| Objective : The preliminary clinical observation,in which Dangui Buxue decoction(DB)was applicated in early stage of ONFH(Osteonecrosis of the Femoral Head)patients,showd that availability of treatment based on syndrome differentiation for “Qi”,“Xue” in ONFH.In this study liquid nitrogen freezing early stage of ONFH model in rat was made,imaging,tissue Structure,signaling molecules expression were observed after DB intragastric administration(IG)in rat model and the possible mechanism of this procedure was explored.Methods : 100 six months old,male SPF SD rats were divided into four groups randomly : group control(control),group model(M),250 mg/ml DB IG group(DB250),500 mg/ml DB IG group(DB500),each groups were feeding in SPF laboratory.After a week of adaptive feeding,Group M,DB250,DB500 were involved in operation of Surgical ONFH modeling.Surgical procedure: intraperitoneal anaesthesia(20% Chloral Hydrate,500mg/kg),skin antisepsis,skin-sterilizing,located middle piont between the right iliac spine middle/lower 1/3 piont and the greater trochanter,cut open vertical,anatomical incision layer by layer,open capsule,exposed the fomoral head,cut off ligament of femoral head,put the Swab rod inside Liquid Nitrogen,5S,then rob touched fomoral head Immediately,3S,15 times,then seamed layer by layer,Antibiotics injection/day,3d,sterilized and feed respectivly.250 mg / ml 、500 mg / ml DB was prepared,gavaged every rat of group DB250 and DB500 4ml once a day after surgery.12 rats were randomly selected all groups(all the rats were secrificed in no pain),completed X-ray,HE staining and empty Lacuna rate calculation,TRAP staining and counting number of osteoclast,Westernblot,immunohistochemistry and Real time PCR 2 weeks after IG,and 2 weeks later the rest of all Groups ’s rats,complete X-ray,MRI imaging,Micro CT imaging and data collection,HE staining and empty Lacuna rate calculation,the TRAP staining and counting number of osteoclast,Westernblot,immunohistochemical and Real time PCR.Results:X-ray,MRI image :The image of DB250,DB500 groups after IG 2 weeks and 4 weeks showed a few improvement to the image of group M within the femoral head edema,femoral head osteoid hyperplasia,hip joint space and smoothness of joint surface.Micro CT and data analysis : there were statistically differences in BMD(P<0.05),Tb.Sp(P<0.05)to between group DB250 and group M at 4 weeks and statistically differences between the group DB500 and group M in TMD(P<0.05),Tb.Sp(P<0.01),and SMI(P<0.05).HE staining and the ratio of empty lacuna : Group M showd trabecular bone was sparse,trabecula direction was irregular,trabecula space increased,epiphyseal was not clear,part of the regional bone tissue was absent,a small amount of fibrous tissue hyperplasia,bone marrow tissue hyperplasiaed with fat cells,vascular tissue decreased.The performance of Group DB250 and DB500 were better than group M in result of tissue HE staining.After 4 weeks IG There was a statistically significant difference between DB250 group M group(P<0.01)and a statistically difference between the DB500 group and the M group(P<0.05)in the ratio of empty lacuna.TRAP staining and the count of multi-nucleus TRAP staining positive cell : The number of positive cells in multi-nucleus TRAP staining was decreased in the two groups which DB was intervened compared with the group M(P<0.05)after 4 weeks IG.Westernbolt: 2 weeks after IG,OPG expression of femoral head of DB250 group(P<0.01)and DB500 group(P<0.01)were higher than group M;4 weeks after IG,the difference between the DB250 group(P<0.01)and the DB500 group(P<0.01)was even biger;2 weeks and 4 weeks after IG,there was no statistical difference between the DB250 group and the DB500 group RANKL expression;4 weeks after IG,the RANK expression of DB250 group(P<0.01)and DB500 group(P<0.01)were statistically different from that of group M;The ratio of RANKL/OPG in DB250 group(P<0.01)and DB500 group(P<0.01)was statistically different from that of group M after 2 weeks and 4 weeks IG.Immunohistochemistry: 2 weeks after IG,group DB250(P<0.05)MOD values of VEGF164 expression were statistically different with group M;4 weeks after IG,DB250 group(P<0.05)and group DB500(P<0.05)MOD values of VEGF164 expression were statistically higher than M group;after IG,each groups express VEGF164 had no difference.Real time PCR: 2 weeks after IG,group DB500(P<0.05)OPG m RNA expression was statistically different from group M;4 weeks after IG,group DB250(P<0.01)and group DB500(P<0.01)OPG m RNA expression was significantly different from group M;2 weeks after IG,DB250 group(P<0.01)and group DB500(P<0.01)RANKL m RNA expression was significantly different from group M;4 weeks after IG,DB250 group(P<0.05)and group DB500(P<0.01)RANKL m RNA expression was statistically different from M group;after IG,each group m RNA expression had no significant difference;2 weeks after IG,DB250 group(P<0.01)and group DB500(P<0.01)VEGF164 m RNA expression were significantly different from M group;4 weeks after IG,DB250(P<0.05)VEGF164 expression of m RNA group was statistically different from M group;after IG Each groups VEGFR2 m RNA expression had no different.Conclusion : Base on the thought of treatment based on syndrome differentiation for “Qi”,”Xue” and simultaneous the therapeutic principl of tonifying “Qi”,“Xue” and Supplement of “Xue”,“Sui”,this study investigated the mechanism that how the Dang Gui Buxue Decoction act on early stage of ONFH in rat.The result confirmed that DB could suppresse the femoral bone marrow edema,improve femoral head pathology morphology,decrease the rate of empty bone lacuna,reduce the number of osteoclast,enhance bone density,upregulate OPG,VEGF164 expression,downregulate RANKL expression,reduce the RANKL / OPG ratio.Its mechanism may be that DB could improve the bone marrow edema,reduce excessive bone cell death and inhibit activity of osteoclast by regulating OPG /RANKL /RANK,VEGF164 / VEGFR2 signalling pathway,thus DB may play a role of prevention in this disease.Experimental results combined with TCM theoretical analysis,intervention of Dang Gui Buxue Decoction had a protective effect to early stage of ONFH in rats.To a certain extent,it proved the feasibility of the thought of treatment based on syndrome differentiation for “Qi”,“Xue”,and the therapeutic principl of tonifying “Qi”,“Xue” and Supplement of “Xue”,“Sui”,which could be applicated in the prevention and treatment of ONFH in early stage. |
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