RESUMEN

AOS alleviates inflammatory responses; however, whether it exerts an effect on the rumen or regulates rumen inflammatory reaction remains unknown. In this study, firstly, the ovine ruminal epithelial cells (ORECs) were treated with 0, 200, 400, 600, and 800 µg/mL AOS, hoping to explore whether AOS hurt cell health. The results showed that compared with the AOS-0 group, the AOS-400 group could significantly increase (p < 0.05) cell viability, reduce (p < 0.05) reactive oxygen species (ROS) and interleukin (IL)-6 content, and have no adverse effect on cells. Secondly, we used LPS to construct an in vitro inflammatory model of rumen epithelial cells and then explored the protective role of AOS on rumen epithelial cells. The study was divided into three groups: the control group (CON), LPS, and LPS + AOS. The results demonstrated that the LPS + AOS group significantly increased the cell viability and reduced the ROS level in comparison with the LPS group (p < 0.05). Pretreatment with AOS also repressed (p < 0.05) the secretion of IL-1ß, IL-6, IL-8, and immunoglobulin (Ig)A from ORECs in the culture medium following LPS. In terms of tight junction (TJ) proteins, AOS treatment also significantly increased (p < 0.05) the zonula occludens 1 (ZO-1) and Occludin expression. The apoptosis rate, Caspase3, Caspase9, BAD, and BCL-2/BAX were decreased (p < 0.05) after AOS treatment, and the expression of BCL-2 was increased (p < 0.05). In addition, the expressions of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) were inhibited (p < 0.05) with the addition of AOS. At the protein level, pretreatment of AOS decreased (p < 0.05) the expression of MyD88 and the phosphorylation level of inhibitor κB α (IκBα) after the LPS challenge. Taken together, our results indicated that AOS could alleviate the LPS-induced apoptosis and inflammatory response of rumen epithelial cells through the NF-κB signaling pathway, which may be a promising strategy for treating apoptosis and inflammation in sheep breeding.

RESUMEN

BACKGROUND: Untargeted metabolomics and proteomics were employed to investigate the intracellular response of yak rumen epithelial cells (YRECs) to conditions mimicking subacute rumen acidosis (SARA) etiology, including exposure to short-chain fatty acids (SCFA), low pH5.5 (Acid), and lipopolysaccharide (LPS) exposure for 24 h. RESULTS: These treatments significantly altered the cellular morphology of YRECs. Metabolomic analysis identified significant perturbations with SCFA, Acid and LPS treatment affecting 259, 245 and 196 metabolites (VIP > 1, P < 0.05, and fold change (FC) ≥ 1.5 or FC ≤ 0.667). Proteomic analysis revealed that treatment with SCFA, Acid, and LPS resulted in differential expression of 1251, 1396, and 242 proteins, respectively (FC ≥ 1.2 or ≤ 0.83, P < 0.05, FDR < 1%). Treatment with SCFA induced elevated levels of metabolites involved in purine metabolism, glutathione metabolism, and arginine biosynthesis, and dysregulated proteins associated with actin cytoskeleton organization and ribosome pathways. Furthermore, SCFA reduced the number, morphology, and functionality of mitochondria, leading to oxidative damage and inhibition of cell survival. Gene expression analysis revealed a decrease the genes expression of the cytoskeleton and cell cycle, while the genes expression associated with inflammation and autophagy increased (P < 0.05). Acid exposure altered metabolites related to purine metabolism, and affected proteins associated with complement and coagulation cascades and RNA degradation. Acid also leads to mitochondrial dysfunction, alterations in mitochondrial integrity, and reduced ATP generation. It also causes actin filaments to change from filamentous to punctate, affecting cellular cytoskeletal function, and increases inflammation-related molecules, indicating the promotion of inflammatory responses and cellular damage (P < 0.05). LPS treatment induced differential expression of proteins involved in the TNF signaling pathway and cytokine-cytokine receptor interaction, accompanied by alterations in metabolites associated with arachidonic acid metabolism and MAPK signaling (P < 0.05). The inflammatory response and activation of signaling pathways induced by LPS treatment were also confirmed through protein interaction network analysis. The integrated analysis reveals co-enrichment of proteins and metabolites in cellular signaling and metabolic pathways. CONCLUSIONS: In summary, this study contributes to a comprehensive understanding of the detrimental effects of SARA-associated factors on YRECs, elucidating their molecular mechanisms and providing potential therapeutic targets for mitigating SARA.

