【作者】 敖明章；

【导师】 余龙江；

【作者基本信息】 华中科技大学 ， 生物制药工程， 2012， 博士

【摘要】 类风湿性关节炎(RA)是一种慢性、炎性、系统性的自身免疫性疾病,以关节和关节周围组织非化脓性炎症为主要特征,成为继缺血性心脏病后导致工作能力丧失的第二位疾病,因其高度致残率WHO将其列为重点关注和治疗的疾病之一。传统的治疗RA的药物大多作用途径或靶点单一,同时有一定的毒副作用,因此开发高效、低毒、多靶点作用的新药就显得尤为重要和迫切。竹节参(Panax japonicum C.A.Mey.)五加科植物,中医记载其具有滋补强壮、散瘀止痛、止血、祛痰功能。用于病后虚弱、肺结核咯血、咳嗽痰多、跌打损伤。本实验将系统开展其抗炎抗风湿作用及机理研究。1) TSPJ对小鼠常规的炎症反应的影响：(1) TSPJ对角叉菜胶所致小鼠足肿胀具有抑制作用,TSPJ中高剂量均能明显减轻角叉菜胶所致小鼠右后足的红、肿等症状,50 mg·kg-1 TSPJ组在致炎后1-3 h,100 mg·kg-1 TSPJ组在致炎后0.5-4 h减轻足肿胀,与control组相比,有明显差异(P<0.05,P<0.01)。(2)TSPJ对巴豆油所致小鼠耳肿胀具有抑制作用,TSPJ高中组均能明显减轻巴豆油所致小鼠右耳的红、肿等症状,显著抑制巴豆油所致的小鼠耳肿胀,100mg·k-1 TSPJ组的抑制率可达33.33%,作用强度与剂量呈正相关,与control组比较,差异有显著性(P<0.05,P<0.01)。(3)TSPJ对冰醋酸所致小鼠腹腔毛细血管通透性增高有抑制作用,各组均能明显抑制冰醋酸所致小鼠腹腔毛细血管通透性的增高,100 mg·kg-1 TSPJ组的抑制率可达52.02%,作用强度与剂量呈正相关,与control组比较,差异有显著性(P<0.05,P<0.01)。(4) TSPJ具有提高血清中GSH-PX、SOD活性及降低MDA的作用,TSPJ各组血清GSH-PX, SOD活性增高,血清MDA含量下降,与control组比较,差异具有显著性(P<0.05)。(5) TSPJ使炎症小鼠血清中IL-1, TNF-α含量下降,TSPJ中高剂量组小鼠血清IL-1水平下降(P<0.05), TSPJ高剂量组小鼠血清TNF-α水平下降,差异性显著(P<0.01)。2) TSPJ对CIA大鼠组织病理学及超微结构改变的影响(1) TSPJ各剂量组AI评分也随着继发性炎症的产生而升高,但与同期的模型组比较有显著性差异,提示竹节参总皂苷具有抗CIA的作用,且具有量效关系,但治疗效果比雷公藤治疗组稍差。(2)模型组大鼠致炎后d12起的足爪开始肿胀,d24-d28达到高峰,至d36仍有一定程度的肿胀。TSPJ各剂量组肿胀程度也随着继发性炎症的产生而增加,但与同期的模型组比较有显著性差异,提示TSPJ具有抗CIA的作用,且具有量效关系,但治疗效果比雷公藤治疗组稍差。(3)CIA模型组进入继发性炎症阶段(d18以后)体重增长速度减慢或停止,TPT治疗组体重增长速度也出现减慢现象,这可能是TPT在治疗关节炎的同时产生了一定的胃肠道刺激导致大鼠进食量下降所致,TSPJ各组体重增长速度基本正常。(4)CIA模型可导致机体免疫功能亢进；给药组大鼠的脾脏指数、胸腺指数与模型组比较均有下降,其中TPT组差异具有显著性(p<0.05),其余组有下降趋势但无明显差异,表明TSPJ具有使亢进的免疫功能恢复正常的作用。(5) TSPJ具有改善CIA关节病变作用：TSPJ各剂量组均能不同程度的减轻关节软组织的充血水肿,足趾容积及关节评分均低于模型组；减轻滑膜充血、水肿的程度；明显抑制CIA大鼠滑膜增生；抑制炎性细胞浸润；抑制大鼠软骨细胞增生。(6) ConA诱导的CIA大鼠脾脏T淋巴细胞增殖反应明显低于正常对照组(P<0.01),产生的IL-2水平明显降低,TSPJ给药可以恢复CIA大鼠脾淋巴细胞功能,TPT对CIA大鼠低下的脾淋巴细胞增殖反应及产生IL-2没有明显影响。(7)CIA组PMΦIL-1β、IL-6和TNF-α等致炎细胞因子的表达量均明显高于正常组(P<0.01),表明在CIA模型产生过程中,各种致炎因子大量产生参与炎症反应。TSPJ大中剂量能明显抑制PMφ产生致炎细胞因子IL-1β、IL-6和TNF-α水平。(8)CIA滑膜超微结构显示：滑膜层细胞内膜明显增厚,多达5-6层,滑膜细胞增生肥大,排列紊乱。细胞器变形,甚至消失,胞质内充满大量空泡,胞核染色质分布不均。有的细胞因变性、坏死至萎缩,仅见到细胞碎块与大量胶原纤维交错排列。TSPJ可明显改善CIA滑膜超微结构,滑膜层细胞性内膜无增厚,细胞排列均匀,偶见少量淋巴细胞浸润,巨噬细胞型细胞以初级溶酶体多见、线粒体稍增多,成纤维细胞型细胞的胞浆内富含粗面内质网及核糖小体,各型细胞接近正常。(9)CIA模型组关节滑膜在炎症过程中,ICAM-1产量(阳性细胞总数、平均黑度值、面积总和、积分光密度值)明显增加,TSPJ各剂量组关节滑膜ICAM-1表达数目、面积总和、积分光密度值及平均黑度值均低于模型对照组,且有着性差异(P<0.05或P<0.01)。(10)正常以及CIA滑膜细胞培养和细胞因子的研究表明：正常滑膜细胞在未受到刺激时并无细胞因子如IL-1、IL-6、IL-8和TNF-α分泌,细胞增长速度正常；当正常滑膜细胞接受LPS刺激后,细胞因子如IL-1、IL-6、IL-8和TNF-α分泌量增加,细胞增殖速度加快,表明LPS是一种良好微生物抗原刺激剂,可以诱导正常细胞的增殖分化。CIA滑膜细胞未受刺激也会有少量细胞因子分泌,当受到LPS刺激后,细胞增殖速度明显加快,细胞因子分泌量成倍或数十倍增加,表明CIA滑膜细胞本身已经处于一种敏感状态,对外来抗原刺激极为敏感,诱发炎症反应的恶性循环。雷公藤显示出强大的抗炎作用；TSPJ竹节参在滑膜细胞培养试验中未观察到细胞损伤现象,并无刺激细胞因子分泌作用,仅使处于“高敏”的CIA滑膜细胞状态恢复正常。从本实验结果来看,竹节参具有以下作用：(1)提高机体抗氧化能力,减少自由基对炎症组织的损伤；(2)具有减轻炎症组织中炎症介质PGE和LTB的作用；(3)使炎症组织中细胞因子如IL-1B、IL-6、TNF-α等明显减少；(4)减轻炎症组织中ICAM-1的产生,使炎症部位不产生粘连；(5)对免疫系统具有“双向调节作用”,在治疗过程中使亢进的免疫功能下调至正常,不会产生类似雷公藤和其他免疫抑制剂导致的免疫功能减退。因此,竹节参极有可能开发成为一种有效、无毒或低毒的抗炎抗风湿的新药。更多还原

