单位代码:10222 学号:158043019 分类号: 佳木斯大学 硕士学位论文 论文题目:巴戟天环烯醚萜苷类成分抗 类风湿性关节炎的作用研究 研 究 生 姓 名: 张建花 学科(专业): 生药学 导师姓名、职称: 杜娟 副教授 论文答辩日期: 2018 年 6 月 2 日 万方数据 佳木斯大学硕士学位论文 论文题目: 巴戟天环烯醚萜苷类成分抗 类风湿性关节炎的作用研究 研究生: 张建花 学科: 生药学 指导教师: 杜 娟 单位: 佳木斯大学 协助指导教师: 刘 娟 单位: 佳木斯大学 张巧艳 单位: 第二军医大学 学习期限:2015 年 9 月至 2018 年 6 月 学位授予单位:佳木斯大学 万方数据 学 术 诚 信 承 诺 诺 本人郑重声明:所呈交的论文是我个人在导师指导下进行的研究工作及取得研究成果。尽我所知,除了文中特别加以标注和致谢的地方外,论文中不包含其他人已经发表或撰写的研究成果,也不包含为获得佳木斯大学或其他教育机构的学位或证书所使用过的材料。与我一同工作的同志对本研究所做的任何贡献均已在论文中做了明确的说明并表示了谢意。 签名:___________ 日期:____________ 关于论文使用授权的说明 本人完全了解佳木斯大学有关保留、使用学位论文的规定,即:学校有权保留送交论文的复印件,允许论文被查阅和借阅;学校可以公布论文的全部或部分内容,可以采用影印、缩印或其他复制手段保存论文。 (保密的论文在解密后应遵循此规定) 签名:___________ 导师签名:___________ 日期:_______ 万方数据 目 录 摘要 ....................................................................................................................................... 1 Abstract .................................................................................................................................. 3 前言 ....................................................................................................................................... 5 第 1 章文献综述 ................................................................................................................... 7 1.1 巴戟天的研究概况 ................................................................................................. 7 1.1.2 巴戟天的化学成分 ....................................................................................... 7 1.1.3 巴戟天的药理活性 ....................................................................................... 8 1.2 类风湿性关节炎的研究概况 ............................................................................... 10 1.2.1 类风湿性关节炎的现状 ............................................................................. 10 1.2.3 中医药治疗类风湿性关节炎 ...................................................................... 11 1.3 环烯醚萜苷对类风湿性关节炎的治疗 ............................................................... 12 1.4 巴戟天治疗类风湿性关节炎的应用 ................................................................... 12 第 2 章 巴戟天环烯醚萜苷的含量测定与稳定性研究 ................................................. 14 2.1 仪器与材料 ......................................................................................................... 14 2.1.1 仪器 .............................................................................................................. 14 2.1.2 试剂 .............................................................................................................. 14 2.1.3 材料 ............................................................................................................. 15 2.2 实验方法 ............................................................................................................... 15 2.2.1 巴戟天环烯醚萜苷的含量测定方法 ......................................................... 15 2.2.2 巴戟天总环烯醚萜苷的制备方法 ............................................................. 17 2.2.3 巴戟天总环烯醚萜苷的稳定性实验 .......................................................... 17 2.3 结果与讨论 ........................................................................................................... 17 万方数据 2.3.1 巴戟天中 4 种主要环烯醚萜苷的含量测定 ............................................. 17 2.3.2 巴戟天总环烯醚萜苷的含量 ..................................................................... 21 2.3.3 巴戟天总环烯醚萜苷的稳定性结果 ......................................................... 21 2.4 小结 ....................................................................................................................... 21 第 3 章 巴戟天环烯醚萜苷抗炎作用的研究 ................................................................. 23 3.1 仪器与材料 ........................................................................................................... 23 3.1.1 仪器 ............................................................................................................. 23 3.1.2 试剂 .............................................................................................................. 