ELABELA ameliorates hypoxic/ischemic-induced bone mesenchymal stem cell apoptosis via alleviation of mitochondrial dysfunction and activation of PI3K/AKT and ERK1/2 pathways

Fig. 1. Interference efficiency of siRNA-APJ in MSC detected by immunoblot analysis. The representative image and quantified expression of APJ were shown (N = 3). *P < 0.05. MSC, mesenchymal stem cell; siRNA, small interfering RNA

Fig. 2. ELA promoted MSC proliferation (a) and viability (b) under H/I exposure. Cell viability = (OD value in each group − OD value of CCK8 and medium/OD value in control group − OD value of CCK8 and medium) × 100%. All experiments were performed in triplicate. *P < 0.05. ELA, ELABELA; MSC, mesenchymal stem cell; H/I, hypoxic/ischemic; CCK8, cell counting kit-8

Fig. 3. Effect of ELA on H/I-induced MSC apoptosis. a MSC apoptosis results obtained from flow cytometry of various experimental groups. b Apoptotic cell rates were statistically presented as mean ± SD. Three parallel experiments were conducted independently. *P < 0.05. ELA, ELABELA; MSC, mesenchymal stem cell; H/I, hypoxic/ischemic

Fig. 4. ELA treatment ameliorated H/I-induced mitochondrial dysfunction in MSCs. a Representative confocal images of JC-1 staining. Scale bar = 100 μm. b Quantitative analysis of mitochondrial membrane potential with JC-1 staining. c ATP content was evaluated using a luminescence enzymatic assay. The ATP level in each group was expressed as the bioluminescence intensity that was normalized to the control group (N = 3). *P < 0.05. ELA, ELABELA; MSC, mesenchymal stem cell; H/I, hypoxic/ischemic; ATP, adenosine triphosphate

Fig. 5. Effects of ELA on Caspase 3 activity and balance of Bcl-2 and Bax expression in MSC under H/I injury. Representative immunoblots and quantitative data were provided. a Cleaved Caspase 3/Caspase 3 expression in different experimental groups. b The ratio of Bcl-2/Bax in different experimental groups (N = 3). *P < 0.05. ELA, ELABELA; MSC, mesenchymal stem cell; H/I, hypoxic/ischemic

Fig. 6. ELA activated the ERK1/2 signaling pathway in reducing the apoptosis of MSCs caused by H/I condition. Representative immunoblots and quantitative data were provided. a t-ERK1/2 and p-ERK1/2 expression in separate groups. b Utilization of U0126 further verified the role of ELA in activating ERK1/2 phosphorylation (N = 3). *P < 0.05. ELA, ELABELA; MSC, mesenchymal stem cell; H/I, hypoxic/ischemic; t-ERK1/2, total ERK1/2; p-ERK1/2, phosphorylated ERK1/2

Fig. 7. PI3K/AKT signaling pathway was related to ELA effects on MSC anti-apoptosis under H/I injury. Representative immunoblots and quantitative data were provided. a t-AKT and p-AKT expression in separate groups. b Utilization of LY294002 further identified the role of ELA in activating AKT phosphorylation (N = 3). *P < 0.05. ELA, ELABELA; MSC, mesenchymal stem cell; H/I, hypoxic/ischemic; t-AKT, total AKT; p-AKT, phosphorylated AKT