复方杜仲汤干预终板软骨退变作用机制的实验研究  

The mechanism of Fufang Duzhong Tang(复方杜仲汤)against degeneration of cartilage endplate:an experimental study

作　　者：王庆敏[1] 沈毅弘[1] 李毅嵩 谢强[1] 郑庆丰[1] 沈鸿辉 WANG Qingmin;SHEN Yihong;LI Yisong;XIE Qiang;ZHENG Qingfeng;SHEN Honghui(Zhangzhou Traditional Chinese Medical Hospital,Zhangzhou 363000,Fujian,China)

机构地区：[1]福建省漳州市中医院,福建漳州363000

出　　处：《中医正骨》2024年第7期1-9,16,共10页The Journal of Traditional Chinese Orthopedics and Traumatology

基　　金：福建省卫健委科技计划项目(2021lzyjc25);福建省中医学术流派传承工作室建设项目(闽卫中医函〔2019〕129号)。

摘　　要：目的:探讨复方杜仲汤干预终板软骨退变的作用机制。方法:取4周龄SD大鼠80只,雌雄各半,随机分为中药低剂量组、中药中剂量组、中药高剂量组和空白组,中药低、中、高剂量组大鼠每日灌胃相应剂量的复方杜仲汤药液,空白组每次用与中药低剂量组等体积的蒸馏水灌胃,早晚各1次,共灌胃7 d。灌胃干预结束后24 h,抽取各组大鼠腹主动脉血,制备相应药物浓度的含药血清和空白血清。取4周龄SD大鼠60只,雌雄各半,摘取终板软骨组织,分离、提取终板软骨细胞。取对数生长期的终板软骨细胞,分为空白对照组、模型组、空白血清组、低剂量含药血清组、中剂量含药血清组和高剂量含药血清组。除空白对照组外,其他5组细胞均加入白细胞介素(interleukin,IL)-1β进行诱导。诱导后,低、中、高剂量含药血清组和空白血清组分别加入制备的低、中、高剂量的含药血清和空白血清进行干预。观察复方杜仲汤对终板软骨细胞活性、氧化应激和炎症因子水平,以及Kelch样环氧氯丙烷相关蛋白1(kelch-like ECH-associated protein 1,Keap1)-核转录因子红系2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)/抗氧化响应元件(antioxidant response element,ARE)信号通路的影响。采用细胞计数试剂盒检测各组终板软骨细胞的活性,计算细胞存活率。采用酶联免疫吸附法检测终板软骨细胞中丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、肿瘤坏死因子-α(tumor necrosis factor,TNF-α)、IL-1β、IL-6水平。采用实时定量PCR扩增法检测终板软骨细胞中Nrf2、Keap1、p53、Runt相关转录因子2(runt-related transcription factor 2,Runx2)、基质金属蛋白酶13(matrix metalloproteinase 13,MMP13)和Y染色体性别决定区-盒转录因子9(sex-determing region of Y chromosome-box transcription factor 9,Sox9)的mRNA相对表达量。采用蛋白�Objective:To explore the mechanism of Fufang Duzhong Tang(复方杜仲汤,FFDZT)against cartilage endplate(CEP)degeneration.Methods:Eighty 4-week-old Sprague-Dawley(SD)rats,half in males and females,were selected and randomized into low-dose FFDZT(L-FFDZT)group,medium-dose FFDZT(M-FFDZT)group,high-dose FFDZT(H-FFDZT)group,and blank group.The rats in L-FFDZT group,M-FFDZT group,and H-FFDZT group were intragastric administrated with FFDZT in daily dosages of 20.92,41.84,83.68 mL/kg,respectively,while the ones in blank group with an equal volume of distilled water as that of L-FFDZT group,once in the morning and evening,respectively,for consecutive 7 days.On hour 24 after the end of drug intervention,the blood was drawn from the abdominal aorta of rats in each group for making blank serum and medicated serum with the corresponding concentrations.Additionally,604-week-old SD rats,half in males and females,were selected for harvesting the CEP tissues,and then isolating and extracting the CEP chondrocytes.The CEP chondrocytes in the logarithmic growth phase were selected and divided into the blank control group,model group,blank serum group,low-,medium-,and high-dose medicated serum group.All the CEP chondrocytes but the ones in blank control group were intervened by interleukin(IL)-1βfor inducing degeneration.After successful inducing,the degenerated CEP chondrocytes in blank serum group,low-,medium-,and high-dose medicated serum group were further intervened with the prepared blank serum and medicated serum in their corresponding concentration,respectively.After the end of intervention,the effects of FFDZT on the viability of CEP chondrocytes,le-vels of oxidative stress(OS)and inflammatory factors,and kelch-like ECH-associated protein 1(Keap1)-nuclear factor-erythroid 2-related factor 2(Nrf2)/antioxidant response element(ARE)signaling pathway were observed.Moreover,the viability of CEP chondrocytes in each group was detected by using the cell counting kit-8(CCK8)assay,and the chondrocyte survival rate was calculated

关 键 词：软骨疾病 终板软骨 复方杜仲汤 软骨细胞 大鼠 Sprague-Dawley 实验研究 

分 类 号：R285.5[医药卫生—中药学]