硝石雄黄散对血管内皮细胞缺氧损伤的保护机制_马梓珊.pdf

广西医科大学学报JOURNAL OF GUANGXI MEDICAL UNIVERSITY2023 Jan；40（1）硝石雄黄散对血管内皮细胞缺氧损伤的保护机制*马梓珊1,2，李畅1,2#，黄汕梅1,2，银帮巧2，陈枝凡1,2，聂莎1,2，张子谦1,3，李力1,3，刘鹰1，唐耀平1,2,3,4（1.广西中医药大学，南宁530200；2.广西中医药大学附属瑞康医院，南宁530011；3.广西中医药科学实验中心，南宁530200；4.广西高发传染病中西医结合转化医学重点实验室，南宁530200）摘要目的：探索硝石雄黄散对血管内皮细胞缺氧损伤的保护机制。方法：采用Griess法检测中药硝石（XS）、雄黄（XH）、硝石雄黄散（XSXH）中硝酸盐（NO3-）和亚硝酸盐（NO2-）含量及各组小鼠血清一氧化氮（NO）水平；制备硝石雄黄散各组分药的小鼠血清，将人冠状动脉内皮细胞（HCAEC）随机分为空白（Blank）组、缺氧（Hypoxia）组、Hypoxia+XS组、Hypoxia+XH组和Hy-poxia+XSXH组，Blank常氧处理，其余组别予缺氧24 h制备细胞缺氧模型；利用小鼠血清干预HCAEC，采用四甲基罗丹明甲酯（TMRM）和线粒体超氧化物指示剂（MitoSox）检测各组HCAEC线粒体膜电位及活性氧（ROS）水平；Griess法检测各组HCAEC中NO水平及ELISA法检测ET-1、IL-6和TNF-水平。结果：XSXH组NO2-含量高于XS组而NO3-含量较XS组低（P0.001），XH组NO3-和NO2-含量最低（P0.001，P0.01）；与Blank组相比，XS组、XH组和XSXH组均能提高小鼠血清NO水平（P0.05，P0.01，P0.001）；细胞结果显示，Hypoxia组能显著降低细胞NO生物活性、升高ET-1和炎症因子（IL-6、TNF-）水平、线粒体膜电位降低及生成大量ROS（P0.001）；与Hypoxia组相比，Hypoxia+XS组、Hypoxia+XH组和Hypoxia+XSXH组对上述指标均有明显改善（P0.05，P0.01，P0.001），其中Hypoxia+XSXH组作用最强，Hypoxia+XS组强于Hypoxia+XH组（P0.05）。结论：XS、XH和XSXH均能不同程度地提高NO生物活性，通过降低ET-1水平和炎症因子水平，恢复细胞线粒体膜电位以及减少ROS生成来保护血管内皮细胞缺氧损伤，其中XSXH作用最强。关键词硝石雄黄散；一氧化氮；线粒体；活性氧；炎症因子中图分类号：R-33文献标志码：A文章编号：1005-930X（2023）01-0046-08DOI：10.16190/ki.45-1211/r.2023.01.008Protective mechanism of Xiaoshi Xionghuang San on hypoxia injury of vascular endothelialcellsMa Zishan1,2,Li Chang1,2,Huang Shanmei1,2,Yin Bangqiao2,Chen Zhifan1,2,Nie Sha1,2,Zhang Ziqian1,3,Li Li1,3,LiuYing1,Tang Yaoping1,2,3,4.(1.Guangxi University of Traditional Chinese Medicine，Nanning 530200,China;2.Ruikang Hospital Affiliated to Guangxi University of Traditional Chinese Medicine,Nanning 530011,China;3.Guangxi Scientific Research Center of Traditional Chinese Medicine,Nanning 530200,China;4.Guangxi KeyLaboratory of Translational Medicine for Treating High-Incidence Infectious Diseases with Integrative Medicine,Nanning 530200,China)AbstractObjective:To explore the protective mechanism of Xiaoshi Xionghuang San on vascular endothelialcell hypoxia injury.Methods:Griess method was used to detect the content of nitrate(NO3-)and nitrite(NO2-)inChinese medicine Xiaoshi(XS),Xionghuang(XH),and Xiaoshi Xionghuang San(XSXH),and the level of se-rum nitric oxide(NO)in mice of each group.Mouse serum of each component of Xiaoshi Xionghuang San wasprepared and human coronary crtery endothelial cells(HCAEC)were divided into Blank,Hypoxia,Hypoxia+XS,Hypoxia+XH and Hypoxia+XSXH groups.Blank group was treated with normoxia,and the other groups weregiven hypoxia for 24 h to prepare cell hypoxia mo-dels.Serum of mice was used to intervene HCAEC;tetramethylrhodamine(TMRM)and mitochondrialsuperoxide indicator(MitoSOX)were used to detectmitochondrial membrane potential and reactive oxy-gen species(ROS)levels of HCAEC in each group.