OBJECTIVETo observe the regulation of Youguiyin (YGY, ) on the gene expression profile of the rat with steroid-induced femoral head necrosis (sFHN), for the sake of investigating its molecular mechanism of sFHN prevention and treatment.

METHODSAll the 30 rats were randomly divided into three groups, the normal control group (A), the model control group (B), and the YGY treated group (C), 10 in each group. After rats in Groups B and C were being made into FHN models with steroid injection, they received a daily intragastric administration of saline and YGY respectively in equal volume for a total of 6 weeks, while to the unmodeled normal rats in Group A, saline was administered instead. The rats were sacrificed at the terminal of administration; their mRNA from femoral head tissue was extracted and prepared to cDNA probe through inverse transcription for detecting gene expression profile by microarray, outcomes of which was passing fluorescence quantitative PCR verification, and the differential expressed genes were analyzed adopting gene ontology (GO) method.

RESULTSCompared with Group A, the numbers of differential genes found in Groups B and C were 190 and 92, respectively, but the changing trend in the two groups was opposite, mainly manifested as down-regulating in Group B/Group A (GB/GA) and up-regulating in Group C/Group B (GC/GB). The analysis showed that these differential genes were mainly assigned to cell apoptosis, signal transduction, metabolism, cell proliferation and differentiation, cell cycle, blood coagulation, antioxidant activity, etc.

CONCLUSIONSsFHN was regulated by various genes; the regulation of YGY on expressions of these genes and the intra/extra-cellular signaling processes was possibly the molecular mechanism of YGY for preventing/treating sFHN. This study gave an explanation to the effectiveness of Chinese medicine in preventing/treating FHN from aspects of gene expression and enriched the Chinese medicine theory of "Kidney (Shen) governing bones".

Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Femur Head ; drug effects ; metabolism ; pathology ; Femur Head Necrosis ; chemically induced ; drug therapy ; genetics ; Gene Expression Profiling ; Male ; Rats ; Rats, Wistar ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; Steroids ; Treatment Outcome

PURPOSE: To explore the value of transplanting peripheral blood-derived mesenchymal stem cells from allogenic rabbits (rPBMSCs) to treat osteonecrosis of the femoral head (ONFH). MATERIALS AND METHODS: rPBMSCs were separated/cultured from peripheral blood after granulocyte colony-stimulating factor mobilization. Afterwards, mobilized rPBMSCs from a second passage labeled with PKH26 were transplanted into rabbit ONFH models, which were established by liquid nitrogen freezing, to observe the effect of rPBMSCs on ONFH repair. Then, the mRNA expressions of BMP-2 and PPAR-γ in the femoral head were assessed by RT-PCR. RESULTS: After mobilization, the cultured rPBMSCs expressed mesenchymal markers of CD90, CD44, CD29, and CD105, but failed to express CD45, CD14, and CD34. The colony forming efficiency of mobilized rPBMSCs ranged from 2.8 to 10.8 per million peripheral mononuclear cells. After local transplantation, survival of the engrafted cells reached at least 8 weeks. Therein, BMP-2 was up-regulated, while PPAR-γ mRNA was down-regulated. Additionally, bone density and bone trabeculae tended to increase gradually. CONCLUSION: We confirmed that local transplantation of rPBMSCs benefits ONFH treatment and that the beneficial effects are related to the up-regulation of BMP-2 expression and the down-regulation of PPAR-γ expression.
Animals ; Blood Cells/*cytology ; Bone Morphogenetic Protein 2/genetics ; *Cell- and Tissue-Based Therapy ; Femur Head Necrosis/metabolism/*pathology/*therapy ; Gene Expression Regulation ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/*cytology ; Osteonecrosis/*pathology/*therapy ; PPAR gamma/genetics ; Rabbits ; Transplantation, Homologous

OBJECTIVETo investigate the effect of Huogu II Formula (II) with medicinal guide Radix Achyranthis Bidentatae (Ach) on bone marrow stem cells (BMSCs) homing to necrosis area after osteonecrosis of the femoral head (ONFH) frozen by liquid nitrogen in rabbit as well as to explore the mechanism of prevention and treatment for ONFH.

