阿维来司他对主动脉瓣膜间质细胞成骨分化的作用及机制研究

Effect of alvelestat on osteogenic differentiation of aortic valve interstitial cells and its mechanism

目的:探讨中性粒细胞弹性蛋白酶(NE)抑制剂阿维来司他(alvelestat;又称AZD9668)对猪主动脉瓣膜间质细胞(VICs)成骨分化的作用及其潜在机制。方法:收集3例患钙化性主动脉瓣膜疾病(CAVD)和1例非CAVD患者的瓣膜组织,采用免疫组织化学染色和Western blot检测NE、骨桥蛋白(OPN)、α-平滑肌肌动蛋白(α-SMA)和Runt相关转录因子2(RUNX2)的表达。分离猪主动脉VICs,采用细胞免疫荧光染色进行表型鉴定。MTT法检测alvelestat对VICs活力的影响。将对数生长期的VICs分为正常对照组(blank组)、成骨培养液(OM)组及OM+5、10、20和40 nmol/L alvelestat组。采用Western blot、碱性磷酸酶染色和茜素红染色等方法检测早期纤维化指标α-SMA和I型胶原(collagen I),晚期成骨分化指标OPN和RUNX2,以及NE的水平;Western blot检测alvelestat对细胞外信号调节激酶1/2(ERK1/2)信号通路的作用情况,并设置blank组、OM组、OM+alvelestat组及OM+PD98059(ERK1/2抑制剂)组,检测NE、OPN和RUNX2的表达。结果:钙化的瓣膜组织中NE、OPN、RUNX2、α-SMA及pERK的表达上调(P<0.05)。分离出的猪主动脉VICs中α-SMA及vimentin阳性,CD31阴性;40 nmol/L以上的alvelestat影响VICs的活力(P<0.05);40 nmol/L alvelestat可显著抑制纤维化指标α-SMA和collagen I,成骨分化指标RUNX2和OPN,以及NE的表达(P<0.05);碱性磷酸酶染色和茜素红染色结果显示,alvelestat可以抑制成骨诱导的VICs早期和晚期钙盐沉积;alvelestat降低VICs中ERK1/2的磷酸化水平(P<0.01),且alvelestat和ERK1/2抑制剂PD98059均能降低钙盐诱导的NE、RUNX2及OPN蛋白表达(P<0.05)。结论:NE抑制剂alvelestat通过抑制成骨诱导的NE和ERK1/2信号通路而抑制VICs的成骨分化。

AIM:To investigate the effect of neutrophil elastase(NE)inhibitor alvelestat(also known as AZD9668)on the osteogenic differentiation of porcine aortic valve interstitial cells(VICs)and its underlying mechanism.METHODS:The valve tissues of 3 patients with calcific aortic valve disease(CAVD)and 1 patient with non-CAVD were collected,and then the expression of NE,osteopontin(OPN),α-smooth muscle actin(α-SMA)and Runt-related transcription factor 2(RUNX2)were detected by immunohistochemical staining and Western blot. Porcine aortic VICs were isolated,and cellular immunofluorescence staining was used for phenotypic identification. The MTT method was used to detect the effect of alvelestat on the viability of VICs. The VICs in logarithmic growth phase were divided into normal control group(blank group),osteogenic medium(OM)group,and OM+5,10,20 and 40 nmol/L alvelestat groups. Western blot,alkaline phosphatase staining and alizarin red staining were used to detect the expression levels of early fibrosis indicators α-SMA and type I collagen(collagen I),late osteogenic differentiation indicators OPN and RUNX2,and NE. Western blot was used to detect the effect of alvelestat on extracellular signal-regulated kinases 1/2(ERK1/2)signaling pathway,and blank group,OM group,OM+alvelestat group and OM+PD98059(ERK1/2 inhibitor)group were set to detect the expression of NE,OPN and RUNX2.RESULTS:The protein levels of NE,OPN,RUNX2,α-SMA and p-ERK in calcified valve tissue were increased(P<0. 05). The isolated porcine aortic VICs were positive for α-SMA and vimentin,but negative for CD31. Alvelestat above 40 nmol/L affected the vialility of VICs(P<0. 05),and 40 nmol/L alvelestat significantly inhibited the expression of fibrosis indexes α-SMA and collagen I,osteogenic indexes RUNX2 and OPN,and NE(P<0. 05). The results of alkaline phosphatase staining and alizarin red staining showed that alvelestat inhibited early and late calcium deposition in VICs. Alvelestat reduced the phosphorylation level of ERK1/2 in VICs(P<0. 01),and both alvelestat and ERK1/2 inhibitor PD98059 reduced the protein expression of NE,RUNX2 and OPN induced by osteogenesis(P<0. 05).CONCLUSION:Alvelestat inhibits the osteogenic differentiation of VICs by inhibiting the NE and ERK1/2 signaling pathway induced by osteogenesis.