Mechanism Of Metformin On Sepsis Related Liver Injury Through AMPK-FOXO1 Pathway

Sepsis is a kind of life-threatening organ dysfunction caused by the host's maladjustment to infection.As the main cause of admission and death of patients in intensive care unit,the death rate is as high as one-third to one-half of the inpatient death rate.As the high mortality of sepsis is still a major medical problem,it is particularly important to explore the mechanism of its occurrence and development.Up to now,the exact mechanism is not clear,but it is generally believed that inflammatory storm and immune reaction disorder caused by inflammatory factors released by immune cells and somatic cells play an important role in the pathogenesis of sepsis.Interestingly,recent studies have shown that active molecular reactions in inflammatory reactions require strong metabolic support,and regulation of metabolic pathways may be a new strategy to limit inflammatory diseases.Metformin is a broad and strong metabolic regulatory agent,which is typically used as a first-line antidiabetic for type 2 diabetes.In addition to its well-known hypoglycemic activity,there is increasing evidence that metformin inhibits the expression of pro-inflammatory factors in vitro and reduces inflammatory damage in vivo.The mechanism of the pharmacological action of metformin is not clear.It has been suggested that metformin inhibits the activity of mitochondrial respiratory complex,thereby reducing the production of adenosine triphosphate and activating adenosine monophosphate activated protein kinase(AMPK).Some studies have proved that AMPK can prevent experimental septicemia in animals by activating drugs or small molecular compounds,indicating that AMPK plays an irreplaceable role in the pathogenesis of septicemia.In addition,AMPK is considered to be the main target of metformin.Fork head transcription factor(fork head box protein O,FOXO)protein family is a large class of transcription factors,which can directly participate in gene transcription and expression and cooperate with other transcription factors for transcription regulation.It was first found in Drosophila.FOXO is a subgroup of FOXO family,which has four subtypes in mammals.FOXO1 is expressed in all tissues in vivo,and plays an important role in cell apoptosis,oxidative stress,DNA damage/repair,sugar metabolism and inflammatory diseases.At present,it has been observed that the expression of FOXO1 is low and the degree of lung injury is significant in LPS induced sepsis mice.FOXO1 is one of the main targets of AMPK,so it has been reported that metformin can improve the prognosis of cervical endometrial cancer by activating AMPK-FOXO1;there is no relevant report in sepsis and other serious diseases.It is known that FOXO1 can improve the inflammatory response of sepsis by regulating the early inflammatory factors such as NF-?B and TNF-?.However,it has not been reported whether FOXO1 has an effect on the late lethal inflammatory factors such as high mobility group protein 1(HMGB1)in sepsis.Whether metformin improves the prognosis of sepsis by activating AMPK,decreasing the FOXO1 expression,thereby inhibiting the release of HMGB1 by hepatocytes.To sum up,this study is based on the results of meta-analysis and clinical significance of metformin in improving the prognosis of sepsis patients before hospital use.The purpose of this study is to build a sepsis mouse model with LPS and verify whether metformin can improve the prognosis of sepsis,so as to further explore the potential mechanism of metformin in improving the prognosis of sepsis.Part ? Meta-analysis of metformin in improving the prognosis of septic patients with diabetes mellitusMethods1.Database retrieval:MEDLINE,EMBASE and Cochrane Central databases searched from the beginning to September 30,2018.2.Participants:included in the cohort study,whether metformin was used in sepsis patients with diabetes.3.Quality assessment included in the study:the newcastle Ottawa scale(NOS)was used to assess the outcome quality.4.The inverse variance method with random effect model is used to calculate the odds ratio(OR)and 95%confidence interval(CI)of the combination.5.Sensitivity analysis and funnel chart were used to analyze the heterogeneity source of total combined effect.Result1.Five observational cohort studies were included in this study,involving 