The Study Of Hydrostatin-SN10 From Sea Snake On The Anti-rheumatoid Arthritis

BackgroundRheumatoid arthritis is a kind of autoimmune diseases characterized by joint synovitis.The genetic and environmental factors are closely related to the occurrence and development of the disease.The degree of inflammation in rheumatoid arthritis is affected by inflammatory factors and the main inflammatory factors are the tumor necrosis factor-?,interleukin-6,interleukin-17,interleukin-1 and so on.A large number of studies show that tumor necrosis factor-? plays an important role in the pathogenesis of RA system.At present,the TNF-? monoclonal antibodies such as adalimumab,infliximab achieve good clinical effects,which not only can improve symptoms,but also can inhibit the destruction of joints.Due to completely close the biological function of TNF-?,the immune homeostasis and immune surveillance function are affected by this kind of anti-TNF-? monoclonal antibody drugs,which bring the prone to tuberculosis infection,produce new autoimmune diseases even induce tumor to many patients.The over expression of TNF-? in vivo and the abnormal activation of TNF-?/TNFRs signaling pathway are closely related to the occurrence and development of autoimmune diseases such as rheumatoid arthritis.With the study of the pathogenesis of RA,the research is now turning the target of treatment to TNFRs.However,compared with TNF-? monoclonal antibodies and other biological agents,the progress of TNFRs based small molecule antagonists is slowly.From the anti-inflammatory perspective,TNFR1 is mainly transfer the signal of proinflammatory and apoptosis.Selectively blocking the signal pathway of TNFR1 to close the biological function of TNF-? has become a hot topic in the drug development.At present,there has no highly selective TNFR1 antagonist for clinical application.Our group constructs a Hydrophis Cyanocinctus venom gland phage display library.Using phage display technology,we get a snake venom peptides(Hydrostatin-SN1,22AA)with anti-inflammatory activity.With the active peptide Hydrostatin-SN1 as the leading peptide.An anti-inflammatory peptide(Hydrostatin-SN10,10AA)was obtained by further structural optimization.Surface Plasmon Resonance(SPR)and microcalorimetry(MST)results showed that Hydrostatin-SN10 could interact with TNFR1 directly but not combine with TNFR2 and TNF-?.Moreover,Hydrostatin-SN10 could inhibit the interaction of TNFR1-TNF-? competitively and the intracellular NF-?B and MAPKs signaling pathway.In this study,we evaluated the anti-inflammatory effects of Hydrostatin-SN10 and PEG-SN10 which modified by PEG2000 on model of rheumatoid arthritis(CIA mice),Which will lay the foundation for the research and development of Hydrostatin-SN10 as a new type of selective TNF-? receptor inhibitor peptide drugs with independent intellectual property rights and target specific marine organisms.ObjectiveThrough the establishment of collagen-induced arthritis(CIA)model,studying the anti-inflammatory effects of Hydrostatin-SN10 and PEG-SN10 in vivo,which will lay the foundation for the development of new selective TNF-? receptor inhibitor peptide drugs.Methods1.Establishment of CIA model: Bovine II collagen type(4mg/ml)was mixed with equal volume of Freund's adjuvant CFA(4mg/ml)for zeroth days,which was not dispersed in water.Experimental mice were injected intracutaneously with 100?l of emulsion.Twenty-first days after immunization.The same concentration of CII was used as for the primary immunization.However,CII was emulsified in IFA for this immunization.The same volume of emulsion was injected intracutaneously.The model was successful at Twenty-ninth days.The mice were divided into normal group,model group,Hydrostatin-SN10 group,PEG-SN10 and infliximab.Drugs were injected intraperitoneally from that day and the model group and the normal group were injected PBS;Hydrostatin-SN10 group were injected intraperitoneally with Hydrostatin-SN10(1.6mg/kg);PEG-SN10 group were injected intraperitoneally with PEG-SN10(1.6mg/kg)and Infliximab group received intraperitoneal injection of infliximab(4mg/kg).Once a day,mice were killed on the fifty-third day.2.Observing the activity and hair condition of mice every day.Animals should be evaluated arthritis incidence per 3 days.Each paw should be scored individually on a scale of 0-4,with 4 indicating the most severe inflammation.The anti-inflammatory effects of Hydrostatin-SN10 and PEG-SN10 were analyzed apparently.3.The concentration of collagen specific antibody IgG and inflammatory factorsIL-17 in the serum were detected by ELISA and