Muscone ameliorates myocardial ischemia‒reperfusion injury by promoting myocardial glycolysis.
Muscone ameliorates myocardial ischemia‒reperfusion injury by promoting myocardial glycolysis.
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DOI：
10.1016/j.heliyon.2023.e22154
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发表时间：
2023-11
期刊：
影响因子：
4
通讯作者：
Wang, Guangyan
中科院分区：
文献类型：
--
作者：
Gu, Xin;Bao, Neng;Zhang, Jing;Huang, Guangyi;Zhang, Xiaodong;Zhang, Zhixuan;Du, Yinqiang;Meng, Haoyu;Liu, Jiabao;Wu, Peng;Wang, Xiaoyan;Wang, Guangyan
急性心肌梗死（AMI）的发病率逐年上升。随着溶栓或经皮冠状动脉介入治疗（PCI）的应用，急性心肌梗死的死亡率显著降低。然而，再灌注可引起额外的心肌损伤。目前仍缺乏治疗I/R损伤的有效药物，迫切需要寻找新的治疗药物。在这项研究中，网络药理学被用来预测潜在的靶点和参与muscone介导的急性心肌梗死治疗的生物学过程。为了模拟缺血再灌注损伤，我们建立了缺氧再氧合模型和体内缺血再灌注损伤C57BL/6小鼠模型。小鼠灌胃4周。第28天检测心功能。免疫印迹法检测AC16细胞Muscone处理后及体内凋亡蛋白Caspase-3、Bax及抗凋亡蛋白Bcl-2的表达水平。此外，通过qRT-PCR分析PUMA和p53基因的表达水平。利用分子对接方法评估Muscone与nlrp3相关蛋白之间的结合能。采用免疫印迹法和qRT-PCR法分别检测NLRP3信号通路相关蛋白（NLRP3、ASC和Caspase-1）和NLRP3基因的表达水平。此外，使用Seahorse系统测量AC16细胞的细胞外酸化率，以评估Muscone处理后的糖酵解水平。采用免疫印迹和qRT-PCR分析糖酵解关键酶PKM2的表达。最后，通过ChIP-qPCR检测PKM2启动子区域组蛋白修饰（H3K4me3、H3K27me3和H2AK119Ub）的水平。氧化石墨烯功能富集分析表明，muscone参与AMI的生物过程（BP）调控，主要包括细胞凋亡信号通路的负调控、对脂多糖的反应、血压调节等。参与肌孔介导的AMI调控的细胞组分（CC）主要包括脂筏、膜微域和膜区。muscone介导的AMI调控的分子功能（MF）主要包括氧化还原酶活性、核受体活性和转录因子活性。体外实验结果表明，muscone处理可抑制缺血再灌注后AC16细胞Bax和Caspase-3的表达水平，同时提高抗凋亡蛋白Bcl-2的表达水平。Muscone显著抑制AC16细胞中p53和PUMA的转录水平。分子对接表明，muscone可以很好地结合NEK7（PDB ID:6NPY）的Cryo-EM结构。进一步对炎症通路的研究发现，muscone可以抑制AC16细胞NLRP3的表达水平，降低Caspase-1和Caspase募集域的表达水平。荧光定量PCR实验显示，muscone显著抑制NLRP3的转录。此外，我们发现muscone可以提高AC16细胞的糖酵解效率，这可能与AC16细胞中PKM2蛋白表达增加有关。荧光定量PCR结果显示，muscone可提高PKM2的转录水平。染色质免疫沉淀实验显示，muscone处理增加了PKM2启动子区域H3K4me3的表达水平，抑制了PKM2启动子区域H3K27me3和H2AK119Ub的表达水平。Muscone促进心肌糖酵解，抑制NLRP3通路激活，改善心肌缺血再灌注损伤。
The incidence of acute myocardial infarction (AMI) is increasing yearly. With the use of thrombolysis or percutaneous coronary intervention (PCI), the mortality rate of acute myocardial infarction has been significantly reduced. However, reperfusion can cause additional myocardial injury. There is still a lack of effective drugs to treat I/R injury, and it is urgent to find new therapeutic drugs. In this study, network pharmacology was used to predict potential targets and biological processes involved in Muscone-mediated treatment of acute myocardial infarction. To model ischemia‒reperfusion injury, a hypoxia-reoxygenation model and in vivo ischemia‒reperfusion injury C57BL/6 mice model was constructed. Mice were treated with Muscone i.p. for 4 weeks. We detected the cardiac function on day 28.The expression levels of the apoptotic proteins Caspase-3 and Bax and the anti-apoptotic protein Bcl-2 were detected by immunoblotting after Muscone treatment of AC16 cells and in vivo. Additionally, the gene expression levels of the PUMA and p53 were analyzed by qRT‒PCR. Molecular docking was used to evaluate the binding energy between Muscone and NLRP3-related proteins. Immunoblotting and qRT‒PCR were