1282 patients.According to the results of NOS quality assessment table,these studies were judged as low risk of bias.In this meta-analysis,metformin use was associated with a significant reduction in mortality(OR,0.59;95%CI,0.43-0.79,P=0.001).SummaryMetformin could improve the mortality of septic patients with diabetes mellitus.Part ? Mechanism of metformin on sepsis related liver injury through AMPK-FOXO1 pathwayMethod1.Construction of sepsis mouse modelThe sepsis mice model was established by intraperitoneal injection of LPS(12.5mg/kg)in male C57BL/6J mice at 7-8 weeks.The symptoms and manifestations of the mice were observed at 12,24 and 48 hours after modeling.The liver tissues of the mice at 24 and 48 hours after modeling were stained with H&E and tested with TUNEL.The results were analyzed.The degree of liver injury and the number of apoptosis were observed and compared with the control group.2.To explore the effect of metformin on the prognosis of sepsis miceSixty male C57BL/6J mice were randomly divided into three groups:control group(20 mice),LPS group(20 mice),LPS+MET group(20 mice).LPS group was given intraperitoneal injection of 12.5mg/kg LPS,then intraperitoneal injection of the same amount of saline one hour later,and then intraperitoneal injection of saline once a day until the fifth day;LPS+MET group mice were given intraperitoneal injection of 12.5mg/kg LPS,then intraperitoneal injection of metformin 1 hour later(25mg/kg),and then intraperitoneal injection of metformin once a day until the fifth day,and the state and death of mice in each group were observed and recorded every day mortality.3.To explore the effect of metformin on systemic inflammatory response and liver injury in sepsis mice(1)To observe the effect of metformin on systemic inflammatory response in sepsis miceAt 12,24 and 48 hours after modeling,the mice in each group were collected by eyeball extraction,and collected by coagulation promoting tube.After two hours of room temperature standing,the supernatant was centrifuged at 2500rpm,which was serum.The changes of TNF-?,HMGB1 and IL-1? in serum were detected.(2)To observe the effect of metformin on liver injury and inflammation related to sepsisAt 12,24 and 48 hours after modeling,the precooled normal saline was used for perfusion through the left ventricle of the heart,and the livers of each group of mice were taken respectively.Part of the livers were collected with labeled cryopreservation tubes and placed in liquid nitrogen tanks for cryopreservation,and then a complete liver tissue was taken and placed in 4%paraformaldehyde loaded with precooled for fixation.Western blotting(WB),real-time PCR(RT-PCR),he staining and tunnel test were used to detect the changes of inflammatory indexes such as TNF-?,HMGB1 and the degree of liver injury.4.Study on the protective effect and mechanism of metformin on sepsis miceThe expression of AMPK,p-AMPK,FOXO1 and the changes of TNF-? and HMGB1 were observed by WB and RT-PCR.ResultCompared with the control group,the inflammatory exudation and apoptosis of liver tissue in LPS group were significantly increased,and metformin could improve these LPS induced abnormalities.Compared with the control group,the levels of TNF-?,HMGB1,IL-1? and other inflammatory factors in LPS group were significantly higher(P<0.05),while those inflammatory factors were significantly lower(P<0.05)after metformin treatment.The results of WB and RT-qPCR showed that the expression of FOXO1,TNF-? and HMGB1 in LPS group was higher than that in control group,while the level of p-AMPK in LPS group was lower than that in control group;metformin treatment could reverse the high expression of pro-inflammatory factors and the low expression of p-AMPK in LPS group(P<0.05).In addition,the mortality of LPS group was higher than that of control group,while metformin treatment significantly decreased the mortality of sepsis mice(P<0.05).SummaryMetformin could improve the liver injury and inflammatory response of sepsis mice by activating AMPK and inhibiting FOXO1 level,thereby improving the mortality of septic mice.Conclusion1.Metformin can reduce the mortality of sepsis patients with diabetes mellitus.2.Metformin may improve the liver injury and inflammatory response of sepsis mice by activating AMPK and inhibiting FOXO1 level,thereby improving the mortality of septic mice.