从线粒体凋亡通路探讨参附注射液对脓毒症小鼠心肌损害的影响  

Discussion on effect of Shenfu injection on myocardial damage of sepsis mice through mitochondria apoptotic pathway

作　　者：徐坡[1] 谢靖 彭媛 陆士奇[4] XU Po;XIE Jing;PENG Yuan;LU Shiqi(Department of Intensive Care Unit,Jingjiang Chinese Medicine Hospital,Jingjiang 214500,China)

机构地区：[1]靖江市中医院重症医学科,214500 [2]南京中医药大学第一临床医学院 [3]昆山市第一人民医院重症医学科 [4]苏州大学附属第一医院急诊科

出　　处：《环球中医药》2021年第7期1197-1203,共7页Global Traditional Chinese Medicine

基　　金：昆山市社会发展科技专项(KS18060)

摘　　要：目的观察参附注射液对脂多糖(Lipopolysaccharides, LPS)所致脓毒症小鼠心肌线粒体凋亡及Caspase-9蛋白表达的影响,从线粒体凋亡通路探讨参附注射液对脓毒症心肌细胞的保护机制。方法选用56只6周龄SPF级雄性B6小鼠,随机分为正常对照组(NC)、假脓毒症组(Sham)、脓毒症模型组(LPS)、参附注射液组(SFI)4组。采用腹腔注射LPS法构建脓毒症小鼠模型,分别于造模后6小时、12小时、24小时取小鼠心肌组织,电镜观察小鼠心肌超微结构变化,苏木精-伊红染色(HE)及心肌细胞凋亡TUNEL检测,Western-blot法测定心肌组织B细胞淋巴瘤/白血病-2基因(B-cell lymPhoma-2,Bcl-2)、BH3介导的死亡域激动剂(BH3 interacting-domain death agonist, Bid)、截短型Bid(truncated Bid, tBid)、半胱胺酸-天门冬胺酸酶(cysteine-dePendent asPartate-sPecifc Proteases-9,Caspase-9)蛋白表达。结果电镜可见脓毒症小鼠心肌线粒体肿胀明显,部分出现"空泡样变"。造模后12小时LPS模型组小鼠心肌线粒体改变最严重,经过参附注射液处理后小鼠心肌空泡样变以及线粒体肿胀均有明显缓解。HE染色可见LPS组心肌结构破坏、局部坏死、间质水肿、炎性细胞浸润以及空泡样变;而SFI组心肌结构基本完整,间质水肿以及炎性细胞浸润明显改善;TUNEL染色可见参附组凋亡水平有明显改善(P<0.05);Western-blot检测造模后LPS组小鼠心肌Bcl-2表达较Sham组明显降低,SFI组与LPS组相较表达明显增强,差异具有统计学意义(P<0.01),同时SFI组Bid、tBid、Caspase-9表达较LPS组明显降低,差异有统计学意义(P<0.01)。结论参附注射液可通过下调脓毒症小鼠心肌Bid、tBid、Caspase-9表达,上调Bcl-2表达,保护心肌线粒体结构和功能,抑制心肌线粒体凋亡,对脓毒症小鼠心肌损伤起保护性作用。Objective To observe the effect of Shenfu injection on the mitochondria apoptosis and the expression of caspase-9 protein in sepsis mice induced by lipopolysaccharide(LPS),and to explore the protective mechanism of Shenfu injection on myocardial cells of sepsis mice through the pathway of mitochondria apoptotic.Methods A total of 56 male B6 mice with 6 weeks old were chosen,and they were randomly divided into 4 groups,including the normal control(NC)group,the sham sepsis(Sham)group,the sepsis(LPS)model group,and the Shenfu injection(SFI)group.The mice sepsis model was established by intraperitoneal injection of LPS.Myocardial tissue of mice was collected at 6,12 and 24 hours after modeling,respectively.Ultrastructural changes of myocardium in mice,hematoxylin-Eosin staining(HE)and TUNEL detection of myocardial cell apoptosis were observed by electron microscopy.Western-blot method was used to determine the proteins expression levels of B-cell lymPhoma-2(Bcl-2)of myocardial tissue,BH3 interacting-domain death agonist(Bid),truncated Bid(tBid)and cysteine-dePendent asPartate-sPecifc Proteases-9(caspase-9).Results Electron microscopy showed that the myocardium mitochondria of sepsis mice were swollen,and some of them were vacuolated.The myocardial mitochondria of the LPS model group were the most seriously altered 12 hours after modeling,and the myocardial vacuolation and mitochondrial swelling of the mice were significantly relieved after Shenfu injection.HE staining showed myocardial structural damage,local necrosis,interstitial edema,inflammatory cell infiltration and vacuolation in LPS group.In the SFI group,the myocardial structure was basically intact,interstitial edema and inflammatory cell infiltration were significantly improved.TUNEL staining showed the apoptosis level has significant improvement in the SFI group(P<0.05).After Western-blot detection,the expression level of Bcl-2 in myocardial tissue of mice in LPS group was significantly lower than that in Sham group,and the expression of Bcl-2 in SFI group

关 键 词：参附注射液 脓毒症 线粒体 凋亡 心肌损害 小鼠 

分 类 号：R285.5[医药卫生—中药学]