| Background:Acute Myocardial Infarction(AMI)is the most important reason for Heart Failure(HF),which severely affects human health.The aspect about how to reduce the apoptosis rate of Myocardial ischemia after AMI,protect normal and ischemic myocardium around the infarcted area,delay ventricular remodeling and improve heart function,is a major subject of current cardiovascular disease research.In recent years,study found that inflammatory response regulated by NLRP3 inflammatory body after myocardial infarction is the main reason for inducing myocardial apoptosis and cardiac fibrosis,promoting ventricular remodeling.Thus,controlling inflammatory response after myocardial infarction and inhibiting myocardial damage exposed to inflammatory cytokines may be an effective measure for poor ventricular remodeling after myocardial infarction.NLRP3 inflammatory body,a component of innate immune system,is a macromolecular protein complex.It can precisely regulate activation of caspase-1 and the production and secretion of strong pro-inflammatory cytokines such as IL-18 and IL-1β.MCC950,a specific inhibitor of NLRP3 inflammatory,can specifically inhibit the activation of NLRP3 inflammatory body and decrease the secretion of inflammatory cytokines IL-18 and IL-1β.Therefore,in this study we tried to identify the effect of MCC950 on cardiac function protection and ventricular remodeling after myocardial infarction in mice,illuminate the concrete mechanism and provide new clinical treatment strategies in order to improve the clinical prognosis of patients after myocardial infarction,reduce myocardial fibrosis and improve ventricular remodeling.Part I: The function of NLRP3 inflammatory body on myocardial infarction(MI)in mice Objective:Observe the role of NLRP3 inflammatory body on MI in mice.Methods:Six to eight week C57BL/6J male mice were assigned to either the MI group or sham group.Delect the levels of IL-18 and IL-1β at postnatal 1,3,7,14 in the serum of mice.Western blot and RT-PCR detect the levels of related inflammatory factors NLRP3,Cleaved IL-18 and Cleaved-1β.Results:(1)Compared with the Sham group,the IL-18 and IL-1β level in the serum of mice increased significantly in MI group,the difference was statistically significant(p<0.05).Compared with the Sham group,the level of IL-18(p<0.05)and IL-1β(p<0.05)at postnatal 1,3,7 in the serum of mice increased significantly,peaks at postnatal 3 and 7.(2)Western blot and RT-PCR results show that the content of NLRP3,Cleaved IL-18 and Cleaved IL-1β in the myocardia increased significantly,the difference was statistically significant(p < 0.05).Compared with the Sham group,the level of NLRP3(p<0.05),Cleaved IL-18(p<0.05)and Cleaved IL-18(p<0.05)at postnatal 1,3,7 in the myocardia increased significantly,peaks at postnatal 3 and 7.Summary:(1)After myocardial infarction in mice,myocardial inflammatory response is induced,and a large number of inflammatory factors NLRP3,IL-18 and IL-1β are secreted.(2)The content of inflammation-related protein NLRP3,Cleaved IL-18 and Cleaved IL-1β first increased and then decreased in mouse myocardium,and reached a peak at 3 and 7 days.Part II: Effect of MCC950 on cardiac function and ventricular remodeling after myocardial infarction in mice and its mechanism Objective:Observe the effect of MCC950 on cardiac function and ventricular remodeling after infarction in mice,and explore the underlying mechanism.Methods:Six to eight week C57BL/6J male mice were divided into three groups: SHAM group,MI group and MI+MCC950 group.Left ventricular ejection fraction(LVEF),left ventricular shortening fraction(LVFS),cardiac output(CO)and heart rate(HR)were detected by color Doppler ultrasound in mice.Detection of creatine kinase isoenzyme MB(CK-MB)and lactate dehydrogenase(LDH)levels in mouse serum.HE staining to observe myocardial inflammation infiltration and histomorphological changes.Western blot and RT-PCR detect the content of fibrosis related protein Collagen-I,Collagen-III and αSMA and inflammation related protein NLRP3,Cleaved caspase-1,Cleaved IL-18 and Cleaved IL-1β.Results:(1)Echocardiography to detect heart function in mice:compared with SHAM group,there was no significant difference between the HR of MI group and MI+MCC950 group.Compared with the SHAM group,the heart function of the mice in the MI group was worsened,and LVEF(p<0.05),LVFS(p<0.05)and CO(p<0.05)was significantly reduced.Compared with the MI group,the cardiac function in the MI+MCC950 group was significantly improved,and LVEF,LVFS and CO were significantly improved,which was statistically significant(p<0.05).(2)Compared with SHAM group,the serum levels of CK-MB and LDH in the MI group increased significantly,the difference was statistically significant(p <0.05).Compared with the MI group,the serum levels of CK-MB(p<0.05)and LDH(p<0.05)in the MI+MCC group were significantly reduced.(3)Masson staining results showed that compared with the SHAM group,the MI myocardial fibrosis was significantly increased(p<0.05).Compared with the MI group,the MI + MCC950 group myocardial fibrosis level was reduced,the difference was statistically significant Significance(p<0.05).Western blot and RT-PCR detection of fibrosis-related proteins Collagen-I,Collagen-III and αSMA in myocardium further confirmed that,compared with the SHAM group,the degree of myocardial fibrosis in the MI group increased,while compared with the MI group,MI The degree of myocardial fibrosis was reduced in the + MCC950 group,and the differences were statistically significant(all p<0.05).