| Objective: To explore the effect and distribution of serum exosome(Acupuncture Exosome,Acu-exo)on the survival rate of sepsis mice.Methods:Content 1.Study on the effect of Acu-exo on improving the survival rate of sepsis mice.(1)Experiment 1:study on the establishment of effect platform for electroacupuncture at ST36 acupoint to pretreat sepsis in mice.30 healthy male C57BL/6J mice were randomly divided into model group(n = 15)and electroacupuncture group(n = 15).Acupuncture needle(0.25×13 mm)was inserted into ST36point(3~5mm)and pretreated with electroacupuncture.The parameters of electroacupuncture were as follows: frequency 10 Hz,continuous wave,15 min,intensity 5.At the end of electroacupuncture,the sepsis mouse model was established by intraperitoneal injection of lipopolysaccharide(12mg/kg).The survival rate of sepsis mice within 7 days(168 h)was observed.(2)Experiment 2 : extraction and identification of Acu-exo in normal mice after electroacupuncture at ST36 for different days.60 healthy male C57BL/6J mice were divided into blank group(E0),electroacupuncture1-day group(E1),electroacupuncture 7-day group(E7),electroacupuncture 14-day group(E14)and electroacupuncture 21-day group(E21),with 12 mice in each group.After acupuncture for 1 day,7 days,14 days and 21 days,once a day,Acu-exo,was extracted by Exo Quick and identified by Western blotting,transmission electron microscopy and nanoparticle tracking analysis,and the markers,shape and size of the exocrine body were identified by Western blotting,transmission electron microscopy and nano-particle tracking analysis,the mice in each group were not acupuncture,acupuncture for 1 day,7 days,14 days and 21 days,once a day.Content 2.Study on the dose-effect effect of Acu-Exo on sepsis mice(1)Experiment 1:effect of reinfusion of Acu-exo for different days on the survival rate of sepsis mice in experiment 3.72 healthy male C57BL/6J mice were divided into model group(n = 12),E0 group(n =12),E1 group(n = 12),E14 group(n = 12),E7 group(n = 12)and E21 group(n = 12).The Acu-exo(4mg/kg)of E0,E1,E7,E14 and E21 in experiment 2 was injected intraperitoneally into the above groups,and no reinfusion was given in the model group.The sepsis mouse model was established by intraperitoneal injection of lipopolysaccharide(12mg/kg)after30min.The survival rate of sepsis mice within 7 days(168 h)was observed.(2)Experiment 2 : the effect of re-infusion of different concentrations of Acu-exo on the survival rate of sepsis mice.72 healthy male C57BL/6J mice were divided into 6 groups,one group was model group,and the other 5 groups were Acu-exo groups with 5 different concentrations(2mg/m L,0.8mg/m L,0.4mg/m L,0.2mg/m L,0.08mg/m L).According to the results of experiment 3,Acu-exo,with the best stimulation time to improve the survival rate of mice was infused into the above groups with 5 different concentrations,and no reinfusion was given in the model group.The sepsis mouse model was established by intraperitoneal injection of lipopolysaccharide(12mg/kg)after 30 min.The survival rate of sepsis mice within 7 days(168 h)was observed.Content 3.Study on the aging effect of Acu-Exo on sepsis miceExperiment :study on the time effect of Acu-exo intervention.60 healthy C57BL/6J male mice were divided into model group(n = 12),model +E7-Acu-exo 1 group(n = 12),model + E7-Acu-exo 2 group(n = 12),model + EA 1 group(n= 12)and model + EA 2 group(n = 12).According to the results of experiment 4,the best concentration of Acu-exo,model + Acu-exo 1 group and model + Acu-exo 2 group were selected.Before and after modeling,30 min was injected intraperitoneally with Acu-exo,model + EA 1 group and model + EA 2 group were treated with electroacupuncture at bilateral ST36 points before and after modeling,respectively.The survival rate of sepsis mice within 7 days(168 h)was observed.Study 4.Observation on the distribution of Acu-exo in the organs and tissues of sepsis mice and the effect of electroacupuncture on targeted chemotaxis.(1)Experiment 1:study on the distribution of Acu-exo in the organs and tissues of sepsis mice.6 healthy C57BL/6J male mice were divided into simple fluorescence group(Di R group)and fluorescence labeled exocrine group(Di R-Acu-exo group)with 3 mice in each group.The sepsis mouse model was established by intraperitoneal injection of lipopolysaccharide(12mg/kg).60μL of Di R and 60μL of Di R-Acu-exo were injected into the tail vein respectively,and the fluorescence intensity of different parts of the body at different time points was analyzed by small animal in vivo optical imager,and the enrichment of Acu-exo in target organs related to severe inflammation was observed.(2)Experiment 2 : preliminary observation on the effect of electroacupuncture on targeted chemotaxis of Acu-exo in experiment 2.9 healthy C57BL/6J male mice were divided into three groups: simple fluorescence group(Di R group),fluorescence labeled exocrine group(Di R-Acu-exo group)and fluorescence labeled exosome plus electroacupuncture group(Di R-Acu-exo+ EA group),with3 mice in each group.The sepsis mouse model was established by intraperitoneal injection of lipopolysaccharide(12mg/kg).60μL of Di R,Di R-Acu-exo and Di R-Acu-exo were injected into the tail vein respectively,and the Di R-Acu-exo + EA group was treated with electroacupuncture(15min)1 hour after injection.The promoting effect of electroacupuncture on targeted chemotaxis of Acu-exo was observed by in vivo optical imager.Results:1.Electroacupuncture at ST36 point can