*基金项目：国家自然科学基金资助项目（No.81774115；No.82160856）；广西中医药大学博士科研启动基金项目资助（No.2020BS035）通信作者，唐耀平，E-mail：；刘鹰，E-mail：#共同第一作者收稿日期：2022-11-14 46The levels of nitric oxide(NO)in HCAEC of each group were detected by Griess and the levels of ET-1 and IL-6and TNF-were detected by ELISA.Results:The content of NO2-in XSXH group was higher than that in XSgroup and the content of NO3-was lower than that in XS group(P0.001),and the content of NO3-and NO2-wasthe lowest in XH group(P0.001,P0.01).Compared with Blank group,levels of serum NO of mice in XSgroup,XH group and XSXH group increased(P0.05,P0.01,P0.001).Cell results showed that Hypoxiagroup could significantly reduce the biological activity of NO in cells,increase the levels of ET-1 and inflammato-ry factors(IL-6,TNF-),decrease mitochondrial membrane potential and generate a large number of ROS(P0.001).Compared with Hypoxia group,the above indicators in the Hypoxia+XS,Hypoxia+XH and Hypoxia+XSXH groups significantly improved(P0.05,P0.01,P0.001),of which the Hypoxia+XSXH group hadthe strongest effect and the Hypoxia+XS group was stronger than the Hypoxia+XH group(P0.05).Conclu-sion:XS,XH and XSXH can all increase the biological activity of NO to varying degrees and protect the hypox-ia injury of vascular endothelial cells by reducing the levels of ET-1 and inflammatory factors,restoring mito-chondrial membrane potential and reducing the generation of ROS,among which XSXH has the strongest effect.KeywordsXiaoshi Xionghuang San;nitric oxide;mitochondria;reactive oxygen species;inflammatory factor冠状动脉粥样硬化性心脏病（coronary arterydisease，CAD）的死亡率及发病率至今仍居高不下1，严重威胁人类健康与生存。血管内皮功能障碍是 CAD 发生发展的重要原因，一氧化氮（nitricoxide，NO）已被证明在血管内皮细胞中发挥重要作用2，当血管内皮细胞受损会使NO产生减少或活性降低、内皮素1（ET-1）升高、线粒体膜电位受损、活性氧（reactive oxygen species，ROS）水平升高和炎症反应3-4。线粒体是细胞产生ROS并造成氧化损伤的主要场所，而ROS水平增加也会进一步导致NO水平降低和内皮细胞损伤发生5。研究表明，提高NO生物活性、维持线粒体稳态、减少ROS生成都有助于对缺氧诱导的内皮细胞功能损伤的保护6-7。中医早在 辅行诀脏腑用药法要 就记载了通过将硝石与雄黄研为极细末，令急心痛（冠心病）患者含服舌下并随涎咽下，以此在缓解急性心绞痛中取得显著疗效8。本课题组前期研究发现，硝石、雄黄和硝石雄黄散均能不同程度地调控小鼠血清NO水平、降低心肌梗死标志物水平并减小心肌梗死面积来改善小鼠心肌缺血损伤，以硝石雄黄散作用效果最明显9，然而硝石雄黄散是否可以保护血管内皮缺氧损伤及其机制尚不清楚。因此，本研究通过检测硝石雄黄散各组分药的硝酸盐（nitrate，NO3-）和亚硝酸盐（nitrite，NO2-）含量、含服硝石雄黄散后对小鼠血清NO水平的影响以及硝石雄黄散对人冠状动脉内皮细胞（human coronary artery endothelialcells，HCAEC）NO和ET-1水平、线粒体功能、ROS水平及炎症因子白细胞介素（IL）-6、肿瘤坏死因素-（TNF-）水平的影响，初步探索硝石雄黄散对缺氧诱导血管内皮细胞损伤的保护作用及潜在的机制。1材