摘要
类风湿性关节炎(rheumatoid arthritis, RA)是一种以关节及关节周围组织慢性炎症病变为主要表现的自身免疫性疾病,是临床多发病。
RA发病特点是反复发作,病情缠绵,且有较高的致残率,严重危害人类健康。近年来虽在RA发病机制及病理变化等研究领域取得了显著进展,但是长期应用西药治疗,毒副作用大,疗效不佳。中医药治疗具有疗效较好、毒副作用小的特点。因而,利用丰富的中医药资源,开发治疗RA有效的方法和药物,明确其治疗机制,有重要的科学意义和社会意义。
RA属于中医顽痹范畴,其病因为外感寒湿之邪,气血阴阳失调,脏腑亏损,寒湿凝滞,气血运行不畅导致邪盛正虚、瘀滞挟杂引起关节肿胀、疼痛变形、晨僵等症状。中医认为,气行则血行,气滞则血凝,不通则痛,通则不痛。人体正气的强弱,对类风湿性关节炎的演变和预后起着重要作用。故治疗原则应以整体出发,针对病因病机,扶正祛邪,寒热并用,以祛风除湿,通络散寒,扶正固本为基本治法。而对类风湿关节炎疗效最为突出的中药首推雷公藤(TriPteyrgimu Wilofulii Hook F, TWHF)。
雷公藤系卫矛科雷公藤属木质藤本植物,最早记载雷公藤功效的是公元1476年明朝兰茂所著的滇南本草,书中称其味辛,性温,有毒,入肝脾十二经,行十二经络,具有治筋骨疼痛,风寒湿痹,麻木不仁,瘫痪痿软,湿气流痰等功效,能缓解肢体的疼痛与拘挛等症状。雷公藤作为一种传统中药,具有活血化瘀、清热解毒、消肿散结、杀虫止血等功效,可以起到扶正以御邪气之作用,使RA之邪、虚、瘀得以治疗,直中病机。
雷公藤作为一种的免疫抑制剂应用于临床已有近30年。其中,环氧二萜内酯化合物—雷公藤甲素(triptolide, TP)被认为是最主要的抗炎和免疫抑制成分。药理和临床试验表明其具有免疫抑制、抗炎等多种生物活性,为常用的改善病情类抗风湿药,能缓解RA骨侵蚀,是治疗RA的有效药物。
大量的临床实验证实,TP具有较好的抗炎作用,特别是对RA有很好的疗效。TP治疗RA(RA)起效时间较快(平均2.57d),用药后能明显减轻关节肿痛,改善关节功能障碍,降低血沉,控制发热,并能部分或全部代替激素和/或非甾体抗炎药物,且较之为优(近期有效率达100%,显效率为46.67%)。TP在剂量范围0.05-0.3mg/kg时,无论是皮下注射、腹腔注射还是灌胃均能明显抑制大鼠腹腔毛细血管通透性增加,对角叉菜胶性和甲醛性前足肿胀具有明显的抑制作用,表明药物对渗出性和增殖性炎症均有疗效
RA主要病理特点是滑膜细胞增生,衬里层增厚,多种炎性细胞浸润,血管翳形成,以及软骨和骨组织的破坏,最终导致关节畸形和功能丧失。
大量资料表明T淋巴细胞、巨噬细胞和增生的滑膜细胞在这种疾病的发病机制中起主要作用,其中滑膜成纤维细(Fibroblast-like synoviocyte, FLS)表型改变的机制成为RA发生机制研究的新热点。
现在已有大量的证据提示,FLS是RA的关键效应细胞,它们释放多种效应分子作用于各种细胞(淋巴细胞、单核细胞、间充质细胞),降解细胞外基质并且向间充质细胞和浸润免疫细胞提供趋化和活化信号。抑制FLS效应的治疗策略可以显著地减轻关节炎和预防骨的吸收。
因此,TP治疗RA疗效确切。然而,TP治疗RA的机制仍旧处于起步阶段,详细作用机制仍需深入探讨。其中,TP抑制RA滑膜成纤维细胞增殖机制研究很少。在本研究中,我们对TP抑制RA滑膜成纤维细胞增殖作用进行验证并利用蛋白质组学方法比较RA滑膜成纤维细胞受到TP刺激后发生上调或下调的蛋白质,这将有利于揭示TP抑制RA滑膜成纤维细胞增殖的作用机制,将有助于为其临床应用提供理论基础,并有利于理解RA发生发展机制,为其临床治疗提供候选靶点。
本课题采用的具体研究方案如下：
(1)制作RA大鼠模型。
本课题采用新的模型制作方法,热杀死结核杆菌(Heat-killed myeobaeterium tuberculosis H37Ra, Mtb)诱导建立大鼠佐剂性关节炎模型,该模型造模成功率高(可达100%),关节炎症发生时间早(注射Mtb后8天左右发生,15天达高峰),实验周期较短,是较为理想的研究药物治疗类风湿关节炎疗效及其作用机理的模型。
(2)研究TP对RA有关指标的影响。
实验随机分为3组：空白对照组、模型对照组、TP组。采用HE染色法进行滑膜组织病理学检查,观察RA程度；在前期的基础上,进一步应用LiquiChip蛋白液相芯片系统检测RA的多种相关因子TGF、IGF-1、IFN-γ、TNF-α、MCP-1、 MIP-2、IL-1、IL-4、IL-6、IL-12、IL-13、IL-17、VCAM-1、ICAM-1等含量变化。
