| Objective: To investigate the anti-inflammatory effects of electroacupuncture and postacupuncture serum Exosome(Acu-exo)in Sepsis and the key genes and network mechanisms of Acu-exo.Methods:Research Content One Study on effect platform of the prevention and treatment of sepsis mice by electroacupuncture at Zusanli24 healthy male C57BL/6J mice were randomly divided into Sepsis group(n=12)and Sepsis+EA group(n=12).The specification of filiform needle was 0.25×13s,stabing Zusanli point for about 3-5mm,and then connecting Huatuo brand electroacupuncture instrument.Parameter setting included continuous wave,frequency 10 Hz,intensity 5,15 min.Fifteen minutes after the end of electroacupuncture,the two groups were given intraperitoneal injection of lipopolysaccharide(LPS)(30mg/kg)to prepare the sepsis mouse model.The survival time and mouse sepsis score(MSS)within 24 hours were observed and recorded.Fifteen healthy male C57BL/6J mice were randomly divided into PBS group(n=5),Sepsis group(n=5)and Sepsis+EA group(n=5).15 minutes after electroacupuncture,the Sepsis group and Sepsis+EA group were given intraperitoneal injection of LPS(24mg/kg)to prepare sepsis mouse model.The PBS group was intraperitoneally injected with the same amount of PBS solution.The expression levels of TNF-α,IL-6 and IL-1β in serum of the three groups were detected by ELISA at 2h after modeling.Research Content Two Study on the effect of secondary infusion of Acu-exo on sepsis miceExperiment 1 Extraction and identification of exosomes from normal mice after electroacupuncture at Zusanli for 7 daysEighteen healthy male C57BL/6J mice were treated with electroacupuncture once a day for 7 days.After the electroacupuncture on the 7th day,Acu-exo was extracted by Exo Quick method.Western Blot(WB),Transmission electron microscope(TEM)and Nanoparticle tracking analysis(NTA)were used to identify the markers,morphology and size of exosome.Experiment 2 Effect of reinfusion of Acu-exo on survival time of septic miceTwenty four healthy male C57BL/6J mice were divided into Sepsis group(n=12)and Sepsis+Acu-exo group(n=12).Acu-exo(0.4mg/m L,0.1m L/10g)prepared in Experiment 1 was injected intraperitoneally,and the Sepsis group was not given reinfusion.30 minutes later,the two groups were given intraperitoneal injection of LPS(30 mg/kg)to prepare the sepsis mouse model,and the survival time and MSS score within 24 hours were observed and recorded.Experiment 3 observation on anti-inflammatory effect of back infusion of Acu-exo on septic miceFifteen healthy male C57BL/6J mice mice were randomly divided into PBS group(n=5),Sepsis group(n=5)and Sepsis+Acu-exo group(n=5).The Sepsis group and Sepsis+Acu-exo group were given intraperitoneal injection of LPS(24mg/kg)to prepare sepsis mouse model.Intraperitoneal infusion of Acu-exo(0.4mg/m L,0.1m L /10g)in the Sepsis+Acu-exo group was performed 30 min before the Sepsis modeling.The PBS group was intraperitoneally injected with the same amount of PBS solution.The expression levels of TNF-α,IL-6 and IL-1β in serum of the three groups were detected by ELISA at 2h after modeling.Experiment 4 observation on the law of organ protection of Acu-exo reinfusion in septic miceFifteen healthy male C57BL/6J mice mice were randomly divided into PBS group(n=5),Sepsis group(n=5)and Sepsis+Acu-exo group(n=5).The Sepsis group and Sepsis+Acu-exo group were given intraperitoneal injection of LPS(24mg/kg)to prepare sepsis mouse model.Intraperitoneal infusion of Acu-exo(0.4mg/m L,0.1m L /10g)in the Sepsis+Acu-exo group was performed 30 min before the Sepsis modeling.The PBS group was intraperitoneally injected with the same amount of PBS solution.HE staining was used to observe the effects of Acu-exo on the injury of heart,lung,liver and kidney in septic mice for 2h and 24 h.The expression levels of inflammatory cytokines TNF-α,IL-6 and IL-1β were detected by real-time fluorescence quantification method 24 hours