Nrf2基因敲除加剧单侧输尿管梗阻肾纤维化模型中巨噬细胞介导的炎症损伤作用

Aggravation of macrophage-mediated inflammatory damage by knockdown of Nrf2 in a unilateral ureteral obstruction renal fibrosis model

目的:研究Nrf2基因敲除对小鼠单侧输尿管梗阻(UUO)肾纤维化模型中炎症损伤的作用及其机制。方法:将实验小鼠分为4组:Nrf2野生型UUO组(Nrf2Wild-type UUO)、Nrf2野生型假手术组(Nrf2Wild-type Sham)、Nrf2敲除型UUO组(Nrf2KO UUO)和Nrf2敲除型假手术组(Nrf2KO Sham),每组6只。PAS与Masson染色分别用于检测肾组织损伤和总胶原累积程度;免疫组织化学染色检测巨噬细胞标志物CD68、IRF5表达;ELISA检测iNOS水平;qRT-PCR检测促炎症基因IL-1β、IL-6和TNF-α的mRNA表达;Western blot检测IRF5蛋白的表达。结果:PAS染色显示,Nrf2基因敲除没有明显引起肾组织损伤,但构建UUO模型后,其肾组织损伤明显加剧。Masson和免疫组织化学染色结果显示,Nrf2基因敲除可加重UUO模型中肾间质纤维化程度,并促进CD68阳性的巨噬细胞大量浸润,且ELISA结果显示iNOS表达水平也明显升高(P<0.05)。深入研究发现,Nrf2KO UUO组与Nrf2Wild-type UUO组相比,肾组织中促炎基因IL-1β、IL-6和TNF-αmRNA的表达水平明显升高(P<0.05)。同时,Western blot和免疫组织化学染色结果显示,Nrf2基因敲除可增加IRF5蛋白的表达(P<0.05)。结论:Nrf2基因敲除加剧了UUO肾纤维化模型中的炎症损伤作用,其机制可能是通过促进IRF5介导的炎症型巨噬细胞浸润,进而增加炎症因子的合成与释放。

Objective:To study the mechanism of Nrf2 gene knockout and its effect on inflammatory injury in unilateral ureteral obstruction(UUO)renal fibrosis model.Methods:The experimental mice were randomly divided into four groups,namely Nrf2 wild-type UUO group(Nrf2Wild-type UUO),Nrf2 knockout UUO group(Nrf2KO UUO),Nrf2 wild-type sham operation group(Nrf2Wild-type Sham)and Nrf2 gene knockout sham operation group(Nrf2KO Sham),with 6 animals in each group.PAS and Masson staining was used to detect tissue damage and total collagen accumulation;immunohistochemical staining was used to detect the expression of macrophage marker CD68 and IRF5;ELISA was used to detect the level of iNOS;qRT-PCR was used to detect the mRNA expression of pro-inflammatory genes IL-1β,IL-6 and TNF-α.The protein expression of IRF5 was determined by Western blot.Results:PAS staining showed that Nrf2 gene knockout did not cause renal tissue damage,but after the UUO model was constructed,the renal tissue damage significantly increased.The results of Masson and immunochemical staining showed that Nrf2 gene knockout aggravated the degree of interstitial fibrosis in the UUO model,and promoted the infiltration of CD68-positive macrophages,accompanied with the enhancement of iNOS level.Further study found that compared with Nrf2Wild-type UUO group,the mRNA expression levels of IL-1β,IL-6,and TNF-αin the kidney tissues in the Nrf2KO UUO group were significantly increased(P<0.05).At the same time,Western blot and immunochemical staining results showed that Nrf2 gene knockout increased the expression of IRF5 protein(P<0.05).Conclusion:Nrf2 gene knockout aggravates the inflammatory damage in the UUO renal fibrosis model,and its mechanism may be related to the promotion of IRF5-mediated infiltration of inflammatory macrophages,thereby increasing the synthesis and release of inflammatory factors.