MiR-214-3p Over-expression Prevents Sepsis-induced Myocardial Dysfunction Through The HDAC9 Pathway

Sepsis refers to a life-threatening multiple organ dysfunction caused by the host’s dysregulation of response to bacterial,fungal,or viral infections.The heart is one of the most vulnerable organs in sepsis,often manifested clinically as impaired myocardial systolic and diastolic function,and degeneration and necrosis of myocardial cells.According to statistics,40%-50%of sepsis patients experience myocardial damage,manifested as myocardial cell degeneration and necrosis,impaired myocardial systolic and diastolic function,etc.Severe cases may experience cardiac exhaustion,shock,arrhythmia,etc.Sepsis induced myocardial dysfunction(SIMD)is one of the primary cause of sepsis related deaths.SIMD is a serious complication of sepsis and is directly related to the high mortality rate of sepsis.Its main manifestations include left ventricular dilation,inhibition of ventricular systolic and diastolic function,leading to a decrease in patient ejection fraction and shock.Research has found that multiple mechanisms are involved in regulating the occurrence of myocardial dysfunction in sepsis,including cell apoptosis,abnormal release of inflammatory mediators,mitochondrial dysfunction,oxidative stress,and abnormal calcium regulation.miRNAs,as endogenous non coding small RNAs,are widely present in sweat,urine,or blood.They exert an inhibitory and regulatory effect on post transcriptional gene expression by promoting the degradation of target mRNA,and control the occurrence and development of sepsis through immune suppression,inflammatory response,immune cell differentiation,or apoptosis.Research has found that up-regulation of miR-214-3p expression can reduce inflammatory response,reduce free radical damage,inhibit cell apoptosis and autophagy,and improve cardiac function in sepsis induced myocardial injury.However,the role of miR-214-3p in sepsis induced myocardial dysfunction is not yet clear,and further research is necessary.HDAC9 is located on human chromosome 7 and is highly expressed in the heart.It has been found that HDAC9 can mediate the inflammatory damage by oxidized low density lipoprotein(oxLDL)to cause atherosclerosis by regulating the phosphorylation level of P38-MAPK.Based on the cellular protection,inhibition of apoptosis,and inhibition of inflammatory cytokines release of HDAC9 in ischemic injury,we speculate that HDAC9 may have a similar effect in sepsis induced myocardial dysfunction.Combined with the myocardial protective effect of miR-214-3p in sepsis induced myocardial dysfunction,we hypothesize that miR-214-3p can improve sepsis induced myocardial dysfunction and reduce cell apoptosis and inflammatory cytokines response by regulating the expression of HDAC9.The website prediction indicated that the 3’UTR of HDAC9 has a target binding site of miR-214-3p.We observed the effects of miR-214-3p and HDAC9 on sepsis induced myocardial dysfunction at the levels of in vitro cells,in vivo animal models,and clinical samples,and explored their potential molecular mechanisms.This study includes of three parts.Part Ⅰ The study of miR-214-3p and HDAC9 in LPS induced H9C2 myocardial cells injuryObjective:1.To observe the expression of miR-214-3p and HDAC9 in H9C2 myocardial cells induced by LPS.2.By knocking down HDAC9,to observe the effect on the injury of H9C2 myocardial cells induced by LPS.3.Demonstrate the relationship between miR-214-3p and HDAC9.Methods:1.Purchase rat H9C2 myocardial cells.With different concentrations of LPS(0,0.1,0.2,0.5,1,2μg/ml)to treat the myocardial cells.After 24h,Real-time PCR was used to detect the expression of HDAC9 mRNA and miR-214-3p.Experimental groups:0,0.1,0.2,0.5,1,2μg/ml group.The H9C2 myocardial cells were induced by optimal concentration of LPS for different times(0,6,12,24h),and then the Real-time PCR was used to detect the expression of HDAC9 mRNA and miR-214-3p.Experimental groups:0,6,12,24h group.2.Two siRNAs of HDAC9 and control siRNA(siNC)were designed and purchased and transfected into H9C2 myocardial cells respectively.After 48h,H9C2 myocardial cells were induced by LPS at the optimal concentration.After 24 hours,Real-time PCR was used to detect the expression of HDAC9 mRNA and miR-214-3p,and Western blot was used to detect the expression of HDAC9 protein in cells;The levels of cardiac troponin(cTnI),myocardial kinase isozyme(CK-MB)and lactate dehydrogenase(LDH)in the supernatant were detected by the kit;The level of TNF-α,IL-6 and IL-1β in supernatant of H9C2 myocardial cells were detected