The Mechanism Study Of Sinomenine Alleviates Sepsis-induced Myocardial Dysfunction By Regulating SOCS3

Part Ⅰ.IntroductionSepsis is a life-threatening disease resulting from a dysregulated host response to infection.Sepsis-induced myocardial dysfunction(SIMD)is a common complication of sepsis,characterized by decreased myocardial contractility,impaired ventricular diastole,and decreased cardiac output.These changes lead to hypotension,decreased tissue perfusion,and ultimately organ failure.SIMD is reversible in the early stage,so timely identification and management of myocardial injury caused by sepsis is very important to improve patient prognosis and reduce the risk of death.Several biomarkers are currently associated with myocardial injury in sepsis,including creatine kinase isoenzyme(CK-MB),troponin I,B-type natriuretic peptide,and C-reactive protein,etc.,but lack specificity.Therefore,finding new biomarkers is of great significance for early,timely,and accurate diagnosis of SIMD.SIMD occurs due to a complex interplay of multiple mechanisms,including inflammation,oxidative stress,and cardiomyocyte apoptosis.These mechanisms form a vicious cycle that eventually leads to myocardial damage.Therefore,SIMD can be treated by reducing inflammation,oxidative stress,and cardiomyocyte apoptosis.High-throughput sequencing(HTS)technology has revolutionized the field of genomics by enabling the rapid and cost-effective sequencing of large amounts of DNA or RNA.In recent years,HTS has been increasingly used in the research of various diseases.Bioinformatics analysis plays a crucial role in the analysis of HTS data,as the large amount of sequence information generated by these techniques requires complex computational methods to interpret.The integration of HTS and bioinformatics analysis can provide insight into the underlying molecular mechanisms involved in sepsis-induced myocardial injury,improve our understanding of SIMD at the gene level,and help identify potential therapeutic targets.Sinomenine is a natural alkaloid extracted from the traditional Chinese medicine Sinomenium acutum.Current research has found that it has anti-inflammatory effects in various inflammatory diseases.Sinomenine can modulate various signaling pathways related to inflammation and oxidative stress.Previous studies have shown that sinomenine can reduce myocardial ischemia-reperfusion injury by preventing oxidative stress,inflammation,and cardiomyocyte apoptosis while reducing arrhythmia caused by myocardial ischemia-reperfusion injury.However,whether sinomenine has a protective effect on SIMD is still unknown.Studies have shown that sinomenine has anti-inflammatory,immunomodulatory,and antioxidant properties that may help alleviate pathophysiological changes associated with SIMD.Sinomenine has potential as a SIMD therapeutic,but further studies are needed to fully elucidate its action mechanism.The suppressor of cytokine signaling 3(SOCS3)plays a critical role in the regulation of inflammation and immune responses.SOCS3 regulates inflammatory responses by inhibiting signal transduction and activation of STAT signals and also regulates apoptosis by regulating the expression of pro-apoptotic and anti-apoptotic proteins.SOCS3 has been shown to regulate the levels of pro-inflammatory cytokines during sepsis.Studies have reported that IL-33 inhibits the IL-17 pathway by activating SOCS3,plays a negative regulatory role in the progression of sepsis,and reduces organ damage caused by sepsis.On the other hand,inhibition of SOCS3 can increase the levels of pro-inflammatory cytokines and worsen sepsis outcomes.These findings suggest that targeting SOCS3 may represent a novel therapeutic strategy to prevent and treat sepsis-induced myocardial injury.Further studies are needed to fully elucidate the mechanisms underlying SOCS3’s role in SIMD and to explore its potential as