The self-renewal and differentiation ability of BMSCs makes them play an important role in the maintenance, renewal, and reconstruction of bone tissue. However, more and more evidence shows that the aging of BMSCs will affect their normal function, which may eventually lead to bone loss, bone deficiency, bone tissue defect repair and reconstruction difficulties, and other problems. Surprisingly, based on previous studies, we further found that CHNQD-00603 can promote autophagy through AKT/ERK/mTOR signaling pathway, inhibit the senescence of BMSCs, and promote the osteogenic differentiation of BMSCs (Figure.6).
Due to its special environment of high salt, high pressure, low oxygen, and oligotrophic, the ocean has nurtured many marine organisms whose structure and function are different from those of terrestrial products. The relationship between marine products and diseases and the underlying molecular mechanism has generated tremendous interest over the years. In this experiment, we used the marine product derivative CHNQD-00603 for the first time, which is a series of 4-phenyl-2 (1H)-quinolinone natural products isolated from gorgonian-derived fungus Scopulariopsis sp. by our co-operation group15. In our previous experiments, we not only found that CHNQD-00603 can regulate autophagy in BMSCs but also predicted possible target genes through the CHNQD-00603 structural formula on the Swiss TargetPrediction database platform. In this experiment, we performed GO enrichment analysis on the DAVID database and found that CHNQD-00603 may be related to aging, cytoplasm, nucleus, etc. Therefore, we speculate that CHNQD-00603 may affect the aging of BMSCs.
Cell senescence is a cell cycle arrest leading to decreased cell function and resilience. This is not only reflected in the increased expression of aging-related molecules including p16, p21, and β-galactosidase at the molecular level. At the cellular level, it can be manifested as a weakened or subsided function33, 34. For example, the expression of osteogenic-related factors RUNX2, ALP, OPN, and OCN decreased in BMSCS. In our study, we observed that the number of SA-β-gal positive cells increased, and the mRNA expression of p16 and p21 increased significantly in O-BMSCs induced by D-gal. However, ALP staining and mRNA expression of RUNX2, ALP, OPN, and OCN were significantly reduced in O-BMSCs. These results indicate that the osteogenic differentiation ability of senescent BMSCs is weakened. Interestingly, in senescent BMSCs after CHNQD-00603 treatment, the number of SA-β-gal positive cells decreased, the mRNA expression of p16 and p21 decreased, ALP staining and the mRNA expression of RUNX2, ALP, OPN, and OCN decreased. This indicates that CHNQD-00603 inhibits the senescence of BMSCs to some extent. Based on these findings, we conclude that CHNQD-00603 can inhibit the senescence of BMSCs and enhance osteogenic differentiation ability.
Autophagy is a self-degrading system with multiple functions and plays an important role in maintaining the balance of bone metabolism35. Many signaling molecules play a role in autophagy. When cells are induced by various intracellular and extracellular stimuli, ATG13 induces ULK1 to the pre-autophagosome structure (PAS), and then almost all autophagy-related (Atg) proteins are aggregated on the PAS36. Some mammalian cells have homologs of yeast Atg8, such as LC3, GATE16, GABARAP, and ATG8L. In mammal cells, LC3 has been studied and characterized as a marker of autophagosomes16, 37. The P62, also known as sequestosome 1 (SQSTM1), is a selective substrate of autophagy. p62/SQSTM1 can directly interact with LC3, leading to the specific degradation of p62 by autophagy. Therefore, the p62 level has been used as a marker of autophagy inhibition or autophagy degradation defects38, 39. 3-MA (3-methyladenine) is an inhibitor of class I and class III PtdIns 3-kinase, which results in autophagy inhibition due to the suppression of class III PtdIns 3-kinase32. Our data showed that CHNQD-00603 triggered autophagy in senescent BMSCs, inducing autophagosome formation, increasing LC3II expression, and decreasing P62 expression. Nevertheless, autophagy inhibitor 3-MA could attenuate the effect of CHNQD-00603 on aging BMSCs. These results demonstrate that CHNQD-00603 inhibits BMSCs senescence by autophagy. Nonetheless, the mechanism of CHNQD-00603 to regulate autophagy in senescence BMSCs is not clear.
Several molecular and signaling pathways play a crucial role in regulating autophagy. We focused on AKT and ERK. AKT is a key signaling molecule in the PI3K/AKT/mTOR signaling pathway, which has a pivotal role in the regulation of cell proliferation, differentiation, and survival under normal physiologic and pathophysiological processes30, 31. Constitutively active ERK1/2 also traffics to autophagy32. CHNQD-00603 treated O-BMSCs decreased the phosphorylated expression of AKT and ERK, and finally activated autophagy. To further verify the role of AKT/ERK/mTOR in the regulation of autophagy by CHNQD-00603, SC79(AKT agonist) and TBHQ (ERK agonist) were used to interfere with senescent BMSCs, respectively. Our results demonstrated that AKT/ERK/mTOR was regulated by CHNQD-00603 and that their specific agonist SC79 and ERK mitigated CHNQD-00603-elicited autophagy, thereby verifying the AKT/ERK/mTOR molecular network's positive participation in the regulation of autophagy by CHNQD-00603 in aging BMSCs.