23 3.1.3 材料 ............................................................................................................. 23 3.2 实验方法 ............................................................................................................. 24 3.2.1 巴戟天总环烯醚萜苷的急性毒性实验 ..................................................... 24 3.2.2 小鼠醋酸扭体镇痛实验 ............................................................................. 24 3.2.3 小鼠气囊肉芽肿实验 ................................................................................. 24 3.2.4 大鼠棉球肉芽肿实验 ................................................................................. 25 3.2.5 巴戟天环烯醚萜苷对 RAW264.7 细胞的作用 ......................................... 26 3.3 实验结果与讨论 .................................................................................................... 27 3.3.1 巴戟天环烯醚萜苷的急性毒性实验 .......................................................... 27 3.3.2 巴戟天环烯醚萜苷对小鼠醋酸扭体反应的影响 ...................................... 28 3.3.3 巴戟天环烯醚萜苷对小鼠气囊肉芽肿的影响 ......................................... 29 3.3.4 巴戟天环烯醚萜苷对大鼠气棉球肉芽肿的影响 ..................................... 30 3.3.5 巴戟天环烯醚萜苷对 RAW264.7 细胞影响的结果 ................................. 31 3.5 小结 ....................................................................................................................... 36 第 4 章 巴戟天环烯醚萜苷抗类风湿性关节炎作用的研究 ......................................... 37 4.1 仪器与材料 ........................................................................................................... 37 万方数据 4.1.1 仪器 ............................................................................................................. 37 4.1.2 试剂 ............................................................................................................. 37 4.1.3 材料 ........................................................................................................... 38 4.2 实验方法 ............................................................................................................... 38 4.2.1 大鼠佐剂性多发性关节炎(AA)模型 ................................................... 38 4.2.2 小鼠肾虚痹症模型 ..................................................................................... 40 4.2.3 巴戟天环烯醚萜苷对滑膜细胞(FLS)的作用 ...................................... 41 4.2.4 数据统计分析 ............................................................................................. 42 4.3 实验结果与讨论 ................................................................................................... 42 4.3.1 巴戟天总环烯醚萜苷对 AA 模型大鼠的治疗作用 ................................. 42 4.3.2 巴戟天总环烯醚萜苷对肾虚痹症小鼠的治疗作用 ................................. 48 4.3.3 巴戟天环烯醚萜苷对关节炎患者滑膜细胞的作用 ................................. 63 4.4.小结 ........................................................................................................................ 65 结论 ..................................................................................................................................... 67 英文缩写 ............................................................................................................................. 74 附图 ..................................................................................................................................... 75 攻读学位期间取得的学术成果 ......................................................................................... 77 万方数据 佳木斯大学硕士学位论文 -1- 摘 要 类风湿性关节炎(Rheumatoid arthritis, RA)是一种慢性进行性的自身免疫性疾病,其病理特点是多关节受累,滑膜炎持久反复发作,病情缠绵难愈,可导致关节内软骨和骨的破坏。中医理论认为 RA 是由于肝肾亏损及寒湿阻络引起的,其治疗应采用补肝肾、祛风除湿、活血通络的方法。中药巴戟天为茜草科植物巴戟天 Morinda officinalis How.的根,性温,味辛、甘,归肝、肾经;具有补肾阳,强筋骨,祛风湿的作用。研究表明巴戟天环烯醚萜苷类成分具有显著的抗炎活性。课题组前期研究发现巴戟天乙醇提取物能有效抑制佐剂性关节炎大鼠踝关节的急性炎症反应,降低血清IL-1β 和 TFN-α 的水平,而环烯醚萜苷类成分为巴戟天抗炎和防治大鼠关节炎的有效成分。 目的:明确巴戟天环烯醚萜苷类成分抗炎和抗类风湿新关节炎的作用及机制,为巴戟天资源的开发利用及新药研究开发提供科学依据。 方法:1. 以巴戟天环烯醚萜苷类成分水晶兰苷、去乙酰基车叶草苷酸、车叶草苷酸和车叶草苷为对照品,建立HPLC法测定巴戟天环烯醚萜苷类成分的方法。采用大孔吸附树脂富集巴戟天中环烯醚萜苷类成分,以获得巴戟天总环烯醚萜苷。 