Screening The Antagonist Of PGE2 Receptor Using Phage Displaying And Study On Its Anti-inflammation Activity And Mechanism

Rheumatoid arthritis (RA), an autoimmune disorder, is a chronic relapsing inflammatory disease that affects mainly the diarthroidal joints which affects 0.5-1% of the adult. The aberrant autoimmune response in RA is centered within the synovium membrane lining the joint. In response to the inflammatory cytokine milieu, the mesenchymal and fibroblast-like synovial cells proliferate and become a locally invasive malignancy that, if unchecked, can irreversibly destroy cartilage and bone.RA is characterized by systemic and local inflammation resulting in cartilage and bone destruction. Nonsteroidal anti-inflammatory drugs (NSAIDs), which represent an effective therapy for treating RA, elicit their effects by inhibiting cyclooxygenase (COX) activity and blocking the downstream production of prostanoid, including prostaglandin E2. PGE2 is the most important molecule in the pathogenesis of rheumatoid arthrititis, which can be secreted by a lot of cells including macrophage cells and synovial fibroblast and so on. Moreover, PGE2 is a major cyclooxygenase product in a number of physiological settings. Phage displaying technology is a high-throughout screening (HTS) assay for studying the binding site of ligands and receptors, searching for the ligand molecules with high affinity and detecting the dimensional structure epitopes of unknown protein, which have abroad application respect in the study of protein molecules reorganization, development of new vaccine and new drug. Moreover, it plays an important role on the simulating epitopes, identifying the major animos between proteins interaction. The Ph.D-C7C is to link the genotype of short peptide displaying on the phage surface with the phenotype closely. A big advantage of phage display, accounting for its widespread use, is the convenience with which libraries can be selected for target-specific binders. Rapid enrichment of library clones encoding binding polypeptides is achieved by phage library incubation with a target followed by removal of the non-reacting phage and amplification of binder clones in the host bacteria. Usually, three to five rounds of panning are sufficient to enrich for binding peptide sequences.In our study, we screened the specified peptide with PGE2 as a target using phage displaying, because of high specific and affinity of this technology, we obtained the circular 7-mer peptide which can bind specifically with PGE2 and have anti-inflammation in the rat adjuvant arthritis and play an antagonist role on EP4. Through the rat adjuvant arthritis models, we found that the circular 7-mer peptide could reduced arthritis severity and footpad swelling and its anti-inflammation mechanism is on one hand to inhibit the expression and migration into nucleus of NF-κB and on the other hand to inhibit the proliferation of synoviacyte and induce their apoptosis. Because of the advantages of this drug such as small molecule, high specific, and fewer doses, it might be taken into the clinic assay, and open a new therapy method for treating RA. The project includes three parts as following.PartⅠScreening the antagonist of PGE2 receptor1. The rate of recovery of phageAfter five rounds screening, we can found that recovery rate increased from 1.5×10-8 in the first round to 1.8×10-5, 1.8×10-4, and 4.1×10-4, until the fifth round, the rate only increase one time than the fourth round. There have no notable change, which demonstrates that this affinity screening has been intended to saturation and the enrichment result is very good, and we can obtain the high affinity circular 7-mer peptides by this method.2. The identification of positive cloneIn order to compare the binding activity of the different peptide sequences, ELISA was performed using 96 well plates coated with phage library. If the absorption of the hole coated with phage is 2.1 times to negative control, we consider positive. A total of 23 individual clones were randomly selected from the firth round of panning with 22 of these clones being found to be positive in both the simple ELISA assays. We can conclude that this screening process is highly specific and affinity.3. The sequencing and analysis of phage positive cloneThe 8 positive phage clones in the ELISA assay were analyzed by DNA sequencing. Among them, 4 clones have the same sequence and the others have the conserved motif, which can certify that the method of obtaining the PGE2 binding peptides is applicable and the circular 7-mer peptides also have certain conversed characters. Considered the factor of EP4 in RA, we select the sequence