He Jieqiong, Wang Chao, Sun Yunpeng, Lu Bo, Cui Jinjin, Dong Nana, Zhang Maomao, Liu Youbing, Yu Bo
The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Harbin, Heilongjiang, P.R. China.
Department of Cardiology, The First Hospital of Qiqihar, Qiqihar, Heilongjiang, P.R. China.
Int J Mol Med. 2016 Apr;37(4):889-900. doi: 10.3892/ijmm.2016.2509. Epub 2016 Feb 29.
Exendin-4 (ex-4) is a long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist which exerts beneficial effects on glycemic control and promotes cell viability. In the present study, we investigated the anti-apoptotic effects of ex-4, as well as the potential mechanisms responsible for these effects in rat bone marrow-derived mesenchymal stem cells (BM-MSCs) under conditions of oxygen, glucose and serum deprivation (OGD). The apoptosis of the MSCs was induced by subjecting the cells to OGD conditions for 4 h and was detected by Annexin V/PI and Hoechst 33258 staining. The MSCs were pre-conditioned with ex-4 for 12 h prior to being subjected to OGD conditions, and the expression levels of an apoptotic marker (cleaved caspase-3), endoplasmic reticulum (ER) stress markers [phosphorylated (p-)protein kinase RNA-like endoplasmic reticulum kinase (PERK), PERK, binding immunoglobulin protein (BIP), activating transcription factor 4 (ATF-4) and C/EBP homologous protein (CHOP)], as well as those of a survival marker (Bcl-2) were measured by western blot analysis. Furthermore, the mRNA levels of ATF-4 and CHOP were determined by RT-qPCR. ELISA was used to examine the activity of intracellular cAMP. Moreover, the GLP-1R antagonist, exendin9-39 (ex9-39), the protein kinase A (PKA) inhibitor, H89, and small interfering RNA (siRNA) targeting rat ATF-4 and CHOP were co-incubated with the MSCs. The apoptotic rate was markedly diminished following pre-conditioning with ex-4 in a dose‑dependent manner (P<0.05). The ER stress markers, p-PERK, BIP, ATF-4 and CHOP, were upregulated in the cells subjected to OGD conditions. Ex-4 pre-conditioning significantly decreased the mRNA and protein levels of ATF-4 and CHOP (P<0.05), and increased the activity of intracellular cAMP (P<0.05). Furthermore, the anti-apoptotic effects of ex-4 were almost reversed by treatment with either H89 or ex9-39 (P<0.05); transfection with siRNA-CHOP significantly reduced the apoptotic rate of the MSCs and did not impair the cytoprotective effects of ex-4. Taken together, these findings suggest that ex-4 protects rat BM-MSCs from OGD-induced apoptosis through the activation of the PKA/cAMP pathway and the attenuation of the ER stress signaling pathway. Ex-4 may thus prove to be a therapeutic agent with the potential to improve the viability of MSCs in the ischemic milieu, and consequently, to optimize the therapeutic effects of MSC therapy in acute myocardial infarction.
艾塞那肽-4（ex-4）是一种长效胰高血糖素样肽-1受体（GLP-1R）激动剂，对血糖控制具有有益作用并能促进细胞活力。在本研究中，我们研究了ex-4在氧、葡萄糖和血清剥夺（OGD）条件下对大鼠骨髓间充质干细胞（BM-MSCs）的抗凋亡作用及其潜在机制。通过将细胞置于OGD条件下4小时诱导MSCs凋亡，并通过Annexin V/PI和Hoechst 33258染色进行检测。在将MSCs置于OGD条件之前，先用ex-4预处理12小时，然后通过蛋白质印迹分析测量凋亡标志物（裂解的半胱天冬酶-3）、内质网（ER）应激标志物[磷酸化（p-）蛋白激酶RNA样内质网激酶（PERK）、PERK、结合免疫球蛋白蛋白（BIP）、活化转录因子4（ATF-4）和C/EBP同源蛋白（CHOP）]以及存活标志物（Bcl-2）的表达水平。此外，通过RT-qPCR测定ATF-4和CHOP的mRNA水平。ELISA用于检测细胞内cAMP的活性。此外，将GLP-1R拮抗剂艾塞那肽9-39（ex9-39）、蛋白激酶A（PKA）抑制剂H89以及靶向大鼠ATF-4和CHOP的小干扰RNA（siRNA）与MSCs共同孵育。用ex-4预处理后，凋亡率以剂量依赖性方式显著降低（P<0.05）。在OGD条件下的细胞中，ER应激标志物p-PERK、BIP、ATF-4和CHOP上调。ex-4预处理显著降低了ATF-4和CHOP的mRNA和蛋白水平（P<0.05），并增加了细胞内cAMP的活性（P<0.05）。此外，用H89或ex9-39处理几乎逆转了ex-4的抗凋亡作用（P<0.05）；用siRNA-CHOP转染显著降低了MSCs的凋亡率，并且不损害ex-4的细胞保护作用。综上所述，这些发现表明ex-4通过激活PKA/cAMP途径和减弱ER应激信号通路来保护大鼠BM-MSCs免受OGD诱导的凋亡。因此，ex-4可能被证明是一种具有改善缺血环境中MSCs活力潜力的治疗剂，从而优化MSCs治疗在急性心肌梗死中的治疗效果。
