Department of Cardiology, Ruijin Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Luwan Branch), Shanghai, China.
J Biochem Mol Toxicol. 2022 Jun;36(6):e23034. doi: 10.1002/jbt.23034. Epub 2022 Mar 21.
Catalpol (CA) is widely used in the protection of cardiomyocytes. Nevertheless, the mechanism of CA in alleviating ischemia-reperfusion-induced injury of cardiomyocytes remains unclear. Human cardiomyocyte AC16 cells were subjected to hypoxia/reoxygenation (H/R) injury. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis were applied to detect tumor necrosis factor-alpha (TNF-α) mRNA, interleukin-6 (IL-6) mRNA, interleukin-1beta (IL-1β) mRNA, microRNA-22-3p (miR-22-3p), dipeptidyl peptidase 4 (DPP4) mRNA, and DPP4 protein expressions. The cell viability and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Lactate dehydrogenase (LDH) and creatine kinase (CK-MB) were examined by enzyme-linked immunosorbent assay (ELISA) kits. A dual-luciferase reporter gene assay was performed to confirm the binding sequence between miR-22-3p and DPP4 mRNA 3'-untranslated region (3'UTR). CA promoted the viability and reduced cell apoptosis of AC16 cells and repressed the release of inflammatory cytokines TNF-α, IL-6, and IL-1β, and inhibited the leakage of myocardial injury markers LDH and CK-MB. Furthermore, CA enhanced the expression of miR-22-3p in cardiomyocytes, and DPP4 was validated to be the target gene of miR-22-3p. The inhibition of miR-22-3p and augmentation of DPP4 reversed the above effects of CA. CA protects A16 cells from H/R injury by regulating the miR-22-3p/DPP4 axis.
梓醇 (CA) 广泛用于保护心肌细胞。然而，CA 缓解心肌细胞缺血再灌注损伤的机制尚不清楚。将人心肌细胞 AC16 细胞进行缺氧/复氧 (H/R) 损伤。应用实时定量聚合酶链反应 (qRT-PCR) 和 Western blot 分析检测肿瘤坏死因子-α (TNF-α) mRNA、白细胞介素-6 (IL-6) mRNA、白细胞介素-1β (IL-1β) mRNA、微小 RNA-22-3p (miR-22-3p)、二肽基肽酶 4 (DPP4) mRNA 和 DPP4 蛋白表达。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐 (MTT) 测定法和流式细胞术分别测定细胞活力和细胞凋亡。通过酶联免疫吸附测定 (ELISA) 试剂盒测定乳酸脱氢酶 (LDH) 和肌酸激酶同工酶 (CK-MB)。通过双荧光素酶报告基因实验验证 miR-22-3p 与 DPP4 mRNA 3'-非翻译区 (3'UTR) 的结合序列。CA 促进 AC16 细胞的活力并减少细胞凋亡，抑制炎症细胞因子 TNF-α、IL-6 和 IL-1β 的释放，并抑制心肌损伤标志物 LDH 和 CK-MB 的漏出。此外，CA 增强了心肌细胞中 miR-22-3p 的表达，并且验证了 DPP4 是 miR-22-3p 的靶基因。miR-22-3p 的抑制和 DPP4 的增加逆转了 CA 的上述作用。CA 通过调节 miR-22-3p/DPP4 轴来保护 A16 细胞免受 H/R 损伤。
