Research Of 5-hydroxymethyl-2-furaldehyde Extract From Eucommia Ulmoides On The Adipogenic Differentiation Of Adipose Stem Cells And The Signaling Pathways

Purpose:To make the effective monomeric substance,5-hydroxymethyl-2-furaldehyde,5-HMF, extracted from Eucommia Ulmoides, to be revulsive of isolated culturing rat’s adipose tissue-derived stromal cells, differentiating into fat cells. To observe the changes in the signaling pathway in the process. To further explore the effect and mechanism of traditional Chinese medicine Eucommia Ulmoides for the prevention and treatment of osteoporosis.Medthod:a. Rat’s ADSCs were cultured and purified in vitro.b.5-HMF was made by salt Eucommia Ulmoides’s watering, concentration, extraction, precipitation and HPLC methods.c. Dexamethasone, indomethacin, insulin and 3 isobutyl 1 methylxanthine were made as revulsive of differentiating rat’s ADSCs into fat cells. Oil red O staining was used to show the formation of lipid droplets in cells. Lipid droplets were used as the observation index to determine the degree of differentiation of induced cells.d. Effect of 5-HMF on ADCs’s fat differentiation.Rat’s ADSCs, purified and cultured was made into 5-HMF experimental group and differentiated into adipocytes. Fat cells were induced into positive control group and negative control group. Cells were inducted and then morphologically observed and taken photographs by inverted microscope.e. Study of the signal pathway of 5-HMF inhibiting ADSCs fat differentiation.The purified ADSCs group was divided into positive control group, negative control group and 5-HMF positive control group. After two weeks of induction, the total DNA extracted was conducted reverse transcription, and detected expression of Fzd3, MEK1/2, ERK1/2, Raf, b-Catenin, c-myc mRNA by real-time fluorescence quantitative PCR method. Protein extracted was detested expression of P38, p-P38, b-Catenin, p-b-Catenin, ERK1/2, p-ERK1/2 by Western blot.To mix signaling pathway inhibitor DKK1 and MAPK signal pathway inhibitor U0126, with ADSCs, induced by 5-HMF.To detect the change of expression of PPARg, Adipoq and ABP4 mRNA by real-time fluorescence quantitative PCR method. The protein expression changes of p-PPARg, FABP4 and PPARg were detected by Western blotting.Rsults:a. The quantity of fat and the expression of adipocyte marker protein such as PPARg, Adipoq, FABP4 and FABP4 decrease significantly, in the course of the differentiation of ADSCs into fat.b. During the differentiation of adipose derived mesenchymal stem cells into adipocytes, Wnt signaling pathway and MAPK signaling pathway are activated or inhibited. Inhibitors of the two signaling pathways weaken the effect of 5-HMF on the differentiation of ADSCs into adipocytes.c.5-HMF, the effective monomer, extracted from Eucommia Ulmoides can significantly inhibit the differentiation of adipose derived mesenchymal stem cells into fat cells.