Tianle Xu

Hepatic TLR4 signaling is activated by LPS from digestive tract during SARA, and epigenetic mechanisms contribute to enforced TLR4 expression

Subacute ruminal acidosis (SARA) is known to trigger a systemic inflammatory response that is possibly caused by the translocationof lipopolysaccharides (LPS) from the gastrointestinal tract into the bloodstream. The aim of this study is to investigate this causal relationship between the increases of circulating LPS and liver inflammation. Here we found that SARA goats exhibited significantly increased LPS concentrations in both the rumen and portal vein. The livers of these goats exhibited increased mRNA concentrations of pro-inflammatory genes that indicated inflammation. Meanwhile, the occurrence of liver inflammation was further validated by the enhanced protein expression of those cytokines in the livers of SARA goats. These increased expressions of detected pro-inflammatory genes were likely mediated by enforced TLR4 signaling because SARA increased the concentrations of TLR4 mRNA and protein in the liver and the abundance of both the NF-kB-p65 factor and its active phosphorylated variant. We also verified that the enhanced TLR4 expression was accompanied by chromatin decompaction and demethylation of the proximal TLR4 promoter. Hence, epigenetic mechanisms are involved in the enforced expression of immune genes during SARA, and these findings open innovative routes for interventions via the modulation of these epigenetic mechanisms.

Lipopolysaccharide derived from the digestive tract provokes oxidative stress in the liver of dairy cows fed a high-grain diet

The aims of this study were to measure oxidative stress parameters and to investigate the molecular mechanism triggered by grain-induced subacute ruminal acidosis in mid-lactation cows. Twelve Holstein-Friesian cows with an average weight of 455±28kg were divided into 2 groups and subjected to 2 diets over 18wk: either a low-grain (forage-to-concentrate ratio=6:4) or a high-grain (forage-to-concentrate ratio=4:6) diet based on dry matter. Being fed a long-term high-grain diet resulted in a significant decrease in rumen pH and a significant increase in ruminal lipopolysaccharide (LPS) at 4 h postfeeding in the morning. The increase was also observed in LPS concentrations in the portal vein, hepatic vein, and jugular vein blood plasma as well as reduced milk yield in a high-grain diet. Cows fed a high-grain diet had lower levels of catalase and glutathione peroxidase (GPx) activity and total antioxidant capacity than cows fed a low-grain diet; however, super oxide dismutase (SOD) activity and malondialdehyde (MDA) levels were higher in both the liver and the plasma of high-grain than in low-grain cows. Positive correlations were observed between plasma LPS versus hepatic MDA, plasma MDA, and hepatic SOD activity, whereas hepatic GPx and plasma GPx were negatively correlated with plasma LPS. The relative mRNA abundances of GPX1 and CAT were significantly lower in the liver of cows fed a high-grain diet than those fed a low-grain diet, whereas SOD1 was significantly higher in cows fed a high-grain diet than cows fed a low-grain diet. The expression levels of Nrf2, NQO1, MT1E, UGT1A1, MGST3, and MT1A were downregulated, whereas NF-kB was upregulated, in cows fed a high-grain diet. Furthermore, nuclear factor E2-related factor 2 (Nrf2) total protein and mRNA levels were significantly lower than in low-grains. Our results demonstrate the relationship between the translocated LPS and the suppression of cellular antioxidant defense capacity, which lead to increased oxidative stress and suggests that the Nrf2-dependent antioxidant response may be affected by higher levels of LPS translocated to the bloodstream.

Lipopolysaccharide derived from the digestive tract activates inflammatory gene expression and inhibits casein synthesis in the mammary glands of lactating dairy cows.

To meet the nutrition requirements of lactation, dairy cows are usually fed a high concentrate diet (HC). However, high-grain feeding causes subacute ruminal acidosis (SARA), a metabolic disorder that causes milk protein depression. This study aimed to investigate the effect of lipopolysaccharide (LPS) released in the rumen on inflammatory gene expression and casein synthesis in mammary glands of lactating dairy cows fed a HC diet. We found that milk protein was significantly decreased in the HC group after 15 weeks of feeding. Overall, LPS concentrations in the rumen fluid, lacteal artery and vein were increased in the HC group. Transcriptome microarray was used to evaluate alterations in the signaling pathway in mammary glands. Signaling pathways involved in inflammatory responses were activated, whereas those involved in protein synthesis were inhibited in the HC group. mRNA expression involved in inflammatory responses, including that of TLR4, NF-кB and pro-inflammatory genes, was increased in the HC group, while αs1-casein (CSN1S1), β-casein (CSN2), mTOR and S6K gene expression were decreased. Moreover, protein expression was consistent with the corresponding gene expression. After feeding with an HC diet, LPS derived from the rumen increased inflammatory gene expression and inhibited casein synthesis in the mammary glands of lactating dairy cows fed a HC diet.

