淫羊藿苷联合地塞米松对人髓核细胞增殖及分泌表达的影响

Effect of icariin in combination with Dexamethasone on cell proliferation and secretion expression of human nucleus pulposus cells

目的探讨淫羊藿苷(ICA)与地塞米松(Dex)联合作用对人髓核细胞(h NPs)增殖及聚集蛋白聚糖(Aggrecan)、II型胶原蛋白(Col2a)、IL-6、IL-8和基质金属蛋白酶(MMP-13)表达的影响。方法取生长状态良好的h NPs按不同处理因素分为:control组(只含培养基)、ICA组(20μmol/L ICA)、Dex组(0.2μmol/L Dex)、ICA+Dex组(20μmol/L ICA+0.2μmol/L Dex)、IL-1β组(10 ng/ml IL-1β)、ICA+IL-1β组(20μmol/L ICA+10 ng/ml IL-1β)、Dex+IL-1β组(0.2μmol/L Dex+10 ng/ml IL-1β)、ICA+Dex+IL-1β组(20μmol/L ICA+0.2μmol/L Dex+10 ng/ml IL-1β),各组作用48 h;CCK-8法检测细胞的增殖;Western blot、RT-PCR以及ELISA检测Aggrecan、Col2a、IL-6、IL-8和MMP-13表达。结果 CCK-8结果显示:与control组比较,低浓度的ICA(≤20μmol/L)和Dex(≤0.2μmol/L)单独作用均提高h NPs存活率但差异无统计学意义(P>0.05)。然而,10μmol/L ICA和0.2μmol/L Dex联合用药对h NPs的存活率大于单独用药(P<0.05);Western blot、RT-PCR以及ELISA结果表明:与单独用药组相比,ICA和Dex联合作用促进Aggrecan、Col2a蛋白和m RNA表达(P<0.05);另外,ICA和Dex联合处理抑制IL-1β诱导的h NPs炎症介质IL-6、IL-8的分泌表达(P<0.05),同时降低MMP-13蛋白、m RNA水平(P<0.05)。结论 ICA与Dex联合作用对促进h NPs增殖、Aggrecan和Col2a表达有效果;同时抑制IL-1β诱导的h NPs炎症介质IL-6、IL-8和MMP-13的表达,为椎间盘退行性病变的药物治疗提供理论依据。

Objective To investigate the effect of icariin （ICA） in combination with Dexamethasone （Dex） on cell proliferation and secretion expression of Aggrecan, type Ⅱcollagen （Col2a）, IL-6, IL-8 and MatrixMetalloproteinase-13 （MMP-13） in human nucleus pulposus cells （hNPs）. Methods The experiment was divided into different groups based on different treatments： control group （only culture medium）, ICA group（20 μM/L ICA）, Dex group（0.2 μM/L Dex）, ICA ＋ Dex group （20 μM/L ICA ＋ 0.2 μM/L Dex）, IL-1β group （10 ng/mL IL-1β）, ICA ＋ IL-1β group （20 μM/L ICA ＋ 10 ng/mL IL-1β）, Dex ＋ IL-1β group （0.2 μM/L Dex ＋ 10 ng/mL IL-1β）, ICA＋Dex ＋ IL-1β group （20 μM/L ICA ＋ 0.2 μM/L Dex ＋ 10 ng/mL IL-1β）. Each group was treated for 48 h. Cell proliferation was detected by CCK-8 assay. The expression levels of Aggrecan, Col2a, IL-6, IL-8 and MMP-13 were examined by Western blotting, RT-PCR and ELISA methods. Results ICA（20 μM/L ） or Dex （≤ 0.2 μM/L） at low concentrations promoted hNPs proliferation, but there was no significant difference when compared with the control group （P 〉 0.05）. However, 20 μM/L ICA in combination with 0.2 μM/L Dex significantly increased the cell survival rate of hNPs when compared with the control group, the ICA and teh Dex single treatment group （P 〈 0.05）. The protein and mRNA expression of Aggrecan and Col2a in hNPs were also markedly increased by ICA in combination with Dex when compared with the control group, the ICA and the Dex single treatment group （P 〈 0.05）. In addition, the secretion expression of IL-6, IL-8 and MMP-13 in IL-1β-induced hNPs were significantly inhibited by ICA in combination with Dex when compared with IL-1β, ICA ＋ IL-1β or Dex ＋ IL-1β groups（P 〈 0.05）. Conclusions These findings suggest that icariin in combination with Dexamethasone significantly promotes cell proliferation and the expression of Aggrecan and Col2a in hNPs, and inhibites the secretion expression of IL-6, IL-8 and MMP-13 in IL-1β-induced hNPs, providing a preliminary experimental basis for drug treatment of intervertebral disc degeneration.