| BackgroundRheumatoid Arthritis (RA) is a kind of autoimmune disease characterized by Joint synovial immune pathological inflammation, whose main pathological change is synovial hyperplasia and erosion of the articular cartilage and bone, eventually leading to joint destruction and malformation. Its Pathophysiological basis is the proliferation of the Synovial fibroblasts (fibroblast-like synoviocyte, FLS) and infiltration of inflammatory cells combined with angiogenesis, and the proliferation of FLS is the core event in the progression of RA. At present, there are two main mechanisms of synovial hyperplasia, the one is the passive hyperplasia, which is driven by a variety of inflammatory factors, where the tumor necrosis factor a (TNF-a) plays a major role; the other one is active hyperplasia, which is due to disorder of the cell cycle regulation of the FLS, leading to the synovial tissue transforming to a tumor-like growth. The RA-FLS, compared with the normal and other arthritis synovial cells, has demonstrated obvious characteristics of transformed cells. The treatment targets of RA in recent years have gradually focused on inhibiting the synovial hyperplasia and bone erosion, so as to suppress the progression of the RA. In recent years, the biological treatments of RA have made great achievements, which is targeting the inflammatory factors promoting the passive hyperplasia. TNF-a antagonists, widely used in treatment of RA, could significantly control symptoms of RA, and partially block the synovial hyperplasia; Recombinant IL-l,a small-molecule antagonists, has been applied in the clinical treatment of RA, Other biological medicines, such as recombinant human IL-6and IL-17,are still in clinical trials, also achieved good therapeutic effects. At the same time, the transgenic therapies aimed to inhibit the active hyperplasia of RA FLS are in the phase of experiments, which also showed good therapeutic effects. For example, the proliferation of RA-FLS and cartilage erosion can be effectively inhibited and the abnormal synovial tissues can be eliminated, when transfecting the apoptosis-associated genes, such as the genes of fas and fasl associated death decided protein(FADD),inhibition protein of NF-kB (IkappaB) or negative mutants of proto-oncogene raf and myc.Rheumatoid arthritis (RA) belongs to the category of "bi disease" and "lijie" in TCM. The pathogenesis of active RA is defined as the pattern of hot and damp-heat impediment or accumulation of phlegm and blood stasis. Based on its pathogenesis and our clinical experiences, we developed the SanShui BaiHu Decoction(SSBH),which is evolved from Rhinoceros horn powder in<Qian Jin> and BaiHu formulae in<Guang Ji>.Its major active constitutes containing three functions:the components with the function of removing toxicity and dampness are rhizome rehmanniae, cornu bubali, cortex moutan, gypsum rubrum, anemarrhenae, artemisia apiacea; Components with heat dewetting benefits of water swelling are olygonum cuspidatum, coix seed, semen brassicae, lovage, asarum; Components with activating meridians to stop pain are Ramulus Mori, spatholobus suberctu, centipede, scorpion, earthworm, dulcamara, glycyrrhiza.The SSBH decoction has been widely applied in our department to treat the patients with active RA,OA,adult Still’s disease and juvenile rheumatoid arthritis (including system type and partial joints type). and achieved good therapeutic effects. Our team have conducted an extensive experimental study on the pharmacological mechanism of SSBH. our previous studies demonstrated that SSBH has an effects on immune inflammatory reaction of RA in a manner of multi-channel and multi-level, which not only has the effects of anti-inflammatory and analgesia and immunosuppression, also has an important function of inhibiting the proliferation of synovial fibroblasts. Our previous researches demonstrated that SSBH can suppress the expression of TNF-a and inhibit its downstream signal pathway of NF-kβand p38/MAPK, which may be the potential pharmacological mechanism of SSBH in inhibiting the proliferation of RA-FLS. However, SSBH may have several pathway in inhibiting proliferation of RA-FLS, its accurate pharmacological mechanism still need further research.Proteomics refers to explore the biological properties of proteins in the large scale, including protein expression, post-translational modification, protein and protein interactions. so as to comprehensively understand the mechanism of diseases and processes of cell metabolism. proteomics attempts to compare the differences of protein expression between the physiological and pathological conditions, and to classify and identify the related proteins. More importantly, proteomics can analyze the interaction between proteins and their functions.Proteomics technology is a series of newly developed research methods based on development of the proteomics research, which have been widely used in exploring protein biochemistry properties. This emerging technology have facilitated us to clarify the occurrence, development and outcome of the diseases and find new therapeutic targets. There are three main techniques in proteomics research including two-dimensional electrophoresis(2-DE), mass spectrometric, computer image analysis and large-scale data processing technology, which have bring a new research method for RA.. In recent years, scholars at home and abroad have carried out extensive researches on the type and