A pH probe inhibits senescence in mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BMSCs) are gradually getting attention because of its multi-directional differentiation potential, hematopoietic support, and promotion of stem cell implantation. However, cultured BMSCs in vitro possess a very limited proliferation potential, and the presence of stem cell aging has substantially restricted the effect together with the efficiency in clinical treatment. Recently, increasing attention has been paid to the connection between cellular aging and lysosomal acidification as new reports indicated that vacuolar H⁺-ATPase (v-ATPase) activity was altered and lysosomal pH was dysregulated in the process of cellular aging. Therefore, promoting lysosomal acidification might contribute to inhibition of cell senescence. Our previous studies showed that a novel small molecule, 3-butyl-1-chloro imidazo [1, 5-a] pyridine-7-carboxylic acid (SGJ), could selectively and sensitively respond to acidic pH with fast response (within 3 min), but whether SGJ can promote lysosomal acidification and inhibit senescence in BMSCs is unknown.

Rat BMSCs were cultured based on our system that had been already documented. BMSCs were treated with SGJ and/or Bafilomycin-A1 (Baf-A1). The co-localization between SGJ and lysosomes was assessed by a confocal microscope. Acridine orange (AO) staining and the Lysosensor™ Green DND-189 reagents were used for indicating changes in lysosomal concentration of H⁺. Changes of senescence were detected by immunoblotting of p21 and senescence-associated beta-galactosidase (SA-β-gal) staining as well as immunofluorescence assay of senescence-associated heterochromatin foci (SAHF). Changes of autophagy were detected by immunoblotting of MAP1LC3 (LC3B) and SQSTM1 (p62). Cell proliferation was determined by flow cytometry. Cell viability was calculated by sulforhodamine B assay (SRB). The V0 proton channel of v-ATPase was knocked down by transfecting with its small interfering RNA (si-ATP6V0C).

Our work showed that SGJ can promote lysosomal acidification and inhibit senescence in BMSCs. Firstly, SGJ and lysosomes were well co-located in senescent BMSCs with the co-localization coefficient of 0.94. Secondly, SGJ increased the concentration of H⁺ and the protein expression of lysosome-associated membrane protein 1 (LAMP1) and lysosome-associated membrane protein 2 (LAMP2). Thirdly, SGJ suppressed the expression of p21 in the senescent BMSCs and reduced SA-β-gal positive cells. Fourthly, SGJ promoted senescent BMSCs' proliferation and protein level of LC3B but reduced the p62/SQSTM1 protein level. Furthermore, experimental group pretreated with 20 μM SGJ showed a stronger red fluorescent intensity, thinner cell morphology, less SA-β-gal positive cell, and less p21 protein level as well as higher cell viability in the presence of Baf-A1. Notably, ATP6V0C knockdown decreased the activity of v-ATPase and SGJ increased the concentration of H⁺.

Our work showed that SGJ could inhibit senescence in BMSCs and protect lysosomes by promoting expression of LAMP1 and LAMP2. Meanwhile, SGJ could promote autophagy. Furthermore, our study also suggested that SGJ was a new Baf-A1 antagonist because SGJ could target and occupy the V0 proton channel of v-ATPase.