| PART 1 :The protective effects of metformin on endotoxin-induced acute myocarditis in mice Objective: To investigate whether metformin could have protective effects on endotoxin-induced acute myocarditis and whether metformin could have the protective effects in a concentration dependent manner.Methods: Male BALB/c mice were randomly divided into 6 groups(n=8): group A(control group),group B(metformin treated group),group C(LPS treated group),group D(the combination of LPS plus metformin at a dose of 100mg/kg treated group),group E(the combination of LPS plus metformin at a dose of 200mg/kg treated group),F group(the combination of LPS plus metformin at a dose of 400mg/kg treated group).Group A received normal saline,group B received metformin(400 mg/kg;i.p),and group C received LPS(10.0 mg/kg;i.p).Based on all receiving LPS(10 mg/kg;i.p)at start time,30 min before LPS injection group D? E ?F respectively received metformin at the doses of 100 mg/kg,200 mg/kg,400 mg/kg.18 hours later,all of mice were killed.The concentrations of CK-MB and BNP in the serum were measured by ELISA kits.The activity of MPO in the heart tissue and the levels of TNF-??IL-1??IL-6 were tested by ELISA kits.The hearts were removed,paraformaldehyde-fixed heart pieces were embedded in paraffin and stained with hematoxylin and eosin(H&E)for the histopathological examinations.Results: 1.In contrast to control group,the concentrations of CK-MB and BNP in the plasma in other 5 groups were elevated significally.The concentrations of CK-MB and BNP in the plasma in groups that respectively treated with LPS at the doses of 100 mg/kg,200 mg/kg,400 mg/kg were elevated significally compared with LPS group(p<0.05),among them the 400 mg/kg group decreased most obviously.2.Histological evaluation by light microscopy demonstrated that the myocardium in the control group were clear and well-distributed,contiguously aligned,and no inflammatory cell infiltration were observed.However,LPS administration significantly changed morphology and structure of the myocardium and the myofibrils displayed a disrupted and disordered arrangement,myocardial cell diaplayed edema and deterioration,myocardium spaces were widened obviously,and much inflammatory cell infiltration were also found.Following treatment with metformin,the histopathological abnormalities described above were alliviated significally compared to LPS group(P<0.05).In addition,the number of infiltrating inflammatory cells and degeneration of myocardial fibers were significantly decreased compared to LPS group(P<0.05).3.After LPS administration by intraperitoneal injection,the activity of MPO in myocardium increases obviously compared to control group.Following treatment with metformin at the dose of 400mg/kg,the activity of MPO in myocardium decreased significally in contrast with LPS group(p<0.05).4.After LPS administration by intraperitoneal injection,the levels of inflammation factors such as TNF-??IL-1??IL-6 in myocardial tissue increased obviously compared to control group.Following treatment with metformin at the dose of 400mg/kg,the levels of TNF-??IL-1??IL-6 in myocardial tissue decreased significally in contrast with LPS group(p<0.05).Conclusions: 1.Metformin treatment significally decreased the concentrations of CK-MK and BNP in plasma of endotoxin-induced septic mice,and obviously alleviated pathological changes such as adisrupted and disordered arrangement of the myofibrils,edema and deterioration of myocardial,increased width of myocardium spaces,as well as the number of infiltrating inflammatory cells and degeneration of myocardial fibers.Results showed that metformin had protective effects on endotoxin-induced acute myocarditis in a concentration dependent manner.2.Metformin treatment significally decreased the activity of MPO and the concentrations of TNF-? ? IL-6 ? IL-1?in heart tissues of endotoxin-induced septic mice.The results of the present study demonstrated that metformin could significantly alleviate cardiac inflammatory responses in LPS-induced septic mice,which was the important mechanisim by which metformin presented the protective effects on the injury of cardiac function in sepsis.PART 2 : Metformin attenuated endotoxin-induced acute myocarditis and its signal transduction mechanism Objective: To investigated whether activation of AMPK pathway by metformin could alleviate endotoxin-induced acute myocarditis in septic mice Methods:Male BALB/c mice were randomly divided into 7 groups(n=8): group A(control group),group B(metformin treated group),group C(AICAR treated group),group D(LPS treated group),group E(the combination of LPS plus AICAR treated group),F group(the combination of LPS plus metformin treated group),G group(the combination of LPS,metformin plus CC treated group).Group A received normal saline,group B received metformin(400mg/kg;i.p),and group C received AICAR(30.0 