The rumen immune system often suffers when challenging antigens from lysis of dead microbiota cells in the rumen. However, the rumen epithelium innate immune system can actively respond to the infection. Previous studies have demonstrated G protein-coupled receptors 41 (GPR41) as receptors for short chain fatty acids (SCFAs) in human. We hypothesized that SCFAs, the most abundant microbial metabolites in rumen, may regulate the immune responses by GPR41 in bovine rumen epithelial cells (BRECs). Therefore, the objective of study was to firstly establish an immortal BRECs line and investigate the regulatory effects of SCFAs and GPR41 on innate immunity responses in BRECs. These results showed that long-term BRECs cultures were established by SV40T-induced immortalization. The concentrations of 20 mM SCFAs significantly enhanced the levels of GPR41, IL1β, TNFα, chemokines, and immune barrier genes by transcriptome analysis. Consistent with transcriptome results, the expression of GPR41, IL1β, TNFα, and chemokines were markedly upregulated in BRECs treated with 20 mM SCFAs by qRT-PCR compared with control BRECs. Remarkably, the GPR41 knockdown (GPR41KD) BRECs treated with 20 mM SCFAs significantly enhanced the proinflammatory cytokines IL1β and TNFα expression compared with wild type BRECs treated with 20 mM SCFAs, but reduced the expression of CCL20, CXCL2, CXCL3, CXCL5, CXCL8, CXCL14, Occludin, and ZO-1. Moreover, GPR41 mRNA expression is positively correlated with CCL20, CXCL2, CXCL3, CXCL8, CXCL14, and ZO-1. These findings revealed that SCFAs regulate GPR41-mediated levels of genes involved in immune cell recruitment and epithelial immune barrier and thereby mediate protective innate immunity in BRECs.
Approximately 15 to 50% of short-chain fatty acids (SCFA) reach the ruminant small intestine. Previous research suggests that activation of small intestinal gluconeogenesis induced by propionate has beneficial effects on energy homeostasis. However, the regulatory effect of propionate on key gluconeogenic genes in enterocytes of the bovine small intestine remains less known. Therefore, the purpose of this study was to establish the long-term cultures of bovine intestinal epithelial cells (BIEC) from bovine jejunum tissue using SV40T (1:200; Santa Cruz, Shanghai, China) and investigate the regulatory effect of propionate on the key gluconeogenic genes in BIEC. Our study showed that long-term BIEC cultures were established by SV40Tinduced immortalization. Immortal BIEC were distinguished by the expression of cytokeratin 18, villin, fatty acid binding protein 2, and small intestine peptidase. The mRNA expression of genes involved in the SCFA transporters, monocarboxylate transporter 4, and Na + / H + exchanger isoforms 1 were significantly elevated with 20 mM SCFA compared with untreated controls. In addition, BIEC exhibited significant uptake of propionate and butyrate from the culture medium. Remarkably, 3 mM propionate induced profound changes in mRNA level of key genes involved in gluconeogenesis, including phosphoenolpyruvate carboxykinase 2, pyruvate carboxylase, fructose-1,6-bisphosphatase 1, and peroxisome proliferator-activated receptor-γ coactivator 1α. Additionally, 3 mM propionate enhanced the expression of PGC1A mRNA at 3, 6, 12, and 24 h of incubation. These findings suggest that propionate controls the mRNA expression of genes involved in key enzymes for gluconeogenesis in the enterocytes of bovines.
Short-chain fatty acids (SCFAs) produced by microbial fermentation of dietary fibers are utilized by intestinal epithelial cells to provide an energy source for the ruminant. Although the regulation of mRNA expression and inflammatory response involved in SCFAs is established in other animals and tissues, the underlying mechanisms of the inflammatory response by SCFAs in goat jejunum epithelial cells (GJECs) have not been understood. Therefore, the objective of the study is to investigate the underlying mechanisms of the effects of SCFAs on SCFA transporters and inflammatory response in GJECs. These results showed that the acetate, butyrate, and SCFA concentration were markedly reduced in GJECs (p < 0.01). In addition, the propionate concentration was significantly decreased in GJECs (p < 0.05). The mRNA abundance of monocarboxylate transporter 1 (MCT1), MCT4, NHE1, and putative anion transporter 1 (PAT1) was elevated (p < 0.05) by 20 mM SCFAs at pH 7.4 compared with exposure to the pH group. The anion exchanger 2 (AE2) was increased (p < 0.05) by 20 mM SCFAs at pH 6.2. The mRNA abundance of vH ATPase B subunit (vH ATPase) was attenuated by SCFAs. For inflammatory responses, IL-1β and TNF-α were increased with SCFAs (p < 0.05). In addition, IκBα involved in NF-κB signaling pathways was disrupted by SCFAs. Consistently, p-p65 signaling molecule was enhanced by adding SCFAs. However, IL-6 was attenuated by adding SCFAs (p < 0.05). Furthermore, p-ErK1/2 mitogen-activated protein kinase (MAPK) signaling pathway was downregulated by adding SCFAs. In conclusion, these novel findings demonstrated that mRNA abundance involved in SCFA absorption is probably associated to SCFAs and pH value, and mechanism of the inflammatory response by SCFAs may be involved in NF-κB and p-ErK1/2 MAPK signaling pathways in GJECs. These pathways may mediate protective inflammation response in GJECs.
