Luteolin Pretreatment Ameliorates Myocardial Ischemia/Reperfusion Injury by lncRNA-JPX/miR-146b Axis

IF 2.6 4区医学 Q3 CELL BIOLOGY

Analytical Cellular Pathology Pub Date : 2023-12-02 DOI:10.1155/2023/4500810

Tongda Xu, Yuanyuan Zhang, Gege Liao, Haochen Xuan, Jie Yin, Jieli Bao, Yang Liu, Dongye Li

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Western blot was utilized to examine apoptosis-related protein expression levels, including cleaved caspase-9, caspase-9, cleaved caspase-3, caspase-3, Bcl-2, Bax, and BAG-1. Apoptosis was analyzed by Annexin V-APC/7-AAD dualstaining, Hoechst 33342 staining, as well as flow cytometry. Animal echocardiography was used to measure cardiac function (ejection fraction (EF) and fractional shortening (FS) indicators). <i>Results</i>. miR-146b was demonstrated to bind and recognize the JPX sequence site by dual luciferase reporter gene assay. The expression level of miR-146b was corroborated to be enhanced by H/R using RT-qPCR (<span><svg height=\"9.2729pt\" style=\"vertical-align:-0.6370001pt\" version=\"1.1\" viewbox=\"-0.0498162 -8.6359 19.289 9.2729\" width=\"19.289pt\" xmlns=\"http://www.w3.org/2000/svg\" xmlns:xlink=\"http://www.w3.org/1999/xlink\"><g transform=\"matrix(.013,0,0,-0.013,0,0)\"></path></g><g transform=\"matrix(.013,0,0,-0.013,11.658,0)\"></path></g></svg><span></span><svg height=\"9.2729pt\" style=\"vertical-align:-0.6370001pt\" version=\"1.1\" viewbox=\"22.8711838 -8.6359 28.182 9.2729\" width=\"28.182pt\" xmlns=\"http://www.w3.org/2000/svg\" xmlns:xlink=\"http://www.w3.org/1999/xlink\"><g transform=\"matrix(.013,0,0,-0.013,22.921,0)\"></path></g><g transform=\"matrix(.013,0,0,-0.013,29.161,0)\"></path></g><g transform=\"matrix(.013,0,0,-0.013,32.125,0)\"><use xlink:href=\"#g113-49\"></use></g><g transform=\"matrix(.013,0,0,-0.013,38.365,0)\"><use xlink:href=\"#g113-49\"></use></g><g transform=\"matrix(.013,0,0,-0.013,44.605,0)\"></path></g></svg></span> vs. Con). Moreover, JPX could reduce the expression of miR-146b, whereas inhibiting JPX could reverse the alteration (<span><svg height=\"9.2729pt\" style=\"vertical-align:-0.6370001pt\" version=\"1.1\" viewbox=\"-0.0498162 -8.6359 19.289 9.2729\" width=\"19.289pt\" xmlns=\"http://www.w3.org/2000/svg\" xmlns:xlink=\"http://www.w3.org/1999/xlink\"><g transform=\"matrix(.013,0,0,-0.013,0,0)\"><use xlink:href=\"#g113-81\"></use></g><g transform=\"matrix(.013,0,0,-0.013,11.658,0)\"><use xlink:href=\"#g117-91\"></use></g></svg><span></span><svg height=\"9.2729pt\" style=\"vertical-align:-0.6370001pt\" version=\"1.1\" viewbox=\"22.8711838 -8.6359 28.182 9.2729\" width=\"28.182pt\" xmlns=\"http://www.w3.org/2000/svg\" xmlns:xlink=\"http://www.w3.org/1999/xlink\"><g transform=\"matrix(.013,0,0,-0.013,22.921,0)\"><use xlink:href=\"#g113-49\"></use></g><g transform=\"matrix(.013,0,0,-0.013,29.161,0)\"><use xlink:href=\"#g113-47\"></use></g><g transform=\"matrix(.013,0,0,-0.013,32.125,0)\"><use xlink:href=\"#g113-49\"></use></g><g transform=\"matrix(.013,0,0,-0.013,38.365,0)\"><use xlink:href=\"#g113-49\"></use></g><g transform=\"matrix(.013,0,0,-0.013,44.605,0)\"><use xlink:href=\"#g113-50\"></use></g></svg></span> vs. H/R, respectively). Western blot analysis demonstrated that Lut pretreatment increased BAG-1 