| BackgroundRheumatoid arthritis (RA) is a systemic autoimmune disease that mainly shown as an erosive arthritis, which the most prominent feature is the synovitis induced synovial hyperplasia and erosion of the articular cartilage and bone, leading to joint destruction and deformity eventually. In recent years, with deeper understanding in the key pathogenesis of RA, a large number of studies have showed that the formation of synovial fibroblasts (SFs) is the key to the occurrence, development and metastasis of RA, and the migration of RASFs plays an importaint role in RA multi-joint inflammation and cartilage destruction. So does it exist in the serious stage of osteoarthritis (OA).When the joint is injuried, it’s a kind of physiological function for synovial membrane that SFs migrate to the extracellular matrix (ECM) of the exposed lesion to repair the wound tissue. This process is thought to be an important mechanism for the invasion of cartilage and bone in the pathogenesis of RA. Since the establishment of RA-SCID rat model by Muller-Ladner, a series of studies found that RASFs in the body can pass through the blood circulation to migrate to the spleen, synovial joints or the transplantated bovine cartilage, and keep the erosion characteristics for cartilage no matter subcutaneous, kidney or intraperitoneal transplantation. At the same time, study shows that SFs can hardly adhere to intact-cartilage, so the loss of cartilage matrix components (such as proteoglycans) constitutes as an important step in cell adhesion. Proteoglycan loss induced by cytokines such as interleukinl, happened before in inflammatory synovial tissue adhesion and invasion into cartilage, is an important precondition for the inflammation of synovial tissue adhesion and subsequent cartilage destruction.Bone damage in RA is mediated by osteoclasts in synovial pannus. Similarly, after cartilage damage and ECM exposure, RASFs migrate toghter to secrete large amounts of matrix metalloproteinases (MMPs) to further increase the degration of cartilage matrix, and ultimately resulted in the exposure of subchondral bone cortex, then the synovial tissue (mainly RASFs, inclued macrophages, osteoclasts) could erode the bone cortex of cartilage. The contact of synovial tissue to the cartilage and bone is a basic on RASFs proliferation and migration, which declares that cartilage and bone destruction is also dependent on the migration of RASFs from the synovial lining of bottom layer to the cartilage and bone.What’s more, the Epithelial-mesenchymal transition (EMT), as a key step in the process of invasion and migration for tumor, has been confirmed to exists in RASFs. In view of the important effect of the migration of RASFs in tissues and blood in the pathogenesis of RA, inhibiting the migration of RASFs will become a new target in the treatment of RA. To prevent the migration will undoubtedly contribute to block the important pathological changeing in RA-the progression of structural damage of joints and multi-joint propagation, even to avoid the adverse effects of immun osuppressants. In recent years, the common treatment of TNF-α biological agents in vivo cann’t block the migration of RASFs and its erosion on the cartilage, and the clinical choice of optional anti rheumatic drugs is limited, but the traditional Chinese medicine has its unique advantages.Rheumatoid arthritis (RA), which belongs to Traditional Chinese Medical (TCM) "Bi disease," " Li-jie disease " category, Sanshui Baihu decoction(SSBH) is as the clinical experience side for Hot Bi-based arthritis(pyretic arthralgia, one of the syndromes of Bi disease). Preliminary series of studies has demonstrated that its containing serum could significantly inhibited the proliferation of arthritis synovial fibroblasts (SFs) in vitro. SSBH can not only inhibit the local expression of MMP-1, MMP-3, NF-κB mRNA in joint, but also inhibit the production of TNF-a, reduced activation and expression intraarticular RASFs p38MAPK, reduce cartilage destruction. We recently discovered that, there were different expressions of 45 proteins in RASFs cultured with serum of SSBH containing compared to that cultured with normal serum, and the expressions of centrosome protein and vinculin related with cell migration were significantly reduced after cultured with serum of SSBH containing. The actin-binding protein (DST protein) and cadherin screened by phage display technology are closely related to cell structure, movement and migration. It suggests that SSBH’s pharmalogical mechanism involves not only anti-inflammatory analgesic, immunosuppression, suppression of synovial fibroblast proliferation, and may affect the synovial fibroblast migration, which will undoubtedly prompt the prospects of SSBH to block the progression of RA prospects. Because of the complex formula of SSBH, in order to reduce it, which components inhibiting cell migration would be got clear. This experiment performs study on simplified separated prescription based on different effects:Antipyresis and detoxication group (raw radix rehmanniae, buffalo horn, cortex moutan, gypsum rubrum, rhizoma anemarrhenae, artemisia annua), dehumidification and swelling group (polygonum cuspidatum, coix seed, semen brassicae, heracleum, asarum, tangerine peel), group of activating meridians and stopping pain (ramulus mori, caulis spatholobi, centipede, scorpion, lumbricus, celastrus