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Objective:To discuss the inhibitory effect of chelerythrine(CHE)on the migration,invasion,and epithelial-mesenchymal transition(EMT)of the human ovarian cancer SKOV3 cells,and to clarify the associated mechanism.Methods:The SKOV3 cells were cultured in vitro and divided into control group and 2.5,5.0,10.0,20.0,and 40.0 μmol·L-1 CHE groups.Methylthiazolydiphenyl-tetrazolium(MTT)assay was used to detect the inhibitory rates of proliferation of the cells in various groups.The SKOV3 cells were cultured in vitro and divided into control group,transforming growth factor-β1(TGF-β1)group,TGF-β1+5 μmol·L-1 CHE group,and TGF-β1+10 μmol·L-1 CHE group.Cell scratch assay was used to detect the migration rates of the cells in various groups;Transwell chamber assay was used to detect the numbers of migration and invasion cells in various groups;Western blotting method was used to detect the expression levels of E-cadherin,N-cadherin,and Vimentin proteins in the cells in various groups;immunofluorescence staining method was used to detect the fluorescence intensities of E-cadherin and N-cadherin in the cells in various groups.Results:The MTT assay results showed that compared with control group,the inhibitory rates of proliferation of the cells in 5.0,10.0,20.0,and 40.0 μmol·L-1 CHE groups were significantly increased(P<0.05 or P<0.01).The cell scratch assay results showed that compared with control group,the migration rate of the cells in TGF-β1 group was increased(P<0.01);compared with TGF-β1 group,the migration rates of the cells in TGF-β1+5 μmol·L-1 CHE group and TGF-β1+10 μmol·L-1 CHE group were significantly decreased(P<0.01).The Transwell chamber assay results showed that compared with control group,the numbers of migration and invasion cells in TGF-β1 group were significantly increased(P<0.05);compared with TGF-β1 group,the numbers of migration and invasion cells in TGF-β1+5 μmo·l L-1 CHE group and TGF-β1+10 μmo·l L-1 CHE group were significantly decreased(P<0.01).The Western blotting results showed that compared with control group,the expression level of E-cadherin protein in the cells in TGF-β1 group was significantly decreased(P<0.01),while the expression levels of N-cadherin and Vimentin proteins were increased(P<0.05 or P<0.01);compared with TGF-β1 group,the expression levels of E-cadherin protein in the cells in TGF-β1+5 μmol·L-1 CHE group and TGF-β1+10 μmol·L-1 CHE group were significantly increased(P<0.01),and the expression levels of N-cadherin and Vimentin proteins were significantly decreased(P<0.01).The immunofluorescence staining results showed that compared with control group,the fluorescence intensity of E-cadherin in the cells in TGF-β1 group was decreased,and the fluorescence intensity of N-cadherin was increased;compared with TGF-β1 group,the fluorescence intensities of E-cadherin in the cells in TGF-β 1+5 μmol·L-1 CHE group and TGF-β1+10 μmol·L-1 CHE group were significantly increased,and the fluorescence intensities of N-cadherin were decreased.Conclusion:CHE can inhibit the proliferation,migration,invasion,and EMT of the human ovarian cancer SKOV3 cells.
