外泌体在电针抑制脓毒症小鼠炎症反应及提高生存率中的作用

Effect of exosomes on inhibiting inflammatory response and improving survival rate of septic mice by electroacupuncture

目的:探讨外泌体在电针抑制脓毒症小鼠炎症反应及提高生存率中的作用。方法:选取雄性C57BL/6J小鼠,检测电针介入时间[设模型组(Sepsis组)、电针(EA)预处理组、EA后处理组]、回输外泌体[设对照组(E0)、电针1 d(E1)、电针4 d(E4)及电针7 d(E7)回输外泌体组]、拮抗外泌体(设Sepsis+DMSO组、Sepsis+DMSO+EA组、Sepsis+GW4869+EA组)对小鼠生存率的影响,并检测小鼠血清炎症因子变化[设PBS对照组、模型组(Sepsis组)和EA预处理组]。各组小鼠造模处理后分别观察小鼠7 d生存率;采用透射电镜观察外泌体并进行纳米颗粒跟踪分析与鉴定;于处理2 h、4 h、6 h取小鼠血清,ELISA检测炎症因子TNF-α、IL-6和IL-1β含量。结果:与Sepsis组相比,EA预处理组小鼠生存率提高(P<0.05),2 h、4 h、6 h血清IL-6、IL-1β均明显降低(P<0.05,P<0.01),2 h、4 h血清TNF-α明显降低(P<0.001),6 h未见明显变化;电针处理前后生存率差异无统计学意义(P>0.05);与Sepsis组相比,回输E7组生存率差异有统计学意义(P=0.0269,P<0.05),回输E0、E1、E4组生存率差异无统计学意义;Sepsis+GW4869+EA组较Sepsis+DMSO+EA组死亡率提高30%(P=0.2868)。各组小鼠血清外泌体粒径集中在100 nm,小部分偏离范围可能与颗粒间黏着有关。结论:电针及回输电针正常小鼠后血清外泌体预处理均可提高脓毒症小鼠生存率,电针预处理可抑制血清炎症因子水平,回输不同电针天数的小鼠血清外泌体防治脓毒症可能存在一定的量效关系。

Objective:To explore the effect of electroacupuncture in inhibiting inflammatory response of septic mice and improving survival rate of mice.Methods:Male C57BL/6J mice were selected,and detected effect of electroacupuncture intervention time[Model group(Sepsis group),electroacupuncture(EA)pretreatment group,EA post-treatment group],reinfusion exosomes[control(E0)group,electroacupuncture 1-day(E1)group,electroacupuncture 4-day(E4)group,electroacupuncture 7-day(E7)group],antagonistic exosomes[Sepsis+DMSO group,Sepsis+DMSO+EA group,Sepsis+GW4869+EA group]on survival rate,and detected changes of inflammatory factors[PBS control group,model group(Sepsis group),EA pretreatment group].Seven-day survival rate of mice in each group was observed after modeling treatment;exosomes were observed by transmission electron microscope and identified by nanoparticle tracking analysis;serum of mice that treated 2 h,4 h and 6 h were collected,and TNF-α,IL-6 and IL-1βcontents were detected by ELISA.Results:Compared with Sepsis group,survival rate of EA pretreatment group was increased(P<0.05),serum IL-6 and IL-1βin EA pretreatment group at 2 h,4 h and 6 h all decreased significantly(P<0.05,P<0.01),and TNF-αin 2 h and 4 h were significantly decreased(P<0.05,P<0.01),there was no significant change at 6 h;there was no significant difference in survival rate before and after electroacupuncture treatment(P>0.05);compared with Sepsis group,there was significant difference in survival rate of E7 group(P=0.0269,P<0.05),while there was no difference in survival rate of E0,E1 and E4 groups;mortality of Sepsis+GW4869+EA group was 30%higher than that of sepsis+DMSO+EA group(P=0.2868).Particle size of serum exosomes of mice in each group was concentrated at about 100 nm,and a small part of the deviation may be related to the adhesion between particles.Conclusion:Electroacupuncture pretreatment and serum exosome pretreatment after electroacupuncture to normal mice can improve survival rate of septic mice,and electroacupuncture pretreatment can inhibit level of serum inflammatory factors.There may be a certain dose-effect relationship between the prevention and treatment of sepsis by reinfusion of serum secretion of mice with different electroacupuncture days.