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

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。采用蛋白质印迹法检测终板软骨细胞中Nrf2、Keap1、P53、Runx2、MMP13和Sox9蛋白相对表达量。结果:①复方杜仲汤对终板软骨细胞活性影响的检测结果。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终板软骨细胞存活率均低于空白对照组。低、中、高剂量含药血清组细胞存活率均高于模型组和空白血清组。中、高剂量含药血清组细胞存活率均高于低剂量含药血清组。高剂量含药血清组细胞存活率高于中剂量含药血清组。②复方杜仲汤对终板软骨细胞氧化应激和炎症因子水平影响的检测结果。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终板软骨细胞中MDA、TNF-α、IL-1β和IL-6水平均高于空白对照组,SOD、CAT水平均低于空白对照组。低、中、高剂量含药血清组终板软骨细胞中MDA、TNF-α、IL-1β、IL-6水平均低于模型组,SOD、CAT水平均高于模型组和空白血清组。中、高剂量含药血清组终板软骨细胞中MDA、TNF-α、IL-1β和IL-6水平均低于低剂量含药血清组,SOD、CAT水平均高于低剂量含药血清组。高剂量含药血清组MDA、TNF-α、IL-1β和IL-6水平均低于中剂量含药血清组,SOD、CAT水平均高于中剂量含药血清组。③复方杜仲汤对终板软骨细胞中Keap1-Nrf2/ARE信号通路影响的检测结果。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终板软骨细胞中Keap1、p53、Runx2、MMP13的mRNA相对表达量和蛋白相对表达量均高于空白对照组,Nrf2、Sox9的mRNA相对表达量和蛋白相对表达量均低于空白对照组。低、中、高剂量含药血清组终板软骨细胞中Keap1、p53、Runx2、MMP13的mRNA相对表达量和蛋白相对表达量均低于模型组和空白血清组,Nrf2、Sox9的mRNA相对表达量和蛋白相对表达量均高于模型组和空白血清组。中、高剂量含药血清组终板软骨细胞中Keap1、p53、Runx2、MMP13的mRNA相对表达量和蛋白相对表达量均低于低剂量含药血清组,Nrf2、Sox9的mRNA相对表达量和蛋白相对表达量均高于低剂量含药血清组。高剂量含药血清组终板软骨细胞中Keap1、p53、Runx2、MMP13的mRNA相对表达量和蛋白相对表达量均低于中剂量含药血清组,Nrf2、Sox9的mRNA相对表达量和蛋白相对表达量均高于中剂量含药血清组。结论:复方杜仲汤可能通过调节Keap1-Nrf2/ARE信号通路相关基因的表达,提高终板软骨细胞的活性,降低细胞内氧化应激和炎症因子水平,从而起到干预终板软骨退变的作用,且其干预效果呈剂量依赖性。

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,meanwhile,the levels of malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT),tumor necrosis factor-α(TNF-α),IL-1β,and IL-6 in CEP chondrocytes were detected by using enzyme-linked immunosorbent assay(ELISA),and the relative mRNA and protein expression levels of Nrf2,Keap1,p53,runt-related transcription factor 2(Runx2),matrix metalloproteinase 13(MMP13),and sex-determining region of Y chromosome-box transcription factor 9(Sox9)in CEP chondrocytes were detected by using real-time quantitative PCR(RT-qPCR)and Western blotting,respectively.Results:①The survival rate of CEP chondrocytes was lower in model group,blank serum group,low-,medium-,and high-dose medicated serum group compared to blank control group,and it was lower in model group and blank serum group compared to low-,medium-,and high-dose medicated serum group,was lower in low-dose medicated serum group compared to medium-,and high-dose medicated serum group,and was lower in medium-dose medicated serum group compared to high-dose medicated serum group.②The levels of MDA,TNF-α,IL-1β,and IL-6 in CEP chondrocytes were higher,while the levels of SOD and CAT were lower in model group,blank serum group,low-,medium-,and high-dose medicated serum group compared to blank control group.The levels of MDA,TNF-α,IL-1β,and IL-6 in CEP chondrocytes were lower in low-,medium-,and high-dose medicated serum group compared to model group,while the levels of SOD and CAT were higher in low-,medium-,and high-dose medicated serum group compared to model group and blank serum group.The levels of MDA,TNF-α,IL-1β,and IL-6 in CEP chondrocytes were lower,while the levels of SOD and CAT were higher in medium-and high-dose medicated serum group compared to low-dose medicated serum group.The levels of MDA,TNF-α,IL-1β,and IL-6 in CEP chondrocytes were lower,while the levels of SOD and CAT were higher in high-dose medicated serum group compared to medium-dose medicated serum group.③The relative mRNA and protein expression levels of Keap1,p53,Runx2,and MMP13 in CEP chondrocytes were higher,while the relative mRNA and protein expression levels of Nrf2 and Sox9 were lower in model group,blank serum group,low-,medium-,and high-dose medicated serum group compared to blank control group.The relative mRNA and protein expression levels of Keap1,p53,Runx2,and MMP13 in CEP chondrocytes were lower,while the relative mRNA and protein expression levels of Nrf2 and Sox9 were higher in low-,medium-,and high-dose medicated serum group compared to model group and blank serum group.The relative mRNA and protein expression levels of Keap1,p53,Runx2,and MMP13 in CEP chondrocytes were lower,while the relative mRNA and protein expression levels of Nrf2 and Sox9 were higher in medium-and high-dose medicated serum group compared to low-dose medicated serum group.The relative mRNA and protein expression levels of Keap1,p53,Runx2,and MMP13 in CEP chondrocytes were lower,while the relative mRNA and protein expression levels of Nrf2 and Sox9 were higher in high-dose medicated serum group compared to medium-dose medicated serum group.Conclusion:FFDZT may enhance the viability of CEP chondrocytes and reduce the levels of intracellular OS and inflammatory factors by regulating the expression of Keap1-Nrf2/ARE signaling pathway-related genes.Therefore,it can intervene the degeneration of CEP,with a dose-dependence intervention effect.