摘要
该研究利用间歇性低氧(intermittent hypoxia,IH)细胞模型,观察当归补血汤(Danggui Buxue Decoction,DBD)含药血清对H9c2心肌细胞损伤的作用及机制。将H9c2细胞分为5组:对照(control,CON)组、间歇性低氧(IH)组、间歇性低氧加DBD含药血清处理(IH+DBD)组、间歇性低氧加自噬增强剂雷帕霉素(rapamycin,RAPA)组(IH+RAPA)、间歇性低氧加DBD含药血清处理和自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)(IH+DBD+3-MA)组。丹酰尸胺(monodansylcadaverine,MDC)染色检测细胞自噬小体变化情况;细胞计数试剂盒-8(cell counting kit-8,CCK-8)、乳酸脱氢酶(lactate dehydrogenase,LDH)和肌酸激酶(creatine kinase,CK)试剂盒检测心肌细胞活性、细胞培养液中LDH和CK的含量,观察细胞损伤情况;原位末端转移酶标记(TdT-mediated dUTP nick-end labeling,TUNEL)染色法检测心肌细胞凋亡情况;JC-1荧光探针法检测线粒体膜电位变化情况;Western blot方法检测线粒体自噬相关蛋白微管相关蛋白轻链3Ⅱ型(microtubule-associated proteins light chain 3Ⅱ,LC3Ⅱ)、微管相关蛋白轻链3Ⅰ型(microtubule-associated proteins light chain 3Ⅰ,LC3Ⅰ)、P62、Parkin和凋亡相关蛋白半胱天冬酶-3前体(pro caspase-3)、半胱天冬酶-3(caspase-3)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)基因、Bcl-2-associated X(Bax)蛋白的表达变化情况。结果显示,与CON组比较,IH组MDC染色荧光强度、LC3Ⅱ/LC3Ⅰ及Parkin表达显著降低,P62表达显著升高,细胞存活率显著下降,培养液中LDH及CK含量显著升高,TUNEL阳性细胞数量显著增加,pro caspase-3/caspase-3、Bcl-2/Bax比值显著下降,线粒体膜电位显著下降。与IH组比较,IH+DBD组及IH+RAPA组MDC染色荧光强度、LC3Ⅱ/LC3Ⅰ及Parkin表达显著升高,P62表达显著降低,细胞存活率显著增加,培养液中LDH及CK含量显著减少,TUNEL阳性细胞数量显著减少,pro caspase-3/caspase-3、Bcl-2/Bax显著升高,线粒体膜电位显著升高;IH+DBD+3-MA组差异
This study aims to explore the effect and mechanism of Danggui Buxue Decoction(DBD)-containing serum in alleviating the H9c2 cell injury caused by the exposure to intermittent low oxygen.H9c2 cells were assigned into five groups:control(CON) group,intermittent low oxygen(IH) group,intermittent low oxygen plus DBD-containing serum(IH+DBD) group,intermittent low oxygen plus the autophagy enhancer rapamycin(IH+RAPA) group,and intermittent low oxygen plus DBD-containing serum and the autophagy inhibitor 3-methyladenine(IH+DBD+3-MA) group.Monodansylcadaverine(MDC) staining was employed to detect the changes of autophagosomes.Cell counting kit-8(CCK-8) assay was employed to determine the activity of myocardial cells,and lactate dehydrogenase(LDH) and creatine kinase(CK) kits were used to measure the LDH and CK levels in the cell culture,which would reflect the degree of cell damage.TdT-mediated dUTP nick-end labeling(TUNEL) staining was used to detect the apoptosis of myocardial cells,and JC-1 fluorescence probe to detect the changes in mitochondrial membrane potential.Western blot was employed to determine the expression levels of the autophagy-related proteins microtubule-associated proteins light chain 3Ⅱ(LC3Ⅱ),microtubule-associated proteins light chain 3Ⅰ(LC3Ⅰ),P62,Parkin and apoptosis related proteins pro caspase-3,caspase-3,B-cell lymphoma-2(Bcl-2),Bcl-2-associated X(Bax).The results showed that compared with the CON group,the IH group showed decreased fluorescence intensity of MDC staining,decreased LC3Ⅱ/LC3Ⅰ ratio,down-regulated Parkin expression,and up-regulated expression of P62.In addition,the IH group showed decreased cell survival rate,increased content of LDH and CK in the culture medium,increased number of TUNEL positive cells,and decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential.Compared with the IH group,the IH+DBD and IH+RAPA groups showed increased fluorescence intensity of MDC staining,increased LC3Ⅱ/LC3Ⅰ ratio,up-regulated Parkin expression
作者
李亭亭
陈捷
吉恩生
郭亚净
LI Ting-ting;CHEN Jie;JI En-sheng;GUO Ya-jing(Hebei University of Chinese Medicine,Shijiazhuang 050200,China;Hebei Technology Innovation Center of Traditional Chinese Medicine Combined Hydrogen Medicine,Shijiazhuang 050091,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2023年第21期5881-5887,共7页
China Journal of Chinese Materia Medica
基金
河北省医学科学研究课题(20220184)
河北中医学院省属高校基本科研业务费项目(YXTD2023004)。
