Experimental Study Of The Effects Of Ginsenoside Rg1 On Adipogenic Differentiation Of Adipose-derived Stem Cells

Background and purpose:Adipose-derived stem cells（ASCs）are adipose-derived cells with regenerative capacity.Compared with mature adipose tissue or bone marrow stem cells,ASCs have the advantages of accessibility,high proliferation rate and easy differentiation.Therefore,ASCs cell therapy or regenerative adipose tissue reconstruction has attracted more and more researchers’attention.Soft tissue defects resulting from complex trauma,tumor resection,and congenital defects are commonly seen presently,which has a greater negative impact on patients’mental health,appearance,and quality of life.Stable,efficient,and low-immunogenic grafts are the key to treating huge volumes of soft tissue defects.However,there is still a lack of graft materials that can fully meet clinical needs,which makes it difficult to achieve stable results for huge volume soft tissue defect transplantation.Cell-assisted Lipotransfer（CAL）is one of the effective treatment methods for huge volume soft tissue defects.Compared with simple fat transplantation,this therapy can reduce the absorption rate of fat grafts and reduce the occurrence of complications.However,the fat graft absorption rate varies greatly among CAL applied studies,which may be related to the different survival rates and cell status of ASCs.Optimizing the proliferation,differentiation,and paracrine functions of ASCs can effectively improve the survival rate,state,and function of their cells in vivo,and is of great significance for improving the long-term efficacy of CAL.Ginsenoside Rg1（Ginsenoside-Rg1,G-Rg1）is a natural active steroidal saponin isolated from ginseng.It has been shown that G-Rg1 can promote the proliferation and multipotent differentiation of ASCs or other adult stem cells,and has certain potential application value in soft tissue reconstruction therapy;but the impact on the proliferation of adipose stem cells and adipogenic differentiation is not clear.This study intends to explore the effects of G-Rg1 on the proliferation and adipogenic differentiation function of ASCs in both vivo and vitro environment,and to explore the practical function and significance of ASCs adipogenic differentiation in soft tissue reconstruction,and provide theoretical and experimental basis for the clinical application of ASCs in regenerative medicine.Materials and methods:1.Isolation,culture and identification of human adipose-derived stem cells（hASCs）.Redundant adipose tissue was obtained from 6 female patients who underwent liposuction surgery,and P0 hASCs were isolated by collagenase digestion.The adipogenic,osteogenic and chondrogenic differentiation potentials of P3 hASCs were assessed after the third-passage.Flow Cytometry Analysis was used to identify hASCs-specific cell surface markers,as well as the expression of CD34,CD45,CD73,CD90 and CD105.2.Explore the effect of G-Rg1 on the proliferation,adipogenic differentiation and paracrine function of hASCs in vitro.（1）The hASCs were cultured in the following five groups of medium conditions,control group:basal medium（BM）only,0.5μM G-Rg1 group:0.5μM G-Rg1 BM culture,1.0μM G-Rg1 group:1.0μM G-Rg1 BM culture,2.5μM G-Rg1 group:2.5μM G-Rg1 BM culture,5.0μM G-Rg1 group:0.5μM G-Rg1 BM culture.Each group was cultured under corresponding conditions for 10-14 days,then CCK-8 test was performed to detect the effect of G-Rg1 on the proliferation of hASCs in vitro and ELISA to detect the effect of G-Rg1 on the paracrine function of hASCs.（2）The hASCs were cultured in the following five groups of medium conditions:control group:Adipogenic induction medium（AIM）only,0.5μM G-Rg1group:0.5μM G-Rg1 AIM culture,1.0μM G-Rg1 group:1.0μM G-Rg1 AIM culture,2.5μM G-Rg1 group:2.5μM G-Rg1 AIM culture,5.0μM G-Rg1 group:5.0μM G-Rg1 AIM culture.Each group was cultured under corresponding conditions for 14 days.The effect of G-Rg1 on the ability of hASCs to differentiate into fat was proved by Oil Red O staining experiment,cell density and lipid concentration detection.