RBPJ Influences the Development of Clinical Mastitis in Dairy Cows (Bos taurus) by Regulating Mammary Gland Morphogenesis
CHEN Na1,3, ZHANG Bo-Hao1,2,3, CAI Nong1,3, LI Jian-Fu2,3, GAO Yu-Meng1,3, ZHAO Xing-Xu1,2,3, ZHANG Yong1,2,3, ZHANG Quan-Wei1,2,3,*
1 College of Life Science and Biotechnology, Gansu Agricultural University, Lanzhou 730070, China; 2 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 3 Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation, Lanzhou 730070, China
Abstract：Abnormality in the morphological structure of the mammary gland (MG) is a core pathological alteration in clinical mastitis (CM) in dairy cows (Bos taurus). However, the molecular regulatory mechanisms of morphogenesis remain incompletely understood in MG of dairy cows with CM. In this study, the biological processes (BPs) related to mammary morphogenesis, key differentially expressed proteins (DEPs), and their involved KEGG pathways were identified based on data-independent acquisition (DIA) proteomics and GO enrichment analyses of lactating MG tissues from healthy (control, Con) and CM-affected Holstein cows. Morphological changes were observed using hematoxylin-eosin (H&E) staining. The distribution characteristics and expression patterns of key candidate DEPs were analyzed using immunohistochemistry (IHC), immunofluorescence (IF), qRT-PCR, and Western blot. The results showed that a total of 11 morphogenesis-associated BPs and 40 DEPs were identified based on GO enrichment analysis. Among these, recombination signal binding protein for immunoglobulin kappa J region (RBPJ) was identified as a key protein regulating morphogenesis in CM. Pathway analysis indicated that RBPJ was involved in signaling pathways such as Notch signaling, viral carcinogenesis, and Epstein-Barr virus (EBV) infection. Compared with the Con group, the CM group exhibited collapsed mammary alveoli, disorganized epithelial cell distribution, and concomitant inflammatory cell infiltration. RBPJ was primarily localized in the cytoplasm of mammary epithelial cells. RBPJ mRNA and protein expression levels in the CM group were extremely significantly higher than those in the Con group (P<0.01). These results demonstrated that RBPJ participated in regulating MG morphogenesis during CM via morphogenesis-related pathways, and its upregulated expression was positively correlated with the occurrence and progression of CM. This study provides a theoretical foundation for elucidating the function and molecular mechanisms of RBPJ in bovine CM.
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