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Objective:To explore the effect and mechanism of pilose antler different components on the bone tissue of ovariectomized osteoporosis model rats and ascertain the material basis of pilose antler. Method:fifty-six SD rats were divided randomly into seven groups:normal group,model group,Xianling Gubao group(468 mg·kg-1),Bujiale group(80 mg·kg-1),polysaccharide group(50 mg·kg-1),polypeptides group(175 mg·kg-1),polysaccharide and polypeptide mixture group(50 mg·kg-1+175 mg·kg-1). Osteoporosis mode was established through ovary resection of female rats,meanwhile,the rats were given different components of pilose antler for consecutively 12 weeks. Subsequently, using absorptiometry to measure the rats' bone mass density. The activities of bone alkaline phosphatase(BALP),osteocalcin (OT),bone morphogenetic protein2(BMP-2),Smad1,Smad5,Runt-related transcription factor 2 (RUNX2) were detected by enzyme-linked immuno sorbent assay (ELISA). The expression of BMP-2,Smad1,Smad5,Runx2 protein was examined by Western blot and Real-time polymerase chain reaction (Real-time PCR). Morphological assay for bone tissue were detected by htoxylin eosin(HE) staining. Result:After 12 weeks, Compared with the normal group, the osteoporosis model group showed significantly decrease in bone mineral density(PPPConclusion:Pilose antler different components has therapeutic effect on ovariectomized osteoporosis model rats.The mechanism may be related to up-regulat the expression of BMP-2/Smad1,Smad5/Runx2 signal pathways.
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Objective:: To investigate the effects of pilose antler polypeptide on the abilities of proliferation and collagen secretion of mouse embryonic fibroblasts NIH/3T3, and to clarify the relevant mechanisms. Methods: The NIH/3T3 cells were treated with different doses 1.56, 3.13, 6.25, 12.50, 25.00, 50.00, 100.00, and 200.00 mg • L_ 1) of pilose antler polypeptide as experimental groups, the cells treated with 0 mg • L_ 1 pilose antler polypeptide were used as blank control group, and the cells treated with 50.00 fig • L-1 basic fibroblast growth factor (bFGF) were used as positive control group. MTT assay was used to detect the survival rates of NIH/3T3 cells in various groups. ELISA assay was used to detect the collagen secretion of NIH/3T3 cells in various groups. Wound healing assay was used to detect the migration abilities of NIH/3T3 cells. Western blotting method was performed to detect the expression levels of p-ERK 1/2 in the NIH/3T3 cells in various groups. Immunofluorescence method was used to detect the expression levels of transforming growth factor-fil (TGF-J31) in the NIH/3T3 cells in various groups. Results: Compared with blank control group, the survival rates of NIH/3T3 cells in positive control group and 6.25, 12.50, 25.00, 50.00, 100.00, 200.00 mg • L_ 1 pilose antler polypeptide groups were markedly increased (P < 0 . 05 or P < 0 . 01). Compared with blank control group, the levels of type I collagen protein in the culture solution of the NIH/3T3 cells in positive control group and 6. 25, 12. 50, 25. 00, and 50. 00 mg • L_ 1 pilose antler polypeptide groups were markedly increased (P < 0 . 05 or P < 0 . 01), and the levels of type IE collagen protein in the culture solution of the NIH/3T3 cells in positive control group and 12. 50 and 25.00 mg • L_ 1 pilose antler polypeptide groups were markedly increased (P < 0 . 05). Compared with blank control group, the scratch healing rates of NIH/3T3 cells, and the expression levels of p-ERK 1/2 in the NIH/3T3 cells, and the expression levels of TGF-J31 in the NIH/3T3 cells in positive control group and 12. 50 mg • L_ 1 pilose antler polypeptide groups were markedly increased (P < 0 . 05 or P < 0 . 01). Conclusion: Pilose antler polypeptide can promote the proliferation, and collagen secretion of NIH/3T3 cells and increase the migration ability, which may be achieved by activating the phosphorylation of ERK 1/2 and increasing the expression of TGF-J31.