which has arginine and threonine to sythesis because the major interactional aminos between PGE2 and EP4 are arginine and threonine according with the reference at presently.PartⅡAnti-inflammation activities of circular 7-mer peptide on rat adjuvant arthritis1. The level of joint swellingTo examine whether circular 7mer peptide therapy could be effective in the treatment of AA, rats were given a daily intraperitoneal injection with various dosages of circular 7mer peptide or saline for 21 days. All animals were treated on the seventh day after immunization. Although disease started on different days after immunization, we did not observe a relation between clinical response and time of onset of disease. All groups treated with circular 7-mer peptide showed decrease in paw swelling compared to controls.2. The effect on the cytokineSynovial fluid level of IL-1βand TNF-αwere determined by ELISA. IL-1βreduced from 1133.89pg/ml in model to 534.35pg/ml, almost to the normal level after treating with screened peptide. While TNF-αreduced from 59.41pg/mlto 37.63pg/ml (P<0.05), and there has no dose dependent which accord with the results of joint swell which suggests that all dosages were in the therapeutic range with regard to effects on clinical signs of arthritis. 3. The effect of circular 7mer peptide treatment on synovial inflammation was also assessed by histology.Wild-type mice show no inflammation and destruction at all by H&E from histopathology of the ankle joints, while peptide treatment groups showed fewer destruction and infiltration compared with model groups. A remarkable reduction in the number of inflammatory cells was found for all circular 7mer peptide treated groups statistically significant (P< 0.01) reduction of inflammatory cells was observed in rats treated with the 20μg/kg or 40μg/kg doses; there was also a tendency towards decreased cell inifiltration in the rats treated with the 10μg/kg dose.PartⅢThe anti-inflammatory mechanism of circular 7-mer peptide1. Effect on SplenocytesThrough analysis the spleen/weight (g/g), thymus/weight (g/g), we can find that circular 7-mer peptide may reduce the thymus index. With regardness to spleen index, it has no reduction at any dose, but promotes spleen hyperplasia. Moreover, splenocytes proliferation response was measured by MTT incorporation methods, and the proliferation ability was expressed as stimulation index (SI). Results indicate that circular 7-mer peptide supress splenocyte proliferation induced by ConA, but have a suppress activity at lower dose without ConA and gently promote at high dose.2. Effect and mechanism of circular 7mer peptideInhibitors on Synovial Fibroblast Proliferation Through MTT assays, we observe that circular 7mer peptide could inhibit the synovial fibroblast proliferation compared with non-circular 7mer peptide. It was the evident that PGE2 may be a mitogen which can promote synovial fibroblast proliferation. Therefore, this peptide through screening phage library with PGE2 as a target can inhibit PGE2 to interact with its receptor EP4. The result of cycle detected using FACS also supports this suppose. We also observe that this peptide can induce the rabbit synovial fibroblast apoptosis with Hoechst staining. Therefore, the results stat that the anti-inflammation mechanism of this peptide at one hand is to inhibit synovial fibroblast prolifertion, at other hand is to induce its apoptosis.3. The expression of NF-κB by immunohistochenmistry analysisThrough immunohistochemistry staining, Results indicated that the expression of NF-κB decrease after peptide treatment and it mostly lies in the cytoplasm, while in the model group; the expression of NF-κB mainly lies in the nucleus. Therefore, this peptide can inhibit the expression and nuclear translocation of NF-κB through competition with EP4 to bind with PGE2.In this study, we obtained a circular 7-mer peptide which can bind with PGE2 through phage displaying technology and exert antagonist of EP4. We use the rat adjuvant arthritis to analysis the cytokines concentration and histology by H&E staining, indicates directly and indirectly that this peptide can exert an anti-inflammatory effect. Moreover, we also prove that the mechanisms of anti-inflammation is to inhibit the function of synoviol fibroblasts and also to intervene the NF-κB and AKT signal transduction. With respect to its potential as a drug target in treating RA, our findings indicate that selective inhibition of EP4 of PGE2 will be effective. It is of interest to see if future studies designed to investigate the effects. However, many problems still need to be solved. There is a long way to go before practical use in clinic.