RESUMO
The Sequence Alignment/Map (SAM) format file is the text file used to record alignment information. Alignment is the core of sequencing analysis, and downstream tasks accept mapping results for further processing. Given the rapid development of the sequencing industry today, a comprehensive understanding of the SAM format and related tools is necessary to meet the challenges of data processing and analysis. This paper is devoted to retrieving knowledge in the broad field of SAM. First, the format of SAM is introduced to understand the overall process of the sequencing analysis. Then, existing work is systematically classified in accordance with generation, compression and application, and the involved SAM tools are specifically mined. Lastly, a summary and some thoughts on future directions are provided.
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Alinhamento de SequênciaRESUMO
High-grain (HG) feeding can trigger subacute ruminal acidosis (SARA) and subsequent liver tissue injury. This study investigated pyroptosis and NLRP3 inflammasome activation in SARA-induced liver injury, and the role of mitophagy during this process. Twelve mid-lactating Holstein cows equipped with rumen fistulas were randomly divided into 2 groups: a low-grain (LG) diet group (grain:forage = 4:6) and a HG diet group (grain:forage = 6:4). Each group had 6 cows. The experiment lasted for 3 wk. The ruminal fluid was collected through the rumen fistula on experimental d 20 and 21, and the pH immediately measured. At the end of the experiment, all animals were slaughtered, and peripheral blood and liver tissue were collected. The ruminal pH was lower in the HG group than that in the LG group at all time points. In addition, the ruminal pH in the HG group was lower than 5.6 at 3 consecutive time points after feeding (4, 6, and 8 h on d 20; 2, 4, and 6 h on d 21), indicating that HG feeding induced SARA. The content of lipopolysaccharide, IL-1ß, and apoptosis-related cysteine protease 1 (caspase-1) and the activity of alanine aminotransferase and aspartate aminotransferase in the blood plasma of the HG group were all significantly increased. Hepatic caspase-1 activity was increased in the livers of the HG group. The increased expression levels of pyroptosis- and NLRP3 inflammasome-related genes IL1B, IL18, gasdermin D (GSDMD), apoptosis-associated speck-like protein containing a card (ASC), NLR family pyrin domain-containing 3 (NLRP3), and caspase-1 (CASP1) in liver tissue of the HG group were detected. Furthermore, western blot analysis showed that HG feeding led to increased expression of pyroptosis- and NLRP3 inflammasome-related proteins GSDMD N-terminal (GSDMD-NT), IL-1ß, IL-18, cleaved-caspase-1, ASC, NLRP3, and cleaved-caspase-11 and upregulated expression of mitophagy-related proteins microtubule-associated protein 1 light chain 3 II (MAP1LC3-II), beclin 1 (BECN1), Parkin, and PTEN-induced kinase 1 (PINK1) in liver tissue. Collectively, our results revealed that SARA caused increased mitophagy and activated the NLRP3 inflammasome, causing pyroptosis and subsequent liver injury in dairy cows fed a HG diet.
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Acidose , Ração Animal , Dieta , Fígado , Mitofagia , Piroptose , Rúmen , Animais , Bovinos , Acidose/veterinária , Acidose/metabolismo , Feminino , Dieta/veterinária , Rúmen/metabolismo , Fígado/metabolismo , Fígado/patologia , Inflamassomos/metabolismo , Doenças dos Bovinos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Grão Comestível , LactaçãoRESUMO
This study aimed to investigate the effect and mechanism of a high concentrate (HC) diet on the inflammatory response and cellular tight junctions (TJs) in the mammary gland of dairy cows. Twelve lactating Holstein dairy cows were randomly assigned into low concentrate (LC) and HC groups (n = 6), which were fed with LC diet and HC diet respectively for 3 weeks. The HC diet lead to subacute ruminant acidosis with a rumen pH < 5.6 more than 3 h daily. The HC diet triggered an inflammatory response with increased levels of inflammatory cytokines in the lacteal vein, upregulated expression of inflammation-related genes, elevated activity of myeloperoxidase, and inflammatory cells infiltration in the mammary gland. Furthermore, the HC diet induced the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways with enhanced phosphorylation ratios of NF-κB P65, inhibitor of NF-κB (IκB), P38 and extracellular signal-regulated kinase 1/2 (ERK1/2) as well as decreased ratios of DNA methylation and chromatin compaction of genes coding for proinflammatory cytokines, which contributed to the upregulation of proinflammatory cytokine expression. The HC diet also destroyed the integrity of TJ with discontinuous and decreased expression levels of zonula occludens-1, Occludin, Claudin-4 and increased expression level of Claudin-1 in the mammary epithelial cells compared with LC group. Conclusively, the HC diet induced the activation of NF-κB and MAPK signaling pathways and epigenetic modifications, promoted the transcription of proinflammatory cytokines, and finally caused inflammatory response and TJ disruption in the mammary gland of dairy cows.
