本研究旨在探讨同型半胱氨酸（Hcy）诱导血管平滑肌细胞（VSMC）脂质沉积和泡沫细胞形成的作用机制，为动脉粥样硬化（As）的防治提供理论依据。通过 C1q/肿瘤坏死因子相关蛋白 9 (CTRP9) 启动子区域高甲基化负调节内质网应激 (ER)。因此，将载脂蛋白E缺陷(ApoE-/-)小鼠随机分为对照组[ApoE-/-  正常饮食(NC)]和高蛋氨酸组[ApoE-/-  (正常饮食添加1.7%蛋氨酸(HMD)]组(n = 6只小鼠/组)，饲养15周后，采用自动生化分析仪测定血清同型半胱氨酸(Hcy)、总胆固醇(TC)、甘油三酯(TG)水平，HE染色和油红O染色。主动脉根部观察病理变化，同时免疫荧光染色检测CTRP9、葡萄糖调节蛋白78 kD（GRP78）、磷酸化蛋白激酶RNA样ER激酶（p-PERK）、源自小鼠主动脉根的 VSMC 中的激活转录因子 6a (ATF6a)、磷酸化肌醇需要酶 -1α (p-IRE1α)、甾醇调节元件结合蛋白 -1c (SREBP1c) 和甾醇调节元件结合蛋白 -2 (SREBP2)。在体外，VSMC 用 100 μmol/l Hcy 刺激。转染过表达并干扰CTRP9的质粒后，给予ERs激动剂（TM）和抑制剂（4-PBA）刺激VSMC细胞。采用HE染色和油红O染色观察Hcy刺激对VSMC脂质沉积的影响。此外，通过RT-qPCR和蛋白质印迹分析分别检测VSMC中CTRP9、GRP78、PERK、ATF6a、IRE1α、SREBP1c和SREBP2的mRNA和蛋白表达水平。最后，研究了CTRP9启动子区的甲基化修饰。利用NCBI数据库搜索CTRP9基因的启动子区域，利用CpG岛预测甲基化位点。 Hcy 刺激 VSMC、过度表达 DNMT1 并用 5-Azc 干预后，通过亚硫酸氢盐测序 PCR (BSP) 评估 CTRP9 启动子的甲基化水平。结果显示，ApoE-/-  HMD组血清Hcy、TC、TG水平较ApoE-/-  NC组显着升高。另外，HE染色和油红O染色显示血管壁有明显的AS斑块形成，VSMC中有大量脂肪沉积，表明高同型半胱氨酸血症动物模型建立成功。此外，ApoE-/-  HMD组主动脉VSMC中CTRP9表达下调，而GRP78、p-PERK、ATF6a、p-IRE1α、SREBP1c、SREBP2表达上调。与体内结果一致，Hcy可以抑制VSMC中CTRP9的表达并诱导VSMC中ER和脂质沉积。同时，CTRP9表达增加可以减少ERs并保护Hcy诱导的VSMC中的脂质沉积。此外，ERs可以促进Hcy诱导的VSMC脂质沉积，抑制ERs可以减少Hcy诱导的VSMC脂质沉积，CTRP9可能通过ERs的负调节在Hcy诱导的VSMC脂质沉积和泡沫细胞转化中发挥保护作用。此外，Hcy组中的CTRP9启动子显示出高甲基化。在Hcy干预的同时，过表达DNMT1会增加CTRP9启动子的甲基化水平，而5-Azc可以降低CTRP9启动子的甲基化水平。最后，Hcy可以上调DNMT1的表达并下调CTRP9的表达。 DNMT1过表达后，CTRP9的表达进一步降低。 5-Azc抑制DNMT1后，DNMT1的表达降低，而CTRP9的表达增加。提示Hcy抑制CTRP9表达的分子机制与Hcy诱导、DNMT1调控的CTRP9启动子高甲基化有关。 5-Azc可以抑制DNMT1的表达并逆转DNMT1对CTRP9的调节作用。总体而言，本研究结果表明，Hcy 通过上调 DNMT1 表达诱导 CTRP9 启动子区域 DNA 高甲基化，并负向调节 ER 介导的 VSMC 脂质沉积和泡沫细胞形成。 CTRP9 可能是治疗高同型半胱氨酸血症和 As 的潜在治疗靶点。© 2023。作者。

To provide a theoretical basis for the prevention and treatment of atherosclerosis (As), the current study aimed to investigate the mechanism underlying the effect of homocysteine (Hcy) on inducing the lipid deposition and foam cell formation of the vascular smooth muscle cell (VSMC) via C1q/Tumor necrosis factor-related protein9 (CTRP9) promoter region Hypermethylation negative regulating endoplasmic reticulum stress (ERs). Therefore, apolipoprotein E deficient (ApoE-/-) mice were randomly divided into the control [ApoE-/- + normal diet (NC)] and high methionine [ApoE-/- + (normal diet supplemented with 1.7% methionine (HMD)] groups (n = 6 mice/group). Following feeding for 15 weeks, the serum levels of Homocysteine (Hcy), total cholesterol (TC), and triglyceride (TG) were measured using an automatic biochemical analyzer. HE and oil red O staining were performed on the aorta roots to observe the pathological changes. Additionally, immunofluorescence staining was performed to detect the protein expression levels of CTRP9, glucose-regulated protein 78 kD (GRP78), phosphorylated protein kinase RNA-like ER kinase (p-PERK), activating transcription factor 6a (ATF6a), phosphorylated inositol-requiring enzyme-1α (p-IRE1α), sterol regulatory element binding proteins-1c (SREBP1c) and sterol regulatory element binding proteins-2 (SREBP2) in VSMC derived from murine aortic roots. In vitro, VSMC was stimulated with 100 μmol/l Hcy. After transfection of plasmids with