摘要
目的探讨阿魏酸(Ferulic acid,FA)对脂多糖(LPS)诱导的OA软骨细胞Wnt/β-catenin信号通路的影响。方法于4月龄新西兰大白兔膝软骨提取软骨细胞,体外培养后分为4组,空白对照组、OA模型组、OA+FA低剂量组和OA+FA高剂量组,将2μg/ml的LPS加入培养基中培养软骨细胞,构建OA软骨细胞模型,对照组加入等体积的细胞培养液,选取合适的阿魏酸浓度,确定低剂量组和高剂量组,低剂量组和高剂量组则分别加入5μmol/ml、20μmol/ml的阿魏酸进行共培养,甲苯胺蓝染色观察正常软骨细胞形态,qRT-PCR法检测4组软骨细胞Ⅱ型胶原基因的表达(COL2)、软骨细胞基质金属蛋白(MMP-13)基因的表达,CCK-8法检测软骨细胞增殖情况,Western blot检测Wnt2、GSK-3β、β-catenin、COL2、MMP-13、Bcl-2、Bax蛋白的表达。结果甲苯胺蓝染色法鉴定软骨细胞,软骨细胞在显微镜下呈现形态不规则、梭形透光度良好的多层细胞,细胞核红染,呈球形;qRT-PCR和Western blot结果显示OA模型组COL2基因和蛋白的表达量显著低于空白组(P<0.05)。低剂量组、高剂量组COL2的基因和蛋白的表达情况显著高于模型组(P<0.05),且高剂量组的表达水平更高(P<0.05);模型组的MMP-13基因和蛋白的表达显著高于空白组(P<0.05),低剂量组、高剂量组MMP-13基因和蛋白的表达显著低于模型组(P<0.05),且低剂量组的MMP-13蛋白的表达水平更高(P<0.05);CCK-8法检测软骨细胞的增殖细胞情况显示,模型组软骨细胞的增殖情况显著低于空白组(P<0.05),低剂量组、高剂量组软骨细胞的增殖情况显著高于模型组(P<0.05),且高剂量组的增殖水平更高(P<0.05);模型组的Bcl-2/Bax显著低于空白组(P<0.05),低剂量组、高剂量组Bcl-2/Bax显著高于模型组(P<0.05),且高剂量组的Bcl-2/Bax水平更高(P<0.05);与空白组相比,模型组Wnt2、GSK-3β、β-catenin水平明显升高,阿魏酸呈剂量依赖性抑制Wnt2、GSK-3β、β-catenin蛋白表达(P<0.
Objective To investigate the effects of ferulic acid(FA)on the Wnt/βcatenin signaling pathway of the OA chondrocytes induced by lipopolysaccharide(LPS).Methods The chondrocytes were extracted from the knee cartilage of New Zealand white rabbits with 4 months of age.After cultured in vitro,the chondrocytes were divided into four groups:blank control group,OA model group,OA+FA low dose group,OA+FA high dose group.The LPS 2μg/ml LPS was added into the culture medium to establish the OA chondrocyte model.The equal volume of cell culture medium was added into the culture medium in control group,moreover the appropriate concentration of ferulic acid was selected to determine the concentrations in low dose group and high dose group.The low dose group chondrocytes and the high dose group chondrocytes were co cultured by adding 5μmol/ml and 20μmol/ml of ferulic acid,respectively.The morphous of normal chondrocytes was observed by toluidine blue staining.The expression levels of typeⅡcollagen gene(COL2)and chondrocyte matrix metal protein 13 gene(MMP-13)were detected by qRT PCR.In addition the chondrocytes proliferation was detected by CCK-8 method,and the expression levels of Wnt2,GSK-3β,β-catenin,COL2,MMP-13,Bcl-2 and Bax protein were detected by Western Blot.Results The chondrocytes were identified by toluidine blue staining.The chondrocytes were multi layered in irregularly shaped and the spindle shaped with well translucency under the microscope.The cell nuclei were red stained and spherical.The results of qRT PCR and Western blot showed that the expression levels of COL2 gene and protein in OA model group were significantly lower than those in blank group(P<0.05).The expression levels of COL2 gene and protein in low dose group and high dose group were significantly higher than those in model group(P<0.05).Moreover the expression levels of COL2 gene and protein in high dose group were significantly higher than those in low dose group(P<0.05).The expression levels of MMP-13 gene and protein in model group were
作者
刘新宁
曹滨滨
刘杰
毕建年
赵亮
LIU Xinning;CAO Binbin;LIU Jie(Department of Orthopedics,Weihai Stomatological Hospital,Shandong,Weihai 264200,China)
出处
《河北医药》
CAS
2020年第11期1610-1614,共5页
Hebei Medical Journal
