Screening The Target Of Sanshui Baihu Decoction Affecting On Rheumatoid Arthritis Synovial Fibroblasts By Phage Display Technology

BackgroundRheumatoid Arthritis (Rheumatoid Arthritis, RA) is a kind of autoimmune disease with the pathological center of joint synovitis, which is characterized by pathology of synovial hyperplasia, erosion of articular cartilage and bone, and leading to joint destruction or deformity at last. Proliferation of RA synovial fibroblasts (rheumatoid arthritis synovial fibroblasts, RASFs), accompanied inflammatory cell infiltration and neovascularization are the basis of synovial hyperplasia. RA joint destruction occurs mainly in the cartilage site of proliferative pannus invasion. Synovial pannus tissue can be divided into two parts, respectively immune function and erosion. The former is located in the synovial lower lining tissue, including T cells, B cells, macrophages and dendritic cells. It mainly have effects on antigen presentation, production of antibodies and cytokines, and synovial inflammation and hyperplasia related. The latter mainly refers to synovial tissue and cells close to the bone and damaged cartilage surroundings. It is composed of excessive proliferated synovial fibroblasts and pannus. In the pathological progress of RA, RASFs is activated and can produce large amounts of inflammatory cytokines, matrix proteins and tissue enzymes, causing local inflammation and destruction of articular cartilage, which in turn causes the entire joint damage. Proliferation mechanisms of RASFs are mainly divided into two types. First, inflammatory hyperplasia (passive hyperplasia) is driven by a variety of proinflammatory cytokines, among them, tumor necrosis factor-α (TNF-α) play a major role. Widely used TNF-α antagonists can significantly control the symptoms of RA, and partially blocked the process of synovial hyperplasia. Second, due to the change of biological characteristics and obstacles of cells growth cycle regulatory mechanisms, RASFs is induced into transformed cells with the characteristics of similar tumor-like transformation (initiative hyperplasia). Now, it has been found that the activated RASFs possess characteristics below:①It invades and grows similar to abnormal and limited proliferation of tumor cells.②It may be formed like a cancer tumor nests with the loss of contact inhibition in vitro.③Because of the high expression of proto-oncogenes, they can get away with normal growth control mechanisms and obtain the characteristics of excessive proliferation and invasion.④The tumor suppressor gene of mutant P53is highly expressed, and PTEN expression is significantly reduced and apoptosis is inhibited.⑤The activity of protein tyrosine kinase(PTK) is greatly increased, and it is the key enzyme in normal cells and tumor cells to transform into each other.⑥It can leave cytokines dependent pathway alone. MAPK molecule L1signaling molecules p38δ can activate RASFs only guided by the endogenous retrovirus, and secrete matrix metalloproteinases continuely, thereby leading to degradation of cartilage.⑦The invasion degree in human stromal of long term cultured RASFs is four times than the normal synovial cells. Transplantation of cultured alone passaged RASFs in immunodeficient mice still showed a strong characteristics of cartilage erosion, while osteoarthritis and normal fibroblasts does not have this feature. The evidence suggests that, RASFs have a significant feature of the biological characteristics of transformed cells, compared to other arthritic and normal synovial cells. The treatment goal of RA has increasingly focused on the blocking synovial hyperplasia and bone erosion directly in recent years, which is so-called blocking disease progression. It can delay bone erosion, block the progression of RA, and reach the therapeutic targets in RA by blocking synovial hyperplasia.RA is divided into the category of "Bi disease" in Traditional Chinese Medicine. Clinically, SSBH (Sanshui Baihu decoction, SSBH) is used to treat paralysis disease in type of wet and heat. Previous studies showed that, SSBH can alleviate RA symptoms, decrease markers of inflammation, and prevent and delay the progress of the X-ray bone invasion levels of RA. All these were proved in a rat collagen-induced arthritis (CIA) model. SSBH suppress the inflammatory process of development of synovial hyperplasia, fibrosis, inflammatory cell infiltration, cartilage and bone in CIA. It can significantly