after modeling.Research Content Three high throughput sequencing of Acu-exo miRNA and the network mechanism of prevention and treatment of sepsisExperiment 1 high throughput sequencing and bioinformatics analysis of Acu-exo miRNAThirty healthy male C57BL/6J mice were divided into NC group(n=15)and EA group(n=15).The NC group was not given any intervention.In the EA group,Acu-exo was extracted by exoquick method for 7 days,and the expression of miRNA in Acu-exo was detected by highthroughput sequencing technology.The differential genes were screened,and the differential target genes were predicted for go and KEGG enrichment analysis.Experiment 2 network biological analysis of important targets and mechanism of Acu-exo in the prevention and treatment of sepsisThe differential target genes predicted by high-throughput sequencing were transformed into human genes through gene mapping,and the data were coupled with the human sepsis targets retrieved from Gene Cards database,Dis Ge NET database and OMIM database to obtain the important targets of Acu-exo for the prevention and treatment of sepsis.The interaction genes,signaling pathways and sources of key targets were analyzed using STRING database and Cytoscape software to explore the possible biological mechanism of Acu-exo in the prevention and treatment of sepsis.Result:1.Electroacupuncture can prolong the survival time of sepsis mice,reduce MSS score and inhibit the expression of inflammatory factorsAfter 24 h of modeling,12 mice in the Sepsis group died and none survived,with a survival rate of 0%.The death time of 12 dead mice were 10.29 h,13.11 h,16.04 h,11.44 h,12.53 h,14.14 h,14.31 h,13.8h,14.45 h,15h,15.14 h and 15.44 h respectively.In the Sepsis+EA group,9 mice died and 3 survived,with a survival rate of 25%.The death time of 9 dead mice were 14.77 h,23.30 h,19.16 h,19.49 h,18.29 h,15.20 h,20.32 h,15.30 h and 17.42 h respectively,indicating that electroacupuncture can prolong the survival time of septic mice.Ten hours after intraperitoneal injection of LPS(30mg/kg),the mice in the Sepsis group showed tremor,decreased movement,disorientation,hair erection,white secretion around the eyes and dry stool,while the mice in the Sepsis+EA group were significantly better than those in the Sepsis group,and the MSS score was lower than that in the Sepsis group(P <0.001).Compared with the PBS group,the serum levels of inflammatory factors TNF-α and IL-6 in the 2h Sepsis group were significantly increased(P <0.01),and the IL-1β showed an upward trend.Compared with the Sepsis group,serum inflammatory factors TNF-α and IL-6 in the 2h Sepsis+EA group were significantly decreased(P <0.05,P <0.01),and IL-1β showed a decreasing trend.2.Infusion of Acu-exo can prolong the survival time of sepsis mice,reduce MSS score and inhibit the expression of inflammatory factorsBCA protein quantitative results showed that the extracted Acu-exo protein concentration was 7.713087mg/m L.Electrophoresis showed that there were positive bands of CD63 and CD9,indicating that exosomes were extracted well.TEM observation showed that the extracted serum exosomes were all round or like round vesicles,and membranous structures were clearly seen around the vesicles.In the NTA detection report,the MODE curve was linear and smooth,indicating that the impurities contained were few.The arithmetic average diameter was 90.6nm,and the peak diameter was 89.6nm,which was consistent with the theoretical exosome diameter(20-200nm).After 24 h of modeling,12 mice in the Sepsis group died and none survived,with a survival rate of 0%.The death time of 12 dead mice were 10.29 h,13.11 h,16.04 h,11.44 h,12.53 h,14.14 h,14.31 h,13.8h,14.45 h,15h,15.14 h and 15.44 h respectively.In Sepsis+Acu-exo group,11 mice died and 1 survived with a survival rate of 8%.The death time of 11 dead mice were 14.28 h,15.48 h,16.21 h,17.01 h,17.29 h,18.32 h,18.53 h,19.32 h,20.15 h,20.54 h and 23.13 h