expression level and Bcl-2/Bax ratio, but diminished the ratio of cleaved caspase 9/caspase 9 and cleaved caspase 3/caspase 3 (<span><svg height=\"9.2729pt\" style=\"vertical-align:-0.6370001pt\" version=\"1.1\" viewbox=\"-0.0498162 -8.6359 19.289 9.2729\" width=\"19.289pt\" xmlns=\"http://www.w3.org/2000/svg\" xmlns:xlink=\"http://www.w3.org/1999/xlink\"><g transform=\"matrix(.013,0,0,-0.013,0,0)\"><use xlink:href=\"#g113-81\"></use></g><g transform=\"matrix(.013,0,0,-0.013,11.658,0)\"><use xlink:href=\"#g117-91\"></use></g></svg><span></span><svg height=\"9.2729pt\" style=\"vertical-align:-0.6370001pt\" version=\"1.1\" viewbox=\"22.8711838 -8.6359 28.182 9.2729\" width=\"28.182pt\" xmlns=\"http://www.w3.org/2000/svg\" xmlns:xlink=\"http://www.w3.org/1999/xlink\"><g transform=\"matrix(.013,0,0,-0.013,22.921,0)\"><use xlink:href=\"#g113-49\"></use></g><g transform=\"matrix(.013,0,0,-0.013,29.161,0)\"><use xlink:href=\"#g113-47\"></use></g><g transform=\"matrix(.013,0,0,-0.013,32.125,0)\"><use xlink:href=\"#g113-49\"></use></g><g transform=\"matrix(.013,0,0,-0.013,38.365,0)\"><use xlink:href=\"#g113-49\"></use></g><g transform=\"matrix(.013,0,0,-0.013,44.605,0)\"><use xlink:href=\"#g113-50\"></use></g></svg></span> vs. H/R, respectively). Moreover, the cell apoptosis change trend, measured by Annexin V-APC/7-AAD dualstaining, Hoechst 33342 staining, along with flow cytometry, was consistent with that of apoptosis-related proteins. Furthermore, pretreatment with Lut improved cardiac function (EF and FS) (<span><svg height=\"9.2729pt\" style=\"vertical-align:-0.6370001pt\" version=\"1.1\" viewbox=\"-0.0498162 -8.6359 19.289 9.2729\" width=\"19.289pt\" xmlns=\"http://www.w3.org/2000/svg\" xmlns:xlink=\"http://www.w3.org/1999/xlink\"><g transform=\"matrix(.013,0,0,-0.013,0,0)\"><use xlink:href=\"#g113-81\"></use></g><g transform=\"matrix(.013,0,0,-0.013,11.658,0)\"><use xlink:href=\"#g117-91\"></use></g></svg><span></span><svg height=\"9.2729pt\" style=\"vertical-align:-0.6370001pt\" version=\"1.1\" viewbox=\"22.8711838 -8.6359 28.182 9.2729\" width=\"28.182pt\" xmlns=\"http://www.w3.org/2000/svg\" xmlns:xlink=\"http://www.w3.org/1999/xlink\"><g transform=\"matrix(.013,0,0,-0.013,22.921,0)\"><use xlink:href=\"#g113-49\"></use></g><g transform=\"matrix(.013,0,0,-0.013,29.161,0)\"><use xlink:href=\"#g113-47\"></use></g><g transform=\"matrix(.013,0,0,-0.013,32.125,0)\"><use xlink:href=\"#g113-49\"></use></g><g transform=\"matrix(.013,0,0,-0.013,38.365,0)\"><use xlink:href=\"#g113-49\"></use></g><g transform=\"matrix(.013,0,0,-0.013,44.605,0)\"><use xlink:href=\"#g113-50\"></use></g></svg></span> vs. I/R, respectively), as indicated in animal echocardiography. <i>Conclusion</i>. Our results demonstrated that <i>in vitro</i> and <i>in vivo</i>, Lut pretreatment inhibited apoptosis via the JPX/miR-146b axis, ultimately improving myocardial I/R injury.","PeriodicalId":49326,"journal":{"name":"Analytical Cellular Pathology","volume":null,"pages":null},"PeriodicalIF":2.6000,"publicationDate":"2023-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytical Cellular Pathology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1155/2023/4500810","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"CELL BIOLOGY","Score":null,"Total":0}