Asunto(s)

RESUMEN

OBJECTIVE: Rutin, also called vitamin P, is a flavonoids from plants. Previous studies have indicated that rutin can alleviate the injury of tissues and cells by inhibiting oxidative stress and ameliorating inflammation. There is no report on the protective effects of rutin on goat rumen epithelial cells (GRECs) at present. Hence, we investigated whether rutin can alleviate lipopolysaccharide (LPS)-induced damage in GRECs. METHODS: GRECs were cultured in basal medium or basal medium containing 1 µg/mL LPS, or 1 µg/mL LPS and 20 µg/mL rutin. Six replicates were performed for each group. After 3-h culture, the GRECs were harvested to detect the relevant parameters. RESULTS: Rutin significantly enhanced the cell activity (p<0.05) and transepithelial electrical resistance (TEER) (p<0.01) and significantly reduced the apoptosis rate (p<0.05) of LPSinduced GRECs. Rutin significantly increased superoxide dismutase, glutathione peroxidase, and catalase activity (p<0.01) and significantly decreased lactate dehydrogenase activity and reactive oxygen species and malondialdehyde (MDA) levels in LPS-induced GRECs (p<0.01). The mRNA and protein levels of interleukin 6 (IL-6), IL-1ß, and C-X-C motif chemokine ligand 8 (CXCL8) and the mRNA level of tumor necrosis factor-α (TNF-α) and chemokine C-C motif ligand 5 (CCL5) were significantly increased in LPS-induced GRECs (p<0.05 or p<0.01), while rutin supplementation significantly decreased the mRNA and protein levels of IL-6, TNF-α, and CXCL8 in LPS-induced GRECs (p<0.05 or p<0.01). The mRNA level of toll-like receptor 2 (TLR2), and the mRNA and protein levels of TLR4 and nuclear factor κB (NF-κB) was significantly improved in LPS-induced GRECs (p<0.05 or p<0.01), whereas rutin supplementation could significantly reduce the mRNA and protein levels of TLR4 (p<0.05 or p<0.01). In addition, rutin had a tendency of decreasing the protein levels of CXCL6, NF-κB, and inhibitor of nuclear factor kappa-B alpha (0.05< p<0.10). Rutin could significantly decreased interferon regulatory factor 3 mRNA expression in LPS-induced GRECs (p<0.05), whereas interferon induced protein with tetratricopeptide repeats 3 (IFIT3) and toll-interacting protein (TOLLIP) mRNA expression was not significantly different between the groups. LPS reduced the tight junction protein zonula occludin 1 (ZO-1) level in GRECs whereas rutin enhanced it. Rutin significantly improved tight junction protein Claudin-1 mRNA expression in LPS-induced GRECs (p<0.01), but could not affect tight junction protein Occludin mRNA expression. CONCLUSION: Rutin alleviated LPS-induced barrier damage in GRECs by improving oxidation resistance and anti-inflammatory activity, which may be related to TLR/NF-κB signaling pathway inhibition.

RESUMEN

The aim of this experiment was to investigate the effects of Ageratina adenophora on the expression of epithelium tight junction proteins and inflammatory factors in the rumen of goats. Twelve goats were randomly divided into three groups. The first group was the blank control group (n = 3, C) which was fed normal diet. The second group was fistulas control group (n = 3, RFC), which was fitted with rumen fistulas, and fed normal diet. The third group was the A. adenophora test group (n = 6, AA), which was fitted with rumen fistulas and fed a mixture of 60% of normal diet and 40% of A. adenophora grass powder. The feeding experiment lasted for 90 d, after which all goats were sacrificed and samples were collected from the rumen dorsal sac and ventral sac. The relative expression of mRNA of inflammatory factors in the rumen epithelium (tumor necrosis factor alpha [TNF-α], interferon gamma [IFN-γ], interleukin 1 beta [IL-1ß], IL-2, IL-4, IL-6, and IL-10) and tight junction protein genes (occludin, claudin-1, and ZO-1) was measured by quantitative real-time fluorescence PCR. Expression of tight junction proteins in the rumen epithelium was measured by Western blot. A correlation was established between the expression of inflammatory factors and tight junction protein genes using Graph Pad Prism. The results showed that A. adenophora caused a significant increase in the mRNA expression levels of TNF-α, IFN-γ, IL-1ß, IL-2, IL-6, and IL-10 in the rumen epithelial (P < 0.05 or P < 0.01). The expression of tight junction proteins at both gene and protein levels was significantly decreased (P < 0.05 or P < 0.01). Furthermore, the correlation analysis revealed that the changes in tight junction protein expression in the test group were closely related to the upregulation of the expression of inflammatory factors TNF-α and IFN-γ in rumen epithelial cells. In conclusion, the expression of inflammatory factors was increased and the expression of tight junction proteins was decreased in goats after feeding on A. adenophora, which caused some damage to the rumen epithelium.