a therapeutic target for sepsis-related cardiovascular complications.Part Ⅱ.Bioinformatics analysis identifies hub differentially expressed genes in SIMD Purpose:1.Use HTS data for bioinformatics analysis to screen and identify hub differentially expressed genes(DEGs)of SIMD at the gene level.2.Establish SIMD models(including mouse cardiomyocyte HL-1 and human cardiomyocyte AC16),and verify the expression of SIMD hub DEGs in the cell model.Method:1.Identification of DEGs in SIMD by comprehensive bioinformatics analysis of SIMD-related microarray datasets GSE53007(composed of mouse heart tissue samples)and GSE79962(composed of human heart tissue samples)downloaded from the GEO database.2.In-depth exploration of DEGs using bioinformatics analysis,including GO and KEGG pathway enrichment analysis,PPI network construction,identification of hub DEGs,etc.3.After determining the top ten hub DEGs,the cardiomyocyte SIMD model was constructed by treating HL-1 cells and AC16 cells with LPS(1μg/ml),and q PCR experiments was performed to check the effect of the top ten hub DEGs in the cardiomyocyte SIMD model.Result:1.After bioinformatics analysis,a total of 423 DEGs were identified from GSE53007,including 294 up-regulated genes and 129 down-regulated genes.A total of 121 up-regulated genes and 70 down-regulated genes were identified from GSE79962.In both datasets,22 upregulated genes and 1 downregulated gene overlap.2.After an in-depth exploration of DEGs,the top ten hub DEGs of SOCS3,STAT3,CCL2,IL1R2,TIMP1,JUNB,S100A9,OSMR,ZFP36,and HAMP were identified.3.After the expression was verified by q PCR experiments,it was found that the expression of nine hub DEGs,SOCS3,STAT3,CCL2,IL1R2,JUNB,S100A9,OSMR,ZFP36,and HAMP,was significantly increased in the established cardiomyocyte SIMD model(P<0.05).Conclusion:After bioinformatics analysis and q PCR experiment verification,it was confirmed that the expressions of hub differentially expressed genes SOCS3,STAT3,CCL2,IL1R2,JUNB,S100A9,OSMR,ZFP36,and HAMP were significantly increased in the established cardiomyocyte SIMD model,and these genes may play an important role in SIMD.Part Ⅲ.Protective effect of sinomenine on SIMD Purpose:1.To explore whether sinomenine has a protective effect on SIMD mice.2.To observe the changes of hub differentially expressed gene SOCS3 after using sinomenine in SIMD mice.Method:1.Use C57BL/6J male mice to establish a mouse SIMD model by cecal ligation and puncture.The mice were randomly divided into 3 groups.Sham operation group(Sham group,8 mice): only open and close the abdomen,without other operations.Cecum ligation and puncture group(CLP group,8 mice): the mouse SIMD model was established by cecal ligation and puncture,and normal saline was injected 6hours after operation.Sinomenine treatment group(SIN group,8 mice): A mouse SIMD model was established by cecal ligation and puncture,and sinomenine was injected 6 hours after operation.Mice were euthanized 24 h after surgery.2.Before the operation,6 hours after the operation,and 24 hours after the operation,a Doppler ultrasound was performed to measure the left ventricular diastolic diameter,left ventricular systolic diameter,left ventricular ejection fraction and left ventricular fractional shortening to evaluate the cardiac function of the mice.3.Measure the levels of inflammatory cytokines(TNF-α,IL-1β,and IL-6)and CK-MB in mouse serum by enzyme-linked immunosorbent assay(Enzyme-Linked Immunosorbent Assay,ELISA),and determine the levels of myocardial SOD and MDA levels.4.HE staining to detect pathological changes in myocardial tissue.5.TUNEL staining was used to detect cardiomyocyte apoptosis.6.Detect the expression of SOCS3 gene and protein by q PCR and immunohistochemistry.7.The protein expression levels of inflammatory pathway-related proteins TLR4 and p-NF-κB P65,hub differentially expressed genes SOCS3 protein,and