METHODSThe animal model of ONFH was established by liquid nitrogen frozen on the rabbit left hind leg. Forty-eight Japanese White rabbits were randomly assigned to sham-operated group, model group, Huogu II group, and Huogu II plus Ach group, with 12 rabbits in each. During the course of ONFH animal model establishment, all rabbits were subcutaneously injected with recombinant human granulocyte colony-stimulating factor [rhG-CSF, 30 μg/(kg·day) for continuous 7 days]. Meanwhile, normal saline and decoction of the two formulae were administrated by gavage, respectively. White blood cells (WBC) were counted in peripheral blood before and after injection of rhG-CSF. Materials were drawn on the 2nd and 4th weeks after model built; bone glutamine protein (BGP) and bone morphogenetic protein 2 (BMP2) levels in serum were tested. Histopathologic changes were observed by hematoxylin and eosin (HE) staining. BMP2 mRNA levels were detected with in situ hybridization (ISH) staining. 5-Bromo-2'-deoxyuridine (BrdU) and stromal cell derived factor 1 (SDF-1) were measured by immunohistochemical assay in femoral head of the left hind leg.

RESULTSCompared with the shamoperated group, the ratio of empty lacuna, serum BGP, and SDF-1 level in the model group increased significantly, and BMP2 in both serum and femoral head decreased significantly. However, in comparison with the model group, the empty lacuna ratio of Huogu II group and Huogu II plus Ach group decreased obviously in addition to the levels of serum BGP and BMP2, and the expressions of BMP2 mRNA, BrdU, and SDF-1 increased significantly. Above changes were particularly obvious in Huogu II plus Ach group. BGP and SDF-1 on the 2nd week and empty lacuna rate and serum BMP2 level on the 4th week in Huogu II group significantly exceeded their counterparts. On the 2nd week, only in Huogu II plus Ach group that the BrdU counting rose significantly. On the 4th week, empty lacuna rate and serum BMP2 level in Huogu II plus Ach group exceeded those in Huogu II group distinctively.

CONCLUSIONSTo a certain extent, the medicinal guide Ach improves the preventive and therapeutic effects of Huogu II Formula on experimental ONFH model. The possible mechanism of this is related to its promoting effect on directional homing of BMSCs to the necrosis area.

Achyranthes ; Animals ; Bone Marrow Cells ; cytology ; drug effects ; Bone Morphogenetic Protein 2 ; blood ; genetics ; Bromodeoxyuridine ; metabolism ; Cell Movement ; Chemokine CXCL12 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Enzyme-Linked Immunosorbent Assay ; Femur Head ; drug effects ; pathology ; Femur Head Necrosis ; blood ; genetics ; pathology ; therapy ; Gene Expression Regulation ; drug effects ; Granulocyte Colony-Stimulating Factor ; administration & dosage ; pharmacology ; Humans ; Leukocyte Count ; Male ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Radioimmunoassay ; Stem Cell Transplantation ; Stem Cells ; cytology ; drug effects

OBJECTIVEOn the basis of YouGui drink ([Chinese characters: see text]) to act on the normal rats in vitro osteoblast proliferation and differentiation, further observe YooGui drink ([Chinese characters: see text]) inductive effect for steroid induced necrosis of femoral head rats in vitro on cultivation of osteoblast proliferation and differentiation.

METHODSPrednisolone acetate 49 mg/kg x d were injected into gluteal of 20 male SD rats (body weight in 100-120 g), continuously 6 days for model. After 7 days, under sterile conditions from the model rats bone marrow stromal cells were induced and cultured to osteoblast, and then randomly divided into 4 groups, respectively, add the high, medium and low concentration YouGui drink ([Chinese characters: see text]) containing serum (group R1, R2, R3) and control serum (group R4). After 72 h, osteoblast proliferation, alkaline phosphatase activities (APA) were detected, and expression level of osteoprotegerin (OPG) and osteoclast differentiation factor (RANKL) were detected by real-time fluorescence quantitative PCR.

