An Experimental Study Of The Effects And Mechanism Of FN On The Alcohol-Induced Osteoblast Apoptosis

Objective To investigate the effects and mechanisms of bovine plasma fibronectin on the alcohol-induced osteoblast apoptosis.Methods Calvariaosteoblast cells were extracted from the SD neonatal rat and cultured until the third generation.Osteoblastic cells were randomly divided into three groups: the control group,the alcohol-induced group and the FN group.In the control group,no interventions were performed.With regards to the alcohol-induced group,osteoblastic cells were intervened by adding 100mmol/L alcohol while cultured.In the FN group,different concentrations of fibronectin(20?g/ml,40?g/ml,60?g/ml,80?g/ml)were added as well as100mmol/L alcohol.Osteoblastic cells were assessed by MTT colorimetric method after24 h and 48 h cell culture.In addition,apoptosis rates of osteoblastic cells in all groups were examined by the Flow Cytometry after 48 h cell culture.The change of apoptosis-related genetic expression(Bcl-2 m RNA and Bax m RNA)was tested by the RT-PCR after 24 h cell culture.Results Osteoblastic cells were successfully isolated by enzymatic digestion.After purified osteoblastic cells were obtained by differential adhesion method,analysis of alkaline phosphatase staining results showed that positive cells were identified.Their cell cytoplasm was dyed brown and the variation in the size of the nucleoli was obvious.This confirmed that osteoblastic cells were successfully isolated and cultured.Using the Flow Cytometry method,we found that there was a statistically significant difference between the alcohol-induced group and the control group(14.42±1.26% VS 3.86±0.98%;P<0.05).In the FN group,apoptosis rates of osteoblastic cells decreased with the increasing concentrations of fibronectin(12.80 ±1.08%;8.51 ±1.02%;5.75±0.86%;5.52±0.82%),with a statistically significance(P<0.05).In terms of the change of genetic expression,RT-PCR results indicated that compared with the control group,the intensity of genetic expression of Bcl-2 m RNA decreased whereas the intensity of Bax m RNA increased in the alcohol-induced group(P<0.05).When comparing the FN group with alcohol-induced group,the intensity of genetic expression in the FN group showed an increasing gradient for Bcl-2 m RNA and a descending gradient for Bax m RNA(P<0.05).Conclusions (1)A certain concentration of alcohol(100mmol/L)could facilitate the apoptosis process of rat osteoblastic cells.(2)With the increase of the concentration of FN,apoptosis rates decreased among alcohol-induced rat osteoblastic cells.FN could be a protecting factor that influences the effects of alcohol on osteoblast apoptosis.(3)The mechanisms of alcohol-induced osteoblast apoptosis and fibronectin(FN)protection are related to the genetic expression of Bcl-2 m RNA and Bax m RNA.The protective effect of Fibronectin(FN)on alcohol-induced osteoblast apoptosis may be taken by regulating apoptosis-related genes of Bcl-2 m RNA and Bax m RNA,which may become an effective treatment strategy for alcohol-induced bone disease in future.