Impact Of Inflammatory Microenvironment On The Senescence Of Mesenchymal Stem Cells And Its Mechanisms

Backgrounds:Mesenchymal stem cells(MSCs)are a kind of stem cells which existed in tissues and organs.MSCs have the capable of self-renewal,they can obtain from bone marrow,fat,placental and umbilical cord tissue and so on.Under certain conditions,they are capable of differentiating into multilineage cells,as osteoblasts,adipocytes,and chondrocytes.MSCs have several abilities,for example,they have the multi-directional differentiation ability,they can regulate immune cells and support bone marrow hematopoietic.In view of the important functions of MSCs,they were seen as promising cells for clinical cell therapy;MSCs have the immunoregulation so they can effectively inhibit a variety of immune cells in vivo and in vitro,thus can be used for the prevention and treatment of inflammatory and immune related diseases,such as transplantation rejection and autoimmune diseases.However,to exert good curative effects,MSCs should be guaranteed to be in a good state before use.Stem cell microenvironment is a complex environment which affected the function of stem cells.Different microenvironment has different effects on biological functions of MSCs.In recent years,the experiments in vivo and in vitro found that the immunoregulation of MSCs are different or even had opposite results: the MSCs could mediate immune suppression,in addition,they can promote the progress of inflammation.Above all,the microenvironment of MSCs is crucial on whether it can play an important role in clinical.Cell senescence is an important factor that seriously affects cell activity and function and impaired the effect of cell therapy.Inflammation is one of the causes of cell senescence.So,what are the effects of the inflammatory microenvironment on the biological function of MSCs in vivo and in vitro? Whether it can induce cellular senescence and what's the machanism? The first part: impact of inflammatory microenvironment on the biological functions and senescence of BMSCs and ADSCs and its mechanisms Objective:Our study intended to explore the effect of inflammatory microenvironment on the biological functions of MSCs and cell senescence in vitro,and further explore its potential molecular mechanism.Methods:1.Bone marrow MSCs(BMSCs)and adipose-derived mesenchymal stem cells(ADSCs)were isolated and expanded.Healthy 4~6-week old Sprague-Dawley rats were used to obtain the MSCs.MSCs of passage 2-4 were used in the following experiments unless otherwise specified.Using flow cytometry(FCM)to detect cell surface molecule markers,and through the osteogenic,adipogenic and chondrogenic differentiation to identify BMSCs and ADSCs.2.Inducing endotoxemia model in rats.Lipopolysaccharide(LPS)(10 mg/kg)were injected peritoneally into rats to induce endotoxemia.the same volume of PBS was injected as control.Animals were sacrificed in 24 hours.Levels of inflammatory factors as IL-1??IL-10?TGF-?and TNF-?in serum were measured through ELISA,another part of serum was filtered,inactivated and stored for subsequent experiments.Liver and lung of rats were fixed with paraformaldehyde and then stained with HE to observe the pathological changes of each tissue,so as to determine whether the establishment of endotoxemia model in rats was successful.3.We co-cultured BMSCs and ADSCs with inflammatory serum or control serum respectively,then detected the migration capacity of MSCs by transwell,cell cycle and cell apoptosis by FCM,which discussed the biological functions of inflammatory microenvironment on BMSCs and ADSCs.4.We co-cultured BMSCs and ADSCs with inflammatory serum or control serum respectively,then detect the ?-galactose glucoside enzyme activity(?-gal),senescence-associated secretory phenotype(SASP)and cell cycle inhibitors(p16,p21)to explore the effects of inflammatory serum on MSCs senescence.5.Reactive oxygen species(ROS)level was detected by FCM and the cell mitochondrial membrane potential(? ? m)was investigated by confocal to analysis the relationship of mitochondrial function and senescence of MSCs.6.Western blot(WB)was used to detect the expression levels of Notch1 and its downstream target gene Hes1 in MSCs at different time points(2 d,4 d and 6 d)after inflammatory serum stimulation.Notch1 is one signaling molecule of Notch pathway and DAPT is a inhibitor of Notch signaling pathway,which was used to inhibit the classical Notch1 signaling pathway,to detect its influence on senescence