The TRPV1 Ligand Capsaicin Regulates The Proliferation And Senescence Of Adipose-derived Mesenchymal Stem Cells

Objective:Due to its abundant source,simple material,immunogenic and low tumors,ADSCs are important seed cells for stem cell therapy in regenerative medicine and tissue engineering research,but they are prone to aging and decreased proliferation ability in high-sugar environment or in vitro expansion,which seriously affects the therapeutic effect of ADSCs and limits the application of ADSCs cell treatment in diabetic patients.It has been found that both rat and human ADSCs express the transient receptor potential vanilloid 1(TRPV1),but its physiological function has not been elucidated.Therefore,our study aimed to observe whether the effect of TRPV1 ligand capsaicin on ADSCs proliferation in 2 D and 3 D culture and against high glucose-induced ADSCs senescence.Thus improved in vitro culture methods guarantee clinical access to sufficient quantity and high-quality ADSCs.Methods:(1)Primary cultured rat ADSCs,after immunophenotype identification(positive CD29,CD90 and negative CD31,CD45)of ADSCs by flow cytometry,the experiments were performed for 3 to 5 generations,using 2D and 3D culture modes,the latter were cultured in 3D cell pellets using the agarose microplate method,to observe the effect of different concentrations of capsaicin(1μM,3μM,10μM)on the proliferation of ADSCs in both dimensions.The proliferative index of 2D cultures measured cell viability by CCK-8,cell cycle by flow cytometry,and protein levels of proliferating cell nuclear antigen PCNA by Western Blot.Cell viability of ADSCs in3 D culture was determined by Celltiter-Glo 3D Cell Viability Assay.The stemness of ADSCs was determined by Western Blot for the levels of Nanog,Oct4 and Sox2 in the 2D and 3D states.The expression of the cell cycle inhibitory protein P21 as well as the phosphorylation of ERK was measured by Western Blot,and the MAPK/ERK signaling pathway blocker PD98059 was administered to determine the effect of this pathway on the proliferation of ADSCs.Lentiviral transfection with sh RNA knockdown of TRPV1 expression further explored the effect of TRPV1 on ADSCs proliferation.(2)We incubated ADSCs from passages 3 to 5 with high glucose(33m M Glucose)medium for ADSCs 4 days to establish a high glucose-induced stem cell aging damage model,treated cells with different concentrations of capsaicin(1μM,3μM,10μM).Cell viability was measured by CCK-8,Senescent cells were detected by β-galactosidase staining,detection of cell cycle arrest,reactive oxygen species(ROS)levels,and apoptosis by flow cytometry,SIRT1 and P21 by Western Blot,and inflammatory factor IL-6 levels by Elisa,to determined the effect of capsaicin on high glucose-induced ADSCs senescence.cells were transfected with TRPV1-sh RNA lentivirus to further determine whether the anti-ADSCs senescence effect of capsaicin affected after silencing TRPV1.Results:(1)Capsaicin significantly promoted the proliferation of ADSCs in 2D cultures,it showed that increased cell viability,upregulated the protein level of the proliferating cell nuclear antigen PCNA,the effect of 3μM capsaicin was the most significant and increased the proportion of S and G2/M phases in the cell cycle.(2)In contrast to the 2D culture,the pluripotency index,Nanog and Oct4 and Sox2 were more expressed in ADSCs cell sphere in 3D culture,but the proliferative capacity of cells in 3D culture state was much lower than in 2D.Celltiter-Glo 3D Cell Viability Assay determined that capsaicin levels significantly increased cell viability in 3D,and Western Blot showed that capsaicin did not reduce Nanog and Oct4 and Sox2 stemness gene expression in ADSCs in 3D.(3)The Western Blot results showed that capsaicin dose-dependent increased the phosphorylation level of ERK and significantly downregulated the expression of the cell cycle inhibitory protein P21.However,prior administration of the MAPK/ERK signaling pathway blocker,PD98059,not only abolished the pro-proliferative effect of capsaicin,but also reduced cell viability.(4)The Western Blot and q PCR results determined that TRPV1-sh RNA significantly downregulated TRPV1 expression,whereas TRPV1 knockdown inhibited PCNA levels,reduced cell viability in the 2D and 3D culture states,and abolished the proproliferative effect of capsaicin.Further studies revealed that 3μM capsaicin can upregulate the expression level of TRPV1.(5)Capsaicin significantly inhibited high glucose-induced ADSCs senescence,it showed reduced the number of high glucose-induced cellular senescence,relieved G1/G0 phase inhibition,decreased high glucose-induced ROS generation,and decreased IL-6 levels.The Western Blot results showed that capsaicin upregulated the longevity protein SIRT1 expression and downregulated the senescence protein P21 expression,while the SIRT1 inhibitor EX527 abolished the inhibitory ADSCs senescence effect of capsaicin.Furthermore,Flow assays also confirmed that capsaicin reduced the rate of high glucose-induced apoptosis.(6)The β-galactosidase staining and CCK-8 showed that TRPV1-sh RNA increased cellular senescence in the high glucose state,and abolished the effect of capsaicin.Conclusion:(1)Capsaicin had a significant pro-proliferative effect on ADSCs cultured at both 2D and 3D,and the mechanism may be related to the activation of the MAPK/ERK signaling pathway and the downregulation of the cell cycle inhibitory protein P21 levels.(2)Capsaicin inhibited high glucose-induced senescence of ADSCs and apoptosis,and the mechanism may be related to upregulation of longevity protein SIRT1 expression,downregulation of cellular senescence-related protein P21 expression,and reduction of high glucose-induced ROS and IL-6 production.