| Defensins are important part of innate immunity in mammals.Recent studies have found that defensins have various biological functions such as anti-bacterial,pro-inflammatory and anti-inflammatory,adaptive immunity,which can be used as alternatives to antibiotics and as targets to improve disease resistance of animals.The porcine defensin 114（PBD114）was discovered in 2012 by comparative analysis of pig genomes.So far,the regulatory mechanism of PBD114 expression,biological function and its mechanism of PBD114 have not been reported.Therefore,five experiments were conducted in this study.Firstly,cloning and analyzing PBD114 gene sequence,comparing the differential expression of PBD114 gene in different breeds of pigs,exploring the regulatory mechanism of PBD114 expression.On this basis,PBD114 protein was successfully expressed,and then mice and IPEC-J2 cells were used to study the effects and mechanism of PBD114 on alleviating inflammatory damage of the intestinal barrier.The main results are as follows: Exp 1 Cloning and sequence analysis of PBD114 and differential expression of PBD114 in different breeds of pigsTibetan pig（TP）is one of the special pig species in Tibet and western of sichuan.Compared with foreign pigs,it has the advantages of resistance to crude feeding and disease.CDS sequences of PBD114 gene in tibetan pig（TP）and DLY were cloned using cDNA of TP and DLY as templates respectively.Comparative study of beta defensin 114 amino acids in Sus scrofa,Homo sapiens,Pan troglodytes and Bos indicus were analyzed by phylogenetic analysis.Results: The CDS sequence of PBD114 gene in TP and DLY pig is identical.Homology of beta defensin 114 among Sus scrofa,Homo sapiens,Pan troglodytes and Bos indicus were close;To study the expression of PBD114 in different breeds of pigs,6 60-day old TP and 6 60-day old DLY were performed to measure the expression abundance of PBD114 in pig tissues.Results: The expression of PBD114 was highest in the duodenum of DLY and in the jejunum of TP.The expression of PBD114 in jejunum,colon and lung of TP were significantly higher than DLY（P < 0.05）,however,the expression of PBD114 in the kidney of TP was significantly lower than DLY（P < 0.05）.The results indicated that PBD114 was highly conserved in different breed pigs and species,The expression level of PBD114 was significantly higher in TP than DLY pig.It is suggested that PBD114 may play an important role in the health and immune regulation of pigs and is one of the potential disease-resistant genes.Exp 2 Regulation and mechanism of PBD114 expressionTo explore the role of PBD114 in immune regulation,7-day old piglets and IPEC-J2 cell were used as in vivo and in vitro models,and challenged by E.coli K88,to explore the expression of PBD114 under immune activation.Results: E.coli K88 significantly increased the expression of PBD114 in the duodenum and ileum of DLY（P < 0.05）,and E.coli K88 also significantly increased the expression of PBD114 in IPEC-J2（P < 0.05）.The results showed that the expression of PBD114 was up-regulated under immune activation.Because TLRs are important receptors that mediate immune system activation,to explore which signal pathway regulates the expression of PBD114,IPEC-J2 cells were further incubated with different TLRs activators Pam3CSK4,Poly（I:C）,LPS and R837（TLR2,TLR3,TLR4 and TLR7）.Results: Pam3CSK4 significantly decreased the expression of PBD114 in IPEC-J2（P < 0.05）.Poly（I:C）,LPS and Imiquimod-R837 significantly increased the expression of PBD114 in IPEC-J2（P < 0.05）.The results indicated that the expression of PBD114 may be regulated by TLR2/3/4/7;NF-κB and MAPK are the downstream signal patyways of TLRs signal patyway.As we all know,the expression of genes were regulated by transcription factors.The transcription factor binding site of NF-κBp65（RELA）and AP-1（FOS and JUN）on the promoter region of PBD114 were predicted by JASPAR online.Double luciferase reporter assay comfirmed