引用次数: 0

Abstract

Background. In the present study, we aimed to find out whether luteolin (Lut) pretreatment could ameliorate myocardial ischemia/reperfusion (I/R) injury by regulating the lncRNA just proximal to XIST (JPX)/microRNA-146b (miR-146b) axis. Methods. We established the models in vitro (HL-1 cells) and in vivo (C57BL/6J mice) to certify the protection mechanism of Lut pretreatment on myocardial I/R injury. Dual luciferase reporter gene assay was utilized for validating that JPX could bind to miR-146b. JPX and miR-146b expression levels were determined by RT-qPCR. Western blot was utilized to examine apoptosis-related protein expression levels, including cleaved caspase-9, caspase-9, cleaved caspase-3, caspase-3, Bcl-2, Bax, and BAG-1. Apoptosis was analyzed by Annexin V-APC/7-AAD dualstaining, Hoechst 33342 staining, as well as flow cytometry. Animal echocardiography was used to measure cardiac function (ejection fraction (EF) and fractional shortening (FS) indicators). Results. miR-146b was demonstrated to bind and recognize the JPX sequence site by dual luciferase reporter gene assay. The expression level of miR-146b was corroborated to be enhanced by H/R using RT-qPCR ( vs. Con). Moreover, JPX could reduce the expression of miR-146b, whereas inhibiting JPX could reverse the alteration ( vs. H/R, respectively). Western blot analysis demonstrated that Lut pretreatment increased BAG-1 expression level and Bcl-2/Bax ratio, but diminished the ratio of cleaved caspase 9/caspase 9 and cleaved caspase 3/caspase 3 ( vs. H/R, respectively). Moreover, the cell apoptosis change trend, measured by Annexin V-APC/7-AAD dualstaining, Hoechst 33342 staining, along with flow cytometry, was consistent with that of apoptosis-related proteins. Furthermore, pretreatment with Lut improved cardiac function (EF and FS) ( vs. I/R, respectively), as indicated in animal echocardiography. Conclusion. Our results demonstrated that in vitro and in vivo, Lut pretreatment inhibited apoptosis via the JPX/miR-146b axis, ultimately improving myocardial I/R injury.

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木犀草素预处理通过lncRNA-JPX/miR-146b轴改善心肌缺血/再灌注损伤

背景。在本研究中，我们旨在研究木犀草素(Lut)预处理是否可以通过调节XIST (JPX)/microRNA-146b (miR-146b)轴近端lncRNA来改善心肌缺血/再灌注(I/R)损伤。方法。我们建立了体外(HL-1细胞)和体内(C57BL/6J小鼠)模型，验证了Lut预处理对心肌I/R损伤的保护机制。利用双荧光素酶报告基因试验验证JPX可以与miR-146b结合。RT-qPCR检测JPX和miR-146b的表达水平。Western blot检测凋亡相关蛋白表达水平，包括cleaved caspase-9、caspase-9、cleaved caspase-3、caspase-3、Bcl-2、Bax和BAG-1。Annexin V-APC/7-AAD双染色、Hoechst 33342染色及流式细胞术检测细胞凋亡。采用动物超声心动图测量心功能(射血分数(EF)和缩短分数(FS)指标)。结果。通过双荧光素酶报告基因试验证明miR-146b结合并识别JPX序列位点。RT-qPCR证实miR-146b表达水平升高(与Con相比)。此外，JPX可以降低miR-146b的表达，而抑制JPX可以逆转这种改变(分别vs. H/R)。Western blot分析显示，Lut预处理提高了BAG-1的表达水平和Bcl-2/Bax比值，但降低了裂解型caspase 9/caspase 9和裂解型caspase 3/caspase 3的比值(分别比H/R)。Annexin V-APC/7-AAD双染色、Hoechst 33342染色及流式细胞术检测细胞凋亡变化趋势与凋亡相关蛋白变化趋势一致。此外，如动物超声心动图所示，Lut预处理可改善心功能(EF和FS)(分别vs. I/R)。结论。我们的研究结果表明，在体外和体内，Lut预处理通过JPX/miR-146b轴抑制细胞凋亡，最终改善心肌I/R损伤。

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来源期刊

Analytical Cellular Pathology ONCOLOGY-CELL BIOLOGY

CiteScore

4.90

自引率

3.10%

发文量

审稿时长

16 weeks

期刊介绍： Analytical Cellular Pathology is a peer-reviewed, Open Access journal that provides a forum for scientists, medical practitioners and pathologists working in the area of cellular pathology. The journal publishes original research articles, review articles, and clinical studies related to cytology, carcinogenesis, cell receptors, biomarkers, diagnostic pathology, immunopathology, and hematology.