Subacute ruminal acidosis induces inflammation, autophagy, and apoptosis by disrupting Ca2+ homeostasis via STIM1/ORAI1 upregulation in mammary gland tissues.

Subacute ruminal acidosis (SARA) is a prevalent metabolic disorder in ruminants that induces mastitis, while Ca²⁺ homeostasis disruption is closely linked to mammary gland diseases. This study aimed to uncover the role of Ca²⁺ signaling in SARA-induced mammary gland injury by investigating its regulation of inflammation, autophagy, and apoptosis. In this study, we induced SARA by feeding a high-concentrate (HC, 70% concentrate) diet to Hu sheep (pH<5.6 for more than 3 h). The diets were administered over a period of 56 d. We collected samples of blood and mammary gland tissues from the animals at the end of the experiment. Compared with the low-concentrate (LC, 30% concentrate) diet, HC diet increased the mRNA and protein expression of STIM1 and ORAI1, and Ca2+ content in mammary gland tissues. Elevated Ca2+ activated the CaMKKβ-NF-κB pathway, as evidenced by increased p-IκB, p-p65, upregulation of pro-inflammatory cytokines (IL-6, IL-8, IL-1β, TNF-α) in mammary gland tissues, and enhanced interaction between ORAI1 and p65. Concurrently, HC diet activated the CaMKKβ-AMPK pathway, which in turn upregulates the expression of autophagy-related proteins and induces autophagy. Finally, the HC diet promoted apoptosis by upregulating the expression of pro-apoptotic proteins (Cytochrome C, Cleaved-Caspase3 and BAX) and downregulating anti-apoptotic proteins (Bcl-2). In summary, SARA disrupts Ca²⁺ homeostasis via STIM1/ORAI1 upregulation, triggering a cascade of inflammation, autophagy, and apoptosis in Hu sheep mammary glands through the CaMKKβ-mediated NF-κB and AMPK-mTOR pathways.