上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2021 , Vol. 41 >Issue 11: 1446 - 1453

DOI: https://doi.org/10.3969/j.issn.1674-8115.2021.11.007

论著 · 基础研究

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
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  • 上海交通大学医学院附属同仁医院心内科，上海 200336
陈露露（1994—），女，硕士生；电子信箱：118732910690@sjtu.edu.cn。
邱朝晖，电子信箱：qzh3503@shtrhospital.com。

网络出版日期: 2021-12-03

基金资助

上海市长宁区心血管内科重点专科项目(20161001)

收起

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
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  • Department of Cardiology, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200336, China
QIU Zhao-hui, E-mail: qzh3503@shtrhospital.com.

Online published: 2021-12-03

Supported by

Key Specialist Project of Cardiovascular Medicine in Changning District, Shanghai(20161001)

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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

本文引用格式

陈露露 , 徐勋龙 , 陈婉岚 , 邱朝晖 . 多肽PDSORBS2对缺氧/复氧诱导的H9C2细胞凋亡的影响[J]. 上海交通大学学报（医学版）, 2021 , 41(11) : 1446 -1453 . DOI: 10.3969/j.issn.1674-8115.2021.11.007

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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