apoptosis-related proteins Caspase3,Bcl-2,and Bax were detected by WB.Result:1.The results of the Doppler ultrasound showed that at 6 hours after the operation,the cardiac function of the mice in the CLP group and the SIN group was significantly lower than that in the Sham group.It shows that 6h after CLP,the mice have sepsis-induced myocardial injury.However,after treatment with sinomenine,the cardiac function of mice in the SIN group was partially restored.2.Compared with the Sham group,the inflammatory factors in the CLP group and SIN group were increased.Compared with the CLP group,the levels of TNF-α,IL-1β,IL-6,and CK-MB in the serum of the mice in the SIN group were significantly decreased(P<0.05),indicating that sinomenine has an anti-inflammatory effect.3.Compared with the CLP group,the SOD level and the MDA level in the myocardium of the mice in the SIN group were increased(P<0.05),indicating that sinomenine has an anti-oxidative stress effect.4.The results of HE staining showed that compared with the Sham group,the CLP group had myocardial fiber rupture,myocardial striations blurred,interstitial edema,and inflammatory infiltration.After treatment with sinomenine,the arrangement of myocardial cells was more disordered,the structure was clearer,and there was slight bleeding.Although there was still a small amount of rupture of myocardial fibers in the SIN group,the injury of myocardial tissue cells was obviously improved.5.TUNEL results showed that the CLP group had obvious green fluorescence and increased cell apoptosis.After sinomenine administration,compared with the CLP group,the green fluorescence and cell apoptosis in the SIN group were significantly reduced.It shows that sinomenine can reduce cardiomyocyte apoptosis in SIMD and has an anti-apoptotic effect.6.The results of q PCR and immunohistochemistry suggested that the use of sinomenine in the mouse SIMD model would cause the up-regulation of the key differentially expressed gene SOCS3(P<0.05).7.WB results showed that compared with the Sham group,the protein expressions of TLR4,p-NF-κB P65,SOCS3,Bax,and Caspase3 in the CLP group were significantly increased(P<0.05),and the protein expression of Bcl-2 was significantly decreased(P<0.05).Compared with the CLP group,the expressions of SOCS3 and Bcl-2 proteins in the SIN group were significantly increased(P<0.05),while the expressions of TLR4,p-NF-κB P65,Bax and Caspase3 were significantly decreased(P<0.05).Conclusion:1.Sinomenine can up-regulate the expression of hub differentially expressed gene SOCS3 in SIMD mice.2.Sinomenine can improve the cardiac function of SIMD mice,reduce the release of inflammatory factors,reduce the level of oxidative stress in cardiac tissue,and alleviate cardiomyocyte apoptosis.Sinomenine has a protective effect on SIMD.Part Ⅳ.The protective effect of sinomenine on SIMD by regulating SOCS3Purpose:1.Construction of Si RNA-mediated SOCS3 gene silencing HL-1 mouse cardiomyocytes.2.Using the HL-1 SIMD model to confirm that sinomenine has a protective effect on sepsis myocardial injury by regulating SOCS3.Method:1.Culture HL-1 cardiomyocytes.When the cell density reaches 70%,prepare for transfection;48h later,detect the expression level of SOCS3 by WB.Silenced SOCS3 was constructed for subsequent rescue experiments.2.Divide HL-1 cardiomyocytes into 6 groups for subsequent experiments.(1)Control group(Control group): HL-1 cardiomyocytes were cultured normally without special treatment.(2)Sepsis-induced myocardial injury group(LPS group): add LPS(1μg/ml)and incubate for 24 hours.(3)Sinomenine control group(SIN group): add sinomenine(0.2 m M)and incubate for 24 hours.(4)Sinomenine treatment of sepsis-induced myocardial injury group(LPS+SIN group): add sinomenine(0.2 m M)and LPS(1μg/ml)and incubate for 24 hours.