内质网应激参与高磷诱导的血管平滑肌细胞钙化的作用机制

The mechanism of endoplasmic reticulum stress on vascular smooth muscle cell calcification induced by high phosphorus

目的探讨内质网应激是否参与高磷诱导血管平滑肌细胞钙化及其作用机制。方法体外培养人胸主动脉血管平滑肌细胞(VSMC),分为对照组、正常磷组(1.3 mmol/L磷,NP组)、高磷组(2.6 mmol/L磷,HP组),培养10 d。茜素红染色后观察细胞钙盐沉积情况;实时定量PCR和Western blotting检测肌醇需求因子1(IRE1)、RNA依赖的蛋白激酶样内质网激酶(PERK)、内质网应激标志蛋白C/EBP同源蛋白(CHOP)、c-Jun氨基末端激酶(JNK)、骨形态发生蛋白2(BMP-2)及Runt相关转录因子2(Runx-2)的表达;应用CHOP siRNA转染高磷培养VSMC敲低CHOP表达后,检测BMP-2及Runx-2表达;应用JNK抑制剂SP600125(DMSO溶解),终浓度10μmol/L预处理30 min后加入高磷培养基培养10 min,茜素红染色观察钙沉积情况。结果与对照组及NP组比较,HP组内质网应激蛋白(IRE1、PERK、JNK、CHOP)及钙化蛋白(BMP-2、Runx-2)均明显增高(均P<0.01)。敲低CHOP表达后钙化蛋白BMP-2及Runx-2表达均下调(均P<0.05)。应用JNK抑制剂SP600125预处理的高磷培养VSMC钙沉积减轻。结论内质网应激参与高磷诱导的VSMC钙化,其作用机制可能是激活了pPERK-CHOP和IRE1-p-JNK两条途径。

Objective To study the mechanism by which endoplasmic reticulum stress(ERS)participates in the process of vascular smooth muscle cell calcification induced by high phosphorus.Methods Human thoracic aorta smooth muscle cells were divided into three groups:control,normal phosphorus(1.3 mmol/L,NP),and high phosphorus(2.6 mmol/L,HP),and cultured in vitro for 10 days.Calcium deposition in vascular smooth muscle cells was distinguished by alizarin red staining.The protein and mRNA expression of inositol-requiring enzyme 1(IRE1),RNA-dependent protein kinase-like endoplasmic reticulum kinase(PERK),ERS characteristic protein C/EBP homologous protein(CHOP),c-Jun N-terminal kinase(JNK),bone morphogenetic protein 2(BMP-2),and Runt-related transcription factor 2(Runx-2)were detected using RT-PCR and Western blotting.CHOP knockdown was induced by CHOP siRNA transfection cells cultured in high phosphorus,and BMP-2 and Runx-2 protein expression were detected.A JNK inhibitor SP600125(DMSO as dissolvent,final concentration 10μmol/L)was applied for 30 min to pretreat cells,which were then cultured in high phosphorus for 10 days.Calcium deposition was detected by alizarin red staining.Results Compared with the control and NP group,cells cultured in high phosphorus overexpressed characteristic proteins of ERS(IRE1,PERK,JNK,and CHOP)and calcification proteins(BMP-2 and Runx-2)significantly increased(P<0.01).Knockdown CHOP expression,BMP-2,and Runx-2 expression significantly decreased(P<0.05).Pretreatment with SP600125 alleviated calcium deposition.Conclusion ERS stress may play a role in the process of high phosphorus-induced vascular smooth muscle cell calcification.Its underlying mechanism of action may be through the activation of pPERK-CHOP and IRE1-P-JNK pathways.