2. 按照最大给药容量、最大药物溶解浓度,小鼠单次灌胃给予巴戟天总环烯醚萜苷,观察给药后小鼠14天内的毒性反应及死亡情况。通过小鼠醋酸扭体实验、小鼠气囊肉芽肿实验和大鼠棉球肉芽肿实验,研究巴戟天总环烯醚萜苷的抗炎镇痛作用。以LPS刺激的RAW264.7细胞为模型,初步研究巴戟天总环烯醚萜苷抗炎的作用机制。 3. 应用弗氏佐剂诱导大鼠关节炎模型,以去卵巢联合Ⅱ型胶原诱导小鼠肾虚痹症模型,通过观察小鼠和大鼠的足趾肿胀度、关节炎指数、血清生化指标和滑膜组织的病理变化,评价巴戟天总环烯醚萜苷抗类风湿性关节炎的作用。以TNF-α刺激的人成纤维滑膜细胞为模型,初步探讨巴戟天总环烯醚萜苷的作用机制。 结果:1. 所建立的HPLC方法可用于巴戟天中环烯醚萜苷类成分的含量测定。水晶兰苷、去乙酰车叶草苷酸、车叶草苷酸和车叶草苷在0.375-12 μg,0.13-4.16 μg,0.016-0.516 μg和0.012-0.384 μg范围内,与峰面积的线性关系良好;重复性、稳定性、精密度和加样回收率试验结果均符合要求。经大孔树脂富集,可得到含量为62%的巴戟天总环烯醚萜苷。 万方数据 佳木斯大学硕士学位论文 -2- 2. 急性毒性结果表明,巴戟天总环烯醚萜苷对小鼠没有明显的毒副作用。巴戟天总环烯醚萜苷在50mg/kg,100mg/kg和200mg/kg的剂量下,均可显著减少醋酸扭体试验中小鼠的扭体次数,减少小鼠气囊肉芽肿和大鼠棉球肉芽肿实验中肉芽肿的干重和湿重,表明巴戟天总环烯醚萜苷具有显著的抗炎和镇痛作用。巴戟天总环烯醚萜苷及其主要成分水晶兰苷显著抑制LPS刺激的RAW264.7产生IL-1β、IL-6和NO,抑制RAW264.7细胞中iNOS和COX-2 蛋白的表达,及NF-ƙB通路中的蛋白表达,表明巴戟天环烯醚萜苷通过NF-ƙB通路产生抗炎作用。 3. 巴戟天总环烯醚萜苷能够减轻弗氏完全佐剂诱导的大鼠关节炎症状,显著降低大鼠血清炎症细胞因子IL-6、IL-1β和IL-17a的水平,显示出良好的抗类风湿性关节炎的作用。在去卵巢和Ⅱ型胶原联合诱导的小鼠肾虚痹症关节炎模型中,巴戟天总环烯醚萜苷能够改善小鼠的关节炎症状,显著降低小鼠血清炎症细胞因子IL-6 和IL-10的水平,减缓关节炎小鼠滑膜组织的增生,显示出良好的抗类风湿性关节炎作用。同时,巴戟天总环烯醚萜苷能够增加肾虚痹症关节炎小鼠血清骨形成指标骨钙素的含量,降低血清碱性磷酸酶的活性,显著降低血清骨吸收指标DPD和RANKL的水平,显著增加小鼠股骨的骨密度和骨矿物质含量,增加骨小梁数目,降低骨小梁分离度,改善骨组织的微结构,具有良好的改善肾虚痹症小鼠骨丢失和骨质破坏的作用。 巴戟天总环烯醚萜苷及水晶兰苷能显著抑制TNF-α 刺激的滑膜成纤维细胞的增殖及IL-6、IL-8的产生,具有抑制滑膜增生的作用。 结论:巴戟天总环烯醚萜苷具有确切的抗炎和抗类风湿性关节炎作用,可为巴戟天资源的开发利用和中药新药研制提供科学的依据。 关键词:巴戟天;环烯醚萜苷;抗炎;抗类风湿性关节炎 万方数据 佳木斯大学硕士学位论文 -3- Anti-rheumatoid Arthritis Effects of Iridoid Glycosides from the root of Morinda officinalis How. Abstract Rheumatoid arthritis (RA), which is a chronic progressive autoimmune disease, are characterized as multiple joints lesions, sustained synovial inflammation with recurrence and difficulty to cure, leading to destruction of cartilage and bone in the joints. In traditional Chinese medicine, the etiology and pathogenesis of RA are related with loss of liver and kidney and also cold-dampness blocking collaterals. Therefore, the treatment methods of RA often include tonifying liver and kidney, removing wind and dampness, and promoting blood circulation. The Bajitian, a commonly used traditional Chinese medicine, comes from the root of Morinda officinalis How. (MO), is naturally warm, pungent, taste sweet, contribute to liver and kidney, and has the action of tonifying kidney-yang, strengthening bones and muscles and removing wind and dampness. The Iridoid glycosides from MO have been shown to possess the activities of anti-inflammation. Our previous study showed that ethanol extract of MO can effectively inhibit acute inflammatory reaction of ankle joint in adjuvant arthritis rats, significantly reduce the levels of serum IL-1β and TFN-α, while the Iridoid glycosides from MO are responsible for the activities of anti-inflammation and anti-rheumatoid arthritis. Objective: To elucidate the anti-rheumatoid arthritis effects and mechanism of iridoid glycosides from MO to provide scientific basis for the utilization and development of new drugs for anti-rheumatoid arthritis. Methods 1. HPLC method was developed to determine the content of iridoid glycosides, including monotropein, deacetyl asperulosidic acid, asperulosidic acid and asperuloside, in mo and total iridoid glycosides (MOIG). The macroporous adsorption resins were used to enrich iridoid glycosides from MO to obtain total iridoid glycosides (MOIG). 2. The acute toxitic effects of MOIG were evaluated on mice with single intragastric administration maximum volume and the maximum concentration of drug dissolved through observing the toxicity and mortality of mice within 14 days after administration. 