当归多糖组分APS-3c软骨保护作用的体外研究
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摘要
目的:探究当归多糖组分ASP-3c抗骨关节炎的分子机制。方法:不同浓度ASP-3c(2～50μg/mL)单独或联合人重组白细胞介素1β(IL-1β,10 ng/mL)处理人原代软骨细胞48 h。MTT法检测软骨细胞活力;倒置显微镜观察细胞形态;DMB法检测培养上清蛋白多糖含量;实时定量PCR及Western Blot检测IL-1β、IL-6及糖基转移酶基因表达。结果:ASP-3c处理人原代软骨细胞48 h不影响其增殖活性,并可改善IL-1β所致的软骨细胞退变表型。ASP-3c可浓度依赖性地抑制软骨细胞内IL-1β、IL-6的基因表达,并阻断外源性IL-1β对软骨细胞IL-1β、IL-6的上调作用。同时,无论是否存在外源性IL-1β处理,ASP-3c均可促进蛋白多糖合成,上调糖基转移酶mRNA水平。结论:ASP-3c可通过抑制软骨细胞IL-1β、IL-6等炎症因子的合成减轻软骨局部炎症;并促进软骨基质蛋白多糖合成恢复基质稳态,进而起到抗骨关节炎功效。
Objective: To uncover the underlying mechanism of the anti-osteoarthritis of APS-3 c, one of the components of Angelica Sinensis polysaccharides. Methods: Human primary chondrocytes were treated with 2-50 μg/mL APS-3 c, alone or with 10 ng/mL interleukin 1 beta(IL-1β), for 48 hours.Then, cell viability was detected with MTT assay, while inverted microscope was used for the morphological observation. Meanwhile, DMB assay was applied to test the glycosaminoglycan(GAG)concentration of the chondrocyte cultures. Moreover, mRNA and/or protein levels of IL-1β, IL-6 and glycosyltransferases were detected using RT-PCR assay or Western Blot assay. Results: APS-3 c didn't affect cell viability of human primary chondrocytes, whether in the presence of IL-1β or not,while it alleviated the degenerative features caused by IL-1β and promoted GAG synthesis and secretion of the chondrocytes. Meanwhile, APS-3 c inhibited mRNA and protein expression of IL-1β and IL-6, but induced gene expression of glycosyltransferases, both in the absence and presence of exoge-nous IL-1β. Conclusion: APS-3 c presented its anti-osteoarthritis activities though inhibiting gene expression of pro-inflammatory factors like IL-1β and IL-6, as well as promoting GAG synthesis by inducing gene expression of glycosyltransferases.
Objective: To uncover the underlying mechanism of the anti-osteoarthritis of APS-3 c, one of the components of Angelica Sinensis polysaccharides. Methods: Human primary chondrocytes were treated with 2-50 μg/mL APS-3 c, alone or with 10 ng/mL interleukin 1 beta(IL-1β), for 48 hours.Then, cell viability was detected with MTT assay, while inverted microscope was used for the morphological observation. Meanwhile, DMB assay was applied to test the glycosaminoglycan(GAG)concentration of the chondrocyte cultures. Moreover, mRNA and/or protein levels of IL-1β, IL-6 and glycosyltransferases were detected using RT-PCR assay or Western Blot assay. Results: APS-3 c didn't affect cell viability of human primary chondrocytes, whether in the presence of IL-1β or not,while it alleviated the degenerative features caused by IL-1β and promoted GAG synthesis and secretion of the chondrocytes. Meanwhile, APS-3 c inhibited mRNA and protein expression of IL-1β and IL-6, but induced gene expression of glycosyltransferases, both in the absence and presence of exoge-nous IL-1β. Conclusion: APS-3 c presented its anti-osteoarthritis activities though inhibiting gene expression of pro-inflammatory factors like IL-1β and IL-6, as well as promoting GAG synthesis by inducing gene expression of glycosyltransferases.
引文
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[2] Qin J,Liu YS,Liu J,et al. Effect of angelica sinensis polysaccharides on osteoarthritis in vivo and in vitro:a possible mechanism to promote proteoglycans synthesis[J]. Evid Based Complement Alternat Med,2013,2013:794761.
[3] Neogi T,Zhang Y. Epidemiology of osteoarthritis[J].Rheum Dis Clin North Am,2013,39(1):1-19.
[4] Fernandes JC,Martel-Pelletier J,Pelletier JP. The role of cytokines in osteoarthritis pathophysiology[J].Biorheology,2002,39(1-2):237-246.
[5] Goldring SR,Goldring MB. The role of cytokines in cartilage matrix degeneration in osteoarthritis[J]. Clin Orthop Relat Res,2004(427Suppl):S27-S36.
[6] Khair M,Bourhim M,Barre L,et al. Regulation of xy-losyltransferaseⅠgene expression by interleukin 1beta in human primary chondrocyte cells:mechanism and im-pact on proteoglycan synthesis[J]. J Biol Chem,2013,288(3):1 774-1 784.
[7] Gouze JN,Bordji K,Gulberti S,et al. Interleukin-1be-ta down-regulates the expression of glucuronosyltrans-feraseⅠ,a key enzyme priming glycosaminoglycan bio-synthesis:influence of glucosamine on interleukin-1beta-mediated effects in rat chondrocytes[J]. Arthritis Rheum,2001,44(2):351-360.
[8] Lindahl U,Kusche M,Lidholt K,et al. Biosynthesis of heparin and heparan sulfate[J]. Ann N Y Acad Sci,1989,556:36-50.
[9] Cao W,Li XQ,Wang X,et al. Characterizations and anti-tumor activities of three acidic polysaccharides from Angelica sinensis(Oliv.)Diels[J]. Int J Biol Macro-mol,2010,46(1):115-122.