Study On The Relationship Between Antibacterial Peptide S100A7 In Goat Milk Somatic Cells And Mastitis

Goat mastitis has caused certain economic losses to the goat industry.Early diagnosis of mastitis,especially subclinical mastitis,is great significant to prevent and treat for goat mastitis.The antimicrobial peptide S100A7 has many advantages including rapid synthesis and secretion,sensitivity to pathogenic microorganisms.Therefore,S100A7 is considered as a potential early diagnosis marker for goat mastitis.Moreover,the antimicrobial peptide S100A7 is a type of small molecule polypeptide synthesized by the organism,and is an inherent component of the non-specific immune system.In addition to broad-spectrum antimicrobial activity with no drug resistance,S100A7 also participates in immune regulation.Recently,the role of antimicrobial peptide S100A7 in prevention and treatment for goat mastitis has attracted more and more attention.However,the correlation between the antimicrobial peptide S100A7 and goat mastitis is still unclear.The expression of antimicrobial peptide S100A7 in goat mammary epithelial cells and whether it can be induced by pathogenic microorganisms to participate in the mammary innate immune response is still controversial.In present study,a method for extracting total RNA from goat milk somatic cells was established to study the differential expression of antimicrobial peptide S100A7 in goat milk somatic cells of healthy goats,subclinical mastitis and clinical mastitis and its correlation with the pathogenesis of mastitis.The goat mammary epithelial cells were isolated to further explore the expression and regulation mechanism of antimicrobial peptide S100A7 in the mammary epithelial cells.The main results are as follows:(1)The quality of total RNA extracted from goat milk somatic cells by optimizated method is eligible enough for the further test.The internal reference gene GAPDH and the target gene S100A7 can be amplified by RT-PCR.(2)The somatic cells count in the milk of healthy goats is the least.The somatic cells count in the milk of suspected subclinical mastitis,subclinical mastitis and clinical mastitis goats increases successively.There is a significant difference between healthy,subclinical mastitis and clinical mastitis goat in somatic cells count.(3)The antimicrobial peptide S100A7 expression was the lowest in the milk somatic cells of healthy goats.The expression level of antimicrobial peptide S100A7 in the milk somatic cells of suspected subclinical mastitis goats,subclinical mastitis goats and clinical mastitis goats increased successively.The expression of S100A7 in goat milk somatic cells increased with the severity of the clinical symptoms of mastitis.The expression level of S100A7 mRNA in goat milk somatic cells has a significant correlation with somatic cells count,and can reflect the severity of clinical symptoms of mastitis within a certain range.(4)In healthy goats and mastitis goats,antimicrobial peptide S100A7 is expressed in teat duct epidermis,teat skin epidermis,sweat glands,teat gland ducts,mammary alveolar epithelial cells and gland ducts.In mastitis goats,the expression of S100A7 in mammary alevolar epithelial cells was significantly higher than that in healthy goat.(5)Lipopolysaccharide(LPS)induced goat mammary epithelial cells to synthesize and secrete antimicrobial peptide S100A7.This inductive effect was dependent on LPS concentration and treatment duration.However,LPS alone could not induce the continuous secretion of S100A7 in vitro.(6)LPS induced the up-regulation of mRNA expression levels of TLR4 and My D88 genes.Moreover,LPS treatment promoted the phosphorylation of p-65 and transfer location from cytoplasm into nuclear indicating that LPS could activated the TLR4 and NF-κB signaling pathways in goat mammary epithelial cells.In summary,this study successfully improved a PCR method for detecting S100A7 gene in goat milk somatic cells.The expression level of S100A7 significantly correlated with the occurrence of mastitis.LPS could induce the expression and secretion of S100A7 in goat mammary epithelial cells by activating the TLR4 and NF-κB signaling pathways.