【Abstract】 Rheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmune disease, which joints and joint’s surrounding tissue non-suppurative inflammations are its main features. It becomes the second cause of loss ability to work behind the ischemic heart disease. The traditional treatment of RA drugs are mostly single pathway or target, while there are certain side effects, so development of efficient, low toxicity, the role of multi-target drug is particularly important and urgent.Panax japonicus (Panax japonicum CA Mey.),a traditional chinese medical herb, belongs to Araliaceae. It has strong tonic, dissipate blood stasis and alleviate pain, bleeding and expectorant functions in Chinese medicine record.So it used totreated weakness after diseases, tuberculosis hemoptysis, cough, sputum, and bruises. In this study, its anti-inflammatory effects and anti-rheumatic mechanisms will be carried out syetemic researched.1) The effect of TSPJ on normal inflammatory response in mice:(1) TSPJ 50 mg/kg and 100 mg/kg group could significantly reduce the paw edema, foot redness swelling and other symptoms induced by carrageenan after inflammation 0.5～4 h (P<0.05)Vs control group.(2) TSPJ could inhibit the mouse ear edema induced by croton oil, the ear’s redness, swelling and other symptoms are significantly inhibited.The high dose group of TSPJ’s inhibition rate is 33.33% compared with the control group. The inhibition rate is positively correlated with the dose (P<0.05).(3) TSPJ could inhibit the increased capillary permeability induced by acetic acid on mice inhibition rate of TSPJ’s high dose group(100 mg/kg) is 48.6% compared with the control group. The inhibition rate is positively correlated with the dose (P<0.05).(4) TSPJ could increasethe levels of serum GSH-PX, SOD activity and reduce serum MDA content. The inhibition rate is positively correlated with the dose(5) TSPJ could reduce the inflammatory factors (IL-1, TNF-α) in mice serum, the differences are significant (P<0.05).2) The effect of TSPJ on CIA rats’histopathological and ultrastructural stractures (1) The AI scores in CIA group are higher than normal group, three dose group’s AI of TSPJ were also increased compare with the normal group at the same period, but the difference were significant compare with the CIA group at the same period.(2) The model group’ feet swelling begins from d12 to the end, peak time was at d24-d28. The swelling degree of TSPJ groups were also increased compare with the normal group at the same period, but the difference was significant compare with the CIA group at the same period.(3) CIA model rats’weight-gain rate slow down or stoped at the secondary inflammatory phase (after d18), TPT treatment group also shown the weight-gain rate, TSPJ groups’ growth rate were normal.(4) The immune function of CIA model could lead to higher compared with model group; the administration of TPT could decreasethe higher s spleen index and thymus index in CIA rats, the difference was significant,and TSPJ group alos decline differences but no significant.(5) TSPJ can improve joint damage of CIA:thre dose groups of TSPJ can reduce the levels of congestion and edema of soft tissue joint, toe joint volume and scores; reduce synovial hyperemia, edema of the degree; significantly inhibit the CIA rats’ synovial hyperplasia; inhibit the inflammatory cell’ infiltration; inhibit chondrocytes proliferation.