连翘苷通过磷脂酰肌醇3激酶-蛋白激酶信号通路减轻缺氧诱导心肌细胞损伤的研究  

Effect of forsythin on hypoxia induced injury of cardiomyocyte by phosphatidylinositol 3kinase protein kinase signaling pathway

作　　者：杨宁[1] 柏勇平[2] YANG Ning;BAI Yong-ping(Department of Emergency,Xiangya Hospital,Central South University,Changsha 410008,Hunan Province,China;Department of Geriatrics,Xiangya Hospital,Central South University,Changsha 410008,Hunan Province,China)

机构地区：[1]中南大学湘雅医院急诊科,湖南长沙410008 [2]中南大学湘雅医院老干科,湖南长沙410008

出　　处：《中国临床药理学杂志》2023年第1期22-26,共5页The Chinese Journal of Clinical Pharmacology

基　　金：湖南省科技创新重大基金资助项目(2020SK1013);北京协和医学基金-睿E(睿意)急诊医学科研专项基金资助项目(2021-22)。

摘　　要：目的观察连翘苷对缺氧诱导h9c2细胞存活、氧化应激及凋亡的影响,并探究其与磷脂酰肌醇3激酶-蛋白激酶(PI3K/Akt)信号通路之间的关系。方法不同浓度连翘苷处理h9c2细胞,筛选最适浓度10μmol·L^(-1);建立缺氧诱导h9c2细胞设为模型组,正常培养的细胞设为对照组;10μmol·L^(-1)连翘苷处理的模型细胞设为连翘苷组;10μmol·L^(-1)连翘苷联合Akt信号通路激动剂SC79(5μg·mL^(-1))处理的模型细胞设为连翘苷+SC79组。以细胞计数试剂盒-8(CCK-8)检测细胞存活率,以酶联免疫吸附(ELISA)法检测乳酸脱氢酶(LDH)、丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-Px)的含量;以免疫荧光实验检测细胞活性氧(ROS)含量;以蛋白质印迹实验检测裂解的胱天蛋白酶(cleaved capase-3)、B细胞淋巴瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、磷酸化磷脂酰肌醇3激酶(p-PI3K)、PI3K、磷酸化蛋白激酶B(p-Akt)和Akt蛋白表达;以流式细胞术检测细胞凋亡率。结果对照组、模型组、连翘苷组、连翘苷+SC79组细胞的存活率分别为(100.01±8.53)%,(60.56±6.27)%,(84.51±7.25)%,(72.48±6.43)%;LDH分别为(864.86±64.47),(1554.17±142.42),(935.84±82.83),(1217.56±101.74)U·L^(-1);ROS分别为(40.01±5.16),(110.56±11.12),(71.51±5.84),(94.48±10.64)MFI;MDA分别为(5.84±0.76),(11.76±1.33),(7.26±0.95),(10.57±1.02)nmol·mg^(-1)prot;GSH-Px分别为(0.94±0.08),(0.59±0.07),(0.72±0.08),(0.62±0.07)U·μg^(-1)prot;凋亡率分别为(4.77±0.72)%,(15.42±2.13)%,(7.64±1.28)%,(14.30±2.54)%;p-PI3K分别为0.56±0.06,0.21±0.03,0.39±0.04,0.28±0.05;p-Akt分别为0.66±0.05,0.31±0.06,0.49±0.07,0.38±0.04。以上指标,模型组与对照组相比,连翘苷组与模型组相比,连翘苷+SC79组与连翘苷组相比,差异均有统计学意义(均P<0.05)。结论连翘苷促进缺氧诱导h9c2细胞存活,抑制细胞氧化应激和凋亡,与激活PI3K/Akt信号通路的活性有关。Objective To observe the effects of forsythin on hypoxia induced survival,oxidative stress and apoptosis of H9 c2 cells,and to explore the relationship between forsythin and protein phosphatidylinositol-3-kinase/protein kinase(PI3 K/Akt)signaling pathway.Methods H9 c2 cells were treated with different concentrations of forsythin,and the optimum concentration was 10μmol·L^(-1).Hypoxia induced H9 c2 cells were established as the model group,the normal cultured cells were set as control group.The model cells treated with10μmol·L^(-1)forsythin were set as forsythin group.The model cells treated with 10μmol·L^(-1)forsythin combined with Akt signaling pathway agonist SC79(5μg·mL^(-1))were set as forsythin+SC79 group.Cell counting kit-8(CCK-8)was used to detect the cell survival rate,enzyme-linked immunosorbent assay(ELISA)was used to detect the contents of lactate dehydrogenase(LDH),malondialdehyde(MDA)and glutathione peroxidase(GSH-Px).Immunofluorescence assay was used to detect the content of reactive oxygen species(ROS).Western blotting(WB)was used to detect the expression of cleaved cysteine protease-3,B-cell lymphoma-2(Bcl-2),Bcl-2 related X protein(Bax),phosphorylated phosphatidylinositol kinase(p-PI3K),PI3K,phosphorylated protein kinase B(p-Akt)and Akt.Flow cytometry was used to detect the apoptosis rate.Results The survival rates of cells in control group,model group,forsythiin group and forsythiin+SC79 group were(100.01±8.53)%,(60.56±6.27)%,(84.51±7.25)%,(72.48±6.43)%;LDH were(864.86±64.47),(1554.17±142.42),(935.84±82.83),(1217.56±101.74)U·L^(-1);ROS were(40.01±5.16),(110.56±11.12),(71.51±5.84),(94.48±10.64)MFI;MDA were(5.84±0.76),(11.76±1.33),(7.26±0.95),(10.57±1.02)nmol·mg^(-1)prot;GSH-Px were(0.94±0.08),(0.59±0.07),(0.72±0.08),(0.62±0.07)U·μg^(-1)prot;apoptosis rate were(4.77±0.72)%,(15.42±2.13)%,(7.64±1.28)%,(14.30±2.54)%;p-PI3K were 0.56±0.06,0.21±0.03,0.39±0.04,0.28±0.05;p-Akt were 0.66±0.05,0.31±0.06,0.49±0.07,0.38±0.04,respectively.Compared between control gr

关 键 词：连翘苷 磷脂酰肌醇3激酶-蛋白激酶信号通路 缺氧诱导h9c2细胞 氧化应激 凋亡 

分 类 号：R28[医药卫生—中药学]