摘要
目的 探讨肾小管低氧诱导因子-1α(HIF-1α)/MicroRNA-23a(miR-23a)通路在脓毒血症急性肾损伤(SA-AKI)中的作用及相关作用机制.方法 体外培养人近曲小管上皮细胞(HK-2 细胞),采用脂多糖(LPS)处理HK-2细胞构建SA-AKI细胞模型.LPS处理的HK-2 细胞分为LPS组、NC siRNA 组、HIF-1α siRNA组、anti-miR-NC组、anti-miR-23a组、HIF-1α siRNA+miR-NC组、HIF-1α siRNA+miR-23a组,以正常培养的HK-2 细胞作为空白对照组(Control组).采用qRT-PCR法检测细胞中HIF-1α、miR-23a基因表达;CCK-8法检测细胞活力;流式细胞术检测细胞凋亡;ELISA法检测细胞中炎性因子[白介素-1β(IL-1β)、白介素-6(IL-6)、肿瘤坏死因子α(TNF-α)]水平;Western blot法检测细胞中HIF-1α蛋白、NF-κB通路蛋白表达.结果 与Control组比较,LPS组HK-2 细胞中HIF-1α蛋白和mRNA表达水平、miR-23a mRNA表达水平均升高,细胞活力降低,细胞凋亡率、细胞中IL-6、IL-1β和TNF-α水平均升高(P<0.05).与LPS组比较,HIF-1α siRNA组HK-2细胞中HIF-1α蛋白和mRNA表达水平、miR-23a mRNA表达水平均降低,细胞活力升高,细胞凋亡率、细胞中IL-6、IL-1β和TNF-α水平均降低(P<0.05).与LPS组比较,anti-miR-23a组HK-2细胞中miR-23a mRNA表达水平降低,细胞活力升高,细胞凋亡率、细胞中IL-6、IL-1β和TNF-α水平均降低(P<0.05).与HIF-1α siRNA组比较,HIF-1α siRNA+miR-23a组HK-2 细胞中miR-23a mRNA表达水平升高,细胞活力降低,细胞凋亡率、细胞中IL-6、IL-1β和TNF-α水平均升高(P<0.05).与Control组比较,LPS组HK-2 细胞p-NF-κB-p65/NF-κB-p65、p-IκBα/IκBα比值均升高(P<0.05).与LPS组比较,HIF-1α siRNA组和anti-miR-23a组HK-2细胞p-NF-κB-p65/NF-κB-p65、p-IκBα/IκBα比值均降低(P<0.05).与HIF-1α siRNA组比较,HIF-1α siRNA+miR-23a组HK-2细胞p-NF-κB-p65/NF-κB-p65、p-IκBα/IκBα比值均升高(P<0.05).结论 肾小管HIF-1α通过调控miR-23a表达调节NF-κB信号通路,从而参与LPS诱导的肾小管上皮细胞损伤.
Abstract
Objective To investigate the role of renal tubules hypoxia inducible factor-1α(HIF-1 α)/MicroRNA-23a(miR-23a)pathway in sepsis-associated acute kidney injury(SA-AKI)and its related mechanisms.Methods Human proximal convoluted tubules epithelial cells(HK-2 cells)were cultured in vitro and treated with lipopolysaccharide(LPS)to construct the SA-AKI cell model.LPS-treated HK-2 cells were divided into LPS group,NC siRNA group,HIF-1α siRNA group,anti-miR-NC group,anti-miR-23a group,HIF-1α siRNA+miR-NC group,and HIF-1α siRNA+miR-23a group.HK-2 cells treated with blank control were considered as the control group.Relative expressions of HIF-1α,miR-23a in cells were detected by quantitative reverse transcriptase PCR(qRT-PCR).Cell viability and apoptosis ere detected by CCK-8 assay and flow cytometry,respectively.The levels of inflammatory factors,including interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor α(TNF-α)in cells were detected by enzyme-linked immunosorbent assay(ELISA).Protein expressions of HIF-1α,and those in the nuclear factor kappa B(NF-κB)pathway were detected by Western blot.Results Compared with those of control group,HK-2 cells in LPS group presented significantly higher protein and mRNA levels of HIF-1α,mRNA level of miR-23a,apoptotic rate and relative levels of IL-6,IL-1β and TNF-α,but significantly lower cell viability(P<0.05).Compared with those of LPS group,HK-2 cells in HIF-1α siRNA group presented significantly lower protein and mRNA levels of HIF-1α,mRNA level of miR-23a,apoptotic rate and relative levels of IL-6,IL-1β and TNF-α,but significantly higher cell viability(P<0.05).Compared with those of LPS group,HK-2 cells in anti-miR-23a group presented significantly lower mRNA level of miR-23a,apoptotic rate and relative levels of IL-6,IL-1β and TNF-α,but significantly higher cell viability(P<0.05).Compared with those of HIF-1α siRNA group,HK-2 cells in HIF-1α siRNA + miR-23a group presented significantly higher mRNA level of miR-23a,apoptotic rate and relative levels of IL-6,IL-1β and TNF-α,but significantly lower cell viability(P<0.05).Compared with those of control group,HK-2 cells in LPS group presented significantly higher levels of p-NF-κB p65/NF-κB p65 and p-IκBα/IκBα(P<0.05).Compared with those of LPS group,p-NF-κB p65/NF-κB p65 and p-IκBα/IκBα were significantly lower in the HIF-1α siRNA group and anti-miR-23a group(P<0.05).Compared with those of HIF-1α siRNA group,p-NF-κB p65/NF-κB p65 and p-IκBα/IκBα were significantly higher in the HIF-1α siRNA + miR-23a group(P<0.05).Conclusion Renal tubules HIF-1α regulates the NF-κB signaling pathway by regulating the expression level of miR-23a,thus participating in LPS-induced renal tubular epithelial cell injury.
基金项目
宁夏回族自治区自然科学基金(2021AAC03309)
维普
万方数据