Ex Vivo Pro-proliferation Effect And Molecular Mechanism Of Tanshinone ⅡA On Human Bone Marrow Mesenchymal Stem Cells

AimsBone marrow mesenchymal stem cells（BMSCs）have been used in clinical research as seed cells for cell therapies.However,the clinical application of BMSCs is largely hampered by the fact that the number of BMSCs harvested from bone marrow cells is limited,as well as its proliferative activity is easily affected by age and/or pathological conditions.Therefore,the methods or strategies that focused on promoting the capacity of BMSCs proliferation ex vivo to obtain a sufficient number of BMSCs for clinical transplantation become more and more important.Salvia Miltiorrhiza Bge.（also known as Danshen）,the traditional Chinese herbal medicine,has been reported that some of the active ingredients have the capacity to promote BMSCs proliferation ex vivo,but the molecular mechanism has not yet been elucidated.This study aims to investigate the pharmacological effects and molecular mechanism of Salvia Miltiorrhiza Bge.ingredients in promoting the proliferation of BMSCs,and to provide a new way to effectively promote the proliferation of MSCs ex vivo.MethodsIn this present study,BMSCs from human were used as the research object.Firstly,the active ingredients of Salvia Miltiorrhiza Bge.were screened by network pharmacology,and the data sets of gene targets related to active ingredients were constructed as well as the key components and targets and the signaling network of Salvia Miltiorrhiza Bge.regulating the proliferation of BMSCs were screened and analyzed,then the enrichment analysis of signaling pathways involved in the key targets were conducted.Secondly,MTT methods were used to screen the results of network pharmacology,then Ed U labeling method and Western blot technique were further used to verify the screen results to determine the components of Salvia Miltiorrhiza Bge.which could promote the proliferation of BMSCs,and to determine the optimal drug concentration and culture time.Afterwards,quantitative proteome analysis was applied furtherly to identify the key regulatory proteins in Salvia Miltiorrhiza Bge.compotents treated BMSCs.Finally,ELISA kit,immunocytochemistry,Western blot technique and flow cytometry were employed to verify the results of proteomics and to determine the key molecules and mechanism of Salvia Miltiorrhiza Bge.components in promoting BMSCs proliferation.Results1.The network pharmacological results showed that there were 37 main potential active ingredients in Salvia Miltiorrhiza Bge.that could regulate the proliferation of BMSCs,including Cryptotanshinone,Luteolin,Tanshinone ⅡA,Dihydrotanshinone,etc.;and there were 135 potential targets which the top mainly included AKT1,tumor protein 53,tumor necrosis factor（TNF）,estrogen receptor 1,interleukin 6,vascular endothelial growth factor A,epidermal growth factor receptor,JUN,FOS and signal transducer and activator of transcription 3.KEGG enrichment analysis showed that TNF signaling pathways,calcium signaling pathways,T cell receptor signaling pathways,hypoxia-inducible factor 1 signaling pathways,PI3K-AKT signaling pathways,etc.may play an important role in the process of Salvia Miltiorrhiza Bge.components regulating the proliferation of BMSCs.2.The results of cell viability experiments showed that compared with the control group,the cells viability in h BMSCs were significantly elevated after exposure to Tanshinone ⅡA at the concentration of 0.1～10μM（P<0.05）,indicating that Tanshinone ⅡA had the promotional effect of cell growth.Result from Edu assay further showed that the proliferation rate in Tanshinone ⅡA-treated groups（0.1～10μM）were markedly increased after 24 h exposure（P<0.05）,and the proliferation rate increased most obviously at the concentration of 1μM（P<0.01）;in addition,the protein expression of proliferating cell nuclear antigen（PCNA）in h BMSCs were significantly increased after24 h exposure to Tanshinone ⅡA（1μM,P<0.01）and consistent well with PCNA,the protein expression of CD44,the stemness marker was also markedly increased after 12h and 24 h exposure to Tanshinone ⅡA（P<0.05）,these results all indicated that Tanshinone ⅡA