| Sitagliptin attenuates hypoxia-induced apoptosis and autophagy in mesenchymal stem cells by suppressing JNK/c-Jun signaling pathwayBACKGROUND:Mesenchymal stem cells (MSCs) had been widely applied to post-infarct cardiac repair in regenerative medicine. However, MSCs encounter an ischemic microenvironment which may endanger the viability of tissue repair after in vivo transplantation into myocardial necrosis area. DPP-4inhibitors, a new class of oral anti-hyperglycemic agents, have pleiotropic effects on cardiovascular protection besides its antidiabetic property. It is reported that DPP-4inhibitors have exhibited the potential to mediate these effects through various pathways and mechanisms. It has been demonstrated that systemic pretreatment with DPP-4inhibitors enhanced mesenchymal stem cells recruitment into ischemic myocardium. However, it is unclear that whether there is protective effects of DPP-4inhibitors on MSCs in hypoxia condition so far. In this study, our aim was to explore the protective effect of sitagliptin (one DPP-4inhibitor) on MSCs during H/SD and potential mechanisms of action. Moreover, we investigated the relationship between apoptosis and autophagy in sitagliptin-induced protective effects.METHODS:Bone marrow was harvested from the femurs of Sprague-Dawley rats (60-80g, male), and seeded into cell culture flasks with complete medium. And P3MSCs were exposed to different concentrations of sitagliptin (0.001μM-10μM)for6h in H/SD condition. And we assessed the effect of sitagliptin on cell apoptosis and autophagy during H/SD. For later studies, the cells were incubated with the JNK inhibitor SP600125(10μM) before the addition of sitagliptin (1μM) to investigate the role of JNK/c-Jun signaling pathway in these protective effects. To further test the relationship between autophagy inhibition and anti-apoptosis effects of sitagliptin, we exposed MSCs to1μM sitagliptin with rapamycin (autophagy activator),3-MA (autophagy inhibitor), or ABT-737(Bcl-2inhibitor) under H/SD for6h, and then assessed the level of cell apoptosis and autophagy. Cell apoptosis was assessed using Annexin V-FITC/PI Kit, bcl-2and bax. And cell autophagy was assessed by detecting acidic vesicular organelles using acridine orange staining, Beclin1, light chain3(LC3), and transmission electron microscopy. And the phosphorylation of JNK and c-Jun were analyzed by Western Blot.RESULTS:Stained with Annexin V/propidine iodine (PI), we found sitagliptin (0.001μM-10μM) reduced apoptosis of rat bone marrow-derived MSCs cultured in H/SD condition. And sitagliptin suppressed the autophagic activity observed in MSCs exposed to H/SD as identified by Beclin1, type Ⅱ of light chain3(LC3-Ⅱ) expression, and autophagosome formation. However, this effect was obstructed by JNK inhibitor SP600125. Bcl-2protein, an anti-apoptosis protein, increased, while bax protein, a pro-apoptosis protein decreased in sitagliptin treated cells as showed by Western blotting. Meanwhile, MSCs treated with sitagliptin decreased phosphorylation of JNK and c-Jun. The trend was partially inhibited by SP600125. Meanwhile, we exposed MSCs to1μM sitagliptin with3-MA, rapamycin, or ABT-737under H/SD for6h. The result showed that3-MA or rapamycin had no significant influence on the rate of cell apoptosis. However, ABT-737increased autophagic activity observed in MSCs identified by beclin1, light chain3expression, acidic vesicular organelles, and autophagosome formation.CONCLUSIONS:We demonstrated for the first time that sitagliptin attenuated hypoxia-induced apoptosis and autophagy in MSCs by suppressing JNK/c-Jun signaling pathway. Furthermore, autophagy inhibition, is regulated by anti-apoptosis effects of sitagliptin protecting MSCs from hypoxia. And this study provides a novel explanation for the protective effect of sitagliptin on MSCs. Sitagliptin attenuates hypoxia-induced mitochondrial apoptosis in mesenchymal stem cells by activating JAK2/PI3K/AKT/STAT3signaling