prevent CIA index destruction of the joints in X-ray pictures. Its mechanism of action includes that: It inhibits the local expression of matrix metalloproteinases (MMP-1, MMP-3), NF-κB mRNA in the joints. It inhibits the production of TNF-a, and reduces the order of p38MAPK activation in synovial fibroblasts, resulting in reduced joint tissue expression of p38MAPK. Because of TNF-a and its signal transduction pathways in cells is inhibited by p38MAPK, synovial inflammation and hyperplasia are suppressed. Matrix-metalloproteinase enzymes is an important cause in cartilage damage and SSBH can reduce cartilage damage. Proliferation-related proteins of RASFs mainly IL-1Ra, OS-9, sorcin, Centrosomal so on were inhibited by SSBH with Bidirectional protein electrophoresis. Mechanism of SSBH are undoubtedly multi-channel, multi-level intervention multiple aspects of immune and inflammatory responses in RA. SSBH has effect not only on anti-inflammatory and analgesic, but also suppressing the immune and play an important role on inhibiting the proliferation of synovial fibroblasts. And inhibiting the proliferation of synovial fibroblasts becomes the main direction of RA treatment, which suggests the prospect of SSBH is directly blocking RA progression, and still need for further research and development.Phage display technology is a kind of gene technology using peptide of the exogenous recombinant protein fused to the phage coat protein, which is widely used in drug screening targets. The synthetic oligonucleotide fragments were cloned into a random sequence of the phage surface protein genes by phage display random peptide library. A mixture of random peptides consisting of a length of random peptide phage library theoretically contains the length of all possible amino acid sequence information. With a specific target molecule by affinity panning, it can efficiently, quickly and easily select phage peptide binding to specific target molecules from phage random peptide library, and greatly simplify the screening and identification of protein expression.In this paper, RASFs proliferation inhibiting protein targets treated with SSBH was panned by subtractive screening. The screening proteins will be used to explore the mechanism of SSBH in treatment of RA, the scientific group of streamlining and clinical applications positioning (eg. positioning on improving the condition of DMARDs drugs). Precise positioning targets for blocking RA synovial proliferation mechanisms with SSBH provides a more scientific basis. It explores new ideas in the mechanism of traditional Chinese medicine syndrome treatment of rheumatoid arthritis. The efficacy of treatment of RA will be formulated as an index when the target protein activity could be detected in clinical serum or synovial fluid in the future. Meanwhile, the study also found that the new hyperplasia mechanism of RASFs. Part1The isolation and culture of RASFs and intervention of SSBHPurposeRASFs was isolated and cultured in vitro and SSBH containing serum was prepared. To determine the optimal concentration and time of SSBH inhibiting proliferation of RASFs.Method1. Synovial tissue of clinical RA knee involved patients was selected to primary culture and passage to3-4generations for experimental studies.2. SD rats were fed with boiled and concentrated SSBH for7days, and serum was centrifuged and inactivated. Then Traditional Chinese medicine SSBH serum containing was prepared.3. Direct counting method was used to explore suitable concentration of SSBH (5%,10%,20%) and days (Id,3d,5d) inhibiting RASFs proliferation. Statistical analysis was performed.ResultThree stable cell lines were obtained by tissue culture method. B-type synovial fibroblasts were the majority of fusiform cells. Direct counting method showed that SSBH drug containing serum concentration of10%,20%in the third day can significantly inhibit the proliferation of RASFs.ConclusionCell morphology gradually stabilized after the third generation of cultured RASFs. Concentration of20%SSBH containing serum in third days can significantly inhibit the proliferation of RASFs. Containing serum is dependent on the reliability of the source of serum, route of administration, dosage, standardization of blood and serum preservation and so on.Part2To screen the peptide binding to RASFs treated with SSBH by method of BRAZILPurposeTo find the polypeptide that specifically binding to Rheumatoid Arthritis synovial fibroblasts (RASFs) cultured