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ObjectiveTo compare the effects of Taxillus chinensis from different hosts with different meridian affinity on bone microstructure and bone metabolism in ovariectomized osteoporotic rats， and investigate its mechanism of action. MethodEighty-eight specific-pathogen-free （SPF）-grade female Sprague-Dawley （SD） rats were selected and randomly divided into 11 groups： sham-operated group， model group， low-， medium- and high-dose groups of T. chinensis from Morus alba （2.5， 5， and 10 g·kg-1）， low-， medium- and high-dose groups of T. chinensis from Cinnamomum cassia （2.5， 5， and 10 g·kg-1）， and low-， medium- and high-dose groups of T. chinensis from C. burmannii （2.5， 5， and 10 g·kg-1）. After 12 weeks of drug intervention， the rats were examined for proximal femur bone density and bone microstructure using dual-energy X-ray absorptiometry （DXA） and micro-computed tomography （Micro-CT）. Histopathological changes in rat femur were observed by the hematoxylin-eosin staining （HE）. Contents of serum estradiol （E2）， bone Gla protein （BGP）， bone alkaline phosphatase （BALP）， tartrate-resistant acid phosphatase 5b （TRACP-5b） and pre-collagen type Ⅰ amino-terminal protopeptide （PINP） were measured by the enzyme-linked immunosorbent assay （ELISA）. Real-time quantitative polymerase chain reaction （Real-time PCR） was employed to detect the messenger ribonucleic acid （mRNA） expressions of bone morphogenetic protein-2 （BMP-2）， Smad1， Smad9 and recombinant runt-related transcription factor 2 （Runx2） in rat humerus. Western blot was used to detect the protein expressions of BMP-2， p-Smad1/5/9 and Runx2 in rat humerus. ResultCompared with that in the sham-operated group， the femur microstructure of rats in the model group was significantly disrupted， with significant decreases in bone mineral density （BMD） value， bone volume fraction （BV/TV）， trabecular number （Tb.N）， and trabecular thickness （Tb.Th） （P<0.01）， and significant increases in trabecular separation （Tb.Sp） and structure model index （SMI） （P<0.01）. The serum levels of BGP， BALP， TRACP-5b and PINP were significantly increased （P<0.05， P<0.01）， and E2 levels were significantly decreased （P<0.01）. The mRNA expressions of BMP-2， Smad1， Smad9， and Runx2 were significantly decreased in rat humerus （P<0.01）， and the protein expressions of BMP-2， p-Smad1/5/9， and Runx2 were significantly reduced （P<0.01）. Compared with the model group， the administration groups of T. chinensis from different hosts all elevated the BMD， BV/TV， Tb.N， Tb.Th， Tb.Sp， and SMI levels in the femur， improved bone microstructure， increased serum E2 levels （P<0.05， P<0.01）， lowered the levels of serum BGP， BALP， TRACP-5b， and PINP， upregulated the mRNA expression of BMP-2， Smad1， and Runx2 and upregulated the mRNA expression levels of Smad9 （P<0.05， P<0.01）， and upregulated the protein expressions levels of BMP-2， p-Smad1/5/9， and Runx2 （P<0.01）. The best effect was observed in the group of T. chinensis from C. cassia. ConclusionT. chinensis from different hosts improved osteoporosis in ovariectomized rats， with the group of T. chinensis from C. cassia being the most potent among the administered groups， and its treatment of osteoporosis may regulate the balance of bone conversion by regulating BMP/Smad signaling pathway.
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Aim To investigate the relationship between Wnt11 and the BMP9-induced osteogenic differentiation, and the possible molecular mechanism underlying this process as well. Methods qPCR, histochemical staining and Western blot were used to detect the changes of osteogenic differentiation markers and activities of BMP/Smad and p38 MAPK signal. Luciferase reporter assay was used to detect the activity of BMP/Smad signal. Results BMP9 increased the alkaline phosphatase(ALP) activity, mineralization, expression of osteopontin (OPN) and Wnt11 in C3H10T1/2 cells. Wnt11 was detectable in C3H10T1/2, MEFs, MC3T3-E1 and C2C12 cells. In C3H10T1/2, Wnt11 promoted the effect of BMP9 to increase ALP activity, mineralization, and the expression of Runx-2 and OPN. Wnt11 enhanced the effect of BMP9 on increasing BMP/Smad reporter plasmid transcriptional activity and phosphorylation of Smadl/5/8. Wnt11 also increased the BMP9-induced phosphorylation of p38 MAPK. Inhibition of p38 MAPK attenuated the BMP9-induced ALP activity, mineralization and expression of OPN, but these effects were partially reversed by Wnt11. Conclusions BMP9 can up-regulate Wnt11 in MSCs. Wnt11 can promote the BMP9-induced osteogenic differentiation, which may be mediated by increasing the activity of BMP/Smad and p38 MAPK signaling..