（3）The hASCs were divided into G-Rg1stimulation group and control group which are respectively cultured in AIM containing 1μM G-Rg1 and AIM without G-Rg1 for 14 days.Then conduceted q PCR and WB tests to detect the expression levels of PPARγ2,C/EBPα,and ADD1.3.Explore the effect of G-Rg1 on the adipogenic differentiation function of hASCs in vivo.（1）Adenovirus infection or green fluorescent labeled（GFP-labeled）.Transfecting adenovirus（Ad-GFP）into hASCs and observing for 72h,then construct GFP-hASCs with the appropriate MOI value selected according to the fluorescence brightness for the next experiment.（2）Set up G-Rg1 pretreatment group and control group.G-Rg1 pretreatment group:GFP-hASCs were cultured and pretreated in AIM containing 1μM G-Rg1 for one week to make 1.0 ml cell suspension（2×10⁷/ml hASCs）,and infiltrated in 0.5×0.5×0.5cm³Cell-loaded collagen scaffold.Control group:GFP-hASCs were cultured in AIM for one week and implanted in collagen scaffolds.The density,number and scaffold volume of planted cells in both groups were the same.Then implant materials from these two groups into the left and right sides of the back of 20 nude mice.12 weeks later,sacrificed animals,dissected their regenerated tissue at the transplant site and measured wet weight.And Oil Red O staining,q PCR,WB and other experiments were carried out to prove the effect of G-Rg1 on the fat differentiation ability of hASCs in vivo.Results:1.Homogeneous hASCs were observed after the initial isolation and expansion（P1）and fulfilling up to 80–90%confluency during the 7^th–14^thdays.The cells grew in a spindle-shaped monolayer and showed a strong proliferation ability.Positive staining by Oil Red O,Alizarin Red and Alpha Blue confirmed that hASCs can differentiate into adipocytes,bone cells,and chondrocytes.the results of flow cytometry analysis of cellular immunophenotypic showed high expression of CD73,CD90 and CD105,while low expression of CD34 and CD45.2.（1）G-Rg1 can significantly promote the proliferation of hASCs in a concentration-dependent manner,the higher the stimulation concentration of G-Rg1 within a certain range（0.5-5.0μM）,the higher the proliferation ability of hASCs（P<0.05）.（2）On the 7th and 14th days stimulated by G-Rg1,the concentrations of VEGF,FGF-2,HGF,IGF-1,GM-CSF,MCP-1,TGF-β1 and IL-8 secreted by hASCs were higher than those of the control group（all P<0.05）,while the concentration of IL-6 was significantly lower than the control group（all P<0.05）（3）After adipogenic induction of hASCs stimulated by G-Rg1,larger Oil Red O staining-positive lipid droplets were observed in the cytoplasm compared with cells cultured with AIM only.The number and lipid concentration of adipocytes in different concentrations of G-Rg1 group were not only higher than the control group（P<0.05）,but also showed a positive dose-dependent effect.q PCR and WB tests showed that the expression levels of PPARγ2,C/EBPαand ADD1 in the Rg1-stimulated group were significantly higher than those in the control group（P<0.001）,which further proved the ability of G-Rg1 to promote adipogenic differentiation.3.72 hours after being infected with adenovirus,hASCs in group MOI=50,group MOI=100,and group MOI=200 all showed visible fluorescence.Group MOI=200 was used to construct GFP-hASCs for which had the strongest fluorescence and the best infection efficiency.The GFP-hASCs+collagen scaffold with or without G-Rg1 pretreatment was transplanted to the left and right sides of the back of 20 nude mice.The volume of new adipose tissue in the G-Rg1 pretreatment group was larger than that of the control group,and the wet weight was significantly increased（P<0.001）.The detection results of cell density and lipid concentration suggest that the number and lipid concentration of G-Rg1 pretreated componentized adipocytes were significantly increased compared with the control group（P<0.001）.q PCR test showed that the expression levels of PPARγ2 and ADD1 in the G-Rg1 pretreatment group were significantly higher than those in the control group（P<0.001）.Conclusion:G-Rg1 can optimize the proliferation,adipogenic differentiation and paracrine functions of hASCs;hASCs can be transformed into adipose cells in the in vivo environment;G-Rg1 can promote the survival and adipogenic differentiation of hASCs in vivo,there by improving the survival rate of regenerated tissues assisted by hASCs transplantation.