(6) ConA-induced spleen T lymphocyte proliferation in CIA group was significantly lower than the control group, and IL-2 levels were also significantly lower, TSPJ groups can restore the CIA rat spleen lymphocytes and production of IL-2, but TPT group has no effect on proliferation of lymphocytes and production of IL-2.(7) The productions of IL-1β, IL-6 and TNF-αwere significantly higher in CIA group’s PMΦthan those of normal group (P<0.05), TSPJ medium and large doses groups can inhibit the production of proinflammatory cytokines IL-1β, IL-6 and TNF-αlevels in CIA’s PMΦ.(8) The synovial membrane ultrastructure:at the CIA group, the synovial cell lining layers were thickened up to 5-6 layers and disarrangement, organelle deformation, or even disappear, the cytoplasm filled with large vacuoles, the uneven distribution of nuclear chromatin. Some cells due to degeneration and necrosis to atrophy, only to see a large number of cell fragments with staggered collagen fibers. TSPJ can significantly improve the CIA synovial ultrastructure of synovial lining cell layer and cell arrangement, only occasionally a small amount of lymphocytic infiltration can be seen, A-type cells to primary lysosomes, mitochondria slightly increased, B-type Cytoplasm of cells rich in rough endoplasmic reticulum and ribosome bodies, various types of cells close to normal.(9) The ICAM-1 production (the total number of positive cells, the average black level value of the total area and integral optical density) were significantly increased in CIA model group’s synovial membrane during the inflammatory process, TSPJ groups can reducethe ICAM-1 expression in synovial number, size Sum, integral optical density and average black levels, the differences are significant (P<0.05).(10) synovial cell culture and cytokine researches showed:normal synovial cells’ cytokines productions are very low even no detected, cell growth rate are normal; after stimulated by LPS, the speed of cell proliferation and cytokines such as IL-1, IL-6, IL-8 and TNF-αsecretions are increased, this suggest that LPS is a good stimulant of microbial antigens, can induce normal cell proliferation and differentiation. CIA synovial cells’ proliferation rate and cytokines secretions were significantly higher after LPS stimulation. Tripterygium showed a strong anti-inflammatory effects; TSPJ didn’t show cell-damage in the synovial cell culture experiments,and didn’t stimulate cytokine secretion, only regulate the CIA synovial cells from "super- sensitivity" status to normal.From the experimental results, TSPJ has the following parameters:(1) increasing the antioxidant capacity, reduce inflammation, tissue damage induced by free radicals; (2) to relieve inflammatory mediators such as PGE and LTB in inflamed tissue; (3) significantly decreased cell factors such as IL-1B, IL-6, TNF-αinf lammated tissues; (4) reduce the ICAM-1 generation in inflammated tissues; (5) TSPJ has ’two-way adjustment’ on the immune system, so at the process of treating RA it can reduce hyperthyroidism immune function to normal, but don’t produce immune dysfunction as triptolide and other immunosuppressive agents do. Therefore, TSPJ is likely to be developed as an effective, non-toxic or low toxic anti-inflammatory and anti- rheumatic drug.更多还原