by ELISA;Detection of the expressions of IκBα,p-IκBα,p-p65 and p65 by Western blot;The number of p65-(+)cells in the nucleus was calculated by immunofluorescence staining.CCK-8 was used to detect cell viability;Apoptosis was detected by flow cytometry;The expressions of cleaved Caspase-3,Bcl-2 and Bax were detected by Western blot.Experimental groups:Control,LPS,LPS+siNC,LPS+siHDAC9-1,LPS+siHDAC9-2 group.3.Purchase rno-miR-214-3p mimics and control NC mimics.H9C2 myocardial cells were transfected with NC mimics or miR-214-3p mimics.After 48h,H9C2 myocardial cells were treated with optimal concentration of LPS.After 24h,the Real-time PCR was used to detect the expression of miR-214-3p and HDAC9 mRNA.The expression of HDAC9 protein was detected by Western blot.Experimental groups:Control,LPS,LPS+NC mimics,LPS+miR-214-3p mimics group.The wild type HDAC9-3’UTR luciferase report vector(HDAC9-wt)and mutant vector(HDAC9-mut)containing the miR-214-3p binding site were constructed and transfected with NC mimics or miR-214-3p mimics into H9C2 myocardial cells,respectively.Double Luciferase experiments verified the targeting relationship between miR-214-3p and HDAC9.Experimental groups:HDAC9-wt+NC mimics,HDAC9-wt+miR-214-3p mimics,HDAC9-mut+NC mimics,HDAC9-mut+miR-214-3p mimics group.Results:1 In LPS induced H9C2 myocardial cells injury,HDAC9 expression was up regulated and miR-214-3p expression was down regulated.In H9C2 myocardial cells injury induced by different concentrations of LPS,with the increased of LPS concentration,the expression of HDAC9 was gradually up regulated,but the miR-214-3p was gradually down regulated.After treating H9C2 myocardial cells with 1μg/ml LPS,the expression of miR-214-3p gradually decreased and the expression of HDAC9 gradually increased with the prolongation of time point.2 The effect of knocking down HDAC9 on LPS induced injury in H9C2 myocardial cells2.1 In LPS induced H9C2 myocardial cells injury,the HDAC9 mRNA level was significantly increased in LPS group compared to control group,and the difference was statistically significant(P<0.001).Compared with LPS+siNC group,the expression of HDAC9 mRNA was significantly down regulated in LPS+siHDAC9-1 and LPS+siHDAC9-2 group,and the difference was statistically significant(P<0.001).The protein expression trend of HDAC9 is consistent with that of HDAC9 mRNA.The expression of miR-214-3p was significantly increased in the control group and decreased in the other four groups,the difference was statistically significant(P<0.001).2.2 In LPS induced H9C2 myocardial cells injury,the LPS group showed significantly higher expression of myocardial injury markers of cTnI,CK-MB,and LDH compared to control group,with significant statistical significance(P<0.001).The LPS+siHDAC9-1 and LPS+siHDAC9-2 groups showed significant lower expression of myocardial injury markers of cTnI,CK-MB,and LDH compared to the LPS+siNC group,with significant statistical significance(P<0.01 or P<0.001).2.3 In LPS induced H9C2 myocardial cells injury,LPS group showed an obvious decrease in cell viability compared to control group,with a significant difference(P<0.01).The LPS+siHDAC9-1 and LPS+siHDAC9-2 groups showed a significant increase in cell viability compared to LPS+siNC group,with a significant difference(P<0.01).2.4 In LPS induced H9C2 myocardial cells injury,the LPS group showed significantly higher expression of cell apoptosis,cleaved caspase-3 and Bax compared to control group,while the expression of Bcl-2 decreased significantly,with a statistically significant difference(P<0.001).Compared to LPS+siNC group,the expression of cell apoptosis,cleaved caspase-3 in LPS+siHDAC9-1 and LPS+siHDAC9-2 groups were significantly decreased,while the expression of Bcl-2 was significantly increased,with a statistically significant difference(P<0.001).The expression level of the Bax was significantly decreased,with a statistically significant difference(P<0.05 or P<0.01).2.5 In LPS induced H9C2 myocardial cells injury,compared to control group,the LPS group showed increased levels of inflammatory cytokines of TNF-α.IL-6 and IL-1 p,and the difference was statistically significant(P<0.001).Compared to LPS+siNC group,in LPS+siHDAC9-1 and LPS+siHDAC9-2 group,TNF-α and IL-1β were down regulated with statistically significant differences(P<0.05 or P<0.01),IL-6 was down regulated with statistically significant differences(P<0.01 or P<0.001).2.6 In LPS induced H9C2 myocardial cells injury,compared to the control group,the p-IκBα/IκBα and p-p65/P65 in LPS group were significantly increased,and the proportion of