(3)研究类风湿发生过程中的信号传导网络。
取上述实验各组的动物组织,提取总蛋白,应用蛋白质双向凝胶电泳技术(Two-dimensional electrophoresis,2-DE)、质谱仪等蛋白组学相关技术和方法,筛选差异蛋白表达谱,然后应用免疫组化、Western Blot、Q-PCR等方法鉴定关键蛋白质,进一步弄清RA发生过程中的信号传导网络。
本课题结果表明：
(1)TP治疗后,RA大鼠病变关节肿胀有显著的消退。
(2)TP治疗后,RA大鼠细胞因子谱发生变化：模型组IL-1β、IL-6、TNF-α MIP-1α血清水平明显升高,而TP治疗后显著下降,模型组血清IL-4、IL-10显著升高,而TP治疗组相对于模型组显著升高。
(3)经过蛋白质组学分析,筛选得到同时满足下列条件的差异点5个：1)模型组与正常对照组之间存在差异；2)TP治疗组上述1)差异点变化趋势逆转；3)变化趋势1.5倍以上。差异点分别为：Annexin A3、Cytoplasmic aconitate hydratase、Peroxiredoxin6、Transgelin、LIM and SH3domain protein1。
(4)经过Q-PCR、western-blot对感兴趣的蛋白质(Peroxiredoxin6、Transgelin)进行验证,表明其表达水平的变化与蛋白质组学分析一致。
Annexin A3也被称作lipocortin3或placental anticoagulant protein3是膜联蛋白(annexin)家族的一员。
Annexin家族是Ca2+依赖性磷脂结合蛋白,是细胞内高丰度蛋白,约占细胞总蛋白的1%～2%; annexin是一种普遍存在的细胞溶质蛋白,其以可溶形式和以通过与细胞骨架组成蛋白或调控细胞与细胞外基质相互作用蛋白的稳定结合或可逆结合的形式存在。Annexin家族包括A,B,C,D和E5类,脊椎(哺乳)动物的annexin属A类,被报道的有12种(annexin Al-All和A13)；无脊椎动物的属B类；真菌和部分单细胞真核生物的属C类；植物的annexin属于D类：原核生物的annexin则属于E类。Annexin家族成员参与抗凝血、抗炎、出胞入胞作用、信号转导、细胞增殖、分化和凋亡等多种生理活动。
Annexin表达失调与人类疾病相关,其表达水平的改变或其定位的改变可引起病态或产生疾病。Annexin在肿瘤的发生发展中,可介导细胞信号通路、细胞运动、肿瘤的侵袭转移、血管生成、细胞凋亡及抗药性。
我们推测：Annexin A3可能在RA关节滑膜细胞表达释放细胞因子、MMP等方面发挥作用,导致滑膜细胞增殖、侵袭能力增加等。
Transgelin是一种未知功能的22kDa蛋白质,也被命名为SM22。该蛋白质曾在多种样本中被鉴别出,并在鼠p27、WS3-10、胶转蛋白和SM22的一些不同命名下对其进行了研究。物种间的基因和蛋白质序列高度保守,且在果蝇和秀丽隐杆线虫中鉴别出同源性。胶转蛋白是肌动结合蛋白钙调蛋白家族中的一种,存在于细胞骨架中。
Transgelin是一种肌动蛋白压力纤维素相关的蛋白质,与凝胶剂起反应并能稳定体外的肌动蛋白凝胶剂。它还与平滑肌中的激动蛋白丝有相关性。考虑到胶转蛋白是一种最早的平滑肌细胞分化标志物且其与肌动蛋白有关,很久之前就假设它有调节这些细胞发育和收缩的功能。但是,无胶转蛋白(SM22-/-)的小鼠能正常发育、繁殖,且没有出现血压、心率、组织学或组织形态学上的任何改变。不过,对无胶转蛋白小鼠血管平滑肌的进一步检查发现小鼠对兴奋剂诱导的、钙独立收缩的收缩性反应度增加。胶转蛋白可能也涉及到细胞的转移。胶转蛋白也可能与细胞的移动有关。已证明其与肌动蛋白丝束的一种特异性亚群有关联,而肌动蛋白丝束足体会形成足体。该事实表明钙调蛋白的缺失能引起钙调蛋白与胶转蛋白转化率的降低,从而有利于足体的形成并致使细胞向侵略性、恶性细胞方向发展。
最近的证据表明某些细胞中的肿瘤抑制功能与胶转蛋白有关,而与细胞骨架本身没有关系。在前列腺肿瘤细胞中,已显示胶转蛋白能通过阻止雄性激素受体联合活化剂与雄性激素受体的结合来抑制雄性激素所刺激的细胞生长,以及随后向细胞核的移位。胶转蛋白也有抑制间质金属蛋白酶MMP-9表达的作用。MMP-9涉及到组织的重组过程,而组织的重组导致癌症细胞的移动和对周围组织的侵犯。
由于我们的实验结果表明胶转蛋白在TP治疗RA大鼠关节滑膜组织中表达发生改变,而胶转蛋白可能与MMP-9的表达相关,因此,我们推测TP作用于关节滑膜组织,可能影响转胶蛋白表达,通过影响MMP-9表达,而发挥抑制骨质破坏的作用。