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Peptides from Pilose antler aqueous extract (PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanisms are not well understood. Here, PAAE was isolated and purified to explore the molecular mechanisms underlying PAAE's effects on BMSCs as well as its osteoprotective effects in ovariectomized rats. Our results showed that PAAE promoted proliferation and differentiation of BMSCs to become osteoblasts by enhancing ALP activity and increasing extracellular matrix mineralization. The trabecular microarchitecture of ovariectomized rats was also found to be protected by PAAE. Quantitative reverse transcription-polymerase chain reaction (Quantitative RT-PCR) results suggest that PAAE also increased the expression of osteogenic markers including, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2), and collagen I (COL-I). Immunoblotting results indicated that PAAE upregulated the levels of BMP-2 and Runx2 and was associated with Smad1/5 phosphorylation. PAAE A at the concentration of 200 μg·mL showed the strongest effect on proliferation and osteogenic differentiation of BMSCs after 48 h. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), we identified the molecular weight of PAAE A and found that it is less than 3000 Da and showed several significant peaks. In conclusion, PAAE activates the BMP-2/Smad1, 5/Runx2 pathway to induce osteoblastic differentiation and mineralization in BMSCs and can inhibit OVX-induced bone loss. These mechanisms are likely responsible for its therapeutic effect on postmenopausal osteoporosis.
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Objective: To explore the effect of differentiation of cardiac stem cells (CSC) mediated by pilose antler polypeptides on the expressions of terminal myocardial differentiation genes atrial natriuretic polypeptide (ANP) and myosin light chain 2v (MLC-2v), and to clarify the mechanism of repairing the damaged myocardium Methods: The healthy male Wistar rats born 2 d were selected to extract the CSC. The surface marker c-Kit and the purity of CSC were identified by immunofluorescence and flow cytometry. The culture dish was used as the experimental unit and the extracted cells were divided into the following four groups: blank control group (the same amount of buffer was added for induction), 5-azacytidine group (induced with 3 jumol · L-1 5-azacytidine), pilose antler polypeptides group (induced with 800 mg · L-1 pilose antler polypeptides) and combined group (induced with 800 mg · L-1 pilose antler polypeptides and 3 μmol · L-1 5-azacytidine); the cells were incubated for 48 h in the condition of 37°C and 5% CO2. The expression levels of ANP and MLC-2v in supernatant of the cells in various groups were detected by ELISA method. The expression levels of ANP and MLC-2v mRNA in the cells in various groups were detected by RT-PCR method. Results: CSC were prepared with the purity0.05). The result of RT-PCR showed that the expression levels of ANP and MLC-2v mRNA in 5-azacytidine, pilose antler polypeptides and combined groups were significantly increased compared with blank control group (P<0.05). Conclusion: Pilose antler polypeptides can induce the differentiation of CSC into cardiac cells by promoting the expressions of ANP and MLC-2v, and they can reduce the cytotoxicity induced by 5-azacytidine in some degrees.
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Objective:To explore the effect of differentiation of cardiac stem cells(CSC)mediated by pilose antler polypeptides on the expressions of terminal myocardial differentiation genes atrial natriuretic polypeptide (ANP)and myosin light chain 2v(MLC-2v),and to clarify the mechanism of repairing the damaged myocardium.Methods:The healthy male Wistar rats born 2 d were selected to extract the CSC.The surface marker c-Kit and the purity of CSC were identified by immunofluorescence and flow cytometry.The culture dish was used as the experimental unit and the extracted cells were divided into the following four groups:blank control group(the same amount of buffer was added for induction),5-azacytidine group(induced with 3 μmol·L-15-azacytidine),pilose antler polypeptides group(induced with 800 mg·L-1pilose antler polypeptides)and combined group(induced with 800 mg·L-1pilose antler polypeptides and 3 μmol·L-15-azacytidine);the cells were incubated for 48 h in the condition of 37℃ and 5% CO2.The expression levels of ANP and MLC-2v in supernatant of the cells in various groups were detected by ELISA method.The expression levels of ANP and MLC-2v mRNA in the cells in various groups were detected by RT-PCR method.Results:CSC were prepared with the purity>95%.The results of ELISA showed that the expression levels of ANP and MLC-2v in 5-azacytidine,pilose antler polypeptides and combined groups were significantly increased compared with blank control group(P<0.05).The expression levels of ANP and MLC-2v in combined group were increased compared 5-azacytidine and pilose antler polypeptides groups,but there were no significant differences(P>0.05).The result of RT-PCR showed that the expression levels of ANP and MLC-2v mRNA in 5-azacytidine,pilose antler polypeptides and combined groups were significantly increased compared with blank control group(P<0.05).Conclusion:Pilose antler polypeptides can induce the differentiation of CSC into cardiac cells by promoting the expressions of ANP and MLC-2v,and they can reduce the cytotoxicity induced by 5-azacytidine in some degrees.
