| Background and ObjectiveCardiovascular disease is the leading cause of death worldwide.Myocardial infarction(MI)has a higher risk of mortality.In China,the prevalence of MI is higher in individuals under 45 years of age.Its incidence is increasing yearly.Myocardial cell death caused by MI is permanent,with irreparable scars developing during healing.Therefore,a better understanding of underlying pathophysiological mechanisms can reduce myocardial injuries and provide new therapeutic strategies to repair the infarcted hearts.The body’s innate immune system can recognize exogenous infection or endogenous cell damages and produce a defensive inflammatory response.The defense system stimulates pattern recognition receptors(PRRs)to recognize danger-associated molecular patterns(DAMPs)and subsequently activate the inflammatory pathways.The NLRP3 inflammasome of the nod-like receptor(NLRs)family are common PPRs,involved in the regulation of cardiovascular inflammation.They can separate the precursor protein of IL-1β/IL-18 into their active forms,mediating effective intracellular inflammation.NLRP3 inflammasome are widely expressed in all kinds of cells,mainly in macrophages.Recent studies have shown that NLRP3 inflammasome activation also exists in non-immune cells,including endothelial cells and fibroblasts.Previous studies have confirmed that NLRP3 inflammasome is involved in the process of coronary atherosclerosis,MI,ischemia/reperfusion injury,and cardiac remodeling.Inflammation is a prerequisite for the healing of heart injury,but excessive inflammation may lead to ventricular remodeling.Therefore,inflammatory response is of great significance for the prevention and treatment for myocardial injury.NLRP3 inflammasome can be indirectly activated by a variety of stimuli.But its specific activation mechanism after MI is still unclear.Glycogen synthase kinase-3β(GSK-3β)is a key enzyme of glycogen metabolism,which is related to a variety of cell functions and signal transduction pathways.GSK3β regulates the different physiological and pathological functions of the body.It is a key molecule in regulating cellular inflammation and participates in the occurrence and development of a variety of diseases,including inflammatory diseases,neurodegenerative diseases,diabetes,and cancer.Infection can regulate the activity of GSK-3β and affect host cell adaptation,immune cell infiltration,and cytokine expression.Inhibition of GSK-3β can reduce the secretion of IL-1β and relieve inflammation.Based on MI-induced inflammation and the regulation of GSK-3β on inflammation,we speculate that GSK-3β may participate in the activation of NLRP3 inflammasome after MI,regulating the occurrence and development of cardiac cells inflammation,as well as the process of cardiac cells death.This study will be carried out in the following two aspects.Fisrtly,to investigate the mechanism of GSK-3βmediated NLRP3 inflammasome activation after MI.Secondly,to explore the regulatory pathway of GSK-3 β-induced NLRP3 inflammasome activation in cardiomyocyte death.This study will provide a new strategy for the clinical treatment of MI.Methods1.Exploreing the molecular mechanism of GSK-3β-mediated the activation of NLRP3 inflammasome after MI1)Establishment and experimental grouping of acute MI model in ratsSPF grade-male Sprague Dawley rats(220-240 g)were randomly divided into three groups:sham operation group(only threading without ligation),operation(MI)group(ligation),and GSK-3β inhibitor intervention(MI+SB)group(SB216763,administered intravenously through the tail vein 1 h before surgery at a dose of 0.6 mg/kg,once daily for 7 days).Serum and tissue samples were collected from 12,17 and 12 animals in each group at 2,7 and 28 days after operation.Five samples were used for histochemical detection,seven samples were used for mRNA and protein extraction for molecular biological detection.Five samples were used to detect the infarct size on 7 days.2)Determination of cardiac function and serum markers in ratsThe heart functions of rats in each group were determined by transthoracic echocardiography;the serum contents of lactate dehydrogenase(LDH),cardiac troponin I(cTn-I)and interleukin-1β(IL-1β)were determined by enzyme linked immunosorbent assay(ELISA).3)Changes of cardiac structure detecting by immunohistochemistryTTC staining was used to measure the ischemic area of MI;paraffin sections were prepared,and the pathological differences of the heart were observed by H&E staining,Masson staining and Sirius red staining.TUNEL staining was used to detect cells apoptosis.4)Expression of GSK-3β and NLRP3 inflammasome in rat heartsqPCR and western blot were used to detect the expression of GSK-3β,NLRP3,ASC,caspase-1,and IL-1β at mRNA and protein