总结：通过蛋白质组学技术,我们找到一些可能参与TP治疗RA的蛋白质,一些蛋白目前已有文献报道与RA发生发展有一定联系,另一些蛋白目前并无相关报道。这些蛋白的发现,为我们深入研究TP治疗RA的机制提供了新的靶点,需要我们进行进一步的研究。
Rheumatoid arthritis (RA) is a chronic inflammatory disease of the joints and joints surrounding tissue as the main manifestation of autoimmune diseases, clinical frequently-occurring disease.
The pathogenesis of RA is characterized by recurrent, lingering, and have a higher morbidity, seriously harm to human health. In recent years, there are remarkable progress has been achieved in the pathogenesis and pathological changes of RA, but the efficacy is poor and the side effects are serious by long-term application of western medicine. Troditional Chinese medicine treatment has better efficacy and low toxicity. Thus, using of abundant resources of Chinese medicine, there are important scientific and social significance to develop effective treatment and drugs for RA, and to clarify its therapeutic mechanism.
RA belong to Chinese Bi syndrome. The cause of this disease is the exogenous cold and dampness, the imbalance of yin and yang of qi and blood, the function decline of organs, the stagnation of cold and dampness, the sluggish of qi and blood. So, the symptom of RA is pathogen retreating with asthenia healthy qi, joint swelling, pain and deformation, morning stiffness. Chinese medicine believes that the gas running causes the blood running, the qi stagnation causes blood clotting, the block of qi causes the pain. The strength of the body qi, plays an important role in the evolution and prognosis of rheumatoid arthritis. The principles of treatment should proceed to the overall etiology and pathogenesis, to get rid of cold and dampness, to activate collaterals and dispell cold to reinforce the vital energy and consolidate the constitution. The most prominent Chinese medicine for treating RA is TriPteyrgimu Wilofulii Hook F (TWHF).
