补体C1q/肿瘤坏死因子相关蛋白13通过盐诱导激酶1/环磷酸腺苷调节转录共激活因子2通路参与高糖诱导的人肝窦内皮细胞滤过功能障碍

C1q/tumor necrosis factor related protein 13 is involved in high glucose-induced filtration dysfunction in human liver sinusoidal endothelial cells via the salt-inducible kinase 1/cAMP regulated transcriptional co-activators 2 pathway

目的探讨血清补体C1q/肿瘤坏死因子相关蛋白13(CTRP13)参与高糖诱导的人肝窦内皮细胞(HLSEC)滤过功能障碍的分子机制。方法体外培养HLSEC,构建慢病毒CTRP13过表达载体(LV-CTRP13)并感染HLSEC,依据不同处理条件将细胞分为正常对照(5.5 mmol/L葡萄糖,NC)组、高糖(25 mmol/L,HG)组、NC+LV-CTRP13组、HG+LV-CTRP13组、NC+空载对照(LV-CON)组、HG+LV-CON组、HG+LV-CTRP13+盐诱导激酶1(SIK1)抑制剂(HG-9-91-01)组、HG+LV-CON+HG-9-91-01组、HG+LV-CTRP13+环磷酸腺苷调节转录共激活因子2(CRTC2)抑制剂(GW4064)组、HG+LV-CON+GW4064组。采用实时荧光定量PCR(qRT-PCR)和Western blotting法检测CTRP13、SIK1、CRTC2、P130 Crk相关底物(P130Cas)的mRNA及蛋白表达水平。采用单因素方差分析进行组间比较。结果与NC组相比,HG组CTRP13、SIK1、P130Cas的mRNA和蛋白表达降低,CRTC2的mRNA和蛋白表达升高(P<0.05)。与HG+LV-CON组相比,HG+LV-CTRP13组CTRP13、SIK1、P130Cas的mRNA和蛋白表达升高,CRTC2的mRNA和蛋白表达降低(P<0.05)。与HG+LV-CTRP13组相比,HG+LV-CTRP13+HG-9-91-01组CRTC2的mRNA和蛋白表达升高,而SIK1、P130Cas的mRNA和蛋白表达降低(P<0.05)。与HG+LV-CTRP13组相比,HG+LV-CTRP13+GW4064组CRTC2的mRNA和蛋白表达降低,P130Cas的mRNA和蛋白表达升高(P<0.05)。结论CTRP13、SIK1、CRTC2、P130Cas参与了高糖诱导的HLSEC滤过功能障碍,CTRP13过表达可以抑制高糖诱导的HLSEC滤过功能障碍,这一机制是通过SIK1/CRTC2通路实现的。

Objective To investigate the molecular mechanism of serum complement C1q/tumor necrosis factor-related protein 13(CTRP13)involvement in high glucose-induced filtration dysfunction in human hepatic sinusoidal endothelial cells(HLSEC).Methods HLSEC were cultured in vitro,lentiviral CTRP13 overexpression vector(LV-CTRP13)was constructed and infected with HLSEC,and the cells were divided into normal control(5.5 mmol/L glucose,NC),high glucose(25 mmol/L,HG),NC+LV-CTRP13,HG+LV-CTRP13,NC+empty control(LV-CON)group,HG+LV-CON group,HG+LV-CTRP13+salt-inducible kinase 1(SIK1)inhibitor(HG-9-91-01)group,HG+LV-CON+HG-9-91-01 group,HG+LV-CTRP13+cyclic adenosine monophosphate-regulated transcriptional co-activator 2(CRTC2)inhibitor(GW4064)group,and HG+LV-CON+GW4064 group.The mRNA and protein expression levels of CTRP13,SIK1,CRTC2,P130 Crk-related substrate(P130Cas)were detected by quantitative real-time fluorescence PCR(qRT-PCR)and Western blotting.One-way analysis of variance(ANOVA)was used for comparison between groups.Results Compared with the NC group,mRNA and protein expression of CTRP13,SIK1,and P130Cas were decreased in the HG group,and mRNA and protein expression of CRTC2 were increased(P<0.05).Compared with the HG+LV-CON group,the mRNA and protein expression of CTRP13,SIK1,and P130Cas were elevated in the HG+LV-CTRP13 group,and the mRNA and protein expression of CRTC2 were decreased(P<0.05).Compared with the HG+LV-CTRP13 group,the mRNA and protein expression of CRTC2 was elevated in the HG+LV-CTRP13+HG-9-91-01 group,while the mRNA and protein expression of SIK1 and P130 Crk-associated substrate were decreased(P<0.05).Compared with the HG+LV-CTRP13 group,the mRNA and protein expression of CRTC2 was decreased in the HG+LV-CTRP13+GW4064 group,and the mRNA and protein expression of P130Cas was increased(P<0.05).Conclusion CTRP13,SIK1,CRTC2,and P130Cas are involved in high-glucose-induced HLSEC filtration dysfunction,and CTRP13 overexpression inhibits high-glucose-induced HLSEC filtration dysfunction through a mechanism mediated through the SIK1/CRTC2 pathway.