TCDCA对佐剂性关节炎模型大鼠的治疗作用及其作用机制研究
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摘要
类风湿性关节炎(Rheumatoid arthritis, RA)是一种慢性自身免疫性疾病,伴随着复杂的炎症介质导致关节的损害、滑膜炎症、软骨和骨的损坏,导致人类和动物严重的机体功能障碍。尽管RA的病因及发病机制尚未完全明确,但其主要的病理过程环节基本清楚,即免疫功能失调、关节滑膜组织增殖增厚、软骨与骨破坏等三个方面。目前尚缺乏真正有效而副作用小的治疗药物,因此,寻找作用显著、副作用小的天然活性成分逐渐成为RA药物的研究热点。
牛磺鹅去氧胆酸(Taurochenodeoxycholic Acid, TCDCA)是动物胆汁酸中具有生物活性的主要有效成分之一,从动物胆汁酸中分离提取纯化的TCDCA对动物机体的急、慢性炎症均有显著的抑制作用；对机体的体液免疫、细胞免疫和吞噬功能均有显著的调节作用。在前期研究的基础上,本研究以佐剂性关节炎(adjuvant arthritis, AA)大鼠为对象,观察了具有较高抗炎免疫调节活性的TCDCA的抗类风湿性关节炎作用,同时从影响AA模型发展的关键细胞因子和核转录因于-κB (NF-κB)活性方面探讨了其治疗AA模型可能的药理作用机制,为将其开发为治疗RA的新药奠定实验基础。
本课题的主要研究内容包括以下五个方面：
(1)AA模型大鼠的建立与评价
通过比较常用关节炎动物模型的诱导方法,本研究优化筛选出了高成功率、临床症状典型、稳定的诱导AA模型的方法。通过关节炎动物模型的评价标准,本试验建立的AA模型大鼠,造模15d时模型大鼠的对侧脚踝关节、足跖部出现明显的肿胀、四肢爪子表面出现红斑、尾根明显肿胀结节,对侧脚的肿胀率达到25.15%,随着时间的延长对侧脚踝关节、足跖部肿胀率不断增大；造模30d时大鼠的四肢严重肿胀、尾巴出现明显结节,关节炎指数达到14.25,对侧脚的肿胀率达到48.09%,X-ray拍片显示骨骼之间界限模糊,骨密度降低,踝关节部和趾部软组织明显增厚。通过3次重复试验显示,本实验建立的AA模型较为稳定,成功率均在80%以上。
(2) TCDCA对AA模型大鼠的治疗作用
AA模型大鼠连续灌胃给药高低剂量TCDCA (0.2g/kg.b.w、0.1g/kg.b.w)14d后,根据模型评价标准对给药前后的关节炎动物模型进行了评价。结果：模型大鼠锐减的体重得到显著性的恢复,增重率分别由25.57%提高到44.41%、46.02%；模型大鼠四肢及尾根呈现的红斑、肿胀得到一定程度的缓解,关节炎指数分别由13.38降为10.44、10.03；模型大鼠对侧脚肿胀率分别由50.17%降为47.96%、45.83%。通过X-ray观察,模型大鼠两后肢踝关节和趾部骨骼的明显肿大,趾部各骨骼之间界限模糊,骨密度降低,踝关节部和趾部的软组织明显增厚等特征均得到一定程度的减轻。以上结果显示TCDCA对AA模型大鼠呈现出良好的治疗作用。
(3) TCDCA对AA模型大鼠体内关键细胞因子的影响
通过ELISA法和RT-PCR技术分别检测了AA模型大鼠血清和滑膜组织中细胞因子TNF-α、IL-1β、IL-6和IL-10蛋白和mRNA水平的表达。结果,分别灌胃给药AA模型大鼠高低剂量TCDCA (0.2g/kg.b.w,0.1g/kg.b.w)14d和31d后,TCDCA可显著的抑制AA大鼠血清中过度分泌的TNF-α、IL-1β、IL-6蛋白和滑膜组织中mRNA水平的表达(P<0.05)；高剂量(0.2g/kg.b.w) TCDCA连续给药31d后可显著的提高模型大鼠血清中IL-10的含量和滑膜组织mRNA水平的表达量(P<0.05)。
(4) TCDCA对AA模型大鼠成纤维样滑膜细胞(FLS)和腹腔巨噬细胞(PMΦ)中关键细胞因子的影响
