黄芪多糖调控微RNA-16/NF-κB轴对阿霉素肾病大鼠多药耐药基因1和P-糖蛋白170的影响

Astragalus polysaccharides affects multidrug resistance gene 1 and P-glycoprotein 170 in adriamycin nephropathy rats via regulating microRNA-16/NF-κB axis

目的:肾病综合征是一种常见的泌尿系统疾病。本研究拟探讨黄芪多糖(astragalus polysaccharides,APS)通过调控微RNA(miR)-16/NF-κB轴,对阿霉素肾病(adriamycin nephropathy,AN)大鼠多药耐药基因1(multidrug resistance gene 1,MDR1)及其表达产物P-糖蛋白170(P-glycoprotein 170,P-gp170)的影响。方法:将72只Wistar雄性大鼠分成对照组、模型组、APS低剂量组、APS高剂量组、APS+miR-16拮抗剂组和APS+miR-16拮抗剂对照组,每组12只。使用尿液分析仪检测各组大鼠的尿液中尿蛋白(urine protein,UP)含量;全自动生化分析仪检测血清中胆固醇(cholesterol,CHOL)、白蛋白(albumin,ALB)、血尿素氮(blood urea nitrogen,BUN)和肌酐(creatinine,SCr)水平;ELISA试剂盒检测血清白介素(interleukin,IL)-6、IL-1β、肿瘤坏死因子α(tumor necrosis factorα,TNF-α)水平;HE染色试剂盒观察肾组织的形态学变化;real-time RT-PCR检测肾组织中miR-16和MDR1 mRNA水平;蛋白质印迹法检测肾组织中NF-κB p65、磷酸化NF-κB(p-NF-κB)p65和P-gp170蛋白表达水平;双荧光素酶验证miR-16与NF-κB的靶向关系。结果:对照组大鼠肾组织结构正常,无炎性细胞浸润;模型组大鼠肾组织中肾小球轻度充血,毛细血管狭窄或闭塞,炎性细胞浸润明显;APS低剂量组和高剂量组大鼠肾小球无明显充血,系膜细胞增殖明显减少,炎性细胞减少;与APS高剂量组和APS+miR-16拮抗剂对照组相比,APS+miR-16拮抗剂组大鼠肾组织结构损伤较为严重。与对照组相比,模型组大鼠UP、CHOL、BUN、SCr、IL-6、IL-1β、TNF-α水平及MDR1 mRNA、p-NF-κB p65、P-gp170蛋白质水平均明显升高(均P<0.05),ALB和miR-16水平均显著降低(均P<0.05)。与模型组相比,APS低剂量组和高剂量组大鼠UP、CHOL、BUN、SCr、IL-6、IL-1β、TNF-α水平及MDR1 mRNA、p-NF-κB p65、P-gp170蛋白质水平均明显降低(均P<0.05),ALB和miR-16水平均显著升高(均P<0.05)。与APS+miR-16拮抗剂对照组相比,APS+miR-16拮抗剂组大鼠UP、CHOL、BUN、SCr、IL-6、IL-1β、TNF-α水平及MDR1 mRNA、p-NF-κB p65、Pgp170蛋白质水平均明显升高(均P<0.05),ALB和miR-16水平均显著降低(均P<0.05)。MiR-16与NF-κB具有靶向调节关系。结论:APS可调节miR-16/NF-κB信号通路,影响MDR1、P-gp170水平,从而可减轻AN大鼠肾组织的炎症反应。

Objective:Nephrotic syndrome is a common disease of the urinary system.The aim of this study is to explore the effect of astragalus polysaccharides(APS)on multidrug resistance gene 1(MDR1)and P-glycoprotein 170(P-gp170)in adriamycin nephropathy rats and the underlying mechanisms.Methods:A total of 72 male Wistar rats were divided into a control group,a model group,an APS low-dose group,an APS high-dose group,an APS+micro RNA(mi R)-16 antagomir group and an APS+mi R-16 antagomir control group,with 12 rats in each group.Urine protein(UP)was detected by urine analyzer,and serum cholesterol(CHOL),albumin(ALB),blood urea nitrogen(BUN),and creatinine(SCr)were detected by automatic biochemical analyzer;serum interleukin-6(IL-6),IL-1β,tumor necrosis factorα(TNF-α)levels were detected by ELISA kit;the morphological changes of kidney tissues were observed by HE staining;the levels of mi R-16 and MDR1 m RNA in kidney tissues were detected by real-time RT-PCR;the expression levels of NF-κB p65,p-NF-κB p65,and Pgp170 protein in kidney tissues were detected by Western blotting;and dual luciferase was used to verify the relationship between mi R-16 and NF-κB.Results:The renal tissue structure of rats in the control group was normal without inflammatory cell infiltration.The renal glomeruli of rats in the model group were mildly congested,capillary stenosis or occlusion,and inflammatory cell infiltration was obvious.The rats in the low-dose and high-dose APS groups had no obvious glomerular congestion,the proliferation of mesangial cells was significantly reduced,and the inflammatory cells were reduced.Compared with the high-dose APS group and the APS+mi R-16 antagomir control group,there were more severe renal tissue structure damages in the APS+mi R-16 antagomir group.Compared with the control group,the levels of UP,CHOL,BUN,SCr,IL-6,IL-1β,TNF-α,and MDR1 m RNA,and the protein levels of p-NF-κB p65 and P-gp170 in the model group were significantly increased(all P<0.05);the levels of ALB and mi R-16 were significantly decreased(both P<0.05).Compared with the model group,the levels of UP,CHOL,BUN,SCr,IL-6,IL-1β,TNF-α,and MDR1 m RNA,and the protein levels of p NF-κB p65 and P-gp170 in the low-dose and high-dose APS groups were significant decreased(all P<0.05);and the levels of ALB and mi R-16 were significantly increased(both P<0.05).Compared with APS+mi R-16 antagomir control group,the UP,CHOL,BUN,SCr,IL-6,IL-1β,and TNF-αlevels,MDR1 m RNA,and the protein levels of p-NF-κB p65 and P-gp170 were significantly increased(all P<0.05).The levels of ALB and mi R-16 were significantly decreased in the APS+mi R-16 antagomir group compared with the APS+mi R-16 antagomir control group(both P<0.05).Conclusion:APS can regulate the mi R-16/NF-κB signaling pathway,thereby affecting the levels of MDR1 and P-gp170,and reducing the inflammation in the kidney tissues in the adriamycin nephropathy rats.