miR-24-3p/S1PR2信号轴对大鼠RMECs损伤的作用与机制研究

Role and mechanism of MIR-24-3P/S1PR2 signaling axis for rat renal microvascular endothelial cell injury

目的研究miR-24-3p/1-磷酸鞘氨醇受体2(S1PR2)信号轴对肾小球内皮细胞(RMECs)损伤的作用及影响机制。方法原代分离获得大鼠RMECs,制备急性肾损伤(AKI)模型。采用CCK8检测细胞增殖能力,流式细胞术检测细胞凋亡情况、活性氧(ROS)水平,酶联免疫吸附试验(ELISA)检测肿瘤坏死因子(TNF)-α和白细胞介素(IL)-1β的表达;生化试剂盒检测超氧化物歧化酶(SOD)、丙二醛(MDA)水平;实时荧光定量PCR检测细胞中S1PR2(NM_017192.2)的mRNA表达水平,Western blot检测S1PR2和凋亡相关基因caspase-3和caspase-9、血管内皮细胞损伤相关因子细胞分裂周期蛋白20(CDC20)和血管内皮生长因子(VEGF)的表达水平。结果LPS组TNF-α、IL-1β水平高于control组,miR-24-3p inhibitor+LPS组TNF-α、IL-1β水平高于inhibitor NC+LPS组,差异有统计学意义(P<0.05)。LPS组MDA、SOD水平高于control组,miR-24-3p inhibitor+LPS组MDA水平高于inhibitor NC+LPS组,但SOD水平低于inhibitor NC+LPS组,差异有统计学意义(P<0.05)。LPS组S1PR2 mRNA表达水平高于control组,miR-24-3p inhibitor+LPS组S1PR2 mRNA表达水平低于inhibitor NC+LPS组,差异有统计学意义(P<0.05)。LPS组S1PR2、caspase-9、caspase-3蛋白表达水平高于control组,CDC20、VEGF蛋白表达水平低于control组,差异有统计学意义(P<0.05);miR-24-3p inhibitor+LPS组S1PR2、caspase-3、caspase-9蛋白表达水平低于inhibitor NC+LPS组,CDC20、VEGF蛋白表达水平高于inhibitor NC+LPS组,差异有统计学意义(P<0.05)。结论miR-24-3p inhibitor通过联动抑制S1PR2表达,下调caspase-3、caspase-9表达,上调CDC20、VEGF表达,实现抑制TNF-α、IL-1β与MDA表达、降低AKI模型对细胞造成损伤的作用。

Objective To investigate the effect of miR-24-3p/S1PR2 signal axis on the renal microvascular endothelial cell(RMECs)injury and its influencing mechanism.Methods The rat RMECs were obtained by primary isolation and the acute renal injury model was established.The cell proliferation ability was detected by CCK8;the cellular apoptosis and reactive oxygen species(ROS)level were detected by flow cytometry;the TNF-αand IL-1βexpressions were detected by ELISA;the SOD and MDA levels were detected by the biochemical reagent kit;the mRNA expression level of S1PR2(NM_017192.2)in cells was detected by real time qPCR,and the expression level of S1PR2 and apoptosis related genes caspase-3,caspase-9,CDC20 and VEGF were detected by Western blot.Results The levels of TNF-αand IL-1βin the LPS group were higher than those in the control group(P<0.05),while which in the miR-24-3p inhibitor+LP group were higher than those in the inhibitor NC+LPS group,and the differences were statistically significant(P<0.05).The MDA and SOD levels in the LPS group were higher than those in the control group,the MDA level in the miR-24-3p inhibitor+LPS group was higher than that in the inhibitor NC+LPS group,but the SOD level was lower than that in the inhibitor NC+LPS group,and the differences were statistically significant(P<0.05).The SIPR2 mRNA expression level in the LPS group was higher than that in the control group,the SIPR2 mRNA level in the miR-24-3p inhibitor+LPS group was lower than that in the inhibitor NC+LPS group,and the differences were statistically significant(P<0.05).The SIPR2,caspase-3 and caspase-9 protein expression levels in the LPS group were higher than those in the control group,the CDC20 and VEGF protein expression levels were lower than those in the control group,and the differences were statistically significant(P<0.05).the SIPR2,caspase-3 and caspase-9 protein levels in the miR-24-3p inhibitor+LPS group were lower than those in the inhibitor NC+LPS group,the CDC20 and VEGF protein expression levels were higher than those in the inhibitor NC+LPS group,and the differences were statistically significant(P<0.05).Conclusion miR-24-3p inhibi-tor inhibitsthe expression of S1PR2 by linkage,down-regulates the caspase-3 and caspase-9 expressions,up-reg-ulates the CDC20 and VEGF expressions,and realizes to inhibit the TNF-α,IL-1βand MDA expressions and reduce the injury effect of the AKI model on the cells.