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NF-kappa B , Junções Íntimas , Feminino , Animais , Bovinos , NF-kappa B/metabolismo , Junções Íntimas/metabolismo , Lactação , Dieta/veterinária , Citocinas/metabolismo , Ração Animal/análiseRESUMO
High-concentrate diet can cause metabolic diseases, such as subacute ruminal acidosis (SARA), and secondary mastitis. To investigate the effect of SARA induced by high-concentrate diet on the lysine lactylation (Kla) and inflammatory responses in the mammary gland of dairy cows and the mechanism between them, we selected twelve mid-lactation Holstein cows with similar body conditions for modelling. They were randomly divided into two groups, fed a low-concentrate diet (LC) and a high-concentrate diet (HC) for 21 days. Our results showed that high-concentrate diet feeding significantly reduced ruminal pH, and the pH was below 5.6 for more than 3 h per day, indicating successful induction of the SARA model. Lactic acid concentrations in mammary gland and plasma were higher in the HC group than that in the LC group. HC diet feeding significantly up-regulated the expression levels of the Pan Kla, H3K18la, p300/CBP and monocarboxylate transporter 1 (MCT1) in the mammary gland. In addition, the mRNA expression levels of inflammatory factors were significantly regulated, including IL-1ß, IL-1α, IL-6, IL-8, SAA3, and TNF-α, while the anti-inflammatory factor IL-10 was down-regulated. The mammary gland of HC group was structurally disorganized with incomplete glandular vesicles, with a large number of detached mammary epithelial cells and inflammatory cells infiltration. The up-regulation of TLR4, TNF-α, p-p65, and p-IκBα indicated that the TLR4/NF-κB signaling pathway was activated. In conclusion, this study found that HC diet feeding can induce SARA and increase the concentration of lactic acid in mammary gland and plasma. Then, lactic acid could be transported into cells by MCT1 and up-regulate the expression level of histone lactylation mediated by p300/CBP, and subsequently promote the activation of TLR4/NF-κB signaling pathway, ultimately causing inflammatory responses in the mammary gland.
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Doenças dos Bovinos , NF-kappa B , Feminino , Animais , Bovinos , NF-kappa B/metabolismo , Regulação para Cima , Histonas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Lactação , Dieta/veterinária , Dieta/métodos , Concentração de Íons de Hidrogênio , Leite/metabolismo , Doenças dos Bovinos/metabolismoRESUMO
Pseudomonas aeruginosa is a common pathogenic bacteria in canine ophthalmology. Lipopolysaccharide (LPS), a component in the cell wall of gram-negative bacteria, is released following bacterial lysis and causes pathology and inflammation of the cornea. Antibiotics are used to treat bacterial keratitis, and the reuse of antibiotics can easily cause bacterial resistance. Research has shown that glutamine (GLN) has anti-inflammatory and antioxidant biological functions. Herein, we explored the effects and underlying mechanisms of GLN and established an LPS-induced cornea inflammation model. Treatment groups comprised: control check (CK), LPS, LPS + GLN, and Sham groups. Topical GLN treatment alleviated corneal opacity, reduced corneal injury, and accelerated corneal wound healing. Furthermore, GLN treatment altered the uniform distribution of corneal epithelial cells and transformed the healing approach of these cells in the corneal wound from crawling to filling. The expression of Toll-like receptor 4 (TLR4), IL-6, TNF-α, and p-p65 and the activity of myeloperoxidase and superoxide dismutase decreased while the content of malondialdehyde increased in the LPS + GLN group compared with those in the LPS group. Thus, our study suggests that LPS-induced inflammation and oxidative stress may be suppressed via the TLR4/NF-κB signaling pathway by GLN and that GLN could be used as an adjunct therapy to reduce antibiotic use.
