| Background and aims:Obese metabolic syndrome is a complicated metabolic disorder caused by obesityinduced abnormal glucose and lipid metabolism,insulin resistance and hepatic steatosis.It is also a risk factor for diabetes,obstructive apnea-hypopnea syndrome,tumor and cardio-cerebrovascular diseases.Human amniotic mesenchymal stem cells(hAMSCs)are pluripotency stem cells with no ethical controversy,no tumorigenesis,low immunogenicity,multilineage differentiation potential and strong paracrine effects,which have a great potential in clinical application.hAMSC-exosomes produced by their paracrine are rich in bioactive substances such as miRNA and proteins,which have some advantages such as having no risky of tumorigenesis,easy to store and realizing industrial production compared with cell therapy.Our previous studies showed that hAMSCs and their conditional medium/secretions(hAMSCs-CM)efficiently repaired hot water-induced skin wounds,significantly inhibited hepatocellular carcinoma induced by Hep G2 cells and obviously alleviated high fat diet(HFD)-induced hyperglycemia,and miR-181 and miR-199 derived from hAMSC-exosomes prominently suppress pathological melanosis.Our previous results demonstrated that hAMSCs-CM significantly alleviated HFD-induced obesity in mice by effectively attenuating visceral adipose tissue hypertrophy,impaired glucose tolerance and hepatic dysfunction.However,the in detailed mechanism has not been explored.Whether the active substances such as hAMSCs-CM-exosomes and included miRNA play a role has not been reported.Based on these results,we used the HFD-induced obese mice to monitor energy expenditure,and combine hAMSC-exosomes-miRNAs sequencing to discover and verify the effects of specific miRNAs in hAMSC-exosomes on adipocyte maturation,differentiation and lipid metabolism and molecular mechanism.In addition,we explored the effect and mechanism of hAMSC-exosomes on the polarization of adipose tissue macrophages.This project aims to provide an insight in prevention and treatment of obesity clinically.Methods:1.Isolation and identification of hAMSCs and hAMSC-exosomes:1)hAMSCs from amnion were isolated using trypsin and collagenase digestion for cell culture;2)Identification of hAMSCs immunophenotype by RT-PCR,flow cytometry and immunofluorescence;3)Isolation of exosomes by ultracentrifuge;4)The morphology and diameter of exosomes were measured by transmission electron microscopy,and exosomes surface molecular markers CD63,TSG101 and HSP70 were confirmed by western blot.2.Effects of hAMSCs-CM on energy expenditure dysfunctions in HFD induced obese mice: Obese mice were induced by HFD feeding for 15 weeks,and mice were assigned into 3 groups,normal diet group(CHOW-D),HFD group and hAMSCs-CM treated group fed with HFD(HFD-CM).1)To investigate the role of hAMSCs-CM/exosomes in oxygen consumption(VO2),carbon dioxide production(VCO2),food and water intake in obese mice by metabolic chambers;2)The effect of hAMSCs-CM on lipid accumulation and glucose absorption in liver was respectively detected by oil red O staining and PAS staining;3)The effect and mechanism of hAMSCs-CM on lipid and glucose metabolism: the expressions of the proteins related to lipid metabolism such as PPARα,PGC1α and the proteins related to glucose metabolism protein GLUT4,AMPK,the proteins related to IGFR/AKT/GSK3β signaling pathway and the proteins related to insulin sensitivity such as IGFR in adipose tissue/adipocytes were detected by western blot analysis;4)The expression of thermogenic protein UCP1 in BAT was analyzed by immunofluorescence,RT-qPCR and western blot.3.Effects of hAMSC-exosomes and exosomes-derived miR-199b-5p on adipogenesis in vitro: 1)the effect of hAMSC-exosomes on adipogenesis was examined by oil red O staining,and western blot analysis;2)By combined with exosomes miRNA sequencing results,and further integrated KEGG enrichment pathway,GO enrichment and bioinformatics analyzed the possible miRNA related to adipogenesis;3)RT-qPCR was used to analyze miR-199b-5p and miR-27a-3p in white adipose tissue and different induction time during 3T3L1 preadipocyte differentiated to mature adipocyte;4)Effect of miR-199b-5p and miR-27a-3p on adipogenesis was determined by oil red O staining during the process of 3T3L1 preadipocytes differentiated to mature adipocytes,and western blot and RT-qPCR were used to detect the expressions of the proteins related to adipogenesis such as FAS,PPARγ,SREBP1 and C/EBPα;5)Dual-luciferase reporter assay was used to determine whether GSK3β is the possible target gene of miR-199b-5p,the effect of GSK3β inhibitor SB216763 on adipogenesis was examined by oil red O staining during the process of 3T3L1 preadipocyte differentiated to mature adipocyte,western blot and RT-qPCR were used to detect the expressions of the proteins related to adipogenesis such as FAS,PPARγ,SREBP1 and C/EBPα.4.Effects of hAMSCs-CM/exosomes on the inflammation of white adipose tissue in HFD-induced obese mice: 1)the expressions of macrophage markers F4/80 was analyzed by immunofluorescence,and the m RNA expressions of the inflammation