参附注射液调控巨噬细胞自噬对脓毒症小鼠免疫功能影响  

Effect of Shenfu Injection on Immune Function in Septic Mice by Regulating Macrophage Autophagy

作　　者：赵锋利[1] 罗苑苑[1] 赵馥[1] 张先进[1] 林新锋[1] 冼绍祥[1] ZHAO Fengli;LUO Yuanyuan;ZHAO Fu;ZHANG Xianjin;LIN Xinfeng;XIAN Shaoxiang(The First Affiliated Hospital of Guangzhou University of ChineseMedicine,Guangzhou 510405,Guangdong,China)

机构地区：[1]广州中医药大学第一附属医院

出　　处：《辽宁中医药大学学报》2019年第8期34-37,共4页Journal of Liaoning University of Traditional Chinese Medicine

基　　金：广东省科技计划发展专项基金项目(2017B020247062)

摘　　要：目的:观察参附注射液对脓毒症小鼠免疫功能的影响,探讨相关机制。方法:将90只10周龄雄性SDF级C57BL6小鼠使用随机数字表法分为对照组(Control,n=30)、脓毒症组(CLP,n=30)、参附注射液治疗组(SF,n=30)。脓毒症小鼠模型制备采用国际公认的盲肠结扎穿孔法。SF组造模后第1天开始腹腔注射参附注射液(4 mL·kg^-1·d^-1,共3 d);Control组和CLP组注射生理盐水(4 mL·kg^-1·d^-1,共3 d)。比较各组小鼠造模死亡率,通过ELISA检测炎症因子IL-1β、IL-6、TNF-α和抗炎因子IL-10水平;使用流式细胞法检测外周血CD3^+CD4^+和CD3^+CD8^+比值;免疫印记检测巨噬细胞P62、LC3Ⅱ/Ⅰ以及cleaved caspase-3的表达水平;电镜检测评价巨噬细胞线粒体损伤情况。结果:Control组无小鼠死亡,CLP组小鼠死亡率为70.0%,而SF组小鼠死亡率明显下降为30.9%(P<0.05)。Control组小鼠外周血CD3^+CD4^+/CD3^+CD8^+为2.12,CLP组明显下降为1.13(P<0.05);SF组小鼠明显升高为1.56(P<0.05)。CLP组小鼠血清IL-1β、IL-6和TNF-α水平较Control组均明显升高(P<0.05),抗炎因子IL-10水平显著下降(P<0.05),而经过参附注射液治疗后上述炎症因子下降(P<0.05),抗炎因子升高(P<0.05)。免疫印迹结果提示CLP组的小鼠脾巨噬细胞中的自噬蛋白P62和LC3Ⅱ/Ⅰ表达水平均低于control组,凋亡蛋白cleaved caspase-3明显升高;与CLP组比较,SF组P62和LC3Ⅱ/Ⅰ表达增高,cleaved caspase-3降低(P<0.05)。电镜证实CLP组小鼠脾脏巨噬细胞线粒体损伤较为严重,SF组明显减轻。结论:参附注射液能够明显调节免疫抑制脓毒症小鼠的免疫系统,抑制炎症反应与调节小鼠脾巨噬细胞自噬相关。Objective:To observe the effect of Shenfu Injection on immune function in septic mice and to explore related mechanisms.Methods:90 mice were divided into control group(n=30),sepsic model group(CLP group,n=30)and Shenfu Injection treatment group(SF group,n=30)using random number table method.The septic model was established by cecal ligation and puncture.The SF group was injected with Shenfu Injection(4 mL/kg·d)intraperitoneally.The control group and CLP group were injected with the same volume of normal saline for 3 consecutive days.The mortality of the mice was evaluated after 72 hours of modeling,and the levels of serum inflammatory factors IL-1β,IL-6 and TNF-αwere detected by ELISA.The ratio of CD3^+CD4^+and CD3^+CD8^+in peripheral blood was detected by flow cytometry;immunoblotting detection expression levels of macrophage P62,LC3Ⅱ/Ⅰand cleaved caspase-3;electron microscopy to evaluate mitochondrial damage in macrophages.Results:The mortality rate of mice in the CLP group was 70.0%,while that in the SF group was significantly reduced to 30.9%(P<0.05).The peripheral blood CD3^+CD4^+/CD3^+CD8^+of the control group was 2.12,and the CLP group was significantly decreased to 1.13(P<0.05).The SF group was significantly increased to 1.56(P<0.05).Compared with the control group,the inflammatory factors IL-1β,IL-6 and TNF-αin the CLP group were significantly increased(P<0.05),but the inflammatory factors were significantly decreased after the treatment with Shenfu Injection(P<0.05).The results of immunoblotting indicated that the expression levels of autophagy proteins P62 and LC3Ⅱ/Ⅰin the spleen macrophages of the CLP group were lower than that of the control group,and the caspase-3 was significantly increased.Compared with the CLP group,the expression of P62 and LC3Ⅱ/Ⅰwas increased in the SF group,and cleaved caspase-3 was decreased in the SF group(P<0.05).Electron microscopy confirmed that mitochondrial damage of spleen macrophages in CLP group was more serious,and SF group was significantly relieve

关 键 词：脓毒症 参附注射液 巨噬细胞自噬 免疫调节 

分 类 号：R285.5[医药卫生—中药学]