柚皮苷对白介素-1β诱导的兔关节软骨细胞炎性反应的调节作用

Regulatory Effect of Icariin on Interleukin-1β-induced Inflammatory Response in Rabbit Articular Chondrocytes

目的观察不同浓度柚皮苷(naingin,NG)对白介素-1β(interleukin-1β,IL-1β)诱导的兔关节软骨细胞炎性反应的影响,探讨NG治疗骨关节炎的潜力。方法体外正常培养的兔关节软骨细胞用IL-1β进行炎性诱导,造软骨关节炎细胞模型。用荧光倒置显微镜观察及吖啶橙、甲苯胺蓝染色鉴定软骨细胞,运用CCK-8检测细胞活性来筛选NG浓度;取第2代生长良好的软骨细胞,根据所加培养物的不同将实验分为5组:空白组(C)、模型组(M)、观察组(OH、OM、OL),连续干预72 h,于RNA和蛋白提取前24 h加入IL-1β。通过qRT-PCR和Western Blot检测蛋白聚糖(Aggrecan)、Ⅱ型胶原(CoL-Ⅱ)、基质金属蛋白酶-13(MMP-13)和SOX9的表达。结果 (1)吖啶橙及甲苯胺蓝染色结果呈阳性,体外分离培养的确为软骨细胞。(2)CCK-8表明,NG浓度在大于10-5mol/L和小于10-9mol/L时,细胞活性较空白组减弱(P<0.05),NG浓度在10-6mol/L到10-8mol/L时,细胞活性增强(P<0.05,P<0.01)。(3)qRT-PCR显示:与空白组相比,模型组CoL-Ⅱ、Aggrecan和SOX9表达量显著降低(P<0.05,P<0.01),MMP-13表达量升高(P<0.05);与模型组比,CoL-Ⅱ、SOX9和Aggrecan明显升高(P<0.05,P<0.01),MMP-13表达量显著降低(P<0.05,P<0.01)。(4)Western Blot显示,与正常组比,模型组CoL-Ⅱ、SOX9和Aggrecan表达量均显著降低(P<0.05,P<0.01),MMP-13表达量升高(P<0.05);与模型组比,NG不同浓度的CoL-Ⅱ、SOX9和Aggrecan表达量均上升;MMP-13下降,且有浓度依赖趋势。结论柚皮苷可以调节软骨细胞基质和炎性微环境,促进软骨细胞CoL-Ⅱ、SOX9、Aggrecan的表达,并抑制MMP-13,进而逆转IL-1β诱导的软骨细胞炎性退变。

Objective To observe the effects of different concentrations of Naingin(NG) on interleukin-1β(IL-1β)-induced inflammatory response in rabbit articular chondrocytes and explore the potential of NG in the treatment of osteoarthritis. Methods Rabbit articular chondrocytes cultured in vitro were induced by inflammatory induction with IL-1β, and a chondrogenic arthritis cell model was established. The chondrocytes were identified by fluorescence inverted microscope and acridine orange and toluidine blue staining, and the NG concentration was screened by CCK-8 cell viability. The second generation well-growth chondrocytes were obtained, and the experiment was divided according to the culture. There were 5 groups: blank group(C), model group(M), observation group(OH, OM, OL), continuous intervention for 72 h, and IL-1β was added 24 h before RNA and protein extraction. The expressions of proteoglycan(aggrecan), type II collagen(CoL-Ⅱ), matrix metalloproteinase-13(MMP-13) and SOX9 were detected by qRT-PCR and Western blot. Results The results of acridine orange and toluidine blue staining were positive, and the chondrocytes were isolated and cultured in vitro. CCK-8 showed that when the NG concentration was greater than 10-5mol/L and less than 10-9mol/L, the cell viability was weaker than that of the blank group(P<0.05), and the NG concentration was between 10-6mol/L and 10-8mol/L. The cell activity was enhanced(P<0.05, P<0.01). The qRT-PCR showed that compared with the blank group, the expression levels of CoL-Ⅱ, Aggrecan and SOX9 in the model group were significantly decreased(P<0.05, P<0.01), and the expression of MMP-13 was increased(P<0.05). Compared with those of the model group, The colII, SOX9 and Aggrecan were significantly increased(P<0.05, P<0.01), and the expression of MMP-13 was significantly decreased(P<0.05, P<0.01). Western blot showed that compared with those of the normal group, the expression levels of colII, SOX9 and Aggrecan in the model group were significantly decreased(P<0.05, P<0.01), and the expression of MMP-13 was increased(P<0.05). Compared with those of the model group, the expression levels of colII, SOX9 and Aggrecan increased at different concentrations and MMP-13 decreased. There was a concentration-dependent trend. Conclusion Naingin can regulate the chondrocyte matrix and inflammatory microenvironment, promote the expressions of CoL-Ⅱ, SOX9 and Aggrecan in chondrocytes and inhibit MMP-13, thereby reversing the inflammatory degeneration of chondrocytes induced by IL-1β.