Lipopolysaccharide (LPS) is the dominating endotoxin of Gram-negative bacteria, which can cause mastitis. Bovine mammary epithelial cells (BMECs), as major components of the mammary gland, usually suffer LPS challenge. Cis-9, trans-11 conjugated linoleic acid (CLA) has been reported to have anti-inflammatory characteristics, while its anti-oxidative ability to maintain cellular homeostasis in BMECs under LPS challenge is limited. Therefore, we studied whether cis-9, trans-11 CLA can restore the disturbance of cellular homeostasis indicated by the redox status and autophagy level caused by LPS and have an effect on cellular function- milk fat metabolism. For oxidative stress, LPS challenge promoted the formation of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) and decreased the concentration of glutathione. Anti-oxidative signaling regulated by transcription factor nuclear factor, erythroid 2 like 2 (Nrf2) was also depressed by LPS at the mRNA and protein level. However, cis-9, trans-11 CLA pretreatment downregulated the formation of ROS and TBARS and upregulated the expression of antioxidative enzymes. As a part of innate immunity, autophagy was also motivated by LPS challenge, while CLA decreased the autophagy level. LPS and H₂O₂ inhibited milk fat synthesis-related transcription factor sterol regulatory element binding protein (SREBP1), peroxisome proliferator activated receptor gamma (PPARG) and their downstream enzymes. Furthermore, 50 uM cis-9, trans-11 CLA promoted the mRNA and protein abundance of milk fat synthesis-related genes and lipid droplet formation in BMECs. In conclusion, LPS challenge disturbed the cellular homeostasis and depressed milk fat synthesis in BMECs; while cis-9, trans-11 CLA alleviated oxidative stress and decreased autophagy level, thus promoting milk fat synthesis, which offers a natural therapeutic strategy for mastitis.

Dietary structure in ruminants is closely connected with the composition of gastrointestinal microbiota. Merging study has shown that dietary induced SARA causes the alteration of microbial community in the cecum leading to the local inflammation. However, the mechanisms of cecum inflammation elicited by the shift of microbial flora in ruminants are largely unknown, and whether the development of this inflammation is modified by epigenetic modifications. Ten multiparous lactating goats were randomly seperated into two groups and received either a low concentrate diet (LC, 40% concentrate, n = 5) or a high concentrate diet (HC, 60% concentrate) to induce subacute ruminal acidosis (SARA). Compared with LC, HC-induced SARA altered the predominant phyla and genera, thereby increasing the concentration of lipopolysaccharide (LPS) and short chain fatty acids (SCFAs). Meanwhile, HC-induced SARA enhanced the mRNA expression of cytokines and chemokines and the expression of mRNA and protein of GPR41, GPR43, p38 and ERK1/2, while HC-induced SARA had no effect on TLR4 and p65. Furthermore, HC-induced SARA decreased the percentage of chromatin compaction and DNA methylation at the area of the promoters of GPR41 and GPR43. This study indicated that HC diet induced SARA resulted in the alteration in the composition of cecal microbiota. This alteration increased the concentration of LPS, but failing to activate TLR4 signaling pathway due to the tolerance effect of intestinal epithelial cell to certain level of LPS, as well as elevated the concentration of SCFAs, thereby activating GPR41 and GPR43 signaling pathway to produce cytokines and chemokins and cause the cecal inflammation. And epigenetic mechanisms contributed to the development of this inflammation in the lactating goats suffering from SARA.

(1) Background: The basic mechanism of store-operated Ca²⁺ entry (SOCE) in bovine hepatocytes (BHEC) is related to the activation of STIM1 and Orai1. The effect of STIM1- and Orai1-dependent calcium ion signaling on the NF-κB signaling pathway is unclear. (2) Methods: In this study, the expression of STIM1 and Orai1 in BHEC was regulated. RT-qPCR, Western blotting, and an immunofluorescence antibody (IFA) assay were performed to elucidate the effect of inflammation and endoplasmic reticulum stress (ERS) in BHEC. (3) Results: First of all, in this study, RT-PCR and Western blotting were used to detect the levels of IκB, NF-κB, and inflammatory factors (IL-6, IL-8, and TNF-α) and the expression of genes and proteins related to ERS (PERK, IRE1, ATF6, GRP78, and CHOP), which reached peak levels simultaneously when BHEC were treated with 16 μg/mL LPS for 1 h. For STIM1, we overexpressed STIM1 in BHEC by using plasmid transfection technology. The results showed that after overexpression of STIM1, the gene and protein expression of STIM1 levels were significantly upregulated, and the expression of Orai1 on the cell membrane was also upregulated, which directly activated the SOCE channel and induced inflammation and ERS in BHEC. The overexpression group was then treated with LPS, and it was found that the overexpression of STIM1 could enhance LPS-induced BHEC inflammation and ERS in BHEC. For Orai1, BHEC were pretreated with 8 μg/mL of the specific inhibitor BTP2 for 6 h. It was found that BTP2 could inhibit the expression of mRNA in Orai1, significantly reduce the gene expression of STIM1, inhibit the activation of the NF-κB signaling pathway, and alleviate inflammation and ERS in BHEC under LPS stimulation. (4) Conclusions: In conclusion, STIM1/Orai1 can intervene and exacerbate LPS-induced inflammation and ERS in bovine hepatocytes through SOCE.