| 李 伟,王龙涛,陈川斌,林 朋,李小蕤.基于NF-κB通路探讨IRAK3基因表达对脂多糖诱导的心肌细胞损伤的作用及机制[J].现代生物医学进展英文版,2022,(22):4242-4246. |
| 基于NF-κB通路探讨IRAK3基因表达对脂多糖诱导的心肌细胞损伤的作用及机制 |
| Based on NF-κB Pathway to Explore the Effect and Mechanism of IRAK3 Gene Expression on Lipopolysaccharide Induced Cardiomyocyte Injury |
| Received:May 06, 2022 Revised:May 28, 2022 |
| DOI：10.13241/j.cnki.pmb.2022.22.008 |
| 中文关键词: IRAK3 LPS 心肌细胞损伤 NF-κB通路 |
| 英文关键词: IRAK3 LPS Myocardial cell injury NF-κB pathway |
| 基金项目:海南省卫生健康行业科研项目（20A200521） |
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| 中文摘要: |
| 摘要 目的：探讨白介素1受体相关激酶3（IRAK3）基因表达对脂多糖（LPS）诱导的心肌细胞损伤的作用及机制。方法：分离培养SD乳鼠原代心肌细胞，随机分为对照组、LPS组、siIRAK3组和siIRAK3+LPS组。siIRAK3组和siIRAK3+LPS组心肌细胞转染IRAK3沉默核糖核酸（siRNA），对照组和LPS组转染阴性对照siRNA。转染48 h后LPS组和siIRAK3+LPS组分别用LPS（10 μg/mL）处理心肌细胞6 h，对照组和siIRAK3组加入等量的PBS溶液。采用免疫蛋白印迹法（Western blot）检测LPS对心肌细胞IRAK3表达的影响，采用细胞计数试剂盒-8（CCK-8）法检测四组心肌细胞增殖，采用原位末端转移酶标记技术（TUNEL）检测四组心肌细胞凋亡。采用酶联免疫吸附测定（ELISA）检测四组心肌细胞上清液中炎症因子白介素（IL）-6、肿瘤坏因子（TNF）-α的水平。Western blot检测核转录因子-κB（NF-κB）蛋白和NF-κB抑制蛋白α（IκB-α）的表达。结果：Western bolt结果显示，LPS使原代心肌细胞IRAK3的蛋白表达升高（P<0.05），CCK-8和TUNEL结果显示，与对照组相比，LPS组心肌细胞活力降低，心肌细胞凋亡比例升高（P<0.05）；siIRAK3组细胞活力和细胞凋亡比例与对照组相比均无明显差异（P>0.05）。与LPS组相比，siIRAK3+LPS组心肌细胞活力升高，心肌细胞凋亡比例降低（P<0.05）。与对照组相比，LPS组心肌细胞分泌的IL-6、TNF-α升高，NF-κB蛋白表达升高而IκB-α蛋白表达降低（P<0.05）。与LPS组相比，siIRAK3+LPS组心肌细胞炎症因子分泌减少，NF-κB蛋白表达降低，IκB-α蛋白表达升高（P<0.05）。结论：干扰IRAK3基因表达通过负向调控NF-κB通路减轻LPS诱导的大鼠原代心肌细胞损伤。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate the effect and mechanism of interleukin-1 receptor-associated kinase 3 (IRAK3) gene expression on lipopolysaccharide (LPS)-induced cardiomyocyte injury. Methods: Primary cardiomyocyte of SD suckling mice were isolated and cultured, and they were divided into the control group, LPS group, siIRAK3 group and siIRAK3 combined with LPS group. The cardiomyocyte in the siIRAK3 group and siIRAK3 combined with LPS group were transfected with IRAK3 silencing ribonucleic acid (siRNA). Control group and LPS group were transfected with negative control siRNA. 48 h after transfection, cardiomyocyte in the LPS group and siIRAK3 combined with LPS group were treated with LPS (10 μg/mL) for 6h respectively, and the control group and siIRAK3 group were added with the same amount of PBS solution. The Western blot (Western bolt) was used to detect the effect of LPS on cardiomyocyte IRAK3 expression, The Cell Counting Kit-8 (CCK-8) was used to detect the proliferation of four groups of cardiomyocyte, and Terminal deoxynucleotidyl transferasedUTP nick end labeling (TUNEL) was used to detect the apoptosis of four groups of cardiomyocyte. Enzyme linked immunosorbent assay (ELISA) was used to detect the inflammatory factors interleukin (IL)-6 and tumor necrosis factor (TNF) -α levels in the supernatant of the four groups of cardiomyocyte. The expressions of nuclear factor-κB (NF-κB) protein and NF-κB inhibitor protein α (IκB-α) were detected by Western Blot. Results: Western Blot results showed that LPS increased the protein expression of IRAK3 in primary cardiomyocyte (P<0.05), CCK-8 and TUNEL results showed that compared with the control group, the viability of cardiomyocyte in the LPS group was decreased, and the cardiomyocyte apoptosis ratio was increased (P<0.05). There were no significant differences in cell viability and cell apoptosis ratio between siIRAK3 group and control group (P>0.05). Compared with the LPS group, the viability of cardiomyocyte in the siIRAK3 combined with LPS group was increased, and the cardiomyocyte apoptosis ratio was decreased (P<0.05). Compared with the control group, the secretion of IL-6 and TNF-α in the cardiomyocyte of LPS group was increased, the protein expression of NF-κB was increased, and the protein expression of IκB-α was decreased (P<0.05). Compared with the LPS group, siIRAK3combined with LPS group showed decreased secretion of inflammatory cytokines, decreased expression of NF-κB protein, and increased expression of IκB-α protein (P<0.05). Conclusion: Interference of IRAK3 gene expression attenuates LPS-induced injury of primary rat cardiomyocyte by negatively regulating NF-κB pathway. |
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