Mechanism Of C-type Natriuretic Peptide Regulating The Expression Of Antimicrobial Peptide S100A7 In Goat Mammary Epithelial Cells

Mastitis is an inflammatory disease caused by the destruction of blood milk barrier that stimulated by internal or external cause,which make the dairy goat industry suffer a certain economic loss.At present,antibiotics are commonly used to treat and prevent mastitis,but the use of antibiotics not only causes the emergence of drug-resistant bacteria,but also causes antibiotic residues in goat’s milk,affecting the quality of goat’s milk.Antimicrobial peptides(AMPS)are a class of small molecular peptides synthesized by the organism,and are inherent components of the non-specific immune system.Compared with antibiotics,AMPS not only has broad-spectrum antimicrobial activity,but also participates in immune regulation and has no drug resistance.Therefore,more and more attention has been paid to the role of antimicrobial peptides in the prevention and treatment of mastitis in dairy goat.Antimicrobial peptide S100 family is a small molecular polypeptide synthesized in vivo,belonging to important components of innate immune system.As an important regulatory protein of innate immunity,the role of antimicrobial peptide S100A7 in the pathological process of goat mastitis is unclear.C-type natriuretic peptide(CNP)is a member of natriuretic peptide family.CNP can catalyze the second messenger cyclic guanosine monophosphate(c GMP)by binding with natriuretic peptide receptor-B(NPR-B).CNP/NPR-B signaling pathway is involved in regulating many biological effects such as cell proliferation and apoptosis.It has been suggested that CNP itself may also be an immunomodulatory factor and participate in inflammatory reaction.However,the expression of CNP and its receptor NPR-B in goat udder has not been reported,and the mechanism of CNP regulating the synthesis and secretion of antibacterial peptide S100A7 in goat mastitis is still unclear.In this study,goat mammary epithelial cells(GMECs)were used as an in vitro research model to study the effects of CNP signaling pathway on S100A7 synthesis and secretion in GMECs,reveal the mechanism of CNP signaling pathway regulating S100A7 synthesis and secretion in GMECs,and provide a new theoretical basis for studying S100A7 expression regulation mechanism.It provided new evidence to prove that CNP is an immunoregulatory factor,and also provided theoretical and experimental evidence for S100A7 as a candidate polypeptide for diagnosis and treatment of mastitis in dairy goats.The research contents and results are as follows:(1)Immunohistochemistry showed that CNP is mainly expressed in the epidermis of teat skin and teat canal,but not in mammary gland tissue.NPR-B is mainly expressed in the epidermis of teat skin and teat canal,and also expressed in alveolar epithelial cells of mammary gland.(2)GMECs were isolated.GMECs have typical mammary epithelial cell morphology.The growth curve of GMECs was drawn,and the growth curve was "s" shape in 6generations,which was in line with the growth characteristic of normal somatic cells.Immunofluorescence staining showed that GMECs expressed Cytokeratin-18(CK-18)of luminal epithelial cells but not cytokeratin-14(CK-14)of myoepithelial cells.RT-PCR results showed that the isolated GMECs could express β-casein and accumulate lipid droplets after induced by lactation medium proving that the isolated cells were GMECs with strong proliferation and continuous lactation ability.(3)Immunofluorescence staining,RT-PCR and Western Blotting were used to study the cell expression and localization of natriuretic peptide system in GMECs.RT-PCR results further proved that GMECs expressed NPR-B and BNP(B-type natriuretic peptide),but did not express NPR-A,NPR-C,ANP(A-type natriuretic peptide)and CNP.Immunofluorescence staining and Western Blotting results showed that GMECs could stably express NPR-B protein in at least 10 generations.(4)CCK-8 test and flow cytometry were used to determine the effect of CNP on the proliferation of GMECs cells.CCK-8 results showed that CNP did not affect the vitality of GMECs cells,while flow cytometry results showed that CNP did not affect the cell cycle distribution.TUNEL staining and Annexin V-FITC/PI double staining were used to detect the effect of CNP on apoptosis of GMECs cells.the results showed that CNP did not affect apoptosis of GMECs cells.The effect of CNP on the lipid synthesis of GMECs was detected by oil red O staining and absorbance analysis.The results showed that CNP did not affect the lipid synthesis of GMECs in the culture system of lactation medium.The effect of CNP on S100A7 expression was detected by Western Blotting and ELISA.The results showed that CNP upregulated the expression and secretion of S100A7,and CNP upregulated the secretion of GMECs antibacterial peptide S100A7 in a time-dependent manner.(5)Immunohistochemical technique was used to detect the expression and localization of CNP,NPR-B and S100A7 in udder tissues of healthy and mastitis goat.The results showed that there was no expression of CNP in alveolar epithelial cells of healthy and mastitis dairy goats;NPR-B and S100A7 are mainly expressed in the epidermis of teat skin,teat canal and alveolar epithelial cells of mammary gland tissue,and the expressions of NPR-B and S100A7 in mammary gland tissue of mastitis goats are significantly higher than those of healthy dairy goats.(6)ELISA was used to detect the effect of CNP on c GMP content in GMECs.The results showed that CNP could up-regulate c GMP level in GMECs in a time-and dose-dependent manner.ELISA and Western Blotting were used to detect the effect of CNP on the synthesis and secretion of S100A7 in the presence or absence of KT5823(PKG specific antagonist).the results further proved that CNP affected the synthesis and secretion of S100A7 through CNP/NPR-B/PKG signaling pathway.