could promote h BMSCs proliferation and maintain the stemness,and the ideal drug concentration was 1μM,the best culture time was 24 h.3.After treated h BMSCs by Tanshinone ⅡA（1μM）for 24 h,quantitative proteome analysis showed that 6574 proteins were identified,of which 84 proteins were differentially expressed proteins（DEPs）and among them,51 upregulated proteins and33 downregulated proteins were identified.The subcellular localization analysis showed that the identified DEPs belong to nucleus（39.29%）,cytoplasm（17.86%）,extracellular（16.67%）,mitochondria（14.29%）,plasm membrane（9.52%）and endoplasmic reticulum（2.38%）;GO functional classification showed that DEPs are classified into biological process,cellular components and molecular functions.The interaction analysis of the DEPs showed that in the upregulated DEPs,these proliferation-related proteins,such as fibroblast growth factor 1 and 2（FGF1 and FGF2）were significantly upregulated,indicating that Tanshinone ⅡA treatment may activate the proliferation-related proteins FGF1/2 to promote the proliferation of h BMSCs.Besides,the enrichment analysis of KEGG revealed that PI3K/AKT signaling pathways and MAPK signaling pathways may play an important role in Tanshinone ⅡA’s effect on h BMSCs proliferation.4.ELISA results showed that compared to the control,the content of FGF2 level in the culture medium and cell lysates both significantly increased after exposure to Tanshinone ⅡA（P<0.01）;immunocytochemical results showed that the nucleus staining of FGF2 was presented in h BMSCs,while FGF receptor（FGFR）expression was found located in the membrane of h BMSCs;results from Western blot assay indicated that the protein expression of FGF2,but not FGFR,was markedly increased in h BMSCs after exposure to Tanshinone ⅡA（P<0.05）;furthermore,Tanshinone ⅡA-mediated h BMSCs proliferation was significantly and dose-dependently abolished（P<0.01）by the preincubation of FGFR inhibitor Debio-1347,and the increased protein expression of PCNA and CD44 in Tanshinone ⅡA-treated h BMSCs were also significantly reversed in the presence of Debio-1347（P<0.05）.All these results indicated that Tanshinone ⅡA could promote the autocrine of FGF2,FGF2 played an important role in Tanshinone ⅡA-mediated h BMSCs proliferation.5.Western blot assay results showed that the expression of phosphorylated c AMP-response element binding protein（p-CREB）was significantly increased（P<0.05）,while the expression of protein phosphatase 2A（PP2A）was markedly decreased after Tanshinone ⅡA treatment（P<0.05）;at the same time,the protein expression of p-PI3K and p-AKT were significantly increased after Tanshinone ⅡA treatment（P<0.05）,while the total protein expression of PI3K and AKT did not show alteration.Moreover,the expression of p-p27 and cyclin D1,were also significantly increased after Tanshinone ⅡA treatment（P<0.05）.Flow cytometry analysis showed that the percentage of S phase cells was significantly increased after 12 h and 24 h exposure to Tanshinone ⅡA（P<0.05）.All these results indicated that Tanshinone ⅡA could not only up-regulate the expression of PI3K/AKT signaling molecules by regulating PP2A to promote the cell cycle progression from G₁to S phase,but also enhance the function of PI3K/AKT signaling by regulating p-CREB,which may be the main molecular mechanism of Tanshinone ⅡA in promoting h BMSCs proliferation.ConclusionTanshinone ⅡA could promote h BMSCs proliferation and maintain the stemness,and the ideal drug concentration for promoting the proliferation was 1μM,the best culture time was 24 h.Moreover,Tanshinone ⅡA could promote the autocrine of FGF2,on the one hand to down-regulate the expression of AKT signal inhibitory molecule PP2A to regulate the cell cycle through FGF-PI3K/AKT signal to promote the proliferation of h BMSCs;on the other hand to enhance the function of PI3K/AKT signal by regulating p-CREB to promote the survival of h BMSCs.Our study,to some extent,clarified the mechanism of Tanshinone ⅡA’s effect on promoting the ex vivo proliferation of BMSCs,will provide a certain theoretical basis and new ideas for the study of the mechanism of traditional Chinese medicine regulating the proliferation of BMSCs.