the concentration of inflammatory factors TNF-??IL-6?IFN-??IL-10 were detected by flow detection.Observing the anti-inflammatory effect of Hydrostatin-SN10 and PEG-SN10 on inflammatory factors.4.Micro-CT was used to observe the morphological changes,the degree of bone destruction and the changes of bone parameters of trabecular bone.observing the effect of Hydrostatin-SN10 and PEG-SN10 on bone destruction.5.Flow cytometry was used to detect the expression of Foxp3 in Treg cells,T helper 1(Th1),T helper 2(Th2),T helper 17(Th17)in spleen,observing the effect of Hydrostatin-SN10 and PEG-SN10 on immune aspect.6.HE staining was used to observe the changes of hind limb joints injury and inflammatory cell infiltration in synovitis.The expression changes of TNF-? in hind limb joints were observed by immunohistochemistry and the changes of osteoclasts were investigated by the phosphatase staining.Observing the effects of Hydrostatin-SN10 and PEG-SN10 on bone destruction in joint pathology.Results1.Normal mice were in good spirits,normal activities and the hair color was light and no hair removal phenomenon.After second immunization,the swelling joint of model mice gradually obvious,the mental state was bad and the activities were significantly affected.Besides,the coat color was lack luster and had the depilation phenomenon.The mental activities,hair color gloss of Hydrostatin-SN10,PEG-SN10 and infliximab were significantly reduced.Hydrostatin-SN10 and PEG-SN10 can effectively reduce the paw swelling and arthritis index in CIA mice.2.The results of serum inflammatory factors showed that the collagen specific antibody IgG and proinflammatory factor IL-17,TNF-?,IL-6,IFN-? in serum of model mice were significantly higher than that in normal mice.The group of Hydrostatin-SN10,PEG-SN10 and infliximab could not only effectively reduce the collagen specific antibody IgG and the proinflammatory factors like IL-17?TNF-??IL-6?IFN-? but also increase the anti-inflammatory like IL-10 in serum of mice.3.The results of Th cells in spleen showed Hydrostatin-SN10,PEG-SN10 and infliximab could significantly increase the expression of Foxp3 and IL-4 and reduce the expression of IL-17 and IFN-? in spleen.Hydrostatin-SN10 and PEG-SN10 may play an anti-inflammatory role by regulating the changes of Treg/Th17 and Th1/Th2,so as to inhibit the occurrence and development of rheumatoid arthritis.4.Micro-CT results showed that the articular surface was smooth and the joint structure was clearly visible in the normal group.However,The joint surface of the CIA model group was rough and the bone destruction was serious;Hydrostatin-SN10,PEG-SN10 and infliximab could significantly improve the degree of bone destruction in mice.Micro-CT of bone trabecular showed that normal trabecular structure was integrity,and CIA model group had severe bone destruction.Hydrostatin-SN10,PEG-SN10 and infliximab could significantly reduce bone trabecular bone destruction.the parameters results showed that Hydrostatin-SN10,PEG-SN10 and infliximab could significantly increase the parameters like bone mineral density(BMD),bone volume / tissue volume(BV/TV),bone area / tissue volume(BS/TV),the number of trabecular(Th.N)and the trabecular thickness but decreased the other parameters like bone surface / bone volume(BS/BV),trabecular bone pattern factor the(Tb.pf),trabecular separation(Tb.sp)parameters.These results indicated that Hydrostatin-SN10 and PEG-SN10 have significant anti-inflammatory effects.5.The results of HE staining showed that in the normal group,the joint cavity structure was integrity,no cartilage injury and no obvious inflammatory cell infiltration in the synovial joints and tissues.the CIA model group showed joint structure destruction and loss of normal joint structure,the joint gap disappeared,synovial hyperplasia and cartilage injury were serious and severe inflammatory cell infiltration was seen.The joint cavity structure was integrity and the joint damage was significantly reduced in the group of Hydrostatin-SN10,PEG-SN10 and infliximab.6.Immunohistochemical results showed that the expression of TNF-? was decreased in the group of Hydrostatin-SN10,PEG-SN10 and infliximab and showed significant anti-inflammatory effect.7.TRAP results showed that The tartrate resistant acid phosphatase decreased in the group of Hydrostatin-SN10,PEG-SN10 and infliximab.Hydrostatin-SN10 and PEG-SN10 could reduce the numbers of osteoclasts,thus reducing the erosion of bone tissue.ConclusionOur findings reveal that Hydrostatin-SN10 and PEG-SN10 have significant anti-inflammatory effects in the model of CIA.