Effect Of Melatonin On Myocardial Injury Induced By Sepsis And Its Mechanism

Background:Sepsis is an organ dysfunction caused by host response disorder caused by infection.Aggravation can further develop into septic shock,accompanied by multiple organ dysfunction or failure.The heart is one of the most damaged organs.The mortality of myocardial injury caused by sepsis is as high as 50%,which is related to the poor prognosis of septic shock.At present,the pathogenesis of septic myocardial injury has not been thoroughly studied,which limits the application of clinical treatment methods or drugs.Melatonin(MEL)is an endogenous indoleamine that regulates the circadian rhythm.Previous studies have reported that MEL plays a protective role in a variety of cardiovascular diseases.It is worth noting that MEL can play the role of anti-septic myocardial injury through anti-oxidation,anti-inflammatory and anti-endoplasmic reticulum stress.However,there are few studies on the comparison of the efficacy of MEL before and after administration,and the combined application of MEL with traditional antibiotics such as azithromycin(AZI)and vancomycin(VAN),which need to be further elucidated.AMPK is a member of the Ser/Thr kinase family.Studies have confirmed that it plays important a role in regulating energy metabolism in pathological processes such as myocardial ischemia-reperfusion injury,myocardial hypertrophy and myocardial fibrosis.In addition,AMPK also plays an active role in sepsis-induced myocardial injury.An important study confirmed that in LPS-induced sepsis mice,MEL can play a cardioprotective effect by activating AMPK-dependent autophagy pathways.However,the potential mechanism of MEL in septic myocardial injury by regulating AMPK signaling pathway has not been fully elucidated,and further research is urgently needed.In summary,we will use a series of experimental methods to detect and analyze related indicators to explore:(1)MEL alone or in combination with classic antibiotics for the treatment of septic myocardial injury;(2)The specific role and potential molecular mechanism AMPK pathway in MEL against septic myocardial injury.Method: Taking BALB/c mice as the research object,a septic myocardial injury model induced by CLP was constructed,and the groups were set respectively:(1)Sham group,CLP group,MEL+CLP group(MEL pretreatment);(2)Sham group,CLP group,MEL+CLP group,MEL+CC+CLP group,CC+CLP group(CC(compound C,AMPK inhibitor),MEL pretreatment);(3)Sham group,CLP group,CLP+MEL group(MEL posttreatment);(4)Sham group,CLP group,CLP+AZI group(AZI post-treatment);(5)CLP+AZI group,CLP+AZI+MEL group(AZI,MEL combined post-treatment);(6)Sham group,CLP group,CLP+VAN group(VAN post-treatment);(7)CLP+VAN group,CLP+VAN+MEL group(VAN,MEL combined post-treatment);using a series of experimental instruments and methods such as temperature,small animal ultrasound system,blood routine analyzer,blood biochemical analyzer,histopathological staining,q RT-PCR,Western Blot,transcriptome sequencing to clarify:(1)the protective effect of MEL pretreatment on septic myocardial injury;(2)the protective effect of MEL on septic myocardial injury by regulating AMPK signaling pathway and its mechanism;(3)the therapeutic potential of Mel postconditioning and combined with classical antibiotics on septic myocardial injury.Results:1.Compared with the CLP group,the survival rate of the MEL+CLP group was significantly increased(P<0.05),the sepsis score of the MEL+CLP group was significantly lower,and the rectal temperature was significantly increased(P<0.05);the blood routine indicators: WBC,LYM,MON,GRA,PLT was significantly increased,while RBC was significantly decreased(P<0.05);the blood biochemical indicators: LDH,CK,AST,BUN levels were significant decreased,while the level of ALB increased significantly(P<0.05);the cardiac function parameters: SV,CO,LVEDV,LVESV increased significantly,while LVPWd,LVPWs and HR decreased significantly(P<0.05);H&E staining showed that myocardial fiber breakage and myofibrillar interstitial edema was significantly reduced;Masson staining showed that the degree of myocardial fibrosis was reduced;IHC staining showed that the expressions of MPO,TNF-α,IL-6 and NOX2 were significantly reduced;DHE staining showed that the production of ROS in myocardial tissue was significantly reduced;q RT-PCR results showed that the m RNA expression levels of TNF-α,IL-6,IL-8,CXCL2,NLRP3,IL-1β and Caspase1 in myocardial tissue were significantly reduced(P<0.05).The above results prove that MEL pretreatment can improve the survival rate of septic mice,improve