摘要
目的探讨苯甲酰乌头碱(benzoylaconitine,BAC)对H9c2心肌细胞氧糖剥夺(oxygen glucose deprivation,OGD)/复氧(reoxyenation,R)损伤的保护作用。方法体外培养建立H9c2心肌细胞OGD/R损伤模型,通过不同浓度(0、25、50、75、100μmol/L)BAC处理,确定最佳BAC治疗浓度。根据BAC最佳剂量,将心肌细胞分为对照组、OGD/R组、BAC处理组(OGD/R+75μmol/L BAC组)、PI3K/Akt抑制剂组(OGD/R+LY294002组)以及BAC和PI3K/Akt抑制剂组(OGD/R+LY294002+75μmol/L BAC组)。通过相关试剂盒评估细胞活力及细胞乳酸脱氢酶(lactate dehydrogenase,LDH)水平;检测细胞肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素(interleukin,IL)-6、IL-1β、丙二醛(malondialdehyde,MDA)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)的表达。蛋白质免疫印迹试验(Western Blot)检测磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)通路蛋白和微管相关蛋白1轻链3(light chain 3,LC3)Ⅱ/LC3Ⅰ、苄氯素1(Beclin1)和自噬降解底物(P62)等自噬相关蛋白的表达情况。结果与对照组比较,OGD/R组细胞活力显著下降,LDH显著升高(P<0.01);与OGD/R组比较,OGD/R+75μmol/L BAC组H9c2心肌细胞活力显著升高(0.87±0.06 vs 0.49±0.06,P<0.01),ODG/R+75μmol/L BAC组LDH水平显著下降(86.75±7.79 U/L vs 234.42±6.20 U/L,P<0.01)。与对照组比较,OGD/R组TNF-α、IL-6、IL-1β、MDA、LC3Ⅱ/LC3Ⅰ、Beclin-1表达显著升高(P<0.01),OGD/R组GSH-Px、p-PI3K、p-Akt、P62表达显著降低(P<0.01);与OGD/R组比较,OGD/R+75μmol/L BAC组p-PI3K、p-Akt、P62蛋白表达显著升高(0.90±0.07 vs 0.58±0.04、1.02±0.02 vs 0.49±0.01、1.48±0.05 vs 0.87±0.04),LC3Ⅱ/LC3Ⅰ、Beclin-1表达显著降低(0.52±0.01 vs 1.24±0.04、0.12±0.01 vs 0.32±0.02,P<0.01)。结论BAC能够降低OGD/R后心肌细胞炎性反应和氧化应激水平,调节细胞自噬稳态,降低心肌细胞损伤,其调节心肌自噬稳态的作用可能与PI3K/Akt通路的激活有关。
Objective To investigate the protective effect of benzoylaconine(BAC)on H9c2cardiomyocytes after oxygen glucose deprivation/reoxyenation(OGD/R)injury.Methods After an in vitro model of OGD/R injury was established in H9c2cells,the cells were treated with BAC at different concentrations(0,25,50,75,100μmol/L)to determine its optimal dose.Then,H9c2 cells were randomly divided into control group,OGD/R group,OGD/R+BAC group(75μmol),OGD/R+LY294002group(PI3K/Akt inhibitor),and OGD/R+LY294002+BAC group.Corresponding reagent kits were used to determine cell viability and LDH level,as well as the expression levels of TNF-α,IL-6,IL-1β,MDA and GSH-Px in the cells.Western blotting was applied to detect the expression of the PI3K/Akt pathway proteins,as well as autophagic proteins such as LC3,Beclin1,and P62.Results Compared to the control group,the cell viability was significantly decreased,and LDH level was obviously increased in the OGD/R group(P<0.01).Treatment of 75μmol/L BAC significantly increased the cell viability(0.87±0.06 vs 40.49±0.06,P<0.01)and decreased the LDH level(86.75±7.79U/L vs 234.42±6.20U/L,P<0.01)when compared to the levels of the OGD/R group.OGD/R injury induced notable increases in TNF-α,IL-6,IL-1β,and MDA expression levels,while decrease of GSH-Px expression level(P<0.01),and downregulation of p-PI3K,p-Akt and P62and up-regulation of LC3Ⅱ/LC3Ⅰand Beclin-1(P<0.01)when compared with the control group.Treatment of 75μmol/L BAC increased the levels of p-PI3K,p-Akt,and P62proteins(0.90±0.07 vs 0.58±0.04,1.02±0.02 vs 0.49±0.01,1.48±0.05 vs 0.87±0.04)and decreased those of LC3Ⅱ/LC3Ⅰand Beclin-1(0.52±0.01 vs 1.24±0.04,0.12±0.01 vs 0.32±0.02)when compared with the OGD/R group(P<0.01).Conclusions BAC attenuates the inflammatory response and oxidative stress of myocardial cells after OGD/R injury,regulates autophagy homeostasis,and reduces myocardial cell damage.Its regulatory effect on myocardial autophagy homeostasis may be related to the activation of the PI3K/Akt pathway.
作者
周伍明
郎胜坤
葛新
蒋薇
贾迪
姚浩
宦陟榕
Zhou Wuming;Lang Shengkun;Ge Xin;Jiang Wei;Jia Di;Yao Hao;Huan Zhirong(Department of Emergency and Critical Care Medicine,Wuxi Ninth People's Hospital Affiliated to Soochow University,Wuxi 214062,Jiangsu Province,China)
出处
《中华老年心脑血管病杂志》
北大核心
2025年第2期211-216,共6页
Chinese Journal of Geriatric Heart,Brain and Vessel Diseases
基金
无锡市卫生健康委重大科研项目(Z202105)
无锡市第二届“双百”中青年医疗卫生拔尖人才项目(BJ2023108)。
作者简介
通信作者:葛新,Email:gexin2021@suda.edu.cn。
