Mechanism Of Hmgcs2 Regulating M2 Polarization Of Macrophages In The Repair Of Sepsis Induced Cardiomyopathy

Background:Sepsis usually rapidly leads to multiple organ failure,with the respiratory and cardiovascular systems most commonly affected.When combined with septic shock,the incidence of sepsis-induced cardiomyopathy is 18% to 40%,the mortality rate increases by 70% to 90%,and the global annual cost is increasing year by year and occupying a large amount of medical resources.Therefore,there is an urgent need to find effective strategies for sepsis-related myocardial injury.The systemic inflammatory response syndrome(SIRS)of sepsis,caused by infection,is characterized by cardiac dysfunction that is a result of a variety of biological processes,such as inflammation,oxidative stress damage,mitochondrial dysfunction,and apoptosis.Ketones are produced by the oxidation of non-esterified fatty acids in the liver,which is a form of energy supply for the human body,and the oxidation of fatty acids in the liver to form acetyl-Co A can be generated,and the oxidation can be continued to generate ketone bodies.3-hydroxy-3 methylglytaryl-Co A synthetase 2(Hmgcs2)is a rate-limiting enzyme in the ketone body production pathway which leads to the production of ketone bodies including β-hydroxybutyric acid.Many studies have confirmed that elevated ketone levels are a protective mechanism for the heart in failing hearts.Patients with severe heart failure have significantly reduced myocardial cell insulin sensitivity,and the oxidation of fatty acids and glucose is significantly reduced,while ketones have become the main energy supply.In addition to this,ketones also play a cardioprotective role in other ways.Ketone bodies have potentially beneficial effects in heart,which have potential cardioprotective effects in the case of experimental ischemia/reperfusion injury,possibly due to increased cardiac mitochondrial abundance or upregulation of key oxidative phosphorylation mediators.Immune inflammatory stability is the foundation of sepsis,and macrophages,a vital cell of the innate immune system,are highly diverse and adjustable.M1 type macrophages,capable of suppressing the infiltration of pathogenic bacteria by releasing a great deal of inflammatory mediators,are the primary source of sepsis in its early stages.In contrast,M2 type macrophages,which can reduce the release of inflammatory factors and perform anti-inflammatory,angiogenesis,and tissue repair functions,are the main contributors to the late stage of sepsis.PI3K/AKT,is expressed in a variety of metabolic pathways throughout the human body and performs a range of biological functions.PI3K/AKT signal transduction works through mammalian/molecular target of rapamycin(m TOR)complex to control the response of innate immune cells(including macrophages).PI3K/AKT/m TOR signal regulates cell metabolism,translation,cytokine response,macrophage polarization and migration.In the study of sepsis,PI3K/AKT can regulate the polarization phenotype of macrophages,thus affecting the expression of inflammatory factors.However,the regulatory effect of different macrophage phenotypes on cardiomyopathy in septic cardiomyopathy has not been confirmed.We have established the relationship between Hmgcs2 and sepsis from the perspective of bioinformatics through the screening of differential genes based on time series,and determined that Hmgcs2 is the key gene to regulate cardiomyopathy.This research seeks to investigate the influence of Hmgcs2 on myocardial injury caused by sepsis,to delve deeper into the particular mechanism of Hmgcs2/PI3K/AKT’s involvement in sepsisrelated cardiomyopathy,and to investigate the correlation between macrophage polarization and this pathway in this process.It aims to improve the understanding of the underlying pathogenesis of sepsis-induced cardiomyopathy and provide theoretical basis and potential targets for clinical treatment and new drug research and development.Methods:1.Through the GEO public data mining sepsis related cardiomyopathy transcriptome data set(GSE171546),we explored the time-related differential m RNA of cecal ligation and puncture(CLP)mice for the m RNA gene expression profile of model mice based on time sequence.To construct a CLP-induced sepsis mouse model and used the targeted inhibitor of Hmgcs2 ASO for intervention.The pathological damage of myocardial tissue was detected by H&E method.Echocardiography was employed to assess the cardiac activity of mice by analyzing the left ventricular ejection fraction(LVEF)and left ventricular shortening fraction(LVSF).ELISA revealed the presence of cardiac troponin I(c Tn I),creatine kinase isoenzyme(CK-MB),lactate dehydrogenase(LDH)and other indicators of myocardial injury,and further measured the release of inflammatory factors IL-1β,IL-6,IL-10 and TNF-α in the mice serum.2.We used bioinformatics to screen the key pathways in the process of sepsis by using gene