Tripterygium belongs to Celastraceae Tripterygium woody vine, the earliest record is the Yunnan Herbal of1476AD, Ming Dynasty, written by Lan Mao. This book told it was spicy, warm, toxic, going into the twelve jing of liver and spleen, with treatment to pain of arthralgia and myalgia, wind, cold, dampness, apathetic, soft paralysis atrophy, wet airflow sputum, can alleviate the symptoms of limb pain and Spasm. As a traditional Chinese medicine, Tripterygium has the function of blood-activating and stasis-dissolving, heat-toxin clearing, swellingreducing, and other effects, can play a centralizer to the role of the imperial evil.
The Tripterygium has been used clinically for nearly30years as a new immunosuppressive agents. A epoxy diterpenoid lactones-triptolide (TP) is considered to be the most important anti-inflammatory and immunosuppressive component of the Tripterygium. Pharmacological and clinical trials have demonstrated its immunosuppressive, anti-inflammatory and other bioactive. TP is an effective drug for the treatment of RA with anti-rheumatic and ease of RA bone erosion.
A large number of clinical trials confirmed that the TP has a better anti-inflammatory effects, especially for RA. TP has a rapid onset (mean2.57d) when treating RA. TP can significantly reduce joint swelling and pain, improve joint function, lower Erythrocyte Sedimentation Rate (ESR), control fever, and can replace partly or all of hormones and/or non-steroidal anti-inflammatory agent, and has a better effect (the effective rate of TP was100%, markedly effective rate was46.67%). In the dose range0.05-0.3mg/kg, regardless of subcutaneous injection, intraperitoneal injection or orally, TP significantly inhibited rat peritoneal capillary permeability increasing, foot swelling caused by carrageenan and formaldehyde, showing that the drugs have effect on the exudative and proliferative inflammatory.
The pathology of RA is characterized by synovial cell hyperplasia, thickening of the lining layer, and a variety of inflammatory cell infiltration, pannus formation and destruction of cartilage and bone tissue, eventually leading to joint deformity and loss of function.
Large amounts of data indicate that T lymphocytes, macrophages and proliferation of synovial cells play a major role in the pathogenesis of the disease. The phenotypic changes of fibroblast-like synoviocyte (FLS) became a new hot spot of RA mechanism.
There are already plenty of evidence prompted that the FLS is the key effector cells in RA. They release a variety of effector molecules, which act on a variety of cells (lymphocytes, monocytes, mesenchymal cells), degradate extracellular matrix and apply chemotaxis and activation signals to mesenchymal cells and infiltrating immune cells. The treatment strategies of inhibition of FLS effect can significantly reduce the arthritis and prevent bone resorption.
Therefore, TP has the good efficacy of treatment for RA. However, the mechanism of the TP treatment of RA is not clear. There are few studies on the proliferation inhibition of RA synovial fibroblast by TP. In the present study, inhibition of RA synovial fibroblast cell proliferation by TP was validated and proteomics method was used to identify the proteins upward or downward of synovial fibroblasts by TP stimulate, which will be helpful to reveal the the mechanism of TP on fibroblast proliferation, will be helpful to provide a theoretical basis for its clinical application, and provide candidate targets for clinical treatment.
The programs of this study are as follows:
(1) The model of rat RA. The model of this study was Heat-killed myeobaeterium tubercuolsis H37Ra (Mtb) induced rat adjuvant arthritis model. The model is characterized by a high success rate (up to100%), early occurrence of joint inflammation (occurred about8days, and reached the peak about15days after the injection of Mtb), and the short experimental cycle. This model is an ideal model for rheumatoid arthritis drug treatment.
(2) The effect of TP to RA. The experiments were randomly divided into three groups:control group, model control group, the TP group. Synovial histopathological examination by HE staining to observe the serious of RA. LiquiChip was then used to detecte a variety of related factors TGF and IGF-1, IFN-gamma and TNF-a MCP-1, MIP-2, IL-1, IL-4, IL-6, IL-12, IL-13, IL-17, VCAM-1and ICAM-1.
(3) The signal transduction networks invoved into RA. The total proteins of rat synoviocyte tissue were abtained by lysis. Protein two-dimensional gel electrophoresis technique (2-DE), mass spectrometry ware taken to identify differential proteins among every groups. Immunohistochemistry, western blot, Q-PCR were used to verify the key proteins involved into the mechanism of RA.
Our results showed:
(1) After TP treatment, the swelling of RA rat joint significantly relieved.
