| 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. |
PDF Full Text Request