采用组织块培养法获得形态典型、单一的FLS细胞。采用ELISA法检测了AA模型大鼠FLS细胞上清液中细胞因子TNF-α、IL-1β、IL-6和IL-10蛋白的含量,同时采用RT-PCR法测定了相应细胞因子mRNA水平表达的变化。结果：TCDCA (300μg/mL、400μg/mL、500μg/mL)均可显著的抑制AA模型FLS细胞上清液中TNF-α、IL-1β、IL-6蛋白的含量(P<0.05),而对IL-10蛋白则无显著性的影响(P>0.05); TCDCA (300μg/mL、400μg/mL、500μg/mL)可显著抑制FLS细胞中TNF-α、IL-1β和IL-6基因的表达量(P<0.05), TCDCA (500μg/mL)显著上调IL-10基因的表达量(P<0.05)。
采用PBS腹腔灌洗法获得高纯度的PMΦ细胞；采用ELISA法测定了AA大鼠PMΦ细胞上清液中细胞因子TNF-α、IL-1β、IL-6和IL-10蛋白的含量；采用RT-PCR技术检测了相应细胞因子mRNA水平表达的变化。结果：TCDCA (150μg/mL、180μg/mL、200μg/mL)可剂量依赖性的显著抑制AA模型PMq)细胞上清液中TNF-α、IL-1β、IL-6蛋白的含量(P<0.05), TCDCA(150μg/mL、180μg/mL)显著提高IL-10蛋白的含量(P<0.05)。TCDCA (150μg/mL、180μg/mL、200μg/mL)可剂量依赖性的显著抑制AA模型PMΦ细胞中TNF-α、IL-1β、IL-6基因的表达量(P<0.05),而对IL-10基因的表达量则无显著性的影响(P>0.05)。
(5) TCDCA对AA模型大鼠FLS和PMΦ中NF-κB活性表达的影响
通过ELISA法和Western-blot法分别检测了FLS和PMΦ细胞核内NF-κB p65蛋白和细胞内IκBα蛋白的表达量。结果：TCDCA (300μg/mL、400μg/mL500μg/mL)对FLS细胞中活化的NF-κB p65蛋白的表达呈现出剂量依赖性的抑制作用,并显著的提高细胞内IκBα蛋白的表达和抑制磷酸化IκBα蛋白的表达；TCDCA (150μg/mL、180μg/mL、200μg/mL)对PMΦ细胞中活化的NF-κB p65蛋白的表达呈现出剂量依赖性的抑制作用,并显著性提高细胞内IκBα蛋白的表达和TCDCA (200μg/mL)显著抑制磷酸化IκBα蛋白的表达。
总体结论：
(1)通过完全弗氏佐剂成功建立了临床症状典型、稳定的AA大鼠模型,建模成功率达到80%以上；
(2) TCDCA对AA模型大鼠呈现出良好的治疗作用；
(3) TCDCA通过抑制AA模型大鼠FLS和PMΦ中NF-κB的活性,进而降低大鼠血清和滑膜组织中、FLS和PMΦ中TNF-α、IL-1β、IL-6蛋白和mRNA水平的表达,进而发挥其良好的抗关节炎作用。
Rheumatoid arthritis (RA) is a chronic autoimmune disease associated with multiple inflammatory mediators that lead to joint damage, synovial inflammation and cartilage and bone damage, which leads to serious dysfunction. Although the etiology and pathogenesis of RA have not been completely cleared, numerous studies have confirmed that immune function disorder, Synovial tissue of joint hyperblastosis, and articular cartilage and bone damage are key pathologic processes in RA development. Although there are a few anti-rheumatic drugs showing effectiveness on treating RA, their side effects and toxicity call for new and more effective natural drugs.
