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NLRP3介导的细胞焦亡在心肌细胞缺氧/复氧损伤中的作用 被引量:9

Effect of NLRP3 mediated pyroptosis in myocardial cells undergoing hypoxia/deoxygenation injury
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摘要 目的探讨核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)介导的细胞焦亡在心肌细胞缺氧/复氧损伤中的作用。方法为明确缺氧/复氧是否会引起细胞焦亡,采用抓阄法将H9c2心肌细胞随机分为2组:对照组(细胞不予缺氧/复氧处理)和缺氧/复氧组(细胞进行缺氧/复氧处理);为了明确焦亡在缺氧/复氧损伤中的作用,采用抓阄法将细胞随机分为4组:对照组(细胞正常培养)、YVAD组[用含有20 μm Z-缬氨酸-丙氨酸-消旋天冬氨酸(甲酯)-氟甲基酮(Z-YVAD-FMK)的培养基预处理4 h后,换普通培养基正常培养]、缺氧/复氧组(细胞进行缺氧/复氧处理)和YVAD+缺氧/复氧组(用含有20 μm Z-YVAD-FMK的培养基处理4 h后,再进行缺氧/复氧处理);为明确缺氧/复氧诱导的细胞焦亡是否与NLRP3有关,采用抓阄法将细胞随机分为4组:对照组[培养基中加入非特异性小干扰RNA(si-RNA)]、si-NLRP3组(培养基中加入NLRP3的si-RNA)、缺氧/复氧组(对培养基中加入非特异性si-RNA的细胞进行缺氧/复氧处理)和si-NLRP3+缺氧/复氧组(对培养基中加入NLRP3 si-RNA的细胞进行缺氧/复氧处理)。采用碘化丙啶(PI)染色检测细胞膜上的孔隙形成,采用CCK8法检测细胞活力,采用Western blot检测NLRP3、焦亡相关蛋白Caspase-1和白细胞介素1β(IL-1β)的蛋白表达水平。结果(1)缺氧/复氧组PI染色阳性率[(26.46±5.15)%比(1.69±0.73)%]、NLRP3(0.57±0.16比0.23±0.06)、Caspase-1(1.07±0.13比0.37±0.08)和IL-1β(0.38±0.08比0.16±0.05)蛋白表达水平均高于对照组(P均<0.01)。(2)YVAD+缺氧/复氧组细胞活力高于缺氧/复氧组[(87.31±9.05)%比(73.30±7.19)%,P<0.05],PI染色阳性细胞率低于缺氧/复氧组[(18.12±4.36)%比(26.45±4.60)%,P<0.05],Caspase-1(0.72±0.12比1.07±0.15,P<0.05)和IL-1β(0.29±0.07比0.39±0.06,P<0.05)蛋白表达水平均低于缺氧/复氧组。(3)si-NLRP3+缺氧/复氧组细胞活力高于缺氧/复氧组[(85.46±7.71)%比(72.41±5.53)%,P<0.05],PI染色阳性率低于缺氧/复氧组[(18.22±4.20)%比(26.73±3.26)%,P<0.05],Caspase-1(0.87±0.07比1.15±0.15,P<0.05)和IL-1β(0.41±0.07比0.58±0.10,P<0.05)蛋白表达水平均低于缺氧/复氧组。结论NLRP3介导的细胞焦亡与心肌细胞缺氧/复氧损伤有关。 Objective To investigate the effect of NACHT-LRR-PYD- containing proteins 3 (NLRP3) mediated pyroptosis in myocardial cells undergoing hypoxia/deoxygenation (H/R) injury.Methods In order to observe whether H/R-treatment could cause pyroptosis,H9c2 cells were divided into 2 groups randomly using the lottery method:control group(without H/R-treatment) and H/R group (in which the H9c2 cells were underwent H/R-treatment).In order to clarify the role of pyroptosis in H/R-injury,H9c2 cells were divided into 4 groups randomly using the lottery method:control group(in which the H9c2 cells were cultivated with normal medium);YVAD group(in which the H9c2 cells were pretreated with z-Val-Ala-Asp(Ome)-fluoromethylketone (Z-YVAD-FMK) 20 μm for 4 hours,then replaced with normal medium);H/R group(H9c2 cells underwent H/R-treatment);YVAD+H/R group (in which the H9c2 cells were pretreated with 20 μm Z-YVAD-FMK for 4 hours before H/R-treatment).To determine whether H/R-induced cell pyroptosis is associated with NLRP3,H9c2 cells were divided into 4 groups