基于NF-κB通路探讨杜仲皮、叶醇提物对胶原诱导型关节炎大鼠炎症性骨破坏的影响

目的研究杜仲皮、叶醇提物对胶原诱导型关节炎(collagen-induced arthritis,CIA)大鼠骨破坏的治疗作用及机制。方法随机选取6只雌性Wistar大鼠作为对照组,其余大鼠于背部多点及尾根部sc 0.1 mL牛Ⅱ型胶原乳剂建立CIA模型,第14天将CIA大鼠随机分为模型组,杜仲皮醇提物低、高剂量(2、4 g/kg)组,杜仲叶醇提物低、高剂量(2、4 g/kg)组及甲氨蝶呤(1 mg/kg)组,各给药组ig相应药物,1次/d,连续4周。观察各组大鼠足肿胀情况;采用苏木精-伊红(HE)染色法观察各组大鼠踝关节病理变化;采用ELISA法检测各组大鼠血清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)水平;采用qRT-PCR法检测各组大鼠脾脏中炎性因子如TNF-α、TNF受体相关因子-6(TNF receptor-associated factor-6,TRAF-6)、IL-1β、IL-17、IL-1βmRNA表达,膝关节组织中破骨细胞标志物如抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)、活化T细胞的核因子c1(nuclear factor of activated T cells cytoplasmic 1,NFATc1)、组织蛋白酶K(cathepsin K,CTSK)、原癌基因c-Fos、核转录因子-κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)mRNA表达,膝关节组织中血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)、缺氧诱导因子-1(hypoxiainduciblefactor-1,HIF-1)mRNA表达及成骨细胞标志物如金属蛋白酶组织抑制剂1(tissueinhibitor1of metalloproteinases,TIMP1)、骨钙素(osteocalcin,OCN)、骨保护素(osteoprotegerin,OPG)mRNA表达;采用Western blotting法检测各组大鼠膝关节组织中磷酸化NF-κB抑制蛋白激酶α/β(phosphorylated inhibitor of NF-κB kinaseα/β,p-Iκκα/β)、磷酸化NF-κB抑制蛋白α(phosphorylated inhibitorαof NF-κB,p-IκBα)、磷酸化p65(p-p65)蛋白表达情况。结果与模型组比较,杜仲皮、叶醇提物可显著降低CIA大鼠足肿胀度(P<0.05、0.01);改善踝关节组织病理;显著降低CIA大鼠血清中TNF-α、IL-6水平(P<0.01);显著降低CIA大鼠脾脏中TNF-α、TRAF-6、IL-1β、IL-17 mRNA表达水平(P<0.05、0.01),显著降低CIA大鼠膝关节组织中TRAP、NFATc1、CTSK、c-Fos、RANKL、VEGF、HIF-1 mRNA表达水平(P<0.01),显著升高CIA大鼠膝关节组织中TIMP1、OCN、OPG mRNA表达水平(P<0.05、0.01),显著降低CIA大鼠膝关节组织中p-Iκκα/β、p-IκBα、p-p65蛋白表达水平(P<0.01)。结论杜仲皮、叶醇提物可以减轻CIA大鼠关节炎症,抑制促血管生成因子的表达,延缓关节内软骨与骨的破坏,其机制与调控NF-κB通路相关的炎症性骨代谢有关。

Effects of ethanol extracts of barks and leaves from Eucommia ulmoides on inflammatory bone destruction in collagen-induced arthritis rats based on NF-κB pathway

Objective To study the therapeutic effect and mechanism of alcohol extract of barks and leaves from Duzhong(Eucommia ulmoides)on bone destruction of collagen-induced arthritis(CIA)rats.Methods Six female Wistar rats were randomly selected as control group,the rest of rats were sc 0.1 mL bovine typeⅡcollagen emulsion at multiple points on back and tail to establish CIA model.On 14 th day,CIA rats were randomly divided into model group,alcohol extract of barks from E.ulmoides low-and high-dose(2 and 4 g/kg)groups,alcohol extract of leaves from E.ulmoides low-and high-dose(2 and 4 g/kg)groups,and methotrexate(1 mg/kg)group,rats were ig corresponding drugs,once a day for 4 weeks.Foot swelling situation of each group was observed.Hematoxylin-eosin(HE)staining method was used to observe the pathological changes of ankle joint in each group of rats.Levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in serum were detected by ELISA.qRT-PCR was used to detect mRNA levels of inflammatory factors such as TNF-α,TNF receptor related factor-6(TRAF-6),IL-1β,IL-17,and IL-1βin spleen,osteoclast markers such as tartrate resistant acid phosphatase(TRAP),nuclear factor of activated T cells cytoplasmic 1(NFATc1),cathepsin K(CTSK),proto-oncogene c-Fos,and receptor activator of NF-κB ligand(RANKL)mRNA expressions,vascular endothelial growth factor(VEGF),and hypoxia inducible factor-1(HIF-1)mRNA expressions,and osteoblast markers such as tissue inhibitor 1 of metalloproteinases(TIMP1),osteocalcin(OCN)and osteoprotegerin(OPG)mRNA expressions in knee joints;Western blotting was used to detect expressions of phosphorylated inhibitory of NF-κB kinaseα/β(p-Iκκα/β),phosphorylation inhibitorαof NF-κB(p-IκBα),phosphorylated p65(p-p65)in knee joints.Results Compared with model group,swelling of feet was significantly reduced(P<0.05,0.01),pathology of ankle joint was improved,levels of TNF-αand IL-6 in serum were significantly reduced(P<0.01),TNF-α,TRAF-6,IL-1β,IL-17 mRNA levels in spleen were significantly reduced(P<0.05,0.01),TRAP,NFATc1,CTSK,c-Fos,RANKL,VEGF,HIF-1 mRNA levels in knee joints were significantly reduced(P<0.01),TIMP1,OCN,OPG mRNA levels in knee joints were significantly increased(P<0.05,0.01),expressions of p-Iκκα/β,p-IκBα,and p-p65 in knee joints were significantly reduced(P<0.01)in alcohol extract of barks and leaves from E.ulmoides groups.Conclusion Barks and leaves from E.ulmoides can relieve joint inflammation in CIA rats,inhibit expressions of pro-angiogenic factors,and delay the destruction of cartilage and bone in the joints,of which mechanism is related to the regulation of inflammatory bone metabolism related to NF-κB pathway.