levels in the ischemic region,marginal region,and distal region of the three groups.5)In vitro culture of rat primary cells to determine the site of GSK-3β mediated activation of NLRP3 inflammasomePrimary new-born rat cardiomyocytes(RCMs)/cardiac fibroblasts(RCFs)were extracted by enzyme digestion.The two type cells were co-cultured in immunofluorescence dish at a ratio of 4:1,and then incubated with SB216763(10μmol/l)for 1 h,LPS(1 μg/mL)for 12 h and ATP(5 mmol/L)for 1 h.The localization of IL-1β protein was observed by confocal laser scanning microscope,which was to determine the activated cell site of NLRP3 inflammasome mediated by GSK-3β.6)Construction of cell hypoxia model to explore the molecular mechanism of GSK-3β regulating NLRP3 inflammasome activationTo explore the mechanism of GSK-3β regulating the activation of NLRP3 inflammasome,an in vitro hypoxia model was established by pharmacological inhibition(SB216763)and knockdown(specific shRNA)of GSK-3β.7)Identification of molecular proteins interacting with GSK-3β by immunoprecipitationAfter LPS/ATP stimulation,the specific NLRP3 inflammasome components(NLRP3,ASC and pro-caspase-1)affected by GSK-3β were determined by immunoprecipitation,and the cells were crosslinked with disuccinidyl substrate(DSS).The effects of GSK-3β on the phosphorylation and oligomerization of these components were observed by western blot.2.GSK-3β mediates the activation of NLRP3 inflammasome and regulates cardiomyocyte death1)Establish the inflammation model of RCFs in vitro and observe the apoptosis and pyroptosis of RCFs and RCMsEstablished an in vitro inflammatory model of RCFs(LPS/ATP)and detect the levels of NLRP3,ASC,caspase-1,IL-1β and IL-18 by western blot.The supernatant of RCFs cells was transferred to RCMs cells after TLR1(40 μmol/L)was added.Detected apoptosis-related proteins(Bax,Bcl-2,caspase-3)and pyroptosis-related protein(N-GSDMD).Observed the effects of secreted IL-1β on apoptosis and pyroptosis of RCMs cells.2)Observe the effect of IL-1β on H9c2 cell apoptosis and pyroptosisH9c2 cells were cultured and pretreated with SB216763 or TLR1.H9c2 cells were stimulated with recombinant IL-1β(40 ng/mL).The apoptotic rate of each group was observed by flow cytometry,and the percentage of pyroptosis was determined by propidium iodide(2 μg/mL)staining and LDH release.3)H9c2 cells apoptosis and pyroptosis were observed after GSK-3β knockdownAfter knockdown of GSK-3β in H9c2 cells with specific siRNA,western blot was used to detect the changes of apoptosis and pyroptosis related proteins to verify the effect of GSK-3β mediated NLRP3 inflammasome activation on cell death.4)Determined the expressions of apoptosis and pyroptosis proteins in rat heartsExpressions of GSDMD in ischemic area,border area and remote area were detected by immunohistochemistry.The expressions of apoptosis and pyroptosis proteins in ischemic area were detected by western blot.Statistical analysisIBM SPSS 21.0 software was used to process the data.The data were expressed as mean ± standard deviation.T-test was used to compare the mean values between the two groups of data with bivariate normal distribution and homogeneous variance.Oneway ANOVA was used to compare the mean values between multiple groups.Dunnett t was used to analyze the data that did not conform to bivariate normal distribution and had uneven variance.Two tailed test showed that P<0.05 was statistically significant.Results1.GSK-3β participated in the activation of NLRP3 inflammasome after MI1)GSK-3β inhibitor improved cardiac dysfunction and remodeling after MICompared with sham group,the infarct size of MI group increased from 4.6±0.7%to 46.9±3.0%(P<0.01)on the 7th day after operation.In MI+SB group,the infarct size was significantly reduced and the cardiac pathological injury was significantly improved to 14.6±1.7%(P<0.05).The arrangement structure of heart was destroyed with obvious interstitial hemorrhage and fiber rupture.The deposition of type I/III collagen,the area of cardiac fibrosis and the proportion of apoptosis were significantly increased in MI group(P<0.01).The left ventricular ejection fraction(LVEF)decreased from 69.3±5.3%to 43.9±4.9%(P<0.01)and the short axis shortening rate(LVFS)decreased from 45.9±3.7%to 24.1±2.3%(P<0.01),suggesting the occurrence of cardiac dysfunction.While they were recovered in MI+SB group as 53.5±3.2%and 31.7±2.1%(P<0.05).In addition,LDH activity increased from 584.5±102.3 U/L to 1385.2±182.7 U/L(P<0.01);the cTn-I increased from 64.5±4.9 pg/mL to 223.8±38.8 pg/mL after MI(P<0.05).SB216763 significantly reduced the contents of LDH and cTn-I to 768.1±112.6 U/L and 144.7±18.5 pg/mL(P<0.05).At the same time,GSK-3β in MI group increased in mRNA and protein levels in different