荷叶碱对脂多糖诱导的大鼠软骨细胞炎症的抗炎作用研究

Anti-inflammatory effect of nuciferine in lipopolysaccharide-induced chondrocyte inflammation in rats

目的探讨荷叶碱对软骨细胞的保护作用及对软骨细胞炎症的抗炎作用。方法取体外分离培养3~5天的SD大鼠膝关节软骨细胞,应用10μg/ml LPS诱导软骨细胞建立体外骨关节炎模型。将实验分为3组,即空白组、模型组(LPS)和实验组(LPS+荷叶碱)。通过活/死细胞(Calcein-AM/EthD-I)双染色,实时荧光定量聚合酶链反应(qRT-PCR),HE染色(苏木素-伊红),番红O染色,免疫荧光染色,检测荷叶碱抑制软骨细胞外基质降解作用及缓解软骨细胞炎症的作用。结果荷叶碱浓度为10μM时,无明显毒性,Calcein-AM/EthD-I染色表明该浓度对炎症诱导的软骨细胞有保护作用。qRT-PCR表明,与模型组相比,实验组的Col2al表达升高,MMP-13和IL-6表达下降。HE染色(苏木素-伊红)、番红O染色、免疫荧光染色结果表明荷叶碱能够维持软骨细胞的形态,促进软骨细胞蛋白多糖的分泌,抑制MMP-13的表达。结论浓度为10μM的荷叶碱对LPS诱导的软骨细胞有较好保护作用及抗炎作用,无毒副作用。

Objective To investigate the protective effect of nuciferine on chondrocytes and the anti-inflammatory effect on lipopolysaccharide-induced chondrocyte inflammation.Methods Chondrocytes were extracted from 3-5-day-old SD rat knees and cultured in vitro.LPS(10μg/ml)was used to induce chondrocytes to establish an in vitro osteoarthritis model,and the experiment was divided into blank group,model group(LPS)and experimental group(LPS+nuciferine).Live dead cells(Calcein-AM/EthD-I)staining,real-time fluorescent quantitative polymerase chain reaction(qRT-PCR),(hematoxylin-eosin)HE staining,safranin O staining,immunofluorescence staining was used to detect the effects of nuciferine in inhibiting the degradation of extracellular matrix of chondrocytes and relieving chondrocyte inflammation.Results There was no obvious toxicity for chondrocytes at a concentration of 10μM of nuciferine.Calcein-AM/EthD-I staining showed that this concentration had a protective effect on the inflammation of chondrocytes.The qRT-PCR showed that,compared with the model group,the expression of Col2al in the experimental group increased,however the expression of MMP-13 and IL-6 decreased.(Hematoxylin-Eosin)HE staining,Safranin O staining,and immunofluorescence staining showed that nuciferine can maintain the morphology of chondrocytes,promote the secretion of chondrocyte proteoglycan,and inhibit the expression of MMP-13.Conclusion Nuciferine(10μM)has a good protective and anti-inflammatory effect on chondrocytes induced by LPS and has no side effects.