Peptide hormone ELABELA promotes rat bone marrow-derived mesenchymal stem cell proliferation and migration by manipulating the cell cycle through the PI3K/AKT pathway under the hypoxia and ischemia microenvironment

Abstract

Background: Mesenchymal stem cells (MSCs) are emerging as a potential candidate for stem cell transplantation to repair myocardial tissue in myocardial infarctions (MI). However, there are some pivotal limitations such as poor survival and low migration capacity of MSCs in hypoxic and ischemic microenvironments of MI. Our previous work verified that ELABELA (also abbreviated as ELA), a peptide hormone, could play a role as a growth factor and prolong the life span of rat bone marrow-derived mesenchymal stem cells (RAT BM-MSCs) under hypoxic and ischemic conditions. Nevertheless, the influence of ELA on the cell cycle, proliferation, and migration remains elusive. This study will further explore the improvement of the biological functions of ELA-treated RAT BM-MSCs, so as to provide a reference for improving the efficacy of RAT BM-MSCs in MI.

Methods: Rat BM-MSCs were isolated from 80 to 120 g Sprague Dawley rats by flushing femurs and tibias under the aseptic condition. RAT BM-MSCs of the third passage were divided into control group, hypoxic/ischemic (H/I) group, ELA group, ELA-LY group and LY group. RAT BM-MSCs were cultured under normoxia in control group. In H/I group, RAT BM-MSCs were exposed to hypoxia (1% O2) and serum deprivation for 24 h. RAT BM-MSCs in ELA group were treated with 5 µM ELA prior to the H/I exposure for 24 h. The PI3K/AKT inhibitor, LY294002 (50 µM), was used in ELA-LY group and LY group to observe the effect of ELA on PI3K/AKT activation. Cell proliferation ability was examined by CCK-8. Cell cycle was assessed with flow cytometry. Cell migration was evaluated by Transwell assay. Expression levels of total-AKT, phosphorylated-AKT, and cell cycle-associated proteins were examined by Western blotting.

Results: ELA-treated RAT BM-MSCs exhibited significantly higher proliferation ability, cell viability, and migration under H/I conditions. The cell cycle analysis showed that an increased proportion of cells in the S and G2/M phases of the cell cycle were observed in ELA-treated RAT BM-MSCs. The addition of ELA activated the PI3K/AKT signaling pathway. Additionally, upon treating with the inhibitor of the PI3K/AKT signaling pathway, ELA-triggered proliferation, cell viability, and migration were abrogated.

Conclusions: ELA can be used to enhance the proliferation ability, cell viability, and migration of RAT BM-MSCs through the PI3K/AKT signaling pathway and alleviate cell cycle arrest at the G0/G1 phase under hypoxic and ischemic injury. Thus, this study provides a promising strategy that ELA may help to optimize the mesenchymal stem cell-based therapy in MI.

Keywords: Cell cycle; ELABELA; Migration; Proliferation; Rat bone marrow-derived mesenchymal stem cells.

Fig. 1

ELA promotes RAT BM-MSCs proliferation (A) and cell viability (B) and PI3K/AKT is required in this effect under H/I injury at 0 h, 24 h, 48 h and 72 h. The treatment time of ELA (at a concentration of 5 μM) was 12 h prior to H/I injury. *P < 0.05. ELA, ELABELA; RAT BM-MSCs, Rat bone marrow-derived mesenchymal stem cells; H/I, hypoxic-ischemic; LY, LY294002

Fig. 2

The impact of ELA on the cell cycle progression under H/I conditions for 24 h. The treatment time of ELA (at a concentration of 5 μM) was 12 h prior to H/I injury. A The representative results of the cell cycle. B The percentage of S and G2/M phases of RAT BM-MSCs. C The percentage of cell cycle distributions of RAT BM-MSCs involving G0/G1, S, and G2/M. *P < 0.05. ELA, ELABELA; RAT BM-MSCs, Rat bone marrow-derived mesenchymal stem cells; H/I, hypoxic-ischemic; LY, LY294002

Fig. 3

ELA promotes RAT BM-MSCs migration within 10 h of H/I injury. The treatment time of ELA (at a concentration of 5 μM) was 12 h prior to H/I injury. A The representative figures in different experimental groups (Scale bar = 100 μm). B Cell counts of RAT BM-MSCs migration. *P < 0.05. ELA, ELABELA; RAT BM-MSCs, Rat bone marrow-derived mesenchymal stem cells; H/I, hypoxic-ischemic; LY, LY294002

Fig. 4

ELA activates the PI3K/AKT signaling pathway in RAT BM-MSCs under H/I conditions for 24 h. The treatment time of ELA (at a concentration of 5 μM) was 12 h prior to H/I injury. A The representative blots showed the protein level of total-AKT and p-AKT (Ser 473) in RAT BM-MSCs. B The quantitative data were calculated with the ratio of p-AKT and t-AKT. *P < 0.05. ELA, ELABELA; RAT BM-MSCs, Rat bone marrow-derived mesenchymal stem cells; H/I, hypoxic-ischemic; LY, LY294002; t-AKT, total-AKT; p-AKT, phosphorated-AKT

Fig. 5

ELA modulates the cell cycle-related proteins Cyclin D1 and Cyclin E in RAT BM-MSCs under H/I conditions for 24 h. The treatment time of ELA (at a concentration of 5 μM) was 12 h prior to H/I injury. A, C The protein levels of Cyclin D1 and Cyclin E in different groups. B, D The quantitative data were calculated with the ratio of Cyclin D1/GAPDH and Cyclin E/GAPDH. *P < 0.05. ELA, ELABELA; RAT BM-MSCs, Rat bone marrow-derived mesenchymal stem cells; H/I, hypoxic-ischemic; LY, LY294002