A Mechanism Study Of The Protection By Resveratrol Against Sepsis Induced Cardiac Injuries In Mice

Background: Sepsis is the leading cause of death in critical ill patients,and cardiovascular dysfunction in sepsis is associated with a significantly increa sed mortality rate of 70-90% as compared to 20% in septic patients without exhibitions of cardiovascular impairment.Thus,sepsis induced cardiac damages,also called sepsis-induced cardiomyopathy(SIC)is a decisive factor in determining survival or death in septic patients.Recently,a lot of studies have found a reduced activity of cardiac sarcoplasmic endoplasmic reticulum Ca2+ ATPase isoform(SERCA2a)under septic conditions,and thus improving SERCA2 a activity may be a potential stragety to protect heart from sepsis.While,the mechanism that how the SERCA2 a got inhibited in sepsis remains unclear.The phospholamban(PLB)is a SERC A2 a regulator protein and exists in the two forms of pentamer and monomer,which are kept in a dynamic balance in the myocytes.The pentamer is thought as a stock form of PLB with no biological function,while the monomer has a direct inhibitory effect on SERC A2 a.However,up to now,the role of sepsis on the transition of PLB remains unclear.Resveratrol(RSV),a natural small molecular compound found in grape and wine has been shown to have a prophylactic effect against sepsis induced cardiac damages and protect heart under many pathological conditions.So this study examined whether RSV could protect against SIC by improving the SERC A2 a activity and explore its corresponding mechanisms.This study will give a novel insight into SIC and is helpful to design SERC A2a-based treatment against SIC in clinic.Objective: 1.Whether the RSV could treat SIC.2.Whether the therapeutic effect of RSV against SIC is related to regulate the SERC Aa-PLB axis.3.To explore the mechanism that how the RSV modulates the SERCAa-PLB axis.Methods: In our study,we gave mice intraperitoneally injection of LPS(Sigma corporation,E.coli O55:B5 serotype)at a dose of 6 mg/kg to set up an experimental sepsis induced cardiac dysfunction model to examine whether resveratrol could protect heart from sepsis.The 8-week male C57BL/6J mice were randomly divided into 4 groups,defined as control group,RSV group,LPS group and LPS treated by RSV group respectively.Six hours after LPS administration,echocardiography tests were performed to measure cardiac parapeters,such as: cardiac ejection fraction(EF%),fraction shortening(FS%)and left ventricular end-diastolic dimension(LVEDD).Parallel in vitro cellular studies were performed in isolated cardiomyocytes to measure myocyte contractility,calcium transient and reactive oxygen species(ROS).H-E staining was used to observe histological structure of ca rdiac section of different groups.Immunohistochemistry was used to measure the amount of CD45+ cells in heart.ELISA was used to assess the levels of plasma Tn I and TNF-α,cardiac ATP and MDA contents,as well as cardiac SERCA2 a ATPase activity.Western blot and or real time PCR were used to measure the expression levels of cardiac 4-HNE,SERC A2 a,NCX,Ry R2,IP3 R,TNF-α,PLB,cleaved Caspase 3,Pgc-1α,Nrf-2 and its downstream proteins(C AT and NQO-1).Results: 1.LPS could massively decrease cardiac EF% >50%,companied by enlarged LVEDD in our study.RSV treatment(two hours after LPS administration)could alleviate these damages.LPS challenge significantly decreased ce llular contractile amplitude and the peak of Ca2+ transient and largely increased their time return to 90% baseline.While,RSV treatment significantly improved these malignant altercations.2.The levels of plasma troponin I and cardiac cleaved caspase 3 were not increased,The levels of plasma TNF-α was highly increased in LPS-treated mice,companied with increase in infiltration of CD45 positive cells in cardiac sections,while the TNF-α levels of cardiac tissues were comparable among different groups.RSV treatment did not reduce plasma TNF-α levels,and also did not reduced the amount of CD45 positive cells in cardiac sections.The expression levels of cardiac NCX,IP3 R and Ry R2 were not different among different groups.In LPS-treated mice,the expression level of SERCA2 a was decreased and its ATP hydrolytic activity was reduced,while RSV treatment could increase the SERCA2 a expression and improve its activity.3.LPS increased the ratio of monomer phospholamban(PLB)and decreased the ratio of pentamer PLB,which was related to disturbed redox status induced by LPS.LPS could massively increase the contents of 4-hydroxynonenal(4-HNE)and malondialdehyde(MDA)in cardiac tissues,and down-regulate the expression levels of nuclear factor erythroid 2-related factor(Nrf-2)and its downstream genes.RSV treatment could significantly improve these altercations.In vitro studies further found that RSV worked similarly with N-acetylcysteine(NAC),a type of ROS scavenger,reduced intracellular ROS levels,improved inhibited SERCA2 a activity and promoted the transition of PLB from monomer into pentamer.Conclusion: 1.RSV can treat SIC.2.The therapeutic effect of RSV against SIC is related to regulate the SERC Aa-PLB axis via promoting the transiton of PLB from monomer to pentamer.3.The mechanism that RSV modulates the SERCAa-PLB axis is related to modulate intracellular redox status by activating Nrf-2 and its downstream proteins.Innovation: 1.In the study,we found that RSV could treat SIC by enhanc ing SERC A2 a activity via promoting the transition of PLB from monomer to pentamer,which is related to activating Nrf-2 and its downstream proteins.2.Our study provide a theory that RSV(or direct Nrf-2 activator)can be a potential drug used to correct SIC in clinic.In all,our study firstly found a novel mehcanism that RSV protected heart under septic condition: RSV could activate Nrf-2 and its downstream genes,regulate intracellular redox status,promote transition of PLB from monomer into pentamer and improve inhibited SERC A2a.