| Sepsis is a systemic inflammatory response syndrome characterized by multiple organ failure and cardiovascular suppression,with high mortality,poor prognosis and high treatment costs,and has become the most common death in non-cardiovascular intensive care units the reason.The study of sepsis around the world has focused on the initiation,enlargement and regulation of proinflammatory,antiinflammatory,oxidative-antioxidant and coagulation-fibrinolysis.Although a number of new drugs that modulate immune function,block lipid peroxidation,and restore coagulation / fibrinolysis balance are found,their clinical efficacy is poor.Moreover,septic shock is a clinical emergency.Cardiovascular system is an important circulatory system,early sepsis that there are abnormal cardiovascular function,especially severe sepsis and sepsis shock patients,50%of left ventricular systolic / diastolic dysfunction.Clinical studies have shown that cardiac dysfunction significantly increases the mortality of patients with sepsis.Septic heart dysfunction has been widely concerned,but its underlying mechanism is not well explained.Leukotriene B4(LTB4)is a potent inflammatory mediator and chemokine that exerts a biological effect by binding to its corresponding receptor,and its potential inflammatory lipid regulation can be involved in bronchial asthma,chronic obstructive Lung disease,cystic fibrosis,acute respiratory distress syndrome,rheumatoid arthritis and ischemic heart disease and other diseases of the pathophysiological process.In addition,LTB4 plays an important role in sepsis by white blood cell aggregation mediated by leukotriene B4 receptor 1(BLT1).At present,foreign studies have shown that LTB4/BLT1 pathway is involved in the regulation of innate immunity and acquired immune system immune cell function.LTB4-mediated inflammation has demonstrated increased susceptibility to sepsisin patients with type 1 diabetes mellitus.The study found that LTB4 in sepsis increased vascular endothelial dysfunction,increased serum LTB4 concentration in sepsis,the use of BLT1 receptor antagonists can reduce the degree of injury caused by sepsis,indicating that LTB4 and BLT1 may play a role in lipopolysaccharide-induced acute cardiac dysfunction.AMP-activated protein kinase(AMPK)is a silk,threonine protein kinase,the main role is to coordinate the needs of metabolism and energy.Known as the "cell energy regulator".AMPK can initiate catabolic pathways,increase ATP production,and turn off the metabolic pathway,reducing ATP consumption.It has been found that activation of AMPK activity contributes to the protection of myocardial function and reduction of myocardial infarct size.AMPK signaling pathway is associated with lipopolysaccharide-induced acute cardiac dysfunction.It is noteworthy that AMPK in the body’s energy metabolism process is also associated with cerebral ischemia,heart ischemia and reperfusion,diabetes,certain cancers,vascular remodeling and so on.It has been found that AMPK signaling pathway plays an important role in the increase of energy demand when ischemic injury is on the heart.Activated AMPK can increase glucose uptake and glycolysis,accelerate fatty acid oxidation,increase the energy supply of myocardial tissue and inhibit myocardium apoptosis protects myocardium and plays an important role in the process of cardiovascular remodeling,which affects the proliferation and energy metabolism of vascular smooth muscle cells,endothelial cells.However,the relationship between AMPK and BLT1 in lipopolysaccharide-induced septic cardiac dysfunction is unclear.In conclusion,we suggest that the BLT1 inhibitor U75302 may play an anti-septic cardiac dysfunction by activating the AMPK pathway and ultimately play a cardioprotective role by mitigating mitochondrial oxidative stress injury and anti-apoptotic pathways.The project will use a series of research models and experimental