RESULTSThe high and medium concentration YouGui drink ([Chinese characters: see text]) containing serum group compared with the control group (R1, R2 vs. R4), the rate of osteoblast proliferation was significantly high (P < 0.01), APA significantly increased (P < 0.01), relative expression level of osteoblast OPG mRNA significantly increased (P < 0.01), and with the concentration of a certain dose was positively correlated; RANKL mRNA significantly was lower (P < 0.01). The low concentration YouGui drink (t 91 t) containing serum group compared with the control group (R3 vs R4), there was no significantly different in the rate of osteoblast proliferation, ALP and relative expression level of osteoblast OPG mRNA, RANKL mRNA.

CONCLUSIONThe high concentration of YouGui drink ([Chinese characters: see text]) containing serum can obviously promote the proliferation and differentiation of steroid induced necrosis of femoral head rats in vitro osteoblast, and plays a clear role in promoting and enhance the relative expression of osteoblast OPG mRNA and an inhibitory effect on RANKL mRNA. The effect of YouGui drink ([Chinese characters: see text]) containing serum acting on induced in vitro cultured osteoblasts have relevance with dose, at a certain concentration range it can promote osteoblast differentiation and proliferation.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drinking ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Femur Head Necrosis ; chemically induced ; drug therapy ; metabolism ; pathology ; Gene Expression Regulation ; drug effects ; Male ; Osteoblasts ; drug effects ; metabolism ; pathology ; Osteoprotegerin ; genetics ; RANK Ligand ; genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Steroids ; pharmacology

OBJECTIVETo observe the effect of Huogu II formula compatible with different channel ushering drugs on the homing of bone marrow stem cells of osteonecrosis of the femoral head (ONFH) induced by liquid nitrogen freezing in rabbits and discuss the mechanism for preventing and treating ONFH.

METHODThe ONFH model was established by liquid nitrogen freezing of 84 rabbits. They were randomly assigned to the model group and the Huogu II formula group and groups of Huogu II formula combining with Achyranthis Bidentatae Radix, Asari Radix et Rhizoma, Angelicae Pubescentis Radix, Platycodonis Radix. The remaining 14 rabbits were sham-operated. During the course of ONFH modeling, all of the rabbits were subcutaneously injected with recombinant human granulocyte colony-stimulating factor (rhG-CSF)(30 microg x kg(-1) x d(-1), for consecutively 7 days). Meanwhile, normal saline and decoction of the formulae were orally administrated respectively. WBC was counted in peripheral blood before and after the injection of rhG-CSF. HE stainings at the 2nd and the 4th weeks after the modeling were adopted to observe histopathological changes, vascular morphology was observed by ink perfusion, BrdU and SDF-1 were determined by immunohistochemical assay in femoral heads of the left hind leg.

RESULTCompared with the sham-operated group, the Huogu II formula group showed decrease in the ratio of empty lacuna and increase in vessel area, number of BrdU positive cells and SDF-1 level. In comparison with the model group, the Achyranthis Bidentatae Radix group displayed decreasing empty lacuna ratio and increasing vessel area at the 4th week and increasing SDF-1 at the 2nd week; the Platycodonis Radix group revealed a notably increasing empty lacuna ratio and a sharp decrease in the number of BrdU positive cells at 4th week; Asari Radix et Rhizoma and Angelicae Dubescentis Radix groups showed no remarkable change.

CONCLUSIONHuogu II formula can promote the directional homing of bone marrow stem cell to the necrosis area. Channel ushering drug achyranthes can further boost above effects of Huogu II formula.

Animals ; Bone Marrow Cells ; cytology ; Bromodeoxyuridine ; metabolism ; Chemokine CXCL12 ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Femur Head Necrosis ; blood ; drug therapy ; metabolism ; pathology ; Gene Expression Regulation ; drug effects ; Humans ; Leukocyte Count ; Male ; Rabbits ; Stem Cells ; cytology ; drug effects