Feeding a High Concentrate Diet Down-Regulates Expression of ACACA, LPL and SCD and Modifies Milk Composition in Lactating Goats

High concentrate diets are fed to early and mid-lactation stages dairy ruminants to meet the energy demands for high milk production in modern milk industry. The present study evaluated the effects of a high concentrate diet on milk fat and milk composition, especially, cis-9, trans-11 CLA content in milk and gene expression of lactating goats. Eight mid-lactating goats with rumen fistula were randomly assigned into a high concentrate diet (HCD) group and low concentrate diet (LCD) group. High concentrate diet feeding significantly increased lipopolysaccharides (LPS) in plasma and decreased milk fat content, vaccenic acid (VA) and cis-9, trans-11 CLA in milk of the lactating goats. The mRNA expression levels of sterol regulatory element binding protein B 1c (SREBP1c), lipoprotein lipase (LPL), fatty acid synthetase (FASN) and acetyl-CoA carboxylase α (ACACA, ACCα) involving in lipid metabolism were analyzed, and ACACA and LPL all decreased in their expression level in the mammary glands of goats fed a high concentrate diet. DNA methylation rate of stearoyl-CoA desaturase (SCD) was elevated and decreased, and SCD mRNA and protein expression was reduced significantly in the mammary glands of goats fed a high concentrate diet. In conclusion, feeding a high concentrate diet to lactating goats decreases milk fat and reduced expression of SCD in the mammary gland, which finally induced cis-9, trans-11 CLA content in milk.

Stearoyl-CoA desaturase 1 expression is downregulated in liver and udder during E. coli mastitis through enhanced expression of repressive C/EBP factors and reduced expression of the inducer SREBP1A

BackgroundStearoyl-CoA desaturase 1 (SCD1) desaturates long chain fatty acids and is therefore a key enzyme in fat catabolism. Its synthesis is downregulated in liver during illnesses caused by high levels of circulating lipopolysaccharide (LPS). SCD1 expression is known to be stimulated under adipogenic conditions through a variety of transcription factors, notably SREBP1 and C/EBPα and −β. However, mechanisms downregulating SCD1 expression during illness related reprograming of the metabolism were unknown. Escherichia coli elicited mastitis is an example of such a condition and was found to downregulates milk and milk fat synthesis. This is in part mediated through epigenetic mechanisms. We analyzed here mechanism controlling SCD1 expression in livers and udders from cows suffering from experimentally induced E. coli mastitis.ResultsWe validated with RT-qPCR that SCD1 expression was reduced in these organs of the experimental cows. They also featured decreased levels of mRNAs encoding SREBP1a but increased levels for C/EBP α and −β. Chromatin accessibility PCR (CHART) revealed that downregulation of SCD1 expression in liver was not caused by tighter chromatin compaction of the SCD1 promoter. Reporter gene analyses showed in liver (HepG2) and mammary epithelial (MAC-T) model cells that overexpression of SREBP1a expectedly activated the promoter, while unexpectedly C/EBPα and −β strongly quenched the promoter activity. Abrogation of two from among of the three C/EBP DNA-binding motifs of the promoter revealed that C/EBPα acts in cis but C/EBPβ in trans. Overexpressing truncated C/EBPα or −β factors lacking their repressive domains confirmed in both model cells the direct action of C/EBPα, but not of C/EBPβ on the promoter.ConclusionsWe found no evidence that epigenetic mechanism remodeling the chromatin compaction of the SCD1 promoter would contribute to downregulate SCD1 expression during infection. Instead, our data show for the first time that C/EBP factors may repress SCD1 expression in liver and udder rather than stimulating as it was previously shown in adipocytes. This cell type specific dual and opposite function of C/EBP factors for regulating SCD1 expression was previously unknown. Infection related activation of their expression combined with downregulated expression of SREBP1a explains reduced SCD1 expression in liver and udder during acute mastitis.

Sodium Butyrate Supplementation Alleviates the Adaptive Response to Inflammation and Modulates Fatty Acid Metabolism in Lipopolysaccharide-Stimulated Bovine Hepatocytes

