大黄酚介导TLR4/NF-κB通路对IgA肾病大鼠肾损伤和免疫反应的调控作用

Regulation of Chrysophanol-mediated TLR4/NF-κB Pathway on Renal Injury and Immune Response in IgA Nephropathy Rats

目的探讨大黄酚(CP)对IgA肾病大鼠肾损伤和免疫反应的调控作用。方法将大鼠随机分为5组:对照组,IgA肾病模型(IgA)组, IgA+CP低、中、高剂量(2.5、5、10 mg/kg)组,除对照组外,其余组的大鼠采用脂多糖+牛血清蛋白+四氯化碳法建立IgA肾病模型,造模完成后,IgA+CP组腹腔注射不同剂量的大黄酚,对照组和模型组给予等容生理盐水,每天1次,连续给药4周。留取24 h尿液检测尿蛋白含量,取血检测血清肌酐和尿素氮;肾脏组织HE染色和TUNEL染色检测各组大鼠病理损伤和细胞凋亡;RT-PCR和Western blot检测肾组织Caspase-3和Caspase-9表达水平;酶联免疫吸附实验(ELISA)检测丙二醛(MDA)、过氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(Gpx)含量;ELISA和Western blot检测血清和肾组织白介素-1β、-6 (IL-1β、IL-6)和肿瘤坏死因子-α(TNF-α)蛋白表达;并采用RT-PCR和Western blot检测Toll样受体4(TLR4)、核转录因子-κB P65(NF-κB P65)表达水平和下游血管细胞黏附分子-1(VCAM-1)蛋白表达水平。结果 CP能剂量依赖性的降低IgA肾病大鼠尿蛋白、血清肌酐和尿素氮水平(P<0.01);改善模型大鼠肾脏组织病理损伤、降低细胞凋亡率(P<0.01),降低凋亡相关蛋白Caspase-3和Caspase-9的mRNA及蛋白表达水平(P<0.01);抑制MDA产生的同时,增加抗氧化酶Gpx、SOD活性(P<0.01);降低IL-1β、IL-6、TNF-α的血清水平和蛋白表达水平(P<0.01);下调TLR4、NF-κB P65和VCAM-1的表达水平(P<0.01)。结论大黄酚对IgA肾病大鼠起到保护作用,其机制可能是通过TLR4/NF-κB P65信号通路调节IgA肾病的免疫反应,减轻肾损伤。

Objective To investigate the regulatory effect and its mechanism of chrysophanol(CP)on renal injury and immune response in immunoglobin A(IgA)nephropathy rats.Methods IgA nephropathy rat model was established by the method of lipopolysaccharide+ bovine serum protein+carbon tetrachloride.Then the rats were randomly divided into 5 groups:control group,IgA group,IgA+low,medium and high dose of CP groups(2.5,5 and 10 mg/kg for each group respectively).IgA+CP groups were intraperitoneally injected with different doses of chrysophanol once a day for 4 weeks,and the control group and IgA group were given isovolumetric saline.Urine protein content,serum creatinine and urea nitrogen were detected at 24 hafter the administration of drugs.Kidney histopathological damage and apoptosis were measured by HE and TUNEL staining.The expression levels of Caspase-3 and Caspase-9 were detected by RT-PCR and Western blot;The contents of malondialdehyde(MDA),superoxide dismutase(SOD)and(glutathione peroxidase,Gpx)were detected by enzyme-linked immunosorbent assay(ELISA).The expression of interleukin-1β,-6(IL-1β,IL-6)and tumor necrosis factor(TNF-α)in serum and kidney tissue were measured by ELISA and Western blot,respectively.The mRNA and protein expression levels of toll-like receptro 4(TLR4),nuclear factor-κB P65(NF-κB P65)were also detected by RT-PCR and Western blot,and vascular cell adherin molecule(VCAM-1)protein level was deteted by Western blot.Results In IgA nephropathy rats,the administration of CP reduced proteinuria,serum creatinine and urea nitrogen in a dosedependent manner(P<0.01).It also improved the pathological damage of kidney tissue,reduced the apoptosis rate(P<0.01),and decreased the mRNA and protein expression levels of apoptosis-related proteins Caspase-3 and Caspase-9(P<0.01).CP inhibited MDA production while increased the activities of antioxidant enzymes Gpx and SOD(P<0.01),and decreased the levels of serum and protein expression of IL-1β,IL-6 and TNF-α(P<0.01),as well as the expression levels of TLR4,NF-κB P65 and VCAM-1(P<0.01).Conclusion Chrysophanol could play aprotective role in IgA nephropathy rats,and its mechanism may be related to alleviating kidney injury and regulating immune response.