TSB2通过减少脱偶联内皮型一氧化氮合酶氧自由基生成抑制动脉粥样硬化形成
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(1.中山大学附属第一医院高血压血管病科,,广东省广州市 510080;2.血管疾病诊治技术国家地方联合工程实验室(广东) 国家卫健委辅助循环重点实验室(中山大学) 广东省血管疾病诊治工程技术研究中心 广东省脑功能与 脑疾病重点实验室,,广东省广州市 510080;3.中山大学附属第一医院心脏外科,广东省广州市 510080)

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吴方圆,博士研究生,研究方向为趋炎脂质与血管内皮功能,E-mail:wufy5@mail2.sysu.edu.cn。通信作者欧志君,博士,主任医师,博士研究生导师,研究方向为趋炎脂质与血管内皮功能,E-mail:zhijunou@163.com。#为同等贡献。

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国家重点研发计划项目(2021YFA0805100);国家自然科学基金面上和重点项目(81970363、81830013、81370370);广东省基础与应用基础研究基金项目重点项目(2019B1515120092);广州市重点领域研发计划项目(202103000016);中山大学临床医学研究5010计划项目(2014002)


TSB2 inhibits atherosclerosis by reducing the production of superoxide anion of uncoupled endothelial nitric oxide synthase
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1.Division of Hypertension and Vascular Diseases, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510080, China;2.National-Guangdong Joint Engineering Laboratory for Diagnosis and Treatment of Vascular Diseases, NHC Key Laboratory of Assisted Circulation (Sun Yat-sen University) & Guangdong Provincial Engineering and Technology Center for Diagnosis and Treatment of Vascular Diseases & Guangdong Provincial Key Laboratory of Brain Function and Disease, Guangzhou, Guangdong 510080, China;3.Division of Cardiac Surgery, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510080, China)

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    目的]观察TSB2抑制热休克蛋白90(HSP90)与内皮型一氧化氮合酶(eNOS)的结合对动脉粥样硬化形成的影响。 [方法]用TSB2处理人脐静脉内皮细胞,用免疫共沉淀法检测HSP90与eNOS的结合情况。利用C57BL/6小鼠和低密度脂蛋白受体敲除(LDLR-/-)小鼠,普通饮食(ND)或高脂饮食(HFD)喂养12周,同时腹腔注射PBS或TSB2,分为4组:C57BL/6+ND+PBS组、LDLR-/-+ND+PBS组、LDLR-/-+HFD+PBS组、LDLR-/-+HFD+TSB2组。提取主动脉检测HSP90与eNOS的结合情况,检测主动脉和主动脉窦的粥样硬化斑块情况、一氧化氮(NO)和氧自由基(O2·-)生成情况,同时使用左旋精氨酸的竞争性底物L-单甲基-精氨酸(L-NMMA)明确NO的生成和一氧化氮合酶抑制剂左旋硝基精氨酸甲酯(L-NAME)明确O2·-的生成。 [结果]与对照组相比,加入TSB2处理的人脐静脉内皮细胞后,HSP90与eNOS的结合水平减少41.06%(P<0.05)。与LDLR-/-+HFD+PBS组相比,LDLR-/-+HFD+TSB2组小鼠主动脉中HSP90与eNOS的结合水平减少40.95%(P<0.05),主动脉内皮细胞O2·-生成水平减少63.73%(P<0.05)(L-NAME明显抑制LDLR-/-+HFD+PBS组O2·-的生成),但NO生成量未见明显变化(L-NMMA抑制所有组NO的生成),同时主动脉及主动脉窦的斑块形成水平分别减少59.39%和68.86%(P<0.05)。 [结论]TSB2通过抑制主动脉血管内皮细胞HSP90与eNOS的结合,减少血管内皮细胞脱偶联eNOS的O2·-生成,最终抑制动脉粥样硬化形成。

    Abstract:

    Aim To observe the effect of TSB2 inhibiting the combination of heat shock protein 90 (HSP90) and endothelial nitric oxide synthase (eNOS) on the formation of atherosclerosis. Methods Human umbilical vein endothelial cells (HUVEC) were treated with TSB2 and the combination between HSP90 and eNOS was detected by co-immunoprecipitation. C57BL/6 mice and low density lipoprotein receptor knockout (LDLR-/-) mice were fed with normal diet (ND) or high fat diet (HFD) for 12 weeks while injected with phosphate buffered saline (PBS) or TSB2 intraperitoneally. The mice were divided into four groups:C57BL/6+ ND+PBS group, LDLR-/-+ND+PBS group, LDLR-/-+HFD+PBS group, LDLR-/-+HFD +TSB2 group. Then the aorta was isolated. The combination between HSP90 and eNOS in aorta was measured. The atherosclerotic plaque in aorta and aortic sinus were determined. The production of nitric oxide (NO) and superoxide anion (O2·-) were also detected. At the same time, L-monomethyl-arginine (L-NMMA), a competitive substrate of L-arginine, was used to determine the production of NO, and L-nitroarginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, was used to determine the production of O2·-. Results Compared with control group, the combination between HSP90 and eNOS was decreased by 41.06% (P<0.05) in cultured HUVEC treated with TBS2. Compared with LDLR-/-+HFD+PBS group,the combination between HSP90 and eNOS in the mouse aortas was decreased by 40.95% (P<0.05) in LDLR-/- +HFD+TSB2 group, and the production of O2·- was decreased by 63.73% (P<0.05) (L-NAME significantly inhibited the production of O2·- in LDLR-/-+HFD+PBS group), while the production of NO had no significant change in the mouse aortic endothelial cells (L-NMMA inhibited NO production in all groups), and the formation of atherosclerotic lesions in aortas and aortic sinus were significantly decreased by 59.39% and 68.86% (P<0.05) respectively in LDLR-/-+HFD+TSB2 group. Conclusion TSB2 can reduce the O2·- production of uncoupled eNOS in vascular endothelial cells by inhibiting the combination of HSP90 and eNOS in aortic endothelial cells, and finally inhibits the formation of atherosclerosis.

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吴方圆,李艳,宁大晟,陈靖,李尚宣,彭月明,何诗慧,闫凤侠,区景松,欧志君. TSB2通过减少脱偶联内皮型一氧化氮合酶氧自由基生成抑制动脉粥样硬化形成[J].中国动脉硬化杂志,2023,(10):833~840.

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  • 收稿日期:2022-06-29
  • 最后修改日期:2023-02-19
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  • 在线发布日期: 2023-11-08