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Aim To study the relationship between WntlOb and bone morphogenetic protein-9 (BMP9)-induced osteogenic differentiation of mesenchymal stem cells (MSCs), and the molecular mechanisms underlying this process. Methods PCR, Western blot and histochemical staining were used to detect the effect of BMP9 on WntlOb and the effect of WntlOb on BMP9-induced osteogenic differentiation in MSCs. Meanwhile, real-time PCR, Western blot, oil red O staining, and flow cytometry assay were used to analyze the potential mechanism of Wnt10b affecting the function of BMP9. Results Wnt10b could be detected in C3H10T1/2, C2C12, MEFs and MC3T3-E1 cells. BMP9 up-regulated the expression of WntlOb in C3H10T1/2 cells. WntlOb enhanced the capability of BMP9 to increase the level of OCN and mineralization in C3H10T1/2 cells, and silencing Wnt10b attenuated these effects of BMP9. Wnt10b exhibited no substantial effect on cell cycle affected by BMP9, but it enhanced the effect of BMP9 on inducing phosphorylation of Smadl/5/8. While silencing Wnt10b attenuated this effect of BMP9. In addition, Wnt10b inhibited BMP9-induced adipogenic differentiation in C3H10T1/2 cells, and silencing Wnt10b promoted this effect of BMP9. Conclusions Wnt10b can promote BMP9-induced osteogenic differentiation of MSCs, which may be mediated through enhancing BMP/Smad signaling and reducing adipogenic differentiation.
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Dynamic balance of bone metabolism is one of the important factors to maintain normal osseous tissue function. When the balance is broken, it causes bone damage and even bone metabolic disease. However, the mechanism of bone metabolism is still unclear, the signal pathway is complex, and the treatment of diseases is still under study. The research on the mechanism of bone metabolism by Chinese materia medica has become a new direction in the treatment of bone metabolism diseases. At present, common mechanisms in bone metabolism include OPG/RANKL/RANK signal pathway, Wnt/β-catenin signal pathway, TGF-β/BMP/Smad signal pathway, and NF-κB signal pathway. In this paper, the research on the above pathways was reviewed, so as to provide a reference for further exploring the treatment of bone metabolism diseases with Chinese materia medica.
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OBJECTIVE: To observe the effect of guhong injection(GHI) on tibial fracture healing in rats and to explore the mechanism of the action of GHI. METHODS: One hundred and eighty male SD rats were randomly divided into 6 groups with 30 rats in each group: sham operation group, model group, positive drug group(compound ossotide injection, 5 mL•kg-1), low, medium and high dose of GHI groups(2.5, 5, 10 mL•kg-1). In addition to the sham operation group, the other groups established the rat model of tibial fracture. All were given once daily intraperitoneal injections and samples were taken at 1st, 2nd, 4th and 6th week. Blood biochemical analysis and Elisa kit detection were performed on blood samples. X-rays, biomechanical tests, immunohistochemistry and RT-PCR were performed on the tibial samples. RESULTS: ①After administration for one, two and four weeks, the levels of serum calcium(Ca) and phosphorus(P) in medium and high dose of GHI groups were higher than those in model group(P<0.05). ②X-ray showed that the outer callus growth and the disappearance of fracture line in all dose groups of GHI were faster than those in model group. ③Compared with model group, the maximum load and rigidity of medium and high dose of GHI groups were increased at each time point(P<0.05), and the trend of stress line graph were improved obviously. The content of alkaline phosphatase(ALP) in medium and high dose of GHI groups were higher than that in model group at each time point(P<0.05). Compared with model group, the serum levels of PDGF were increased in all dose groups of GHI(P<0.05 or P<0.01). After administration for one, two and four weeks, the serum BMP-2 in all dose groups of GHI were higher than those in model group(P<0.05 or P<0.01). ⑤Compared with model group, the expression of Runx2 mRNA were increased in medium and high dose of GHI groups, as well as Smad5 protein expression(P<0.05 or P<0.01). CONCLUSION: GHI could significantly improve the biomechanical properties of bone in fracture rats. The promotion of fracture healing might be through the upregulation of PDGF and BMP-2 expression in different stages of bone healing, and the regulation of BMP/Smad5/ Runx2 signaling may be one of the mechanisms of promoting fracture healing.