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Objective:To discuss the protective effect of velvet antler peptide(VAP)in the osteoporosis(OP)model rats,and to clarify the possible mechanism.Methods:Sixty 12-week-old SD rats were randomly divided into control group,model group,positive drug group(treated with 1 mg·kg-1·d-1 of alendronate sodium by gavage),low dose of VAP group(treated with 100 mg·kg-1·d-1 VAP),medium dose of VAP group(treated with 200 mg·kg-1·d-1 VAP),and high dose of VAP group(treated with 300 mg·kg-1·d-1 VAP),and there were ten rats in each group.Except for control group,the rats in the other groups were injected with dexamethasone(2 mg·kg-1)to replicate the OP rat model,while the rats in control group were injected with the equivalent volume of saline twice a week for 11 consecutive weeks.Dual-energy X-ray absorptiometry was used to detect the bone mineral density(BMD)of femur tissue of the rats in various groups;enzyme-linked immunosorbent assay(ELISA)method was used to detect the levels of serum calcium(Ca2+),phosphate(P),osteoprotegerin(OPG),alkaline phosphatase(ALP),and osteocalcin(OCN)in serum of the rats in various groups;biochemical method was used to detect the malondialdehyde(MDA)level and superoxide dismutase(SOD)activity in serum of the rats in various groups;HE staining was used to observe the pathomorphology of bone tissue of the rats in various groups;Western blotting method was used to detect the expression levels of silent information regulator 1(SIRT1),catalase(CAT),Runt-related transcription factor 2(RUNX2),and forkhead box protein O1(FOXO1)proteins in bone tissue of the rats in various groups.Results:Compared with control group,the BMD of femoral tissue of the rats in model group was decreased(P<0.05);compared with model group,the BMD of femur tissue of the rats in positive drug group,medium dose of VAP group,and high dose of VAP group were increased(P<0.05 or P<0.01).Compared with control group,the levels of Ca2+,P,OPG,and SOD activities in serum of the rats in model group were decreased(P<0.05),and the levels of ALP,OCN,and MDA were increased(P<0.05);compared with model group,the level of OPG in serum of the rats in low dose of VAP group was significantly increased(P<0.05),the levels of Ca2+,P,OPG,and activities of SOD in serum of the rats in positive drug group,medium dose of VAP group,and high dose of VAP group were significantly increased(P<0.05 or P<0.01),and the levels of ALP,OCN,and MDA in serum of the rats in positive drug group and different doses of VAP groups were decreased(P<0.05 or P<0.01).The HE staining results showed that compared with control group,the rats in model group had fewer bone cells and disordered arrangements in the bone tissue,thinner bone trabeculae with large fractures,and an expanded marrow cavity;compared with model group,the rats in positive drug group,medium dose of VAP group,and high dose of VAP group had thicker bone trabeculae arranged more tightly.The Western blotting results showed that compared with control group,the expression levels of SIRT1,CAT,RUNX2,and FOXO1 proteins in bone tissue of the rats in model group were decreased(P<0.05);compared with model group,the expression levels of SIRT1,CAT,RUNX2,and FOXO1 proteins in bone tissue of the rats in positive drug group,medium dose of VAP group,and high dose of VAP group were significantly increased(P<0.05 or P<0.01).Conclusion:VAP has the protective effect against OP in the rats,and its mechanism may be related to mediating the antioxidant stress action through the SIRT1/FOXO1 signaling pathway.
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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 evaluate effects of Lycium barbarum polysaccharide (LBP) on expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α) in ultraviolet B (UVB)-radiated HaCaT cells.Methods Conventionally cultured HaCaT cells were divided into control group and LBP groups,which were firstly treated with DMEM,12.5,25.0,50.0 and 100 μg/ml LBP solution respectively for 4 hours,and then were irradiated by UVB at different intensity of 0,20,40,60 mJ/cm2 separately.After 24-hour continuing culture,CCK-8 assay was performed to determine the cell survival rate,and an enzymatic-biochemical method to estimate the activity of superoxide dismutase (SOD).RT-PCR and Western blot analysis were conducted to measure the mRNA and protein expression of HIF-1α and VEGF respectively.Results Compared with the control group at the same UVB radiation dose,the 12.5-,25.0-and 100.0-μ,g/ml LBP groups showed different extents of increase in survival rates of UVB-radiated cells (P ＜ 0.05),and the 50.0-μg/ml LBP group showed the highest cell survival rate (P ＜ 0.01).Among all the LBP groups,SOD activity was highest in the 50.0-μg/ml LBP group (P ＜ 0.01).Along with the increase of UVB radiation dose,the mRNA and protein expression of HIF-1α and VEGF all gradually increased.Compared with the control group,the 50.0-μg/ml LBP group could effectively reduce the mRNA and protein expression of HIF-1α and VEGF in HaCaT cells (all P ＜ 0.05).Conclusion LBP may play a role in protecting cells from UVB radiation-mediated damage,likely by influencing the mRNA and protein expression of HIF-1α and VEGF in HaCaT cells.