mg/kg;i.p).Based on receiving LPS(10 mg/kg;i.p)at start time,30 min before LPS injection group E? F ?G respectively received AICAR at a dose of 100 mg/kg,metformin at a dose of 400 mg/kg,the combination of CC at a dose of 15mg/kg plus metformin at a dose of 400 mg/kg by intraperitoneal injection.18 hours later,all of mice were killed.The concentrations of CK-MB and BNP in the plasma were measured by ELISA kits.The activity of MPO and the levels of inflammatory factors such as TNF-??IL-1??IL-6 in the heart tissue were tested by ELISA kits.Results: 1.The intraperitoneal injection of a single dose of metformin could significally increase the ratio of p-AMPK to AMPK in myocardial tissues of mice,and the ratio could decrease remarkbly after treating with pharmacological inhibitor of AMPK(compound C,CC)(p<0.05),which suggested that the activity of AMPK could be increased by metformin and also could be blocked by CC. 2.The intraperitoneal injection of LPS could remarkably increase the ratio of p-AMPK to AMPK in myocardium of mice(p<0.05).In addition,the ratio could increase after treating with metformin in a dose of 400mg/kg,compared with those in LPS group(p<0.05).In contrast to the combination of LPS plus metformin treated group,the ratio in the combination of LPS,metformin plus CC treated group showed an decreasing trend with the addition of CC(p<0.05).These results showed that the administration of meitformin could furtherly activate the AMPK pathway that could be activated in myocardium of septic mice and also increase the ratio of p-AMPK to AMPK,which could be blocked by use of pharmacological inhibitor of AMPK(compound C).3.In contrast to LPS group,the concentrations of BNP and CK-MB in plasma of LPS and metformin group declined remarkably after inference with metformin in a dose of 400mg/kg by intraperitoneal injection(p<0.05).However,the concentrations of BNP and CK-MB in plasma of the combination of LPS,metformin plus CC treated group increased significally compared with the combination of LPS plus metformin group(p<0.05)and there were no significant differences between the combination of LPS,metformin plus CC treated group and the LPS group.4.In contrast to LPS group,the activity of MPO and the concentrations of TNF-? in myocardium of the combination of LPS plus metformin treated group decreased remarkably after inference with metformin in a dose of 400mg/kg by intraperitoneal injection(p < 0.05).Compared with LPS groups,the combination of LPS,metformin plus CC treated group has no obvious difference,but the activity of MPO and the concentrations of TNF-? in myocardium of the combination of LPS,metformin plus CC treated group diclined significaly in contrast with the combination of LPS plus metformin treated group(p<0.05).5.Results of Western blotting showed that the ratio of p-AMPK to AMPK in myocardium of AICAR group were significantly elevated compared with control group(p<0.05),which demonstrated that the activator of AMPK(AICAR)could activate the AMPK pathway of cardiac tissue in mice.In addition,the ratio of p-AMPK to AMPK in cardic tissue of the combination of LPS plus AICAR treated group were significantly increased compared with control group(p<0.05),which suggested that the activator of AMPK(AICAR)could furtherly activate the AMPK pathway of cardiac tissue in septic mice.6.In contrast to the LPS group,the activity of MPO and the concentrations of CK-MB and BNP of the combination of LPS plus AICAR treated group were significantly decreased(p<0.05),which suggested that the activator of AMPK(AICAR)could significantly alleviate the injury of myocardial inflammatory function in septic mice.Conclusions:1.We found that metformin could active the AMPK pathway in cardiac tissues of mice and therefore increase the ratio of p-AMPK to AMPK.Furthermore,the treatment of metformin furtherly activated the AMPK pathway that could be activated in myocardium of septic mice and also increase the ratio of p-AMPK to AMPK,whih could be blocked by the use of pharmacological inhibitor of AMPK(compound C).2.Intraperitoneal injection of metformin could significantly decrease the concentrations of BNP and CK-MB in plasma of the septic mice,as well as the levels of inflammation factors and the activity of MPO in myocardium of the septic mice,this effect could be blocked by the use of pharmacological inhibitor of AMPK(compound C).3.Intraperitoneal injection of the activator of AMPK(AICAR)could increase remarkably the the ratio of p-AMPK to AMPK,and accordingly alleviate the activity of MPO,the concentrations of CK-MB?BNP.Those results demonstrated that activition of AMPK pathway could alleviate the injury of myocardial inflammation in septic mice.4.The results of the present study clearly demonstrated that metformin attenuated endotoxin-induced acute myocarditis via activating AMPK pathway. |
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