| shRNA干扰NLRP3对晚期糖基化终末产物诱导的心肌细胞炎症反应的影响 |
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| 引用本文： | 贾合磊,卢长青,王娟,任冬冬,陈亚奇. shRNA干扰NLRP3对晚期糖基化终末产物诱导的心肌细胞炎症反应的影响[J]. 四川大学学报(医学版), 2019, 50(1): 7-12. DOI: 10.13464/j.scuxbyxb.2019.01.002 |
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| 作者姓名： | 贾合磊 卢长青 王娟 任冬冬 陈亚奇 |
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| 作者单位： | 河南省中医院(河南中医药大学第二附属医院)急诊科 郑州450002;河南省中医院(河南中医药大学第二附属医院)急诊科 郑州450002;河南省中医院(河南中医药大学第二附属医院)急诊科 郑州450002;河南省中医院(河南中医药大学第二附属医院)急诊科 郑州450002;河南省中医院(河南中医药大学第二附属医院)急诊科 郑州450002 |
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| 摘 要： | 目的 探讨沉默NOD 样受体家族pyrin域3 (NOD-like receptor family,pyrin domain containing 3, NLRP3)炎性小体对晚期糖基化终末产物(advanced glycation end products, AGEs)诱导的心肌细胞损伤的调节作用及机制。 方法 H9c2心肌细胞分为4组:对照组、 AGEs组、AGEs+sh-Ctrl组、 AGEs+sh-NLRP3组，后两组细胞首先将短发夹RNA（shRNA）对照（sh-Ctrl）和shRNA干扰NLRP3（sh-NLRP3）质粒分别转染入H9c2细胞，然后后3组细胞用100 mg/L AGEs处理24 h，建立H9c2损伤模型，对照组细胞用溶剂处理24 h。Western blot检测NLRP3、凋亡相关微粒蛋白(apoptosis-associated speck-like protein containing CARD, ASC)、 Caspase-3、 Caspase-9、核转录因子κB(nuclear factor κB, NF-κB) P65和磷酸化P65（p-P65）的表达，酶联免疫吸附实验(ELISA)检测白细胞介素(interleukin, IL)-6、IL-18和IL-1β的水平，流式细胞术检测细胞凋亡，免疫荧光染色检测细胞核中NF-κB P65的水平。结果 AGEs组和AGEs+sh-Ctrl组NLRP3、 ASC、Caspase-3和Caspase-9的表达均高于对照组( P<0.05)。与AGEs组相比，AGEs+sh-NLRP3组NLRP3、ASC、Caspase-3和Caspase-9的表达均下降( P<0.05)。与对照组相比，AGEs组和AGEs+sh-Ctrl组IL-6、IL-18、IL-1β水平升高，细胞凋亡率上升(P<0.05)。AGEs+sh-NLRP3组IL-6、IL-18、IL-1β水平和细胞凋亡率低于AGEs组( P<0.05)。AGEs组和AGEs+sh-Ctrl组NF-κB P65磷酸化水平及细胞核的NF-κB P65水平高于对照组(P<0.05)。与AGEs组相比，AGEs+sh-NLRP3组NF-κB P65磷酸化水平及细胞核的NF-κB P65水平降低( P<0.05)。结论 沉默NLRP3可通过抑制NF-κB P65的活化减轻AGEs诱导的心肌细胞凋亡及炎症反应。
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| 关 键 词： | NLRP3炎性小体 晚期糖基化终末产物 心肌损伤 细胞凋亡 炎症 |
The Effect of Interference of NLRP3 with shRNA on AGEs-induced Inflammatory Response in Myocardial Cell |
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| JIA He-lei,LU Chang-qing,WANG Juan,et al. The Effect of Interference of NLRP3 with shRNA on AGEs-induced Inflammatory Response in Myocardial Cell[J]. Journal of Sichuan University. Medical science edition, 2019, 50(1): 7-12. DOI: 10.13464/j.scuxbyxb.2019.01.002 |
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| Authors: | JIA He-lei LU Chang-qing WANG Juan et al |
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| Abstract: | Objective This study aims to explore the effect of silence of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome on advanced glycation end products (AGEs)-induced myocardial injury. Methods The myocardial injury model was indued by AGEs. NLRP3 was silenced by shRNA. H9c2 cells were divided into four groups: H9c2 (control group); AGEs group; AGEs+sh-Ctrl group; AGEs+sh-NLRP3 group. The latter two groups of cells will first shRNA control （sh-Ctrl） and shRNA-NLRP3 （sh-NLRP3） plasmids were transfected into H9c2 cells, the last 3 cells were then treated for 24 h with 100 mg/L AGEs, establishment of H9c2 damage model, control cells were treated with solvent for 24 h; Apoptosis was measured by Hoechst33258 staining. The levels of interleukin (IL)-6, IL-18 and IL-1β were detected by enzyme-linked immunosorbent assay (ELISA). The protein levels of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), Caspase-3, Caspase-9, nuclear factor κB (NF-κB) P65 and p-P65 were tested by Western blot. The nuclear NF-κB P65 levels were detected by immunofluorescent staining. Results The expressions of NLRP3, ASC, Caspase-3 and Caspase-9 in AGEs group and AGEs+sh-Ctrl group was higher than control group ( P<0.05). Compared with AGEs group, the expressions of NLRP3, ASC, Caspase-3 and Caspase-9 in AGEs+sh-NLRP3 group was decreased ( P<0.05). Compared with control group, the apoptosis and the levels of IL-6, IL-18 and IL-1β in AGEs group and AGEs+sh-Ctrl group were elevated (P<0.05). The apoptosis and the levels of IL-6, IL-18 and IL-1β in AGEs+sh-NLRP3 group were lower than those of AGEs group ( P<0.05). The phosphorylation of NF-κB P65 and nuclear NF-κB P65 in AGEs group and AGEs+sh-Ctrl group were higher than control group (P<0.05). Compared with AGEs group, the phosphorylation of NF-κB P65 and nuclear NF-κB P65 in AGEs+sh-NLRP3 group were reduced ( P<0.05). Conlusion Silence of NLRP3 inflammasome alleviates AGEs-induced apoptosis and inflammatory response in myocardial cell via inhibiting NF-κB P65 activation. |
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| Keywords: | NLRP3 inflammasome Advanced glycation end products Myocardial injury ApoptosisInflammation |
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