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OBJECTIVE:To study the effects of paeonol on Wnt and BMP/Smad pathway in ankylosing spondylitis (AS) model mice,and to investigate prevention and treatment mechanism of paeonol for AS. METHODS:40 mice were randomly divided into normal group,model group,sulfasalazine group(positive control,9 mg/kg)and paeonol group(3 mg/kg),with 10 mice in each group. In addition to the normal group,Freund's adjuvant and proteoglycan were used to establish AS model in each group. After modeling,mice in each administration group were given relevant medicine intragastrically;normal group and model group were given constant volume of distilled water intragastrically,once a day,for consecutive 20 d. After last administration, the mice were killed,TEM was used to observe the ultrastructural pathologic change of synovial cells in articulationes sacroiliaca. The serum contents of TNF-α and DKK-1 were determined by ELISA,and the mRNA expressions of BMP-2,Cbfα1 and Smad1 in synovial tissue were detected by real-time fluorescent quantitative PCR. RESULTS:Compared with normal group,serum content of TNF-α in model group was increased significantly,while serum content of DKK-1 was decreased significantly;mRNA expressions of BMP-2,Cbfα1 and Smad1 in synovial tissue were increased significantly,with statistical significance (P<0.05 or P<0.01). Under the electron microscope,the synoviocytes of mice in model group were proliferated and arranged in disorder,and the secretory activity of active organelles were hyperactivity and the gap among cells became widened. Compared with model group, serum content of TNF-α in sulfasalazine group and paeonol group were decreased significantly;serum content of DKK-1 was increased significantly and mRNA expressions of BMP-2,Cbfα1 and Smad1 in synovial tissue were decreased significantly in paeonol group,with statistical significance (P<0.05 or P<0.01). Electron microscopy showed that mitochondria,lysosome and rough endoplasmic reticulum structure of synovial cell were improved significantly in paeonol group. CONCLUSIONS:The mechanism prevention and treatment of paeonol on AS may be associated with reducing serum content of TNF-α,increasing serum content of DKK-1,down-regulating mRNA expressions of BMP-2,Cbfα1 and Smad1 in synovial cells,inhibiting Wnt and BMP/Smad ossification related signal transduction pathway and reversing osteogenic differentiation of synovial cells.
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Objective To investigate the protective effect of erythropoietin (EPO)on the kidney of renal anemia rats and further explore the renoprotective action and possible mechanisms.Methods A total of 90 SD rats were randomly divided into 5 groups:normal control rats (NC),simple anemia group (SA),iron + EPO group (IR+EPO),erythropoietin group (EPO),and iron group (IR)with 18 in each.The rats were sacrificed after 4 weeks’ treatment with intragastrically-injected adenine.Serum creatinine (Scr),blood urea nitrogen (Bun),hemoglobin (Hb),hematocrit (Hct),and hepcidin were measured with automated hematology analyzer.The expressions of bone morphogenetic protein-6 (BMP-6 ),a serine/threonine kinase receptor-1 (SMAD-1 )and serine/threonine kinase receptor-4 (SMAD-4)in the kidney were detected by immunohistochemistry and Western blot.Results ①The results of general indicators:BUN and Scr in IR + EPO group and EPO group were lower than those in SA group (P <0.01).Hb and Hct in IR + EPO group and EPO group were increased significantly compared with those in SA group (P <0.01).Hepcidin was significantly increased in simple anemia group (P <0.01).② ELISA results showed that Hepcidin was lower in IR + EPO group and EPO group than in SA group (P <0.01).③ The results of pathological observation indicated that EPO could reduce rat renal pathology and function.④ Immunohistochemistry revealed that SMAD-4,SMAD-1 and BMP-6 were expressed in the rat glomerular cells and renal tubular epithelial cells.⑤ Western blot further indicated that the expression of SMAD-4 was significantly decreased in IR + EPO group and EPO group (P < 0.01 ).The expression of SMAD-1 in IR+EPO group and EPO group was decreased (P <0.05)while that of BMP-6 was elevated in IR+EPO group and EPO group (P <0.01 ).Conclusion The expression and secretion of hepcidin were inhibited by EPO via the BMP/SMAD pathway,which further corrects anemia and exerts a renal protective effect.
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Periodontal therapy has dealt primarily with attempts at arresting progression of disease, however, more recent techniques have focused on regenerating the periodontal ligament having the capacity to regenerate the periodontium. Recombinant human bone morphogenetic protein-7(rhBMP-7) can differentiate the osteoprogenitor cells and induce bone formation. The purpose of this study was to evaluate the effect of BMP-7 on rat periodontal ligament cells differentiation, in vitro. In the control group, cells was cultured with DMEM media. In the experimental groups, cells were cultured with rhBMP-7 in concentration of 10, 25, 50 and 100 ng/ml. Each group was characterized by examining alkaline phosphatase activity at 3 and 5 days of culture and the ability to produce mineralized nodules of rat calvarial cells at 14 days of culture. Synthesis of type I collagen(COL-I), osteocalcin(OCN), and bone sialoprotein (BSP) was evaluated by RT-PCR at 7 days of culture. Activation of Smad proteins and p38 MAP kinase was determined by western blot analysis of the cell lysates. Alkaline phosphatase activity was significantly increased in the concentration of BMP-7 50 ng/ml and 100 ng/ml compared to the control(p<0.05). The mineralized bone nodule formation was greater with addition of 50 ng/ml and 100 ng/ml BMP-7 than the control(p<0.01). In 7 days' culture, the expressions of COL-1, BSP, and OCN was increased by BMP-7 in concentration of 10 ng/ml~100 ng/ml. In western blot analysis, BMP-7 treated culture cells expressed Smad 1,5,8 in dose-dependent manner, whereas BMP-7 did not activate phosphorylated form of p38 MAP kinase. These result suggested that BMP-7 stimulate rat periodontal ligament cells to differentiate toward osteoblast phenotype and increase bone matrix production by activation of BMP-Smad pathway.