基于CaMKⅡδ-RIPK1/RIPK3-MLKL通路的黄芪-丹参配伍对缺氧/复氧诱导的H9C2细胞坏死性凋亡的影响  

Effects of Astragali Radix-Salviea Miltiorrhizae Radix et Rhizoma Compatibility on Hypoxia/Reoxygenation-induced H9C2 Cells Necroptosis Based on CaMKⅡδ-RIPK1/RIPK3-MLKL Pathway

作　　者：张丞波 王新东[1,2] ZHANG Chengbo;WANG Xindong(The Third Clinical College of Nanjing University of Chinese Medicine,Nanjing 210028,China;Affiliated Hospital of Traditional Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing 210028,China)

机构地区：[1]南京中医药大学第三临床医学院,江苏南京210028 [2]南京中医药大学附属中西医结合医院,江苏南京210028

出　　处：《中国中医药信息杂志》2023年第7期108-113,共6页Chinese Journal of Information on Traditional Chinese Medicine

基　　金：国家自然科学基金(81403386);全国名老中医药专家传承工作室建设项目(2022年);江苏省研究生科研与实践创新计划项目(SJCX22_0837、SJCX22_0858、SJCX22_0859);江苏省中医药局科技项目(YB201922)。

摘　　要：目的观察黄芪-丹参配伍对缺氧/复氧诱导的H9C2心肌细胞坏死性凋亡的影响,基于CaMKⅡδ-RIPK1/RIPK3-MLKL通路探讨其作用机制。方法体外培养H9C2细胞,建立缺氧/复氧模型。将细胞分为对照组、模型组、黄芪丹参+激活剂(油酸)组、黄芪丹参组和抑制剂(KN-93磷酸盐)组,分别于相应培养基培养。试剂盒检测细胞上清液乳酸脱氢酶(LDH)水平,流式细胞术检测细胞凋亡,RT-qPCR和Western blot检测钙调蛋白激酶(CaMK)Ⅱδ、受体相互作用蛋白激酶(RIPK)1、RIPK3、混合系激酶区域样蛋白(MLKL)mRNA和蛋白表达。结果与对照组比较,模型组细胞上清液LDH水平显著升高(P<0.01),细胞凋亡率显著升高(P<0.01),CaMKⅡδ、RIPK1、RIPK3和MLKL mRNA和蛋白表达显著升高(P<0.01);与模型组比较,黄芪丹参组细胞上清液LDH水平显著降低(P<0.01),细胞凋亡率显著降低(P<0.01),CaMKⅡδ、RIPK1、RIPK3和MLKL mRNA和蛋白表达显著降低(P<0.05,P<0.01)。黄芪丹参作用与CaMKⅡδ抑制剂KN-93磷酸盐相当。结论黄芪-丹参配伍可减轻心肌细胞缺氧/复氧损伤,抑制心肌细胞坏死性凋亡,其机制与下调CaMKⅡδ-RIPK1/RIPK3-MLKL通路有关。Objective To observe the effects of Astragali Radix-Salviea Miltiorrhizae Radix et Rhizoma compatibility on the necroptosis of H9C2 cardiomyocytes induced by hypoxia/reoxygenation;To explore its mechanism based on the CaMKⅡδ-RIPK1/RIPK3-MLKL pathway.Methods Cardiomyocytes H9C2 were cultured in vitro to establish a hypoxia/reoxygenation model.The cells were divided into control group,model group,Astragali Radix-Salviea Miltiorrhizae Radix et Rhizoma+activator(oleic acid)group,Astragali Radix-Salviea Miltiorrhizae Radix et Rhizoma group and inhibitor(KN-93 phosphate)group,and were clutured with corresponding medium.Lactate dehydrogenase(LDH)level was detected by kit,cell apoptosis was detected by flow cytometry,and the mRNA and protein expressions of calmodulin kinase(CaMK)Ⅱδ,receptor interacting protein kinase(RIPK)1,RIPK3 and MLKL were detected by RT-qPCR and Western blot.Results Compared with the control group,the level of LDH in supernatant significantly increased in the model group(P<0.01),the apoptosis rate increased significantly(P<0.01),and the mRNA and protein expressions of CaMKⅡδ,RIPK1,RIPK3 and MLKL significantly increased(P<0.01).Compared with the model group,the level of LDH in supernatant significantly decreased in Astragali Radix-Salviea Miltiorrhizae Radix et Rhizoma group(P<0.01),the apoptosis rate decreased significantly(P<0.01),and the mRNA and protein expressions of CaMKⅡδ,RIPK1,RIPK3 and MLKL significantly decreased(P<0.05,P<0.01).The effect of Astragali Radix-Salviea Miltiorrhizae Radix et Rhizoma and CaMKⅡδinhibitor KN-93 phosphate was equivalent.Conclusion Astragali Radix-Salviea Miltiorrhizae Radix et Rhizoma compatibility can alleviate the hypoxia/reoxygenation injury of cardiomyocytes and inhibit the necroptosis of cardiomyocytes,and the mechanism is related to down-regulation of CaMKⅡδ-RIPK1/RIPK3-MLKL pathway.

关 键 词：黄芪 丹参 H9C2细胞 缺氧/复氧 钙调蛋白激酶Ⅱδ 坏死性凋亡 RIPK1/RIPK3-MLKL通路 

分 类 号：R285.5[医药卫生—中药学]