摘要
目的 观察尾静脉注射衰老标志蛋白30(SMP30)对脓毒症小鼠心肌损伤的抑制作用,并探讨其作用机制.方法 取60只雄性C57BL/6小鼠,随机分为空载对照组、空载模型组、SMP30模型组各20只,空载对照组和空载模型组尾静脉注射空载腺相关病毒载体,SMP30模型组尾静脉注射SMP30腺相关病毒载体.注射2周后,空载模型组和SMP30模型组通过一次性腹腔注射脂多糖(LPS)建立脓毒症模型,空载对照组腹腔注射同等体积生理盐水.于LPS注射24 h后,使用超声心动图仪检测小鼠心功能指标左心室射血分数(LVEF)和左心室缩短分数(LVFS);处死小鼠,收集血清及心脏标本,ELISA法检测血清肌酸激酶同工酶MB(CK-MB)、乳酸脱氢酶(LDH)、心肌肌钙蛋白I(cTnI)水平以及心肌组织超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性、丙二醛(MDA)含量;TUNEL染色法检测心肌细胞凋亡情况;免疫荧光染色法检测心肌组织巨噬细胞浸润情况;二氢乙锭荧光探针检测心肌组织活性氧自由基(ROS)生成量;qRT-PCR法观察心肌组织白细胞介素(IL)-1β、IL-6、肿瘤坏死因子α(TNF-α)mRNA;Western blotting法观察心肌组织SMP30和沉默信息调节因子1(SIRT1)通路相关蛋白SIRT1、乙酰化核因子κB p65(Ac-NF-κB p65)、乙酰化叉头框蛋白O1(Ac-FOXO1).结果 与空载对照组比较,空载模型组LVEF、LVFS降低,血清CK-MB、LDH、cTnI水平升高,心肌细胞凋亡率增加;心肌组织巨噬细胞浸润明显,IL-1β、IL-6、TNF-α mRNA表达升高;心肌组织ROS及MDA生成增加,SOD和GSH-Px活性减弱;心肌组织SIRT1表达下降,Ac-NF-κB p65和Ac-FOXO1表达升高(P均<0.01).与空载模型组比较,SMP30模型组LVEF、LVFS升高,血清CK-MB、LDH、cTnI水平降低,心肌细胞凋亡率降低;心肌组织巨噬细胞浸润减少,IL-1β、IL-6、TNF-α mRNA表达降低;心肌组织ROS及MDA生成减少,SOD和GSH-Px活性增强;心肌组织SIRT1表达升高,Ac-NF-κB p65和Ac-FOXO1表达下降(P均<0.01).结论 尾静脉注射SMP30可上调脓毒症小鼠心肌组织SMP30表达,抑制心肌炎症反应和氧化应激水平,从而减轻心肌损伤,其机制可能是通过激活SIRT1信号通路来发挥作用.
Abstract
Objective To observe the inhibitory effect of caudal vein injection of senescence marker protein 30(SMP30)on myocardial injury in septic mice and to explore its mechanism.Methods Sixty male C57BL/6 mice were randomly divided into the Con+empty vector group,LPS+empty vector group,and LPS+SMP30 group,with 20 mice in each.Mice in the Con+empty vector group and LPS+empty vector group were injected with adeno-null associated virus vec-tor via the tail vein,and mice in the LPS+SMP30 group were injected with SMP30 adeno-associated virus vector via the tail vein.After 2 weeks of adeno-associated virus vector injection,mice in the LPS+empty vector group and LPS+SMP30 group were intraperitoneally injected with lipopolysaccharide(LPS)to establish the sepsis models,while mice in the Con+empty vector group were given intraperitoneal injection of the same volume of normal saline.Left ventricular ejection fraction(LVEF)and left ventricular fraction shortening(LVFS)were measured by echocardiography at 24 h after LPS injection.Serum and heart tissues were collected after the mice were killed,and serum levels of creatine kinase-MK(CK-MB),lac-tic dehydrogenase(LDH)and cTnI,and myocardial levels of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)were detected by ELISA.TUNEL staining was used to detect cardiomyocyte apoptosis.Macrophage infiltration was detected by immunofluorescence staining.The myocardial reactive oxide species(ROS)pro-duction was detected by dihydroethidium fluorescent probe.Interleukin(IL)-1β,tumor necrosis factor(TNF)-α and IL-6 mRNA levels were detected by qRT-PCR.The protein expression levels of myocardial tissues SMP30,silent information regulator 1(SIRT1),Ac-NF-κB p65,and Ac-FOXO1 were detected by Western blotting.Results Compared with the Con+empty vector group,the values of LVEF and LVFS decreased,the levels of serum CK-MB,LDH and cTnI increased,the apoptosis rate increased,the myocardial macrophage infiltration was more obvious,the mRNA levels of IL-1β,IL-6,TNF-α increased,the productions of ROS and MDA increased,the activities of SOD and GSH-Px decreased,the expres-sion of SIRT1 down-regulated,and the expression levels of Ac-NF-κB p65 and Ac-FOXO1 increased in the LPS+empty vector group(all P<0.01).Compared with LPS+empty vector group,the values of LVEF and LVFS increased,the levels of serum CK-MB,LDH and cTnI decreased,the apoptosis rate decreased,the myocardial macrophage infiltration de-creased,the mRNA levels of IL-1β,IL-6,TNF-α decreased,the productions of ROS and MDA decreased,the activities of SOD and GSH-Px increased,the expression of SIRT1 increased,and the expression levels of Ac-NF-κB p65 and Ac-FOXO1 decreased in the LPS+SMP30 group(all P<0.01).Conclusion The caudal vein injection of SMP30 can up-reg-ulate SMP30 expression in mice with sepsis,inhibit myocardial inflammatory response and oxidative stress level,thereby alleviating myocardial injury,and its mechanism may be related to activating the SIRT1 signaling pathway.
基金项目
陕西省重点研发计划(S2022-YF-YBSF-0781)
维普
万方数据