Anti-inflammatory And Wound Healing Function And Mechanism Analysis Of Cathelicidin From Tylototriton Kweichowensis

Cathelicidin is an unique antimicrobial host defence peptide which is peculiar to vertebrates.They have a variety of biological functions,such as anti-microbial,anti-inflammatory,wound healing etc.Tylototriton kweichowensis is a kind of tylototriton belonging to amphibia,caudata,salamandridae.It is known that it is currently only distributed in the western part of Guizhou and Yunnan.It is endemic to China and is a second-level national protected animal.In ethnic medicine,it is often used as medicine to deal with itchy skin,burns and scalds.At present,there is no report on the research of cathelicidin from Tylototriton kweichowensis.In this research,we take Tylototriton kweichowensis as the research object and systematic study were carried out to study TK-CATH of cathelicidin host defense peptides family from Tylototriton kweichowensis.Total RNA was extracted from the skin of Tylototriton kweichowensis,and the Creator^TM SMART^TMc DNA Library Construction Kit was used to construct a c DNA library.By designing the primers based on the conserved region of the precursor sequence of the amphibian cathelicidin molecule and the c DNA sequence encoding the Tylototriton kweichowensis cathelicidin molecule was obtained by PCR,we analyze and blast the c DNA sequence and the mature peptide sequence is determined.Then,the biological function of the synthesized peptide is determined.The antibacterial activity of the peptide was detected by two-fold broth microdilution method.The inhibitory effect of the peptide on pro-inflammatory factors such as tumor necrosis factor-αand interleukin-6 were detected by ELISA;The neutralizing activity of peptide to LPS derived from E.coli was detected by Toxin Sensor^TM LPS detection kit;The effect of peptide on the protein expression of MAPKs inflammatory signaling pathways activated by LPS was determined by Western blot;The proliferation of Ha CAT keratinocyte cells was detected by MTT assay.Migration of Ha CAT was detected by cell scratch assay.Wound healing activity was detected by constructing mouse full-thickness skin wounding model.The expression levels of TNF-α,IL-6,MCP-1,CXCL1 and TGF-βin mouse macrophages（RAW264.7）were detected by ELISA;The protein expression level of MAPKs signaling pathways was detected by Western blot.In addition,its antioxidant activity,cytotoxicity and hemolytic activity were also detected.A novel cathelicidin family host defense peptide coding gene was successfully obtained from the skin of Tylototriton kweichowensis by gene cloning,which encodes a new cathelicidin family host defense peptide,and it was named as TK-CATH（GGQDTGKEGETGKKK KSDNWFMNLLNKFLELIGLKEAGDDSEPFCFTCIFDMFSQ）,with a total of 55 amino acids,the peptide has 3 negative charges,a molecular weight of 6196.99.The results of antibacterial experiments show that TK-CATH did not show direct antimicrobial activity against the tested 10microorganisms including bacteria and fungi at the concentration up to200μg/ml（32.27μM）.Instead,it demonstrated potent anti-inflammatory and wound healing activities.It effectively reduces the secretion of pro-inflammatory cytokines such as TNF-αand IL-6 in mouse macrophages induced by LPS by means of inhibiting the mitogen-activated protein kinase（MAPK）signaling pathway activated by lipopolysaccharide（LPS）,And its weak LPS-neutralizing property implied that it has special anti-inflammatory target and mechanism.Besides,TK-CATH effectively induced the production of diverse cytokines TNF-α,chemokines（MCP-1,CXCL1）and growth factor TGF-βin macrophages by directly activating MAPKs signaling pathways and up-regulate the phosphorylation of ERK,JNK and P38.What’s more,it promotes the motility and proliferation of keratinocytes and exhibited effective wound healing activity in a mouse full-thickness wounding model.This implies that TK-CATH participates in both inflammatory phase and new tissue formation phase of wound repairing process.Meanwhile,TK-CATH exhibited weak but effective free radical scavenging activity,low cytotoxicity and hemolytic activity.In the present study,a novel anionic cathelicidin TK-CATH was identified from the skin of the salamander,Tylototriton kweichowensis.TK-CATH didn’t exhibit direct antimicrobial activity against the tested microorganisms.Instead,it demonstrated potent anti-inflammatory and wound healing activities.Meanwhile,TK-CATH exhibited weak but effective free radical scavenging activity,low cytotoxicity and hemolytic activity.All the results above indicate that TK-CATH is a multifunctional molecule reside in the skin of the salamander.It may play an important role in immune responses against bacterial infection and skin wound healing.