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Ceratite , Lipopolissacarídeos , Cães , Animais , Lipopolissacarídeos/toxicidade , Receptor 4 Toll-Like/metabolismo , Glutamina/farmacologia , Glutamina/metabolismo , Ceratite/tratamento farmacológico , Ceratite/prevenção & controle , NF-kappa B/metabolismo , Inflamação/patologia , Estresse OxidativoRESUMO
Lipopolysaccharide (LPS) is an important stimulus of inflammation via binding to toll-like receptor 4 (TLR4), but the role of TLR4 in LPS-induced cellular homeostasis disruption indicated by the increased level of endoplasmic reticulum (ER) stress, autophagy, and apoptosis is unknown in the liver of dairy cows. Previous studies show that forkhead box protein A2 (FOXA2) is an important transcriptional factor to maintain cellular metabolic homeostasis, but the mechanisms by which FOXA2 mediates cellular homeostasis disruption in response to LPS remains unclear. To achieve the aims, hepatocytes separated from dairy cows at â¼160 d in milk were pretreated with a specific TLR4 inhibitor TAK-242 for 12 h, followed by LPS treatment for another 12 h to investigate the role of TLR4 in LPS-induced disruption of cellular homeostasis. The results indicated that LPS-induced nuclear factor-κB (NF-κB)-mediated inflammatory cascades, ER stress, autophagy, and apoptosis via activating TLR4 and downregulating FOXA2 expression in bovine hepatocytes. The application of TLR4 inhibitor alleviated LPS-induced inflammation through inactivating NF-κB proinflammatory pathway, restored cell homeostasis by decreasing the level of ER stress, autophagy, and apoptosis, and upregulated FOXA2 expression. Furthermore, we also elevated FOXA2 expression with an overexpression plasmid to clarify its molecular role in response to LPS challenge. FOXA2 overexpression reduced LPS-caused inflammation by inhibiting NF-κB signaling pathway. Also, FOXA2 could alleviate ER stress to block unfolded protein response and suppress autophagic flux. In addition, FOXA2 enhanced mitochondrial membrane potential via reducing pro-apoptotic protein BAX, CASPASE3, and Cleaved CASPASE3 expression and elevating anti-apoptotic protein BCL-2 expression to mitigate LPS-induced apoptosis. Taken together, these findings suggested that FOXA2 is a mediator to alleviate TLR4-controlled inflammation, ER stress, autophagy, and apoptosis in LPS-treated bovine hepatocytes, it could serve as a potential target to intervene cell homeostasis disruption caused by LPS in the liver of dairy cows.
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Doenças dos Bovinos , Lipopolissacarídeos , Feminino , Bovinos , Animais , Lipopolissacarídeos/metabolismo , NF-kappa B/metabolismo , Receptor 4 Toll-Like/metabolismo , Apoptose , Inflamação/metabolismo , Inflamação/veterinária , Hepatócitos/metabolismo , Estresse do Retículo Endoplasmático , Autofagia , Fatores de Transcrição Forkhead , Doenças dos Bovinos/metabolismoRESUMO
Subacute rumen acidosis can lead to mastitis in dairy cows. Mitochondrial dysfunction is closely related to the inflammatory response. This experiment was conducted to investigate the effects of a high-concentrate diet on mammary gland inflammation and mitochondrial damage in dairy cows. Twelve Holstein dairy cows in mid-lactation were randomly divided into 2 groups and fed a 40% concentrate (low concentrate, LC) diet or a 60% concentrate (high concentrate, HC) diet. Cows were fed individually, and the experiment lasted for 3 wk. After the experiment, mammary gland tissue, blood, and rumen fluid were collected. Compared with the LC diet, the HC diet significantly decreased rumen pH; the pH was <5.6 for more than 3 h. The HC diet also increased the concentration of LPS in the blood (7.17 ± 1.25 µg/mL vs. 12.12 ± 1.26 µg/mL), which indicated that feeding the HC diet successfully induced subacute rumen acidosis. The HC diet also increased the concentration of Ca2+ (34.80 ± 4.23 µg/g vs. 46.87 ± 7.24 µg/g) in the mammary gland and upregulated the expression of inflammatory factors IL-6 (1,128.31 ± 147.53 pg/g vs. 1,538.42 ± 241.38 pg/g), IL-1ß (69.67 ± 5.86 pg/g vs. 90.13 ± 4.78 pg/g), and tumor necrosis factor-α (91.99 ± 10.43 pg/g vs. 131.75 ± 17.89 pg/g) in mammary venous blood. The HC diet also increased the activity of myeloperoxidase (0.41 ± 0.05 U/g vs. 0.71 ± 0.11 U/g) and decreased the content of ATP (0.47 ± 0.10 µg/mL vs. 0.32 ± 0.11 µg/mL) in the mammary gland. In addition, phosphorylation of JNK (1.00 ± 0.21 vs. 2.84 ± 0.75), ERK (1.00 ± 0.20 vs. 1.53 ± 0.31), and p38 (1.00 ± 0.13 