多肽PDSORBS2对缺氧/复氧诱导的H9C2细胞凋亡的影响

doi:10.3969/j.issn.1674-8115.2021.11.007

摘要/Abstract

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）、p27^Kip1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p27^Kip1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/p27^Kip1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 p27^Kip1 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 p27^Kip1 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/p27^Kip1 signaling pathway.

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

中图分类号:

R965.1

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.

Lu-lu CHEN, Xun-long XU, Wan-lan CHEN, Zhao-hui QIU. Effect of PDSORBS2 peptide on H9C2 cell apoptosis induced by hypoxia and reoxygenation[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(11): 1446-1453.

图/表 5

图1 多肽PDSORBS2对H/R诱导的细胞活性的影响Note： A?C. The cell viability was detected by CCK-8 in H9C2 cells. ①P=0.000, ②P=0.004.

Fig 1 Effect of PDSORBS2 peptide on cell viability induced by H/R

图2 多肽PDSORBS2对H/R诱导的H9C2细胞氧化应激的影响Note： A. The viability of H9C2 cells was detected by Hoechst 33342 staining. B. ROS content in H9C2 cells. The data were analyzed by using one-way ANOVA. ①P=0.005.

Fig 2 Effect of PDSORBS2 peptide on H/R-induced oxidative stress in H9C2 cells

图3 多肽PDSORBS2对H/R诱导的H9C2细胞凋亡和细胞周期阻滞的影响Note： A. The apoptosis of the H9C2 cells was analyzed by using flow cytometer. B. The apoptosis rate of the H9C2 cells. C. The cell cycle was analyzed by using flow cytometer. D. Proportion of cells distributed in the G1 and S phases. ①P=0.000.

Fig 3 Effect of PDSORBS2 peptide on H/R-induced apoptosis and cell cycle arrest in H9C2 cells

图4 WB检测H9C2细胞中凋亡相关蛋白的表达Note： A. WB analysis of the PARP, cleaved-caspase3, Bcl-2 and Bax. B. The ratio of cleaved-PARP and PARP. C. The expression of cleaved-caspase3. D The expression of Bcl-2. E The expression of Bax. ①P=0.017, ②P=0.003, ③P=0.000, ④P=0.005.

Fig 4 Detection of the expression of apoptosis-related proteins in H9C2 cells by WB

图5 多肽PDSORBS2对H/R诱导的H9C2细胞中ERK/AKT/CDK2/p27Kip1蛋白水平的影响Note： A. WB analysis of AKT, p-AKT, ERK, p-ERK, p27Kip1 and CDK2. B. The ratio of p-AKT and AKT. C. The ratio of p-ERK and ERK. D. WB analysis of p27Kip1 and CDK2. E. The expression of CDK2. F. The expression of p27Kip1. The data were analyzed by using one-way ANOVA. ①P=0.033, ②P=0.000, ③P=0.009.

Fig 5 Effect of PDSORBS2 peptide on the protein levels of ERK/AKT/CDK2/p27Kip1 in H9C2 cells induced by H/R

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