摘要
目的研究拔伸松动手法对膝关节骨性关节炎模型兔早中晚期关节软骨中分子量为p38丝裂原活化蛋白激酶(P38MAPK)信号通路及相关因子的影响,从细胞分子水平研究推拿手法对早中晚期膝关节骨性关节炎的作用机制。方法健康成年白兔90只,体质量1.8~2.5 kg,雌雄不限,重量分布均一。随机分为3组,每组10只:拔伸松动法组、模型组、空白组。采用Videman T造模方法,管型石膏伸直位固定兔右后肢膝关节2、4、6周,复制膝关节骨性关节炎早期、中期和晚期的模型。造模完成后3 d,治疗组以拔伸松动手法干预,治疗20次后取标本,采用反转录·聚合酶链反应(RT-PCR)、蛋白质印迹法(Western Blot)、免疫组织化学(IHC)法检测P38MAPK信号通路及相关因子的表达水平。结果经过治疗后早期治疗组关节软骨和模型组比较基质金属蛋白酶1(MMP1)、基质金属蛋白酶13(MMP13)、前列腺素E受体2(PTGER2)、环氧化酶2(COX2)、诱导型一氧化氮合酶(iNOS)、P38MAPK的mRNA表达、蛋白表达水平以及平均光密度值(IOD)略有降低,MMP13的mRNA表达水平和PTGER2蛋白表达水平差异有统计学意义(P<0.05)。经过治疗后中期治疗组关节软骨和模型组比较MMP1、MMP13、COX2、PTGER2、P38MAPK的蛋白表达水平以及平均光密度值(IOD)明显降低,MMP1、MMP13、PTGER2、P38MAPK的蛋白表达水平组间比较差异有统计学意义(P<0.05)。经过治疗后晚期治疗组关节软骨和模型组比较MMP1、MMP13、PTGER2、COX2、iNOS、P38MAPK的mRNA表达、蛋白表达水平以及平均光密度值(IOD)显著降低,MMP1、MMP13、COX2、P38MAPK的mRNA表达、蛋白表达水平差异有统计学意义(P<0.05),PTGER2和iNOS蛋白表达水平无差异。结论拔伸松动法可通过下调兔KOA早中期P38MAPK信号通路的表达,影响MMP1、MMP13、iNOS、COX2、PTGER2等相关因子的表达水平,改善KOA软骨细胞功能,有利于损伤软骨组织的修复。
Objective The study aims to explore the effect of pulling-stretching-loosening manipulation on p38 mitogen activated protein kinase(P38MAPK)signal pathway and related factors in articular cartilage of early,middle and late stage knee osteoarthritis model rabbits and study the mechanism of massage manipulation on early,middle and late stage knee osteoarthritis from the cellular and molecular level.Methods The study selected 90 healthy adult rabbits(1.8-2.5 kg)as the subjects with regardless of gender and homogeneous weight distribution.Three groups were generated randomly(10 for each group),including the pulling-stretching-loosening manipulation group,the model group and the blank group.Based on the classic model preparation method by Videman T,the right rear knees were fixed in continuous extension with plaster casts for 2,4 and 6 weeks respectively to duplicate the models for early,middle and late stage knee osteoarthritis.3 days after the model preparation,the treatment group was intervened with the pulling-stretching-loosening manipulation.Furthermore,the samples were collected after 2 courses and reverse transcription-polymerase chain reaction(RT-PCR),Western Blot and immunohistochemistry(IHC)method were applied to detect P38MAPK signaling pathway and the expression levels of the factors involved.Results The mRNA expressions,protein expressions and average optical density(IOD)values of matrix metalloproteinase 1(MMP1),matrix metalloproteinase 13(MMP13),prostagiandin E receptor 2(PTGER2),cyclooxy-genase 2(COK2)and inducible nitnc oxide synthase(iNOS)were reduced mildly after the treatment and the mRNA expression of MMP13 and the PTGER2 protein expression were discrepant(P<0.05)among the early stage knee osteoarthritis groups.The protein expressions and IOD values of MMP1,MMP13,COX2,PTGER2 and P38MAPK were decreased significantly and the protein expressions of MP1,MIP13,PTGER2 and P38MAPK were highly diverse(P<0.05)among the middle stage knee osteoarthritis groups.The mRNA expressions,protein expressions and IOD values of MMP1,MMP13,COX2,iNOS and P38MAPK were decreased significantly and the mRNA and protein expressions of MMP1,MMP13,COX2 and P38MAPK were highly diverse(P<0.05),with no difference for the protein expressions of PTGER2 and iNOS among the late stage knee osteoarthritis groups.Conclusion The pulling-stretching-loosening manipulation may impact the expressions of MMP1,MP13,iNOS,COX2,PTGER2 or other factors by downgrading the expression of P38MAPK signaling pathway during the early and middle stages to improve chondrocyte functions,thereby contributing to the restoration of the cartilages damaged.
作者
王春林
向勇
田启东
艾健
胡鸾
赵志勇
董有康
张兴宗
WANG Chunlin;XIANG Yong;TIAN Qidong;AI Jian;HU Luan;ZHAO Zhiyong;DONG Youkang;ZHANG Xingzong(Yunnan University of Chinese Medicine,Kunming 650500,Yunnan,China;Yunnan Hospital of Traditional Chinese Medicine,Kunming 650021,Yunnan,China)
出处
《辽宁中医杂志》
CAS
2023年第9期223-228,I0005-I0008,共10页
Liaoning Journal of Traditional Chinese Medicine
基金
国家自然科学基金地区基金项目(81660824,81760899,82260977)
云南省基础研究计划项目中医联合项目-面上项目[202001AZ070001(-022),2019FF002(-024)]
云南省卫生科技计划项目(2018NS0024)
云南中医药大学校院联合基金项目(YXLH202107)。
关键词
拔伸松动手法
兔膝骨性关节炎
P38MAPK信号通路
作用机制
pulling-stretching-loosening manipulation
rabbit with knee osteoarthrosis
P38MAPK signal pathway
mechanism
作者简介
王春林(1968-),男,云南曲靖人,主任医师,学士,研究方向:推拿防治脊柱疾病及骨关节疾病的基础和临床研究。