vs. 1.47 ± 0.41) and protein expression of IL-6 (1.00 ± 0.22 vs. 2.21 ± 0.27) and IL-8 (1.00 ± 0.17 vs. 1.96 ± 0.26) were enhanced in cows of the HC group, indicating that the mitogen-activated protein kinase (MAPK) signaling pathway was activated. Compared with the LC diet, the HC diet reduced the protein expression of mitochondrial biogenesis-related proteins PGC-1α (1.00 ± 0.17 vs. 0.55 ± 0.12), NRF1 (1.00 ± 0.17 vs. 0.60 ± 0.10), TFAM (1.00 ± 0.10 vs. 0.73 ± 0.09), and SIRTI (1.00 ± 0.44 vs. 0.40 ± 0.10). The HC diet promoted mitochondrial fission and inhibited mitochondrial fusion by reducing protein expression of MFN1 (1.00 ± 0.31 vs. 0.49 ± 0.09), MFN2 (1.00 ± 0.19 vs. 0.69 ± 0.13), and OPA1 (1.00 ± 0.08 vs. 0.72 ± 0.07), and by increasing that of DRP1 (1.00 ± 0.09 vs. 1.39 ± 0.10), MFF (1.00 ± 0.15 vs. 1.89 ± 0.12), and TTC1/FIS1 (1.00 ± 0.08 vs. 1.76 ± 0.14), leading to mitochondrial dysfunction. The HC diet increased mitochondrial permeability by upregulating the protein expression of VDAC1 (1.00 ± 0.42 vs. 1.90 ± 0.44), ANT (1.00 ± 0.22 vs. 1.27 ± 0.17), and CYPD (1.00 ± 0.41 vs. 1.82 ± 0.43). Taken together, these results indicated that feeding the HC diet induced mitochondrial damage via the MAPK signaling pathway in the mammary gland of dairy cows.
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Acidose , Doenças dos Bovinos , Feminino , Bovinos , Animais , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Interleucina-6/metabolismo , Transdução de Sinais , Lactação/fisiologia , Dieta/veterinária , Acidose/veterinária , Acidose/metabolismo , Rúmen/metabolismo , Leite/metabolismo , Ração Animal , Doenças dos Bovinos/metabolismoRESUMO
The purpose of this experiment was to investigate high-concentrate feeding-induced changed status of oxidative and autophagy in the livers of dairy cows. Hepatocyte nuclear factor 3ß (FOXA2) was reported in cases of liver fibrosis, glucolipid metabolism, and hepatocyte differentiation, but not in cases liver damage in cows fed a high-concentrate diet. Therefore, we also aimed to explore the potential role of FOXA2 in SARA-induced liver damage. We divided 12 mid-lactating Holstein cows into 2 groups and fed them a high-concentrate (HC group, forage:concentrate = 4:6) and a low-concentrate (forage:concentrate = 6:4) diet. After a 2-wk adaptation period and a 3-wk experimental period, peripheral blood was collected for determination of antioxidant enzyme activity, and liver tissue was collected to examine genes and proteins. On d 20 and 21 of the experiment, rumen fluid was collected, and the pH was measured. A significant difference in rumen fluid pH was found between the 2 groups (low-concentrate: 6.10 ± 0.07 vs. HC: 5.59 ± 0.09). The rumen fluid pH in the HC group was less than 5.6 at 2 time points, indicating that SARA was successfully induced. Lipopolysaccharide (0.24 ± 0.10 vs. 0.42 ± 0.12) and malondialdehyde (1.46 ± 0.25 vs. 2.94 ± 0.65) increased, whereas superoxide dismutase (14.06 ± 0.63 vs. 11.71 ± 0.64), reduced glutathione (14.48 ± 2.25 vs. 6.82 ± 0.67), and the total antioxidant capacity (0.43 ± 0.03 vs. 0.30 ± 0.03) decreased in the peripheral blood of the HC group. Moreover, in liver tissue from the HC group, catalase (0.71 ± 0.03 vs. 0.49 ± 0.03) and superoxide dismutase (27.46 ± 1.90 vs. 20.32 ± 1.54) were decreased, whereas malondialdehyde (0.21 ± 0.03 vs. 0.28 ± 0.03) was elevated. Meanwhile, we observed lower gene expression of CAT (1.00 ± 0.15 vs. 0.64 ± 0.17), NAD(P)H quinone dehydrogenase 1 (NQO1; 1.00 ± 0.09 vs. 0.47 ± 0.14), glutathione peroxidase 1 (GPX1; 1.03 ± 0.27 vs. 0.55 ± 0.09), SOD1 (1.01 ± 0.17 vs. 0.76 ± 0.17), and SOD3 (1.02 ± 0.21 vs. 0.55 ± 0.16) in the liver tissue of the HC group. Furthermore, western blot analysis showed that high-concentrate feeding led to decreased sirtuin-1 (SIRT1) (1.00 ± 0.10 vs. 0.62 ± 0.15) and FOXA2 (1.02 ± 0.19 vs. 0.68 ± 0.18), elevated autophagy-related protein microtubule associated protein 1 light chain 3 II (MAP1LC3-II; 1.00 ± 0.32 vs. 1.98 ± 0.83) and autophagy related 5 (ATG5; 1.00 ± 0.30 vs. 1.80 ± 0.27), and suppressed antioxidant signaling pathway-related protein nuclear factor erythroid 2-like 2 (NFE2L2; 1.00 ± 0.18 vs. 0.61 ± 0.30) and heme oxygenase 1 (HMOX1; 1.00 ± 0.48 vs. 0.38 ± 0.25) in liver tissue. Overall, these data indicated that SARA elevated systematic oxidative status and enhanced autophagy in the liver, and suppressed SIRT1 and FOXA2 may mediate enhanced oxidative damage and autophagy in the livers of dairy cows fed a high-concentrate diet.
