摘要
目的探讨艾叶提取物(Artemisia argyiextract,AAE)对DOX(doxorubicin,Dox)诱导心肌细胞损伤的影响。方法选择不同剂量的艾叶提取物(0.5、1、3 g/ml)处理经DOX预处理的大鼠心肌细胞H9C2细胞,MTT法检测细胞存活率,Hoechst检测细胞凋亡,免疫荧光检测p65的入核情况,蛋白印迹法检测凋亡和自噬相关蛋白的表达以及AMPK/mTOR/ULK1通路相关蛋白磷酸化,ELISA检测炎症因子TNF-α和IL-6的水平,试剂盒检测超氧化物歧化酶(superoxide dismutase,SOD),丙二醛(malondialdehyde,MDA)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)的水平。将SD大鼠分为对照组、AAE组(3 g/kg)、模型组(DOX)、DOX+AAE(0.5 g/kg)、DOX+AAE(1 g/kg)和DOX+AAE(3 g/kg)组。HE染色观察心肌病理损伤,TUNEL染色检测心肌细胞凋亡情况,蛋白印迹法检测自噬相关LC3Ⅱ/LC3Ⅰ蛋白和自噬相关通路AMPK/mTOR/ULK1蛋白磷酸化水平,ELISA检测炎症因子TNF-α和IL-6的水平,试剂盒检测SOD、MDA和GSH-Px的水平。结果体外实验,与对照组相比,DOX组细胞存活率、SOD、GSH-Px、LC3~+、LC3Ⅱ/LC3Ⅰ、AMPK/mTOR/ULK1通路相关蛋白磷酸化水平显著降低(P<0.05),凋亡率、TNF-α、IL-6、p65的入核数、MDA、SQSTM1、cleaved caspase 3和cleaved caspase 9蛋白水平显著升高(P<0.05);与DOX组相比,DOX+AAE(1 g/ml)组和DOX+AAE(3 g/ml)组细胞存活率、SOD、GSH-Px、LC3~+、LC3Ⅱ/LC3Ⅰ、AMPK/mTOR/ULK1通路相关蛋白磷酸化水平显著升高(P<0.05),凋亡率、TNF-α、IL-6、p65的入核数、MDA、SQSTM1、cleaved caspase 3和cleaved caspase 9表达水平显著降低(P<0.05)。体内实验与体外实验结果相一致,AAE抑制DOX诱导的大鼠心肌细胞凋亡、炎症反应、氧化应激水平,促进AMPK/mTOR/ULK1通路介导的心肌细胞自噬。结论 AAE减轻DOX诱导的心肌细胞损伤,这可能与其能抑制DOX诱导的心肌细胞凋亡、炎症反应、氧化应激水平和促进AMPK/mTOR/ULK1通路介导的心肌细胞自噬有关。
This study was performed to investigate the effects of Artemisia argyi extract(AAE)on myocardial cell injury induced by doxorubicin(Dox).Mouse cardiomyocyte H9C2 cells were introduced and pretreated with adriamycin,then treated with different doses of AAE(0.5,1,3 g/ml).MTT and Hoechst methods were used to detect the survival rate and apoptosis of the H9C2 cells,respectively;P65 was detected by immunofluorescence,while the levels of inflammatory factors TNF-αand IL-6 were detected by ELISA;the levels of superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione peroxidase(GSH-Px)were detected by kits.Western blot was used to detect the expression of apoptosis-and autophagy-related proteins and the phosphorylation of AMPK/mTOR/ULK1 pathway-related proteins.As for in vivo trial,SD rats were recruited and divided into control group,AAE(3 g/kg),model group(DOX),DOX+AAE(0.5 g/kg),DOX+AAE(1 g/kg)and DOX+AAE(3 g/kg)groups.Myocardial damage was observed by HE staining,while cardiomyocyte apoptosis was detected by TUNEL staining.Western blot was used to detect the phosphorylation levels of autophagy-related LC3II/LC3I proteins and autophagy-related pathway AMPK/mTOR/ULK1 proteins;the levels of inflammatory cytokines TNF-αand IL-6 were detected by ELISA,and the levels of SOD,MDA and GSH-Px were detected by kits.In vitro experiment data indicated that the cell survival rate,SOD,SOD,GSH-Px,LC3+,LC3 II/LC3 I,AMPK/mTOR/ULK1 pathway-related proteins phosphorylation levels were significantly reduced in the DOX(adriamycin)group compared with the control group(P<0.05),while the apoptosis rate,TNF-α,IL-6,p65 nucleus number,MDA,SQSTM1,cleaved caspase 3 and cleaved caspase 9 expression levels were significantly increased(P<0.05);compared with DOX group,both DOX+AAE groups(1 g/ml,3 g/ml)demonstrated increases in cell survival rate,SOD,GSH-Px,LC3+,LC3 II/LC3 I,AMPK/mTOR/ULK1 pathway-related protein phosphorylation levels(P<0.05),and decreases in apoptosis rate,TNF-α,IL-6,p65 nucleus number,MDA,SQSTM1,cleaved caspase 3 and cleaved caspase 9 expression levels(P<0.05).The data from in vivo experiments were consistent with those from in vitro experiments:AAE inhibited DOX-induced apoptosis,inflammatory response and oxidative stress levels in rat cardiomyocytes,while promoted AMPK/mTOR/ULK1 pathway-mediated autophagy in cardiomyocytes.In conclusion,AAE attenuates DOX-induced cardiomyocyte injury,and the mechanism may relate to its ability of inhibiting DOX-induced cardiomyocyte apoptosis,inflammatory response,oxidative stress levels and promoting AMPK/mTOR/ULK1 pathway-mediated autophagy in cardiomyocytes.
作者
王中晓
杨峰
张义堂
胡清茹
马瑜红
曲震理
WANG Zhongxiao;YANG Feng;ZHANG Yitang;HU Qingru;MA Yuhong;QU Zhenli(Department of Basic Medicine,Nanyang Medical College,Nanyang 473000,China;Department of Pediatrics,Nanyang Central Hospital,Nanyang 473000,China)
出处
《免疫学杂志》
CAS
CSCD
北大核心
2021年第1期17-25,共9页
Immunological Journal
基金
河南省医学科技攻关计划(201504014)
南阳市2019年市级科技计划(KJGG124)。
关键词
艾叶提取物
炎症应答
氧化应激
自噬
凋亡
Artemisia argyi extract
Inflammatory
Oxidative stress
Autophagy
Apoptosis