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OBJECTIVE: To investigate the effects of microwave radiation on learning and memory abilities in mice, and to study pilose antler peptide's intervention. METHODS: Fifty mice were divided into five groups randomly, designated as control group, radiation group, pilose antler peptide (25, 50, and 100 mg·kg-1) groups. Learning and memory impairment model in mice was established by microwave radiation of 2 450 MHz average surface power, 10.0 mW·cm-2 for 90 min every day for 28 d.The radiation rats were treated with low-, mid-, and high-dose (25, 50, and 100 mg·kg-1) pilose antler peptide by sc injection for 28 d. The learning and memory ability of mice was determined by avoiding darkness experiment and Y maze experiment.The contents of S100B, tumor necrosis factor-α(TNF-α), interleukin-10(IL-10), malondialdehyde (MDA), and nitric oxide(NO) in the brain of mice were determined respectively after the behavioral experiments. RESULTS: Compared with control group, radiation group could shorten the latency of avoiding darkness experiments, increase the numbers of errors both in avoiding darkness experiment and in Y maze experiment. Radiation group could rise the contents of S100B, TNF-α, IL-10, MDA and NO in the brain of mice (P<0.05 or P<0.01). Compared with radiation group, pilose antler peptide (50, 100 mg·kg-1) groups could lengthen the latency of avoiding darkness experiments, significantly shorten the numbers of errors both in avoiding darkness experiment and in Y maze experiment, and reduce the contents of S100B, TNF-α, MDA and NO, increase the content of IL-10 in the brain of mice (P<0.05 or P<0.01). CONCLUSION: Pilose antler peptide could significantly perfect the learning and memory ability of mice exposed to microwave radiation. The mechanism may be related to its anti-oxidative actions by anti-inflammatory action, further lowering neurotoxic effects of NO.
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Objective:To investigate the effect of pilose antler polypeptide combined with Schwann cells modified by glial cell line-derived neurotrophic factor (GDNF)gene on the proliferation of human bone marrow mesenchymal stem cells (BMSCs)in vitro .Methods:According to the conventional method,the bone marrow (10 mL)was extracted from the healthy volunteers and was inoculated into the culture flask.The primary cultured cells were completely fused.The BMSCs were harvested at the 3rd generation and the cells were adjusted to 5 × 106 mL-1 . 4 μL GDNF gene modified Schwann cells was added into GDNF group,4 μL (10 mg· L-1 )PAP combined with GDNF gene modified Schwann cells was added into combination group,and only same amount of medium was added into control group.The proliferative activities,cell nuclear antigen (PCNA)levels and apoptotic rates of BMSCs in various groups were detected by enzyme-linked immunosorbent assay,ELISA method and Annexin Ⅴ-FIFC/PI cell apoptosis detection kit,respectively.Results:After primary culture for 48 h,most of the cells adhered to the wall,and the morphology of the cells changed into polygonal shape and few of them showed spindle.The passaged cells showed spindle spindle,the cells were confirmed as BMSCs,and all of them were non-hematopoietic stem cells.Compared with control group,the proliferative activities and the PCNA level of the BMSCs in GDNF group and combination group were increased (P <0.05)and the apoptotic rates were decreased (P <0.05);compared with GDNF group,the proliferative activity and the PCNA level of the BMSCs in combination group were increased (P < 0.05 )and the apoptotic rate was decreased (P < 0.05 ).Conclusion:PAP combined with Schwann cells modified by GDNF gene can promote the proliferation of human BMSCs in vitro .