respectively,indicating that electroacupuncture for 7 days can prolong the survival time of normal mice with sepsis.Ten hours after intraperitoneal injection of LPS(30mg/kg),the mice in the Sepsis group showed tremor,decreased movement,disorientation,hair erection,white secretion around the eyes and dry stool,while the mice in the Sepsis+Acu-exo group were significantly better than those in the Sepsis group,and the MSS score was lower than that in the Sepsis group(P <0.001).Compared with the PBS group,the serum levels of inflammatory factors TNF-α and IL-6 in the2 h Sepsis group were significantly increased(P <0.01),and the IL-1β showed an upward trend.Compared with the Sepsis group,serum inflammatory factors TNF-α and IL-6 in the 2h Sepsis+Acu-exo group were significantly decreased(P <0.01),and IL-1β showed a decreasing trend.3.Infusion of Acu-exo can reduce the degree of lung organ injury and inhibit the expression of inflammatory factors in sepsis miceHE staining showed that there were no significant changes in heart,liver and kidney for2 h and 24 h.The lung injury was the most obvious.Compared with the PBS group,the alveolar wall in the lungs of mice in the 2-hour Sepsis group was slightly thickened,the alveolar cell boundary was clear,and there was no obvious inflammatory cell infiltration.The lung tissue structure of mice in the 24-hour Sepsis group was disordered,the alveolar wall was significantly thickened,and there was obvious inflammatory cell infiltration;Compared with Sepsis group,the lung tissue structure of Sepsis+Acu-exo group at 2 hours and 24 hours was relatively complete,the infiltration of inflammatory cells in mouse lungs was significantly reduced,there was only a small amount of inflammatory cell infiltration,and the thickening of alveolar wall was significantly reduced.PCR results showed that compared with the PBS group,the m RNA expressions of inflammatory factors TNF-α and IL-1β in the lung tissues of mice with the 24 h Sepsis group were significantly increased(P <0.01,P <0.001),and the IL-6 showed an upward trend.Compared with the Sepsis group,the m RNA expressions of inflammatory factors TNF-α and IL-1β in the lung tissues of the mice with the 24 h Sepsis+Acu-exo group were significantly decreased(P <0.05,P <0.001),IL-6 had a decreasing trend.4.Relevant miRNAs in Acu-exo play an important role in the prevention and treatment of sepsisHigh-throughput sequencing results showed that 53 mirnas in Acu-exo were significantly changed.Biological process mainly enriched in regulating cell metabolism,system development,etc.,cell components mainly enriched in collagen trimer compounds,such as inside the cell,molecular function mainly enriched in protein combination,combination,structure of extracellular matrix components,etc.,enrichment of signaling pathway shows associated with inflammation and immunity.KEGG shows that enriched signaling pathways are associated with inflammation and immunity.The results of network biology analysis showed that the targets of Acu-exo for the prevention and treatment of sepsis came from multiple organs and multiple systems,and the interacting genes and signal pathways involved in inflammation,immune regulation,metabolism,and cell proliferation and apoptosis.Conclusion:1.Electroacupuncture can prolong the survival time of sepsis mice and inhibit the expression of inflammatory factors,which has anti-inflammatory effect.2.The return infusion of Acu-exo has a acupuncture like effect,which can prolong the survival time of sepsis mice,inhibit the expression of inflammatory factors and reduce the degree of lung tissue injury.3.The target of Acu-exo in the prevention and treatment of sepsis comes from multiple organs and multiple systems,and the miRNA and target genes carried by Acu-exo have inflammatory,immune and metabolic network regulatory effects in the prevention and treatment of sepsis. |
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