methods to clarify the role of AMPK and BLT1 in lipopolysaccharide-induced septic cardiac dysfunction and the specific mechanism.In this study,we explored the role of LTB4 /BLT1 in lipopolysaccharide-induced acute myocardial injury in rats and elucidated its underlying mechanisms.By studying its mechanism of action and understanding of the treatment target can be better used to guide the clinical,to further improve the mortality ofsepsis patients.Part Ⅰ: To investigate the effect of LTB4 / BLT1 receptor on septic cardiac dysfunction after LPS-induced mouse model of septic cardiac dysfunction Objective:Establishment of septic cardiac dysfunction in mice model,to observe the effect of LTB4/BLT1 receptor on the cardiac function of mice,and to specifically inhibit the effect of LTB4 / BLT1 receptor on LPS-induced cardiac dysfunction in mice.Methods:C57 mice were injected intraperitoneally with lipopolysaccharide(LPS)to establish the sepsis model to observe the effect of LTB4 on cardiac function in septic mice.The animals were divided into 5 groups:(1)Control;(2)LPS;(3)LTB4;(4)LTB4 + LPS;(5)U75302 1μM+ LTB4.To observe the effect of different concentrations of LTB4 / BLT1 receptor inhibitor U75302 on septic cardiac dysfunction in mice.The animals were divided into 5groups:(1)Control;(2)LPS;(3)U75302 0.25μM + LPS;(4)U75302 0.5μM + LPS;(5)U75302 1μM + LPS.LPS(6mg/kg),LPS(6mg/kg)+ U75302(0.25,0.5,1mg/kg),LTB4(10mg/kg),intraperitoneal injection,the control group given PBS.U75302(0.25,0.5,1mg/kg)and 1 hour before LPS injection.After 6 hours of LPS injection,mice were subjected to echocardiography after anesthesia.Record echocardiographic indicators,including:left ventricular ejection fractions(LVEF%),left ventricular fractional shortening(FS%)and the peak velocity ratios of early to late mitral inflow filling(E / A ratio).Results:In this part,we first studied the effect of LTB4 on the cardiac function of septic mice,and then observed whether different concentrations of U75302 had protective effects on the cardiac function of septic mice.Echocardiographic analysis showed that FS% and EF%in LTB4 group were significantly decreased(compared with Control group,P <0.01);FS%and EF% of LPS + LTB4 group were significantly decreased(compared with LPS group,P<0.05);U75302 + LTB4 group of mice in the mild reduction of EF(compared with the Control group,P> 0.05).Indicating that LTB4 can lead to damages in mice with cardiac dysfunction.Conclusion:(1)LPS can induce damage in mice with cardiac dysfunction,LTB4 can exacerbate damages in mice with cardiac dysfunction;(2)LBT4 / BLT1 inhibitor U75302 may have the effect of protecting the heart function injury in septic mice.Part Ⅱ: To investigate the effect of LTB4 / BLT1 receptor on myocardial inflammation,apoptosis,mitochondrial function,survival rate and its possible mechanism in septic mice Objective:To observe the effects of LTB4 / BLT1 receptor inhibitor U75302 on myocardial inflammation,apoptosis and mitochondrial function in mice with septic cardiac dysfunction and the survival rate of mice.Methods:The animals were divided into 5 groups:(1)Control;(2)LPS;(3)U75302 0.25μM + LPS;(4)U75302 0.5μM + LPS;(5)U75302 1μM + LPS.LPS(6mg / kg),LPS(6mg / kg)+U75302(0.25,0.5,1mg / kg),intraperitoneal injection,the control group given PBS.U75302(0.25,0.5,1 mg / kg)and 1 hour before LPS injection.LPS injection 6h,measuredplasma LTB4 levels.Another inhibitor,BL105,696,BLT1,was also used to assess its effect on myocardial inflammation,apoptosis and mitochondrial function in mice with septic cardiac dysfunction.1)The levels of TNF-α and IL-6m RNA were detected by Real-time PCR method.The expression of pNF-kB and IkB-α protein was detected by Western blot to evaluate the effect of different concentrations of BLT1 inhibitor U75302 on myocardial inflammation in septic cardiac dysfunction mice;2)The expression of BCL-2,BAX and cleaved caspase-3 