Inhibiting The Senescence Of Rat Mesenchymal Stem Cells By A PH Probe

Marrow mesenchymal stem cells(MSCs)derived from mesodermal adult stem cells have self-renewal ability and can differentiate into various cells and tissues under certain conditions,such as chondrocytes,osteoblasts,nerve cells,stromal cells and fat cells.MSCs exist in many tissues,including fat,bone marrow,synovium and muscle.Bone marrow-derived MSCs are called bone marrow mesenchymal stem cells(BMSCs).BMSCs have become popular because of their advantages of easy access,no ethical issues,relatively simple isolation and culture,rapid proliferation and genetic stability,multi-directional differentiation potential,low immunogenicity and high portability,which make them the ideal seed cells for clinical application and scientific research,also make them the most popular in basic research and transformation applications.However,BMSCs cultured in vitro have very limited proliferation potential.As the number of passages during culture increases,BMSCs age,and the self-renewal ability and multi-directional differentiation potential of BMSCs decrease.The senescence of stem cell greatly limits its clinical application and efficacy,especially hindering the development of autologous somatic cell therapy in elderly patients.Cell senescence(cell aging)generally refers to replicative senescence.Generally speaking,except for stem cells and cancer cells,all normal cells,whether cells on the body or cells cultured in vitro,have a certain life span,that is,cells do not divide indefinitely.When the number of cell divisions reaches the limits,the cells stop dividing and become aged and die eventually.Autophagy,a process of "self-purification" of cells,has the ability to remove the harmful waste from the body and to recycle the bioenergy and raw materials.A lot of data indicate that autophagy is cytoprotective.For example,when the cells are under adverse conditions such as metabolic stress and insufficient nutrient supply,the cells can adapt to the harsh environment by promoting autophagy and promote cell survival to prolong the lifespan.It is easy to bring the cells bioenergy disorder and death,if autophagy is inhibited at this time.Meanwhile,many studies found that autophagy closely relates to the lifespan of organism.Many studies found that knocking off autophagy-related genes in a model group such as nematode or yeast can significantly reduce its lifespan.It is speculated that the mechanism connected autophagy and aging may be involved in maintainence of the normal activities and homeostasis in cells,various biochemical reactions and processes that are required in the cells as well as renewal of damaged organelles.Normally,the organism has a set of methods to deal with these metabolic wastes and harmful substances-for example,decompose and reuse the metabolic wastes or the secret them outside the cells by autophagy,which maintaining the homeostasis in cells and organisms.However,when cells and organisms aged,the expression of autophagy-related genes decreased,and the efficiency of autophagy also decreased,which caused the accumulation of metabolic waste and in turn damages cells and accelerates the process of senescence in cells.Lysosomes not only play a role in recycling and decomposition,but also participate in the regulation of many other metabolic pathways,such as nutrient availability and composition in cells,emergency stress,programmed cell death,and repair of plasma membrane as well as responses to cell development and cell differentiation.It is easy to draw a conclusion that lysosomes play an important role in regulating the lifespan and death of cells and organisms.Because lysosomal dysfunction has multilateral importances on the survival and death of cells and organisms,lysosomal dysfunction is associated with age-related diseases such as Parkinson's disease and Alzheimer's disease,as well as decreased lifespan.Therefore,targeting the function of lysosomes to achieve longevity has become a very promising means.Lysosomes affects the lifespan in a variety of ways:for example,lysosomes affects aging by affecting autophagy because lysosomes are closely linked to autophagy;some active lipid molecules from lysosomes can enter the nucleus and then regulate the expression of some life-related genes;other studies shown that pH in lysosomes might be an aging-related signal;in the end,many studies found that amino acids,iron,calcium,etc might also affect aging.Among them,the v-ATPase on the membrane of lysosome is essential for its capacity.The v-ATPase pumps H+ into the lysosome to maintain the acidic environment of the lysosome,which is a prerequisite for the hydrolase in the lysosome to function.Studies found that during cell aging,activity of v-ATPase changes and lysosomal pH is disordered.Therefore,it is reasonable to believe that increase lysosomal activity and promote the acidification of lysosomes by targeting v-ATPase is an effective means of inhibiting cell aging.Our previous work showed that a novel small molecule,3-butyl-1-chloro imidazo[1,5-a]pyridine-7-carboxylic acid(SGJ),had been demonstrated as a fluorescent probe to 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.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-?-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 ?M SGJ showed a stronger red fluorescent intensity,thinner cell morphology,less SA-?-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+.The innovation of our work is that we found that the small chemical molecule SGJ could inhibit senescence,which deepens our understanding of cell aging.Meanwhie,it further demonstrates the feasibility of inhibiting cell senescence by targeting lysosomes and shows us novel ideas about developing drugs that inhibit aging.