Screening,Activity Evaluation And The Preliminary Research On The Mechanism Of Action Of Sea Snake Venom Anti-inflammatory Active Peptide Hydrostatin-TL1

Object:Modern medicine has reported the effect of anti-inflammation,anticoagulation,anti-tumor and analgesia about snake venom.In traditional Chinese medicine,these effect has been applied widely.Hydrophis cyanocinctus,as an advantageous snake species,has been reported to exhibit anti-inflammatory effects in rheumatoid arthritis and other autoimmune diseases for a long time.TNF-α is an important mediator of inflammation,and is also involved in the control of autoimmunity.This study aimed to select a specific TNF-α-binding peptide from Hydrophis cyanocinctus through phage display technology,and to value its anti-inflammatory activity and elucidate its anti-inflammatory mechanisms on the in vitro and in vivo levels.Methods and Results:We screened the T7 phage library constructed by Hydrophis cyanocinctus snake venom gland and find a peptide,named Hydrostatin-TL1,which can combined to TNF-α specifically.The result of ELISA show that Hydrostatin-TL1 can combined to TNF-α and inhibition the combination between TNF-α with TNFR1.The result of MTT show that Hydrostatin-TL1 can inhibit the toxic of TNF-α,and the rate of inhibition can reach 80%when the concentration of Hydrostatin-TL1 was 100nM.The result of Western blotting show that Hydrostatin-TL1 can inhibit the activation of NF-κB and MAPKs signaling and when the concentration of Hydrostatin-TL1 was 800nM,we can see obviously down regulation of the level of phosphorylation of the protein which associated with NF-κB and MAPKs signaling.The result of in vivo experiment also show that Hydrostatin-TL1 can relieve the inflammation caused by TNF-α.Conclusion and meaning:We established a protocol to screen the T7 phage display by TNF-α.Our investigation shows that Hydrostatin-TL1 exhibits significant anti-inflammatory activity in vitro and in vivo.This paper provide a new way to find similar active ingredients from the venom of Hydrophis cyanocinctus to us,and also provide a new way of how to find and identify the active ingredients in a complex system to us.