The Protective Effect And Mechanisms Of Sodium Hydrosulfide On Myocardial Mitochondria In Mice With Sepsis

Purpose:This study aimed to build the optimum sepsis model of mice by cecal ligation and puncture(CLP)method at the optimum ligation position and explore the effect of sepsis on mitochondrial biosynthesis of myocardium in mice the first time.Then intervened with different concentration sodium hydrosulfide(NaHS),we aimed to explore the protective effect and mechanisms of NaHS on myocardium mitochondrial in mice with sepsis and the optimum experimental concentration,which would provide theoretical basis for animal experimental study and clinical diagnosis and treatment of sepsis.Method:1.Forty C57BL/6 mice were randomly divided into Sham group,CLP1,CLP2,CLP3 group(respectively ligating the distal 1/4,1/2,3/4 of the cecum),then observing the survival situation of mice and choosing the best model and the time point of collecting specimen.2.Another fifty C57BL/6 mice were randomly divided into CLP group and CLP+ NaHS group(4 subgroup,NaHS concentration was 25,50,100,200?mol/L respectively),then comparing the survival situation of mice.3.Another seventy C57BL/6 mice were randomly divided into Control group,Sham group,CLP group,CLP+ NaHS group(4 subgroup,NaHS concentration was the same as before),detecting and comparing the following index:(1)Observing sepsis symptoms.(2)Plasma endotoxin level.(3)The detection of myocardial damage:the morphology of myocardial cells were detected by HE staining and serum cardiac troponin I(cTnI)levels were detected by ELISA.(4)The detection of myocardial mitochondrial damage:myocardial mitochondrial morphology were observed through transmission electron microscopy;mitochondrial swelling degree were detected by spectrophotography;ATP levels were detected by bioluminescent method.(5)The detection of oxidative stress levels:malondialdehyde(MDA)levels were detected by the thiobarbituric acid(TBA)method;reactive oxygen species(ROS)levels were detected by means of an oxidation-sensitive fluorescent probe(DCFH-DA).(6)The levels of antioxidant:glutathione(GSH)levels were detected by dithionitrobenzoic acid(DTNB)method;Mn-superoxide dismutase(MnSOD)and heme oxygenase-1(HO-1)protein levels were detected by Western blot;.MnSOD?HO-1 mRNA levels were detected by Reverse Transcription Quantitative Polymerase Chain Reaction(RT-qPCR).(7)Mitochondrial biosynthesis related factors levels:peroxisome proliferator-activated receptor-y coactivator-1?(PGC-1a)?nuclear factor-erythroid-2-related factor 2(Nrf2)?mitochondrial transcription factor A(Tfam)mRNA levels were detected by Reverse Transcription Quantitative Polymerase Chain Reaction(RT-qPCR).Results:1.The death time of mice in CLP1,CLP2,CLP3 group was 12h,11h,8h after modeling respectively and the 48 hours survival percentage in CLP1 group was higher than that in CLP2 and CLP3 group.So CLP 1 was selected as the experimental model in this study and 12 hours after modeling as the time point of collecting specimen.2.The 48 hours survival percentage after modeling of mice in NaHS intervention group were higher than that in CLP group and that in 100?mol/L was the highest.3.Mice in sepsis group began to appear sepsis symptoms 6 hours after modeling and aggravated gradually.Sepsis symptoms of mice in different concentrations NaHS intervention group were all alleviated,the higher the NaHS concentration,the lighter the sepsis symptoms.4.The level of plasma endotoxin in CLP group significantly increased comparing with those in control and sham group.5.In CLP group,myocardial tissue showed paramorphia by HE staining,cTnI level increased.After NaHS intervention,the paramorphia of myocardial tissue showed lessened and cTnI level decreased,the higher the NaHS concentration,the more obvious the effect.6.In CLP group,myocardial mitochondria showed paramorphia,the swelling of myocardial mitochondria was significantly aggravated and ATP level decreased.After NaHS intervention,the paramorphia and swelling of myocardial mitochondria were alleviated and ATP level increased,the higher the NaHS concentration,the more obvious the effect.7.Comparing with those in Sham group,ROS and MDA levels all increased in CLP group.Comparing with those in CLP group,MDA levels decreased in all NaHS intervention group,but there was no significant difference between 100 and 200?mol/L NaHS group;ROS levels only decreased in 100,200?mol/L NaHS group.8.In CLP group,GSH level decreased,MnSOD?HO-1 protein and mRNA levels increased comparing with those in control and sham group.After NaHS intervention,MnSOD?HO-1 protein and mRNA levels,GSH levels all increased in a concentration-dependent manner comparing with those in CLP group;MnSOD?HO-1 protein levels in 200?mol/L group were lower than those in 100 ?mol/L group.9.The levels of PGC-la,Nrf2,Tfam mRNA in CLP group significantly increased comparing with those in control group and sham group.After NaHS intervention,PGC-la,Nrf2,Tfam mRNA levels continuously increased comparing with those in CLP group,the higher the NaHS concentration,the higher the PGC-1?,Nrf2,Tfam mRNA level.10.In CLP group and NaHS intervention groups,serum cTnI levels were positively correlated with the swelling of myocardial mitochondria and negatively correlated with ATP levels.In NaHS intervention groups,ROS,MDA levels were negatively correlated with GSH levels,MnSOD and HO-1 mRNA levels;ATP,GSH levels and MnSOD,HO-1,Tfam mRNA levels were all positively correlated with PGC-la,Nrf2 mRNA levels.Conclusion:1.Sepsis could result in oxidative stress damage on myocardial mitochondria in mice.2.NaHS could concentration-dependently alleviate the damage on myocardial mitochondria induced by sepsis,and 100?mol/L was the optimum experimental concentration.3.NaHS could upregulate antioxidant levels and improve mitochondria biosynthesis by PGC-la and Nrf2 so as to protect myocardial mitochondria during sepsis.