Lipopolysaccharide (LPS) is an endotoxin that induces immune and inflammatory responses in the rumen epithelium of dairy cows. It is well-known that flavonoid phloretin (PT) exhibits anti-oxidative, anti-inflammatory and antibacterial activity. The aim of this research was to explore whether PT could decrease LPS-induced damage to bovine rumen epithelial cells (BRECs) and its molecular mechanisms of potential protective efficacy. BRECs were pretreated with PT for 2 h and then stimulated with LPS for the assessment of various response indicators. The results showed that 100 µM PT had no significant effect on the viability of 10 µg/mL LPS-induced BRECs, and this dose was used in follow-up studies. The results showed that PT pre-relieved the decline in LPS-induced antioxidant indicators (T-AOC and GSH-PX). PT pretreatment resulted in decreased interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor-α (TNF-α) and chemokines (CCL2, CCL5, CCL20) expression. The underlying mechanisms explored reveal that PT may contribute to inflammatory responses by regulating Toll-like receptor 4 (TLR4), nuclear transcription factor-κB p65 (NF-κB p65), and ERK1/2 (p42/44) signaling pathways. Moreover, further studies found that LPS-induced BRECs showed decreased expression of claudin-related genes (ZO-1, Occludin); these were attenuated by pretreatment with PT. These results suggest that PT enhances the antioxidant properties of BRECs during inflammation, reduces gene expression of pro-inflammatory cytokines and chemokines, and enhances barrier function. Overall, the results suggest that PT (at least in vitro) offers some protective effect against LPS-induced ruminal epithelial inflammation. Further in vivo studies should be conducted to identify strategies for the prevention and amelioration of short acute rumen acidosis (SARA) in dairy cows using PT.
Tea tree oil (TTO) plays an important role in lipid metabolism, alleviating the inflammatory responses. Fatty liver is associated with lipid accumulation in hepatocytes, leading to inflammation. However, there is very limited information on the effects of TTO on lipid accumulation, and inflammation in bovine hepatocytes. This study aimed to evaluate whether TTO alleviates palmitic acid (PA)-induced lipid accumulation in bovine hepatocytes. Hepatocytes isolated from mid-lactating Holstein cows were pretreated with 100 μM PA for 72 h. Cells were either pretreated with PA alone (PA group) or with PA followed by 0.00625% TTO treatment for 12 h (PT group). Expression of fatty acid oxidant genes increased (P < 0.05) while fatty acid synthesis genes decreased (P < 0.05) in the PT group compared with the PA group. PA treatment resulted in increased (P < 0.05) expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), but these increases were less in the PT group (P < 0.05). Compared to the PA group, expression of phosphorylated (p)-p65 and p-inhibitor κBα (p-IκBα) was suppressed (P < 0.05) by TTO treatment. TTO treatment limited (P < 0.05) the increase in intracellular reactive oxygen species (ROS) and prevented (P < 0.05) a reduction in mitochondrial membrane potential observed in response to PA treatment. Expression of endoplasmic reticulum (ER) stress genes was reduced (P < 0.05) in the PT group compared with the PA group. Our results suggest that TTO treatment attenuates the effects of PA in hepatocytes, leading to fatty acid oxidation, decreased fatty acid synthesis, suppressed inflammatory response, and reduced ER stress. Taken together, the results of this study suggest that TTO treatment may be a promising therapeutic approach to imbalanced lipid homeostasis, inflammation and ER stress in dairy cows shortly before and after calving.
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