Orai1上调促进血管平滑肌钙化表型转化OA

AIM:To investigate the mechanism of Ca2+release-activated calcium channel protein 1(Orai1)in rat aortic calcification.METHODS:Thirty 8-week-old male SD rats(200 to 250 g)were randomized into 5 groups:growth medium(GM)group,calcification medium(CM)group,dimethyl sulfoxide group,Orai1 inhibitor 3,5-bis(trifluo-romethyl)pyrazole derivative(BTP2)group,and endoplasmic reticulum stress(ERS)inhibitor 4-phenylbutyrate(4-PBA)group.Then,isolated aortic rings were prepared and stimulated with 10 mmol/L β-glycerophosphate and 3 mmol/L CaCl2 for 7 d to establish a calcification model.Concurrently,BTP2 or 4-PBA intervention was performed during modeling.The severity of calcification was evaluated via alizarin red and von Kossa staining.Additionally,the expression levels of Orai1,osteogenic differentiation markers[Runt-related transcription factor 2(RUNX2)and bone morphogenetic protein 2(BMP2)],contractile markers[smooth muscle myosin heavy chain(SMMHC)and smooth muscle protein 22α(SM22α)],and key molecules involved in the ERS signaling pathway,especially phosphorylated eukaryotic translation initiation factor 2α(p-eIF2α)and activating transcription factor 4(ATF4),were assessed by Western blot.By utilizing laser confocal microscopy,intracellular free Ca2+concentration([Ca2+]i)in vascular smooth muscle cells(VSMCs)was monitored using Fluo-4 AM fluorescent probes.The effect of BTP2 on store-operated calcium(SOC)channel-mediated va-soconstriction was further evaluated using an in vitro vascular tension assay.RESULTS:The results of alizarin red and von Kossa staining revealed a marked increase in aortic ring calcification following stimulation with high levels of calcium and phosphate(P<0.01).In an in vitro rat aortic ring calcification model,there was significant up-regulation of Orai1 pro-tein expression(P<0.05),accompanied by elevated levels of osteogenic differentiation-related markers RUNX2 and BMP2(P<0.05 or P<0.01).In addition,the expression of ERS pathway markers was markedly elevated,especially p-eIF2α and ATF4(P<0.01 or P<0.05).Treatment with BTP2 resulted in significant attenuation of aortic ring calcification(P<0.05)and reversal of osteogenic differentiation markers,including RUNX2 and BMP2 protein expression(P<0.01),while concurrently down-regulating the ERS pathway proteins p-eIF2α and ATF4(P<0.01).Furthermore,compared with the CM group,treatment with 4-PBA reversed the expression levels of RUNX2 and BMP2(P<0.05 or P<0.01).The as-sessment of[Ca2+]i in VSMCs indicated that BTP2 significantly inhibited SOC channel-mediated calcium influx(P<0.05).In vitro vascular tension assays demonstrated that BTP2 significantly inhibited SOC channel-mediated vasoconstric-tion(P<0.05).CONCLUSION:An aortic ring calcification model is successfully developed,demonstrating up-regula-tion of Orai1 expression.Treatment with Orai1 inhibitor BTP2 effectively suppresses SOC channel-mediated calcium influx and vasoconstriction,consequently inhibiting aortic calcification.This mechanism may be associated with ERS.