of MSCs,and to explore the role of Notch1 in inducing MSCs senescence in inflammatory microenvironment.7.The expression level of Sirtuin 1(SIRT1)was detected by WB.SIRT1 is a protein related to cell senescence,after stimulated with inflammatory serum,the interaction between Notch1 and SIRT1 was detected by co-immunoprecipitation(co-IP).Acetylation levels of Notch1 were further detected by co-IP.8.The expression levels of NF-?B and C/EBP? in MSCs at different time points(2d,4d,6d)were detected by WB.Results:1.MSCs were observed under the microscope after two passage.The morphology of the MSCs was similar to the spindle or the shape of fibroblasts,and the cells were arranged and grew in a spiral shape.CD29 and CD90 were highly expressed on the MSCs surface,CD11 b and CD45 were expressed very low.MSCs can differentiate into osteoblasts,chondrocytes and adipocytes by induction of corresponding differentiation medium.These results suggested that the cultured and amplified cells were MSCs.2.After intraperitoneal injection of LPS for 24 h,the inflammatory factors IL-1,IL-10,TGF-? and TNF-? in the serum of rats were significantly higher than those of the control group.3.Compared with the control serum,the proliferation of the two types of MSCs was significantly reduced when stimulated by inflammatory serum,cell cycle was blocked in G0/G1 phase,and the migration of MSCs was significantly increased,but inflammatory serum had no significant effect on the apoptosis level of MSCs.4.After stimulated with inflammatory serum,the activity of ?-gal in MSCs was increased,the level of SASP(IL-1,IL-6,IFN-gamma,MCP-1)was increased,and the expression of cyclin-related proteins(p16,p21)also increased significantly,indicating that inflammatory serum can induce the senescence of MSCs.5.After stimulated with inflammatory serum,the ROS level of MSCs increased significantly,and the mitochondrial membrane potential of the MSCs decreased.6.With the extension of inflammatory serum stimulation time,the expression of Notch1 and its target gene Hes1 on MSCs were up-regulated,and DAPT could blocking the classical Notch signaling pathway could partially reverse the effect of inflammatory serum stimulation on MSCs,including the increase of cell proliferation,the decrease of ?-gal activity,the decrease of SASP and the decrease of p16 and p21 expression.7.After stimulated with inflammatory serum,the expression of SIRT1 in MSCs was decreased,and the Notch1 binding to SIRT1 was significantly increased,and the acetylation level of Notch1 was significantly increased.8.After stimulated with inflammatory serum,the expression of C/EBP? was increased in MSCs,while the expression of NF-?B was not changed.Conclusion:1.Inflammatory serum from endotoxemia model significantly affected the biological functions of MSCs from different tissues,including cell proliferation and migration ability and cell cycle,inflammatory microenvironment induced senescence of MSCs.2.Inflammatory microenvironment may induce MSCs senescence by inhibiting SIRT1 protein expression,promoting activation of Notch1 signaling pathway,acetylation of Notch1,and C/EBP? expression. The second part Septic inflammatory microenvironment can induce aging of bone marrow mesenchymal stem cells Objectives:To investigate the effects of septic inflammatory microenvironment in vivo on the biological functions and aging of MSCs from bone marrow.Methods:Sepsis was induced in 4-6 week-old SD rats by cecal ligation puncture(CLP)procedure.MSCs from bone marrow(BMSCs)and were isolated and expanded ex vivo.BMSCs from sham-operated rats were cultured as control.Morphology was invetigated by light microscope,scanning electron microscope and flow cytometry.Proliferation was detected through CFU-F test and CCK8.Cell cycle and apoptosis were detected by flow cytometry.Expression of senescence-associated molecules were analyzed through Westen-blot.Capacity of differentiationand activity of ?-gal were detected as well.Results:BMSCs sepsis models showed a very different morphology from MSCs from sham control animals upon culture in vitro.They were smaller in size and round in shape.Their ability of proliferation was declined,and cell cycle was blocked in G1 phase with enhanced expression of p21,which is inhibitor of cell cycle.Furthermore,activity of ?-gal in these cells was significantly enhanced.However,no oobvious apoptosis was detected..Conclusion:Septic inflammatory microenvironment in vivo can induce a senescence-associated phenotype in BMSCs.