cytokines ARG-1,CD206,CD11 C and TNFα were examined by RTPCR,the expressions of STAT3/ARG-1 signaling pathway were detected by western blot analysis;2)To analyze the roles of hAMSCs-CM and hAMSCexosomes in macrophage inflammation,RAW264.7 macrophages were treated with LPS and then the protein expressions of the inflammation related proteins of NFκB and MAPK signaling pathway.Results:1.hAMSCs possess the characteristics of the mesenchymal stem cells:1)hAMSCs from human amniotic membrane were isolated by trypsin and collagenase digestion and hAMSCs have a typical bipolar spindle-like and fibroblastic-shaped morphology and strong reproduction ability;2)RT-PCR assay confirmed that hAMSCs expressed the embryonic stem cells(ESCs)markers(SOX2,Nanog,OCT4)and MSCs markers(CD29,CD90,CD73,CD105),but not express HSCs markers(CD34 CD133,CD45),Flow cytometry further confirmed that hAMSCs expressed mesenchymal stem cells(MSCs)surface markers including CD73,CD90,CD29 and CD105,as well as human leucocyte antigen class I(HLA-I ABC).However,hAMSCs did not express CD40,CD80,HLA-II DR and hematopoietic stem cells(HSCs)markers CD34,CD45.In addition,the expressions of ESCs surface marker SSEA4 and MSCs marker Vimentin were also confirmed by immunofluorescent staining;3)hAMSC-exosomes exhibited spherical with uniform in size and the diameters were 50-150 nm approximately.Western blot results showed that there were exosomes molecular markers CD63,TSG101 and Hsp70,but not endoplasmic reticulum marker Calnexin in hAMSCs-exosomes.2.hAMSCs-CM/exosomes improve HFD-induced metabolic dysfunctions by activating AMPK/PPARα and IGFR/AKT/GSK3β signaling pathways,respectively,to promote lipid and glucose metabolism in vivo: 1)The metabolic chambers results showed that hAMSCs-CM significantly reversed HFD-induced reductions in VO2,VCO2 and water intake in mice,but not food intake;2)hAMSCs-CM markedly reduced HFD-induced lipid droplets deposition and increased glycogen accumulation in liver;3)hAMSCs-CM markedly promoted the expressions of the proteins related to lipid metabolism such as PPARα,PGC1α and glucose transporter GLUT4 in HFD-induced mice,and promoted lipid and glucose metabolism by activating AMPK/PPARα and IGFR/AKT signaling pathways in WAT and AKT/GSK3β signaling pathway in liver,respectively,and hAMSCs-CM and hAMSC-exosomes promoted the phosphorylation of insulin sensitivity related protein IGFR in adipocytes;4)Immunofluorescence,RT-qPCR and western blot results showed that hAMSCs-CM significantly improved thermogenesis protein UCP1.3.hAMSC-exosomes and exosomes-derived miR-199b-5p inhibited adipocytes differentiation in vitro: 1)Oil red O staining and western blot results showed that hAMSC-exosomes inhibited adipocyte differentiation and reduced the expressions of the adipogenesis related proteins PPARγ and C/EBPα;2)The expression of miR-199b-5p and miR-27a-3p of WAT in mice fed with HFD was lower than that in the mice fed with normal diet and the expressions of these miRNAs were decreased during 3T3L1 preadipocytes differentiated to mature adipocytes;3)Oil red O staining,western blot and RT-qPCR results further demonstrated that miR-199b-5p and miR-27a-3p inhibited adipocyte differentiation and decreased the expressions of adipogenesis related proteins such as FAS,PPARγ,SREBP1 and C/EBPα;4)The results from dual-luciferase reporter assay showed that miR-199b-5p directly targeted GSK3β,and oil red O staining and western blot results showed that GSK3βinhibitor SB216763 also attenuated adipocyte differentiation and decreased the expressions of the adipogenesis related proteins of FAS,PPARγ,SREBP1 and C/EBPα.4.hAMSCs-CM/exosomes alleviate the inflammation of white adipose tissues in in HFD-induced obese mice by activating STAT3/ARG-1 signaling pathway and suppressing NFκB and MAPK signaling pathway: 1)Immunofluorescence,RT-qPCR and western blot results showed that hAMSCs-CM significantly inhibited macrophage marker F4/80 expression and reduced the m RNA levels of proinflammatory cytokines CD11 C and TNFα,but enhanced that of antiinflammatory cytokines ARG-1 and CD209 A in WAT,suggesting that hAMSCsCM suppressed WAT inflammation by activating STAT3/ARG-1 signaling pathway;2)hAMSCs-CM and hAMSC-exosomes suppressed LPS-induced phosphorylation of the proteins such as IKKα/ β,P65,JNK,AKT,ERK and P38 in NFκB and MAPK signaling pathway in RAW264.7 cells.Conclusion:1、 hAMSCs used in this study were characterized by expressing mesenchymal stem cell and embryonic stem cell surface markers,non-expressions of hematopoietic stem cell surface markers and showed low immunogenicity.2、 hAMSCs-CM was able to improve HFD-induced metabolic dysfunction by activating AMPK/PPARα and IGFR/AKT/GSK3β signaling pathways,and hAMSC exosomes-derived miR-199b-5p directly targeted GSK3β to inhibit adipocytes differentiation.3、 hAMSCs-CM/exosomes alleviated the inflammation of white adipose tissue in HFD-induced obese mice by activating STAT3/ARG-1 signaling pathway and suppressing NFκB and MAPK signaling pathway to improve macrophage inflammation. |
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