title: Exendin-4 protects bone marrow-derived mesenchymal stem cells against oxygen/glucose and serum deprivation-induced apoptosis through the activation of the cAMP/PKA signaling pathway and the attenuation of ER stress.
abstract: 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.
authors.affiliation: The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Harbin, Heilongjiang, P.R. China.
authors.forename: Jieqiong
authors.initials: J
authors.lastname: He
authors.affiliation: The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Harbin, Heilongjiang, P.R. China.
authors.forename: Chao
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Cardiology, The First Hospital of Qiqihar, Qiqihar, Heilongjiang, P.R. China.
authors.forename: Yunpeng
authors.initials: Y
authors.lastname: Sun
authors.affiliation: The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Harbin, Heilongjiang, P.R. China.
authors.forename: Bo
authors.initials: B
authors.lastname: Lu
authors.affiliation: The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Harbin, Heilongjiang, P.R. China.
authors.forename: Jinjin
authors.initials: J
authors.lastname: Cui
authors.affiliation: The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Harbin, Heilongjiang, P.R. China.
authors.forename: Nana
authors.initials: N
authors.lastname: Dong
authors.affiliation: The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Harbin, Heilongjiang, P.R. China.
authors.forename: Maomao
authors.initials: M
authors.lastname: Zhang
authors.affiliation: The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Harbin, Heilongjiang, P.R. China.
authors.forename: Youbing
authors.initials: Y
authors.lastname: Liu
authors.affiliation: The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Harbin, Heilongjiang, P.R. China.
authors.forename: Bo
authors.initials: B
authors.lastname: Yu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D000077270:Exenatide; D005947:Glucose / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology*; D020011:Protective Agents / Q000494:pharmacology*; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D014688:Venoms / Q000494:pharmacology*
chemical_list: D007004:Hypoglycemic Agents; D010455:Peptides; D020011:Protective Agents; D014688:Venoms; D000077270:Exenatide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D005947:Glucose; D010100:Oxygen