The Molecular Mechanism Of Mammary Gland Fibrosis Induced By Staphylococcus Aureus In Mice And PI3K/Akt Signaling Pathway

Mammary gland fibrosis is a pathological process in the later stage of mastitis which was induced by pathogenic microorganism infection or mechanical stimulation,causing severe economic loss to the dairy industry.Staphylococcus aureus is one of the main pathogenic bacteria of mastitis with persistent infection.To study the molecular mechanism of S.aureus-induced mammary gland fibrosis,72 cases of bovine mammary gland tissue were collected for isolating S.aureus and histopathological examination to observe mammary fibrosis.The sections were stained by immunohistochemical staining to detect the distribution of TGF-?1,PDGF-BB and bFGF in bovine mammary gland tissue.Balb/c mice were infected with different concentrations of S.aureus suspension.Mammary gland tissue in mice were collected after S.aureus infection at different days,and observed for the pathological changes.The model of mammary gland fibrosis in mice was established by10~8 cfu/mL S.aureus and mammary gland tissue was collected after S.aureus infection at different days.To investigate the role of cytokines including TGF-?1,PDGF-BB,bFGF and their receptors in the process of mammary gland fibrosis induced by S.aureus,the relative mRNA and protein expression and distribution of TGF-?1,PDGF-BB,bFGF,T?R?,PDGFR?,FGFR2 were measured by Immunohistochemical staining,real time quantitative PCR(qPCR)and Western blot.To study the role of TLRs-NF-kB/AP-1 signaling pathway in the expression of?-SMA and COL??1S.aureus-induced,the relative mRNA and protein expression of TLR2,TLR4,?-SMA and COL??1 were detected by qPCR and Western blot.Meanwhile,the non-phosphorylated and the phosphorylated expression of NF-kB p65 and AP-1 c-jun protein were detected by Western blot.Bovine mammary fibroblasts were treated by TGF-?1,bFGF and PDGF-BB.Total mRNA and protein of bovine mammary fibroblasts were collected at 12h,24h,48h,72 h after treatments.The mRNA and protein expression of PI3K and Akt in mammary fibroblasts cells were detected by qPCR and Western blot,respectively.Finally,to investigate whether PI3K and Akt were involved in the downstream signaling transduction in the expression of?-SMA and COL I?1 induced by TGF-?1,bFGF and PDGF-BB of bovine mammary fibroblasts,LY294002,the PI3K inhibitor,was used to detect the expression of p-Akt,?-SMA and COL I?1.The mRNA expression of Akt,?-SMA and COL I?1 were detected by qPCR,and the protein expression of p-Akt,?-SMA and COL I?1 were detected by Western blot.The results showed that S.aureus was isolated from 10 cases.Compared to bovine mammary gland tissue with no pathological changes,the protein expression of TGF-?1,PDGF-BB and bFGF was obviously detected and mainly in mammary epithelial cells in S.aureus-isolated mammary gland tissue.Histopathological examination showed that mammary gland fibrosis with mammary gland epithelials degeneration,necrosis,shedding,neutrophil infiltration,gland bubble collapse and hyperplasia of fibroblasts and lymphocytes,collagen fibers increased was induced by 10~8 cfu/mL S.aureus suspension.In conclusion,the model of mammary gland fibrosis in mice was established by 10~8 cfu/mL S.aureus suspension.Results showed that the mRNA and protein expression of TGF-?1,TLR2,TLR4 and mRNA expression of PDGF-BB were upregulated from day 1 to days 3 then decreased from 3 d to 7 d,increased again during 14 d to 21 d.the mRNA and protein expression of bFGF,T?R?,PDGFR?,FGFR2,?-SMA and COL I?1 and the protein expression of PDGF-BB were in the significantly upward trends.TLRs-NF-kB p65 and TLRs-AP-1 c-jun signaling pathways were related to the expression of?-SMA and COL I?1 in mammary gland tissue.The mRNA and protein expression of PI3K and Akt were induced by TGF-?1,PDGF-BB and bFGF.The optimum concentration of inhibitor was 50?M with low protein expression levels of p-Akt.TGF-?1,PDGF-BB and bFGF were used to treat mammary gland fibroblasts after inhibitor LY294002.The results showed that the expression of Akt,p-Akt,?-SMA,COL??1 were significantly inhibited by LY294002 compared with TGF-?1,PDGF-BB and bFGF group(p<0.05).The results demonstrated that TGF-?1,PDGF-BB and bFGF were mainly detected in mammary gland epithelial cells in bovine mammary gland tissue with chronic mastitis and different degrees of fibrosis.The experimental mice model of mammary gland fibrosis was established and induced by bovine mastitis S.aureus,S.aureus not only increased TGF-?1,PDGF BB,bFGF,T?R?,PDGFR?,FGFR2 expression,but also induce the expression of TLR2,TLR4,?-SMA and COL I?1,TLR2/4-NF-?B p65 and TLR2/4-AP-1 signaling pathways were relevant to the expression of?-SMA and COL I?1 in S.aureus-infected mice.PI3K/Akt signaling pathway play a key role in the transcriptional level of?-SMA and expression of COL I?1.