摘要
目的以经典Wnt/β-连环蛋白(β-catenin)信号通路为靶向,观察苗药五藤膏贴敷对膝骨关节炎(KOA)模型兔软骨组织中β-catenin、基质金属蛋白酶(MMP)-13及原癌基因(c-myc)表达的影响。方法将同一批4~5月龄96只健康家兔,随机分为空白组、模型组、巴布膏对照组,五藤膏低量组(约1 g),五藤膏中量组(约2 g),五藤膏高量组(约4 g),每组16只。除空白组外,其余5组均制备兔KOA模型,造模成功后,分别予氟比洛芬巴布膏贴敷及相应剂量的五藤膏贴敷干预;空白组不做任何干预,正常饲养;模型组患膝关节无菌纱布包扎固定。给药后第1、2、3、4 w各处死4只,取患膝关节软骨苏木素-伊红(HE)染色观察软骨组织病理变化;实时荧光定量-聚合酶链式反应(qRT-PCR)检测关节软骨细胞中MMP-13、c-myc mRNA的表达水平;Western印迹检测β-catenin蛋白的表达水平。结果与空白组比较,模型组膝关节面软骨病理显示重度退行性改变,软骨细胞重度变性、萎缩,分布紊乱;模型组软骨细胞中MMP-13、c-myc mRNA的表达量最高,空白组表达量最低,而模型组软骨细胞中β-catenin蛋白表达最低,空白组最高,不同量五藤膏干预后各组β-catenin蛋白表达均不同程度升高;量越高也越接近空白组;五藤膏各量组(1、2、3、4 w)与巴布膏对照组(1、2、3、4 w)c-myc mRNA的表达量均较空白组升高,但五藤膏高量组(1、2、3、4 w)与巴布膏对照组(1、2、3、4 w)MMP-13与c-myc升高均不明显;五藤膏各量组(1、2、3、4 w)与巴布膏对照组(1、2、3、4 w)c-myc mRNA的表达量均较模型组显著降低,五藤膏高量组(1、2、3、4 w)与巴布膏对照组(1、2、3、4 w)MMP-13降低更为显著;五藤膏中、高量组(1、2、3、4 w)MMP-13 mRNA表达量与巴布膏对照组相近;各治疗组组内比较,五藤膏低量组(2、3、4 w)MMP-13 mRNA明显高于五藤膏中量组(1、2、3、4 w)与五藤膏高量组(1、2、3、4 w)的表达,其中与五藤膏高量组差异更显著;五藤膏低量组(1、2、3、4 w)c-myc mRNA明显高于五藤膏中量组(4 w)、五藤膏高量(1、2、3、4 w)的表达,除五藤膏中量组第1w与五藤膏高量组(1、2、3、4 w)c-myc mRNA表达差异显著外,其余各时段五藤膏中量组与五藤膏高量组c-myc mRNA表达均无显著差异。结论五藤膏干预可能通过调控Wnt信号通路,促进兔KOA患膝关节软骨细胞β-catenin蛋白表达,抑制KOA模型兔的关节软骨细胞Wnt信号下游基因MMP-13、c-myc mRNA的表达,延缓疾病进展,从而改善KOA的症状。
Objective Classical Wnt/β-catenin signaling pathway was targeted to observe the effects of the Miao medicine wuteng ointment on the expressions ofβ-catenin,MMP-13 and c-myc in the cartilage tissue of rabbit models with knee osteoarthritis(OA).Methods 96 healthy rabbits aged 4~5 months were randomly divided into blank group,model group,flurbiprofen Cataplasms control group(positive control group),low dose group,medium dose group and high dose group.There were 16 rabbits in each group.Except for the blank group,rabbit knee of OA model was prepared in the other five groups.After successful modeling,flurbiprofen Cataplasms attach and the corresponding dose of Miao medicine wuteng ointment was applied.The blank group was fed normally without any intervention.In the model group,knee joint was fixed with sterile gauze dressing.On the 1st,2nd,3rd and 4th week after administration,4 rabbits were sacrificed respectively.HE staining was performed to observe the pathological changes of cartilage tissue.QRT-PCR detected the expressions of MMP-13 and c-myc mRNA.The expression ofβ-catenin protein was detected by Western blot.Results Compared with the blank group,the pathological changes of the chondrocytes in the model group showed severe degeneration,atrophy and disordered distribution.The expressions ofβ-catenin protein in the model group and the blank group were the highest,However,the expression ofβ-catenin protein in chondrocytes in model group was the lowest,while that in blank group was the highest.The expression ofβ-catenin protein in each group increased to different degrees after the intervention of different doses of Miao medicine wuteng ointment.The higher dose was also close to the blank group.The expression of c-myc mRNA in Miao medicine wuteng ointment each dose group(1,2,3,4 w)and flurbiprofen Cataplasms control group(1,2,3,4 w)were all higher than that in the blank group,but the levels of MMP-13 and c-myc in the high-dose group(1,2,3,4 w)and flurbiprofen Cataplasms control group(1,2,3,4 w)were not significantly increased.Compared with the model group,the expression of c-myc mRNA in each dose group(1,2,3,4 w)and flurbiprofen Cataplasms control group(1,2,3,4 w)were significantly lower than those in the model group,among which the decrease of MMP-13 high dose group(1,2,3,4 w)and flurbiprofen Cataplasms control group(1,2,3,4 w)was more significant.Compared with the positive control group(Babu paste control group),the MMP-13 mRNA expressions of Miao medicine wuteng ointment in medium and high doses(1,2,3,4 w)were similar to those of Babu paste control group.The expression of MMP-13 mRNA at low dose(2,3,4 w)was significantly higher than that at medium dose(1,2,3,4 w)and high dose(1,2,3,4 w),and the difference was more significant.The expression of c-myc mRNA at low dose(1,2,3,4 w)was significantly higher than that at medium dose(4 w)and high dose(1,2,3,4 w).There was no significant difference between the expression of c-myc mRNA at medium dose(1 w)and high dose(1,2,3,4 w).Conclusions Intervention of Miao medicine wuteng ointment might promote the expression ofβ-catenin protein in the chondrocytes of the knee joint affected by knee OA by regulating the Wnt signaling pathway,inhibit the expression of MMP-13 and c-myc mRNA in the downstream genes of Wnt signaling in the articular chondrocytes of KOA model rabbits,delay the disease progression,and thus improve the symptoms of KOA.
作者
申海艳
周静
肖丽娜
马武开
姚血明
陈琳
韩珊
SHEN Hai-Yan;ZHOU Jing;XIAO Li-Na(Department of Orthopedics,Guizhou Provincial Orthopaedic Hospital,Guiyang 550001,Guizhou,China)
出处
《中国老年学杂志》
CAS
北大核心
2021年第15期3271-3276,共6页
Chinese Journal of Gerontology
基金
国家自然科学基金资助项目(81760884)
国家自然科学基金委员会青年项目(81804127)
贵州卫生计生委科学技术基金项目(gzwjkj2018-1-028)
贵州省中医药(民族医药)治疗风湿病医学平台建设项目(黔科合平台人才[2018]5707)
国家重点研发计划中医药现代化研究重点专项(2017YFC1703904)
贵州省中医风湿免疫病临床研究中心(黔科合平台人才[2020]2202号)。
