhixue Decoction combined with tripterygium glycosides on purpura nephritis rats based on nuclear factor-κB signaling pathway

目的:探讨清热止血方联合雷公藤多苷对紫癜性肾炎(HSPN)核转录因子-κB(NF-κB)信号通路的影响及作用机制。方法:72只SD大鼠随机分为空白组12只、造模组60只,采用复合瘀热模型法制备HSPN的动物模型,将成功造模大鼠随机分为模型组、清热组(清热止血方3.17 mg/kg)、雷公藤组(雷公藤多苷片4.69 mg/kg)、联合组(清热止血方3.17 mg/kg+雷公藤多苷片4.69 mg/kg)、激素组(醋酸泼尼松2.34 mg/kg),分别给予相应药物,2次/d,持续4周。采用酶标法检测尿红细胞计数、尿蛋白定量及肝肾功能;采用免疫荧光法检测肾脏组织中IgA沉积;采用透射电镜观察大鼠肾小球系膜区的病变情况;采用Western blot及qRT-PCR检测大鼠肾组织IKKβ、IκBα、P65、P50蛋白及mRNA的表达水平。结果:与空白组比较,模型组尿红细胞计数和尿蛋白定量显著升高(P<0.05);与模型组比较,各药物组尿红细胞计数和尿蛋白定量显著下降(P<0.05)。与空白组比较,模型组可见肾小球系膜区较强的IgA表达,肾小球系膜增生,系膜区电子致密物沉积,基底膜弥漫性增宽,内皮细胞肿胀,足突部分融合;与模型组比较,各药物组肾小球系膜区IgA表达减弱,肾小球系膜增生、基底膜增厚及内皮细胞肿胀情况减轻,仍可见少量足突融合。与空白组比较,模型组肾组织中IKKβ、P65、P50蛋白及mRNA的表达显著升高(P<0.05),IκBα蛋白及mRNA的表达显著降低(P<0.05);与模型组比较,各药物组肾组织中IKKβ、P65、P50蛋白及mRNA的表达显著降低(P<0.05),IκBα蛋白及mRNA的表达显著升高(P<0.05)。结论:清热止血方联合雷公藤多苷可降低HSPN大鼠的血尿及蛋白尿水平,其机制可能是通过抑制NF-κB信号通路的激活,减少炎症反应。

Objective:To investigate the effects and mechanisms of antipyretic hemostasis combined with triptolide on the nuclear factor-κB(NF-κB)signaling pathway in rats with Henoch-Schönlein purpura nephritis(HSPN).Methods:A total of 72 SD rats were randomly divided into blank group(n=12)and modeling group(n=60),and animal models of HSPN were prepared by the composite stasis models method,and the successful models were randomly divided into model group,clear heat group(Qingre Zhixue Decoction 3.17 mg/kg),triptolide group(Triptolide Polyside Tablets 4.69 mg/kg),combined group(Qingre Zhixue Decoction 3.17 mg/kg+Triptolide Polyside Tablets 4.69 mg/kg)and hormone group(Prednisone Acetate 2.34 mg/kg),respectively,given the corresponding drug intervention,2 times/d,for 4 weeks.The microplate method was used to detect urine red blood cell count,urine protein quantification and liver and kidney function;the immunofluorescence method was used to detect the deposition of IgA in kidney tissue;the transmission electron microscopy was used to observe the lesions of the mesangial region of rats,the Western blot and qRT-PCR was used to detect the expression levels of proteins such as IKKβ,IκBα,P65,P50 and other proteins and mRNA in rat kidney tissue.Results:Compared with the blank group,the quantitative urine red blood cell count and urine protein in the SD rats in the model group were increased(P<0.05);Compared with the model group,the urine red blood cell count and urine protein quantification decreased in each drug group(P<0.05).Compared with the blank group,the model group could see the strong IgA expression of the mesangial region of the glomeruli,the mesangial hyperplasia of the mesangial membrane could be seen,the electron compaction deposition could be seen in the mesangial area,the basal membrane was diffusely widened,the endothelial cells were swollen,and the partial fusion of the foot process was still seen;Compared with the model group,the IgA expression of the glomerular mesangial region of each drug group was weakened,and the mesangial hyperplasia of the mesangial membrane,the thickening of the basement membrane and the swelling of endothelial cells were reduced under transmission electron microscopy,and a small amount of foot process fusion can still be seen.Compared with the blank group,the protein and mRNA expressions of IKKβ,P65,P50 in the kidney tissue of the model group were increased(P<0.05),and the protein and mRNA expression of IκBαwas reduced(P<0.05);Compared with the model group,the protein and mRNA expressions of IKKβ,P65,P50 in the kidney tissue of each drug group were reduced(P<0.05),while the protein and mRNA expression of IκBαwas increased(P<0.05).Conclusion:The combination of Qingre Zhixue Decoction and triptolide polyside can reduce the hematuria and proteinuria levels of rats in HSPN rats,and the mechanism might be to reduce the inflammatory response by inhibiting the activation of the NF-κB signaling pathway.