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Acidose , Doenças dos Bovinos , Animais , Bovinos , Feminino , Acidose/veterinária , Antioxidantes/metabolismo , Doenças dos Bovinos/metabolismo , Dieta/veterinária , Concentração de Íons de Hidrogênio , Lactação , Fígado/metabolismo , Leite/metabolismo , Estresse Oxidativo , Rúmen/metabolismo , Sirtuína 1/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Ketosis is a metabolic disease that often occurs in dairy cows postpartum and is a result of disordered lipid metabolism. Acetyl-coenzyme A (CoA) acetyltransferase 2 (ACAT2) is important for balancing cholesterol and triglyceride (TG) metabolism; however, its role in subclinical ketotic dairy cows is unclear. This study aimed to explore the potential correlation between ACAT2 and lipid metabolism disorders in subclinical ketotic cows through in vitro and in vivo experiments. In the in vivo experiment, liver tissue and blood samples were collected from healthy cows (CON, n = 6, ß-hydroxybutyric acid [BHBA] concentration <1.0 mM) and subclinical ketotic cows (subclinical ketosis [SCK], n = 6, BHBA concentration = 1.2-3.0 mM) to explore the effect of ACAT2 on lipid metabolism disorders in SCK cows. For the in vitro experiment, bovine hepatocytes (BHEC) were used as the model. The effects of BHBA on ACAT2 and lipid metabolism were investigated via BHBA concentration gradient experiments. Subsequently, the relation between ACAT2 and lipid metabolism disorder was explored by transfection with siRNA of ACAT2. Transcriptomics showed an upregulation of differentially expression genes during lipid metabolism and significantly lower ACAT2 mRNA levels in the SCK group. Compared with the CON group in vivo, the SCK group showed significantly higher expression levels of peroxisome proliferator-activated receptor γ (PPARγ) and sterol regulator element binding protein 1c (SREBP1c) and significantly lower expression levels of peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyl-transferase 1A (CPT1A), sterol regulatory element binding transcription factor 2 (SREBP2), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Moreover, the SCK group had a significantly higher liver TG content and significantly lower plasma total cholesterol (TC) and free cholesterol content. These results were indicative of TG and cholesterol metabolism disorders in the liver of dairy cows with SCK. Additionally, the SCK group showed an increased expression of perilipin-2 (PLIN2), decreased expression of apolipoprotein B, and decreased plasma concentration of very low-density lipoproteins (VLDL) and low-density lipoproteins cholesterol (LDL-C) by downregulating ACAT2, which indicated an accumulation of TG in liver. In vitro experiments showed that BHBA induced an increase in the TG content of BHEC, decreased content TC, increased expression of PPARγ and SREBP1c, and decreased expression of PPARα, CPT1A, SREBP2, and HMGCR. Additionally, BHBA increased the expression of PLIN2 in BHEC, decreased the expression and fluorescence intensity of ACAT2, and decreased the VLDL and LDL-C contents. Furthermore, silencing ACAT2 expression increased the TG content; decreased the TC, VLDL, and LDL-C contents; decreased the expression of HMGCR and SREBP2; and increased the expression of SREBP1c; but had no effect on the expression of PLIN2. These results suggest that ACAT2 downregulation in BHEC promotes TG accumulation and inhibits cholesterol synthesis, leading to TG and cholesterol metabolic disorders. In conclusion, ACAT2 downregulation in the SCK group inhibited cholesterol synthesis, increased TG synthesis, and reduced the contents of VLDL and LDL-C, eventually leading to disordered TG and cholesterol metabolism.
