miR-199b-5p靶向Klotho对脂多糖诱导的大鼠肾小管上皮细胞损伤的影响

Effects of miR-199b-5p targeting Klotho on the lipopolysaccharide-induced injury of rat renal tubular epithelial cells

目的探究MicroRNA-199b-5p(miR-199b-5p)和Klotho在脂多糖(LPS)诱导的大鼠肾小管上皮细胞损伤中的表达变化,并进一步探讨其作用机制。方法体外培养大鼠肾小管上皮细胞NRK-52E,分为正常对照组(NC组)、LPS组、inhibitor-NC组、miR-199b-5p沉默组、sh-NC组、sh-Klotho组、miR-199b-5p沉默+sh-NC组、miR-199b-5p沉默+sh-Klotho组,除NC组外,其他组采用LPS刺激建立脓毒症细胞模型,诱导细胞损伤。实时荧光定量PCR(RT-qPCR)和Western blot检测细胞中miR-199b-5p、Klotho mRNA和蛋白表达水平;酶联免疫吸附法(ELISA)检测细胞培养上清液中白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平;荧光探针DCFH-DA法检测细胞中活性氧(ROS)含量;试剂盒检测细胞中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)含量;流式细胞术测定细胞凋亡率;Western blot检测细胞中B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、Caspase-3的表达。结果与NC组比较,LPS组细胞中miR-199b-5p表达、TNF-α、IL-6水平、ROS的荧光强度、细胞中MDA含量、细胞凋亡率、Bax、Caspase-3表达显著升高(P<0.05),Klotho mRNA和蛋白表达、GSH-PX和SOD活性、Bcl-2表达显著降低(P<0.05);与LPS组比较,miR-199b-5p沉默组细胞中miR-199b-5p表达、TNF-α、IL-6水平、ROS的荧光强度、细胞中MDA含量、细胞凋亡率、Bax、Caspase-3表达显著降低(P<0.05),Klotho mRNA和蛋白表达、GSH-PX和SOD活性、Bcl-2表达显著升高(P<0.05);且在沉默miR-199b-5p的基础上,下调Klotho后细胞抗氧化能力降低(P<0.05),凋亡增加(P<0.05)。结论miR-199b-5p在LPS诱导的大鼠肾小管上皮细胞损伤中表达升高,沉默miR-199b-5p可能通过靶向上调Klotho的表达,增强细胞的抗氧化能力,从而减少肾小管上皮细胞凋亡。

Objective To investigate the expressions of MicroRNA-199b-5p(miR-199b-5p)and Klotho in lipopolysaccharide(LPS)-induced injury of rat renal tubular epithelial cells,and to explore their action mechanism.Methods Rat renal tubular epithelial cells NRK-52E were cultured in vitro and divided into normal control group(NC group),LPS group,inhibitor-NC group,miR-199b-5p silence group,sh-NC group,sh-Klotho group,miR-199b-5p silence+sh-NC group,and miR-199b-5p silence+sh-Klotho group.Except for the cells in NC group,the cells in the other groups were treated by LPS stimulation to establish a sepsis cell model and to induce cell damage.Real-time fluorescent quantitative PCR(RT-qPCR)and Western blot were used to detect the expression levels of miR-199b-5p,Klotho mRNA and protein in cells;enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in cell culture supernatant;fluorescence probe DCFH-DA method was used to detect the content of reactive oxygen species(ROS)in cells;the kit was used to detect the contents of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)activity and malondialdehyde(MDA)in cells;flow cytometry was used to measure the cell apoptosis rate;Western Blot was used to detect the expression levels of B-cell lymphoma/leukemia-2(Bcl-2),Bcl-2-associated X protein(Bax),and Caspase-3 in cells.Results Compared with those in NC group,the expression levels of miR-199b-5p,TNF-α,IL-6,and the fluorescence intensity of ROS,the content of MDA in cells,and cell apoptosis rate,and the expression levels of Bax and Caspase-3 in LPS group were significantly increased(P<0.05),however,the expression levels of Klotho mRNA and protein,the activities of GSH-PX and SOD,and Bcl-2 levels were significantly decreased(P<0.05).Compared with those in LPS group,the expression levels of miR-199b-5p,TNF-α,IL-6,and the fluorescence intensity of ROS,the content of MDA in cells,cell apoptosis rate,and the expression levels of Bax and Caspase-3 in miR-199b-5p silence group were significantly decreased(P<0.05),however,the expression levels of Klotho mRNA and protein as well as Bcl-2,the activities of GSH-PX and SOD were significantly increased(P<0.05).On the basis of silencing miR-199b-5p,after down-regulating Klotho,the cell’s antioxidant capacity was decreased(P<0.05),but apoptosis rate was significantly increased(P<0.05).Conclusion The expression levels of miR-199b-5p are increased in LPS-induced injury of rat renal tubular epithelial cells.Silencing miR-199b-5p may targetedly up-regulating the expression levels of Klotho to enhance the antioxidant capacity of cells,thereby decreasing the renal tubular epithelial cell apoptosis.