with Sanshui Baihu decoction(SSBH).MethodRASFs that treated with SSBH and normal saline (NS) were respectively used as target cells and adsorption cells for subtraction biopanning from a phage display peptide. The panning were performed three times. After the end of the three filters,32randomly selected phage blue spots in IPTG/X-gal agar plates were amplified and purified.ResultAfter three pannings, positive phage was enriched100times (from4.0×10-5to4.6×10-3) compared with the original phage, and32phage clones were randomly selected for DNA sequence analysis.ConclusionAfter three rounds of panning, phage blue spot is visible in the plate. BRAZIL method eliminates the non-specific binding phage of RASFs, and then screens specific short peptides binding with RASFs treated with SSBH. The specific purpose of the receptor is need not to know. It is a kind of effective means to find unknown cell surface receptor and its ligands. Part3The sequencing and analysis of short peptides specific binding to RASFsPurposeTo obtain and analyze sequences of the short peptide specific binding to RASFs with intervention of SSBH.MethodThe selection of32phages were amplified, purification. Single-stranded M13phage DNA extraction kit was used to prepare ssDNA. Phages DNA were measured by gel electrophoresis. Gene sequencing was performed in Ying-Wei-Jie-Ji company in Shanghai. Sequencing primers was-96g Ⅲ5’-CCCTCATAGTTAGCGTAACG-3’. Sequencing results were analyzed to find specific binding peptide sequences.ResultAfter the selection of the32phages blue spots for protein electrophoresis, the electrophoretic bands appeared. Nucleotide sequence AGTGGTGTGTATAAGGTTG-CGTATGATTGGCAGCAT (protein sequence SGVYKVAYDWQH) that binded specifically to RASFs was highly enriched in about56%.ConclusionPositive phage was enriched effectively. By continuous use of subtractive screening strategy, specific binding targets of RASFs have more occurrences of a set of sequences. Part4Preliminary identification of affinity and homology analysis of specific binding peptides in RASFsPurposeTo identify the affinity of short peptides specific binding to RASFs, look for the protein targets and get related mechanisms of SSBH treating RA.Method1Experiment of Phage affinity was taken after sequencing, and HRP-anti M13antibody was used in ELISA to verify cell affinity.2. Homologous sequence analysis was obtained protein sequence database (BLAST) provided by the National Center for Biotechnology Information (NCBI). To submit human protein sequence data from BLAST to the SignalP3.0for the predicted proteins that RASFs secretes, and screen transmembrane proteins from human protein sequence data, by submitting the data to TMHMM2.0. The proteins related to proliferation of RASFs and pathology of RA, and other potential proteins for RASFs secreted proteins or membrane proteins, transmembrane protein are located as candidate targets.Result1. ELISA was used to identify affinity of RASFs treated with SSBH and NS. SGVYKVAYDWQH was showed the highest affinity (OD value of0.382±0.020). In control group, the affinity of NS was low (OD value of0.206±0.015), p<0.05.2. Submitting the SGVYKVAYDWQH to BLAST search for homologous sequences showed the proteins were mainly human visceral adipose serine protease inhibitor (Vaspin), myotubularin-related protein14(MTMR14), dystonin protein (DST protein), cadherin protein (CAD protein) and so on. ConclusionA high-affinity dodecapeptide specific for RASFs treated with SSBH is obtained using phage display peptide library, and the related proteins is analyzed. Other studies had indicated that Vaspin expressed high levels in the serum of RA patients, and it could be the tip to diagnose RA. But the specificity and sensitivity for diagnosis needed further study. Interestingly, we used two-dimensional electrophoresis preliminary to detect protein expression of RASFs cultured with SSBH. DST protein was low expressed in RASFs cultured with SSBH. And other studies also have shown that, CAD-11protein mediates inflammation of RASFs and cartilage degradation in mouse model of arthritis inflammation. So we think SSBH maybe play an important role in RASFs through inhibiting the expression of Vaspin and CAD protein. Meanwhile, the protein of DST and CAD were associated with cell structure, movement and migration. Whether SSBH can intervene athletic ability, cell migration of RASFs, and block the initiative proliferation of RASFs, requires further study.