| Sepsis ranks first in the causes of death among patients in non-coronary disease intensive care units.There are 3.15 million sepsis patients worldwide each year,leading to a mortality rate of up to 20%.Sepsis is a clinical syndrome in which the pathogen invades the body so as to leading to systemic inflammatory reactions and organ dysfunction.Sepsis,which mechanism of action is immune modulation in the early stage of the body to counter excessive inflammation and protect the body,is mainly infection-induced.Immune disorders are considered as one of the major pathophysiological mechanisms in the course of sepsis.How to make full use of existing medical resources and continue to explore new treatment methods is undoubtedly the most important task now.In recent years,domestic and foreign scholars have found that electroacupuncture treatment has a protective effect on patients of sepsis and cerebral ischemia.And this phenomenon,which has not been fully elucidated,depends on the cholinergic anti-inflammatory pathway to complete.?7n Ach R agonists reduce the release of inflammatory cytokines from macrophages,which occurs at post-transcriptional levels.It has been reported that miRNA146 participates in the expression and suppression of various inflammatory genes and transcription factors in the way of negative feedback during the inflammatory response.However,whether miRNA146 plays a role in the anti-inflammatory mechanism of electro-acupuncture activated cholinergic anti-inflammatory pathway or not? That is a question.Therefore,this study aimed to explore whether miRNA146 upregulation play an important role in the electroacupuncture treatment of sepsis and probe the specific mechanism among them,so as to providing theoretical basis and evidence support for the pathogenesis of sepsis and the development of new clinical treatment.Experiment 1: Verification of the role of cholinergic anti-inflammatory pathway in alleviating inflammatory response in mice with sepsis mediated by electroacupunctureObjective To confirm the protective effect of electroacupuncture on sepsis mice by activating the cholinergic anti-inflammatory pathwayMethod 1.Group: 104 male C57BL/6 mice were divided into sham operation group(Group sham,n=26),CLP group(n=26),electroacupuncture+CLP group(CLP+EA group,n=26),electroacupuncture +CLP+MLA foot(EA+CLP+MLA group,n=26).MLA is an inhibitor of ?7n Ach R,which inhibits the cholinergic anti-inflammatory pathway in body activation.2.Electroacupuncture: Continuous Electroacupuncture for 45 min is operated in the acupoint "zusanli" point before the model of sepsis.Electroacupuncture stimulation parameters were as follows: frequency is 2Hz,and density wave is 1m A.In the CLP+ EA+MLA group,the cholinergic receptor inhibitor MLA 5mg/kg was injected into the abdominal cavity before the electroacupuncture was made.3.Detection: Molding of cecal ligation and puncture were handling on Mice in each group.20 mice in each group were selected to observe the survival rate for 7 days.After 24 h,liver and lung tissues of 6 mice in each group were obtained for histological injury and pathological examination,and the levels of proinflammatory cytokines TNF-,IL-1 and IL-6 were detected by cardiac blood sampling.Results Compared with group CLP,the survival rate of EA+CLP group and sepsis mice increased significantly,and the level of proinflammatory factors decreased significantly.Compared with EA+CLP group,the survival rate of sepsis mice in CLP+ EA+MLA group decreased significantly,and the level of proinflammatory factors increased significantly.Conclusion Electroacupuncture can reduce the inflammatory response in septic mice by activating the cholinergic anti-inflammatory pathway.Experiment 2: Effect of cholinergic anti-inflammatory pathway activation on miRNA146 and proinflammatory cytokines in macrophagesObjective To observe the effect of cholinergic pathway on miRNA146 and proinflammatory cytokines in macrophages.Method 1.Group: RAW264.7 macrophages were randomly divided into three groups: control group,LPS group and LPS + GTS-21 group.LPS concentration was 100ng/ml,and GTS-21 concentration was 10umol/L.2.Test: GTS-21 was administered 2h before LPS stimulation.After 2 hours,the levels of TNF-?,IL-1? and IL-6 in cell culture supernatants were detected by ELISA.The expression of HMGB1 in macrophages was detected by Western Blot 18 h later,and the level of miRNA146 in macrophages was detected by q RT-PCR.GTS-21 is an activator of cholinergic pathway.Results Compared with the control group,the levels of pro-inflammatory cytokines TNF-?,IL-1? and IL-6 in LPS group were significantly increased,as well as the levels of HMGB1 and miRNA146(all P<0.05).Compared with LPS Group,after using the activator of cholinergic anti-inflammatory pathway,the proinflammatory cytokines of the LPS + GTS-21 group,which including TNF-?,IL-1?,IL-6 and HMGB1,were significantly decreased,while miRNA146 was significantly increased High(all P <0.05).Conclusion LPS stimulated the release of proinflammatory cytokines and miRNA146 from macrophages.However,the activation of cholinergic anti-inflammatory pathway decreased the expression of pro-inflammatory cytokines and significantly increased the level of miRNA146.Experiment 3: The effect of cholinergic anti-inflammatory pathway activated by electroacupuncture on miRNA146 level in sepsis miceObjective To