that RELA,FOS and JUN were involved in the transcriptional activation of PBD114,the intensity of regulation: RELA-JUN > RELA > RELA-FOS > FOS > JUN > FOS-JUN.the expression of PBD114 was significantly increased in IPEC-J2 under E.coli K88 challenging.However,the expression of PBD114 was significantly inhibited by the RELA inhibitor PDTC.The results further indicated that the expression of PBD114 was regulated by RELA.The above results showed that immune activation（E.coli K88 challenge）could up-regulate the expression of PBD114 gene in pigs.and TLR2/3/4/7 signal pathway were involved in the expression of PBD114 gene.Transcription factors RELA,FOS and JUN play key regulatory roles in gene expression of PBD114.Exp 3 Expression and characterization of recombinant PBD114 in E.coliTo further study the biological characteristics and functions of PBD114.The DNA sequence of mature PBD114 was inserted into the expression vector pET32（a+）,and then transformed pET32a（+）-PBD114 to Origami B（DE3）to construct the recombinant expression stain（Origami B-pET32-PBD114）for obtaining recombinant PBD114 protein.rPBD114 was induced by optimal condition（The optimal initial OD600 expression was 0.6 0.8 and 1.0,the optimal concentration of IPTG was 0.5 and 1.0 mM,and the optimal induction time was 4,6,8 and 10 h）,and then SDS-PAGE and MS were performed to identify rPBD114.rPBD114 was purified after optimized expression conditions.The protein structure and physicochemical properties of rPBD114 were analyzed online,and then MIC,hemolytic and cytotoxicity were determined by purified rPBD114.rPBD114 was expressed successfully,and the optimal OD600 was 0.6,0.8 and 1.0,the optimal concentration of IPTG was 0.5 and 1.0 mM,and the optimal induction time was 4,6,8 and 10 h.Purity of purified protein was more than 95%,and The results of protein spectrum showed that the amino acid sequence of rPBD114 is same to NP001123445 in NCBI database.online analysis show that rPBD114 a protein which is similar to human beta-defensin 2,has a protein structure composed of alpha helix and beta fold,and is a biparental polypeptide with a +1 charge.In vitro antibacterial tests showed that rPBD114 had significant inhibitory effects on E.coli DH5α and E.coli K88,the MIC valure of rPBD114 against E.coli DH5α and E.coli K88 were 64 and 128 g/mL respectively,Red blood cell hemolysis and cytotoxicity test confirmed that rPBD114 has little hemolysis or cytotoxicity,indicated that rPBD114 is safe and can be used for subsequent studies in vivo and in vitro.Exp 4 Effect and mechanism of recombinant PBD114 on alleviating inflammatory damage of intestinal barrierInflammatory injury of intestinal epithelial cells is one of the important causes of intestinal barrier dysfunction.The results above have confirmed that the expression of PBD114 is significantly increased under immune activation.But the immunomodulatory functions and regulatory mechanisms of PBD114 were still unclear.To explore the role and mechanism of rPBD114 in alleviating inflammatory injury of intestinal epithelial cells,a two-factor experiment was designed.rPBD114 and inhibitors of NF-ΚB（PDTC）,ERK1/2（GDC-0994）and FOS（T-5224）pretreated IPEC-J2 cells for 1 h,then incubated with rPBD114 or LPS for 3 h respectively.The results indicated that the decrease of IPEC-J2 cell activity induced by LPS was significantly increased by rPBD114 and inhibitors（P < 0.05）.The increase of IPEC-J2 cell early,late and total apoptosis were significantly decreased by rPBD114 and inhibitors（P < 0.05）.rPBD114 and inhibitors significantly increased the mRNA expression of Bcl-2,ZO-1,claudin-1 and occludin in IPEC-J2（P < 0.05）,improve Bcl-2/Bax,and protein level of ZO-1 and claudin-1 in IPEC-J2（P < 0.05）.rPBD114 and inhibitors observably down-regulated the expression of Bax,TNFα,IL-1β and IL-6 in IPEC-J2（P < 0.05）,markedly down-regulated protein expression of caspase 8 and caspase 9 in IPEC-J2（P < 0.05）,observably decreased