Taurochenodeoxycholic Acid (TCDCA) is one of main bioactive substances of bile from animal bile and our laboratory successfully obtain simple substance with high purity from chicken bile acid. In previous stuties, TCDCA showed remarkably inhibition on both acute and chronic inflammation and confered good regulation in humoral immunity, cytoimmunity and phagocytosis of macrophages. Based on the immunological feature of TCDCA, we examined whether intragastric administration of TCDCA has anti-arthritis effects on adjuvant arthritis model in rat. In order to clarify the mechanism of anti-arthritis action of TCDCA, we also investigated its effects on key cytokines and nuclear factor-κB (NF-κB) in RA development progress.
This sutdies included five aspects as followed:
(1) Induction of AA model and arthritis evalution
Compare to usually inducing methods of AA model, this study sucessfully optimized and screened out a better method, which induced model showed high achievement ratio, stable and typical clinical symptoms. After adopted evaluation criterion of arthritis model, the AA model which induced by the new method, showed followed characteristics:non-injected hind limb ankle and voix pedis became severely red and edematous and its swelling ration was up to25.15%, there were erythema in four limbs and nodules in tail at days15after immunization; there were serious swelling and distension in the four limbs and obvious nodules in tail, arthritis index was up to14.25and non-injected hind limb swelling ration was up to48.09%, X-ray results showed that there appeared unclear dividing line between bones, bone mineral densities, and parenchyma swelling at days30after immunization. Repeated Experiments results showed that the inducing AA model had stable characteristics and its achievement ratio was reached to80%.
(2) Therapeutic action of TCDCA on AA model rats.
AA rats were administrated with TCDCA (0.1g/kg and0.2g/kg) at days14after immunization, respectively. Evaluted by model evaluation criterion, there were found that the decimated weight of AA rats was remarkably recovered, and the growth rate of was from25.57%up to44.41%and46.02%; The erythema, edematous and nodules was obviously relieveed in four limbs and tail, arthritis index was from13.38down to10.44and10.03, and non-injected hind limb swelling ration was from50.17%down to47.96%and45.83%; X-ray results showed that TCDCA could well ameliorated unclear dividing line between bones, bone mineral densities, and parenchyma swelling. TCDCA confered good anti-adjuvant arthritis activity in rats.
(3) Effects of TCDCA on the key cytokines of AA rats in vivo.
The contents and mRNA expression of TNF-α、IL-1β、IL-6和IL-10were detected by ELISA and RT-PCR in the serum and synovium tissue of AA rats, respectively. These results showed that TCDCA (0.2g/kg.b.w,0.1g/kg.b.w) confered remarkably inhibition in the protein and mRNA expression of TNF-α、IL-1β and IL-6(P<0.05); TCDCA(0.2g/kg.b.w) remarkably increaed the lower content and mRNA expression of IL-10in serum and synovium tissue with continuing treated for31d.