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Doenças dos Bovinos , Cetose , Transtornos do Metabolismo dos Lipídeos , Feminino , Bovinos , Animais , Metabolismo dos Lipídeos/fisiologia , LDL-Colesterol , PPAR alfa/genética , PPAR gama/metabolismo , Doenças dos Bovinos/metabolismo , Transtornos do Metabolismo dos Lipídeos/veterinária , Proteínas de Transporte/metabolismo , Lipoproteínas VLDL/metabolismo , Cetose/veterinária , Coenzima A/metabolismo , Ácido 3-HidroxibutíricoRESUMO
Long-term feeding of a high-concentrate diet can induce subacute ruminal acidosis (SARA) in ruminants, which further leads to systemic inflammatory response. However, few studies have examined the effects of feeding a high-concentrate diet on the hindgut of ruminants. The purpose of this study was to investigate the effects of a high-concentrate diet on the composition of gut microbiota in colonic contents, inflammatory response, and barrier damage in the colon tissue of ruminants. A total of 12 healthy multiparous lactating Hu sheep were randomly allotted into the following 2 groups: a high-concentrate (HC) group (concentrate:forage = 7:3) and a low-concentrate (LC) group (concentrate:forage = 3:7). All sheep were fitted with ruminal fistulas. The formal feeding experiment lasted for 8 wk. After the feeding experiment, rumen fluid, portal vein blood, hepatic vein blood, colonic contents, and colon tissue samples were collected. The results showed that feeding the HC diet induced SARA in Hu sheep and significantly reduced pH in the colonic contents. The abundances of Firmicutes, Verrucomicrobiota, and Actinobacteriota decreased significantly, whereas those of Bacteroidota, Spirochaetota, and Fibrobacterota significantly increased in colonic contents. At the genus level, the relative abundances of 29 genera were significantly altered depending on the different type of diets. Analysis of the 10 bacterial genera with high relative abundance revealed that feeding the HC diet significantly reduced the abundance of UCG-005, Christensenellaceae R-7 group, UCG-010-norank, Monoglobus, [Eubacterium] coprostanoligenes group_norank, and Alistipes, whereas the abundances of Rikenellaceae RC9 gut group, Treponema, Bacteroides, and Prevotella increased. Compared with the LC group, feeding the HC diet significantly increased the concentration of LPS in rumen fluid, portal vein blood, hepatic vein blood, and colonic contents, and significantly upregulated the mRNA expression levels of proinflammatory cytokines in colon tissue, including TNF-α, IL-1ß, IL-6, and IL-8, indicating the occurrence of inflammatory response in the colon tissue. In addition, the structure of colonic epithelial cells was loose, the intercellular space became larger, epithelial cells were exfoliated, and the mRNA and protein abundances of ZO-1, occludin, claudin-1, claudin-3, and claudin-4 were significantly decreased in the HC group, which was consistent with the results of immunohistochemistry. Furthermore, feeding the HC diet increased the ratios of DNA methylation and chromatin compaction in the promoter regions of occludin and claudin-1, which in turn inhibited their transcriptional expression. Therefore, the present study demonstrated that feeding an HC diet induced SARA in Hu sheep, altered the composition and structure of the microbial community in the colonic contents, induced an inflammatory response, and disrupted the intestinal mucosal barrier in the colonic tissue.
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Regulação da Expressão Gênica , Doenças dos Ovinos , Feminino , Animais , Ovinos , Lactação , Claudina-1/metabolismo , Ocludina/análise , Ocludina/metabolismo , Rúmen/metabolismo , Inflamação/veterinária , Inflamação/metabolismo , Dieta/veterinária , Colo/metabolismo , Ruminantes/metabolismo , RNA Mensageiro/metabolismo , Concentração de Íons de Hidrogênio , Ração Animal/análiseRESUMO
Mitochondrial homeostasis is closely associated with cellular homeostasis process, whereas mitochondrial dysfunction contributes to apoptosis and mitophagy. Hence, analyzing the mechanism of lipopolysaccharide (LPS)-caused mitochondrial damage is necessary to understand how cellular homeostasis is maintained in bovine hepatocytes. Mitochondria-associated membranes (MAM), a connection between endoplasmic reticulum (ER) and mitochondria, is important to control mitochondrial function. To investigate the underlying mechanisms of the LPS-caused mitochondrial dysfunction, hepatocytes isolated from dairy cows at â¼160 d in milk (DIM) were pretreated with the specific inhibitors of adenosine 5'-monophosphate-activated protein kinase (AMPK), ER stress, RNA-activated protein kinase-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), c-Jun N-terminal kinase, and autophagy followed by a 12 I1/4g/mL LPS treatment. The results showed that inhibiting ER stress with 4-phenylbutyric acid decreased the levels of autophagy and mitochondrial damage with AMPK inactivation in LPS-treated hepatocytes. The AMPK inhibitor compound C pretreatment alleviated LPS-induced ER stress, autophagy and mitochondrial dysfunction by regulating the expression of MAM-related genes, such as mitofusin 2 (MFN2), PERK, and IRE1α. Moreover, inhibiting PERK and IRE1α mitigated autophagy and mitochondrial dynamic disruption by regulating the MAM function. Additionally, blocking c-Jun N-terminal kinase, the downstream sensor of IRE1α, could reduce the levels of autophagy and apoptosis and restore the balance of mitochondrial fusion and fission by modulating the B cell leukemia 2 (BCL-2)/BCL-2 interacting protein 1 (BECLIN1) complex in the LPS-treated bovine hepatocytes. Furthermore, autophagy blockage with chloroquine could intervene in LPS-caused apoptosis to restore mitochondrial function. Collectively, these findings suggest that the AMPK-ER stress axis is involved in the LPS-caused mitochondrial dysfunction by mediating the MAM activity in bovine hepatocytes.
