C-type Natriuretic Peptide Regulates The Expression Of Antimicrobial Peptide S100A7 In Goat Teat Keratinocytes

Mastitis is one of the most common disease in dairy goats,which has caused economic losses to the dairy goat farming industry.Improving the innate immune defense function of dairy goat udder is an important measure to prevent mastitis.Goat teat skin and teat canal epithelium tissue are the first barrier against the invasion of mastitis-causing pathogens.Keratinocytes,as the most important cell type in the epithelium tissue,produce antimicrobial peptides(AMPs)to protect the udder from pathogen bacteria infection,among them,antibacterial peptide S100A7 has received wide attention.CNP,a member of the natriuretic peptide system,is involved in the regulation of inflammatory response mainly by binding with natriuretic peptide receptor-B.Some studies have found that antimicrobial peptide S100A7 has good antibacterial activity and CNP is associated with inflammatory response,but the correlation between S100A7,CNP content in goat milk with mastitis remain poorly understand.In addition,the regulatory mechanism of S100A7 expression in teat skin and duct,which are the first barrier against the invasion of pathogenic microorganisms in the udder of goats,remains unclear.In this study,we examined and analyzed the correlation between S100A7,CNP levels in milk of dairy goats and mastitis.The effects of CNP treatment on the secretion of antibacterial peptide S100A7 by GTKs and its regulatory mechanism were further studied.This study provides theoretical and experimental basis for the hypothesis that CNP,as a regulatory factor for the secretion of S100A7,participate in the natural immune defense function in goat udder.The main contents and results of this study are as flows:(1)According to plate colony count,somatic cell count and clinical diagnosis results,130 milk samples were divided into three groups.40 dairy goats were healthy(30.8%),70 dairy goats were subclinical mastitis(53.8%),20 dairy goats were clinical mastitis(15.4%);ELISA results showed that the contents of S100A7 and CNP in subclinical mastitis group were higher than those in healthy and clinical mastitis groups;There was a significant correlation between S100A7 and CNP in milk of different mastitis grades,correlation coefficient r = 0.7022;Detection of CNP content in milk was superior to detection of antibacterial peptide S100A7 and somatic cell counting in the diagnosis of recessive mastitis of dairy goats(2)The results of hematoxylin-eosin(HE)staining showed that the teat skin and teat canal epithelium were disordered,the corneum was thinner,and blood cells infiltrated between epithelial cells;Immunohistochemical staining showed that the expression of CNP,NPR-B and S100A7 were mainly located in the teat skin and teat canal epithelial tissues of goats.In addition,the expression levels of antibacterial peptide S100A7 and NPR-B in mastitis teat tissue were significantly higher than those in healthy teat tissue.(3)Goat teat skin keratinocytes(GTSKs)and Goat teat canal keratinocytes(GTCKs)were isolated and cultured in vitro.The morphology of cells was typical pavers,and the growth curve was S-shaped.Immunofluorescence staining and Western Blot showed that the two isolated cells expressed the keratinocyte markers CK5 and CK14,but not the fibroblast marker Vementin,indicating that the isolated cells were GTSKs and GTCKs.RT-PCR showed that NPR-B m RNA was highly expressed in both types of cells.(4)ELISA results indicated that LPS could induce the secretion of GTSKs and GTCKs antimicrobial peptide S100A7 and inflammatory factors IL-6,IL-1β and TNF-α.Meanwhile,CNP could also induce the secretion of antimicrobial peptide S100A7 by GTSKs and GTCKs.CNP could up-regulate the level of c GMP in goat teat canal keratinocytes.The secretion of antimicrobial peptide S100A7 in cells was significantly reduced after the addition of PKG inhibitor compared with CNP alone,indicating that CNP regulates the secretion of antimicrobial peptide S100A7 by activating the NPR-B/c GMP/PKG pathway.The results of Western Blot showed that the phosphorylation levels of Erk1/2 and c-Fos were significantly up-regulated after CNP alone treatment.However,the up-regulation of p-Erk1/2 and p-c-Fos by CNP was significantly inhibited after the addition of KT5823.These results indicate that CNP regulates the secretion of antimicrobial peptide S100A7 in goat teat keratinocytes by activating the NPR-B/c GMP/PKG pathway and further activating the Erk/c-Fos pathway.In conclusion,this study for the first time found that the contents of antibacterial peptide S100A7 and CNP in milk of recessive mastitis were significantly higher than those in healthy group and clinical mastitis group,and CNP could be used as a diagnostic marker of recessive mastitis of dairy goats.It was demonstrated that CNP regulates the secretion of antimicrobial peptide S100A7 in goat teat keratinocytes by activating NPR-B/c GMP/PKG pathway and further activating Erk/c-Fos pathway,thus participating in the innate immune defense function of goat udder.