the sepsis score,rectal temperature,blood routine/blood biochemical indicators and cardiac function parameters,and reduce myocardial tissue structural damage,myocardial inflammation and oxidative stress.2.Compared with the CLP group,Western blot showed that the expression levels of Nrf2,NQO1 and HO-1 were increased in MEL+CLP group(P<0.05);the expression levels of endoplasmic reticulum stress-related proteins CHOP,GRP78 and ATF6 were increased,while the expression levels of p-PERK and p-PERK /PERK decreased(P<0.05);the expression levels of mitochondrial function-related proteins TFAM,UCP2,NRF1,COXIV and SDH5 were increased(P<0.05).The above results prove that MEL pretreatment affects the signal pathways related to inflammatory,oxidative stress,endoplasmic reticulum stress and mitochondrial dysfunction related signaling pathways in septic myocardial injury.3.Compared with the CLP group,the MEL+CLP group significantly reversed the increase in the expression levels of p-AMPK,p-AMPK/AMPK,p-ACC,p-ACC/ACC in the AMPK/ACC pathway and downstream molecules PPARγ(P<0.05),proving that the protective effect of MEL pretreatment on myocardial injury in septic mice may be related to AMPK pathway.4.Compared with the MEL+CLP group,the sepsis score of the MEL+CC+CLP group was more severe(P<0.05);the blood routine indicators LYM,MON,GRA and PLT were decreased,and the RBC level was increased(P<0.05);the blood biochemical indicators ALB were decreased(P<0.05);the cardiac function parameters SV,CO,LVEDV and LVESV were decreased,and HR was increased(P<0.05);p-AMPK,p-AMPK/AMPK,p-ACC and p-ACC/ACC in the AMPK/ACC pathway was down-regulated(P<0.05).The above results prove that CC can reverse the protective effect of MEL on septic myocardial injury by inhibiting AMPK activity.5.The results of transcriptome sequencing showed three different transcriptome patterns of sham,CLP and Mel+CLP group,and the samples of each group were well clustered(PCA analysis);In the three groups,these differential genes were divided into 8 different dynamic expression pattern,of which there were significant changes in 2 pattern groups:578 genes in model group 6 were up-regulated and then unchanged,306 genes in model group 1 were down regulated and then unchanged,and the expression of other models also showed different trends in this process(trend analysis).Compared with the CLP group,the Mel+CLP Group increased 52 genes and reduced 28 genes(volcano map and heat map analysis);the up-regulated differential genes were involved in the regulation of fructose-2,6-diphosphate metabolism,butyric acid metabolism,ketone synthesis and degradation,and the down-regulated differential genes are involved in processes such as glial cell migration and affect inflammatory bowel disease and hypertrophy(GO analysis and KEGG pathway analysis).The above results further prove that MEL pretreatment has a significant effect on the transcriptome of septic myocardial injury.6.Compared with the CLP group,the CLP+MEL group reversed the decrease in sepsis score and survival rate within 72 hours(P<0.05),but only ALB was significantly increased(P<0.05),other biochemical indicators,blood routine indicators and cardiac function parameters had no significant changes(P>0.05).The above results prove that MEL post-treatment has a certain therapeutic effect on septic myocardial injury.7.Compared with the CLP group,the CLP+AZI group and the CLP+VAN group can significantly reduce the sepsis score(P<0.05),significantly increase the survival rate within 72 hours(P<0.05),and improve the part blood routine indicators,blood biochemical indicators and cardiac function parameters(P<0.05).However,compared with the CLP+AZI group,the CLP+AZI+MEL group can only significantly reduce the sepsis score(P<0.05),and has no significant effect on blood biochemical indicators,blood routine indicators and cardiac function parameters(P>0.05);VAN group also showed similar results.The above results prove that MEL combined with AZI or VAN has a certain therapeutic effect on septic myocardial injury.Conclusions:In conclusion,the following conclusions can be drawn:(1)Mel pretreatment has obvious protective effect on septic myocardial injury;(2)Mel pretreatment may regulate AMPK signaling pathway in septic myocardial injury,and affect the related pathways of inflammation,oxidative stress,endoplasmic reticulum stress and mitochondrial dysfunction;(3)Mel post-treatment or combined with classical antibiotics has significant protective effect on septic myocardial injury It provides a theoretical basis for the treatment of myocardial injury.