Inhibition of puerarin in the differentiation of marrow stromal cells into adipocytes induced by alcohol / 中国组织工程研究

ABSTRACT

BACKGROUND: Animal tests verified that alcohol could cause lipid accumulation in femoral head, leading to osteonecrosis. Molecular biology tests verified that alcohol could induce differentiation of marrow stromal cells (MSCs) into adipocytes. If a drug can resist the differentiation of MSCs into adipocytes induced by alcohol, it can prevent and treat alcoholinduced osteonecrosis.OBJECTIVE: To observe the effects of inhibition of puerarin on differentiation of MSCs into adipocytes induced by alcohol from the aspect of cell biology.DESIGN: Randomized controlled experiment.SETTING: Department of Orthopaedics, First Affiliated Hospital,Zhengzhou University and Department of Biochemistry and Molecular Biology, Basic Medical College, Zhengzhou University.MATERIALS The experiment was conducted at the Department of Biochemistry and Molecular Biology, Basic Medical College, Zhengzhou University from April 2000 to December 2002. A total of 50 clean healthy Kunming mice aged 6-8 weeks, of either sex, were selected. Bone marrow cells of the bilateral femur were obtained, with the inoculated density of 1.5×106/cm2, implanted into 6 wells and 24 wells cultured plate, randomized into groups, a well as a specimen and 24 specimens in each group.METHODS: MSCs were separated, cultured and assigned into 3 groups at random model group (cells were treated with 0.09 mol/L alcohol), experimental group (0.09 mol/L alcohol and 0.01 mg/mL final concentration of puerarin) and controlled group (no alcohol and puerarin). Sudan Ⅲ staining was performed and adipose cells was counted under light microscope.Content of triacylglycerol, activity of alkaline phosphatase and content of osteocalcin in cell culture fluid were measured.MAIN OUTCOME MEASURES: ①Result of differentiation of MSCs into adipocytes in micé of the three groups, ②content of triacylglycerol in MSCs of mice of the three groups, ③activity of alkaline phosphatase in MSCs of mice of the three groups, ④content of osteocalcin in cell culture fluid in the three groups.RESULTS: ①After being disposed and cultured for 21 days, number of adipocytes in the experimental group and control group decreased markedly compared with the model group. The number of adipocytes in the model group was 8.9 times of that in the experimental group, and 15.6 times of that in the control group [(319.17±19.92), (35.92±23.77)(20.42±12.15)per cm2,P＜0.001]. ②After being disposed and cultured for 21 days, coutent of triacylglycerol in the experinental group and control group was remarkably lower than that in the model group [(4.15±1.92) and (3.42±1.60),(11.55±4.42) μgin a well, P ＜ 0.001]. ③After being disposed and cultured for 12 days, activity of alkaline phosphatase in the experimental group and control group was distinctly higher than that in the model group [(9.51±2.96),(11.18±3.11), (4.84±2.23) μkat/g,P ＜ 0.001]. ④After being disposed and cultured for 14 days, content of osteocalcin in the experimental group and control group was 2.2 times and 2.7 times of that in the model group, respectively, and the difference was very significant com pared with themodel group [(11.11±4.57),(13.43±5.29), (4.95±2.31) μg/g,P ＜ 0.001].CONCLUSION: Puerarin can inhibit the differentiation of MSCs into adipocytes induced by alcohol, and maintain the osteogenic differentiation,which may prevent the development of osteonecrosis.