improve the survival rate of sepsis mice.After intraperitoneal injection of lipopolysaccharide(12mg/kg),the mice showed trembling,drilling,hair erection and periocular secretion.After 7 days(168 h),11 mice in the model group died and 4 survived.The survival rate of 11 dead mice was 26.67%.The time of death of 11 dead mice was 15.38 h,24.17 h,29.52 h,45.47 h,47.01 h,53.24 h,61.40 h,69.32 h,80.02 h,93.55 h,96.19 h,respectively.In the electroacupuncture group,5 mice died and 10 survived,and the survival rate was 66.70%.The time of death of 5 dead mice was 38.24 h,42.03 h,56.33 h,69.12 h and 78.09 h,respectively.After 7 days of modeling,the survival rate of mice in the electroacupuncture group was significantly higher than that in the model group(P=0.0375,P < 0.05).2.The shape,size and markers of Acu-exo are in line with the requirements and standards for the identification of exosome bodies of the International Society of extracellular vesicles.The total protein concentrations of Acu-exo extracted from normal mice(E0),electroacupuncture 1-day normal mice(E1),7-day electroacupuncture normal mice(E7),14-day electroacupuncture normal mice(E14)and 21-day electroacupuncture normal mice(E21)were quantified by BCA protein.The Acu-exo protein concentrations of E0,E1,E7,E14 and E21 were 14.017mg/m L,15.601mg/m L,16.661mg/m L,11.511mg/m L,15.695mg/m L.respectively.Transmission electron microscopy showed that the diameter of the exosome body was between 100~200nm and showed the shape of round or quasi-round vesicles.The membrane-forming structure was clearly seen in the periphery of the vesicles,either singly distributed or clustered,and substances with low electron density could be seen in the vesicles.The MODE curve in the nano-particle tracking analysis report is linear and smooth,indicating that there are few impurities,and the particle size peaks are 124.8nm,113.6nm,131.4nm,127.6nm and 99.6 nm,respectively,and the particle size is consistent with the theoretical exosome value(30-200nm).The protein spectrum of exosome was analyzed by SDS-PAGE electrophoresis,which showed that the protein bands were consistent,suggesting that the extraction of exosome was not disturbed by miscellaneous proteins,and the purification effect was good.3.Electroacupuncture of Acu-exo for 7 days has a better effect on improving the survival rate of sepsis mice.After 7 days(168 hours),2 mice survived in the model group,3 mice survived in the 1day electroacupuncture group and 3 mice in the normal mouse serum exosome transfusion group,8 mice survived in the electroacupuncture 7 day serum exosome transfusion group,and6 mice survived in the electroacupuncture 14 day serum exosome transfusion group and the electroacupuncture 21 day serum exosome transfusion group.6 mice survived in the electroacupuncture 14-day serum exosome transfusion group and the electroacupuncture21-day serum exosome transfusion group.By log-rank analysis,there was significant difference between the 7-day electroacupuncture group and the model group(p < 0.05).The serum exosome transfusion at 14 days and 21 days after electroacupuncture was higher than that in the model group,and there was no statistical difference(P > 0.05).4.Acu-exo at the concentration of 0.4mg/m L is effective in improving the survival rate of sepsis mice.After 7 days(168 hours),2 rats survived in the model group,3 rats in the exosome 2mg/m L group and 7 days exosome 0.8mg/m L group,7 rats in the exosome 0.4mg/m L group,4 rats in the exosome 0.2mg/m L group and 3 rats in the exosome 0.08mg/m L group after 7days of electroacupuncture.By log-rank analysis,there was significant difference in serum exosome body between the 0.4mg/m L group and the model group after electroacupuncture for7 days(P = 0.0386,P < 0.05).5.The effect of Acu-exo intervention before modeling is better than that after modeling.After 7 days(168 hours),3 rats survived in the model group,7 in the model plus exosome pretreatment group,4 in the model plus exosome post-treatment group,8 in the model plus electroacupuncture pretreatment group,and 7 in the model plus electroacupuncture post-treatment group.Through log-rank analysis,the effect of pretreatment before Acu-exo modeling was better than that after modeling,and there was significant difference compared with the model group(P=0.0464,P<0.05),but there was no significant difference before and after electroacupuncture(P > 0.05).6.The fluorescence intensity of Acu-exo is higher in the lungs of sepsis mice.After Di R and Di R-Acu-exo were injected into the caudal vein of sepsis mice,in vivo imaging showed that the exosome was enriched in the claws of sepsis mice immediately,2h and 4h,and the fluorescence intensity of Di R-Acu-exo in the lungs of sepsis mice was higher than that of other organs after 2h and 4h.There was no significant difference in lung fluorescence intensity between Di R-Acu-exo + EA group and Di R-Acu-exo group in sepsis mice.Conclusion:1.Acu-exo has acupuncture-like effect and can improve the survival rate of sepsis mice.2.The therapeutic effect of Acu-exo on septic mice has a certain dose-effect rule.Electroacupuncture for 7 days,Acu-exo of 0.4mg/m L concentration and 30 min infusion before modeling are the better parameters to improve the survival rate of sepsis mice.3.Acu-exo may have the role of targeted chemotaxis,but the promoting effect of electroacupuncture on targeted chemotaxis of Acu-exo has not been found in sepsis mice. |
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