秦皮苷对脂多糖诱导的软骨细胞氧化应激和炎症因子的影响

Effects of fraxin on oxidative stress and inflammatory factors induced by lipopolysaccharide in chondrocytes

目的:探讨秦皮苷对脂多糖(LPS)诱导的软骨细胞炎症模型的抗炎抗氧化作用。方法:提取SD大鼠的软骨细胞并进行体外培养,并通过苏木精-伊红(HE)染色和番红O染色对软骨细胞进行鉴定。采用MTT法检测秦皮苷对软骨细胞的毒性。将细胞分为对照组、模型组和实验组。LPS诱导软骨细胞构建体外骨关节炎(OA)模型,并用秦皮苷进行干预。钙黄绿素/乙锭均二聚物(Calcein-AM/EthD-I)染色评价细胞活力,实时荧光定量PCR(qPCR)法检测炎症相关基因和软骨特异性基因的表达,免疫荧光染色观察软骨细胞内基质金属蛋白酶(MMP)-13的分泌情况,总抗氧化能力(T-AOC)实验检测秦皮苷的总抗氧化水平,二氯荧光黄双乙酸盐(DCFH-DA)荧光探针检测细胞内活性氧(ROS)水平。结果:HE染色显示细胞形态与软骨细胞一致,番红O染色结果表明提取的细胞为软骨细胞。5μg/mL秦皮苷对软骨细胞无毒性,秦皮苷在该浓度下能维持软骨细胞正常的形态,抑制细胞外基质的降解,促进蛋白多糖的分泌,提高软骨细胞活力,显著下调炎症相关基因白介素(IL)-6、MMP-3、MMP-13和诱导型一氧化氮合酶(iNOS)表达,上调软骨特异性基因Ⅱ型胶原纤维α1(Col2al)表达(P<0.05),减少MMP-13分泌,提高总抗氧化水平,清除ROS。结论:秦皮苷对LPS诱导的OA软骨细胞具有抗炎及抗氧化的作用,其可能是OA潜在的治疗药物。

Objective:To investigate the anti-inflammatory and antioxidant effects of fraxin on lipopolysaccharide(LPS)-induced chondrocyte inflammation model.Methods:The chondrocytes of SD rats were extracted and cultured in vitro,and the chondrocytes were identified by hematoxylin-eosin(HE)staining and safranin O staining.The toxicity of fraxin to chondrocytes was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.The cellswere divided into control group,model group and experimental group.The osteoarthritis(OA)model in vitro was established by LPS induced chondrocytes and intervened by fraxin.Cell viability was assessed by calcein/ethidium homodimer(Calcein-AM/EthD-I)staining.Real-time fluorescent polymerase chain reaction(qPCR)was used to investigate the expression of inflammation-related genes and cartilage specific genes,the secretion of matrix metalloproteinase(MMP)-13 in chondrocytes was studied by immunofluorescence staining,total antioxidant capacity(T-AOC)assay was used to determine the total antioxidant level of fraxin,and the level of intracellular reactive oxygen species(ROS)was detected by DCFH-DA fluorescent probe.Results:He staining showed that the cell morphology was consistent with that of chondrocytes,and the results of saffron O staining showed that the extracted cells were chondrocytes.Fraxin was non-toxic at 5μg/mL.5μg/mLfraxincould maintain cartilage cells of normal shape,inhibit the degradation of extracellular matrix,promote the secretion of protein and polysaccharide,enhance the activity of chondrocytes,down-regulate the expressions of interleukin-6(IL-6),MMP-3,MMP-13 and inducible nitric oxide synthase(iNOS),up-regulate the expression of collagen-type II-alpha 1(Col2al)(P<0.05),reduce the secretion of MMP-13,increase the level of total antioxidant,and scavenge ROS.Conclusion:Fraxin has anti-inflammatory and antioxidant effects on LPSinduced OA chondrocytes,and it may be a potential therapeutic drug for OA.