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 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 PDF全文下载地址:
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