(2) After TP treatment, the cytokine profiling of RA rat changes:the serum levels of IL-1, IL-6, TNF-alpha and MIP-la of the model group significantly increased, while decreased after the TP treatment; the serum levels of IL-4, IL-10of the model group significantly increased, while significantly increased after TP treatment.
(3)5differences point were identified by proteomic analysis, following these conditions:1) it's different between the model group and control group;2) it reversal in TP treatment group;3) the difference is more than1.5times. The5differential points were:Annexin A3, Cytoplasmic aconitate hydratase, Peroxiredoxin6, Transgelin, LIM and SH3domain protein1.
(4) the expression level of the interested proteins (Peroxiredoxin6,Transgelin) by Q-PCR and western-blot analysis were consistent with proteomic analysis.
Annexin A3, also known as lipocortin3or placental anticoagulant protein3, is one member of the annexin family.
Annexin family are Ca2+-dependent phospholipid binding proteins, are high abundant proteins in cells, accounting for1%-2%of the total cellular proteins. Annexin is a ubiquitous cytosolic protein, existed by soluble form, or by form a stability construction with the cytoskeleton protein or the proteins regulatiing cell and extracellular matrix. Annexin family, including A, B, C, D, and E5classes. Mammal annexin is a Class A, reported as12kinds of annexin (Al-All, and A13). Invertebrates annexin is a Class B; fungi and some unicellular eukaryotes annexin is Class C. Plant annexin belongs to Class D. Prokaryotic annexin belong to class E. Annexin family members involved in the role of anticoagulant, anti-inflammatory, exocytosis, endocytosis, signal transduction, cell proliferation, differentiation and apoptosis and other physiological activities.
The disorders of annexin expression related to human diseases. Its changes of the expression level or positioning can cause the pathological or disease. Annexin can mediate cell signaling pathways, cell motility, tumor invasion and metastasis, angiogenesis, apoptosis and drug resistance in tumor development.
We speculate that:Annexin A3may play a role in the releasing of cytokines, MMP, etc., resulting in synovial cell proliferation and invasive ability.
Transgelin is a22kDa protein with unknown function, also named as SM22. The protein was identified in a variety of samples, and was studied as the name of p27, WS3-10, adhesive transfer protein and SM22. The gene and protein sequences are highly conserved between species, and are identified the homology in Drosophila and Caenorhabditis elegans. Transgelin is one member of actin binding protein calmodulin family, exists in the cytoskeleton.
Transgelin is an actin related proteins, responsible to gel and make gels stable in vitro. Transgelin also related with smooth muscle actin filaments. Taking into account that the transgelin is the first smooth muscle cell differentiation markers and related to the actin, it has been assumpted that the function of regulating cell growth and contraction long time ago. However, transgelin knockout (SM22-/-) mice can develop and reproduce normally, without any change in blood pressure, heart rate, histology or tissue morphology. However, further examination to the vascular smooth muscle of (SM22-/-) mice, found that the doping-induced, calcium independent contraction of the contractile response increased. Transgelin may also relate to the transfer of cells and cell migration. It has been proved related to a specific subgroup of the actin, while the actin can form the foot body. This facts indicated that the absence of calmodulin can cause calmodulin and transgelin conversion rate decreasing, and thus conducive to the formation of the foot body and causing the cells to an aggressive, malignant cells.
Recent evidence suggests that the tumor suppressor function of certain cells related to trandgelin, but not the cytoskeleton. In prostate tumor cells, it has been shown that transgelin can inhibit cell growth stimulated by androgen though block of the interaction between androgen receptor activator and androgen receptor, and the subsequent translocation to the nucleus. Transgelin also inhibit the expression of interstitial metalloproteinase MMP-9. MMP-9is related to the restructuring process of the organization, the organization's restructuring led to the movement of cancer cells and invasion to surrounding tissue.
Our results show that the transgelin expression altered in the RA joint synovial tissue of TP treated RA rats, and the transgelin may be associated with the expression of MMP-9. Therefore, we speculate that the TP act on the joint synovial tissue, which may affect transgelin expression, and affecting MMP-9expression, which play a role in inhibition of bone destruction.
In conclusion, using proteomics technology, we find some proteins may be involved in the TP treatment to RA. Some proteins have been reported in the literature that invoved into RA development, some other proteins are currently no related reports. The discovery of this study provides us the new targets to investigete the mechanism of TP treatment to RA, and needs further studies.
引文
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