Screening Of Anti-Inflammatory Di-Peptides Derived From Ovotransferrin By Simulated Peptide-Cut And Study Of Their Anti-Inflammatory Mechnism

The intestinal system is an important barrier to protect and maintain the body's immune balance.Intestinal epithelial cells are the first line of defense of the gut system.When the body's intestinal system is attacked by foreign antigens,microbes and harmful molecular substances,the relevant immune cells are activated to help resist these damages.However,the body's immune system has limited resistance to foreign antigens.When the body can not resist or can not clear these stimuli,it is likely to cause chronic intestinal inflammation or intestinal tissue damage,which will seriously affect human health.In view of the limited therapeutic efficacy and side effects of clinical drugs,it has become a research hot spot to discover new,safe and natural activities with intervention or alleviation of inflammatory bowel disease.Many studies have shown that egg ovotransferrin(OVT)exerts some biological activity,such as antibacterial,anti-viral and anti-inflammatory.Nevertheless,there are relatively few studies on its anti-inflammatory substance basis and anti-inflammatory mechanism.Therefore,in order to provide a basis for the high utilization of egg ovotransferrin and a new anti-inflammatory strategy based on egg ovotransferrin-derived polypeptide,it is necessary to explore the basis of its anti-inflammatory substances and anti-inflammatory mechanism.Firstly,all di-peptide sequences(including MK,IL,GR,CR,TK,LK,VK,SK,FL,GK,CQ,NK,IR,LL,ER,MM,VR,and HC)were obtained by using peptide-cutter software,pepsin and trypsin selected to mimic cleavage of ovotransferrin.Di-peptides sample with a purity of >99% were obtained by artificial synthesis technique.TNF-?-induced Caco-2 cell models was established to screen the anti-inflammatory peptide derived from stimulated peptide-cut OVT,and IL-8 was selected as an indicator.The results showed that OVT-derived dipeptides CR,FL,HC,LL and MK inhibited the secretion of IL-8 in Caco-2 cells at some concentrations(P <0.05).Real-time PCR results showed that CR,FL,HC,LL and MK significantly inhibited the gene expression of IL-8,IL-1?,IL-6,IL-12 and TNF-?,while up-regulate the gene expression of anti-inflammatory factor IL-10 to reduce inflammatory reaction.Western blot analysis showed that CR,FL,HC,LL and MK inhibited phosphorylation of I?B,while CR and HC inhibited phosphorylation of p38 and JNK.These results indicate that CR and HC may inhibit cellular inflammatory response through regulating NF-?B and MAPK signaling pathways,while LL,FL and MK alleviate cellular inflammatory responses through the MAPK signaling pathway.We constructed Dextranssulfatesodium(DSS)to induce acute colitis in Balb/c mice.To evaluate the anti-inflammatory activity of CR,FL,HC,LL and MK in mice,the mice were measured for body weight changes,clinical scores for colitis,colon length,and TNF-? and IL-6 levels in the colon.The results showed that oral administration of these dipeptides(50 mg/kg and 150 mg/kg)significantly alleviated weight loss and clinical signs in DSS-induced Balb/c mice,and significantly decreased TNF-? and IL-6 in colonic tissues,which effectively improves DSS-induced symptoms of colonic inflammation in mice.Taken together,the OVT-derived dipeptides CR,FL,HC,LL and MK have good inhibitory effects on inflammation both in vitro and in vivo.Therefore,the OVT-derived dipeptides CR,FL,HC,LL and MK have good anti-inflammatory biological activity,which can effectively protect and maintain intestinal health.At the same time,the research provides new food-borne active ingredients theoretical basis.