血管紧张素Ⅱ1型受体在氧化三甲胺促进ApoE-/-小鼠动脉粥样硬化中的作用

The role of angiotensin Ⅱ type 1 receptor in trimethylamine oxide-induced atherosclerosis in ApoE-/- mice

摘要:

目的 探讨血管紧张素Ⅱ1型受体（AT1R）在氧化三甲胺（TMAO）促动脉粥样硬化中的作用。方法 用分子对接预测TMAO与AT1R可能的相互作用。将21只6周龄ApoE^-/-小鼠随机分为对照组、TMAO组、TMAO+替米沙坦组，每组7只。TMAO组在饲料中加1%胆碱复制高TMAO血症模型。TMAO+替米沙坦组采用替米沙坦10 mg/（kg·d）灌胃治疗。12周后采血，采用高效液相色谱串联质谱法测定血浆TMAO含量；油红O染色确定主动脉根部斑块面积；免疫组织化学法检测斑块内炎症因子单核细胞趋化蛋白-1（MCP-1）和白细胞介素-6（IL-6）的浸润；构建AT1R表达质粒，转染293T细胞，加入TMAO（200 μmol/L）或TMAO（200 μmol/L）+替米沙坦（1 μmol/L）作用0、5、10、15、30和60 min，检测AT1R下游信号通路ERK、PKC磷酸化活化情况。结果 分子对接预测到TMAO与AT1R之间的存在直接结合位点。3组小鼠主动脉根部斑块面积占比、斑块内MCP-1和IL-6表达比较，差异有统计学意义（P <0.05）；TMAO组较对照组主动脉根部斑块面积占比增加（P <0.05），斑块内MCP-1和IL-6表达升高（P <0.05），而替米沙坦组较TMAO组主动脉根部斑块面积占比下降（P <0.05），斑块内MCP-1和IL-6表达降低（P <0.05）。3组主动脉组织的AT1R、p-ERK1/2、p-PKC蛋白相对表达量比较，差异有统计学意义（P <0.05）；TMAO组较对照组升高（P <0.05），而替米沙坦组较TMAO组下降（P <0.05）。在转染AT1R质粒的293T细胞中，TMAO组（200 μmol/L）不同时间点的p-ERK1/2、p-PKC蛋白相对表达量比较，差异有统计学意义（P <0.05）；与0 min比较，作用15和30 min时p-ERK1/2蛋白相对表达量升高（P <0.05），作用10和15 min时p-PKC蛋白相对表达量升高（P <0.05）。而替米沙坦（1 μmol/L）作用后，各个时间点的p-ERK1/2和p-PKC蛋白相对表达量比较，差异无统计学意义（P >0.05）。结论 TMAO加重ApoE^-/-小鼠动脉粥样硬化机制可能包含了其对AT1R的作用。

Abstract:

Objective To explore the role of angiotensin Ⅱ type 1 receptor (AT1R) in the promotion of atherosclerosis by trimethylamine N-oxide (TMAO).Methods Molecular docking was used to predict the possible interaction between TMAO and AT1R. Twenty-one 6-week-old ApoE^-/- mice were randomly divided into control group, TMAO group, and TMAO + telmisartan group, with 7 mice in each group. The TMAO group was fed with 1% choline in the diet to replicate the high TMAO blood model. The TMAO + telmisartan group was treated with telmisartan 10 mg/(kg·d) by gavage. After 12 weeks, blood was collected, and plasma TMAO levels were determined by high-performance liquid chromatography-tandem mass spectrometry. Oil Red O staining was used to determine the plaque area at the aortic root. Immunohistochemistry was used to detect the infiltration of inflammatory factors monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) in the plaque. AT1R expression plasmids were constructed, transfected into 293T cells, and treated with TMAO (200 μmol/L) or TMAO (200 μmol/L) + telmisartan (1 μmol/L) for 0, 5, 10, 15, 30, and 60 min. The activation of the downstream signaling pathways ERK and PKC was detected.Results Molecular docking predicted a direct binding site between TMAO and AT1R. The proportion of plaque area at the aortic root, expression of MCP-1, and IL-6 in the plaque were statistically different among the three groups (P < 0.05). The TMAO group showed an increase in the proportion of plaque area at the aortic root and an elevation of MCP-1 and IL-6 expression compared to the control group (P < 0.05), while the telmisartan group showed a decrease compared to the TMAO group (P < 0.05). The expression of AT1R, p-ERK1/2, and p-PKC in aortic tissues differed significantly among the three groups (P < 0.05). The TMAO group increased significantly compared to the control group (P < 0.05), while the telmisartan group decreased compared to the TMAO group (P < 0.05). In 293T cells transfected with AT1R plasmids, the relative expression of p-ERK1/2 and p-PKC protein at different time points in the TMAO group (200 μmol/L) was statistically different (P < 0.05). Compared with 0 min, the relative expression of p-ERK1/2 increased at 15 and 30 min (P < 0.05), and the relative expression of p-PKC increased at 10 and 15 min (P < 0.05). After treatment with telmisartan (1 μmol/L), there was no statistically significant difference in the relative expression of p-ERK1/2 and p-PKC at each time point (P > 0.05).Conclusion The mechanism by which TMAO exacerbates atherosclerosis in ApoE^-/- mice may involve its action on AT1R.