山茱萸总苷对MRL/lpr狼疮性肾炎小鼠肾炎症反应的改善作用及机制

Effect and mechanism of cornus officinalis glycosides on renal inflammation in mice lupus-prone MRL/lpr mice

目的探讨山茱萸总苷对MRL/lpr狼疮性肾炎小鼠肾炎症反应的改善作用及可能的作用机制。方法将40只MRL/lpr狼疮性肾炎小鼠按随机数字表法分为模型组、山茱萸总苷低、中、高剂量组(27.5、55、110 mg/kg),每组10只,另取10只C57BL/6小鼠作为对照组。灌胃给药,1次/天,连续4周。于药物干预前及干预后第2、4周,收集24 h尿液,测定尿蛋白含量。药物干预结束后,取小鼠血清及肾脏组织,采用试剂盒检测血清肌酐(Scr)、尿素氮(BUN)和抗双链DNA(dsDNA)抗体水平;ELISA法检测血清中白细胞介素(IL)-1β、IL-6和肿瘤坏死因子-α(TNF-α)表达水平;流式细胞术检测外周血T淋巴细胞亚群变化;HE染色观察肾组织病理学变化;Western blot检测肾组织中Toll样受体4(TLR4)、髓样分化因子-88(MyD88)、核转录因子(NF-κB)p65和p-NF-κB p65蛋白表达。结果与对照组比较,模型组小鼠肾组织损伤严重,24 h尿蛋白、Scr、BUN和抗dsDNA抗体以及IL-1β、IL-6和TNF-α水平均升高(P<0.01),外周血CD4+T/CD8+T淋巴细胞值降低(P<0.01),肾脏组织中TLR4、MyD88和p-NF-κB p65蛋白表达水平均上调(P<0.01);与模型组比较,山茱萸总苷各剂量组小鼠肾组织损伤改善明显,24 h尿蛋白、Scr、BUN和抗dsDNA抗体及IL-6、TNF-α和IL-1β水平均降低(P<0.01),外周血CD4+T/CD8+T淋巴细胞值增加(P<0.01),肾组织中TLR4、MyD88和p-NF-κB p65蛋白表达均下调(P<0.01),其中高剂量组小鼠各指标变化最为显著;而各组NF-κB p65蛋白表达无显著性差异。结论山茱萸总苷对MRL/lpr狼疮性肾炎小鼠肾炎症反应具有一定改善作用,其机制可能与抑制TLR4/NF-κB信号通路有关。

Objective To investigate the improvement effect and possible mechanism of cornus officinalis glycosides on MRL/lpr lupus nephritis.Methods Forty mice lupus-prone MRL/lpr mice were divided into model group,cornus officinalis glycosides low-dose,medium-dose,high-dose group(27.5,55,110 mg/kg),with 10 mice in each group,and 10 C57BL/6 mice were taken as the control group.Gastric administration was carried out 1 time/day for 4 weeks.24-hour urine was collected before drug intervention and 2 and 4 weeks after intervention to determine the content of urine protein.After the drug intervention,mouse serum and kidney tissues were taken and the levels of serum creatinine(Scr),urea nitrogen(BUN)and anti-double-stranded DNA(dsDNA)antibodies were detected by kits.The levels of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in serum were detected by ELISA.The changes in peripheral blood T lymphocyte subsets were detected by Flow cytometry;The changes of renal histopathology were observed by HE staining;The protein expression levels of toll-like receptor 4(TLR4),myeloid differentiation factor-88(MyD88),nuclear transcription factor(NF-κB)p65,and p-NF-κB p65 in kidney tissue were detected by Western blotting.Results Compared with the control group,the changes of renal histopathology of the model group was severe,and the levels of 24-hour urine protein,Scr,BUN and anti-dsDNA antibodies,IL-1β,IL-6 and TNF-αsignificantly increased(P<0.01),the peripheral blood CD4+T/CD8+T lymphocyte ratio was significantly reduced(P<0.01),and the protein expression levels of TLR4,MyD88 and p-NF-κB p65 in kidney tissue were significantly up-regulated(P<0.01);Compared with the model group,the changes of renal histopathology of each dose group of cornus officinalis glycosides were significantly improved,the levels of 24-hour urine protein,Scr,BUN and anti-dsDNA antibodies,IL-1β,IL-6 and TNF-αwere significantly reduced(P<0.01),the peripheral blood CD4+T/CD8+T lymphocyte ratio significantly increased(P<0.01),and the protein expression levels of TLR4,MyD88 and p-NF-κB p65 in renal tissue were significantly down-regulated(P<0.01).Among them,the high-dose group had the most significant changes in each index.While there was no significant difference in the expression of NF-κB p65 protein in the groups.Conclusion Cornus officinalis glycosides have a certain effect on renal inflammatory response in mice lupus-prone MRL/lpr mice,and the mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway.