Effect Of ATRA On Differentiation Of Preadipocytes In Yanbian Yellow Cattle

All-trans retinoic acid（ATRA）regulates adipocyte differentiation and lipid metabolism.It participates in a series of important processes in life activities,including cell proliferation and differentiation,cell apoptosis,fat oxidation,and maintenance of body immunity.A large number of studies have confirmed that ATRA has an important role in the process of fat formation.The experiment was carried out by culturing Yanbian yellow cattle precursor adipocytes and setting the ATRA treatment group concentration to 0,2×10^-7,1×10^-6,2×10^-6,1×10^-5and 2×10^-5mol/L.MTS,flow cytometry,Real-time PCR and Western blot were used to detect the mechanism of ATRA regulating the proliferation and differentiation of Yanbian yellow cattle precursor adipocytes,and provide a theoretical basis for the production of Yanbian yellow cattle high-quality beef.The results are as follows:1.ATRA has an inhibitory effect on the proliferation of Yanbian yellow cattle precursor adipocytesThe Yanbian cattle pre-adipocytes were inoculated into the multi-well plate for24 hours and then ATRA was added.The MTS method was used to measure the cell OD on days 0,2,4,6,and 8.When the cells grow to about 50%,total RNA is extracted,the expression of the proliferation marker genes PCNA,CDK 2,and Cyclin D1 are detected,and the cell cycle is detected by flow cytometry.The results showed that the OD value of the cells treated with ATRA was extremely significantly reduced,the cells remained in the G1/G0 phase,and the expression of PCNA,CDK 2,and Cyclin D1 genes were extremely significantly reduced,indicating that ATRA had a significant inhibitory effect on the proliferation of Yanbian cattle precursors.2.ATRA inhibits the differentiation of Yanbian yellow cattle precursor adipocytesDifferent concentrations of ATRA were added while inducing the differentiation of Yanbian yellow cattle precursor adipocytes.After induction,oil red O staining was performed to extract total cell RNA and protein,and PPARγ,C/EBPa,ERK 2 and ADPN mRNA and protein expression were detected.The results showed that compared with the control group,the lipid droplet content and the expression of PPARγ,C/EBα,ERK 2 mRNA and protein in Yanbian yellow cattle treated with ATRA were inhibited,and the higher the ATRA concentration value,the obvious inhibition effect.ADPN gene mRNA and protein The expression of is on the rise.It shows that ATRA can inhibit the differentiation of Yanbian yellow cattle precursor adipocytes.3.ATRA participates in the regulation of mTOR signaling pathway by inhibiting the expression of AKT 2 gene,and inhibits the differentiation of Yanbian yellow cattle precursor adipocytesThe mTOR signaling pathway is very sensitive to changes in insulin levels in animals,and can regulate the production of animal lipids by sensing insulin signals.AKT 2 is located upstream of the mTOR signaling pathway and has a regulatory effect on it.SREBP1 is an important gene in the mTOR signaling pathway,which can regulate FAS and ACC genes.In this study,different concentrations of ATRA were added during the differentiation of Yanbian yellow cattle precursor adipocytes.After differentiation,the total RNA and protein of the cells were extracted,and the mRNA and protein expression of AKT 2,SREBP1,FAS and ACC were detected.The results showed that compared with the control group,the mRNA and protein expressions of AKT 2,SREBP1,FAS,and ACC all decreased to varying degrees.Among them,with the increase of ATRA concentration,the expression of AKT 2,SREBP1 and FAS genes showed a linear decrease.The ACC gene expression of the 2×10-7 mol/L treatment group decreased significantly,and the other treatment groups significantly decreased.It shows that ATRA participates in the regulation of mTOR signaling pathway by inhibiting the expression of AKT 2 gene,and down-regulates the expression levels of mTOR signaling pathway related genes SREBP1,FAS,ACC,thereby inhibiting the differentiation of Yanbian yellow cattle precursor adipocytes.