Puerarin Inhibits Differentiation Of Marrow Stromal Cells Into Adipocytes Induced By Alcohol

The avascular necrosis of the femoral head (ANFH), among which the most cases were induced by alcohol, is a common disease. It often occurs and causes disabilities in clinic. Previous studies showed that alcohol may induce marrow stromal cells (MSCs) differentiate into adipocytes, and followed systemic changes in lipid metabolism, then osteonecrosis eventually develops. But the study of preventive and treatment modalities about those also have not been reported in the literature. We isolated, obtained and cultured the mice primary MSCs, to which alcohol and puerarin were added. The effects of inhibition of puerarin on differentiation of MSCs into adipocytes induced by alcohol were observed by the methods of cell and molecular biology to further investigate the protective mechanism of medicine to alcohol-induced ANFH.Materials and MethodsThe 6-8 week-old female mice offered by the experimental animal center of Henan province were used in the study. Marrow cells were gained from the midshafts of femur bones. In the same time when cells were planted, The cells were treated with 0.09mol/L alcohol in Group A. The cells were treated with 0.09mol/L alcohol and 0.01mg/ml puerarin in Group B. The cells were treated withoutalcohol or puerarin in Group C as control. MSCs were stained with Sudan III after they cultured for 21 days and then the number of adipocytes were counted; The contents of triglyceride in MSCs cultured for 21 days and alkaline phosphatase activity in MSCs clutured for 12 days respectively were determined by biochemical assay; The contents of osteocalcin(BGP) in the media at 14th day were determined by radioimmunoassay; Protein in the cell layer was measured by the Coomassie blue method; The expression levels of PPARy (adipogenic transcription factor) mRNA and Osteocalcin mRNA in MSCs cultured for 6 days were analyzed by Reverse Transcription-Polymerase Chain Reaction. Result1. MSCs could be isolated from other marrow cells by their tendency to adherent growth.2. The number of adipocytes (number of adipocytes/cm ) were 319.17 + 19.92; 35.92?3.77; 20.42?12.15 respectively after MSCs were cultured 21 days in Group A, B and C. The number of adipocytes in Group C is the fewest, and that in Group A was highly increased because of the addition of alcohol. There were statistically significant differences between Group A and the other 2 groups (P<0.001). But the Group B has more lower quantity of adipocytes than Group A because of the effect of puerarin, and there were not significant differences statistically between Group B and C (P>0.05).3. The contents of triglyceride in the cells in Group A were 2.8 and 3.4-fold higher than that in Group B and Group C at 21days. There were statistically significant differences between Group A and the other 2 groups (PO.001). And there were not statistically significant differences between Group B and C (P>0.05).4. The contents of BGP in the media of Group B were as2.2-times as Group A after MSCs were exposed to alcohol and puerarin 14 days. There were significant differences between Group A and the other 2 groups (P<0.001). And there were not significant differences statistically between Group B and C (P>0.05).5. Alkaline phosphatase (ALP) activity values(U/100mg proteins) in MSCs treated with alcohol and puerarin 12 days of Group B were as 2-times as of Group A. The values were decreased with the addtion of alcohol but increased with the addition of puerarin. There were no significant differences between the Group B and C (P>0.05). But There were significant differences between Group A and the other 2 groups (P<0.001).6. Gene expression: After MSCs were exposed to alcohol and puerarin for 6 days, the results of RT-PCR showed that Group A treatment with alcohol significantly lower expression of osteocalcin mRNA than Group B and C (P<0.01), but there were no significant differences between the Group B and C (P>0.05). And there were differences between Group A and the other 2 groups on expression of PPARy...