the concentration of TNFα and IL-1β in cell culture medium（P < 0.05）.rPBD114 and inhibitors significantly decreased pIκB-α/IκB-α,pNF-κB/NF-κB and pERK1/2 /ERK1/2 in IPEC-J2 which were increased by LPS（P < 0.05）.The above results show that rPBD114 could decrease the phosphorylation levels of IκB-α,NF-κB,ERK1/2,key molecules of NF-κB and MAPK signal path,reduce the expression of inflammatory factors in intestinal epithelial cells,and then down-regulate the expression level of apoptosis-related genes and proteins,and reduce the apoptosis of intestinal epithelial cells.Exp 5 Effect of recombinant PBD114 on alleviating inflammatory damage of intestinal barrier in miceTo further verify the effect of PBD114 on alleviating inflammatory injury of intestinal barrier,a two-factor experiment was designed.A total of 72 three weeks old ICR mice were randomly assigned to six groups according to the principle of similar body weight: CON（normal saline）,LOW（4 mg/kg rPBD114）,HIGH（8 mg/kg rPBD114）,LPS（normal saline +10 mg/kg LPS challenge）,LOW+LPS（4 mg/kg rPBD114+10 mg/kg LPS challenge）and HIGH（4 mg/kg rPBD114+10 mg/kg LPS challenge）.After one week of prefeeding,all mice were injected with equal volume of rPBD114 or normal saline for 6 consecutive days.On the morning of the 7th day,mice were injected with normal saline and rPBD114.1h later,mice were injected with equal volume of normal saline or 10 mg/kg LPS immediately according design,all mice were anesthetized with carbon dioxide after 5 h.The results indicated that LOW group significantly reduced feed intake and average daily feed intake（P < 0.05）,and increased the organ index of spleen（P < 0.05）.LPS significantly increased the levels of D-lactic acid and diamine oxidase in serum,indicated that LPS successfully led to intestinal inflammatory injury in mice.LPS increased the gene and protein levels of inflammatory factors,caused damage to the liver,kidney and intestinal barrier of mice.But rPBD114 decreased inflammatory factors（TNFα,IL-1β and IL-6）level of serum and jejunum,and then reduced the damage of the liver（lower serum ALT）induced by LPS,and by adjusting the expression of apoptosis related gene（increased Bcl-2/Bax and the expression of Bcl-2,reduced the expression of Bax and caspase 3）in jejunum,to improve intestinal tight junction proteins（increase the expression of ZO-1,occludin and claudin-1,increased protein expression of ZO-1）,enhance body immunity（increased serum IgG,IgM,C3 and C4）,and improve intestinal morphology（decreased duodenal crypt depth and increased jejunal flocculus ratio）.The above results indicated that LPS challenge induced systemic inflammation and intestinal injury in mice.On the premise of not affecting the growth performance of mice,rPBD114 can regulate the expression of apoptosis-related genes and proteins,reduce the production of inflammatory factors in serum and jejunum,reduce the apoptosis of intestinal mucosa,improve the immunity and intestinal health of mice.In conclusion,PBD114 was highly conserved in different breeds of pigs and the expression level of PBD114 in various tissues of TP was higher than DLY pig.As an important immune response gene,PBD114 is up-regulated under immune activation,and its expression is regulated by TLR2/3/4/7 and the transcription factor RELA/FOS/JUN;PBD114 not only has antibacterial activity,but also has obvious feedback regulating effect on immune activation.In other words,PBD114 can inhibit the expression of inflammatory factors by reducing the phosphorylation levels of IκBα,NF-κB and ERK1/2 during immune activation,thereby alleviating the inflammatory injury of intestinal epithelial cells.The above results not only theoretically revealed the expression regulation mechanism and biological function of PBD114,but also expected to provide new ideas for the research of new antibiotic substitutes and nutritional measures to improve animal health. |
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