(5)Sinomenine treatment of sepsis-induced myocardial injury plus interference empty group(LPS+SIN+NC group): add sinomenine(0.2 m M)and LPS(1μg/ml)to cells 48 hours after Si RNA-NC transfection and incubate for 24 hours.(6)Sinomenine treatment of sepsis-induced myocardial injury plus SOCS3 interference group(LPS+SIN+Si group): add sinomenine(0.2 m M)and LPS(1μg/ml)cells 48 hours after si RNA-SOCS3 transfection and incubate for 24 hours.3.The levels of inflammatory cytokines(TNF-α,IL-1β,and IL-6)and CK-MB in the supernatant of HL-1 mouse cardiomyocytes were measured by ELISA.4.Measure the levels of SOD and MDA in HL-1 cardiomyocytes by ELISA.5.The apoptosis rate of cardiomyocytes was detected by Flow Cytometry.6.qPCR detection of hub differentially expressed gene SOCS3 gene expression.7.The protein expression levels of inflammatory pathway-related proteins TLR4 and p-NF-κB P65,hub differentially expressed genes SOCS3 protein,and apoptosis-related proteins Caspase3,Bcl-2,and Bax were detected by WB.Result:1.The expression of SOCS3 in the Si-1062 group was significantly decreased,the interference was effective(P<0.05),and Si-1062 was selected for subsequent experiments.2.Inflammation-related indicators:(1)ELISA results showed that compared with the Control group,the levels of inflammatory factors(TNF-α,IL-1β,and IL-6)and CK-MB in the cell supernatant of the SIN group did not change(P>0.05).Compared with the Control group,the LPS group had higher levels of these indexes(P<0.05).Compared with the LPS group,the levels of these indexes decreased in the LPS+SIN group(P<0.05).The results of the rescue experiment showed that after silencing SOCS3,the levels of these indicators in the LPS+SIN+Si group increased again compared with the LPS+SIN group(P<0.05),and there was no difference with the LPS group(P>0.05).(2)WB results showed that compared with the Control group,there was no difference in the expression of TLR4 and p-NF-κB P65 in the SIN group(P>0.05).Compared with the Control group,the expression of TLR4 and p-NF-κB P65 in the LPS group increased(P<0.05).Compared with the LPS group,the expressions of TLR4 and p-NF-κB P65 in the LPS+SIN group decreased(P<0.05).After silencing SOCS3,compared with the LPS+SIN group,the expression of TLR4 and p-NF-κB P65 in the LPS+SIN+Si group increased(P<0.05),and there was no difference with the LPS group(P>0.05).The results show that under physiological conditions,sinomenine will not cause changes in the inflammation-related indicators of normal HL-1 cardiomyocytes.In SIMD,sinomenine can exert an anti-inflammatory effect to protect cardiomyocytes.Silencing SOCS3 can reverse the anti-inflammatory effect of sinomenine in SIMD,indicating that sinomenine plays an anti-inflammatory effect in SIMD by regulating SOCS3.3.Oxidative stress related indicators: ELISA results showed that,compared with the Control group,the SOD and MDA contents in the SIN group had no change(P>0.05).Compared with the Control group,the SOD content in the cardiomyocytes of the LPS group decreased,and the MDA content increased(P<0.05).Compared with the LPS group,the content of SOD in cardiomyocytes of the LPS+SIN group increased,and the content of MDA decreased(P<0.05).Compared with the LPS+SIN group,the LPS+SIN+Si group,SOD content in cardiomyocytes decreased,MDA content increased(P<0.05),and there was no difference with the LPS group(P>0.05).The results show that under physiological conditions,sinomenine will not cause changes in oxidative stress-related indicators of normal HL-1 cardiomyocytes.In SIMD,sinomenine can exert an anti-oxidative stress effect and protect cardiomyocytes.Silencing SOCS3 can reverse the anti-oxidative stress effect of sinomenine in SIMD,indicating that sinomenine plays an anti-oxidative stress effect in SIMD by regulating SOCS3.4.Apoptosis-related indicators.