(4)HE staining results showed that,compared with SHAM group,MI group had significantly increased myocardial inflammatory cell infiltration,and the difference was statistically significant(p<0.05).Compared with MI group,MI + MCC950 group inflammatory cell infiltration Significantly reduced,the difference was statistically significant(p<0.05).Immunohistochemical results showed that MCC950 can inhibit macrophage CD68,neutrophil Ly6 G and T lymphocyte CD4 infiltration.Immunofluorescence results further proved that MCC950 can significantly reduce NLRP3 inflammasome infiltration.(5)Western blot and RT-PCR results showed that compared with SHAM group,inflammation-related proteins NLRP3(p<0.05),Cleaved caspase-1(p<0.05),Cleaved IL-18(p<0.05)and Cleaved IL-1β(p <0.05)was significantly increased,and compared with MI group,MI + MCC950 group myocardial NLRP3,Cleaved caspase-1,Cleaved IL-18 and Cleaved IL-1β content were significantly reduced,the differences were statistically Significance(all p<0.05).Summary:(1)MCC950 can improve the cardiac function after myocardial infarction in mice,reduce the serum myocardial enzyme level,reduce the area of myocardial infarction,reduce myocardial fibrosis,and then improve poor ventricular remodeling.(2)MCC950 can specifically inhibit the expression of NLRP3 inflammatory bodies in the serum and myocardium of mice after myocardial infarction,and then inhibit the expression of caspase-1,IL-18 and IL-1β,and reduce myocardial inflammation.(3)MCC950 plays an anti-inflammatory role through NLRP3-IL-1β signaling pathway to protect cardiac function after myocardial infarction and improve ventricular remodeling.Part III: Effect and mechanism of MCC950 on hypoxic injury of neonatal rat fibroblasts Objective:A hypoxic injury model of neonatal rat fibroblasts was constructed to observe the role of MCC950 at the cellular level,further confirming the mechanism of action of MCC950.Methods:(1)Primary fibroblasts of C57 / B6 suckling rats cultured for 1-3 days,and hypoxia injury models at different time points(0h,6h,12 h,18h,24 h and 36h)are set.Western blot was used to detect the expression of NLRP3,Pro-IL-1β and Cleared IL-1β in fibroblasts.(2)Primary fibroblasts of suckling mice were used to construct hypoxic injury models.After treatment with different concentrations of MCC950(1,10,20,40μM),CCK-8 was used to detect cell viability.Western blot was used to detect the expressions of fibrosis-related proteins Collagen-I,Collagen-III and αSMA in primary fibroblasts,and to determine the secretion of inflammatory factors IL-18 and IL-1β in fibroblast supernatants.In the expression of NLRP3,Western blot was used to detect the contents of inflammation-related proteins NLRP3,Cleaved caspase-1,Cleaved IL-18 and Cleaved IL-1β.Results:(1)Western blot results showed that compared with 0h of hypoxia,the expression of NLRP3 and Cleaved IL-1β in fibroblasts increased significantly after 18 h of hypoxia,and the difference was statistically significant(p<0.05).It showed that after 18 h of hypoxia,the activation of NLRP3 and Cleaved IL-1β was the most obvious in fibroblasts.Therefore,the hypoxia time of fibroblasts is 18 h.(2)The CCK-8 test results showed that compared with the SHAM group,the fibroblast viability was significantly reduced after 18 hours of hypoxia,and the difference was statistically significant(p<0.05).Compared with 18 h of hypoxia,cell viability increased after MCC950 treatment,especially after 10μM MCC950 treatment,cell viability increased significantly,the difference was statistically significant(p<0.05).(3)Compared with the SHAM group,the expression of myocardial fibrosis protein Collagen-I(p<0.05)and αSMA(p<0.05)increased significantly after 18 hours of hypoxia.Compared with 18 h of hypoxia,the expression levels of Collagen-I and αSMA in the MCC950(10μM)treatment group were significantly reduced,and the differences were statistically significant(p<0.05).(4)Compared with SHAM group,the secretion of NLRP3(p<0.05),IL-18(p<0.05)and IL-1β(p<0.05)in the cell supernatant increased significantly after 18 h of hypoxia.Compared with 18 h of hypoxia,the expression levels of NLRP3,IL-18 and IL-1β in the cell supernatant of the MCC950(10μM)treatment group decreased,and the differences were statistically significant(all p<0.05).(5)The results of immunofluorescence co-localization showed that compared with the SHAM group,the expression of NLRP3 in fibroblasts increased significantly after 18 h of hypoxia,and the difference was statistically significant(p<0.05).Compared with 18 h of hypoxia,the expression of NLRP3 in the MCC950(10μM)treatment group decreased significantly,and the difference was statistically significant(p<0.05).Western blot results showed that compared with SHAM group,NLRP3(p<0.05),Cleaned caspase-1(p<0.05),Cleaved IL-18(p<0.05)and Cleaved IL-1β(p<0.05)after 18 h of hypoxia)The amount of expression increased significantly.Compared with 18 h of hypoxia,the expression levels of NLRP3,Cleaved caspase-1,Cleaved IL-18 and Cleaved IL-1β in the fibrocytes of MCC950(10μM)treatment decreased,and the differences were statistically significant(all p <0.05).Summary:(1)After 18 h of fibroblast hypoxia,the cell viability decreased and the degree of myocardial fibrosis increased significantly.After MCC950 treatment,the cell viability increased significantly,and the degree of myocardial fibrosis was reduced,further confirming that MCC950 can improve cell viability and reduce myocardial fibrosis.(2)After 18 h of fibroblast hypoxia,the expression of NLRP3 and Cleaved IL-1β increased significantly.After MCC950 treatment,the content of NLRP3,Cleaved caspase-1,Cleaved IL-18 and Cleaved IL-1β in fibroblasts was significantly reduced.It further confirms that MCC950 plays a role in myocardial protection through NLRP3-IL-1β signaling pathway and improves ventricular remodeling. |
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