Our study preliminarily investigated the change in activitie

Our study preliminarily investigated the change in activities of ERK1/2 and p38 MAPK pathways as ASCs were subjected to cyclic stretch, and found that the phosphorylation of both ERK1/2 and p38 was promoted; treatment with specific inhibitor of each pathway completely blocked the promotion. Moreover, we chose to test the expression levels of BMP-2 and Runx2, because Runx2 plays a central role in the interactions between intracellular signaling pathways and is identified as a key factor in the osteogenic differentiation of MSCs while BMP-2 is essential for the osteogenic commitment of MSCs (Chen et al., 2012), the more these two genes are expressed, the higher the level of osteogenic differentiation. We observed that ASCs treated with either ERK1/2 or p38 inhibitor were less responsive to mechanical stretch in terms of BMP-2 and Runx2 expression at several time points. These findings suggest the role ERK1/2 and p38 play in the transduction of tensile stimuli into intracellular signals that promote osteogenic differentiation of ASCs. The loading protocol adopted in the present study was developed according to our previous investigations; cyclically stretching ASCs for two or six consecutive hours was shown significantly promoting the expression of BMP-2 and Runx2 (Yang et al., 2012). Based on the preliminary results and in order to further investigate the very early response of ASCs to cyclic tensile stretch, we set up the time points as 15 min, 30 min, one hour, two hours, and six hours. The phosphorylation level of both ERK1/2 and p38 were higher than the control group, ERK1/2 phosphorylation peaked after cytochalasin d were loaded for two hours and p38 phosphorylation peaked after 30 min. It is thus safe to conclude from the above data that ERK1/2 and p38 indeed participate in the transduction of mechanical signals in ASCs, which coincides with previous published studies on ASCs or other cell types loaded by different mechanical forces (Xiao et al., 2015; Catalano et al., 2017; Lu et al., 2018). When ASCs were pretreated with ERK1/2 or p38 inhibitors, phosphorylated forms of both proteins were no more detected. And the up-regulation of both BMP-2 and Runx2 by cyclic stretch was canceled by ERK1/2 inhibitor U0126 at 15 min and six hours, suggesting that ERK1/2 pathway participated in stretch-induced osteogenic differentiation of ASCs at both early and late stages. Interestingly, p38 inhibitor significantly blocked the up-regulation of BMP-2 and Runx2 at 15 min only and no significant difference was observed between the inhibitor-treated and non-treated groups at later time points. This observation was in parallel with the pace of p38 as Fig. 4B shows. Zhang et al. reported similar results from rat BMSCs that p38 responded to cyclic mechanical strain in a relatively rapid fashion and subsequently returned to normal level within one hour (Zhang et al., 2013). Conversely Liu et al. reported p38 inhibitor blocked the up-regulation of several osteogenic genes except BMP-2 at day 7 and 14. (Liu et al., 2018). Another study reported that SB203580 inhibited up-regulation of osteogenic genes expression in BMSCs induced by intermittent stretching for 1, 3 and 5 days (Xiao et al., 2015), this discrepancy may be due to differences in the mechanical tension system or in the mechanical elongation, frequency, periodicity or duration.
Acknowledgements This work was supported by the National Natural Science Foundation of China (grant no. 81500895).
Introduction In recent years, depression has been found to be a complex disorder involving multiple body organs and systems, such as the nervous, digestive, and cardiovascular system (Joynt et al., 2003; Marano et al., 2009; Mayer et al., 2001). Historically, the brain-gut axis is seen as responsible for mutual regulation of the digestive and nervous system (Nowakowski et al., 2016; Scott et al., 2013; Tache and Bernstein, 2009). Specifically, ghrelin, a representative molecule of the brain-gut axis and a 28-amino-acid peptide, derived from stomach, works as an important modulator in weight control and energy homeostasis through binding to the growth hormone secretagogue receptor (GHSR) in hypothalamus (Dos et al., 2013; Kojima et al., 1999; Mihalache et al., 2016). Recent studies have suggested, that ghrelin also has some extra-hypothalamic actions, including enhancement of learning and memory, participation in the formation of reward and motivation, as well as mediation of neuroprotection (Andrews, 2011). However, the exact role of ghrelin in depression and anxiety is still being debated, as it was shown to both promote (Hansson et al., 2011) and alleviate (Lutter et al., 2008) depression- and anxiety-like behaviors in animal studies. A review paper by Zarouna et al. mentioned a potential link between ghrelin and leptin in human mood disorders. In this publication, the authors also stated, that the evidence from different studies on the relation between mood disorders and the two hormones is controversial (Zarouna et al., 2015). Thus, the question whether ghrelin has an antidepressant effect or not, still remains open. In our previous study, we have found that ghrelin had an antidepressant effect in rodent model of depression induced by chronic mild stress (Huang et al., 2017). We find, that this needs to be further verified in other models of depression, such as chronic social defeat stress (CSDS). Besides, the intracellular mechanisms underlying the antidepressant- and anxiolytic-like effects of ghrelin still merit further investigation.