Assuntos
Proteínas Quinases Ativadas por AMP , Lipopolissacarídeos , Feminino , Bovinos , Animais , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Hepatócitos/metabolismo , Estresse do Retículo Endoplasmático , Apoptose , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismoRESUMO
γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), a bacterial cell wall component, can trigger an inflammatory response. A mammary inflammatory response causes tight junction (TJ) dysfunction. This study aimed to explore the effects and involved mechanisms of iE-DAP-induced inflammatory response on the TJ integrity in bovine mammary epithelial cells (BMECs). The results showed that iE-DAP-induced inflammatory response and TJ disruption was associated with increased expression levels of inflammatory cytokines and decreased gene expression of ZO-1 and Occludin, as well as a reduction in transepithelial electrical resistance and elevation in paracellular dextran passage. While MLCK inhibitor ML-7 reversed the TJ disruption induced by iE-DAP. NF-κB inhibitor BAY 11-7085 hindered the activation of NF-κB and MLCK signaling pathways, the inflammatory response and TJ disruption induced by iE-DAP. NOD1-specific shRNA also inhibited the activation of the NOD1/NF-κB signaling pathway and reversed the inflammatory response and TJ injury in iE-DAP-treated BMECs. Above results suggest that iE-DAP activated the NF-κB and MLCK signaling pathway in NOD1-dependent manner, which promoted the transcription of inflammatory cytokines and altered the expression and distribution of tight junction proteins, finally caused inflammatory response and TJ disruption. This study might provide theoretical basis and scientific support for the prevention and treatment of mastitis.
Assuntos
NF-kappa B , Junções Íntimas , Feminino , Animais , Bovinos , NF-kappa B/metabolismo , Junções Íntimas/metabolismo , Transdução de Sinais , Citocinas/metabolismo , Células Epiteliais/metabolismoRESUMO
High concentrate (HC) diet feeding leads to the lysis of rumen microbes and the release of hazardous metabolites, which can trigger inflammatory responses, thereby impairing dairy cow health and production. γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), which constitutes the peptidoglycan (PGN) layer of bacteria, is the minimum PGN structure capable of activating inflammatory signaling pathways. This research paper aimed to determine the iE-DAP concentration and investigate the effects of an HC diet on the concentration of iE-DAP in the rumen fluid of dairy cows. However, there are limited studies on the determination of iE-DAP concentration. Hence, we established a high-performance liquid chromatography (HPLC) method combined with pre-column chiral derivatization to detect the concentration of iE-DAP in rumen fluid. Moreover, we conducted an animal experiment that included 12 lactating Holstein cows, which were randomly divided into a low-concentrate (LC) group and an HC group. The results showed that the linear range of iE-DAP was 5-500 µg/mL and that the intra- and inter-day RSDs were lower than 7%. Meanwhile, this method was successfully applied to the analysis of iE-DAP in rumen fluid, and the results revealed that long-term feeding with an HC diet elevated the concentration of iE-DAP in rumen fluid of dairy cows.
Assuntos
Lactação , Rúmen , Feminino , Bovinos , Animais , Rúmen/metabolismo , Cromatografia Líquida de Alta Pressão , Ração Animal , Dieta/veterinária , Leite/químicaRESUMO
The aim of the present study was to evaluate how long-term high-concentrate diet feeding affected rumen epithelium (RE) of dairy cows. So, 12 mid-lactating multiparous cows were divided into two groups randomly fed either with high-concentrate diet (HC, concentrate: forage = 6: 4) or low-concentrate diet (LC, concentrate: forage = 4:6) for 20 weeks. Remarkable upregulation of lipopolysaccharide (LPS) level and depress of pH in rumen fluid were induced by HC compared with LC group. mRNA abundance of interleukin-6 (IL-6), interleukin-8 (IL-8), C-C motif chemokine ligand 5 (CCL5), caspase-3, caspase-8, and caspase-9 were elevated in RE of HC group compared with LC group. Greater protein abundance of phosphorylated NF-κB p65, IL-6, and tumor necrosis factor α (TNF-α) was observed in RE of cows fed HC than that fed LC. Abundance of protein related to proapoptotic response (cytochrome c, BAX and caspase-3) in HC group was greater than that in LC group, while the abundance of anti-apoptotic factor protein (Bcl-2) was lower in HC group than LC group. Therefore, the present study demonstrated that long-term high-concentrate diet feeding upregulated LPS level in rumen fluid and induced the proinflammatory response in the rumen epithelium and apoptosis of rumen epithelial cells.
