Objective To investigate the effect of icariin(ICA)on IL-1β-induced chondrocytes degeneration. Methods Primary chondrocytes isolated from articular cartilage of newborn rats were cultured in vitro and treated with icariin at 1×10-7, 1×10-6and 1×10-5mol/L. The cell proliferation was measured by MTT at 12, 24, 36, 48, 60 and 72 h. The cultured rat chondrocytes were than dividing into control group(N group), IL-1β-stimulated group(M group treated with IL-1β 10 ng/ml), ICA-L(1×10-7mol/L)and ICA-H(1×10-6mol/L)groups(IL-1β-stimulated cells treated with ICA). The concentrations of collagen-Ⅱ and glycosaminoglycan(GAG)in culture media after 72 h of intervention were determined by ELISA and the expressions of Col-Ⅱ,MMP13,Acan,Sox9 mRNA were detected by RT-PCR. Results 1×10-5mol/L ICA inhibited chondrocytes proliferation when intervened for 24 h,while promoted cell proliferation after 72 h;there was no significant difference between groups with and without ICA at 48 h or 60 h.ICA concentration at 1×10-7mol/L and 1×10-6mol/L both significantly promoted cell proliferation from 36 h to 72 h, but ICA at 1×10-7mol/L had no significant effect at 60 h.ICA at both concentration of 1×10-7mol/L and 1×10-6mol/L increased the GAG level in culture supernatant,but there was no significant change in Col-Ⅱlevel in ICA-H group. Compared with M group, the expression of Acan/Sox9 mRNA in ICA-L group and Col-ⅡmRNA in ICA-H group significantly went up,while MMP13 decreased in ICA-H group. Conclusion Icariin can affect the matrix microenvironment of chondrocytes, promote the phenotype gene expression and inhibit MMP13 mRNA expression,and further protects chondrocytes from IL-1β-induced degeneration.%目的 观察不同浓度淫羊藿苷(icariin,ICA)对IL-1β诱导软骨细胞退变的保护作用.方法 原代分离培养新生24 h大鼠四肢长骨干骺端透明软骨的软骨细胞,取P1代细胞,以4×103/孔接种96孔培养板,加入终浓度分别为1×10-7、1×10-6和1×10-5mol/L的ICA,MTT法分别测定12、24、36、48、60、72 h后软骨细胞增殖情况.以1.2×105/孔接种6孔培养板,分别设置空白对照组(N组)、炎症刺激组(M组,加入10 ng/mL IL-1β诱导软骨细胞退变)和淫羊藿苷低、高剂量组(ICA-L组和ICA-H组,炎症刺激组基础上加入ICA,终浓度分别为1×10-7、1×10-6mol/L),连续干预72 h.ELISA法测定培养上清液中Ⅱ型胶原(collagen-Ⅱ, Col-Ⅱ)和糖胺多糖(glycosaminoglycan, GAG)含量,RT-PCR 法检测细胞 Col-Ⅱ、基质金属蛋白酶13(matrix metaloproteinase13, MMP13)、蛋白聚糖(aggrecan,Acan)和Sox9 mRNA表达情况.结果 1×10-5mol/L ICA早期(24 h)表现为抑制软骨细胞增殖(P<0.01),72 h后促进软骨细胞增殖(P<0.01);1×10-7mol/L和1×10-6mol/L ICA干预36~72 h后促进软骨细胞增殖(P<0.05),但其中1×10-7mol/L在60 h与空白对照组相比差异无统计学意义(P>0.05).ICA干预72 h后,与M组比较,ICA-L和ICA-H组,GAG浓度均显著升高(P<0.05),而Col-Ⅱ含量于ICA-L组降低、ICA-H组升高,但差异均无统计学意义(P>0.05).ICA-L组Acan/Sox9 mRNA表达升高(P<0.01),ICA-H组Col-ⅡmRNA水平升高而MMP13 mRNA表达降低(P<0.01).结论 淫羊藿苷能影响软骨细胞基质微环境,促进软骨细胞表型基因表达,抑制MMP13 mRNA表达,进而拮抗IL-1β诱导的软骨细胞退变.
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