Anti-obesity Mechanism Of Ginsenoside F2 And Its Effect On Gut Microbiota In Obese Mice

Obesity has become a public health issues that seriously threat to people’s quality of life.Among various contributing factors of obesity,nutritional obesity caused by unbalanced diet was the most common.Anti-obesity drugs may cause a variety of side effects,and therefore it is still necessary to explore the mechanisms of obesity.Ginsenoside F2(GF2)possesses varieties of biological effects,such as anti-inflamatory,anti-oxidation,and anti-cancer.In this study,the anti-obesity effect of GF2 was systematically explored in vivo and in vitro.In addition,the effects of GF2 on gut microbiota were also discussed.(1)In order to explore the lipid-lowering mechanism of GF2 in vitro,3T3-L1 preadipocytes were induced to producing lipid droplets by a ‘cocktail’ method.MTT cell viability assay and trypan blue cell count analysis showed that GF2 did not affect the viability of 3T3-L1 preadipocytes within a concentration below 100 μM,but it significantly inhibited the proliferation of 3T3-L1 preadipocytes during the differentiation with MDI medium contained 25-100 μM GF2.Oil Red O staining showed that GF2 performed the remarkable lipid-lowering effect in 3T3-L1 adipocytes at the early stage of differentiation and significantly reduced the intracellular TG content in a concentration-dependent manner.RT-q PCR analysis indicated that the inhibitory effect of GF2 may be related with the down regulation of the expression of adipogenesis transcription factors such as PPARγ and C/EBPα,thereby inhibiting the differentiation and lipogenesis of 3T3-L1 preadipocytes.(2)Network pharmocology analysis predicted that the anti-obesity of GF2 involved 35 potential targets,including VEGFA,MAPK2,CASP3,et al.GO enrichment suggested that the potential targets mainly participated in metabolism process and binding and cellular component formation.Moreover,it may play an anti-obesity effect through EGFR tyrosine kinase inhibitor resistance,PI3K-Akt signaling pathway and neuroactive ligand-receptor interaction pathway.Hence,network pharmacology successfully showed the multi-targets and multi-pathways characteristics of GF2.(3)RNA sequence was carried out to detect the gene profile of 3T3-L1 preadipocytes induced by MDI medium with GF2.A total of 975 differentially expressed genes were obtained,of which475 DEGs were up-regulated and 518 DEGs were down-regulated.GO enrichment analysis showed that DEGs were mainly involved in the metabolism of fatty acids,organic acids and lipid.KEGG pathway enrichment indicated that the DEGs were significantly enriched in many biological pathways,such as AMPK signaling pathway.Next,western blot was used to verify the AMPK pathway.The results showed that GF2 markedly promoted the phosphorylation levels of AMPK and ACC.Moreover,the distribution of mitohcondria in adipocytes was also observed by means of immunofluorescence labeling.The results suggested that GF2 increased the number of mitochondria.Consequently,GF2 inhibited differentiation and regulated lipid metabolism of3T3-L1 preadipocytes via activating AMPK signaling pathway and promoting mitochondrial biosynthesis.(4)High-fat-diet induced obese C57BL/6J mice were used to explore the anti-obesity effects of GF2 in vivo.After administered with GF2 for 4 weeks,the weight gain,adipose tissue weight and adipocyte size of obese mice were reduced significantly by GF2,without affecting food intake.Besides,GF2 also regulated the blood lipid profiles by decreasing the serum levels of TC,TG,and AST.H&E analysis showed that GF2 remarkably reduced the size of adipocytes and fat vacuoles of liver.Furthermore,the protein expression in AMPK pathway was consistent with the results in vitro,confirming that the anti-obesity effect of GF2 was mediated through the activation of AMPK signaling pathway.(5)GF2 intervention remodeled the composition of gut microbiota in obese mice induced by high-fat-diet and affected its diversity.At the phylum level,GF2 significantly reduced the abundance ratio of Firmicutes to Bacteroidetes.As the genus level,GF2 significantly increased the relative abundance of Akkermansia.These microorganisms have been reported to be closely related to lipid metabolism.Therefore,GF2 may alleviate obesity by regulating the relative abundance of these gut microbiota in the meanwhile.