The pathogenesis of the alcohol-induced osteonecrosis and the preventive effect of puerarin

Objective To observe the pathogenesis of the alcohol-induced osteonecrosis of the femoral head and the preventive effect of puerarin which inhibits adipogenesis of marrow stromal cells induced by alco- hol.Methods The primary marrow stromal cells(MSCs)were isolated and cultured from mice bone mar- row.MSCs were separated by 3 groups at random:Group A:the cells were treated with increasing(0,0.03, 0.09,and 0.15 mol/L)concentrations of ethanol.Group B:the cells were treated with 0.90 mol/L ethanol and 0.01mg/ml puerarin.Group C:the cells were treated without ethanol or puerarin as control.The cells in culture were stained with SudanⅢ,and then the number of adipocytes were counted on a light microscope. The contents of intracellular triglyceride,alkaline phosphatase activity in cells and the contents of osteocalcin in the media were determined.The expression level of 422(aP2)and type-Ⅰcollagen mRNA's in the cells treated with 0.09 mol/L ethanol and without with ethanol was investigated by means of intact cell RNA dot blot hybridization.The expression levels of the peroxisome proliferator-actived receptor-γ(PPARγ)mRNA and os- teoealcin mRNA in cells were analyzed by reverse transcription-polymerase chain reaction(RT-PCR).Re- sults The cells in culture were treated with increasing concentrations of ethanol for 21 days.The number of adipocytes increased with longer durations of exposure to ethanol and with higher concentrations of ethanol, which showed the time-and dose-dependent relations(P＜0.001).The level of triglyceride in the cells trea- ted with with ethanol was markedly higher than that in the control cells(P＜0.001).ALP activity in the cells decreased with higher concentration of ethanol(P＜0.001).The level of osteocalcin in culture media of the cells treated with 0.15 mol/L of ethanol greatly decreased(P＜0.001 ).The 422(aP2)mRNA contents in the group A were markedly higher than that in the group C.By contrast,the type-1 collagen mRNA contents in the group A were significantly lower than that in the group C.The expression levels of PPARγ,mRNA in the cells of group B and group C were significantly lower than that in the cells of group A(P＜0.05),and there were not significant differences statistically between group B and group C(P＞0.05 ).The expression levels of osteocalcin mRNA in the cells of group B and group C were significantly higher than that in the cells of group A(P＜0.01),and there were not significant differences statistically between group B and group C(P＞0.05).Conclusion Alcohol could induce the differentiation of marrow stromal cells into adipocytes and in- hibit osteogenic differentiation by regulating gene expression,which might associate with the development of the alcohol-induced osteonecrosis.Puerarin could inhibit differentiation of marrow stromal cells into adipocytes induced by alcohol,to maintain the normal procedure of osteogenic differentiation,which may prevent the de- velopment of the osteonecrosis.