TRPV1 Agonist Capsaicin Regulates The Proliferation And Senescence Of Human Placental Mesenchymal Stem Cells Through Hippo Pathway

Objective:Human placental mesenchymal stem cells(hPD-MSCs)are derived from full-term placenta,with a large number of available cells,its function and characteristics are similar to bone marrow mesenchymal stem cell,with multi-directional differentiation potential,low immunogenicity,and they are ideal seed cells for stem cell therapy.However,under in vitro culture conditions,with the increase of the number of passages,stem cells are prone to aging,proliferation and stemness decrease significantly,which greatly affect the therapeutic effect of stem cells.Capsaicin(Cap)is a member of the vanillic acid family,is the main active ingredient of chili pepper,which produces a variety of physiological and pharmacological effects by activating the transient receptor potential vanilloid1(TRPV1).Our previous research found that rat adipose mesenchymal stem cells can express TRPV1,and that TRPV1 activation by Cap can change a variety of biological functions of stem cells.Therefore,this study further observe the effect of Cap on the proliferation and aging of hPD-MSCs,and explore the molecular mechanism of TRPV1 regulating the biological function of MSCs cells.This paper aims to establish a pretreatment method to improve the phenotype of MSCs based on TRPV1 target and provide more high-quality seed cells for clinical stem cell therapy.Methods:(1)Exploring the effect of Cap on the biological function of hPD-MSCs:(1)Effect of Cap on proliferation of hPD-MSCs: Western blot determined that hPD-MSCs of different generations were stable and highly expressed TRPV1.The passage 3-6 hPD-MSCs were selected and treated with different concentrations of Cap(1,3,10 μM)for 24 h,The effect of Cap on the cell viability was measured by CCK-8,protein expression level of proliferating cell nuclear antigen PCNA was measured by Western blot,and cell cycle changes were measured by flow cytometry.(2)Effect of Cap on the aging of hPD-MSCs: the 10 th generation hPD-MSCs were selected and incubated in complete medium at different concentrations of Cap(0,1,3,10μM)for 72 h,the total protein Western blot was extracted to detect the expression levels of longevity protein SIRT1 and aging-related proteins P53 and P21,the number of senescent cells was detected by β-galactosidase staining,The cell culture supernatants were collected,and the ELISA kit was used to detect the levels of senescence-related inflammatory factors IL-6,IL-8,and MMP-9 in the supernatants,Reactive oxygen species(ROS)levels were determined by flow cytometry.(3)Effect of Cap on stemness of hPD-MSCs: hPD-MSCs were treated with different concentrations of Cap(0,1,3,10 μ M),and the total protein was extracted by Western blot to detect the protein expression levels of cell stem transcription factors Sox2,Oct4 and Nanog.(2)Exploring the mechanism and verification of Cap regulating the biological function of hPD-MSCs:(1)Capsaicin(10μM)treated hPD-MSCs,cellular RNA was extracted and RNAseq was used to screen differentially expressed genes,and differential gene enrichment analysis was performed in the metascape database to find possible mechanisms regulating the biological function of hPD-MSCs.(2)To explore the relationship between TRPV1 and Hippo signaling pathway(WWC1/YAP-p): on the basis that RNAseq suggests that Cap can inhibit the Hippo pathway,lentiviral transfection constructs hPD-MSCs with low TRPV1 expression and WWC1 overexpression,respectively,and observed the changes in cell proliferation capacity and senescence-related indexes after knocking down TRPV1 or overexpressing WWC1,as well as the expression of WWC1 upstream of and the phosphorylation level of the downstream effector YAP of Hippo signaling pathway,and to determine whether silencing TRPV1 or overexpressing WWC1 affected the protective effect of capsaicin on hPD-MSCs.Results:(1)Western blot results determined that the 4th,8th,and 12 th generations of hPD-MSCs all expressed TRPV1 stably and highly.Cap promoted the proliferation of hPD-MSCs,as shown by increased cell viability and upregulated proliferating cell nuclear antigen PCNA levels,and significantly increased the proportion of cells in S phase.(2)Cap inhibited the senescence of hPD-MSCs,manifested by a significant reduction in the number of cells positive for β-galactosidase staining.Western blot results showed that capsaicin could significantly downregulate the expression levels of senescence-related proteins P21 and P53,and upregulate the levels of longevity-related protein SIRT1.ELISA results showed that capsaicin inhibited the secretion of senescence-related inflammatory factors IL-8 and MMP-9,and flow cytometry results showed that capsaicin significantly inhibited the generation of reactive oxygen species(ROS)caused by aging.(3)Cap significantly upregulated the expression of hPD-MSCs multi-pluripotent stemness indexes Nanog,Oct4 and Nanog.(4)RNAseq screening of differential expression genes of Cap pretreatment hPD-MSCs: 102 up-regulated genes and 91 down-regulated genes were identified,and the differential genes were mainly related to immune activity,oxidative stress,differentiation and enriched in Hippo,MAPK and other signaling pathways related to cell proliferation and differentiation,suggesting a possible mechanism for activating TRPV1 to regulate the biological function of MSCs.Among them,capsaicin significantly downregulated WWC1,a key regulator upstream of the Hippo pathway.RT-q PCR verified that Cap inhibited the expression of the upstream regulator of the Hippo pathway WWC1,and Western blot showed that Cap downregulated the protein level of WWC1 and inhibited the phosphorylation of YAP,a key factor downstream of the Hippo pathway.(5)Compared with the Ctrl-sh RNA group,TRPV1-sh RNA can inhibit the proliferation of hPD-MSCs and promote their senescence,which is manifested as reducing cell viability and PCNA levels,increasing the number of β-galactosidase stain-positive cells,and upregulating the Hippo pathway,showing that the expression level of WWC1 protein and the phosphorylation level of YAP are increased;TRPV1-sh RNA can remove the protective effect of Cap on hPD-MSCs,as well as the regulation of WWC1 / YAP-p.(6)Overexpression of WWC1 reduced the cell viability of hPD-MSCs,downregulated PCNA protein expression,increased the number of β-galactosidase stained positive cells,promoted phosphorylation of YAP,and canceled the protective effect of capsaicin on hPD-MSCs.Conclusion:(1)Cap can promote the proliferation of hPD-MSCs,inhibit cell senescence,and increase cell stemness.(2)Improvement of hPD-MSCs function by Cap was related to the activation of TRPV1 to downregulate the expression of WWC1,the upstream regulator of the hippo pathway,thereby inhibiting YAP-p.