Anti-inflammatory Activity Of Hydrostatin-SN1 From Sea Snake Venom On Colitis Model Of IL-10 Gene Knock-out Mice

Background: Hydrophis cyanocinctus is one of the traditional Chinese medicines.It is often used for rheumatism,limb numbness,joint pain,hemorrhoids,stroke and other illness.However,the details of the active ingredients and mechanism are still unknown.In the early stage,the T7 phage display technology was performes,andwe selected the human tumor necrosis factor-type I receptor(hrTNFR1)as a substrate,established a phage display library of the genomic DNA of the genus Hydrophis cyanocinctus venom,and finally a peptide containing 22 amino acid was screened.SPR analysis showed that the short peptide(Hydrostatin-SN1)could bind to two related receptors of TNF-α,and the binding dissociation constants with TNFR1 and TNFR2 were 32 μM and 220 μM,respectively.Cell experiments and animal model experiments showed that Hydrostatin-SN1 has anobvious anti-inflammatory activityInflammatory Bowel Disease(IBD)is a chronic inflammatory disease that affects the idiopathic ileum of the ileum,rectum,and colon.It is mainly divided into Ulcerative Colitis(UC)and Crohn’s disease(Crohn’s Disease,CD).The main clinical symptoms are repeated abdominal pain,diarrhea,abdominal mass,mucus and bloody stools,intestinal obstruction,intestinal perforation,weight loss,etc.In addition,various degrees of systemic symptoms can be present.In recent years,the incidence rate in Asia,including China,has shown a linear upward trend,which has seriously affected the quality of life and living standards of patients.IBD has become one of the research hotspots of digestive diseases,but there are still no effective drugs for IBD treatment in clinical treatment.OBJECTIVE: The purpose of this study was to investigate the anti-inflammatory activity of the anti-inflammatory active peptide Hydrostatin-SN1 of the snake venom to the IKB disease model of IL-10 knockout mice.And to provide a research basis for screening candidate drugs for treatment of IBD.Methods: 1.Establishment of colitis model: Establishment of a model of spontaneous colitis in IL-10 knockout mice.2.Administration of the drug: The body weight of the mice is reduced to 80% of the initial body weight,and the animals are randomly divided into groups and administered for treatment.The dose was calculated based on the body weight of the mice,while the symptoms of the mice were observed and recorded daily and the disease activity index was also evaluated.A total of 8 groups: normal control group,model control group,negative control random peptide group,positive control SASP group,positive control IFX group,Hydrostatin-SN110 μg/kg,50 μg/kg,250 μg/kg group.Mode of administration: The model group was intraperitoneally injected with Nacl saline at a dose of 100 μl/20 g/d.Hydrostatin-SN1 group was intraperitoneally injected at doses of 10 μg/kg,50 μg/kg,250 μg/kg;negative control random peptide group,intraperitoneal injection,dose of 400 μg/kg;positive control SASP group,irrigation For gastric administration,the dose was 400 mg/kg;the positive control IFX group was intraperitoneally injected at a dose of 5 mg/kg.The control group was fed normally.3.Sampling: After 40 days,Real-time PCR was used to detect the gene expression of inflammatory factors in the colon of mice.The colon tissue of mice was fixed in paraffin and embedded in paraffin-embedded sections.Hematoxylin-eosin(H&E)staining was performed to observe the inflammatory condition of mice colon tissue.Mice colon tissue was fixed in formalin and embedded in paraffin,and immunohistochemistry was performed to detect the expression of TNF-α and FOXP3.RESULTS: The study showed that in the model of spontaneous colitis in IL-10 knockout mice,mice treated with hydrostatin-SN1 10 μg/kg/d,50 μg/k/d,250 μg/kg/d,in each treatment group and positive control SASP group,the symptoms of the mice in the positive control IFX group were significantly better than the model group.presented a significantly better diseases symptoms compaired with the model group.The body weight changes of mice,the disease activity index(DAI),etc.,and the symptoms of colonitis were significantly better than the model control group.The therapeutic effect of the Hydrostatin-SN1 50 μg/kg/d group was comparable to that of the positive group IFX,and the random group had no disease-reducing symptoms;at the same time,mice treated with Hydrostatin-SN1 10 μg/kg/d,50 μg/kg,250 μg/kg/d,their colon length and spleen index were also significantly relieved.The dose of Hydrostatin-SN1 50 μg/kg/d was the best,which was equivalent to IFX group.The results of H&E staining also showed that the dose of Hydrostatin-SN1 50 μg/kg/d could significantly decrease the degree of inflammation,reduce the extent of the colon;Real-time PCR confirmed Hydrostatin-SN1 50 μg/kg/d dose group had a significant inhibitory effect on the expression of related pro-inflammatory inflammatory factors(TNF-α,IL-6,IL-1β,IFN-γ)in colon tissue,and the inhibitory effect was comparable to that of the IFX group.The results of immunohistochemistry showed that the dose of Hydrostatin-SN1 50 μg/kg/d could significantly reduce the expression of TNF-α and promote the expression of FOXP3.Conclusion: Hydrostatin-SN1 can effectively alleviate the symptoms of colitis in IL-10 knockout mice.