orbiculatus).In vitro cell migrationusually scratches experiments and Transwell experiments.The former is a simple method of in vitro studies of cell migration, the principle of the method is:when the cells grow to merge into single state in the confluent monolayer cells artificially create a blank area, known as the scratch cells in scratch edge will gradually enter the blank area so that the "scratch" healing. Features are as follows: ①This simulates in vivo cell migration process to a certain extent. ②It is ideal for the study of the cells migrate that caused by the interaction between cell and the extracellular matrix (ECM), the cells and cells. Transwell as an experimental technique that involves the main material is Transwell chambers (Transwell chamber, Transwell insert). Put the small room into culture plates, Transwell chamber named upper chamber, or culture plate named lower chamber, then the upper and lower interior were dressed broth, and separated bya polycarbonate membrane. Since the polycarbonate film has permeability, the ingredients of the culture medium in lower chamber may affect the cell that seeded in the transwell chamber, which can be studied the effects that in culture medium composition on cell proliferation, migration and other movement. Application a polycarbonate membrane that are different pore sizes or through different treatments, which can be study of various aspects of co-cultured, cell chemotaxis, cell migration, cell invasion and so on.Western blot, as one of the most common methods to perform semi-quantitative detection and analysis for expression and distribution of protein features in biological tissue, has combined the high resolution gel electrophoresis with immunochemical analysis technology, which is characteristed with a large amountof analysis, strong specificity and high sensitivity, mainly includes gel electrophoresis, sample imprinting and immunological detection. Polymerase Chain Reaction (PCR) is a kind of molecular biology techniques for amplification of specific DNA fragments, its biggest characteristic is to greatly increase trace amounts of DNA. PCR is similar to the basic principle of natural DNA reproduction process, whose specificity is dependent on the complements on both ends ofthe target sequence of oligonucleotide primers, includes three basic steps:modified-annealing-extension. If the source of the template DNA is mRNA, which is to be cDNA through reverse transcription, so it’s called reverse transcription PCR (RT-PCR), and the index amplification of RT-PCR is a very sensitive technique to detect low copy number of RNA. In the process of PCR amplification, to perform the real-time quantitative detection by fluorescent signa, namelythe real time fluorescence Quantitative PCR (qrt-PCR), which is widely used in research such as gene expression, genetically modified, drug efficacy, pathogen detection.This article first launched the survey in RA patients to understand their own understanding of the disease and the clinical application of Chinese medicine treatment. While taking advantage of the classic experiments in vitro cell migration to observe and analyze the impact of SSBH and its decomposed recipes on RASFs migration, OASFs as cell contrast.It has the following significance: ①It will more intuitive clear SSBH RASFs migration inhibition effect from the morphological, and interpretation and perfect the mechanism of pharmacological treatment of RA from the perspective of cell migration, and then may even further found that their OA also have a certain effect, the clinical treatment of arthritis has a high regard for value.② It is to explore the role of the three separated prescriptions for synovial cell migration, and to evaluate the most effective combination of demolition party, then to lay a foundation for future reducing experiments of SSBH. ③c-Jun N-terminal kinase (JNK) pathway is closely related to migration of RASFs. Take the small molecule inhibitors sp600125 of JNK signaling pathway as a positive control to explore the possible mechanism of SSBH inhibiting the cell migration. ④To explore the possible inhibition pharmacological mechanism of SSBH on cell migration from protein and gene level, by detecting protein expression or mRNA of molecules related with cell migration and EMT.Part 1 The culture of RASFs and OASFs and preparation of medicated serumPurposeTo establish stable cell lines of RASFs and OASFs in vitro and modify primitive culture techniques, then to make a preparation of medicated serum with SSBH and separated prescription.Method1. Joint synovial tissues were collected by needle synovial biopsy from knees, which from diagnosised patients with RA and OA, tissue culture method and modified tissue culture method were adopted for primary culture, rate of cell climbing out by two methods was compared, to subcultured for 4-6 generation for follow-up study.2. Traditional Chinese medicine SSBH and decomposed formulas medicated serum preparation:SSBH and three decomposed formulas were decocted and then concentrated, which were feed Wistar rats one time per day, continuously for seven days, then abdominal aortic blood was collected, serum was got by centrifuga, at last stored in cold preservation after inactivation and sterilization.ResultFour stable RA and one OA primary SFs were obtained by two methods, the time of cells climbing out by modified tissue culture method was