High-concentrate diets, which are rich in essential nutrients, effectively meet the nutritional requirements for ruminant production. In the short term, increasing the proportion of concentrates in the diet can yield positive results. Consequently, high-concentrate diets are often preferred for ruminants during the breeding period to enhance productivity and economic profitability. Nevertheless, high-concentrate diets can enhance the productive performance of ruminants in the short term, their long-term or excessive consumption may lead to the accumulation of lactic acid and volatile fatty acids in the rumen. This accumulation can potentially induce subacute ruminal acidosis (SARA), causing significant economic losses in the livestock industry [1,2]. Therefore, achieving a balance between the productive performance and health of animals, especially under high-density husbandry conditions, has become a critical issue that requires immediate attention.

Ca²⁺ acts as second messengers within cells and play a crucial role in various physiological processes. Disruption of intracellular Ca2+ levels can lead to the development of multiple diseases. Store-operated calcium entry (SOCE) channel is the primary pathway for the influx of extracellular Ca2+ into cells and is primarily composed of two key proteins: stromal interaction molecule 1 (STIM1) and calcium release-activated calcium modulator 1 (ORAI1) [3]. Previous research has shown that β-carotene alleviates LPS-induced endoplasmic reticulum stress, mitochondrial damage and apoptosis in bovine mammary epithelial cells by inhibiting STIM1 and ORAI1 [4,5]. Additionally, resveratrol suppresses the expression of STIM1 and ORAI1, key components of the SOCE channel, thereby reducing the influx of extracellular Ca²⁺. This action inhibits the activation of the ERK1/2 signaling pathway and mitigates damage to HEK293 cells [6]. Our prior investigations had demonstrated that a high-concentrate diet upregulated the expression of STIM1 and ORAI1, subsequently provoking inflammation response and apoptosis in the mammary gland tissues of dairy cows [7]. Further in vitro studies had confirmed that LPS increased the intracellular Ca2+ levels by enhancing STIM1 and ORAI1 expression and subsequently induced an inflammatory response by activating NF-κB signaling pathway in bovine mammary epithelial cells [8]. Collectively, these findings reveal the potential mechanisms by which high-concentrate diet induce inflammation and apoptosis in mammary gland tissues through the modulation of calcium signaling and associated pathways.

Intracellular Ca²⁺concentrations are essential for initiating autophagy, with increased levels serving as a potent stimulus for this process. This intricate process is regulated through a complex series of signaling events involving the CaMKKβ-AMPK pathway. This pathway, in turn, suppresses the mTOR signaling pathway, thereby effectively modulating autophagy [[9], [10], [11]]. In cells lacking LKB1, elevated of intracellular Ca²⁺ levels can trigger AMPK phosphorylation via the through the CaMKKβ pathway [12]. This mechanism is supported by research from Zheng et al., who demonstrated that Orai1 played a crucial role in promoting autophagic flux [13]. Specifically, silencing Orai1 significantly reduced LC3-II accumulation induced by angiotensin II and inhibited autophagy in cardiomyocytes, both in vitro and in vivo [13]. Thus, SOCE is pivotal in maintaining intracellular Ca2+ homeostasis. Nonetheless, current research on the Ca²⁺ influx facilitated by the STIM1/ORAI1 complex under SARA conditions and its subsequent effects on the organism via the CaMKKβ signaling cascade remains limited. Therefore, it is imperative to investigate the regulatory mechanisms of STIM1/ORAI1 in SARA-induced mammary gland injury in Hu sheep, with the aim of identifying new therapeutic targets for the treatment of mastitis.

Consequently, we hypothesize that SARA induces the upregulation of STIM1 and ORAI1, thereby disrupting intracellular Ca2+ homeostasis. This disruption subsequently triggers a cascade of cellular responses, including inflammation, autophagy, and apoptosis, in the mammary gland tissues of Hu sheep.