concentration of protein expression in the synovial fluid, blood serum, plasma, synovial tissues and Synovial fibroblasts of the patients with RA applying two-dimensional electrophoresis(2-DE) and mass spectrometry techniques. and have obtained several uniquely expressed proteins in RA patients and elucidated their functions, which may provided potential biomarkers for early diagnosis of RA.Proteomics methods have been widely used in the research of traditional Chinese medicine(TCM). Using these methods to screen the chinese medicine in a multi-target high-throughput manner can reveal the relationship between the protain expression and the pharmacological mechanism of the TCM.On the other hand,elucidate the pharmacological targets and process of the TCM by comparing the protein expression profile,further clarify the active ingredients and the synergistic relationship among the ingredients,so as to achieve the optimal combination of the compounds.Pribvious studies laid a solid foundation for us to explore the pharmacological mechanism of SSBH in treatment of RA using proteomics technologies. In the past experimental researches on the mechanism of TCM in improving the condition of RA, the influences of TCM and its compounds on the proliferation of in vitro cultured synovial cells, cytokines expression and signaling pathways have been observed. However, exact pharmacological targets had not been screened. Our research is purposed to screen the protein targets of SSBH in treatment of RA. The Chinese compound medicines have multi-targets in view of the pharmacological effects, which is the most important difficulty in pharmacological studies of TCM. In order to narrow the screening scope, we focus on the pharmacological process of SSBH in inhibiting the proliferation of RA-FLS to screen the protein targets of SSBH, which not only highlights the advantages of SSBH, also greatly increased the technical feasibility.In this study, we investigate the effects of the traditional Chinese medicine SanShui BaiHu Decoction(SSBH) on the proliferation of fibroblast-like synoviocytes(FLS) derived from the rheumatoid arthritis(RA) and osteoarthritis (OA) patients. The fibroblast-like synoviocytes(FLS) were cultured in DMEM with20%serum containing SSBH or normal saline as a control group. The expression of IL-6and IL-17in the supernatant was measured and the expressed protein images of the FLS were obtained using the technologies of two-dimensional electrophoresis and mass spectrometry after the FLS were treated with SSBH. The unique protein spots compared with the normal saline group were screened and identified, so as to find the therapeutic targets for RA and OA and explore the possible mechanism of the SSBH in treating of the RA.Chapter I:The effects of SSBH on the proliferation of fibroblast-like synoviocytes (FLS) derived from RA patientsPurposeTo investigate the effects of SSBH on the proliferation of cultured fibroblast-like synoviocytes(FLS) and explore its potential mechanism in treating the RAMaterials and MethodsSynovial tissues were collected from6patients suffer from OA or RA diagnosed in Spinal Orthopedic department of southern hospital,and randomly divided into two groups(3cases in each group).Tissue pieces were cultured in DMEM with20%FBS to obtain fibroblast-like synoviocytes(FLS).The FLS were identified by inverted microscope, flow cytometry and immunocytochemistry staining. And then the FLS cultured for3～6generations were recruited in the experiments. The recruited FLS were cultured in DMEM with20%serum containing SSBH with different concentration10%-30%) for different culture period (1,2,3d).then the proliferation of FLS was detected by MTT assay, and the growth curve of FLS was drawn. The FLS cultured with serum containing leflunomide or normal saline as a positive or negative control respectively.Statistical analyses:Data were analysed using the Statistical Package for the Social Sciences(SPSS13.0) for Windows (SPSS Inc., Chicago, IL, USA). Values are expressed as mean±SD. Comparison of the means for different groups, one-way ANOVA was applied, and for the two groups, Bonferroni was applied. A P value of <0.05or less is considered as statistically significant.Results1. Primary culture of FLSThe FLS can be observed at the edge of The tissue when cultured for2-3days, with the characteristics of FLS.7days later the FLS demonstrated a rapid growth, and reached70%-80%confluence in2-3weeks.2. The identification of FLSThe cultured FLS were conformed with its characteristics when observed in inverted microscope, Immunocytochemistry staining with antibody of CD86and vimentin demonstrated that the Vimentin is positive and the CD68is negative, which is consistent with previous reports. Flow cytometry detected the percentage of FLS with the marker of CD55+is (98.4±2.29)%.3.Detection of the proliferation of FLS by MTT assayMTT assay demonstrated that SSBH can inhibit the proliferation of FLS with a dose and time dependent manners. The proliferation of FLS treated with SSBH in concentration of10%,20%and30%for72hours can be significantly inhibited, he inhibition rates of RA-FLS are11.7%,42.9%,42.7%(p<0.001)and the OA-FLS are24.9%,47%,48%respectively(p<0.001). FLS treated with20%SSBH for24,48or72hours, the inhibition rates of RA-FLS are11.3%,28.4%,42.9%and the OA-FLS are11.7%,33.9%,47%respectively(p<0.001); The proliferation of FLS treated with leflunomide(LEF)in concentration of10%,20%and30%for3days also can be significantly