This study aimed to evaluate whether sodium butyrate (SB) attenuates the hepatic response to LPS-induced inflammation in bovine hepatocytes. Hepatocytes isolated from cows at ∼160 days in milk (DIM) were exposed to 0.5 mmol/L SB for 18 h as pretreatment. Cells pretreated with SB were used for the SB group, and those subjected to 4 μg/mL lipopolysaccharide (LPS) challenge for 6 h were used for the lipopolysaccharide pretreated with SB (LSB) group. The LPS-challenged hepatocytes showed increases in TNF-α and IL-6 production in culture medium (37 ± 11, P < 0.05); these increases were attenuated by pretreatment with SB in the LSB group (267 ± 4, P < 0.05). Compared to that in LPS-treated cells, the phospho-p65 and phospho-IκBα protein expression and nuclear translocation were suppressed when SB was added. Genes ( SREBP1c, SCD1, and DGAT1) and proteins (SREBP1c and SCD1) related to fatty acid metabolism were upregulated in LSB cells compared to those in LPS-treated cells ( P < 0.05). The ratios of phospho-AMPKα to AMPKα (0.32 ± 0.03 vs 0.70 ± 0.07) and phospho-ACCα to ACCα were decreased (0.81 ± 0.06 vs 2.06 ± 0.16) ( P < 0.05) in the LSB group. SB pretreatment reversed the histone H3 deacetylation that was increased by LPS stimulation in bovine hepatocytes (0.54 ± 0.02 vs 1.27 ± 0.11, P < 0.05). Our results suggest that SB pretreatment suppresses the hepatocyte changes that occur during the LPS-induced inflammatory response, which is accompanied by enhanced fatty acid synthesis, downregulated fatty acid oxidation, and histone H3 deacetylation, thus neutralizing the negative effects of infection.

Epigenetic mechanisms contribute to decrease stearoyl-CoA desaturase 1 expression in the liver of dairy cows after prolonged feeding of high-concentrate diet

Subacute ruminal acidosis (SARA) of dairy cattle is a widely occurring but not very overt metabolic disorder thought to impair milk composition. The enzyme stearoyl-CoA desaturase 1 (SCD1) is rate-limiting for the formation of Δ-9 unsaturated fatty acids and thus crucially involved in controlling lipid metabolism in the liver. It is known that SCD1 expression is downregulated during SARA, but the underlying molecular mechanisms are unknown. To study these mechanisms, we enrolled 12 healthy multiparous mid-lactation Holstein cows into a diet-induced SARA experiment. Six cows were fed a high-concentrate diet for 18 weeks (60% content of high-concentrate to 40% forage; HC group), whereas the others received a low-concentrate diet ad libitum (40% high-concentrate content to 60% forage; LC group). Sustained low ruminal pH values (pH 5.6 maintained for 4 h/d) and reduced milk yield performance (2.07 kg/d less than LC cows) verified that SARA had been induced in the HC group. Results showed a significantly decreased concentrations of cis-9 monounsaturated long-chain fatty acids in plasma collected from hepatic but not portal veins. This was matched by reduced SCD1 mRNA and protein concentrations in HC livers. The expression levels of genes related to lipid formation (DGAT1 and PLIN2) were downregulated during SARA, whereas those of catabolic genes (CPT1A, CPT2, and ACOX1) and some inflammatory genes were upregulated. Expression of SCD1 was downregulated through reduced transcription and abundance of the transcription factor sterol regulatory element-binding protein 1 (SREBP1c).This effect was augmented by local chromatin tightening and DNA methylation at and around the SREBP1c binding site in the SCD1 promoter. Chromatin immunoprecipitation assays confirmed that SARA reduced SREBP1c binding at the SCD1 promoter; hence, epigenetic mechanisms are involved in regulating the expression of genes related to long-chain fatty acid modification, partially through downregulation of both SCD1 and SREBP1c in the liver. Our results suggest that in addition to inflammatory genes, SCD1 is also involved in SARA-induced epigenetic regulation and its associated metabolic changes. This knowledge might help to provide a target for intervening against the detrimental metabolic effects of SARA.

High Grain Diet Triggers Inflammation in the Goat Uterus: A Comprehensive Regulation Diet Modulates the Immune Response

The aim of the present study was to explore the causal association between the increase in circulating subacute ruminal acidosis (SARA)-induced lipopolysaccharide (LPS) levels and uterine inflammation; moreover this study sought to evaluate the role of a comprehensive regulation diet in combating the inflammatory effects of a high grain diet. Twelve dairy goats were randomly assigned to two groups. One group was provided a high-concentrate buffered diet as the comprehensive regulation group (RG) control while, only a high concentrate powdered diet was fed to the treatment group (TG). The LPS concentrations in the rumen fluid were 428702±34505.6 and 255027±39756.2 EU/mL, in the TG and RG goats, respectively, while, the LPS levels in the peripheral plasma were 0.032±0.003 and 0.012±0.003 EU/mL in the TG and RG goats, respectively. The concentration of TNF-α, IL-1β and IL-6 was significantly higher, and there was enhanced expression of the inflammatory genes TLR4, MyD88, TRAF-6, NF-κB, IL-1β and IL-6 in the uteri of TG goats. SARA increased the NF-κB expression and level of its phosphorylated form in TG goats. The uteri of TG goats showed higher mRNA levels of proinflammatory genes than the RG goats, indicating the presence of inflammation. Thus, SARA-induced LPS derived from a high concentrate diet triggers inflammation in the goat uterus by reinforcing TLR4 signaling.