The article aims to investigate the toxic effects of Ageratina adenophora, an invasive plant on the integrity of the rumen epithelium by measuring the changes in the expression of inflammatory factors and tight junction proteins after the consumption of A. adenophora in goats. The results showed that A. adenophora causes damage to the rumen epithelium by increasing the expression of pro-inflammatory markers like TNF-α and IFN-γ and reducing the expression of tight junction proteins such as occludin and claudin-1 in goats.

Asunto(s)

RESUMEN

Subacute rumen acidosis (SARA) will cause an increase in endotoxin, which will have a negative effect on the bovine rumen epithelial cells (BREC). Flavonoids are effective in treating inflammation caused by endotoxin. Quercetin is a vital flavonoid widely occurring in fruits and vegetables and has received significant interest as a prospective anti-inflammatory antioxidant. Nonetheless, quercetin's protective machinery against such damage to BREC induced by lipopolysaccharide (LPS) remains unclear. A combined quercetin and LPS-induced BREC inflammation model was utilized to elucidate the effect of quercetin protecting BREC from LPS-induced injury. After treating BREC with different doses of LPS (1, 5, and 10 µg/mL) for 6 h or 24 h, the mRNA expression of inflammatory factors was detected. Our experimental results show the establishment of the BREC inflammation model via mRNA high expression of pro-inflammatory cytokines in BREC following 6 h treatment with 1 µg/mL LPS. The promotive effect of 80 µg/mL quercetin on BREC growth via the cell counting kit-8 (CCK8) assay was observed. The expression of pro-inflammatory cytokines and chemokines, notably tumor necrosis factor α (TNF-α), Interleukin 1ß (IL-1ß), IL-6, CC-motif chemokine ligand 2 (CCL2), CCL20, CCL28, and CXC motif chemokine 9 (CXCL9), etc., was significantly reduced by quercetin supplementation. We also analyzed the mRNA detection of related pathways by qRT-PCR. Our validation studies demonstrated that quercetin markedly curbed the mRNA expression of the toll-like receptor 4 (TLR4) and myeloid differentiation primary response protein (MyD88) and the nuclear factor-κB (NF-κB) in LPS-treated BREC. In addition, western blot result outcomes confirmed, as expected, that LPS significantly activated phosphorylation of p44/42 extracellular regulated protein kinases (ERK1/2) and NF-κB. Unexpectedly, this effect was reversed by adding quercetin. To complement western blot results, we assessed p-ERK1/2 and p-p65 protein expression using immunofluorescence, which gave consistent results. Therefore, quercetin's capacity to bar the TLR4-mediated NF-κB and MAPK signaling pathways may be the cause of its anti-inflammatory effects on LPS-induced inflammatory reactions in BREC. According to these results, quercetin may be utilized as an anti-inflammatory medication to alleviate inflammation brought on by high-grain feed, and it also lays out a conceptual foundation regarding the development and utilization of quercetin in the later stage.