inhibited, the inhibition rates of RA-FLS are9.9%,39%,38%and the OA-FLS are26.8%,40%,40.8%respectively (p<0.001),FLS treated with20%leflunomide (LEF) for24,48or72hours, the inhibition rates of RA-FLS are15.5%,22.3%,39%and the OA-FLS are11.2%,26.1%,40%respectively(p<0.001); The proliferation of FLS derived from both RA and OA can be inhibited by SSBH and LEF after treated for48hs and reach the peak inhibition for72hs. There is no significant difference of the inhibition rate between the groups of SSBH and LEF(p>0.05).ConclusionsThe proliferation of fibroblast-like synoviocytes(FLS) can be effectively inhibited by SSBH, which may be the potential mechanism of SSBH in the treatment of rheumatoid arthritis (RA).Chapter Ⅱ:The effects of SSBH on the expression of IL-6and IL-17PurposeTo investigate the effects of SSBH on the FLS in expression of IL-6and IL-17and explore the mechanism of the SSBH in treatment of rheumatoid arthritis(RA).Materials and Methods Tissue pieces were cultured in DMEM with20%FBS to obtain fibroblast-like synoviocytes(FLS).The FLS were identified by inverted microscope, flow cytometry and immunocytochemistry staining. And then the FLS cultured for3-6generations were recruited in the experiments. The recruited FLS were cultured in DMEM with20%serum containing SSBH for72hs and collected the supernatant. The FLS cultured with serum containing leflunomide or normal saline as a positive or negative control respectively. The expression of IL-6and IL-17in the supernatant was detected by ELISA following the instruction.Statistical analyses:Data were analysed using the Statistical Package for the Social Sciences(SPSS13.0) for Windows (SPSS Inc., Chicago, IL, USA). Values are expressed as mean±SD.A p value of <0.05or less is considered as statistically significance.ResultsThe FLS expressed IL-17can be effectively inhibited by SSBH and LEF with a significant difference compare with normal saline group(p<0.05)h. However, there is no significant difference between the SSBH and LEF(p>0.05).There is no significant influence of the SSBH on the OA-FLS in expression of IL-6and IL-17.ConclusionsThe expression of IL-17of the fibroblast-like synoviocytes(FLS) can be effectively inhibited by SSBH, which may be the potential mechanism of SSBH in the treatment of rheumatoid arthritis (RA).Chapter III:The effects of SanShui BaiHu Decoction(SSBH) on the proteomics changes of fibroblast-like synoviocytes(FLS)Purpose To investigate the effects of the traditional Chinese medicine SanShui BaiHu Decoction(SSBH) on the proteomics changes of fibroblast-like synoviocytes(FLS) derived from the RA and OA patients using the technologies of two-dimensional electrophoresis and mass spectrometry and explore the molecular mechanism of the SSBH.Materials and MethodsTissue pieces were cultured in DMEM with20%FBS to obtain fibroblast-like synoviocytes(FLS).The FLS in3-6generations were cultured in DMEM with serum containing20%SSBH and cultured with serum containing leflunomide or normal saline as a positive or negative control respectively. The total cell protein was extracted after72hs and separated by two-dimensional electrophoresis (2-DE).the silver stained gels were analyzed PDQUEST2-DE to distinguish the differences in protein spots, and the different protein spots were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).ResultsThe total cell protein was extracted and separated by two-dimensional electrophoresis (2-DE).after two-dimensional electrophoresis(2-DE), the silver stained gels were analyzed PDQUEST2-DE to distinguish the differences in protein sites, and the different protein sites were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).There are833and859protein spots in the2-DE image of the RA-FLS treated with SSBH or normal saline and833and859protein spots in the OA-FLS and control group respectively. We screened16abnormal expressed protein spots in RA-FLS, including7up-regulated proteins and9down-regulated proteins, when compared the2-DE images. There are24abnormal expressed proteins were screened in OA-FLS, including11up-regulated and13down-regulated. All the screened protein spots were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS),there are26protein spots were successfully identified. In the RA-FLS treated with SSBH, the Centrosomal, GSS, Microtubule-associated serine/threonine-protein,Desmoplakin,Interleukin-3receptor, DST and Exocyst, Centrosome-associated were down-regulated, and the Protein OS-9, Sorcin, Interleukin-1receptor antagonist and so on were up-regulated; In the OA-FLS treated with SSBH, the Sulfatase-modifying factor2,Coiled-coil domain-containing protein, Desmoplakin, Centromere-associated, Sickle tail protein homolog and acidic ribosomal were down-regulated; and the Myosin-Ic, RABGAP1L,Centrosomal,Rootletin,inosine triphosphate pyrophosphatase, Myosin-Ⅲ, Translocon-associated protein subunit delta, dioxygenase, Coiled-coil domain-containing and so on were up-regulated.ConclusionsThere are11abnormal protein spots in RA-FLS and15abnormal spots in OA-FLS after treated with SSBH. These proteins including structural proteins, signal transmission proteins, signal molecular and metabolic related proteins and so on. we speculated that these molecular may be associated with the inhibition effect of the SSBH on the proliferation of FLS and have certain relationship with the pharmacological mechanism of SSBH in treatment of RA. |
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