was detected by Western blot analysis to evaluate the effect of different concentrations of BLT1 inhibitor U75302 on myocardial apoptosis in septic cardiac dysfunction mice;3)Isolation and extraction of mitochondria from cardiomyocytes and evaluate the expression of complex I(Nd)and complex IV(Co)subunit mRNAs by real-time PCR method to detect nuclear DNA(Cox4i and Cox5a)or mtDNA(mt-Nd1,mt-Nd2,mt-Co1,and mt-Co2),the effects of different concentrations of BLT1 inhibitor U75302 on the mitochondrial biology and function of septic cardiac dysfunction mice were evaluated by Western blot analysis of ComplexⅠ,Complex Ⅱ and OPA1 protein expression;4)Western blot analysis of pAMPK,AMPK,pACC,ACC and PGC1α protein expression to assess the different concentrations of BLT1 inhibitor U75302 to protect sepsis mice cardiac dysfunction may be the role of access5)Based on previous studies,we selected U75302 1 mg / kg as the optimum concentration.The animals were divided into three groups:(1)PBS;(2)LPS;(3)LPS + U75302,LPS(30mg/ kg),LPS(30mg / kg)+ U75302(1mg / kg),intraperitoneal injection,PBS was given in the control group.U75302(1 mg / kg),1 hour before LPS injection.After 6 h of LPS injection,the survival rate of mice was observed from 6 h to 3d and the Kaplan-Meier survival curve was drawn.Results:1)LPS pretreatment significantly increased TNF-α and IL-6 mRNA levels and pNF-kB expression(compared with control group,P <0.05),but IkB-α expression decreased(compared with Control group,P <0.05).However,U75302 significantly reduced the expression of TNF-α,IL-6 and pNF-κB and increased IkB-α expression(P <0.05 compared with LPS group).BLT1 another inhibitor CP105,696 can reverse pNF-kB increase and Ik B-α decrease(compared with LPS group,P <0.05).2)LPS pretreatment can lead to myocardial apoptosis,and the expression of Bcl-2 was decreased and the expression of Bax and cleaved caspase-3 were increased(compared with control group,P <0.05).However,U75302 can reduce apoptosis in a concentration-dependent manner(compared with LPS group,P <0.05).BLT1 Another inhibitor CP105,696 can reduce apoptosis,Bax expression decreased,Bcl-2 expression increased.(compared with LPS group,P <0.05).3)LPS pretreatment can reduce the expression of complex Ⅰ,complex Ⅱ and OPA1(compared with control group,P <0.05),nuclear DNA and mtDNA were deletioned(compared with control group,P <0.05);U75302 can weaken these changes and keep complex I,complex II and OPA1 expression(Compared with LPS group,P <0.05).U75302 mild reversal of mRNA changes in mitochondria(compared with LPS group,P<0.05).Another inhibitor of BLT1,CP105,696,can reduce mitochondrial damage by maintaining complex I,complex II and OPA1 expression.4))LPS pretreatment resulted in a significant increase in pAMPK,pACC and PGC1α(compared with Control group,P <0.05).U75302 inhibited the expression of pAMPK,pACC and PGC1α after inhibition of BLT1 receptor(compared with LPS group,P <0.05),indicating that the AMPK signal transduction pathway was further activated.5)Kaplan-Meier survival curve showed that LPS + U75302 group had a significant reduction in mortality at 72 h after LPS injection(compared with LPS group,P <0.01).Conclusion:Inhibition of LTB4 / BLT1 receptor can protect the sepsis mouse cardiomyocyte inflammation,apoptosis and mitochondrial damage,thus clear LTB4 / BLT1 inhibitor U75302 on the protection of damage in sepsis mice with cardiac dysfunction.Part Ⅲ: To investigate whether AMPK signaling pathway is involved in the protective effect of BLT1 inhibitors on septic cardiac dysfunction and to explore its mechanism Objective:To further explore the possible role of AMPK signaling pathway in LPS-induced sepsis cardiac dysfunction in mice and to elucidate its mechanism.Methods:1)The animals were divided into 5 groups:(1)Control;(2)LPS;(3)U75302 0.25μM + LPS;(4)U75302 0.5μM + LPS;(5)U75302 1μM + LPS.LPS(6mg / kg),LPS(6mg / kg)+U75302(0.25,0.5,1mg / kg),intraperitoneal injection,the control group given PBS.U75302(0.25,0.5,1mg / kg),1 hour before LPS injection,intraperitoneal injection.The expression of pAMPK / AMPK,pACC / ACC and PGC1α was detected by Western blot to evaluate whether AMPC pathway blocker Compound C had an effect on U75302anti-septic cardiac dysfunction.2)The animals were divided into four groups:(1)LPS;(2)LPS + Compound C;(3)LPS +U75302;(4)LPS + U75302 + Compound C.LPS(6 mg / kg),LPS(6 mg / kg),LPS(6 mg /kg),LPS(6 mg / kg)+ U75302(1 mg / kg)Compound C(20mg / kg),intraperitoneal injection.U75302(1 mg / kg),1 hour before LPS injection.Compound C(20 mg / kg),30 min before LPS injection.(1)After 6 hours of LPS injection,the mice were subjected to echocardiography after anesthesia.To observe the effect of Compound C on the anti-septic cardiac dysfunction of AMPK pathway blocker.Include: Left ventricular ejection fractions,(LVEF%),left ventricular fractional shortening(FS%),and the peak velocity ratios of early to late mitral inflow filling,E / A ratio).(2)The expression of TNF-α and IL-6mRNA was detected by Real-time PCR method.Theexpression of pNF-kB and IkB-α protein was detected by Western blot to evaluate the effect of Compound C on the myocardial inflammation of U75302 against septic cardiac dysfunction.(3)The expression of Bcl-2,BAX and cleaved caspase-3 was detected by Western blot to evaluate the effect of AMPK pathway blocker Compound C on the myocardial apoptosis of U75302 against septic cardiac dysfunction.(4)Isolation and extraction of myocardial mitochondria.The expression of Cox4 i,Cox5amRNA and mt-Nd1,mt-Nd2,mt-Co1 and mt-Co2 mRNA were detected by Real-time PCR.The expression of ComplexI,ComplexⅡ and OPA1 protein was detected by Western blot.To evaluate the effect of AMPK pathway blocker Compound C on U5302anti-septic cardiac dysfunction in myocardial mitochondrial biology and function.Results:1)U75302 treatment significantly increased the expression of pAMPK / AMPK,pACC /ACC and PGC1α protein(compared with control or LPS group,P <0.05).2)Compound C did not result in FS%,E / A ratio or LVEF% improvement(compared with LPS group,P> 0.05).However,Compound C significantly reduced FS% and LVEF%,increasing E / A ratio(compared to LPS + U75302,P <0.05).3)The expression levels of TNF-α,IL-6 mRNA,pNF-κB and IκB-α protein were significantly changed after treatment with Compound C(compared with LPS group,P>0.05).However,Compound C up-regulates the expression of TNF-α,IL-6 m RNA and pNF-κB protein level,down-regulation of IκB-α protein expression reversed the anti-inflammatory effect of U75302(compared with LPS + U75302,P <0.05).4)Compound C significantly inhibited AMPK and ACC phosphorylation,mild down-regulation of PGC1αexpression(compared with LPS group,P <0.05).Furthermore,Compound C reversed the up-regulated expression of PGC1α,AMPK and ACC phosphorylation after treatment with U75302(compared to LPS + U75302,P <0.05).5)Compound C treatment did not affect the expression of apoptosis-dependent protein(compared with LPS group,P> 0.05).However,the increase in Bcl-2 and the decrease ofBax and cleaved caspase-3 were observed by Compound C can reversed the effort of U75302 treatment(P <0.05 compared with LPS + U75302).6)Compound C treatment did not affect complex I,complex II and OPA1 expression(compared with LPS group,P> 0.05).However,Compound C treatment downregulated complex I,complex II and OPA1 expression(compared with LPS + U75302 group,P<0.05).The results of Mt-Nd1,mt-Nd2,mt-Co1,mt-Co2,Cox4 i and Cox5 a mRNA levels and Western blot showed a continuous trend.Conclusion:Inhibition of LTB4/BLT1 receptors attenuates cardiomyocyte inflammation and apoptosis by activating AMPK,which may mediate the protective effect of anti-septic cardiac dysfunction by inhibiting NF-κB signaling and mitochondrial damage. |
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