pathwayBACKGROUND:Our preliminary experiments indicated that Sitagliptin could attenuate the apoptosis of bone marrow mesenchymal stem cells under hypoxia. And previous studies showed that DPP-4inhibitors reduced infarct size by activating the cardioprotective RISK (reperfus ion-induced salvage kinase) pathway. It has been demonstrated that the DPP-4inhibitor is able to stabilize cardiac electrophysiology by preventing the cardiac mitochondrial dysfunction that is caused by severe oxidative stress during ischemia-reperfusion injury, thus attenuating the vulnerability to arrhythmia. Therefore, DPP-4inhibitors may play the anti-apoptotic role by protecting myocardium mitochondrial function. It is well-known that JAK2/STAT3signaling pathway is an important intracellular signal transduction pathways involved in cell growth, proliferation, differentiation, inflammation, apoptosis and so on. Whether this signaling pathway is associated with the effects of sitagliptin inhibiting mitochondrial apoptosis of mesenchymal stem cells (MSCs) under hypoxic conditions is not yet clear. DPP-4inhibitors could adjust NO release by the PI3K-AKT pathway, thereby improving vascular status. However, the relationship between JAK2/STAT3and PI3K-AKT signaling pathway in sitagliptin anti-apoptotic effects remains unclear so far. In this study, our aim was to explore the role of JAK2/STAT3signaling pathway in the protective effect of sitagliptin on MSCs during hypoxia and serum deprivation (H/SD). Moreover, we investigated the relationship between JAK2/STAT3and PI3K/AKT pathway in sitagliptin-induced anti-apoptosis effects.METHODS:Bone marrow was harvested from the femurs of Sprague-Dawley rats (60-80g, male), and seeded into cell culture flasks with complete medium. And P3MSCs were exposed to different concentrations of sitagliptin (0.001μM-10μM)for6h in H/SD condition. And we assessed the effect of sitagliptin on cell mitochondrial apoptosis and phosphorylation of JAK2/STAT3and PI3K/AKT proteins during H/SD. For later studies, the cells were incubated with the JAK2inhibitor AG490(10μM) before the addition of sitagliptin (1μM) to investigate the role of JAK2/STAT3signaling pathway in the protective effects. To further test the relationship between JAK2/STAT3and PI3K/AKT signaling pathway in sitagliptin protective effects, we exposed MSCs to JAK2inhibitor AG490or PI3K/AKT inhibitor LY294002with or without1μM sitagliptin under H/SD for6h, and then assessed the level of cell mitochondrial apoptosis and the phosphorylation of JAK2、STAT3、 PI3K and AKT. The early changes of cell mitochondrial apoptosis were assessed using mitochondrial membrane potential (JC-1), mitochondrial permeability transition pore opening (MPTP). Bcl-2, Bax, Cytochrome C and the phosphorylation of JAK2, STAT3, PI3K, AKT were analyzed by Western blot.RESULTS:We found sitagliptin (0.001μM-10μM) reduced mitochondrial apoptosis of MSCs cultured in H/SD condition. And sitagliptin increased the level of JAK2/STAT3and PI3K/AKT protein phosphorylation. However, this effect was obstructed by JAK2inhibitor AG490. When we added AG490to block JAK2signaling pathway, the effect of sitagliptin enhancing STAT3, PI3K, AKT protein phosphorylation and inhibition of mitochondrial apoptosis was weakened, suggesting that JAK2/STAT3signaling pathway played an important role in anti-apoptotic effects of sitagliptin. To explore the relationship between JAK2/STAT3and PI3K/AKT pathway, we added PI3K inhibitor LY294002 in MSCs. The results showed that the phosphorylation level of AKT and STAT3weakened. However, there is not significant differences on the phosphorylation of JAK2, suggesting that JAK2is the upstream molecules of PI3K/Akt/STAT3.CONCLUSIONS:Sitagliptin attenuates hypoxia-induced mitochondrial apoptosis in mesenchymal stem cells by activating JAK2/PI3K/AKT/STAT3signaling pathway. And this study provides a novel explanation for the protective effect of sitagliptin on MSCs. |
PDF Full Text Request