万方数据 佳木斯大学硕士学位论文 -4- The acetic acid writhing test in mice, bladder granuloma test in mice, cotton ball granuloma test in rats were conducted to investigate the anti-inflammatory and analgesic effects of MOIG. The RAW264.7 cell stimulated with LPS were used to preliminary study the mechanism of anti-inflammation of MOIG. 3. Adjuvant rheumatoid arthritis in rats model, and ovariectomized and collagen-induced rheumatoid arthritis model in mice were used to evaluate the anti-rheumatoid arthritis activities of MOIG through examining the swelling of foot, index of arthritis, biochemical parameters in serum and alteration of tissue structure of synovium in joint. The TNF-α stimulated synovial cells were used to explore mechanism of MOIG on the anti-rheumatoid arthritis. Results 1. The developed HPLC method can be used to determine the contents of iridoid glycosides from MO. Monotropein, deacetyl asperulosidic acid, asperulosidic acid and asperuloside showed good linearity (r>0.9995) in the ranges of 0.375-12 μg, 0.13-4.16 μg, 0.016-0.516 μg, 0.012-0.384 μg with their corresponding peak areas,respectively. Repeatability, precision, recovery and stability were validated according to the requirements of China Pharmacopoeia. The content of iridoid glicosides in MOIG obtained through enrichment of macroporous adsorption resin reached to 62%. 2. The results of acute toxicity indicate that MOIG did not show any toxitic effects on mice. MOIG at dose of 50mg/kg,100mg/kg and 200mg/kg significantly decreased the number of writhings in the acetic acid writhing test, reduced the wet and dry weight of granuloma in mouse bladder granuloma test and rat cotton ball granuloma test, indicating that MOIG had anti-inflammatory and analgesic effect. Furthermore, MOIG and monotropein significantlyinhibited the production of the IL-1β, IL-6 and NO, the expression of iNOS and COX-2 protein, the protein expression of NF-ƙB inflammatory pathway in LPS-stimulated RAW264.7 cells, suggesting that MOIG maybe exerted anti-inflammatory effects through NF-κB pathway. 3. MOIG can abate the paw swelling of arthritis rats induced with Freund's complete adjuvant; significantly reduce the levels of serum inflammatory cytokines IL-6, IL-1β and 万方数据 佳木斯大学硕士学位论文 -5- IL-17α, exhibiting that MOIG had potential anti-rheumatoid arthritis effect. In rheumatoid arthritis mice induced with ovariectomy and collagen Ⅱ, MOIG can relieve the paw swelling of mice, significantly reduce the serum levels of inflammatory cytokines IL-6 and IL-10, and decrease the synovial hyperplasia in joint tissue, indicating that MOIG can prevent rheumatoid arthritis. In addition, MOIG can increase the levels of serum bone formation markers osteocalcin, reduce serum alkaline phosphatase activity (ALP), significantly decrease the levels of serum bone resorption parameters, such as DPD (Deoxypyridinoline) and RANKL (receptor activator of NF-κB ligand), significantly increase the femur bone mineral density and bone mineral content, enhance the number of trabecular bone, reduce the degree of trabecular bone separation, and improve bone microstructure, indicating that MOIG can decrease bone loss and alleviate the destruction of bone tissue in joint of rheumatoid arthritis mice induced with ovariectomy and collagenⅡ. MOIG and monoteoprin significantly reduced the production of IL-6 and IL-8, and significantly inhibited the proliferation of Fibrous synovial cells stimulated by TNF-α . Conclusion MOIG had definitely anti-inflammatory analgesic and anti-rheumatoid properties. This will provide scientific basis for the utilization of MO and development of new drugs for the prevention and treatment of rheumatoid arthritis. Key words:Morinda officinalis How.;iridoid glycosides; anti-inflammatory;anti-rheumatoid 前言 类风湿性关节炎(Rheumatoid arthritis, RA)是一种慢性进行性的自身免疫性疾万方数据 佳木斯大学硕士学位论文 -6- 病,其病理特点是多关节受累,滑膜炎持久反复发作,病情缠绵难愈,可导致关节内软骨和骨的破坏。该病在我国发病率很高,以青壮年居多,是成人劳动力丧失的重要原因之一。治疗RA的药物主要包括非甾体抗炎药、抗风湿药、糖皮质激素和生物制剂等。化学药物的长期应用会引起胃肠道不适、肝损伤等不良反应。生物制剂在干预病变细胞的同时,对机体的固有免疫和适应性免疫也会产生干扰,从而产生毒性和破坏机体防御作用的风险。因此,寻求高效低毒的防治 RA 的药物仍是世界医学界面临的一项任务。 RA 属于中医“痹症”范畴,虚实夹杂为该病的主要病机。现代中医对RA病因病机的认识又有所发展,概括而言,肝肾亏损为病之本,寒湿阻络乃病之标。故其治疗应补肝肾、祛风除湿、活血通络。中药巴戟天为茜草科植物巴戟天(Morinda officinalis How., MO)的干燥根,是我国四大南药之一,主产于广东、福建、广西等地,收载于历版《中国药典》中。