randomly using the lottery method:control group (in which cells were transfected with a control nonspecific siRNA);si-NLRP3 group (in which cells were transfected with NLRP3-targeting siRNA);H/R group(in which cells were transfected with a control nonspecific siRNA before H/R-treatment);si-NLRP3+H/R group(in which the H9c2 cells were transfected with NLRP3-targeting siRNA before H/R-treatment).Pore formation on cell membrane was detected by propidium iodide (PI) staining.Cell viability was detected by CCK8 reagent.The protein expression of Caspase-1,interleukin-1β(IL-1β) and NLRP3 was detected by Western blot.Results (1) The positive rate of PI staining ((26.46±5.15)% vs.(1.69±0.73)%,P<0.01),expression of NLRP3 (0.57±0.16 vs.0.23±0.06,P<0.01),expression of Caspase-1 (1.07±0.13 vs.0.37±0.08,P<0.01),and expression of IL-1β(0.38±0.08 vs.0.16±0.05,P<0.01) were significantly higher in H/R group than in control group.(2)The cell vitality was significantly higher in YVAD+H/R group than in H/R group ((87.31±9.05)% vs.(73.30±7.19)%,P<0.05).The positive rate of PI staining was significantly decreased in YVAD+H/R group than in H/R group ((18.12±4.36)% vs.(26.45±4.60)%,P<0.05).The expression of Caspase-1 (0.72±0.12 vs.1.07±0.15,P<0.05) and IL-1β(0.29±0.07 vs.0.39±0.06,P<0.05) were significantly lower in YVAD+H/R group than in H/R group.(3) The cell vitality was significantly increased in si-NLRP3+H/R group than in H/R group ((85.46±7.71)% vs.(72.41±5.53)%,P<0.05).The positive rate of PI staining was significantly lower in si-NLRP3+H/R group than in H/R group ((18.22±4.20)% vs.(26.73±3.26)%,P<0.05).The expression of Caspase-1(0.87±0.07 vs.1.15±0.15,P<0.05) and IL-1β(0.41±0.07 vs.0.58±0.10,P<0.05) were significantly decreased in si-NLRP3+H/R group than in H/R group.Conclusion NLRP3 mediated pyroptosis is involved in H/R injury of myocardial cells.
作者 岳荣川 卢圣忠 罗瑜 曾静 梁豪 王小波 秦丹 杨小利 胡厚祥 曾春雨 Yue Rongchuan;Lu Shengzhong;Luo Yu;Zeng Jing;Liang Hao;Wang Xiaobo;Qin Dan;Yang Xiaoli;Hu Houxiang;Zeng Chunyu(Department of Cardiology,North Sichuan Medical College First Affiliated Hospital,Nanchong 637000,China;Department of Cardiology,Daping Hospital,Third Military Medical University,Chongqing 400042,China)
出处 《中华心血管病杂志》 CAS CSCD 北大核心 2019年第6期471-478,共8页 Chinese Journal of Cardiology
基金 国家自然科学基金项目(81600232) 四川省教育厅重点项目(17ZA0183) 南充市市校合作科研专项(18SXHZ0458,NSMC20170210) 川北医学院博士科研启动基金(CBY15-QD12).
关键词 肌细胞 心脏 缺氧/复氧 焦亡 Myocytes, cardiac Hypoxia/reoxygenation Pyroptosis
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