| Myocardial infarction(MI)is a common clinical critical illness with an increasing trend of morbidity and mortality in the population year by year.Myocardial fibrosis and heart failure after myocardial infarction are among the leading causes of death in patients.Despite significant advances in reperfusion strategies and pharmacological treatment to limit cardiac injury and improve overall prognosis,some patients still develop myocardial fibrosis and negative ventricular remodeling after treatment,which severely affects the quality of patient survival.There are no effective therapeutic drugs for myocardial fibrosis in clinical practice,so it is of great clinical importance to clarify the pathogenesis of myocardial fibrosis and to find drugs that can effectively treat it.A large body of research evidence suggests that post-infarction inflammation plays an important role in the pathogenesis of myocardial fibrosis,and therefore antiinflammatory therapy after myocardial infarction may be a new promising therapeutic direction to improve the prognosis of patients with myocardial infarction.Celastrol,a natural active products extracted from Celastraceae family plants,such as Tripterygium wilfordii and Celastrus orbiculatus,has strong anti-inflammatory effects and has shown promising therapeutic effects in a variety of diseases,including rheumatoid arthritis and systemic lupus erythematosus.Existing studies suggest that Celastrol has some cardioprotective effects,but the mechanism of action in myocardial fibrosis after myocardial infarction is unclear.Therefore,in order to investigate the role and possible regulatory mechanisms of Celastrol in post-infarction myocardial fibrosis in rats,this study first observed and evaluated the role of Celastrol in post-infarction myocardial fibrosis in rats at the animal level.The differentially expressed genes and signaling pathways associated with post-infarction fibrosis treatment with Celastrol were then analyzed by transcriptomic sequencing for enrichment,followed by validation of selected target genes and signaling pathways from transcriptome sequencing results in vivo experiments.Finally,the role and specific molecular mechanisms of Celastrol in lipopolysaccharide(LPS)-induced fibrosis in neonatal rat cardiofibroblast(NRCF)were elucidated in in vitro experiments by si RNA knockdown and adenoviral overexpression and by applying pathway inhibitors to selected target genes and signaling pathways.The main results are as follows:1.Study of the effect of Celastrol on myocardial fibrosis and cardiac function after myocardial infarction in rats.The results of cardiac ultrasound showed a significant decrease in LVEF(45.00±5.93 vs.83.17±4.12,P<0.001)and LVFS(19.67±3.33 vs.47.50±4.51 P< 0.001);and a significant increase in LVEF(72.33±3.01 vs 45.00±5.93,P<0.001)and LVFS(36.67±2.73 vs 19.67±3.33,P<0.001)in the infarction + Celastrol group(MI+Celastrol group),suggesting that Celastrol can attenuate post-infarction in rats with impaired cardiac function.The results of invasive hemodynamic examination showed that compared with the Sham+saline group,the rats in the MI+saline group had significantly higher LVESP(68.88±0.03 vs.90.10±0.60,P<0.001),+dp/dtmax(2844.70±24.06 vs.5082.54±117.49,P<0.001)and-dp/dtmax(2467.51±10.51 vs 4474.87±38.13,P<0.001)were significantly lower hemodynamic parameters,while LVEDP was significantly higher.In addition,H&E staining results showed that Celastrol treatment effectively attenuated the pathological injury caused by myocardial infarction in rats;Masson staining and Sirius red staining showed that Celastrol treatment significantly reduced collagen fibrillogenesis and fibrosis caused by myocardial infarction.Meanwhile,western-blot results showed that Celastrol treatment significantly reduced the protein expression of CollagenⅠ,CollagenⅢ and α-SMA associated with fibrosis in infarcted myocardial tissues of rats.2.Whole-genome sequencing analysis was performed to explore the gene targets and validate the protective effect of Celastrol in post-infarction fibrosis in rats.To investigate the genes or pathways through which Celastrol mediates the inhibition of myocardial fibrosis after myocardial infarction,we performed wholegenome sequencing of myocardial tissues from Sham+saline,Sham+Celastrol,MI+saline,and MI+Celastrol groups of rats.The whole-genome sequencing results showed that there were 2147 