山姜素对脂多糖诱导的软骨细胞损伤的保护作用研究

The protective effect of alpinetin on chondrocyte damage induced by lipopolysaccharide

目的探讨山姜素对脂多糖(LPS)诱导的乳鼠软骨细胞损伤的保护作用及机制。方法提取乳鼠膝关节处的软骨细胞分离并培养,分为对照组、LPS诱导组(模型组)和LPS诱导后加山姜素处理组(山姜素组),模型组和山姜素组均采用LPS诱导软骨细胞损伤模型,山姜素组LPS诱导后再采用不同剂量的山姜素进行处理。CCK-8法检测软骨细胞活性,通过荧光素二乙酸酯/碘化丙啶(FDA/PI)染色、实时荧光定量聚合酶链反应(qPCR)以及免疫荧光染色观察山姜素对软骨细胞的保护作用。结果 CCK-8提示,当山姜素的剂量为5 mg/L时,软骨细胞的吸光度最高;FDA/PI染色结果显示,与对照组比较,模型组活细胞数量减少,死细胞则增加;与模型组比较,山姜素组活细胞数增加,死细胞减少。q PCR结果显示,与模型组比较,山姜素组的白细胞介素(IL)-1β、IL-6、诱导型一氧化氮合酶(iNOS)、肿瘤坏死因子(TNF-α)、基质金属蛋白酶-13(MMP-13)基因表达水平降低,而Ⅱ型胶原酶(Col2a1)基因表达水平增高(P<0.05)。免疫荧光染色提示,与模型组比较,山姜素组中IL-6表达水平降低。结论山姜素能够有效地保护LPS诱导的软骨细胞炎症损伤,促进软骨细胞增殖,为山姜素治疗骨关节炎提供了理论依据。

Objective To explore the protective effect of alpinetin on lipopolysaccharide(LPS)-induced rat chondrocytes damage and its mechanism.Methods The chondrocytes from the knee joints of suckling rats were isolated and cultured.They were divided into control group,LPS induction group(model group)and LPS induction plus alpinetin treatment group(alpinetin group).LPS were used to induce inflammation in the model group and the alpinetin group.The alpinetin group was treated with different concentrations of alpinetin after LPS induction.The CCK-8 method was used to detect chondrocyte activity,and the inflammation protection was observed by fluorescein diacetate/propidium iodide(FDA/PI)staining,real-time PCR(qPCR)and immunofluorescence staining effect.Results CCK-8 suggested that when the concentration of alpinetin was 5 mg/L,the absorbance of chondrocytes was the highest.FDA/PI staining results showed that the number of live cells was reduced in the model group compared with that the control group,while the number of dead cells were increased.Compared with the model group,the number of live cells were increased in the alpinetin group,while the number of dead cells decreased.qPCR results showed that the expression levels of interleukin-1β(IL-1β),IL-6,inducible nitric oxide synthase(iNOS),tumor necrosis factor(TNF-α)and human matrix metalloproteinase-13(MMP-13)gene were decreased in the alpinetin group compared with those of the model group,while the expression level of typeⅡcollagenase(Col2a1)was increased(P<0.05).Immunofluorescence staining indicated that the expression level of IL-6 was significantly reduced in the alpinetin group compared with that of the model group.Conclusion Alpinetin can effectively protect the inflammatory damage of chondrocytes induced by LPS and promote the proliferation of chondrocytes,which provides a theoretical basis for the treatment of osteoarthritis.