| Objective: Obesity plays a key action in the disease of type 2 diabetes and insulin resistance. It is beneficial for prevention and curing the type 2 diabetes by the way of inhibiting appetite as well as promoting energy consuming. The increase of adipocytes is aroused from differentiating of the preadipocytes.The volume of adipocytes reflects the relationship between lipidolysis and lipid synthesis. Lipid metabolic disorder and insulin resistance are the key features of type 2 diabetes, which also the main causes of cardiac-vascular diseases. At the clinic, curing of diabetes usually emerge weight gain. Euglycemic agent such as Thiazolidinediones (TZDs) can increase the insulin sensitivity, induce the differentiating of preadipocytes and cause weight gain by activating the transcription factor Peroxisome prolif erators-activated receptors 2 (PPARγ2).Now it is proved that the puerarin can improve insulin resistance. It is still not clear of the puerarin act in weight gain of type 2 diabetes patients. Above all, it is meaningful to study the influence of puerarin in the differentiating of the preadipocytes.Methods:Preadipocytes were plated in 6-well culture plates at a density of 5×10 5cells/well. After 1 day , it would reach maximal confluence .Then the cells were cultured in 1ml of DMEM containing of 10% FCS at 37℃and 5%CO 2 for 2 days. Then add 0.5mmol/L GABA, 0.25μg/ml Dexamethasone and 1μg/ml insulin in the medium for 48h, then culture the cells in the DMEM containing of 10% FCS again. The 40μg/ml puerarin should be added into the medium on the 1 day of the whole induction, which acts through the whole experiment. Use the cells differentiated for 10~12 days into the experiment. After stained with 0.1 mg/ml Oil Red O solution for 2 hours,observe the results with inverted microscope and take photos. Measure the concentration and degree of purity of RNA.Design and synthesize the primer. Two steps Reverse Transcription Polymerase Chain Reaction(RT-PCR)is selected to detect the expression of PPARγ2mRNA according to instruction. The cells are classified into control group and puerarin group. Extract the total RNA from the complete differentiated cells. Reverse transcription the total RNA into cDNA, then use the agarose gel electrophoresis to identify amplification product, then scan and analyze with FX-MAC. Make immunoblotting assay for the PPARγ2 protein, coloration with DAB, scan and analyze with FX-MAC.Results:1. Induct the preadipocytes differentiate into adipocytes for 10 days. The control group shows typical adipocyte phenotype. More than 90% of the cells contain big lipid droplet, while the puerarin group, only 20~30% of the cells emerge big lipid droplet. For the puerarin group, coloration of Red Oil O is obviously lighter than the control group.2. The MTT results of preadipocytes proliferation indicates that puerarin can promote the proliferation process. The OD results of 10,20,40μg/ml puerarin groups are 0.3848±0.0169,0.4105±0.0201,0.4328±0.0195, P<0.05,when compared with the control group, it is meaningful in statistic; The OD results of 80,160μg/ml puerarin groups are 0.3104±0.0149,0.3201±0.0153,which shows no meaning in statistic.3. The analysis of semi quantitative RT-PCR indicates that adding puerarin during the induction of adipocytes differentiation can depress the express of PPARγ2mRNA significantly. The fluorescent intensity of the control group and 10,40μg/ml puerarin groups are 1.3866±0.02108,1.1794±0.01937,0.7765±0.01877.The 40μg/ml puerarin group is meaningful in statistic, P<0.05.4. The result of Western Blot shows that after being treated by puerarin, the express of protein PPARγ2 depressed. Conclusions: The result of MTT shows that the puerarin can promote preadipocytes proliferation and suppress the differentiation process. The results of Oil Red O staining indicates that the puerarin group can inhibit the preadipocytes differentiate into adipocytes.The results of RT-PCR and Western Blot show that the puerarin can inhibit the expression of PPARγ2mRNA and protein.
|
PDF Full Text Request