Myricetin Attenuated LPS Induced Cardiomyocyte Injuries In Vitro And In Vivo

Background: Sepsis is one of the leading cause of mortality of patients in intensive care unit(ICU).Sepsis induced myocardial dysfunction(SIMD)is one of the most serious complication of patients suffering from sepsis.Plenty of studies have demonstrated that inflammation,oxidative stress and cardiomyocytes apoptosis were the most important factors associated with SIMD.Myricetin have been demonstrated to possess multiple pharmacological properties including anti-inflammation,anti-oxidative stress,anti-ageing and cardioprotective.However,none investigation have exploreed the role and underlying mechansim in SIMD.Object: To investigate the role and relative mechanisms of myricetin in SIMD relying the model of LPS induced cardiomyocytes injuries in cell level and animal model.To explore its action mechanism and provide new drugs for clinical treatment of myocardial diseases.Methods:1)In in vitro experimen,H9C2 were cultured in 37 ℃ temperature CO2 incubator.After 90% of area of culture dish was covered by H9C2,cells were digested with0.25% trypsin for next experiments.CCK-8 were used to test the cytotoxicity and cell viability.2)In in vivo experiment,100 C57/BL mice(weight:24-25g)were randomly allocated into control group(CON,n=25),myricetin treatment group(M,200mg/kg per day,n=25),LPS treatment group(LPS,15mg/kg,n=25)and LPS+M group(n=25).Myricetin were pre-treated in M and LPS+M groups for five days before injection of LPS.The cardiac function of mice were evaluated by echocardiogram after 12 hours treatment of myricetin.Then the mice were killed by cervical dislocation,the mice hearts were collected and randomly allocated into histopathological and molecular biological group.3)Immunofluorescence were performed to determine NF-κB nuclei translocation in frozen section of mice heart and cell climbing of H9C2.TUNEL staining were used to detect the apoptosis in mice heart and H9C2 cells.Western blot was performed to detect the IκBα/NF-κB/p65 pathway,inflammatory proteins(IL-1β,IL-6,TNF-α)and apoptosis related proteins(Bcl2,Bax,C-Caspase3).Real-time reverse transcription quantitative polymerase chain reaction(RT-PCR)were used to detect the inflammatory cytokines at mRNA level.Commercial kit was used to separate the protein from nuclei and cytoplasm for quantitative detection of NF-κB in different compartment of cells.4)Reactive oxygen species(ROS),SOD and GPx were also tested by commercial kits.Results:1)In in vitro experiment,cells were treated with different concentrations of myricetin(0,5,10,20,40,80,and 160μM)for 24 hours.The result showed that concentrations of myricetin less than or equal to 40μM presented no effect on the cell viability.2)In in vivo experiment,echocardiogram presented that myricetin prevented the mouse heart from LPS induced dysfunction evidenced by increasing the left ventricular ejection fraction(EF)and shorting fraction(FS),improving diastolic and systolic function,and attenuating the blood retention in the left ventricle.3)In in vitro experiment,treatment with myricetin for 24 h could significantly block the accumulation of NF-κB/p65 in the nucleus,treatment with myricetin markedly blunted the phosphorylation of p65 and IκBa.In in vivo experiment,myricetin treatment obviously alleviated the translocation of NF-κB/p65,which could be further confirmed by the decreased phorsphoralation of p65 in mouse heart and decreased accumulation of p65 in the nuclei.In in vitro and in vivo experiment,myricetin markedly alleviated the LPS-induced production of proinflammatory cytokines at mRNA level in a concentration-dependent manner(P<0.05 vs.the LPS group),We observed that myricetin also markedly attenuated the protein accumulation of IL-1β,IL-6 and TNF-α.In in vitro experiment,myricetin treatment significantly reduced the percentage of TUNEL-positive cells,treatment with myricetin increased expression of anti-apoptosis protein(Bcl2)and decreased expression of pro-apoptosis protein,consequently recovering the Bcl2/Bax ratio to the normal level.In in vivo experiment,myricetin treatment inhibited the cardiomyocyte apoptosis evidenced by decreased TUNEL positive nuclei,at the same time,myricetin inhibited the expression of apoptosis related proteins including Bax and c-caspase3.4)In in vitro experiment,pre-treatment with myricetin for 2h significantly blocked the up-regulation of ROS,treatment with myricetin could also recover the enzymatic activity of SOD and GPx.In in vivo experiment,myricetin treatment could reverse the decreased activity of SOD and GPx induced by LPS in mouse heart.Conclusion: Both in cell level and animal model,myricetin treatment could alleviated the LPS induced inflammation reaction,and protected the cardiomyocyte or mouse heart from LPS induced injury.The underling mechanism may be at least partly associated with the properties of anti-inflammation,anti-oxidative stress and anti-apoptosis.Further clarifying the pharmacological function and the underling mechanism may provide a novel strategy for the clinical adjunctive therapy.Myricetin may be a potential therapy or adjuvant therapy drug for SIMD.