The Study Of Anti-inflammatory Effect In Vivo/in Vitro And Mechanism Of Hydrostatin-SN10 Derived From Hydrophis Cyanocinctus

Objective:TNF-α is an important inflammatory factor,the TNF-α/TNFR signaling pathway plays an important role in a variety of inflammatory diseases.Previously,this research group obtained an anti-inflammatory peptide,which have target selectivity of TNFR1,named Hydrostatin-SN10 by screening the phage display library constructed by cDNA of the venom gland of Hydrophis cyanocinctus.This study intends to use the macrophage inflammation model and IL-10 knock out mouse colitis model to study the anti-inflammatory effects of Hydrostatin-SN10 in vitro and in vivo,and to study its anti-inflammatory mechanism.Methods:1 Evaluation of anti-inflammatory effect of Hydrostatin-SN10 by cell model RAW264.7 and BMDM in vitro:Proliferation activity of RAW264.7 cells induced by LPS was detected by CCK-8 assay;Production of Nitric oxide and the expression levels of inflammatory factors of RAW264.7 and BMDM cells induced by LPS was detected by Griess reagent and RT-PCR.2 Study on anti-inflammatory mechanism of Hydrostatin-SN10:Levels of IκB-α was detected by Western Blot;The nuclear transfer levels of NF-κB were detected by immunofluorescence;The expression level of the NF-κB regulatory gene was detected by RT-PCR.The unlabeled Hydrostatin-SN10,Recombinant TNF-α,Anti-TNFR1 antibody was incubated separately with the cell which was combined with fluorescence labeled Hydrostatin-SN10 for 1h to observe fluorescence intensity.3 Evaluation of anti-inflammatory effect of Hydrostatin-SN10 in vivo by colitis model in IL-10 knockout mouse:After establishment of IL-10 gene knockout mouse colitis model,the changes of body weight,colonic length and splenic weight index of mice after Hydrostatin-SN10(800μg/kg)administration were observed to evaluate the effects on colitis symptoms in mice.The colonic histopathology of mice was observed by HE staining.The levels of TNFαand Treg cells in colon tissue of mice were observed by immunohistochemistry.Results:1 Anti-inflammatory activity effect of Hydrostatin-SN10 in vitro:The results of RAW264.7 inflammatory cell model showed that Hydrostatin-SN10(100μg/ml)inhibited LPS-induced cell proliferation activity and was not cytotoxic.Hydrostatin-SN10 also inhibits LPS-induced NO production and expression of inflammatory factors.In the primary macrophage BMDM,the level of NO and the expression level of inflammatory factors were inhibited.2 Anti-inflammatory mechanism of Hydrostatin-SN10:Hydrostatin-SN10(100μg/ml)inhibit TNF-α-induced degradation of IκB-α,nuclear transfer of NF-κB and the the expression level of the gene which NF-κB controled.The results of fluorescently labeled Hydrostatin-SN10 indicated that Hydrostatin-SN10 binds to TNFR1 on the cell surface.3 Anti-inflammatory activity effect of Hydrostatin-SN10 in vivo:The results of IL-10 knockout mouse colitis model showed that the body weight of mice increased significantly,the length of colon increased and the spleen weight index decreased after treatment with Hydrostatin-SN10(800μg/kg).The results of HE staining showed that the colonic mucosal integrity of the Hydrostatin-SN10(800μg/kg)treatment group was significantly increased and the crypts were aligned.The results of immunohistochemistry showed that the level of TNF-α in the colon tissue of the mice in the Hydrostatin-SN10(800μg/kg)treatment group was significantly decreased,and the level of Treg cells was increased.Conclusion:Hydrostatin-SN10 has significant anti-inflammatory activity in vitro and in vivo,and Hydrostatin-SN10 inhibits the biological activity of TNF-α by binding to TNFR1 on the cell surface.