(1)After flow cytometry detection,it was found that there was no difference in the apoptosis rate between the Control group and the SIN group(P>0.05).Compared with the Control group,the apoptosis rate in the LPS group was significantly higher(P<0.05).Compared with the LPS group,the apoptosis rate of the LPS+SIN group decreased significantly(P<0.05).Compared with the LPS+SIN group,the apoptosis rate of the LPS+SIN+Si group was significantly increased(P<0.05),and there was no difference with the LPS group(P>0.05).(2)WB results showed that,compared with the Control group,there was no difference in the expression of Caspase-3,Bcl-2,and Bax proteins in the SIN group(P>0.05).Compared with the Control group,the expression levels of Caspase-3 and Bax proteins in the LPS group were significantly increased,and the expression of Bcl-2 protein was significantly decreased(P<0.05).Compared with the LPS group,the LPS+SIN group had significantly lower Caspase-3 and Bax protein expression levels,and a significantly higher Bcl-2 protein expression level(P<0.05).Compared with the LPS+SIN group,the protein expression levels of Caspase-3 and Bax in the LPS+SIN+Si group were significantly increased,and the protein expression of Bcl-2was significantly decreased(P<0.05),and the expression levels of these three proteins had no difference with LPS group(P>0.05).The results show that under physiological conditions,sinomenine will not cause changes in the apoptosis-related indicators of normal HL-1 cardiomyocytes.In SIMD,sinomenine can play an anti-apoptotic role in cardiomyocytes.Silencing SOCS3 can reverse the anti-apoptotic effect of sinomenine in SIMD,indicating that the anti-apoptotic effect of sinomenine in SIMD depends on SOCS3.5.The results of the hub differentially expressed gene SOCS3 expression assay:Both q PCR and WB results showed that compared with the Control group,the expression levels of SOCS3 m RNA and protein in the LPS group were increased(P<0.05).The m RNA and protein expression levels of SOCS3 in the SIN group were not different from those in the Control group(P>0.05),indicating that the use of sinomenine in normal cardiomyocytes would not cause changes in SOCS3.Compared with the LPS group,the m RNA and protein expression levels of SOCS3 in the LPS+SIN group were increased(P<0.05).The results indicated that under physiological conditions,sinomenine would not stimulate the expression of SOCS3 in normal HL-1 cardiomyocytes.The expression of SOCS3 can be upregulated by sinomenine in SIMD.Conclusion:1.The use of sinomenine under physiological conditions will not cause inflammation,oxidative stress,and apoptosis in HL-1 cardiomyocytes,nor will it stimulate the upregulation of SOCS3 expression.2.After silencing SOCS3,the protective effect of sinomenine on SIMD disappeared.Sinomenine plays a protective role against SIMD by attenuating inflammation,oxidative stress,and apoptosis in sepsis-induced myocardial injury by upregulating the expression of SOCS3.Part Ⅴ.Research Conclusions1.After bioinformatics analysis and q PCR experiment verification,it was determined that the expression of hub differentially expressed genes SOCS3,STAT3,CCL2,IL1R2,JUNB,S100A9,OSMR,ZFP36,and HAMP was significantly increased in SIMD.2.Sinomenine can up-regulate the expression of hub differentially expressed gene SOCS3 in SIMD mice.3.Sinomenine can improve the cardiac function of SIMD mice,reduce the release of inflammatory factors,reduce the level of oxidative stress in cardiac tissue,and alleviate cardiomyocyte apoptosis.Sinomenine has a protective effect on SIMD.4.The use of sinomenine under physiological conditions will not cause inflammation,oxidative stress,and apoptosis in HL-1 cardiomyocytes,nor will it stimulate the upregulation of SOCS3 expression.5.After silencing SOCS3,the protective effect of sinomenine on SIMD disappeared.Sinomenine plays a protective role against SIMD by attenuating inflammation,oxidative stress,and apoptosis in sepsis-induced myocardial injury by upregulating the expression of SOCS3.