(4) Effects of TCDCA on the key cytokines of FLS and PMΦ from AA rats
FLS was cultured by adopted tissue mass cell culture method and obtained FLS showed typical fibroblast-like shape and high purity. The contents and mRNA expression of TNF-α、IL-1β、IL-6and IL-10were detected by ELISA and RT-PCR in the cell supernate and FLS of AA rats, respectively. The concentration of TNF-α, IL-1β and IL-6were markedly decreased by TCDCA (300μg/mL,400μg/mL and500μg/mL) on a concentration-dependent manner. However, there was not markedly influence on IL-10secretion in AA synoviocytes with TCDCA (300μg/mL,400μg/mL and500μg/mL). mRNA expression levels of TNF-α, IL-1β and IL-6were markedly inhibited by TCDCA (300μg/mL,400μg/mL and500μg/mL), meanwhile, TCDCA at concentration of400μg/mL and500μg/mL markedly increased mRNA expression level of IL-10in the synoviocytes of AA rats
PMΦ was obtained by PBS peritoneal lavage mehod. The contents and mRNA expression of TNF-α、IL-1β、IL-6和IL-10were detected by ELISA and RT-PCR in the cell supernate and PMφ of AA rats, respectively. The concentration of TNF-a, IL-1β and IL-6were markedly decreased by TCDCA (150μg/mL,180μg/mL and200μg/mL) on a concentration-dependent manner(P<0.05), and TCDCA(150μg/mL and180μg/mL) showed remarkable augment on the concentration of IL-10(P<0.05). Meanwhile, mRNA expression levels of TNF-α, IL-1β and IL-6were markedly inhibited by TCDCA (150ug/mL,180μg/mL and200μg/mL) in PMφ of AA rats.
(5) Effects of TCDCA on the NF-κB p65activation and expression in the FLS and PMφ from AA rats.
The protein expression of activated NF-κB p65and IκBα was detected by ELISA and Western-blot. It was founded that TCDCA (300μg/mL,400μg/mL and500μg/mL) showed remarkably inhibition in the activated NF-κB p65and phosphorylated IκBα protein expression, and confered augment on the non-phosphorylated IκBα protein in FLS on a concentration-dependent manner; TCDCA (150ug/mL,180μg/mL and200μg/mL) showed remarkably inhibition in the activated NF-κB p65and augment on the non-phosphorylated IκBα protein expression on a concentration-dependent manner, and TCDCA (200μg/mL) remarkably inhibited the phosphorylated IκBα protein expression in PMφ.
Conculsions:
(1) AA rats with typical and stable clinical symptoms was induced and its achievement ratio was up to80%.
(2) TCDCA confered good anti-adjuvant arthritis activity in rats.
(3) Anti-arthritis activity of TCDCA could be mediated via that TCDCA directly inhibited the activity of NF-κB p65in FLS and PMφ, and then reduced the protein and mRNA expression of TNF-α、IL-1β and IL-6in AA rats.
牛磺鹅去氧胆酸(Taurochenodeoxycholic Acid, TCDCA)是动物胆汁酸中具有生物活性的主要有效成分之一,从动物胆汁酸中分离提取纯化的TCDCA对动物机体的急、慢性炎症均有显著的抑制作用；对机体的体液免疫、细胞免疫和吞噬功能均有显著的调节作用。在前期研究的基础上,本研究以佐剂性关节炎(adjuvant arthritis, AA)大鼠为对象,观察了具有较高抗炎免疫调节活性的TCDCA的抗类风湿性关节炎作用,同时从影响AA模型发展的关键细胞因子和核转录因于-κB (NF-κB)活性方面探讨了其治疗AA模型可能的药理作用机制,为将其开发为治疗RA的新药奠定实验基础。
