Objective: To investigate the impact of homocysteine inducible endoplasmic reticulum(ER) protein with ubiquitin like domain 1 protein (Herpud1) in the homocysteine (Hcy) -induced phenotypic switching of vascular smooth muscle cells (VSMCs). Methods: VSMCs were derived from thoracic aortic artery of male Sprague Dawley rats and cultured VSMCs (4-7 passage) were treated with various concentrations of Hcy (0, 100, 500 and 1 000 μmol/L) and applied to immunofluorescence to observe the morphological changes of VSMCs via SM-actin staining. Western blot was used to detect the expression of VSMCs phenotypic markers, including Osteopontin, Calponin and smooth muscle myosin heavy chain (SM-MHC) and the expression of endoplasmic reticulum stress (ERS) related proteins, including C/EBP-homologous protein (CHOP), inositol-requiring kinase 1 (IRE-1) and glucose regulating protein 78 (GRP78) in the absence and presence of non-selective inhibitor of ERS, 4-phenylbutyric acid (4-PBA, 2 mg/ml). The Herpud1 mRNA and protein levels were determined in Hcy-stimulated VSMCs treated with 4-PBA or transfected with specific siRNA targeting Herpud1. Results: Compared with the control group, SM-actin staining results showed that the shape of VSMCs treated with different concentrations of Hcy for 24 hours changed from long fusiform into round form, arrangement of myofilament became irregular and the most significant alteration was found in the 500 μmol/L Hcy group. After intervention of 24 hours, various concentration of Hcy increased protein expression of Osteopontin, and reduced Calponin and SM-MHC protein expressions in VSMCs (all P<0.05). In addition, the results showed that Hcy increased the expression of CHOP, IRE-1 and GRP78 in a dose-dependent manner, which could be reversed by 4-PBA treatment (all P<0.05). However, 4-PBA inhibited Hcy induced upregulation of Osteopontin and downregulation of Calponin and SM-MHC, suggesting that ERS was involved in Hcy-induced phenotypic switching of VSMCs. Herpud1 protein was mostly expressed in the cytoplasm and was also expressed in the nucli, both in the control, Hcy and Hcy+4-PBA groups. Moreover, Hcy increased mRNA and protein levels of Herpud1 (P<0.05), whereas treatment with 4-PBA could significantly reduce Hcy-induced upregulation of Herpud1 (P<0.05). Furthermore, knockdown of Herpud1 abrogated the effects of Hcy on VSMCs phenotype markers. Conclusion: Herpud1 plays an important role in Hcy-induced phenotypic switching of VSMCs.
目的： 探讨同型半胱氨酸诱导内质网应激诱导的泛素样结构域1蛋白（Herpud1）在同型半胱氨酸（Hcy）诱导的大鼠主动脉平滑肌细胞（VSMC）表型转化中的作用。 方法： 取雄性Sprague Dawley大鼠胸主动脉，改良组织贴块法分离VSMC并进行培养，取第4~7代的VSMC用于实验。为明确Hcy能否诱导VSMC发生表型转化，将VSMC分为4组，即对照组以及Hcy100、500和1 000 μmol/L组。为明确内质网应激（ERS）是否参与VSMC表型转化，将VSMC分为3组，即对照组、Hcy500 μmol/L组（Hcy组）和Hcy+4-苯基丁酸钠盐（4-PBA）2 mg/ml组（Hcy+4-PBA组）。为进一步验证Herpud1在VSMC表型转化中的作用，将VSMC分为对照组、Hcy组、Hcy+siRNA阴性对照（scrambled）组和Hcy+Herpud1 siRNA组。免疫荧光法观察VSMC形态学变化和Herpud1蛋白改变。Western blot法检测VSMC表型转化相关标志蛋白骨桥蛋白、钙调节蛋白（Calponin）、平滑肌肌球蛋白重链（SM-MHC），ERS相关蛋白C/EBP同源蛋白（CHOP）、丝氨酸/苏氨酸-蛋白激酶/内切核糖核酸酶肌醇需要酶1（IRE-1）、葡萄糖调节蛋白质78（GRP78）以及Herpud1蛋白的表达水平。实时荧光定量PCR法检测VSMC Herpud1 mRNA的表达水平。 结果： （1）不同浓度Hcy对VSMC表型转化的影响：免疫荧光结果示，对照组VSMC成长梭形，肌丝明显，成束状分布，极性明显；干预24 h后，Hcy100 μmol/L组可见VSMC不同程度地变成椭圆形，肌丝分布紊乱，但仍具有一定极性；干预24 h后，Hcy500 μmol/L组VSMC肌丝不明显、极性缺失，而Hcy1 000 μmol/L组VSMC改变则与Hcy100 μmol/L组类似。Western blot结果显示，Hcy100 μmol/L组VSMC骨桥蛋白表达水平高于对照组，SM-MHC表达水平则低于对照组（P均<0.05）；Hcy500 μmol/L组VSMC骨桥蛋白表达水平高于Hcy 100 μmol/L组，Calponin和SM-MHC表达水平则低于Hcy100 μmol/L组（P均<0.05），而Hcy1 000 μmol/L组以上改变趋势不如500 μmol/L明显。（2）ERS在VSMC表型转化中的作用：干预24 h后，Hcy100、500和1 000 μmol/L组VSMC CHOP、IRE-1和GRP78蛋白表达水平均高于对照组（P均<0.05）。Hcy+4-PBA组VSMC CHOP、IRE-1和GRP78蛋白表达水平低于Hcy组（P均<0.05）。而且，Hcy+4-PBA组VSMC骨桥蛋白表达水平亦低于Hcy组（P<0.05），Calponin和SM-MHC蛋白的表达水平则高于Hcy组（P均<0.05）。（3）Herpud1在Hcy诱导的VSMC表型转化中的表达：干预24 h后，Hcy100、500和1 000 μmol/L组VSMC Herpud1蛋白表达水平均高于对照组（P均<0.05），且具有浓度依赖性。干预12 h后，Hcy（500 μmol/L）组VSMC Herpud1升高最为明显，干预48 h后开始下降。Hcy组VSMC Herpud1 mRNA表达水平在0.5、1、2、6、12、24 h均高于对照组（P均<0.05），且具有一定的时间依赖性，以12 h最为明显；Hcy+4-PBA组VSMC Herpud1 mRNA表达水平在上述各时点均低于Hcy组。免疫荧光染色显示，对照组、Hcy组和Hcy+4-PBA组Herpud1均主要分布在细胞浆中的内质网，细胞核也有少量分布。Hcy组VSMC Herpud1蛋白表达水平高于对照组（P<0.05），Hcy+4-PBA组则低于Hcy组（P<0.01）。（4）抑制Herpud1对Hcy诱导的VSMC表型转化的影响：Hcy+Herpud1 siRNA组VSMC骨桥蛋白表达水平低于Hcy+siRNA阴性对照组（P<0.05），Calponin和SM-MHC蛋白表达水平则均高于Hcy+siRNA阴性对照组（P均<0.05）。 结论： Herpud1在Hcy诱导的VSMC表型转化具有重要作用。.
Keywords: Blood vessels; Herpud1; Homocysteine; Myocytes, smooth muscle.