木犀草苷抑制SNHG14表达改善SW1353骨关节炎细胞模型的实验研究

Inhibition of Luteolin on SNHG14 Expression to Improve SW1353 Osteoarthritis Cell Model

目的:观察木犀草苷(Cy)对骨关节炎细胞模型的保护作用,并探讨其可能的作用机制。方法:使用0.1%二甲基亚砜(DMSO)及不同浓度的Cy(0、10、20、30、40、50μmol/mL)处理SW1353细胞,采用CCK-8检测其细胞毒性,确定刺激浓度。将SW1353细胞分为正常对照组、白细胞介素-1β(IL-1β)组、Cy低剂量组、Cy高剂量组。正常对照组SW1353细胞不进行任何处理;IL-1β组SW1353细胞采用10 ng/mL IL-1β处理24 h;Cy低剂量组SW1353细胞采用10 ng/mL IL-1β+10μmol/mL Cy处理24 h;Cy高剂量组SW1353细胞采用10 ng/mL IL-1β+20μmol/mL Cy处理24 h。采用RT-qPCR法检测基质金属蛋白酶-13(MMP-13)mRNA、白细胞介素-6(IL-6)mRNA、Ⅱ型胶原(COL2a1)mRNA、蛋白聚糖(ACAN)mRNA及长链非编码RNA(lncRNA)SNHG14 mRNA的表达水平;采用Western blotting检测MMP-13及细胞外基质COL2a1、ACAN蛋白表达。结果:CCK-8结果表明Cy(10、20μmol/mL)对SW1353细胞活性没有明显的抑制作用。IL-1β组MMP-13 mRNA、IL-6 mRNA相对表达量高于正常对照组,ACAN mRNA、COL2a1 mRNA相对表达量低于正常对照组,差异均有统计学意义(P<0.05);Cy低、高剂量组MMP-13 mRNA、IL-6 mRNA相对表达量低于IL-1β组,ACAN mRNA、COL2a1 mRNA相对表达量高于IL-1β组,差异均有统计学意义(P<0.05)。IL-1β组lncRNA SNHG14 mRNA相对表达量高于正常对照组,差异有统计学意义(P<0.01);Cy低、高剂量组lncRNA SNHG14 mRNA相对表达量低于IL-1β组,差异均有统计学意义(P<0.01)。IL-1β组MMP-13蛋白相对表达量高于正常对照组,ACAN、COL2a1蛋白相对表达量低于正常对照组,差异有统计学意义(P<0.05);Cy低、高剂量组MMP-13蛋白相对表达量低于IL-1β组,ACAN、COL2a1蛋白相对表达量高于IL-1β组,差异均有统计学意义(P<0.01)。结论:Cy能改善IL-1β诱导的SW1353骨关节炎细胞模型,可能机制为抑制SNHG14表达。

Objective:To observe the protective effect of luteolin(Cynaroside,Cy)on osteoarthritis cell models and to explore its possible mechanism.Methods:SW1353 cells were treated with 0.1%DMSO as well as different concentrations of Cy(0,10,20,30,40,50μmol/mL).And CCK-8 was used to detect their cytotoxicity and determine the stimulus concentration.SW1353 cells were divided into normal control group,IL-1βgroup,Cy low-dose group and Cy high-dose group.SW1353 cells were not treated at all in normal control group.SW1353 cells were treated with 10 ng/mL IL-1βin IL-1βgroup,10 ng/mL IL-1β+10μmol/mL Cy in Cy low-dose group,and 10 ng/mL IL-1β+20μmol/mL Cy in luteolin high-dose group for 24 h.RT-qPCR was used to detect the expression levels of metalloproteinase-13(MMP-13)mRNA,interleukin 6(IL-6)mRNA,type II collagen(COL2a1)mRNA,proteoglycan(ACAN)mRNA and long non-coding RNA(lnc RNA)SNHG14 mRNA.Western blotting was used to detect the expression of MMP-13 and extracellular matrix COL2a1 and ACAN proteins.Results:The results of CCK-8 showed that Cy(10,20μmol/mL)showed no significant inhibitory effect on SW1353 cell activity.The IL-1βgroup showed higher relative expression of MMP-13 mRNA and IL-6 mRNA than normal control group,while lower relative expression of ACAN mRNA and COL2a1 mRNA than normal control group,with statistically significant differences(P<0.05).The low and high Cy groups showed lower relative expression of MMP-13 mRNA and IL-6 mRNA than IL-1βgroup,while higher relative expression of ACAN mRNA and COL2a1 mRNA than IL-1βgroup,with statistically significant differences(P<0.05).The IL-1βgroup showed higher relative expression of lncRNA SNHG14 mRNA than normal control group,and the difference was statistically significant(P<0.01).The low and high Cy groups showed lower relative expression of lncRNA SNHG14 mRNA than IL-1βgroup,and the difference was statistically significant(P<0.01).The IL-1βgroup showed higher relative expression of MMP-13 protein than normal control group,while lower relative expression of ACAN and COL2a1 protein than normal control group,with statistically significant difference(P<0.05).The low and high Cy groups showed lower relative expression of MMP-13 protein than IL-1βgroup,while higher relative expression of ACAN and COL2a1 protein than IL-1βgroup,with statistically significant difference(P<0.01).Conclusion:Cy can improve IL-1β-induced SW1353 osteoarthritis,possibly by inhibiting SNHG14 expression.