本课题的主要研究内容包括以下五个方面：
(1)AA模型大鼠的建立与评价
通过比较常用关节炎动物模型的诱导方法,本研究优化筛选出了高成功率、临床症状典型、稳定的诱导AA模型的方法。通过关节炎动物模型的评价标准,本试验建立的AA模型大鼠,造模15d时模型大鼠的对侧脚踝关节、足跖部出现明显的肿胀、四肢爪子表面出现红斑、尾根明显肿胀结节,对侧脚的肿胀率达到25.15%,随着时间的延长对侧脚踝关节、足跖部肿胀率不断增大；造模30d时大鼠的四肢严重肿胀、尾巴出现明显结节,关节炎指数达到14.25,对侧脚的肿胀率达到48.09%,X-ray拍片显示骨骼之间界限模糊,骨密度降低,踝关节部和趾部软组织明显增厚。通过3次重复试验显示,本实验建立的AA模型较为稳定,成功率均在80%以上。
(2) TCDCA对AA模型大鼠的治疗作用
AA模型大鼠连续灌胃给药高低剂量TCDCA (0.2g/kg.b.w、0.1g/kg.b.w)14d后,根据模型评价标准对给药前后的关节炎动物模型进行了评价。结果：模型大鼠锐减的体重得到显著性的恢复,增重率分别由25.57%提高到44.41%、46.02%；模型大鼠四肢及尾根呈现的红斑、肿胀得到一定程度的缓解,关节炎指数分别由13.38降为10.44、10.03；模型大鼠对侧脚肿胀率分别由50.17%降为47.96%、45.83%。通过X-ray观察,模型大鼠两后肢踝关节和趾部骨骼的明显肿大,趾部各骨骼之间界限模糊,骨密度降低,踝关节部和趾部的软组织明显增厚等特征均得到一定程度的减轻。以上结果显示TCDCA对AA模型大鼠呈现出良好的治疗作用。
(3) TCDCA对AA模型大鼠体内关键细胞因子的影响
通过ELISA法和RT-PCR技术分别检测了AA模型大鼠血清和滑膜组织中细胞因子TNF-α、IL-1β、IL-6和IL-10蛋白和mRNA水平的表达。结果,分别灌胃给药AA模型大鼠高低剂量TCDCA (0.2g/kg.b.w,0.1g/kg.b.w)14d和31d后,TCDCA可显著的抑制AA大鼠血清中过度分泌的TNF-α、IL-1β、IL-6蛋白和滑膜组织中mRNA水平的表达(P<0.05)；高剂量(0.2g/kg.b.w) TCDCA连续给药31d后可显著的提高模型大鼠血清中IL-10的含量和滑膜组织mRNA水平的表达量(P<0.05)。
(4) TCDCA对AA模型大鼠成纤维样滑膜细胞(FLS)和腹腔巨噬细胞(PMΦ)中关键细胞因子的影响
采用组织块培养法获得形态典型、单一的FLS细胞。采用ELISA法检测了AA模型大鼠FLS细胞上清液中细胞因子TNF-α、IL-1β、IL-6和IL-10蛋白的含量,同时采用RT-PCR法测定了相应细胞因子mRNA水平表达的变化。结果：TCDCA (300μg/mL、400μg/mL、500μg/mL)均可显著的抑制AA模型FLS细胞上清液中TNF-α、IL-1β、IL-6蛋白的含量(P<0.05),而对IL-10蛋白则无显著性的影响(P>0.05); TCDCA (300μg/mL、400μg/mL、500μg/mL)可显著抑制FLS细胞中TNF-α、IL-1β和IL-6基因的表达量(P<0.05), TCDCA (500μg/mL)显著上调IL-10基因的表达量(P<0.05)。
采用PBS腹腔灌洗法获得高纯度的PMΦ细胞；采用ELISA法测定了AA大鼠PMΦ细胞上清液中细胞因子TNF-α、IL-1β、IL-6和IL-10蛋白的含量；采用RT-PCR技术检测了相应细胞因子mRNA水平表达的变化。结果：TCDCA (150μg/mL、180μg/mL、200μg/mL)可剂量依赖性的显著抑制AA模型PMq)细胞上清液中TNF-α、IL-1β、IL-6蛋白的含量(P<0.05), TCDCA(150μg/mL、180μg/mL)显著提高IL-10蛋白的含量(P<0.05)。TCDCA (150μg/mL、180μg/mL、200μg/mL)可剂量依赖性的显著抑制AA模型PMΦ细胞中TNF-α、IL-1β、IL-6基因的表达量(P<0.05),而对IL-10基因的表达量则无显著性的影响(P>0.05)。
(5) TCDCA对AA模型大鼠FLS和PMΦ中NF-κB活性表达的影响
通过ELISA法和Western-blot法分别检测了FLS和PMΦ细胞核内NF-κB p65蛋白和细胞内IκBα蛋白的表达量。结果：TCDCA (300μg/mL、400μg/mL500μg/mL)对FLS细胞中活化的NF-κB p65蛋白的表达呈现出剂量依赖性的抑制作用,并显著的提高细胞内IκBα蛋白的表达和抑制磷酸化IκBα蛋白的表达；TCDCA (150μg/mL、180μg/mL、200μg/mL)对PMΦ细胞中活化的NF-κB p65蛋白的表达呈现出剂量依赖性的抑制作用,并显著性提高细胞内IκBα蛋白的表达和TCDCA (200μg/mL)显著抑制磷酸化IκBα蛋白的表达。
总体结论：
(1)通过完全弗氏佐剂成功建立了临床症状典型、稳定的AA大鼠模型,建模成功率达到80%以上；
(2) TCDCA对AA模型大鼠呈现出良好的治疗作用；
(3) TCDCA通过抑制AA模型大鼠FLS和PMΦ中NF-κB的活性,进而降低大鼠血清和滑膜组织中、FLS和PMΦ中TNF-α、IL-1β、IL-6蛋白和mRNA水平的表达,进而发挥其良好的抗关节炎作用。
Rheumatoid arthritis (RA) is a chronic autoimmune disease associated with multiple inflammatory mediators that lead to joint damage, synovial inflammation and cartilage and bone damage, which leads to serious dysfunction. Although the etiology and pathogenesis of RA have not been completely cleared, numerous studies have confirmed that immune function disorder, Synovial tissue of joint hyperblastosis, and articular cartilage and bone damage are key pathologic processes in RA development. Although there are a few anti-rheumatic drugs showing effectiveness on treating RA, their side effects and toxicity call for new and more effective natural drugs.
