氨酰4-羟化酶A2通过抑制炎症反应缓解肾纤维化

Prolyl 4-hydroxylase subunit alpha 2 alleviates renal fibrosis by inhibiting inflammatory response

目的:利用体内和体外模型探讨氨酰4-羟化酶A2(P4HA2)对肾间质纤维化的作用及其机制。方法:采用输尿管结扎模型构建动物肾纤维化,使用免疫组化技术检测P4HA2表达水平;采用不同梯度浓度TGF-β刺激HK2细胞构建细胞肾纤维化模型,利用免疫印迹和细胞免疫荧光检测P4HA2蛋白表达变化水平;利用腺病毒转染HK2细胞,使用免疫印迹和酶联免疫吸附试验技术探索P4HA2在细胞水平对NF-κB信号通路和下游炎症因子的影响。结果:与假手术组小鼠比较,模型组小鼠P4HA2表达量显著增加,且集中于肾小管细胞胞质与胞核中。使用50 ng/mL TGF-β刺激HK-2细胞24 h后P4HA2表达水平显著升高,且细胞形态由鹅卵石形变成针尖样长梭形;与转染对照组(scramble)比较,sh-P4HA2抑制NF-κB信号通路相关蛋白表达量,减少下游炎症因子IL-1β、IL-6和TNF-α水平。结论:P4HA2可能调控NF-κB信号通路,抑制机体炎症反应,缓解肾间质纤维化发展。

Objective:To investigate the effect and mechanism of aminoacyl 4-hydroxylase A2(P4HA2)on renal interstitial fibrosis using in vivo and in vitro models.Methods:The ureteral ligation model was used to construct animal renal fibrosis,and the expression level of P4HA2 was detected by immunohistochemistry;Different gradient concentrations of TGF-βwere used to stimulate HK2 cells to construct a cellular renal fibrosis model,and the expression level of P4HA2 protein was detected by Western Blot and immunofluorescence;HK2 cells were transfected with adenovirus(scramble group and sh-P4HA2 group),and Western Blot and enzyme-linked immunosorbent assay were used to explore the effects of P4HA2 on the NF-κB signaling pathway and downstream inflammatory factors at the cellular level.Results:Compared with the mice in the sham operation group,the expression of P4HA2 in the model group was significantly increased,and it was concentrated in the cytoplasm and nucleus of renal tubular cells.After 50 ng/mL,TGF-βstimulated HK2 cells for 24 hours,the expres-sion level of P4HA2 was significantly increased,and the cell morphology changed from cobblestone to needle-like long spindle.Compared with the scramble group,sh-P4HA2 inhibited the expression of proteins related to the NF-κB signaling pathway,and the levels of downstream inflammatory factors,IL-1β,IL-6,and TNF-α,were also downregulated.Conclusion:P4HA2 may regulate the NF-κB sig-naling pathway,inhibit the inflammatory response,and alleviate the development of renal interstitial fibrosis.