Asunto(s)

RESUMEN

Subacute ruminal acidosis (SARA) is a prevalent disease in intensive dairy farming, and the rumen environment of diseased cows acidifies, leading to the rupture of gram-negative bacteria to release lipopolysaccharide (LPS). LPS can cause rumentitis and other complications, such as liver abscess, mastitis and laminitis. Propionate, commonly used in the dairy industry as a feed additive, has anti-inflammatory effects, but its mechanism is unclear. This study aims to investigate whether sodium propionate (SP) reduces LPS-induced inflammation in rumen epithelial cells (RECs) and the underlying mechanism. RECs were stimulated with different time (0, 1, 3, 6, 9, 18 h) and different concentrations of LPS (0, 1, 5, 10 µg/mL) to establish an inflammation model. Then, RECs were treated with SP (15, 25, 35 mM) or 10 µM PDTC in advance and stimulated by LPS for the assessment. The results showed that LPS (6h and 10 µg/mL) could stimulate the phosphorylation of NF-κB p65, IκB, JNK, ERK and p38 MAPK through TLR4, and increase the release of TNF-α, IL-1ß and IL-6. SP (35 mM) can reduce the expression of cytokines by effectively inhibiting the NF-κB and MAPK inflammatory pathways. This study confirmed that SP inhibited LPS-induced inflammatory responses through NF-κB and MAPK in RECs, providing potential therapeutic targets and drugs for the prevention and treatment of SARA.

Asunto(s)

RESUMEN

Strontium (Sr) belongs to the same group in the periodic table as calcium (Ca). Sr level can serve as an index of rumen Ca absorption capacity; however, the effects of Sr on Ca2+ metabolism are unclear. This study aims to investigate the effect of Sr on Ca2+ metabolism in bovine rumen epithelial cells. The bovine rumen epithelial cells were isolated from the rumen of newborn Holstein male calves (n = 3, 1 day old, 38.0 ± 2.8 kg, fasting). The half maximal inhibitory concentration (IC50) of Sr-treated bovine rumen epithelial cells and cell cycle were used to establish the Sr treatment model. Transcriptomics, proteomics, and network pharmacology were conducted to investigate the core targets of Sr-mediated regulation of Ca2+ metabolism in bovine rumen epithelial cells. The data of transcriptomics and proteomics were analyzed using bioinformatic analysis (Gene Ontology and Kyoto Encyclopedia of genes/protein). Quantitative data were analyzed using one-way ANOVA in GraphPad Prism 8.4.3 and the Shapiro-Wilk test was used for the normality test. Results presented that the IC50 of Sr treatment bovine rumen epithelial cells for 24 h was 43.21 mmol/L, and Sr increased intracellular Ca2+ levels. Multi-omics results demonstrated the differential expression of 770 mRNAs and 2436 proteins after Sr treatment; network pharmacology and reverse transcriptase polymerase chain reaction (RT-PCR) revealed Adenosylhomocysteine hydrolase-like protein 2 (AHCYL2), Semaphoring 3A (SEMA3A), Parathyroid hormone-related protein (PTHLH), Transforming growth factor ß2 (TGF-ß2), and Cholesterol side-chain cleavage enzyme (CYP11A1) as potential targets for Sr-mediated Ca2+ metabolism regulation. Together these results will improve the current comprehension of the regulatory effect of Sr on Ca2+ metabolism and pave a theoretical basis for Sr application in bovine hypocalcemia.

Asunto(s)

RESUMEN

Studies have shown that exogenous thiamine (THI) supplementation can alleviate inflammation and promote rumen epithelial development in goats and cows. This research aimed to evaluate the effect of THI supplementation on LPS-induced inflammation and energy metabolic dysregulation in RECs of goats. Cells were stimulated with either 5 µg/mL THI for 18 h (THI group) or with 5 µg/mL LPS for 6 h (LPS group). The CON group was stimulated with DMEM/F-12 medium without THI for 18 h. The LPTH group was pretreated with THI for 18 h, followed by LPS stimulation for 6 h. THI supplementation decreased the ROS content (p < 0.05), as well as the ratios of phosphorylated (p)-p65 to p65 (p < 0.05) and p-AMPKα to AMPKα (p < 0.05). Interestingly, when the p38 gene was overexpressed in the LPTH group, the ratio of p-p65 to p65 and p-AMPKα to AMPKα proteins significantly increased, and ATP content decreased (p < 0.05). Our results suggest that THI possesses anti-inflammatory and metabolic-modulatory effects in RECs. The mechanism is largely related to the suppression of the NF-κB/p38 MAPK/AMPK signaling pathway. Additionally, we also revealed that THI supplementation can inhibit LPS-induced oxidative damage and apoptosis to protect mitochondrial function in RECs.