differentially expressed genes in the MI+saline group compared to the Sham+saline group,with 1651 up-regulated genes and 496 downregulated genes.In contrast,there were 1591 differentially expressed genes between the MI+Celastrol and MI+saline groups,with 608 genes up-regulated and 983 genes down-regulated in expression.The GO and KEGG results showed that Celastrol treatment upregulated pathways related to cell cycle,p53 signaling pathway and cell adhesion.And Celastrol down-regulated pathways related to inflammation,apoptosis and programmed death,such as NOD-like receptor signaling pathway,NFκB signaling pathway and IL-17 signaling pathway;and pathways related to cardiac fibroblast proliferation and collagen production,such as JAK-STAT signaling pathway.We selected the NFκB/NLRP3 pathway for RT-PCR validation,and the results showed that the expression of NLRP3 inflammasome-related genes increased significantly in rats after infarction and could be inhibited by Celastrol;while the m RNA expression of NFκB p65 did not change significantly in rats before and after infarction and Celastrol treatment,suggesting that we Celastrol may affect the nuclear translocation of p65 by The effect of p65 was suggested.3.We investigated the mechanism of inhibition of NLRP3 inflammasome activation in infarcted myocardial tissues of rats by Celastrol.We validated the results of whole-genome sequencing analysis by immunohistochemistry,ELISA and western-blot experiments at the animal level.Immunohistochemical results showed that the expression levels of NLRP3 and IL-1β were significantly upregulated in myocardial tissues of MI+saline rats compared with those of Sham+saline rats,and the upregulation of both was reversed by Celastrol treatment.In addition,immunohistochemical results showed that Celastrol treatment effectively reduced the infiltration of CD68-positive macrophages and Ly6G-positive neutrophils in the infarcted myocardial tissue,and The ELISA results showed that the serum concentrations of IL-1β and IL-18 were significantly higher in the MI+saline group compared with the Sham+saline group,and the treatment with Celastrol was able to reduce the serum levels of the above inflammatory factors.Western-blot results showed that the protein levels of NLRP3,Cleaved-caspase1,Cleaved-IL1β and IL18 were significantly higher in the MI+saline group than in the control group,and the treatment with Celastrol could inhibit the increase in the expression of these proteins.Meanwhile,the Western-blot results showed that the nuclear translocation of p65 was reduced by the treatment of rhodopsin compared with the MI+saline group.4.Mechanistic study of Celastrol alleviating LPS-induced fibrosis in cardiac fibroblasts via NFκB/NLRP3 pathway.We constructed a cellular inflammation model using LPS-induced NRCF to mimic the inflammatory state of post-infarction rats,and confirmed by si RNA knockdown and adenoviral overexpression of NLRP3 that Celastrol could inhibit the expression of NLRP3 inflammasomes in myocardial tissue of infarcted rats,and that adenoviral overexpression of NLRP3 could counteract this cardioprotective effect of Celastrol.In addition,to investigate the mechanism of inhibition of NLRP3 inflammasome activation in NRCF by Celastrol and to further validate the results of whole-genome sequencing,we used the NFκB pathway inhibitor BAY11-7082 in a parallel control assay,and western-blot results showed that both Celastrol and BAY11-7082 could effectively inhibit NFκB pathway activation and reduce the nuclear translocation of p65.In conclusion,the above experiments fully demonstrate that Celastrol may alleviate LPS-induced NRCF fibrosis through the NFκB/NLRP3 pathway.In summary,through the above study we clarified the protective effects of Celastrol in myocardial fibrosis after myocardial infarction in rats,and analyzed and validated the differences in gene expression and signaling pathway enrichment in rats before and after myocardial infarction modeling and with and without Celastrol treatment.We have elucidated the mechanism of action of Celastrol in reducing the expression of NLRP3 inflammatsome by reducing p65 nuclear translocation and thereby reducing fibrosis in vivo and in vitro,providing new ideas and evidence for the use of Celastrol to improve myocardial fibrosis and heart failure prevention after infarction. |
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