used to assess the expression levels of NLRP3 signaling pathway-related proteins (NLRP3, ASC, and Caspase-1) and the NLRP3 gene, respectively. Moreover, the extracellular acidification rate of AC16 cells was measured using the Seahorse system to evaluate glycolysis levels after Muscone treatment. The expression of the key glycolytic enzyme PKM2 was analyzed by immunoblotting and qRT‒PCR. Finally, ChIP‒qPCR was performed to determine the levels of histone modifications (H3K4me3, H3K27me3, and H2AK119Ub) in the PKM2 promoter region. GO functional enrichment analysis revealed that muscone was involved in regulating the biological processes (BP) of AMI, which mainly included negative regulation of the apoptosis signaling pathway, the response to lipopolysaccharide, and blood pressure regulation. The cellular components (CC) involved in muscone-mediated regulation of AMI mainly included lipid rafts, membrane microdomains, and membrane regions. The molecular functions (MF) involved in muscone-mediated regulation of AMI mainly included oxidoreductase activity, nuclear receptor activity, and transcription factor activity. In vitro results indicated that muscone treatment could inhibit the expression levels of Bax and Caspase-3 in AC16 cells after ischemia‒reperfusion while increasing the expression level of the antiapoptotic protein Bcl-2. Muscone significantly suppressed the transcription levels of p53 and PUMA in AC16 cells. Molecular docking suggested that muscone could bind well with the Cryo-EM structure of NEK7(PDB ID:6NPY). Further investigation of inflammatory pathways revealed that muscone could inhibit the expression level of NLRP3 in AC16 cells and reduce the expression levels of Caspase-1 and Caspase recruitment domain. Fluorescent quantitative PCR experiments showed that muscone significantly inhibited the transcription of NLRP3. Moreover, we found that muscone could enhance the glycolytic efficiency of AC16 cells, which may be related to the increased protein expression of PKM2 in AC16 cells. Fluorescent quantitative PCR showed that muscone could increase the transcription level of PKM2. Chromatin immunoprecipitation assays showed that muscone treatment increased the expression level of H3K4me3 in the PKM2 promoter region and inhibited the levels of H3K27me3 and H2AK119Ub in the PKM2 promoter region. Muscone promoted myocardial glycolysis and inhibited NLRP3 pathway activation to improve myocardial ischemia‒reperfusion injury.
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影响因子：
5.6
作者：
Ciążyńska M;Olejniczak-Staruch I;Sobolewska-Sztychny D;Narbutt J;Skibińska M;Lesiak A
通讯作者：
Lesiak A
影响因子：
3.6
作者：
Ramli FF;Ali A;Ibrahim N'
通讯作者：
Ibrahim N'
影响因子：
3.5
作者：
Toldo, Stefano;Marchetti, Carlo;Abbate, Antonio
通讯作者：
Abbate, Antonio
影响因子：
9.3
作者：
Algoet, Michiel;Janssens, Stefan;Oosterlinck, Wouter
通讯作者：
Oosterlinck, Wouter
影响因子：
9.5
作者：
Liu, Yi;Lian, Kun;Tao, Ling
通讯作者：
Tao, Ling