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In this paper, effect and molecular mechanism of sika pilose antler type I collagen(SPC-I) of ROS1728 cell were explored. For the SPC-I provides the theory basis for the treatment of osteoporosis. The adherent method was used to cultivate rat osteosarcoma osteogenesis sample cell line ROS1728. The effect of SPC-I on ROS1728 cells proliferation was tested by CCK-8 method. Runx2, osernix, ALP, Coll-I, OC osteogenesis related genes expression was tested by RT-PCR, and Runx2 protein expression was tested by Western-bolt. Results showed that 5 g•L ⁻¹ SPC-I could inhibit ROS1728 cell proliferation, and significantly promote the expression of ROS1728 cell specific transcription factor Runx2 and osterix mRNA, Runx2 protein and marker gene ALP, Coll-I, OC mRNA expression(P<0.01). 2.5 g•L ⁻¹ and 10 g•L ⁻¹ SPC-I could significantly inhibit the ROS1728 cell proliferation(P<0.01), and inhibit the expression of related genes. In conclusion, 5 g•L ⁻¹ SPC-I could inhibit ROS1728 cell proliferation, obviously enhance ROS1728 cell function, promote ROS1728 cell differentiation, maturation.
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Through referring to relevant works and the latest literature, the application and research progress of the traditional Chinese medicine inducing mesenchymal stem cells were summarized. The researching results focusing on induction and proliferation of mesenchymal stem cell by salvia, notoginseng, tortoise plastron, pilose antler, ginseng, astragalus were introduced.
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10.3969/j.issn.2095-4344.2013.25.013
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Objective: To explore the effect of sika pilose antler type I collagen (SPC-I) on osteoclast and its molecular mechanism. Methods: The osteoclasts and osteoblasts were cultured by the induction method of whole bone marrow cells. The control (with full medium), osteoclasts (with HG-DMEM inducing medium), and SPC-I (2.5, 5, and 10 g/L) groups were set up. Except the control group, others were given the HG-DMEM inducing medium with each 40 ng/mL of both RANKL and macrophage colony-stimulating factor (M-CSF), then conditioned cultured for 7 d, every other 3 d to replace medium for the complement of the drug concentration. By HE and tartrate-resistant acid phosphatase (TRAP) stainings, the cell morphology was observed under inverted microscope. The TRAP activity was detected using spectrophotometer, the gene expression of TRAP, receptor activator of NF-κB (RANK), receptor activator of NF-κB lig and (RANKL), and osteoprotegerin (OPG) was measured by RT-PCR, and the RANK protein expression was detected by Western blotting. Results: Compared with the osteoclast group, SPC-I (5 and 10 g/L) groups could make TRAP positive cells and TRAP activity decreased, TRAP, RANK, and RANKL expression in gene level reduced, and RANK expression in protein level down-regulated also (P<0.01); Compared with the control group, SPC-I (2.5 and 10 g/L) could make the OPG expression in gene level increased and the RANKL/OPG ratio declined (P<0.01). The effect of 5 g/L SPC-I was the most significant (P<0.01). The effect of 2.5g/L SPC-I was not significant. Conclusion: SPC-I has the inhibitory effect on the osteoclast formation and differentiation; The effect of implementation is through RANKL/OPG signal transduction pathway to regulate the expression of TRAP and RANK genes.
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Anti-bone resorption activity of pilose antler blood (Cervus nippon Temminck) were evaluated in ovariectomized Wistar rats. The rats were randomly divided into sham operated group (SHAM), ovariectomized group (OVX) and pilose antler blood treated group. The ovariectomized rats were treated with pilose antler blood orally in 4000l/kg daily doses for 10 weeks. Compared with SHAM group, serum 17 -estradiol level decreased significantly and osteocalcin level increased significantly in OVX group, indicating successful model of osteoporosis. The experiments showed that the bone mineral density of the lumbar spine and left femur in OVX group decreased remarkably compared to SHAM group but normalized by treatment with pilose antler blood. Additionally, serum levels of insulin-like growth factor-1and testosterone were lower obviously in OVX group than those in SHAM group but preserved by pilose antler blood treatment. However, no obvious changes in serum levels of calcium, phosphorus, total alkaline phosphatase and osteoprotegerin were observed among three groups. These results suggested that administration of pilose antler blood was effective in alleviating osteoporosis in ovariectomized rats.
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Objective The lyophilized pilose antler water extract(PAE) was isolated,and their cell proliferation on PC12 cells was observed.Methods S-200 Size-exclusive gel and DEAE negative ion-exchange liquid chromatograph were employed to fractionate the PAE.SDS-PAGE was employed to analyze the proteins composition of PAE.The protein concentration was determined by Folin-Phenol assay.The proliferation rates of PC12 cells were measured by MTT assay.Results The proliferation rate of PAE on PC12 cells at 13.3 mg/mL was 47%(P