demonstrate that electroacupuncture upregulates the level of plasma miRNA146 in sepsis mice by activating the cholinergic anti-inflammatory pathwayMethod1.Group: 24 male C57 BL / 6 mice were divided into sham group(n = 6),CLP group(n = 6),electroacupuncture + CLP group(CLP + EA group,n = 6)and electroacupuncture + CLP + MLA group(EA + CLP + MLA group,n = 6).MLA is a ?7n Ach R inhibitor that inhibits cholinergic anti-inflammatory pathways in vivo.2.Electroacupuncture: Sepsis mice,anaesthetized by isoflurane,were stimulated with electroacupuncture before the molding of sepsis.Continuous electroacupuncture for 45 min was operated at the acupoint "zusanli" point,and electroacupuncture stimulation parameters were as follows: frequency is 2Hz,and density wave is1 m A.5 mg/kg MLA each mice was administered by ways of intraperitoneal injection before electroacupuncture.3.Detection: Plasma miRNA146 level was detected by rt-PCR 24 h after CLP modeling.Results Compared with sham group,miRNA146 level in CLP group increased significantly.Compared with CLP group,miRNA146 level in EA + CLP group increased significantly.Compared with CLP group,miRNA146 level in CLP + EA + MLA group decreased significantly(all P <0.05).Conclusion The level of miRNA146 in sepsis mice was significantly increased,while EA could further significantly increase the level of plasma miRNA in septic mice.Experiment 4: The role of macrophage miRNA146 in the cholinergic anti-inflammatory pathwayObjective To confirm that cholinergic anti-inflammatory pathway depended on the upregulation of miRNA146 to exert anti-inflammatory effectsMethod 1.Group: RAW264.7 macrophages were randomly divided into 4 groups: LPS group,LPS + GTS-21 Group + empty vector group,LPS + GTS-21 + anti-miRNA146 group and LPS + GTS-21 +High expression of miRNA146 group.LPS concentration is 100 ng / m L,GTS-21 concentration is 10 umol / L.2.Transfection: The lentivirus of anti-miRNA146,high expression of miRNA-146 and empty vector,purchased from and constructed by the company,were transfected into cells for 72 h.3.Test: GTS-21 was administered 2h after LPS stimulation.After 2h,ELISA was used to detect the level of TNF-alpha,IL-1 beta and IL-6 in the culture supernatant.After 18 hours,the expression of HMGB1 in macrophages was detected by Western blot,the level of HMGB1 in supernatant of cells by ELISA,and the level of miRNA146 in macrophages by q RT-PCR.Results 1.Compared with the LPS group,after GTS-21 was administrated,the levels of TNF-?,IL-1?,IL-6 and HMGB1 in LPS+GTS-21+empty vector group were significantly decreased,while miRNA146 was significantly increased(all P<0.05).2.Compared with LPS+GTS-2+empty vector group,the levels of TNF-?,IL-1?,IL-6 and HMGB1 in LPS+GTS-21+anti-miRNA146 group were significantly increased,while miRNA146 was significantly reduced(all P<0.05).3.Compared with LPS+GTS-21+empty vector group,the expression of TNF-?,IL-1?,IL-6 and HMGB1 in LPS + GTS-21 +High expression of miRNA146 group were significantly decreased,while miRNA146 was significantly increased(all P<0.05).Conclusion Activation of cholinergic pathways leads to upregulation of miRNA146,which can inhibit the inflammatory response.Experiment 5: MiRNA146 can reduce the effect of electroacupuncture on septic mice by activating the cholinergic anti-inflammatory pathwayObjective To confirm that electroacupuncture can activate cholinergic anti-inflammatory pathway so as to reduce the mortality of septic mice and alleviate systemic inflammatory response and organ damage by up-regulating miRNA146Method 1.Group: 104 mice were randomly divided into 4 groups: CLP group(n = 26),EA group + CLP group(n = 26),EA + CLP + anti-miRNA146 group(n = 26)and EA+ CLP + high expression of miRNA146 group(n = 26).20 rats in each group were selected for the survival rate analysis,and the remaining 6 for other tests.2.Transfection: The lentivirus construction vector of anti-miRNA146 and over-expressed miRNA146 were intraperitoneally injected into mice.After 7 days,these mice were used for electroacupuncture treatment.3.Electroacupuncture: sepsis model were conduct before continuous stimulation of electroacupuncture for 5 days,30 min daily.4.Test: 1)Survival Analysis Statistics:Use the Kaplan-Meier method to analysis survival rate of 7 days.2)Take pathological assessment of important organs: Take mice liver,lung and kidney tissue for HE staining,analysis the results for statistic.3)Western blot was used to detect the expression of HMGB1 in serum,and the levels of TNF-?,IL-1? and IL-6 in serum were detected by q RT-PCR.Results Compared with the CLP group,the survival rate of EA + CLP group increased significantly,and organ injury and systemic inflammatory response significantly decreased.Compared with EA + CLP group,the survival rate of EA + CLP + anti-miRNA146 group decreased significantly,while organ injury and systemic inflammatory response were significantly enhanced.Compared with EA + CLP group,the survival rate of miRNA146 with high expression of miRNA146 increased significantly,while organ injury and systemic inflammatory response were significantly reduced.Conclusion Electroacupuncture can activate the cholinergic anti-inflammatory pathway to upregulate the expression of miRNA146,which significantly improves the survival rate of septic mice and alleviates the organ damage and systemic inflammatory response. |
PDF Full Text Request