尿酸诱导人肾小管上皮细胞炎症损伤中PI3K/AKT/NF-κB信号通路的调控机制

Uric acid induces inflammatory injury in HK-2 cells via PI3K/AKT/NF-κB signaling pathway

目的探讨磷脂酰肌醇3-激酶(phosphoinositide 3-kinase,PI3K)/蛋白激酶B(protein kinase B,AKT)/核因子κB(NF-κB)信号通路在人肾小管上皮细胞(human kideny-2,HK-2)-尿酸性肾病细胞模型中表达水平变化及其作用机制。方法体外培养HK-2细胞,随机分为对照组及实验组。实验组经高尿酸(720μmol/L)浸泡48 h建立体外尿酸性肾病细胞模型,分为高尿酸处理组、过表达蛋白激酶活化受体2(protease activated receptor 2,PAR2)组和敲减PAR2组。实时定量PCR法检测HK-2细胞PAR2、PI3K、AKT、NF-κB的mRNA表达水平,Western印迹法检测HK-2细胞PAR2、PI3K、AKT、NF-κB蛋白表达水平,酶联免疫吸附法检测上清液肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、单核细胞趋化蛋白-1(monocyte chemotactic protein-1,MCP-1)、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1β前体(pro-interleukin-1β,pro-IL-1β)、白细胞介素-1β(interleukin-1β,IL-1β)、转化生长因子-β1(transforming growth factor-β1,TGF-β1)表达水平。结果(1)与对照组相比,高尿酸处理组HK-2细胞中PAR2、PI3K、AKT、NF-κB的mRNA及蛋白表达增加(均P<0.05),上清液中TNF-α、MCP-1、IL-6、pro-IL-1β、IL-1β、TGF-β1增加(均P<0.01)。(2)与高尿酸处理组相比,过表达PAR2组HK-2细胞中PAR2、PI3K、AKT、NF-κB的mRNA及蛋白表达明显增加(均P<0.05),上清液中TNF-α、MCP-1、IL-6、IL-1β、TGF-β1明显增加(均P<0.05)。(3)与高尿酸处理组相比,敲减PAR2组HK-2细胞PAR2、PI3K、AKT、NF-κB的mRNA及蛋白表达明显下降(均P<0.05),上清液中IL-6、pro-IL-1β、IL-1β、TGF-β1明显减少(均P<0.05)。结论尿酸致肾脏HK-2细胞损伤过程中,尿酸通过激活PAR2显著上调PI3K/AKT/NF-κB信号通路表达,导致HK-2细胞炎症损伤明显加重。敲减PAR2抑制PI3K/AKT/NF-κB信号通路,可有效减轻HK-2细胞炎症损伤。

Objective To investigate the effects and underlying mechanisms of phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)/NF-κB signaling pathway in human kidney-2(HK-2)cells of hyperuricemic nephropathy.Methods HK-2 cells were cultured in vitro and randomly divided into control group and experimental group.The experimental group was induced by high uric acid(720μmol/L)immersion for 48 h to establish a cell model of hyperuricemic nephropathy in vitro and subsequently divided into hyperuricemic group,overexpressed protease activated receptor 2(PAR2)and knockdown PAR2 group.The expressions of PAR2,PI3K,AKT,NF-κB mRNA were measured by real-time PCR.The expressions of PAR2,PI3K,AKT and NF-κB protein were measured by Western blotting.The expressions of tumor necrosis factor-α(TNF-α),monocyte chemotactic protein-1(MCP-1),interleukin-6(IL-6),pro-interleukin-1β(pro-IL-1β),interleukin-1β(IL-1β)and transforming growth factor-β1(TGF-β1)were detected by enzyme linked immunosorbent assay(ELISA).Results(1)Compared with the control group,the expressions of PAR2,PI3K,AKT and NF-κB mRNA and protein in hyperuricemic group were significantly increased(all P<0.05),the expressions of TNF-α,MCP-1,IL-6,pro-IL-1β,IL-1βand TGF-β1 in the supernatant in hyperuricemic group were significantly increased(all P<0.01).(2)Compared with the hyperuricemic group,the expressions of PAR2,PI3K,AKT and NF-κB mRNA and protein in overexpressed PAR2 group were significantly increased(all P<0.05),the expressions of TNF-α,MCP-1,IL-6,IL-1βand TGF-β1 in the supernatant were significantly increased(all P<0.05).(3)Compared with the hyperuricemic group,the expression of PAR2,PI3K,AKT and NF-κB mRNA and protein in knockdown PAR2 group were significantly decreased(all P<0.05),the expressions of IL-6,pro-IL-1β,IL-1βand TGF-β1 in the supernatant were significantly decreased(all P<0.05).Conclusions In the process of uric acid-induced HK-2 cell damage,uric acid significantly up-regulates the expression of PI3K/AKT/NF-κB signaling pathway by activating PAR2,leading to a marked increase in inflammatory damage.Knocking down PAR2 inhibits the expression of PI3K/AKT/NF-κB signaling pathway,which can effectively reduce the inflammatory damage of HK-2 cells.