degrees(P<0.01),decreased by SB216763(P<0.05).2)GSK-3β participated in the activation of NLRP3 inflammasome after MIIn the three different time points,the serum IL-1β contents in MI group were significantly increased form 450.2± 126.6 pg/mL to 4683.2± 1125.6 pg/mL(P<0.01),but decreased in MI+SB group to 623.9±190.0 pg/mL(P<0.01).The mRNA levels of NLRP3,ASC,caspase-1 and IL-1β in the border area and ischemic area of MI group were significantly increased(P<0.05),while those in MI+SB group were significantly decreased(P<0.05).Similar to the mRNA results,SB216763 pretreatment also reduced the protein levels of NLRP3,ASC,caspase-1 and IL-1β in the ischemic region after MI(P<0.05).3)GSK-3β participated in the activation of NLRP3 inflammasome in RCFsThe results of immunofluorescence showed that LPS/ATP could induce IL-1βprotein maturation in RCFs in vitro,but no IL-1β was observed in RCMs.The proteins of NLRP3,ASC,caspase-1 and IL-1β and the secretion of IL-1β in RCFs were significantly increased(P<0.05).SB216763 inhibited the activation of NLRP3 inflammasome induced by hypoxia(P<0.05).The expressions of mature IL1β in HCMs and HCFs were also significantly induced by hypoxia,which were blocked by SB216763(P<0.01).After GSK-3β was knocked down by shGSK-3β,the increase of NLRP3,ASC,caspase-1 and IL-1β protein were limited(P<0.05).4)GSK-3β interacted with ASC and oligomerize ASC through phosphorylationAfter LPS/ATP stimulating RCFs,GSK-3β/ASC immunoprecipitation showed that GSK-3β interacted with ASC at protein level,while GSK-3β inhibition reduced the interaction between them.Meanwhile,LPS/ATP induced the increase of ASC oligomerization and phosphorylation in RCFs,which could be inhibited by SB216763.SB216763 also inhibited the binding of GSK-3β to ASC in ischemic myocardium.There was a certain amount of phosphorylated ASC in the ischemic area of MI(P<0.01),but it was significantly decreased in MI+SB group(P<0.05).2.Activation of NLRP3 inflammasome by GSK-3β induced apoptosis and pyroptosis of RCFs and RCMs1)GSK-3β mediated apoptosis and pyroptosis induced by NLRP3 inflammasome activation in RCFsAfter LPS/ATP stimulation,the protein levels of NLRP3,caspase-1 and ASC were significantly increased(P<0.05).The expressions of IL-1β and IL-18 were also significantly increased(P<0.05).SB216763 significantly inhibited the activation of NLRP3 inflammasome(P<0.05)in RCFs.In RCFs,capase-3 and Bax resulting in cells apoptosis whith high epressions,while SB216763 pretreament alleviated apoptosis.The increased content of N-GSDMD in RCFs resulted in cells pyroptosis,and SB216763 pretreament also inhibited pyroptosis.2)GSK-3β participated in apoptosis and pyroptosis of RCMs induced by NLRP3 inflammasome activation in RCFsTo prove whether IL-1β secreted by NLRP3 inflammasome activation in RCFs affect the apoptosis and pyroptosis of RCMs by paracrine pathway,the supernatants of cell culture stimulated by each group of RCFs was transferred to RCMs as conditional medium.The expressions of GSK-3β,Bax/Bcl-2,caspase-3 and N-GSDMD increased in LPS/ATP group(P<0.05).They decreased in SB216763 and TLR1 pretreatment groups(P<0.05).The expressions of GSK-3β,Bax/Bcl-2,caspase-3 and N-GSDMD increased after stimulation with recombinant IL-1β in RCMs and H9c2 cells(P<0.05).Pretreatment with SB216763/TLR1 alleviated these changes(P<0.05).3)GSK-3β mediates caspase-11 dependent pyroptosis in cardiomyocytesAfter recombinant IL-1β was added to H9c2 cells,pro-caspase-1 protein level did not change(P>0.05)and caspase-1(p20)were not produced.But caspase-11 protein levels increased(P<0.01).SB216763/TLR1 pretreatment could reduce the increase of caspase-11 proteins(P<0.01).Caspase-11 inhibitor,Wedelolactone,inhibited the increase of N-GSDMD,but has no obvious effect on the increase of GSK-3β,suggesting that GSK-3β is upstream of caspase-11/GSDMD pathway.The levels of GSK-3β,N-GSDMD,capase-11,capase-3 and Bax/Bcl-2 in H9c2 cells transfected with siGSK-3β were significantly alleviated after stimulating with IL-1β(P<0.05).The positive rate of GSDMD increased from 2.1±0.3%in sham group to 9.3±1.8%in MI group(P<0.05),decreasing in MI+SB group(3.2±0.8%,P<0.05).The expressions of Bax/Bcl-2,caspase-3,N-GSDMD and caspase-11 increased significantly(P<0.05)in MI group and SB216763 decreased the expressions of above proteins(P<0.05).Conclusions1.GSK-3β inhibitor improves cardiac dysfunction after MI.2.GSK-3β mediates the activation of NLRP3 inflammasome in cardiac fibroblasts by interacting with ASC and promoting its phosphorylation.3.The activation of NLRP3 inflammasome in cardiac fibroblasts leads to apoptosis and pyroptosis.The secretion of IL-1β also activates GSK-3β mediated apoptosis and caspase-11 dependent pyroptosis in cardiomyocytes... |
PDF Full Text Request