上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (11): 1446-1453.doi: 10.3969/j.issn.1674-8115.2021.11.007

• 论著 · 基础研究 • 上一篇    下一篇

多肽PDSORBS2对缺氧/复氧诱导的H9C2细胞凋亡的影响

陈露露(), 徐勋龙, 陈婉岚, 邱朝晖()   

  1. 上海交通大学医学院附属同仁医院心内科,上海 200336
  • 出版日期:2021-11-28 发布日期:2021-12-03
  • 通讯作者: 邱朝晖 E-mail:118732910690@sjtu.edu.cn;qzh3503@shtrhospital.com
  • 作者简介:陈露露(1994—),女,硕士生;电子信箱:118732910690@sjtu.edu.cn
  • 基金资助:
    上海市长宁区心血管内科重点专科项目(20161001)

Effect of PDSORBS2 peptide on H9C2 cell apoptosis induced by hypoxia and reoxygenation

Lu-lu CHEN(), Xun-long XU, Wan-lan CHEN, Zhao-hui QIU()   

  1. Department of Cardiology, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200336, China
  • Online:2021-11-28 Published:2021-12-03
  • Contact: Zhao-hui QIU E-mail:118732910690@sjtu.edu.cn;qzh3503@shtrhospital.com
  • Supported by:
    Key Specialist Project of Cardiovascular Medicine in Changning District, Shanghai(20161001)

摘要:

目的·探究多肽PDSORBS2对缺氧/复氧(hypoxia/heoxygenation,H/R)诱导的H9C2细胞凋亡的抑制作用以及作用机制。方法·在前期的工作中,从缺血后的大鼠心肌细胞中发现了1种新型肽PDSORBS2(LPASLNS)。采用细胞计数试剂盒8(cell counting kit-8,CCK-8)检测细胞的活性,以选择合适浓度的多肽和H/R处理的合适时间。随机将细胞分成正常对照组(NC组)、NC+PDSORBS2组、H/R组和H/R+PDSORBS2组,使用荧光显微镜观察细胞核形态的变化,利用流式细胞术分析细胞凋亡的程度和细胞周期,使用试剂盒检测细胞内活性氧(reactive oxygen species,ROS)的含量,通过蛋白质免疫印迹(Western blotting,WB)技术检测H9C2细胞中细胞凋亡相关蛋白聚ADP核糖聚合酶(poly ADP-ribose polymerase,PARP)、cleaved-caspase3、B淋巴细胞瘤2蛋白(B-cell lymphoma-2,Bcl-2)、BCL2-associated X(Bax)蛋白以及胞外信号调节激酶(extracellular signal-regulated kinase,ERK)、蛋白激酶B (protein kinase B,AKT)、周期蛋白依赖性激酶2(cyclin-dependent kinases 2,CDK2)、p27Kip1蛋白的表达水平。结果·与NC组相比,缺氧6 h-复氧2 h的处理明显使H9C2细胞活性下降,而50 μmol/L的PDSORBS2显著增加了H/R诱导的H9C2细胞活性(P=0.004)。与H/R组相比,PDSORBS2的干预改善了H/R诱导的H9C2细胞核的形态变化,使H/R诱导的H9C2细胞的凋亡率下降(P=0.000),细胞周期阻滞减轻(P=0.000),细胞内ROS的含量下降(P=0.005);促凋亡蛋白PARP、Bax、cleaved-caspase3和p27Kip1蛋白的表达水平下调,而Bcl-2、磷酸化细胞外信号调节激酶(phospho-extracellular regulated protein kinases,P-ERK)、磷酸化蛋白激酶B(phospho-protein kinase B,P-AKT)、CDK2等蛋白的表达水平增高。结论·多肽PDSORBS2可能通过ERK/AKT/CDK2/p27Kip1的信号通路抑制H/R诱导的H9C2细胞的凋亡。

关键词: 多肽PDSORBS2, 缺氧/复氧, 凋亡, H9C2细胞

Abstract:

Objective·To explore the inhibitory effect and its mechanism of the PDSORBS2 peptide on H9C2 cell apoptosis induced by hypoxia and reoxygenation (H/R).

Methods·In the previous work of this study, a new peptide PDSORBS2 (LPASLNS) was discovered from rat cardiomyocytes after ischemia. Cell counting kit-8 (CCK-8) was used to detect cell viability to select the appropriate concentration of PDSORBS2 peptide and the appropriate time for H/R treatment. The cells were randomly divided into normal control group (NC group), NC+PDSORBS2 group, H/R group and H/R+PDSORBS2 group. A fluorescence microscope was used to observe the changes in the nucleus morphology. The degree of apoptosis and cell cycle was analyzed by flow cytometry. A kit was used to detect the content of reactive oxygen species (ROS) in cells. The expression levels of apoptosis-related proteins including poly ADP-ribose polymerase (PARP), cleaved-caspase3, B-cell lymphoma-2 (Bcl-2), and BCL2-associated X (Bax), extracellular regulated protein kinases (ERK), protein kinase B (AKT), cyclin-dependent kinases 2 (CDK2), and p27Kip1 protein were analyzed by Western blotting in H9C2 cells.

Results·Compared with the NC group, 6 h of hypoxia and 2 h of reoxygenation significantly decreased the viability of H9C2 cells, while 50 μmol/L PDSORBS2 significantly increased the viability of H9C2 cells induced by H/R (P=0.004). Compared with the H/R group, the pretreatment of PDSORBS2 improved the morphology of H9C2 nuclei induced by H/R and reduced the apoptotic rate of H9C2 cells (P=0.000), cell cycle arrest (P=0.000), and intracellular ROS content (P=0.005). The expression levels of pro-apoptotic proteins (PARP, Bax and cleaved-caspase3) and p27Kip1 protein were down-regulated, and the expression levels of Bcl-2, phospho-extracellular regulated protein kinases (P-ERK), phospho-protein kinase B (P-AKT), CDK2 etc. increased.

Conclusion·The PDSORBS2 may inhibit H/R-induced apoptosis of H9C2 cells through the ERK/AKT/CDK2/p27Kip1 signaling pathway.

Key words: PDSORBS2 peptide, hypoxia and reoxygenation (H/R), apoptosis, H9C2 cells

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