摘要
目的:探讨丹参酚酸B不同给药浓度和不同给药时间窗对缺氧损伤乳鼠心肌细胞TLR4-NFκB-TNFα炎性反应通路的调节作用。方法:分离纯化出生1~3 d Wistar乳鼠心肌细胞、建立体外缺氧细胞模型。丹参酚酸B(Sal B)大、中、小剂量浓度分别为10^(-5)mol/L、10^(-6)mol/L和10^(-7)mol/L并按照缺氧前6 h、缺氧同时和缺氧后6 h 3种给药方式进行干预。qRT-PCR法检测心肌细胞tool样受体4 (TLR4)和核因子κB(NFκB) mRNA的表达,细胞爬片免疫组化法检测TLR4和NFκB蛋白的表达,ELISA法检测细胞上清液中肿瘤坏死因子α(TNFα)的浓度。结果:与正常组比较,缺氧模型组TLR4和NFκB mRNA和蛋白的表达量、细胞上清液中TNFα浓度均显著升高(P <0. 05或P <0. 01)。和缺氧模型组比较,丹参酚酸B预防给药和同时给药大、中剂量组TLR4和NFκB mRNA和蛋白的表达量、细胞上清液中TNFα的浓度均显著下降(P <0. 05或P <0. 01)。预防给药大剂量组下降最显著(P <0. 01)。结论:TLR4-NFκB-TNFα炎性反应通路在缺氧损伤6h即明显激活。SalB预防或同时干预对缺氧损伤的心肌细胞有明显的保护作用,该作用与抑制TLR4-NFκB-TNFα炎性反应损伤通路相关。SalB具有量效和时效性,以大剂量预防给药效果最佳。
Objective To investigate the effects of different concentrations and different administration time of salvianolic acid B （SalB） on regulating TLR4-NFκB-TNFα inflammatory pathway in hypoxia neonatal rat cardiomyocytes. Methods： 1-3 d neonatal cardiomyocytes were isolated and purified, and hypoxia model was established in vitro. SalB high, medium and low dose concentrations were 10^-5 mol/L, 10^-6 mol/L and 10^-7 mol/L, and administered to cells at 6h before hypoxia, same time with hypoxia and 6h after hypoxia respectively. TLR4 and NFκB mRNA expression were detected by qRT-PCR; TLR4 and NFκB protein expression were detected by immunohistochemical method. The concentration of supernatant TNFα was detected by ELISA. Results： Compared with normal group, the expression of TLR4 and NFκB mRNA and protein, the concentration of TNFαin supernatant were significantly increased in hypoxia model group （ P 〈0.05 or P 〈0.01）. Compared with the hypoxia model group, the expression of TLR4 and NFκB mRNA and protein, the concentration of TNFα in supernatant were significantly decreased in large and middle dose of salvianolic acid B administered before and meantime of hypoxia group （ P 〈0.05 or P 〈0.01）. The most significant decrease was observed in the high-dose group administered before hypoxia （ P 〈0.01）. Conclusion： TLR4-NFκB-TNFα inflammatory pathway was significantly activated 6h after hypoxia injury. Salvianolic acid B administered before and same time of hypoxia protected cardiomyocytes tremendously. This effect was related to the inhibition of TLR4-NFκB-TNFα inflammatory pathway. Salvianolic acid B showed dose and time related effects, and the best effect was observed in the high-dose group administered before hypoxia.
作者
张云
张婷
郭丽丽
刘瑞华
吴广均
张盈颖
Zhang Yun;Zhang Ting;Guo Lili;Liu Ruihua;Wu GuangJun;Zhang YingYing(Research Department of Immunology,Guanganmen Hospital,China Academy of Chinese Medical Sciences,Beijing100053,China;Parmacology Department,Guanganmen Hospital,China Academy of Chinese Medical Sciences,Beijing 100053,China;Drug Research and Development Center,Guanganmen Hospital,China Academy of Chinese Medical Sciences,Beijing 100053,China)
出处
《世界中医药》
CAS
2018年第10期2559-2563,共5页
World Chinese Medicine
基金
国家自然科学基金项目(81774168)--从TLR4偶联肠道微生态-黏膜免疫探讨化浊祛湿通心方治疗冠心病机制
广安门医院科研基金项目(2016S349)--miRNA210转染HUVEC中HIF1a-VEGF的变化及SalB的调控
