| Objective To study the mechanism of Qianggu capsule to promote the recovery of rat's denervated fracture.Methods Male SD rats,10%Chloral Hydrate abdominal anesthesia,sever the sciatic nerve in the ischial tuberosity 0.5 cm and cut bone defect in the middle of the femur,establish denervated fracture animal model,randomly divided into Qianggu capsule large dose group(LDG),Qianggu capsule medium dose group (MDG),Qianggu capsule small dose group(SDG),Injectio Salviae Miltiorrhiae group (SMG)and control group(CG),each group have 30 rats.LDG was given Qianggu capsule 180mg/kg/d intragastric administration,once a day;MDG was given Qianggu capsule 90mg/kg/d intragastric administration,SDG was given Qianggu capsule 45mg/kg/d intragastric administration,once a day,SMG was given Injectio Salviae Miltiorrhiae 40mg/kg/d,intragastric administration,once a day;CG no disposal.In each group after one week,two weeks and three weeks cut osteotylus 1 cm from the center of the whole femoral fracture,to measure osteotylus volume.To drawe the materials from osteotylus,conventional HE staining and observe structural changes in osteotylus.NGF,BMP-2 and bFGF immunohistochemical staining,gel image analysis system to calculate each vision positive cells.Results(1)Osteotylus volume result:It is thus evident that the osteotylus volume in every group was increased in the first week of postop.,LDG and MDG increased obviously than SMG(P＜0.05)and CG (P＜0.05),SDG more than CG(P＜0.05)..In the second week of postop.,Neogenesis cartilage volume increased obviously in broken ends of fracture in LDG and SDG,external diameter of Osteotylus hyperplasia.osteotylus volume in every group increased in varied degrees.LDG hyperplasia obviously more than SMG(P＜0.01),CG (P＜0.01)and SDG(P＜0.01),MDG increased obviously than SMG(P＜0.05)and CG(P＜0.01),SDG more than SMG(P<0.05)and CG(P＜0.05).In the third week of postop.,LDG bone coloboma disappeared,extemal diameter of osteotylus more thicken,osteotylus grew more obviously,MDG and SDG have porosis in the place of fracture,SMG and CG hypercallosis not obviously.Osteotylus volume in LDG and MDG achieve peak and exceed SMG(P＜0.01)and CG(P＜0.01),LDG exceed SDG (P＜0.05),SDG exceed SMG(P＜0.05)and CG(P＜0.05).(2)HE staining Results: In the first week postop.,the amount of osteoblast in LDG more than other groups,and other groups hematoma and inflammatory exudation increased,cartilage cells less.In the second week postop.,LDG have cartilage osteotylus and bony osteotylus,cartilage cells surrounding have generated manipulus woven bone,MDG have manipulus newborn trabecular bone,osteoid tissue generated inside and outside broken ends of fracture.Other groups fiber osteotylus generate obviously.In the third week of postop.,LDG have many osteoid,osteotylus immature and low intensity,external osteotylus increased obviously and observed a lot of osteoclast and osteoclasts.MDG trabecular bone were irregular.SDG fracture ends nearly all sclerotization.SMG neovascularization richly,trabecular bone disorder,osteoclast relatively less.CG fiber osteotylus disappeared,replaced by cartilage osteotylus and boney osteotylus.(3) Immunohistochemical staining results:NGF:In the first week postop.,Fibroblast and a small amount of cartilage positive particles NGF irnmunohistochemical staining were pale brown in LDG,the number of positive cells was higher than MDG(P＜0.01)and SDG(P＜0.01).SMG more than CG(P＜0.05).In the second week postop.,MDG and SDG positive cell particles increased significantly in cytosol of osteoblast,MDG more than SMG(P＜0.05)and CG(P＜0.05).The number of positive cell in LDG was significantly more than SMG(P＜0.01)and CG(P＜0.01).In the third week postop.,MDG and SDG intraosteotylus osteoblast cells staining positive and the number of positive cells more than LDG(P＜0.05).LDG osteoblasts showed weak positive staining.BMP-2:The number of BMP-2 positive cells in LDG upward trend during 2weeks,in the second week at the peak,lower in the third weeks.BMP-2 positive cells in MDG higher than other groups,compared with CG and MG the difference was significant(P＜0.01).BFGF:In the first week postop.,LDG and MDG fibrous osteotylus fibroblasts and vascular endothelial cells,undifferentiated mesenchymal cells and naive osteoblast nucleus have uniform granular brown positive expression of bFGF,MDG positive cells was higher than the other groups,significantly higher than SDG(P＜0.05),CG(P＜0.05)and SMG(P＜0.05).In the second week postop.,LDG and MDG in osteoblasts and naive chondrocytes,new bone matrix,periosteal bone cells expression of bFGF positive,positive cells increased up to peak,significantly more than CG(P＜0.01)and SMG(P＜0.01).In the third week postop.,the number of positive cells staining and positive responses in LDG decreased obviously compared with the past,the dgree of coloring weakened than the former,compared with CG and SMG have statistical significance(P＜0.05).Other groups were less positive cells and coloring weakened.(4)Serum Ca,P and ALP results:In the first week postop.,serum Ca in every groups there was no significant difference(P＞0.05).In the second week postop.,serum Ca in MDG was higher than other groups,compared with SMG(P＜0.01)and CG(P＜0.05).compared with LDG no significant difference(P＞0.05).In the third week postop.,serum Ca in SDG was the highest,compared with SMG(P＜0.05)and CG(P＜0.05),the other groups showed no significant difference(P＞0.05).In the first week postop.,serum P in every group there was no significant difference(P＞0.05),In the second week postop.,serum P in SDG decreased obviously,LDG compared with SDG there were significant differences(P＜0.05),In the third week postop.,serum P decreased in every group,MDG decreased obviously,LDG and MDG compared with SDG(P＜0.05).Serum ALP increased in every groups during 1,2 and 3 weeks after operation except for SMG in the first two weeks fell.In the first week postop.,ALP in SDG higher than CG(P＜0.05),while the other group no significant difference (P＞0.05),In the second week postop.,serum ALP in MDG increased obviously compared with SMG(P＜0.01),SDG(P＜0.05)and CG(P＜0.05),compared with LDG the difference was no significant(P＞0.05),LDG compared with SMG(P＜0.05).In the third week postop.,serum ALP increased more significantly in LDG,LDG compared with SMG(P＜0.01),SDG(P＜0.05)and MDG(P＞0.05).There were no significant difference between the other group(P＞0.05).Conclusion1.The recovery of denervation fracture have relate to the expression of cytokine.2.Qianggu capsule can promote the recovery of denervation fracture by inducing the expression of NGF,bFGF and BMP-2.3.Qianggu capsule can improve the microenvironment in the process of neurons recovery and promote osteotylus formation,growth quality,and accelerate the healing of denervation fracture. |
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