| Part One:COX2/NLRP3 signaling is involved in alleviating myocardial ischemia/reperfusion injury in mice of Iguratimod pretreatmentObjective The aim of this study was to determine whether Iguratimod pretreatment could exert cardioprotection against ischemia/reperfusion(I/R)injury in mice and the impact of Iguratimod on inflammatory response mediated by COX2/NLRP3 signaling.Methods Healthy adult male C57BL/6 mice(6-8 weeks of age)were randomly divided into three groups:sham operation group(group Sham),myocardial ischemia/reperfusion group(group I/R),Iguratimod pretreatment and I/R group(group T-614+I/R).The myocardial ischemia/reperfusion model was established by ligation of the left anterior descending coronary artery for 30min,followed by 24h reperfusion.Mice in group T614+I/R received intraperitoneal injection of Iguratimod at a dose of 5 mg/kg one hour before myocardial ischemia.And mice in another two groups were administrated with equal solvent.The myocardial infarct and ischemia area were assessed by Evans Blue and TTC double staining.The concentration of cTnI in the serum was measured by ELISA.HE staining was used to observe the pathological morphology of cardiac tissue.The expression of MPO in the heart was detected by immunohistochemistry.The mRNA levels of COX2,NLRP3 and some inflammatory factors,including IL-1β,IL-6,IL-18 and TNF-α,were measured by real-time quantitative PCR(RT-qPCR).Western Blot was adopted to detect the relative expression levels of COX2,NLRP3,Caspase-1,IL-1β and Cleaved Caspase-1.The cellular localization of NLRP3 protein in cardiac tissue was determined by immunofluorescence staining.Results Compared with group Sham,the myocardial infarct area,the concentration of cTnI in the serum and the expression of MPO in cardiac tissue were all remarkably elevated in group I/R(P<0.01).Moreover,the pathological injury of cardiac tissue was aggravated.The mRNA levels of COX2,NLRP3,IL-1β,IL-6,IL-18 and TNF-α in group I/R were higher than those in group Sham(P<0.05).And the relative protein levels of COX2,NLRP3,Caspase-1,IL-1β and Cleaved Caspase-1 were markedly enhanced(P<0.01).Compared with group I/R,Iguratimod pretreatment significantly reduced the myocardial infarct area,the concentration of cTnI in serum and the expression of MPO in cardiac tissue(P<0.01).It also alleviated the pathological injury of cardiac tissue.The mRNA levels of COX2,NLRP3,IL-1β,IL-18 and TNF-α in group T-614+I/R decreased remarkably(P<0.05).And the relative protein levels of COX2,NLRP3,Caspase-1,IL-1β and Cleaved Caspase-1 were all declined obviously(P<0.01).The result of immunofluorescence staining showed that I/Rinduced NLRP3 protein colocalized mostly with cardiac fibroblast marker.Conclusion Iguratimod pretreatment alleviates myocardial ischemia/reperfusion inj ury in mice.And the mechanism may be associated with the down-regulated inflammatory response,which is mediated by COX2/NLRP3 signaling pathway.Part two:Iguratimod pretreatment attenuates inflammatory response induced by hypoxia/reoxygenation injury of mice primary cardiac fibroblasts by regulating COX2/NLRP3 signalingObjective This study aimed to investigate the effect of Iguratimod pretreatment on hypoxia/reoxygenation(H/R)injury of mice primary cardiac fibroblasts,the impact of Iguratimod on inflammatory response induced by H/R and the relevant molecular mechanism.Methods Murine primary cardiac fibroblasts(CFs)were isolated from C57BL/6 mice aged 1 to 3 days with differential adhesion method.The CFs of second generation were used for cell experiments.And they were divided into three groups according to the random number table method:Control group(Con),hypoxia/reoxygenation group(H/R group),Iguratimod pretreatment+hypoxia/reoxygenation group(T+H/R).The cellular model of H/R was established by hypoxia for 1h and reoxygenation for 3h.Primary CFs in Con group were incubated with 100 ng/ml LPS for 18h.The cells in H/R group were treated with LPS for 18h before H/R.T+H/R group was pretreated with Iguratimod of 5 μmol/L for 6h followed by the same procedure of H/R group.The purity of primary CFs was assessed by immunofluorescence staining.The relative cell viability was determined by CCK-8.2,4dinitrophenylhydrazine colorimetric method was used to detect the relative release ratio of lactate dehydrogenase(LDH)in the supernatant.The mRNA expressions of IL-1β,IL-6,IL18 and TNF-α in CFs were detected by RT-qPCR.Western Blot was used to measure the relative expression levels of COX2,NLRP3,Caspase-1,IL-1β and Cleaved Caspase-1 protein.Results The purity of murine primary CFs was up to 93.3%,which means that these primary CFs could be stably used in subsequent experiments.Compared with Con group,fibroblasts in H/R group showed significantly decreased relative cell viability(P<0.01)and increased LDH release ratio(P<0.01).The mRNA expression levels of COX2,NLRP3,IL1β,IL-6,IL-18 and TNF-α were obviously higher in H/R group(P<0.01).Moreover,the protein expression levels of COX2,NLRP3,Caspase-1,IL-1β and Cleaved Caspase-1 all increased significantly in H/R group(P<0.01).Compared with H/R group,cells in T+H/R group had remarkably higher cell vitality(P<0.05)and lower LDH release ratio(P<0.01).Iguratimod also reduced the mRNA levels of COX2,NLRP3,IL-1β,IL-6,IL-18 and TNFa(P<0.01).And this was the same trend in the protein expressions of COX2,NLRP3,Caspase-1,IL-1β and Cleaved Caspase-1 in T+H/R group(P<0.01).Conclusion Iguratimod pretreatment could attenuate H/R injury of primary cardiac fibroblasts and reduce the inflammatory response induced by H/R injury,the underlying mechanism of which may be related to the inhibition of COX2/NLRP3 signaling pathway. |
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