p65-(+)nucleus was significantly increased,with significant statistical significance(P<0.001).Compared to LPS+siNC group,the p-IκBα/IκBα and p-p65/P65 in LPS+siHDAC9-1 and LPS+siHDAC9-2 group were significantly decreased,the proportion of p65-(+)nucleus was significantly decreased,and the difference was significant(P<0.01 or P<0.001).3 H9C2 myocardial cells were transfected with rno miR-214-3p mimics and their control NC mimics.After 48 hours,H9C2 myocardial cells were treated with 1μg/ml concentration of LPS.The expression of the miR-214-3p in LPS group was obviously decreased compared to control group,and the difference was obviously significant(P<0.001).The expression levels of miR-214-3p in the LPS+miR-214-3p mimics group was obviously higher than that of the LPS+NC mimics group,and the difference was obviously significant(P<0.001).Compared to control group,the HDAC9 mRNA expression level in LPS group was significantly increased,and the difference was significant(P<0.001).The HDAC9 mRNA expression level in LPS+miR-214-3p mimics group was significantly decreased compared to LPS+NC mimics group,and the difference was obviously significant(P<0.001).The changes in HDAC9 protein levels were consistent with the trend of changes in HDAC9 mRNA levels.The results of double luciferase experiment showed that miR-214-3p could target and regulate the expression of HDAC9.Conclusions:1.In LPS induced H9C2 myocardial cells injury,the expression of miR-214-3p decreased and the expression of HDAC9 increased.2.In LPS induced H9C2 myocardial cell injury,blocking the expression of HDAC9 resulted in a decrease in myocardial cell injury markers,increased vitality,reduced apoptosis,and decreased levels of inflammatory factors,the NF-κB signal pathway was inhibited.3.In LPS induced H9C2 myocardial cells injury,miR-214-3p could target and regulate the expression of HDAC9.Part Ⅱ The expression of miR-214-3p and HDAC9 in LPS induced myocardial injury in septic ratsObjective:Using LPS to establish a septic rat model,to observe the expression of miR-214-3p and HDAC9 in septic rats and their effects on cardiac function and myocardial tissues.To establish a LPS induced myocardial injury rat model with HDAC9 silencing,and observe the effects of HDAC9 silencing on cardiac function,myocardial tissue injury markers,and inflammatory cytokines.Methods:1.Male Sprague Dawley rats(220-250g)were randomly divided into Sham group and LPS group,6 rats in each group.LPS group rats were intraperitoneally injected with 10 mg/kg LPS;Sham group was given equal volume sterile phosphate buffered saline(PBS).After 6 hours,the rats were anesthetized and cardiac ultrasound was performed to examine the cardiac function,included left ventricular ejection fraction、fractional shortening、left ventricular internal diameter end diastole and left ventricular internal diameter end systole.Then,the rats were euthanized and left ventricular myocardial tissue was collected.The morphological changes of myocardium were observed by H&E staining.TUNEL staining method was used to observe myocardial apoptosis.IF staining method was used to observe the expression of HDAC9 in myocardial tissue.The expression of HDAC9 protein in myocardial tissue was detected by Western blot.Real-time PCR was used to detect the expression of HDAC9 mRNA and miR-214-3p in myocardial tissue.2.Male Sprague Dawley rats(220-250g)were divided into four groups randomly(6 rats in each group):Sham group,LPS group,LPS+shNC group and LPS+shHDAC9 group.The rats in the latter two groups were treated with corresponding lentivirus particles(1×109 TU/ml)through tail vein injection,with an injection volume of 150μl.After 7 days of injection,10mg/kg LPS was injected intraperitoneally,and equal volume of PBS was used to inject into the control group.After injected of LPS for 6 hours,the left ventricular ejection fraction,left ventricular shortening fraction,left ventricular end diastolic diameter,and left ventricular end systolic diameter were measured.Rats were euthanized,myocardial tissue and serum were collected,and the expression of HDAC9 in each group’s myocardial tissue was detected by Real-time PCR and Western blot;The kit as same as the first part was used to detect the level of cTnI,CK-MB and LDH in myocardial tissue;ELISA was used to detect the level of TNF-α,IL-6 and IL-1β in serum;TUNEL staining method was used to observe the myocardial tissue apoptosis.Results:1.Compared to Sham group,the cardiac ultrasound in LPS group rats showed a obviously increase in LVEDD and LVESD,while EF and FS reduced significantly,with significant statistical