overexpression and interference of CTRP9, ERs agonist (TM) and inhibitor (4-PBA) were given to stimulate VSMC cells. HE staining and oil red O staining were used to observe the effect of Hcy stimulation on lipid deposition in VSMC. Additionally, The mRNA and protein expression levels of CTRP9, GRP78, PERK, ATF6a, IRE1α, SREBP1c, and SREBP2 in VSMC were detected by RT-qPCR and western blot analysis, respectively. Finally, The methylation modification of the CTRP9 promoter region has been studied. The NCBI database was used to search the promoter region of the CTRP9 gene, and CpG Island was used to predict the methylation site. After Hcy stimulation of VSMC, overexpression of DNMT1, and intervention with 5-Azc, assess the methylation level of the CTRP9 promoter through bisulfite sequencing PCR (BSP). The results showed that the serum levels of Hcy, TC, and TG in the ApoE-/- + HMD group were significantly increased compared with the ApoE-/- + NC group. In addition, HE staining and oil red O staining showed obvious AS plaque formation in the vessel wall, and a large amount of fat deposition in VSMC, thus indicating that the hyperhomocysteinemia As an animal model was successfully established. Furthermore, CTRP9 were downregulated, while GRP78, p-PERK, ATF6a, p-IRE1α, SREBP1c, SREBP2 was upregulated in aortic VSMC in the ApoE-/- + HMD group. Consistent with the in vivo results, Hcy can inhibit the expression of CTRP9 in VSMC and induce ERs and lipid deposition in VSMC. Meanwhile, the increased expression of CTRP9 can reduce ERs and protect the lipid deposition in Hcy induced VSMC. Furthermore, ERs can promote Hcy induced VSMC lipid deposition, inhibition of ERs can reduce Hcy induced VSMC lipid deposition, and CTRP9 may play a protective role in Hcy induced VSMC lipid deposition and foam cell transformation through negative regulation of ERs. In addition, The CTRP9 promoter in the Hcy group showed hypermethylation. At the same time as Hcy intervention, overexpression of DNMT1 increases the methylation level of the CTRP9 promoter, while 5-Azc can reduce the methylation level of the CTRP9 promoter. Finally, Hcy can up-regulate the expression of DNMT1 and down-regulate the expression of CTRP9. After overexpression of DNMT1, the expression of CTRP9 is further decreased. After 5-Azc inhibition of DNMT1, the expression of DNMT1 decreases, while the expression of CTRP9 increases. It is suggested that the molecular mechanism of Hcy inhibiting the expression of CTRP9 is related to the hypermethylation of the CTRP9 promoter induced by Hcy and regulated by DNMT1. 5-Azc can inhibit the expression of DNMT1 and reverse the regulatory effect of DNMT1 on CTRP9. Overall, the results of the present study suggested that Hcy induces DNA hypermethylation in the CTRP9 promoter region by up-regulating DNMT1 expression, and negatively regulates ERs mediated VSMC lipid deposition and foam cell formation. CTRP9 may potentially be a therapeutic target in the treatment of hyperhomocysteinemia and As.© 2023. The Author(s).