Effect Of SS31 Peptide On Myocardial Injury In Sepsis And Its Mechanism

Background: Sepsis-induced myocardial dysfunction(SIMD)is one of the major complications of sepsis with high mortality rate and without effective treatment.Mitochondrion-targeted antioxidant peptide SS31 has been reported to be pharmacological myocardial protection in a variety of cardiovascular disease models.However,the role of SS31 in septic myocardial dysfunction is not yet known.Objective: This study sought to explore the effect and mechanism of SS31 in sepsis-induced myocardial dysfunction via in vivo and vitro,providing a novel approach for the treatment of septic patients.Methods: Thirty-five C57BL/6 mice were randomly divided into four groups: sham group(0.9%Nacl 5mg/kg,i.p.,n=8),LPS group(5mg/kg lipopolysaccharide,i.p.,n=9),SS31 group(5mg/kg SS31,i.p.,n=8),and treatment group(n=10).The mice of treatment group were administrated of 5mg/kg SS31 by intraperitoneal injection after one hour of LPS adminstration.Investigations of the level of proinflammatory factors in heart tissues were performed at baseline and 3 different times following administration by real-time quantitative PCR(RT-q PCR).Hematoxylin-eosin(HE)staining was used to observe the morphological changes of myocardial tissue in each group.And myocardial apoptosis was examined by TUNEL staining.We explored the ATP content and oxidoreductase(SOD and GSH-Px)activity of heart tissues through specific kits.In addition,western blot was performed to measure the protein levels of the relevant signaling pathways(p38-MAPK,ERK,JNK,p62).In vitro,H9C2 cells were also divided into four groups: con group,LPS group,SS31 group and LPS+SS31 group.After the treatment of LPS or/and SS31 for 6,12 and 24 h,the total RNA was extracted and the inflammatory factors’ m RNA expressions were detected by RT-PCR.In addition,mitochondrial membrane potential(MMP)and reactive oxygen species(ROS)levels in H9c2 cells were measured by immunofluorescence staining.Cellular ATP content,NLRP3 and redox related indicators were detected from the extracted cellular proteins.Results: 1.The results of cell activity experiments showed that 5～80 μM SS31 had no significant effect on the activity of cardiomyocytes,but greater than or equal to 10 μg/μl LPS significantly inhibited cell viability.Besides,10 μg/μl LPS obviously promoted inflammatory response.2.HE staining revealed that myocardial morphological damage of SS31 intervention group was considerably lower than that of sepsis group.Additionally,SS31 suppressed the myocardial apoptosis induced by LPS in mice as indicated by TUNEL staining.3.Compared with the control group,the levels of myocardial inflammatory factors(IL-6,IL-1β,TNF-α)in septic mice were notably up-regulated within 6 hours,and that lasted for 24 hours.However,the expression in the SS31 intervention group at each time period(6,12 and 24 h)were considerably lower than those in LPS group,and the difference was statistically significant.It was failed to confirm that the effect of SS31 was time-dependent manners.In H9c2 cells,SS31 obviously suppressed inflammatory response induced by LPS within 24 h,which was consistent with animal experiment’ results.4.Compared with sham group,the level of ATP in mice decreased dramatically after 6 hours of LPS stimulation.On the contrary,SS31 remarkably increased the ATP content in each time period(6,12 and 24 hours).In addition,the results in cell experiments were consistent with that in animal experiments.LPS accelerated the depolarization of mitochondrial membrane potential(MMP)and ROS accumulation in cells in a time-dependent manner,and SS31 normalized the levels of ROS and depolarization of MMP and in H9c2 cells.5.With the prolonged action of LPS,the MDA level of myocardial tissue in mice gradually increased,and the activities of SOD and GSH-PX antioxidant enzymes gradually decreased.SOD and GSH-PX activities in the SS31 intervention group were obviously higher than those in LPS group.In vitro,SS31 seemed to up-regulated the activities of SOD and GSH-PX as well.6.Western blot showed that SS31 administration suppressed sepsis-induced activation of NF-κB p65,ERK1/2,JNK as well as NLRP3 inflammatory bodies in the myocardium,while it had poorly effect on p38-MAPK.Conclusion: The cardioprotective effect of antioxidant-peptide SS31 was associated with the repression of NF-κB,ERK1/2,JNK and NLRP3 pathway,which was proved by down-regulating inflammatory cytokines and apoptosis.Meanwhile,SS31 mitigated redox reactions and myocardial energy defect,and maintained mitochondrial function.Thus,SS31 may be a potential drug for cardiac dysfunction caused by sepsis.