Effect Of Huoxuebushen Decoction On Expression Of VEGF And OPG/RANKL In Steroid-induced Osteonecrosis Of The Femoral Head

Objectives:Effection of Huoxuebushen Decoction(HXBSD) on the expression of VEGF and OPG/RANKL in steroid-induced osteonecrosis of the femoral head(SANFH) was studied on molecular and histomorphological level by rabbit experiment. To explore pathogenesis of SANFH and provide an experimental and theoretical basis for the mechanism of HXBSD and new ideas and methods of TCM therapy on SANFH.Methods:1. Effect of HXBSD on the expression of VEGF in SANFH rabbits1.1 130 rabbits were divided into 5 groups:normal control group, Steroid-model group, HXBSD treated groups(High,medium,low dosage group).Steroid-model rabbits were modeled by Matsui's method. Control group and model group were gived saline 10ml by gavage every-day while treated groups gived different concentrations of HXBSD daily.Drawing materials of each group in the 4th,8th,12th week in turn.1.2 Observing histomorphology of rabbit femoral head by microscope.1.3 Detecting VEGF data applying immunohistochemical S-P assay.2. Effect of HXBSD on OPG,RANKL content in SANFH rabbit's serum2.1 Methods of establishment of model and medicine giving were the same as above.2.2 In the 4th,8th,12th week 8 rabbits of each group were drawed blood.Centrifuging to collect serum and saving backup.2.3 Detecting OPG,RANKL content by adopting double antibody sandwich ELISA assay.3. Effect of Serum with HXBSD on expression of OPG/RANKLmRNA in rat osteoblast.3.1 Calvaria of newborn SD rats were applied for primary osteoblasts and subculture. Taking3 generations of growing period osteoblasts and identifying by microscopic observation.3.2 SD rats(n=12) were divided into 4 groups:Saline-control group, High,medium and low dosage HXBSD groups(HDG,MDG,LDG). Saline-control group was gived saline 3ml by gav-age twice a day for 3 days while HXBSD groups gived different concentrations of HXBSD.2 hours after the last gavage,drew blood and centrifuga to collect serum.3.3 Rat osteoblasts were cultivated with HXBSD-containing serum(20%) in incubator.3.4 Using RT-PCR for detection of each group's OPG,RANKLmRNA expression.Results:1.HE observation:Model groups show:trabeculae getting thinner,less trabecular number,bone marrow filled with hypertrophy cells,the number of bone cells reduced and lacunae increased during the experiment; HXBSD treated groups show:trabecular structure was complete basicly and relatively larger number of lacunae in LDG;HDG and MDG show new bone formation in the 8th and 12th week respectively.2. VEGF expression:in the 4th week,HDG,MDG were higher than control groups significantly (P<0.01) and LDG,steroid-model groups were higher than control groups (P<0.05);in the 8th week, Steroid-model group was lower than control group significantly(P<0.01);in the 12th week, Steroid-model group was lower than HDG,MDG, LDG greatly (P<0.01).3.Contents of OPG and RANKL in serum:For OPG content, in the 4th week,HDG,MDG, LDG and control groups were higher than steroid-model group greatly (P<0.01);in the 8th week,Steroid-model group was lower than others greatly (P<0.01) and HDQMDG, LDG higher than control group greatly (P<0.01);in the 12th week,Steroid-model group was lower than others greatly (P<0.01).For RANKL content,Steroid-model group was higher than others greatly (P<0.01) during the experiment.For Ratio of OPG/RANKL,Steroid-model group was lower than others greatly (P<0.01) during the experiment.4.Expressions of OPGmRNA and RANKLmRNA:OPGmRNA expression:HDG,MDG were higher than saline-control group (P<0.05).RANKLmRNA expression:HDG,MDG and LDG were lower than saline-control group significantly (P<0.01).Ratio of OPG/RANKL:HDG, MDG,LDG were higher than saline-control group significantly (P<0.01).Conclusion:1. Decreased expression of VEGF in SANFH can suppress regeneration of bones and vessels.2. HXBSD can increase expression of VEGF in SANFH.3. OPG content was reduced while RANKL increased, and the ratio of OPG/RANKL was reduced in SANFH serum.4. HXBSD can increase OPG content,reduce RANKL content and increase the ratio of OPG/RANKL in SANFH serum.5. HXBSD can upside OPGmRNA expression while reduce RANKLmRNA expression.OPG /RANKL maybe the one of the targets of HXBSD on SANFH.