A high-concentrate diet provokes inflammation, endoplasmic reticulum stress, and apoptosis in mammary tissue of dairy cows through the upregulation of STIM1/ORAI1

Abstract

High-concentrate feeding can induce subacute ruminal acidosis, which leads to mammary tissue injury in dairy cows. Therefore, the purpose of this research was to evaluate the effect of high-concentrate feeding on STIM1 (stromal interaction molecule 1)/ORAI1 (Orai calcium release-activated calcium modulator 1)-mediated inflammation, endoplasmic reticulum stress (ERS), and apoptosis in the mammary tissue of dairy cows. A total of 12 healthy mid-lactating Holstein cows of similar weight were randomly allotted into the following 2 groups: a high-concentrate (HC) group (concentrate: forage = 6:4) and a low-concentrate (LC) group (concentrate: forage = 4:6). The trial lasted for 3 wk. After the feeding experiment, rumen fluid, lacteal vein blood, and mammary tissue samples were collected. The results showed that the HC diet significantly increased blood lipopolysaccharide levels, decreased ruminal pH, and upregulated the concentrations of Ca 2+ and proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, and the enzyme activities of caspase-3, caspase-9, PKC, and IKK. The upregulation of STIM1, ORAI1, PKCα, IKKβ, phosphorylated-IκBα, phosphorylated-p65, TNF-α, and IL-1α proteins in the HC group indicated activation of the STIM1/ ORAI1-mediated inflammatory signaling pathway compared with that in the LC group. The HC diet also induced ERS by increasing the mRNA and protein abundances of GRP78, CHOP, PERK, ATF6, and IRE1α in the mammary tissue. Compared with the LC group, the mRNA expression levels and protein abundances of caspase-3, cleaved caspase-3, caspase-9, and BAX were markedly increased in the HC group. However, the mRNA and protein expression levels of Bcl-2 were significantly decreased in the HC group. Therefore, this study demonstrated that the HC diet can activate the store-operated calcium entry channel by upregulating the expression of STIM1 and ORAI1 and induce inflammation, ERS, and apoptosis in the mammary tissue of dairy cows.

STIM1–Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry

“…RR036A; Takara) according to the manufacturer's instructions, and then the cDNA was diluted fourfold and used for realtime quantitative PCR (RT-PCR). The primer sequences of STIM1/Orai1 and the genes associated with ERS (PERK, IRE1, ATF6, GRP78, CHOP) were designed using sequence data obtained from the National Center for Biotechnology Information with Primer Premier software 5.0 (Premier Bio soft, California, CA, USA), and the primer sequences of the genes for proinflammatory cytokines (IL-6, IL-8, IL-10, IκB, NF-κB, and TNF-α) were directly obtained from previously published articles [21]. The primer sequences of genes were synthesized in Generay equipment (Shanghai, China), and the details are shown in Table 1.…”

“…LPS can stimulate the immune system and enhance innate immunity at low concentrations, while it causes extensive and intense inflammatory reactions at high concentrations [19,20]. Some studies in the livers of cows have reported that LPS affects the nutrient metabolism of the liver and reduces the immune capacity of animals, and may even cause endotoxemia in serious cases [21,22]. The liver, as the largest digestive organ and immune organ of dairy cows, is one of the main target organs attacked by LPS and plays an important immune role for LPS [22].…”

“…Previous studies have shown that LPS binds to receptors on the cell membrane to activate the monocyte-macrophage system inside the liver [23]. LPS induces the release of TNF, IL-6, IL-8, and other inflammatory mediators in the cell through the mitogen-activated protein kinase pathway, the PKC pathway, the NF-κB pathway, and other inflammatory signaling pathways, resulting in inflammatory reactions [21,24]. It has been reported that upregulation of STIM1/Orai1 can promote inflammation and ERS in mammary tissue [21], and this can inhibit the LPS-induced inflammation by regulating ERS in BHEC [20,25].…”

“…LPS induces the release of TNF, IL-6, IL-8, and other inflammatory mediators in the cell through the mitogen-activated protein kinase pathway, the PKC pathway, the NF-κB pathway, and other inflammatory signaling pathways, resulting in inflammatory reactions [21,24]. It has been reported that upregulation of STIM1/Orai1 can promote inflammation and ERS in mammary tissue [21], and this can inhibit the LPS-induced inflammation by regulating ERS in BHEC [20,25]. LPS stimulation could reduce calcium ions in the ER and activate STIM1/Orai1, and thereby conformational activation, resulting in the influx of Ca 2+ from the outer membrane and cell damages [21].…”

“…It has been reported that upregulation of STIM1/Orai1 can promote inflammation and ERS in mammary tissue [21], and this can inhibit the LPS-induced inflammation by regulating ERS in BHEC [20,25]. LPS stimulation could reduce calcium ions in the ER and activate STIM1/Orai1, and thereby conformational activation, resulting in the influx of Ca 2+ from the outer membrane and cell damages [21].…”