RESUMEN

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-1ß (IL-1ß), 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.

Asunto(s)

RESUMEN

Endotoxins play a crucial role in ruminant health due to their deleterious effects on animal health. The study aimed to evaluate whether LPS and LTA can induce an inflammatory response in rumen epithelial cells. For this purpose, epithelial cells isolated from rumen tissue (REC) were stimulated with LPS and LTA for 1, 2, 4, and 24 h. Thereafter, the expression of selected genes of the LPS and LTA pathway and inflammatory response were evaluated. Furthermore, it was assessed whether LPS affects inflammatory response and structural integrity of claw explants. Therefore, claw explants were incubated with LPS for 4 h to assess the expression of selected genes and for 24 h to evaluate tissue integrity via separation force. LPS strongly affected the expression of genes related to inflammation (NFkB, TNF-α, IL1B, IL6, CXCL8, MMP9) in REC. LTA induced a delayed and weaker inflammatory response than LPS. In claw explants, LPS affected tissue integrity, as there was a concentration-dependent decrease of separation force. Incubation time had a strong effect on inflammatory genes in claw explants. Our data suggest that endotoxins can induce a local inflammatory response in the rumen epithelium. Furthermore, translocation of LPS might negatively impact claw health.

RESUMEN

In subacute rumen acidosis (SARA), the rumen epithelium is frequently attacked by endotoxin (LPS), which is caused by the lysis of dead Gram-negative bacteria. However, the rumen epithelium innate immune system can actively respond to the infection. Previous studies have demonstrated that tea tree oil (TTO) has good bactericidal and anti-inflammatory effects. Therefore, the aim of this study was to investigate the effect of TTO on the expression of genes involved in the inflammatory cytokines in goat rumen epithelial cells (GRECs) triggered by LPS. Our study shows that rumen epithelial cells isolated from goat rumen tissue can be cultured in vitro in 0.25% trypsin for a long time. These cells were identified as epithelial cells by the expression of cytokeratin 18, monocarboxylate transporter 4 (MCT4), Na[+]/H[+] hydrogen exchanger 1 (NHE1), putative anion transporter 1 (PAT1), vH+ ATPase B subunit (vH+ ATPase), and anion exchanger 2 (AE2). The mRNA expression of IL-1ß, IL-6, TNF-α, TLR-2, NF-κB, CXCL6 and CXCL8 genes was significantly increased when LPS was used compared to untreated controls. In addition, mRNA expression of IL-1ß, IL-6, TNF-α, TLR-2, NF-κB, CXCL8, CXCL6 and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) genes was also significantly higher in the LPS group compared to the 0.05% TTO group. However, the expression of IL-1ß, IL-6, TNF-α, TLR-2, CXCL6 and IFIT3 genes was significantly lower in the LPS and 0.05% TTO group compared to the 1 µg/mL LPS group. These results suggest that TTO can inhibit LPS-induced inflammatory cytokines expression in GRECs.

RESUMEN

Short-chain fatty acids (SCFAs) play a critical role in regulation of rumen epithelial growth. The mechanisms underlying the regulatory effects of SCFAs on the proliferation of bovine rumen epithelial cells (BRECs) remain unknown; however, SCFAs can bind to G protein-coupled receptor 41 (GPR41); hence, the regulatory effects of SCFAs on BRECs proliferation may be mediated by GPR41. Here, we investigated the molecular mechanisms underlying the effects of SCFAs and GPR41 on BRECs proliferation. We demonstrated that SCFAs activate the expression of GPR41 and inhibit (p < .05) BRECs proliferation, while the GPR41 knockdown (GPR41KD) BRECs exhibited (p < .05) slow proliferation compared with controls. The treatment of BRECs with 10 mM SCFAs significantly enhanced (p < .05) expression of cyclin-dependent kinase inhibitors 1A (CDKN1A), 2A (CDKN2A) and 2B (CDKN2B) and inhibited (p < .05) their transition from G1 to S phase of the cell cycle, compared with controls. Remarkably, the GPR41KD BRECs treated with SCFAs restored high level of CDKN1A, relative to GPR41KD BRECs, but did not affect (p > .05) the expression of CDKN2A and CDKN2B. The GPR41KD BRECs had significantly reduced (p < .05) cyclin-dependent kinase 4 (CDK4) and cyclin D2 mRNA abundance compared with controls. The GPR41KD BRECs treated with SCFAs significantly decreased (p < .05) CDK4, cyclin D2, CDKN2A and CDKN2B mRNA abundance compared with BRECs treated with SCFAs. Overall, our results demonstrated that downregulation of CDK4 and cyclin D2 likely mediates the inhibitory effects of GPR41KD on BRECs proliferation. Additionally, CDKN1A plays a vital role in mediating the inhibitory effect of SCFAs on the BRECs proliferation, and that these changes are not mediated by GPR41.

