CLEC14A在阿霉素诱导FSGS小鼠模型中对足细胞损伤的保护机制

Study on the protective mechanism of CLEC14A against podocyte injury in adriamycin-induced FSGS mouse model

目的:探究CLEC14A在阿霉素诱导局灶节段性肾小球硬化(FSGS)小鼠模型中对足细胞损伤的保护作用,并通过半胱氨酸蛋白酶-3(Caspase-3)、B淋巴细胞癌-2相关蛋白(Bax)和B淋巴细胞瘤-2(Bcl-2)信号通路探讨其机制。方法:将30只C57BL/6小鼠随机分成Sham组(n=10)、Model组(n=10)和CLEC14A siRNA组(siRNA组,n=10)。将小鼠足细胞MPC5分为对照组、FSGS组、FSGS-siCLEC14A组。采用定量聚合酶链反应(qPCR)检测小鼠肾脏中CLEC14A mRNA及血管内皮生长因子(VEGF)mRNA的水平。采用酶联免疫吸附试验(ELISA)检测小鼠肾脏组织中Caspase-3、Bax和Bcl-1水平。结果:FSGS组及FSGS-siCLEC14A组各时间点的细胞增殖能力低于对照组,差异均有统计学意义(均P<0.05)。FSGS-siCLEC14A组的48 h及72 h细胞增殖能力均低于FSGS组,差异均有统计学意义(均P<0.05)。与对照组比较,FSGS组和FSGS-siCLEC14A组的细胞凋亡率、Caspase-3、Bax水平上升,而Bcl-2水平下降,差异均有统计学意义(均P<0.05);与FSGS组比较,FSGS-siCLEC14A组的Caspase-3、Bax水平增加,而Bcl-2水平下降,差异均有统计学意义(均P<0.05)。与对照组比较,FSGS组和FSGS-siCLEC14A组的24 h尿蛋白定量(24 h UTP)、血肌酐(Scr)及血尿素氮(BUN)水平增加,差异均有统计学意义(均P<0.05);与FSGS组比较,FSGS-siCLEC14A组的24 h UTP、Scr及BUN水平增加,差异均有统计学意义(均P<0.05)。与Sham组比较,Model组和siRNA组小鼠的CLEC14A mRNA水平下降,VEGF mRNA上升,差异均有统计学意义(均P<0.05);与Model组比较,siRNA组小鼠的CLEC14A mRNA水平下降,VEGF mRNA上升,差异均有统计学意义(均P<0.05)。与Sham组比较,Model组和siRNA组小鼠肾脏中的Caspase-3、Bax水平上升,而Bcl-2水平下降,差异均有统计学意义(均P<0.05);与Model组比较,siRNA组小鼠肾脏中的Caspase-3、Bax水平升高,而Bcl-2水平下降,差异均有统计学意义(均P<0.05)。结论:CLEC14A可通过抑制Caspase-3、Bax、Bcl-2信号通路起到保护FSGS小鼠足细胞的作用。

Objective To investigate the protective effect of CLEC14A adriamycin-induced focal segmental glomerulosclerosis(FSCS)on podocyte injury in mice and explore its mechanism from the Caspase-3,Bax and Bcl-2 signal pathway.Methods Thirty C57BL/6 mice were randomly divided into sham group(n=10),Model group(n=10),and CLEC14A siRNA group(siRNA group,n=10).Mouse podocytes MPC5 were divided into control group,FSCS group and FSGS-siCLEC14A group.Quantitative polymerase chain reaction(qPCR)was used to detect CLEC14A mRNA and vascularendothelial growth factor(VEGF)mRNA levels in mouse kidneys.Enzyme-linked immunosorbent assay(ELISA)kits were used to detect Caspase-3,Bax and Bcl-2 levels in mouse kidney tissues.Results The cell proliferation capacity of FSCS group and FSCS-siCLEC14A group was lower than that of Sham group at all time points,and the differences were statistically significant(all P<0.05).The cell proliferation ability of FSGS-siCLEC14A group at 48 h and 72 h was lower than that of FSGS group,and the differences were statistically significant(all P<0.05).Compared with control group,apoptosis rate,Caspase-3 and Bax levels in FSGS group and FSCS-siCLEC14A group were significantly increased,while Bcl-2 levels were decreased,with statistical significance(all P<0.05).Compared with FSCS group,Caspase-3 and Bax levels in FSGS-siCLEC14A group were increased,while Bcl-2 levels were decreased,with statistical significance(all P<0.05).Compared with control group,24-hour urinary protein(24 h UTP),serum creatinine(Scr)and blood urea nitrogen(BUN)levels in FSGS group and FSGS-siCLEC14A group were increased,and the differences were statistically significant(all P<0.05).Compared with FSCS group,24 h UTP,Scr and BUN levels in FSGS-si-CLEC14A group were increased,and the differences were statistically significant(all P<0.05).Compared with Sham group,CLEC14A mRNA level in Model group and siRNA group decreased,VEGF mRNA level increased,and the differences were statistically significant(all P<0.05).Compared with Model group,CLEC14A mRNA level in siRNA group decreased,VEGF mRNA level increased,and the differences were statistically significant(all P<0.05).Compared with Sham group,the levels of Caspase-3 and Bax in the kidney of mice in Model group and siRNA group were increased,while the levels of Bcl-2 were decreased,with statistical significance(all P<0.05).Compared with Model group,the levels of Caspase-3 and Bax in kidney of siRNA group were increased,while the levels of Bcl-2 were decreased,with statistical significance(all P<0.05).Conclusions CLEC14A can protect FSCS mouse podocytes through the Caspase-3,Bax and Bcl-2 signal pathway.