significance(P<0.01 or P<0.001).The H&E staining results showed that LPS group rats had edema in myocardial cells and disordered arrangement of myocardial fibers.The TUNEL test results showed that the apoptosis in LPS group rats had a significant increase compared to Sham group.The IF results showed an increase in the expression of HDAC9 in LPS group.Real-time PCR and Western blot showed that compared to Sham group,the expression levels of HDAC9 mRNA and protein were significantly increased in LPS group,with significant statistical significance(P<0.001);the expression level of miR-214-3p in LPS group was also increased significantly in LPS group,and the difference was statistically significant(P<0.001).2.Cardiac ultrasound results showed that silencing HDAC9 expression could reduce LVEDD and LVESD,as well as enhance FS and EF,significantly improve LPS induced cardiac dysfunction.After silencing HDAC9 expression,the levels of myocardial injury markers(cTnI,CK-MB,LDH)and the inflammatory cytokines(TNF-α,IL-6,IL-1β)had obviously decreased.TUNEL staining results indicated that silencing HDAC9 expression could reduce myocardial cell apoptosis.Conclusions:In LPS induced septic rats,the expression of HDAC9 and miR-214-3p increased and morphology showed myocardial injury and cell apoptosis.Cardiac ultrasound showed that the cardiac systolic function was decreased.HDAC9 silencing alleviated myocardial injury,improved cardiac function,reduced inflammatory response,and myocardial cell apoptosis.Part Ⅲ Serum changes of miR-214-3p and HDAC9 in patients with sepsis induced myocardial dysfunctionObjective:To observe the levels of miR-214-3p and HDAC9 in patients with sepsis induced myocardial dysfunction and their clinical importance.Methods:Sixty patients were admitted from the ICU of Cangzhou Central hospital from April 2022 to February 2023,including 30 patients in sepsis myocardial dysfunction group(SIMD group)and 30 patients in sepsis group(NSIMD group).During the same period,20 healthy volunteers(control group)were recruited.After entering the room,all patients were detected by cardiac ultrasound measurement,blood was collected,and inflammatory markers(WBC and PCT)and myocardial injury markers(CK,CK-MB,cTnI,NT-proBNP)were examined in the laboratory.Calculate the patient’s APACHE Ⅱ score.After being selected,2ml of blood was collected from each of the three groups,and the supernatant was centrifuged and stored in a-80℃refrigerator.Real-time PCR was used to determine the expression levels of miR-214-3p and HDAC9 mRNA in serum.Results:1.Compared to patients in NSIMD group,patients in SIMD group showed significant increase in inflammatory factors(WBC and PCT),myocardial injury markers(CK,CK-MB,cTnI,NT-proBNP)and decrease in cardiac function(EF and FS).The difference between the two groups was statistically significant(P<0.001).The APACHE Ⅱ score was higher in SIMD group,and the difference was statistically significant compared to NSIMD group(P<0.001).2.Compared to control group,the expression levels of miR-214-3p and HDAC9 in NSIMD group and SIMD group were significantly increased,and the difference was statistically significant(P<0.001).Compared to NSIMD group,the expression levels of miR-214-3p and HDAC9 in SIMD group were higher,and the difference was statistically significant(P<0.001).3.The HDAC9 expression level was negatively correlated with EF and positively correlated with cTnl in patients with SIMD.miR-214-3p was positively correlated with the expression of HDAC9.Conclusions:The myocardial injury markers and inflammatory indicators increased in the SIMD and NSIMD groups,and the increase was more obvious in the SIMD group.Cardiac function decreased in the SIMD group.The expression level of HDAC9 in the SIMD group was negatively correlated with EF and positively correlated with cTnI.miR-214-3p was positively correlated with the expression of HDAC9.In summary,this study demonstrated that HDAC9 was involved in the development of sepsis induced myocardial injury from three aspects:in vitro cell experiment,in vivo animal experiment,and clinical studies.miR-214-3p and HDAC9 expression were positively correlated.miR-214-3p could target and regulate the expression of HDAC9,and played a protective role in sepsis induced myocardial dysfunction by reducing myocardial cell damage,suppressing NF-κB signal pathway,decreasing inflammatory response and cell apoptosis and enhancing cell vitality though the HDAC9 way.miR-214-3p and HDAC9 were expected to serve as diagnostic markers and therapeutic targets for sepsis and its induced myocardial dysfunction.