Asunto(s)

RESUMEN

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, IL1ß, TNFα, chemokines, and immune barrier genes by transcriptome analysis. Consistent with transcriptome results, the expression of GPR41, IL1ß, 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 IL1ß 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.

Asunto(s)

RESUMEN

BACKGROUND/AIMS: Subacute ruminal acidosis (SARA) is a common disease in high-producing lactating cows. Rumenitis is the initial insult of SARA and is associated with the high concentrations of histamine produced in the rumen of dairy cows during SARA. However, the exact mechanism remains unclear. The objective of the current study is to investigate whether histamine induces inflammation of rumen epithelial cells and the underlying mechanism of this process. METHODS: Bovine rumen epithelial cells were cultured and treated with different concentrations of histamine and pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) cultured in different pH medium (pH 7.2 or 5.5). qRT-PCR, Western-blotting, ELISA and immunocytofluorescence were used to evaluate whether histamine activated the NF-κB pathway and inflammatory cytokines. RESULTS: The results showed that histamine significantly increased the activity of IKK ß and the phosphorylation levels of IκB α, as well as upregulated the mRNA and protein expression levels of NF-κB p65 in the rumen epithelial cells cultured in neutral (pH=7.2) and acidic (pH=5.5) medium. Furthermore, histamine treatment also significantly increased the transcriptional activity of NF-κB p65. High expression and transcriptional activity of NF-κB p65 significantly increased the mRNA expressions and concentrations of inflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin 1 beta (IL-1ß), thereby inducing the inflammatory response in bovine rumen epithelial cells. However, inhibition of NF-κB p65 by PDTC significantly decreased the expressions and concentrations of the inflammatory cytokines induced by histamine in the rumen epithelial cells cultured in the neutral and acidic medium. CONCLUSION: The present data indicate that histamine induces the inflammatory response of bovine rumen epithelial cells through the NF-κB pathway.

Asunto(s)

RESUMEN

Currently, the mechanism(s) responsible for the regulation of urea transporter B (UT-B) expression levels in the epithelium of the rumen remain unclear. We hypothesized that rumen fermentation products affect ruminal UT-B expression. Therefore, the effects of short-chain fatty acids (SCFA), pH, ammonia, and urea on mRNA and protein levels of UT-B were assayed in primary rumen epithelial cell cultures and in rumen epithelium obtained from intact goats. In vitro, SCFA and acidic pH were found to synergetically stimulate both mRNA and protein expression of UT-B, whereas NH4Cl decreased mRNA and protein levels of UT-B at pH 6.8. Treatment with urea increased both levels at pH 7.4. When goats received a diet rich in nitrogen (N) and nonfiber carbohydrates (NFC), their rumen epithelium had higher levels of UT-B, and the rumen contained higher concentrations of SCFA and NH3-N with a lower pH. An increase in plasma urea-N concentration was also observed compared with the plasma of the goats that received a diet low in N and NFC. In a second feeding trial, goats that received a NFC-rich, but isonitrogenous, diet had higher mRNA and protein levels of UT-B, and higher levels of G protein-coupled receptor (GPR) 41 and GPR4, in their rumen epithelium. The ruminal concentrations of SCFA and NH3-N also increased, while a lower pH was detected. In contrast, the serum urea-N concentrations remained unchanged. These data indicate that ruminal SCFA and pH are key factors, via GPR4 and GPR41, in the dietary regulation of UT-B expression, and they have priority over changes in plasma urea.

Asunto(s)