MEDLINE/PubMed Journal Browser Search

Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

Title: [Effect of NLRP3 mediated pyroptosis in myocardial cells undergoing hypoxia/deoxygenation injury].
Author: Yue RC, Lu SZ, Luo Y, Zeng J, Liang H, Wang XB, Qin D, Yang XL, Hu HX, Zeng CY.
Journal: Zhonghua Xin Xue Guan Bing Za Zhi; 2019 Jun 24; 47(6):471-478. PubMed ID: 31262132.
Abstract:
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. 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。.

[Abstract] [Full Text] [Related] [New Search]