Experimental study to prevent alcohol-induced osteonecrosis of the femoral head with puerarin

Objective :To prevent alcohol-induced osteonecrosis of the femoral head,observe the effects of inhibition of puerarin on differentiation of marrow stromal cells into adipocytes induced by alcohol by the methods of cell biology and animal experiment.Methods :(1)Cell biology:Marrow stromal cells(MSCs)were isolated from mice bone marrow,obtained and cultured.MSCs were separated by 3 groups at random;Group A;the cells were treated with 0.09mol/L alcohol.Group B:the cells were treated with 0.09mol/L alcohol and 0.01mg/ml puerarin.Group C:the cells were treated without alcohol or puerarin as control.The cells in culture were stained with Sudan FF3,and then the number of adipocytes were counted on a light microscope.The contents of triglyc-eride and alkaline phosphatase activity in cells were determined by biochemical assay;The contents of osteocalcin in the media were determined by radioimmunoassay.Cells were collected after 10 days,the expression levels of the peroxisome proliferator-actived receptor-γ(PPAR-γ)mRNA,and osteocalcin mRNA in cells were analyzed by Reverse Transcription-Polymerase Chain Reaction(RT-PCR).(2)Animal experiment:300 of 4 week-old mice were divided into 3 groups by random.In group A,the distilled spirit containing 46% of alcohol was administered intragastrically at a dose of 20ml/(kg.d),and the normal saline at dosage of 10ml /(kg.d)by intramuscular injection.In group B,the same dose of the distilled spirit was administered intragastrically,and puerarin at dosage of 0.5g /(kg.d)by intramuscular injection.In group C,the water was administered intragastrically at a dose of 20ml/(kg.d),and the same dose of normal saline were administered as control by same method.The animals were killed in batches in the 4,6,8,10 months.The changes of serology,livers and bilateral femoral heads,adipogenesis and osteogenesis gene expression of the cells in femoral head of mice were detected.Results:(1)Cell biology:In vitro,the number of adipocytes and the contents of triglyceride in the cells treated for 21 days in Group B were significantly lower than that in Group A.The alkaline phosphatase activity values,the cells were treated for 12 days,and the contents of the osteocalcin in the media,the cells were treated for 14 days,in Group B were significantly higher than that in Group A(P 0.001).There were not significant differences statistically between Group B and Group C(P 0.05).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),which were 1.9 and 2.4 fold higher than that in Group A respectively,and there were not significant differences statistically between Group B and Group C(P 0.05).(2)Animal experiment;In vivo,When mice were treated 6 months:(1)The contents of CH0,TG in serum,and ALP in group A,B,C were(6.39 ±0.49),(3.19±0.11),(2.98±0.36)mmol/L;(1.01±0.15),(0.71 ±0.13),(0.68 ±0.22)mmol/L;(161.6 ±32.44),(196.5 ±31.52),(203.4 ±22.83)IU respectively.(2)In group A,the thinner and sparse trabeculae,diminished hematopoiesis,fatty tissue and empty osteo-cyte lacunae increased in the subchondral area of the femoral head,and fatty liver were found.In group B,fatty tissue of the femoral head slightly increased,and near to that in group C that was normal.The mean of the biggest adipocytes diameter and empty osteocyte lacunae in 3 groups was(40.02 ±3.25),(39.15±3.67),(38.51 ± 3.09)μm,(13.5 ± 1.6),(9.8 ±2.2),(10.3 ± 2.7)% respectively.(3)There was high expression of PPARγ mRNA and low expression of osteo-caosin mRNA in the cells of femoral head in group A.There was low expression of PPARγ mRNA(P0.05)and high expression of osteocaosin mRNA(P 0.01)in group B and group C.From 6 months,there were statistically significant differences compared the results in group A to group B and in group C(P 0.01),and not statistically significant differences between group B and group C(P 0.05).Conclusion;Puerarin could against the toxicity of alcohol,inhibit differentiation of marrow stromal cells into adipocytes induced by alcohol,to maintain the normal procedure of osteo-genic differentiation,which may prevent the development of alcohol-induced osteonecrosis of the femoral head.