4-苯基丁酸对同型半胱氨酸诱导的主动脉瓣膜间质细胞成骨分化的作用及机制

Effect and mechanism of 4-phenylbutyric acid on homocysteine-induced osteogenic differentiation of aortic valve interstitial cells

目的探讨内质网应激(ERS)分子伴侣4-苯基丁酸(4-PBA)在同型半胱氨酸(Hcy)诱导的主动脉瓣膜间质细胞(AVICs)成骨分化中的作用及相关机制。方法原代SD大鼠AVICs分离与培养,前钙化培养基(PCM)诱导钙化,为明确Hcy是否诱导AVICs钙化,先采用随机数字法将AVICs分为3组:对照(NC)组,PCM组,PCM+Hcy组;为进一步明确ERS抑制剂4-PBA是否能抑制Hcy诱导的AVICs钙化,采用随机数字法将AVICs分为4组:PCM+Hcy组,4-PBA组,PCM+4-PBA组,PCM+Hcy+4-PBA组。为明确4-PBA是否能通过促进自噬,从而缓解Hcy诱导的AVICs钙化,采用随机数字法将AVICs分别分为NC组,PCM组,PCM+Hcy组;PCM+Hcy组,PCM+Hcy+4-PBA组;PCM+Hcy+4-PBA组,PCM+Hcy+4-PBA+3-甲基腺嘌呤(3-MA)组。Western blot检测内质网跨膜蛋白[蛋白激酶R样内质网激酶(PERK)、活化转录因子6(ATF6)]、成骨因子[Runt相关转录因子2(Runx2)、骨桥蛋白(OPN)]、自噬效应蛋白(Beclin1)的蛋白表达;茜素红染色测量钙化结节。结果在成功建立AVICs钙化模型基础上,茜素红染色显示PCM+Hcy组钙化结节形成较PCM组明显增多,PCM+Hcy组在PCM组基础上进一步上调内质网跨膜蛋白及成骨因子的表达,抑制了自噬;而4-PBA处理后则抑制了Hcy诱导的AVICs成骨分化,促进了自噬;在4-PBA基础上加入自噬抑制剂3-MA后,成骨分化抑制效应被逆转。结论Hcy能促进AVICs成骨分化,而4-PBA可通过抑制ERS,促进自噬,从而延缓Hcy诱导的AVICs成骨分化。

Objective To investigate the role of endoplasmic reticulum stress(ERS)molecular chaperone 4-phenylbutyric acid(4-PBA)in homocysteine(Hcy)-induced osteogenic differentiation of aortic valve interstitial cells(AVICs)and related mechanisms.Methods Primary SD rat AVICs were isolated and cultured,and calcification was induced by pre-calcification medium(PCM).In order to clarify whether Hcy induced calcification in AVICs,AVICs were first divided into three groups using the random number method:control(NC)group,PCM group,and PCM+Hcy group.To further clarify whether the ERS inhibitor 4-PBA could inhibit Hcy-induced calcification in AVICs,the random number method was used to divide the AVICs into 4 groups:PCM+Hcy group,4-PBA group,PCM+4-PBA group and PCM+Hcy+4-PBA group.To clarify whether 4-PBA could alleviate Hcy-induced calcification of AVICs by promoting autophagy,the AVICs were divided into NC group,PCM group,PCM+Hcy group;PCM+Hcy group,PCM+Hcy+4-PBA group;PCM+Hcy+4-PBA group,PCM+Hcy+4-PBA group,PCM+Hcy+4-PBA+3-methyladenosine(MA).Western blot was performed to detect the protein expression of endoplasmic reticulum transmembrane proteins[protein kinase R-like endoplasmic reticulum kinase(PERK),activating transcription factor 6(ATF6)],osteogenic factors[Runt-related transcription factor 2(Runx2),bone bridge protein(OPN)],autophagy effector protein(Beclin1);alizarin red staining was performed to measure calcified nodules.Results Based on the successful establishment of the AVICs calcification model,alizarin red staining showed that calcified nodule formation was significantly increased in the PCM+Hcy group compared with the PCM group,and the PCM+Hcy group further upregulated the expression of endoplasmic reticulum transmembrane proteins and osteogenic factors on top of the PCM group,which inhibited autophagy;whereas 4-PBA treatment inhibited the osteogenic differentiation of AVICs induced by Hcy and promoted autophagy;after adding autophagy inhibitor of 3-MA on top of 4-PBA,osteogenic differentiation was reversed.Conclusions Hcy may promote osteogenic differentiation of AVICs,whereas 4-PBA can retard Hcy-induced osteogenic differentiation of AVICs by inhibiting ERS and promoting autophagy.