significantly shorter than that by tissue culture method(P<0.05). Cell morphology of SFs was fusiform regularly.ConclusionCompared with traditional tissue culture method, rate of SFs grew successful by modified tissue culture method is higher, cell climbs out and subcultures for shorter time. After subcultures to third generation in vitro, RASFs and OASFs represent stable cell morphology, and proliferate quickly. The serum of animal origin, route of administration, dosage, the standardization of collection(including the time of collecting, location of drawing and volume, etc) and serum preservation conditions all influence reliability and repeatability of SSBH medicated serum.Part 2 Inhibitory effects of SSBH on cell migration of SFs observed by wound healing assayPurposeTo investigate the influence of SanShui BaiHu decoction(SSBH) on migration of synovial fibroblasts originated from rheumatoid arthritis and osteoarthritis, and appraise the best simplified separated prescription.MethodScratches plugin instructions provided by the Ibidi company in accordance with Germany, cells were inoculated in chamber, then scratch would formed after the plug-in was removed, the synovial cells’migration was observed by microscopic at different time points (0h,12 h,24 h,36 h,48 h) and photos took at the same time, crawling mobility of cells was analysed and calculated through Image J software. Eight groups were set up, namely. NS control group, drug-contained serum groups of SSBH with different concentrations (10%,20%,30%), decomposed formulas groups of SSBH (Cl, C2 and C3), sp600125 as positive control group. The experiment repeated three times.ResultWound healing assay showed that migration rates of cells in SSBH groups, decomposed formulas groups and sp600125 group were all lower than NS control group (P<0.05), and the inhibitory effects of SSBH groups were positively related to concentrations and treatment time (r=0.883,0.888; P=0.000); sp600125 group was stronger than those of 10%,20%SSBH groups(P<0.05), while there was no significant difference between 30%SSBH and sp600125 group(P>0.05); for the decomposed formulas groups, there was no significant difference between C1 and 10%SSBH group(P>0.05), apart from that, all of them were lower than SSBH groups and sp600125 group(P<0.05).ConclusionSSBH has certain inhibitory effects on the invasion and migration of RASFs and OASFs, which is likely resulted from its inhibiting on JNK signal pathway. Meanwhile, C1 is the strongest disassembled prescription of SSBH inhibiting migration and invasion.Part 3 Inhibited effects of SSBH on cell migration observed by Transwell experimentalPurposeTo investigate the influence of SSBH on migration of SFs originated from RA and OA, and appraise the best simplified separated prescription.MethodTranswell migration assay was performed to count the number of SFs through transwell 12 hours later. Each assay includes eight groups:NS group as control, SSBH groups(concentrations of 10%,20%,30%), decomposed formulas groups(Cl, C2, C3, concentrations of 20%), sp600125 group. All samples were detected in triplicate.ResultCompared with NS group, the number of other seven drug intervention groups passed through the membrane significantly reduced (P<0.05), SSBH group enhanced the inhibitory effect with enrichment of concentration(>=0.935; P=0.000); cell number of sp600125 group was obviously less than SSBH groups (P< 0.05), but there was no significant difference (P> 0.05) compared with 30% SSBH group; Inhibition of dismantle square groups were weaker than SSBH group (P< 0.05), but there was no significant difference (P> 0.05) between the C1 and 10% SSBH group.ConclusionSSBH has certain inhibiting effect on migration both RASFs and OASFs to pass through membrane and the most obvious role shows at concentration of 30%. At the same time, C1 group has the strongest inhibitory effect among the three simplified separated prescriptions.Part 4 Proteins expression related to cell migration and EMTPurposeTo explore the possible mechanism of SSBH inhibiting cell migration by detecting the expression of marker proteins and mRNA level involved in cell migration and EMT.MethodEight groups were set up, namely:NS control group, drug-contained serum groups of SSBH with different concentrations (10%,20%,30%), decomposed formulas groups of SSBH (C1, C2 and C3), sp600125 as positive control group. Total proteins and RNA from RASFs intervented by different treatment groups for 48 h were extracted, then Western blot analysis was carried out to detect proteins expression related to cell migration (integrinpl, cadherin-11) and EMT biomarkers (E-cadherin, N-cadherin, Zo-1, vimentin). Meanwhile qrt-PCR detected mRNA transcription of skeleton proteins (Vinculin, Paxillin, Centrin) and E-cadherin, N-cadherin, Zo-1, vimentin. All samples were detected in triplicate.ResultTotal proteins and RNA from RASFs were extracted successfully, and expression of integrinβ1, cadherin-11, E-cadherin, N-cadherin, Zo-1 and vimentin were qualitative detected by Western blot, but without quantitative results. RNA amplified productions of vimentin and GAPDH were preliminary tested by PCR. ConclusionThe expression on marker proteins and mRNA level related to cell migration and EMT of RASFs has preliminary detected clearly. Farther more, quantitative determination could be performed. |
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