Taurochenodeoxycholic Acid (TCDCA) is one of main bioactive substances of bile from animal bile and our laboratory successfully obtain simple substance with high purity from chicken bile acid. In previous stuties, TCDCA showed remarkably inhibition on both acute and chronic inflammation and confered good regulation in humoral immunity, cytoimmunity and phagocytosis of macrophages. Based on the immunological feature of TCDCA, we examined whether intragastric administration of TCDCA has anti-arthritis effects on adjuvant arthritis model in rat. In order to clarify the mechanism of anti-arthritis action of TCDCA, we also investigated its effects on key cytokines and nuclear factor-κB (NF-κB) in RA development progress.
This sutdies included five aspects as followed:
(1) Induction of AA model and arthritis evalution
Compare to usually inducing methods of AA model, this study sucessfully optimized and screened out a better method, which induced model showed high achievement ratio, stable and typical clinical symptoms. After adopted evaluation criterion of arthritis model, the AA model which induced by the new method, showed followed characteristics:non-injected hind limb ankle and voix pedis became severely red and edematous and its swelling ration was up to25.15%, there were erythema in four limbs and nodules in tail at days15after immunization; there were serious swelling and distension in the four limbs and obvious nodules in tail, arthritis index was up to14.25and non-injected hind limb swelling ration was up to48.09%, X-ray results showed that there appeared unclear dividing line between bones, bone mineral densities, and parenchyma swelling at days30after immunization. Repeated Experiments results showed that the inducing AA model had stable characteristics and its achievement ratio was reached to80%.
(2) Therapeutic action of TCDCA on AA model rats.
AA rats were administrated with TCDCA (0.1g/kg and0.2g/kg) at days14after immunization, respectively. Evaluted by model evaluation criterion, there were found that the decimated weight of AA rats was remarkably recovered, and the growth rate of was from25.57%up to44.41%and46.02%; The erythema, edematous and nodules was obviously relieveed in four limbs and tail, arthritis index was from13.38down to10.44and10.03, and non-injected hind limb swelling ration was from50.17%down to47.96%and45.83%; X-ray results showed that TCDCA could well ameliorated unclear dividing line between bones, bone mineral densities, and parenchyma swelling. TCDCA confered good anti-adjuvant arthritis activity in rats.
