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AIM:To investigate the effect of oleuropein on interleukin-1β( IL-1β)-induced SD rat articular chondrocytes .METHODS:The SD rat articular chondrocytes were isolated by 2 step enzyme digestions .The chondrocytes were cultured in vitro.Inverted microscopic observation was performed during the culture .Alcian blue staining and type II collagen immunohistochemical staining were used to identify the chondrocytes .The effects of oleuropein on the viability of chondrocytes were determined by CCK-8 assay.The cells in 3rd passage were pretreated with oleuropein at 10, 50 or 100 μmol/L and subsequently stimulated with IL-1βat 10 μg/L for 24 h.Production of prostaglandin E 2 ( PGE2 ) and ni-tric oxide (NO) were evaluated by the Griess reaction and an enzyme linked immunosorbent assay (ELISA).The mRNA expression of matrix metalloproteinase ( MMP)-1 and MMP-13 was measured by real-time PCR.The protein levels of in-ducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and nuclear factor-kappa B (NF-κB) were detected by Western blotting .RESULTS:The cell viability of chondrocytes was not significantly impaired by treating with oleuropein at concentration of 10, 50 or 100μmol/L for 24 h compared with control group .Pretreatment with oleuropein significantly in-hibited the production of PGE 2 and NO induced by IL-1β.Oleuropein also significantly decreased the IL-1β-stimulated MMP-1 and MMP-13 mRNA expression in articular chondrocytes .Pretreatment with oleuropein inhibited the IL-1β-media-ted activation of NF-κB by suppressing the degradation of its inhibitory protein IκBαin the cytoplasm .CONCLUSION:Oleuropein inhibits IL-1β-induced inflammatory gene expression by suppressing NF-κB activation at the transcriptional le-vel, suggesting a new mechanism for the anti-inflammatory effects of oleuropein as a novel agent on treating with osteoarthri-tis.
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AIM:Toinvestigatewhetherautophagyisup-regulatedwhenresveratrol(Res)inducesapoptosis in chondrosarcoma , and to study the effects of autophagy inhibitor combined with Res on chondrosarcoma .METHODS:SW1353 cells were divided into 4 groups: control group, Res group, 3-methyladenine (3MA) group, and Res +3MA group.Electron microscopy was used to observe the autophagyosomes in control group and Res group .At the same time, the viability of the cells in the 4 groups was detected by CCK-8 assay.TUNEL staining and Western blotting (for determi-ning the levels of cleaved caspase-3, Bax and Bcl-2) were used to reflect levels of apoptosis in all groups .The expression of autophagy-related proteins Beclin 1, LC3-Ⅱ and p62 was detected by Western blotting .RESULTS: Exposure of the cells to Res resulted in a decrease in cell viability and an increase in the level of apoptosis ( P<0.05 ) .Compared with control group, the level of apoptosis was increased but the autophagy was decreased (P <0.05).Compared with Res group, the cell viability and the level of autophagy were decreased and the level of apoptosis was increased ( P<0.05 ) . CONCLUSION:Resveratrol induces apoptosis and autophagy , and inhibition of autophgay enhances resveratrol-induced apoptosis in chondrosarcoma .
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[ABSTRACT]AIM:Toinvestigatetheinhibitoryeffectsofresveratrolonchondrosarcomaandtherelationwith mitochondrial and PI3K/Akt pathways.METHODS:Chondrosarcoma SW1353 cells were treated with resveratrol at con-centrations of 25, 50 and 100 μmol/L for the time intervals of 24 h, 48 h and 72 h.The viability and apoptosis of the SW1353 cells in the presence or absence of resveratrol were analyzed by CCK 8 assay and Hoechst 33258 staining , respec-tively.The protein levels of Bcl-2, Bax, activated caspase-3, Akt and p-Akt were detected by Western blotting .The cell migration ability was determined by wound scratch assay .RESULTS:Exposure of the cells to resveratrol resulted in a de-crease in the cell viability in a dose-and time-dependent manner (P<0.05).visible nuclei with apoptotic characteristics in resveratrol group were observed .The protein levels of activated caspase-3 and Bax were increased , and Bcl-2 and p-Akt were decreased compared with control group .The total Akt were not significantly changed .Resveratrol also significantly re-duced the migration of tumor cells .CONCLUSION:Resveratrol induces apoptosis of chondrosarcoma , which plays a role of part through mitochondrial and PI 3K/Akt signaling pathways .
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[ABSTRACT]AIM:TostudytheeffectofsmallinterferingRNA(siRNA)ontheexpressionofbeta2-microglo-bulin (β2M) in pre-differentiated bone marrow mesenchymal stem cells (BMSCs).METHODS: The β2M siRNA was transfected into the pre-differentiated BMSCs with Lipofectamine 2000.BMSCs were divided into transfection group , blank control group and negative control group .The expression of β2 M at mRNA and protein levels was determined by real-time qPCR, Western blotting and laser confocal microscopy .The productions of aggrecan and type II collagen in pre-differentia-ted BMSCs were determined by toluidine blue staining and type Ⅱcollagen immunofluorescence .RESULTS:The results of real-time qPCR, Western blotting and laser confocal microscopy showed that siRNA successfully inhibited the expression ofβ2 M at mRNA and protein levels in the pre-differentiated BMSCs .The results of toluidine blue and type Ⅱcollagen im-munofluorescence staining showed that siRNA does not affect the productions of aggrecan and type Ⅱ collagen in the pre-differentiated BMSCs .CONCLUSION:siRNA targeting β2 M reduces the expression of β2 M in the pre-differentiated BM-SCs and does not affect the chondrocyte characteristics of pre -differentiated BMSCs .
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BACKGROUND:Many in vivo and in vitro experiments indicate that hypoxic co-cultures promote stem cells differentiate into chondrocytes. OBJECTIVE:To evaluate the influence of hypoxia on the chondrogenic differentiation of three-dimensional co-cultured adipose-derived stem cells and articular chondrocytes. METHODS:Adipose-derived stem cells and articular chondrocytes were mixed at the ratio of 3:1, then the mixed cells were seeded onto poly(lactic-co-glycolic acid)-gelatin scaffold at the ultimate concentration of 5.0×1010/L. The cells were cultured in normoxia (20%O 2 ) and hypoxic (5%O 2 ) conditions for 6 weeks. After culture, hematoxylin and eosin staining was performed for histological structure analysis, and alcian blue staining was used to evaluate glycosaminoglycan synthesis. Type II col agen expression was detected by immunohistochemistry staining. The content of DNA, glycosaminoglycan and hydroxyproline in the scaffold-cellcomplex was measured. RESULTS AND CONCLUSION:In the hypoxia group, hematoxylin-eosin staining showed the formation of massive cells and extracellular matrix;alcian blue staining showed massive glycosaminoglycan formation;immunohistochemistry staining detected strongly positive expression of col agen type II, the content of DNA, glycosaminoglycan and hydroxyproline was higher than the normoxia group. Hypoxia promotes in vitro chondrogenic differentiation of co-cultured adipose-derived stem cells and articular chondrocytes. .