中介素1-53对高磷诱导人主动脉血管平滑肌细胞ERS-线粒体凋亡通路及钙化的影响

Effects of Intermedin 1-53 on ERS Mitochondrial Apoptosis Pathway and Calcification in Human Aortic Vascular Smooth Muscle Cells Induced by High Phosphorus

目的:探讨中介素1-53(IMD1-53)对高磷诱导人主动脉血管平滑肌细胞(HA-VSMC)内质网应激(ERS)-线粒体凋亡通路及钙化的影响。方法:体外培养HA-VSMC,MTT法检测IMD1-53对HA-VSMC的毒性作用。HA-VSMC中添加10 mmol/Lβ-甘油磷酸盐进行高磷诱导,使用0.1 nmol/L或0.5 nmol/L IMD1-53干预,并在0.5 nmol/L IMD1-53干预基础上添加ERS诱导剂衣霉素(TM),流式细胞仪检测HA-VSMC凋亡率,茜素红S染色观察HA-VSMC中钙盐沉积,酞络合酮比色法测定HA-VSMC中钙含量,分光光度法检测HA-VSMC中碱性磷酸酶(ALP)活性,蛋白质印迹法(Western blot)检测HA-VSMC中钙化以及ERS--线粒体凋亡通路相关蛋白表达。结果:IMD1-53浓度≤2.5 nmol/L时,对HA-VSMC细胞活力无明显影响(P>0.05),故选择对细胞无明显毒副作用的低、高2个剂量(0.1、0.5 nmol/L)进行后续实验。高磷诱导后,HA-VSMC凋亡率、钙含量、ALP活性以及Runt相关转录因子-2(Runx2)、骨桥蛋白(OPN)、Bcl-2相关X蛋白(Bax)、裂解的半胱氨酰天冬氨酸蛋白酶-3(cleaved caspase-3)、葡萄糖调节蛋白78(GRP78)、C/EBP同源蛋白(CHOP)、感受器活化转录因子6(ATF6)、p-需肌醇酶1(IRE1)/IRE1蛋白表达水平升高(P<0.05),橘红色结节明显增多,骨保护素(OPG)、B细胞淋巴瘤/白血病-2(Bcl-2)蛋白表达水平降低(P<0.05)。0.1 nmol/L和0.5 nmol/L IMD1-53干预处理均可降低HA-VSMC凋亡率、钙含量、ALP活性以及Runx2、OPN、Bax、cleaved caspase-3、GRP78、CHOP、ATF6、p-IRE1/IRE1蛋白表达水平(P<0.05),明显减少橘红色结节,升高OPG、Bcl-2蛋白表达水平(P<0.05)。ERS诱导剂TM可升高HA-VSMC中GRP78、CHOP、ATF6、p-IRE1/IRE1蛋白表达水平,并减弱IMD1-53抑制高磷诱导的HA-VSMC ERS、凋亡和钙化的作用。结论:IMD1-53可减轻高磷诱导的HA-VSMC钙化,其作用机制可能与抑制ERS-线粒体凋亡通路激活,减少细胞凋亡有关。

Objective:To investigate the effects of intermedin 1-53(IMD1-53)on endoplasmic reticulum stress(ERS)-mitochondrial apoptosis pathway and calcification in human aortic vascular smooth muscle cells(HA-VSMC)induced by high phosphorus.Methods:HA-VSMC was cultured in vitro,MTT method was used to detect the toxicity of IMD1-53 on HA-VSMC.10 mmol/Lβ-glycerophosphate was added to HA-VSMC for high phosphorus induction,0.1 nmol/L or 0.5 nmol/L IMD1-53 was used for intervention,and the ERS inducer tunicamycin(TM)was added on the basis of 0.5 nmol/L IMD1-53 intervention,the apoptosis rate of HA-VSMC was detected by flow cytometry,Alizarin red S staining was used to observe calcium salt deposition in HA-VSMC,the calcium content in HA-VSMC was determined by phthalein complexing ketone colorimetry,the activity of alkaline phosphatase(ALP)in HA-VSMC was detected by spectrophotometry,Western blot was used to detect calcification and expression of proteins related to ERS mitochondrial apoptosis pathway in HA-VSMC.Results:When IMD1-53 concentration≤2.5 nmol/L,it had no significant effect on the viability of HA-VSMC cells(P>0.05),therefore,low and high doses(0.1 and 0.5 nmol/L)without obvious toxic and side effects on cells were selected for subsequent experiments.After high phosphorus induction,the apoptosis rate of HA-VSMC,calcium content,ALP activity,the expression levels of Runt related transcription factor-2(Runx2),osteopontin(OPN),Bcl-2 associated X protein(Bax),cleaved caspase-3,glucose regulated protein 78(GRP78),C/EBP homologous protein(CHOP),activating transcription factor 6(ATF6)p-inositol-requiring enzyme 1(IRE1)/IRE1 proteins increased(P<0.05),orange nodules increased obviously,and the expression levels of osteoprotegerin(OPG)and B cell lymphoma/leukemia 2(Bcl-2)proteins decreased(P<0.05).0.1 nmol/L and 0.5 nmol/L IMD1-53 intervention treatment were able to reduce the apoptosis rate of HA-VSMC,calcium content,ALP activity,the expression levels of Runx2,OPN,Bax,cleaved caspase-3,GRP78,CHOP,ATF6,p-IRE1/IRE1 proteins(P<0.05),obviously reduce the orange nodules,and increase the expression levels of OPG and Bcl-2 proteins(P<0.05).The ERS inducer TM was able to increase the expression levels of GRP78,CHOP,ATF6,p-IRE1/IRE1 proteins in HA-VSMC,and weaken the inhibition of IMD1-53 on ERS,apoptosis and calcification induced by hyperphosphate in HA-VSMC.Conclusion:IMD1-53 can reduce the calcification of HA-VSMC induced by high phosphorus,and its mechanism may be related to inhibiting the activation of ERS mitochondrial apoptosis pathway,and reducing cell apoptosis.