(3) Effects of TCDCA on the key cytokines of AA rats in vivo.
The contents and mRNA expression of TNF-α、IL-1β、IL-6和IL-10were detected by ELISA and RT-PCR in the serum and synovium tissue of AA rats, respectively. These results showed that TCDCA (0.2g/kg.b.w,0.1g/kg.b.w) confered remarkably inhibition in the protein and mRNA expression of TNF-α、IL-1β and IL-6(P<0.05); TCDCA(0.2g/kg.b.w) remarkably increaed the lower content and mRNA expression of IL-10in serum and synovium tissue with continuing treated for31d.
(4) Effects of TCDCA on the key cytokines of FLS and PMΦ from AA rats
FLS was cultured by adopted tissue mass cell culture method and obtained FLS showed typical fibroblast-like shape and high purity. The contents and mRNA expression of TNF-α、IL-1β、IL-6and IL-10were detected by ELISA and RT-PCR in the cell supernate and FLS of AA rats, respectively. The concentration of TNF-α, IL-1β and IL-6were markedly decreased by TCDCA (300μg/mL,400μg/mL and500μg/mL) on a concentration-dependent manner. However, there was not markedly influence on IL-10secretion in AA synoviocytes with TCDCA (300μg/mL,400μg/mL and500μg/mL). mRNA expression levels of TNF-α, IL-1β and IL-6were markedly inhibited by TCDCA (300μg/mL,400μg/mL and500μg/mL), meanwhile, TCDCA at concentration of400μg/mL and500μg/mL markedly increased mRNA expression level of IL-10in the synoviocytes of AA rats
PMΦ was obtained by PBS peritoneal lavage mehod. The contents and mRNA expression of TNF-α、IL-1β、IL-6和IL-10were detected by ELISA and RT-PCR in the cell supernate and PMφ of AA rats, respectively. The concentration of TNF-a, IL-1β and IL-6were markedly decreased by TCDCA (150μg/mL,180μg/mL and200μg/mL) on a concentration-dependent manner(P<0.05), and TCDCA(150μg/mL and180μg/mL) showed remarkable augment on the concentration of IL-10(P<0.05). Meanwhile, mRNA expression levels of TNF-α, IL-1β and IL-6were markedly inhibited by TCDCA (150ug/mL,180μg/mL and200μg/mL) in PMφ of AA rats.
(5) Effects of TCDCA on the NF-κB p65activation and expression in the FLS and PMφ from AA rats.
The protein expression of activated NF-κB p65and IκBα was detected by ELISA and Western-blot. It was founded that TCDCA (300μg/mL,400μg/mL and500μg/mL) showed remarkably inhibition in the activated NF-κB p65and phosphorylated IκBα protein expression, and confered augment on the non-phosphorylated IκBα protein in FLS on a concentration-dependent manner; TCDCA (150ug/mL,180μg/mL and200μg/mL) showed remarkably inhibition in the activated NF-κB p65and augment on the non-phosphorylated IκBα protein expression on a concentration-dependent manner, and TCDCA (200μg/mL) remarkably inhibited the phosphorylated IκBα protein expression in PMφ.
Conculsions:
(1) AA rats with typical and stable clinical symptoms was induced and its achievement ratio was up to80%.
(2) TCDCA confered good anti-adjuvant arthritis activity in rats.
(3) Anti-arthritis activity of TCDCA could be mediated via that TCDCA directly inhibited the activity of NF-κB p65in FLS and PMφ, and then reduced the protein and mRNA expression of TNF-α、IL-1β and IL-6in AA rats.
引文
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