基于Nrf2/Keap1/HO-1和P38MAPK/P53/P21通路探究大补阴丸对IgAN小鼠的作用机制

Probing the mechanism of action of Dazhuanyinwan in IgAN mice based on Nrf2/Keap1/HO-1 and P38MAPK/P53/P21 pathways

关键词:  关键词：IgA肾病  大补阴丸  Nrf2/Keap1/HO-1通路  P38MAPK/P53/P21通路  氧化应激

Key words:  IgA nephropathy  Dabuyin pill  Nrf2/Keap1/HO-1 pathway  P38MAPK/P53/P21 pathway  oxidative stress

摘要:

目的:观察大补阴丸治疗IgA肾病(IgAN)模型小鼠的疗效，基于Nrf2/Keap1/HO-1和P38MAPK/P53/P21信号通路探究其作用机制。方法:采用“牛血清白蛋白+四氯化碳+脂多糖”联用法构建IgAN小鼠模型，72只小鼠随机分为对照组、模型组、西药组（4 mg·kg-1）、大补阴丸高剂量组（4.68 g·kg-1）、中剂量组（2.34 g·kg-1）、低剂量组（1.17 g·kg-1），每组8只，均连续给药9 w。观察小鼠的一般情况及计算肾脏指数，测定24h-UTP、BUN、血清SCr，采用HE染色观察肾组织病理变化，采用免疫荧光（IF）法测定大补阴丸对IgAN小鼠肾脏系膜区IgA沉积的影响，对小鼠肾脏氧化应激水平与炎症因子进行检测；RT-PCR、Western Blot法测定肾脏Nrf2、Keap1、AKT、HO-1、p-P38MAPK、P53、P21表达。结果：与模型组相比，西药组、大补阴丸中剂量组肾脏指数均升高，具显著差异（P<0.05）。与模型组相比，西药组、大补阴丸高、中、低剂量组24h-UTP、BUN、Scr含量均有不同程度下降（P<0.05）。与对照组比较，模型组小鼠肾小球可见大量炎症细胞浸润，肾组织系膜细胞和基质增生显著，间质纤维化明显，还伴有管腔狭窄、球囊黏连等病理损伤；与模型组比较，西药组和大补阴丸高、中、低剂量组病理损伤均有不同程度减轻；对照组IgA沉积为阴性，较模型组，西药组、大补阴丸中剂量组IgA沉积均明显降低（P＜0.01），大补阴丸高剂量组IgA沉积降低（P＜0.05）。与模型组相比，西药组、大补阴丸高、中剂量组肾脏T-SOD、GSH-PX升高、MDA明显降低；西药组与中剂量MCP-1、IL-1β、IL-18均降低（P＜0.05），大补阴丸高剂量组MCP-1降低（P＜0.05）。与模型组比较，西药组和大补阴丸各剂量组Nrf2、HO-1 mRNA及蛋白表达明显升高（P＜0.05），Keap1、p-P38MAPK、P53、P21mRNA及蛋白表达下降。结论：大补阴丸可能通过作用于Nrf2/Keap1/HO-1和P38MAPK/P53/P21信号通路，改善IgA肾病模型小鼠的肾组织损伤，减轻肾损伤程度。

Abstract:

Objective: to observe the therapeutic effect of Dabu Yin Pill on IgA nephropathy (IgAN) model mice and explore its mechanism based on Nrf2/Keap1//HO-1 and P38MAPK/P53/P21 signal pathway. Methods: the IgAN mouse model was established by the combination of bovine serum albumin, carbon tetrachloride and lipopolysaccharide. 72 mice were randomly divided into control group, model group, western medicine group (4 mg kg-1), high dose group (4.68g kg-1), middle dose group (2.34g kg-1) and low dose group (1.17g kg-1). The general condition and renal index of mice were observed, 24h-UTP, BUN and serum SCr were measured, the pathological changes of kidney were observed by HE staining, the effect of Dabu Yin Pill on IgA deposition in Mesangial area of IgAN mice was determined by immunofluorescence (IF), the level of oxidative stress and inflammatory factors in kidney were detected, and the expressions of Nrf2, Keap1, AKT, HO-1, p-P38MAPK, p53 and P21 in kidney were measured by RT-PCR and Western Blot. Results: compared with the model group, the renal index in the western medicine group and the middle dose Dabuyin pill group increased significantly (P < 0.05). Compared with the model group, the contents of 24h-UTP, BUN and Scr in the western medicine group, high, middle and low dose groups decreased in different degrees (P < 0.05). Compared with the control group, a large number of inflammatory cells infiltration, Mesangial cell and matrix proliferation, interstitial fibrosis, lumen stenosis and balloon adhesion were observed in the model group. Compared with the model group, the pathological damage was alleviated in different degrees in the western medicine group and the high, middle and low dose groups of Dabuyin pills. The IgA deposition in the control group was negative, which was significantly lower than that in the model group, the western medicine group and the middle dose group of Dabuyin pill, and the IgA deposition in the high dose group of Dabuyin pill decreased (P < 0.05). Compared with the model group, T-SOD, GSH-PX and MDA of kidney in western medicine group, high and middle dose groups of Dabuyin pill increased and MDA decreased significantly, MCP-1, IL-1 β and IL-18 decreased in western medicine group and middle dose group, and MCP-1 decreased in high dose group of Dabuyin pill. Compared with the model group, the expression of Nrf2, HO-1 mRNA and protein in western medicine group and Dabuyin pill group increased significantly, while the expression of Keap1, p-P38MAPK, p53, P21mRNA and protein decreased.Conclusion: Dabuyin Pill may improve the renal tissue injury and reduce the extent of kidney damage in IgA nephropathy model mice by acting on Nrf2/Keap1/HO-1 and P38MAPK/P53/P21 signal pathway.