ontology(GO)analysis,KEGG enrichment analysis and more in-depth GSEA analysis for the previous part of differential genes(DEGs).In order to further study the regulatory effect of Hmgcs2 on macrophages,we conducted cell experiments in vitro.We explored the regulatory effect of Hmgcs2 on macrophage polarization through Src/PI3K/AKT signaling pathway by constructing a cell model of Hmgcs2 overexpression and Src inhibitor PP1 administration.The Western blotting technique was employed to ascertain the alterations in protein concentration of the Hmgcs2/Src/PI3K/AKT signal pathway in relation to macrophage polarization.Flow cytometry was employed to gauge the polarization of macrophages in distinct groups.QRT-PCR revealed the transcription of inflammatory factors TNF-α,IFN-β,IL-6 and IL-10 in macrophages of various groups,while ELISA revealed their protein levels.Immunofluorescence technique was employed to further detect the infiltration of M2 macrophages into the heart tissue of CLP model mice.3.Finally,in order to verify the regulatory effect of macrophages with different phenotypes on sepsis cardiomyopathy,we exposed human cardiac cell line AC16 to macrophages conditioned medium with different phenotypes for indirect co-culture.Flow cytometry was employed to ascertain the influence of macrophages-conditioned medium co-culture on cardiac cell activity,and CCK8 was utilized to measure the effect of macrophages on cardiac cell apoptosis.Results:1.Based on the summary of differential genes,ENSMUSG00000027875(Hmgcs2)was identified as the key differential gene.After CLP surgery,it can be seen that the mouse heart is marked by H&E staining of mouse heart sections,with significant edema of cardiomyocytes,vacuoles,cell deformation,necrosis,and exfoliation,and diffuse inflammatory cell infiltration,suggesting myocardial injury and diffuse inflammatory cell infiltration,suggesting myocardial injury,which was further aggravated after the administration of the Hmgcs2 targeted inhibitor ASO.Echocardiography revealed a significant decrease in the left ventricular ejection fraction(LVEF)and LV systolic function(FS)of CLP mice’s hearts,suggesting a decrease in cardiac function,which was further diminished after the application of ASO.The ELISA mice showed a marked rise in serum concentrations of IL-1β,IL-6,IL-10 and TNF-α following CLP surgery,while the inhibition of Hmgcs2 caused a more pronounced rise in L-1β,IL-6,and TNF-α,and the levels of IL-10 decreased in comparison to those in the CLP group.2.In vitro cell experiments,the expression of Hmgcs2 and Src proteins increased in cells overexpressing Hmgcs2,and the phosphorylation levels of PI3 K and AKT increased.The inhibition of Src decreased the expression of Src and the phosphorylation level of PI3 K and AKT,yet had little effect on Hmgcs2,thereby demonstrating the regulatory effect of Hmgcs2 on Src/PI3K/AKT in macrophages.We measured the polarization of macrophages in different groups by flow cytometry,and the results showed that overexpression of Hmgcs2 promoted the polarization of macrophages M1 to M2,and the inhibition of Src reversed this phenomenon,and finally proved that Hmgcs2 promoted the polarization of macrophages M1 to M2 through the Src/PI3K/AKT pathway.In terms of transcriptional activity,the expression of m RNA of inflammatory factors including IL-10,IFN-β,IL-6 and TNF-α of macrophages induced by LPS was increased,while overexpression of Hmgcs2 decreased the expression of m RNA of LPS-induced macrophages TNF-α,IFN-β and IL-6,further increasing the transcriptional activity of IL-10.Src inhibitors reversed the effect of Hmgcs2,indicating that Hmgcs2 can reduce the release of lps-induced M1 macrophage inflammatory factors and increase the release of anti-inflammatory factors.3.The CCK-8 results revealed that AC16 cell activity diminished after being exposed to LPS-induced macrophage-conditioned medium,whereas cardiac myocytes exposed to LPS+Hmgcs2 group cell-conditioned medium regained their activity.Flow cytometry confirmed that AC16 cells exposed to LPS-induced macrophage conditioned medium had a significantly higher apoptosis level,whereas myocardial cells exposed to LPS+Hmgcs2 group had a significantly lower apoptosis level.The results showed that Hmgcs2 could restore the proliferation level of cardiomyocytes and reduce the level of cardiomyocyte apoptosis by inducing the polarization of macrophage M1 to M2.Conclusion:1.Hmgcs2 is one of key genes that regulating the myocardial injury in CLP mice.Inhibition of Hmgcs2 can further aggravate the release of multiple inflammatory cytokines and myocardial function damage in CLP mice.2.Hmgcs2 promotes the polarization of macrophage M1 to M2 through Src/PI3K/AKT pathway.Hmgcs2 can reduce multiple inflammatory cytokines from LPS-induced M1 macrophages and increase the release of some anti-inflammatory factors.3.Hmgcs2 reduces cardiomyocyte apoptosis by inducing macrophages to M2 polarization.