Effects And Mechanism Of Human Amniotic Mesenchymal Stem Cells Conditional Medium On High-Fat Diet Induced Obesity In Mice

Background and aims:Obesity is a chronic metabolic disease caused by a variety of factors,in which it can cause a series of complications such as diabetes,heart disease,dyslipidemia and hypertension.Human amniotic mesenchymal stem cells(hAMSCs)are a kind of pluripotent stem cells with low immunogenicity,no tumorigenicity,self-renewal and multi-directional differentiation potential.Studies have shown that hAMSCs and its secreted cellular factors play an important role in inhibiting inflammation,tissue repair and immunoregulation.However,the effect of the hAMSCs,specially the hAMSCs conditional medium(hAMSCs-CM)on the pathological process of obesity has not been clearly reported.This study aimed to investigate the effect of hAMSCs-CM on high fat diet induced obesity with a mouse model.Meanwhile,the effects of hAMSCs-CM on the maturation and differentiation of 3T3L1 preadipocytes and inflammatory secretion of macrophages were investigated in vitro.Finally,the possible active factors were explored by combining the results of protein chip.The completion of this project will further explore the application potential of hAMSCs-CM and provide new ideas for the prevention and treatment of obesity.Methods:1.Isolation of hAMSCs and preparation of hAMSCs-CM:Fresh and healthy amnion was used for primary hAMSCs isolation as descripted in the protocol of our lab.After digestion with trypsin and collagenase respectively,the cells were collected for cell culture.The CM was collected with serum-free DMEM for 48 hours within the 5th generation.Finally,CM was collected according to the cell density of1.5 x 10~6 and administered every 20g mice.2.HFD induced obesity model in mice:The mice were fed with high-fat diet to establish an obesity model.After intraperitoneal injection,the effects of hAMSCs-CM on obesity were analyzed by detecting the changes of body weight,histomorphology,serum lipid changes and glucose tolerance in mice.3.Mechanism analysis:1)Oil red staining was used to observe the effect of hAMSCs-CM on the differentiation of 3T3L1 preadipocytes into mature adipocytes,and the lipogenesis-associated gene expression such as PPARγ,SREBP1,FASN were detected by western blot or qPCR analysis.2)QPCR was used to detect the effect of hAMSCs-CM on macrophage polarization.RAW264.7 macrophages treated with LPS+IFN-γwere used to detect the expression of M1-type pro-inflammatory protein gene.RAW264.7 macrophages treated with IL4 were used to detect the expression of M2-type anti-inflammatory gene protein.Result:1.hAMSCs-CM improved HFD-induced obesity,fatty liver in mice,and can reduce hyperlipidemia,adipocyte hypertrophy and glucose tolerance in vivo.2.hAMSCs-CM inhibited the maturation and differentiation of preadipocytes into adipocytes in vitro:1)Oil red staining results showed that lipid accumulation was reduced by hAMSCs-CM treatment in mature adipocytes;2)Q-PCR and Western blot showed that hAMSCs-CM significantly inhibited the expressions of lipogenic genes such as PPARγ,SREBP1 and FASN.3.HAMSCs-CM significantly inhibited LPS plus IFN-γ-induced secretions of M1-type proinflammatory factors IL-6,IL-1β,and promoted IL-4-induced secretions of M2-type anti-inflammatory factors ARG-1 and IL-10 in RAW264.7 cells.Conclusion:HAMSCs-CM can significantly inhibit HFD-induced obesity in mice and the mechanism may be related to inhibiting the maturation and differentiation of adipocytes,thereby affecting their lipid accumulation and promoting macrophage M1polarization to M2.