Assuntos
Doenças dos Bovinos , Rúmen , Ração Animal , Animais , Apoptose , Bovinos , Doenças dos Bovinos/metabolismo , Dieta/veterinária , Células Epiteliais/metabolismo , Epitélio/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Inflamação/induzido quimicamente , Lactação/fisiologia , Rúmen/metabolismoRESUMO
The present study investigated the anti-inflammatory effects and potential mechanisms of sodium butyrate (SB) in bovine embryo tracheal cells (EBTr) stimulated with lipopolysaccharide (LPS). EBTr were exposed to either 1 mmol/L SB for 18 h for the SB group (SB) or to 0.4 µg/mL LPS for 6 h for the LPS group (LPS). PBS was added to EBTr for a control group (CON). EBTr were pretreated with SB for 18 h followed by 6 h of LPS stimulation for the LSB group (LSB). Results showed that with LPS stimulation, the gene expression of TLR4, NF-κB, IL6, and IL8, as well as cytokine production of IL6 and TNF-α, were significantly increased compared with the CON group. In contrast, protein expression of IL10 was decreased. However, these inflammatory effects induced by LPS were reversed in the LSB group. Compared with the CON group, protein expression of TLR4, phospho-NF-κB p65, phospho-IκBα, and IL1α were increased in the LPS group and these were decreased in the LSB group. Similarly, increased nuclear translocation of phospho-NF-κB p65 in the LPS group was suppressed with SB pretreatment. In conclusion, SB can reduce inflammation induced by LPS in EBTr, and this positive effect is mediated through the TLR4 and NF-κB signaling pathway.
Assuntos
Doenças dos Bovinos , NF-kappa B , Animais , Bovinos , NF-kappa B/genética , NF-kappa B/metabolismo , Lipopolissacarídeos/farmacologia , Ácido Butírico/farmacologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Interleucina-6/efeitos adversos , Traqueia/metabolismo , Transdução de Sinais , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológicoRESUMO
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 Ca2+ and proinflammatory cytokines, including TNF-α, IL-1ß, and IL-6, and the enzyme activities of caspase-3, caspase-9, PKC, and IKK. The upregulation of STIM1, ORAI1, PKCα, IKKß, 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.
Assuntos
Doenças dos Bovinos , Lactação , Animais , Apoptose , Bovinos , Doenças dos Bovinos/metabolismo , Dieta/veterinária , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Feminino , Inflamação/metabolismo , Inflamação/veterinária , Lactação/fisiologia , Glândulas Mamárias Animais/metabolismo , Proteínas Serina-Treonina Quinases , Rúmen/metabolismo , Molécula 1 de Interação Estromal/análise , Molécula 1 de Interação Estromal/metabolismo , Regulação para CimaRESUMO
Autophagy is a crucial cellular homeostatic process and an important part of the host defense system. Dysfunction in autophagy enhances tissue susceptibility to infection and multiple diseases. However, the role of nucleotide oligomerization domain 1 (NOD1) in autophagy in bovine hepatocytes is not well known. Therefore, our aim was to study the contribution of NOD1 to autophagy during inflammation in response to a specific ligand γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP). To achieve this aim, hepatocytes separated from cows at â¼160 days in milk (DIM) were divided into six groups: the nontreated control (CON) group, the rapamycin-treated (RAP) group as a positive control, the iE-DAP-treated (DAP) group, the 3-MA-treated (MA) group, the rapamycin with 3-MA (RM) group, and the iE-DAP with 3-MA (DM) group. iE-DAP administration significantly increased the mRNA expression of NOD1, ATG16L1, RIPK2, ULK1, AMBRA1, DFCP1, WIPI1, ATG5, ATG7, ATG10, ATG4A, IκBα, NF-κB, CXCL1, IL-8, and STAT6 and significantly decreased PIK3C3. The protein expression of NOD1, p-IκBα, p-NF-κB/p-p65, LC3-II, ATG5, and beclin 1 were significantly upregulated and that of SQSTM1/p62, p-mTOR, and FOXA2 were significantly downregulated in response to iE-DAP. iE-DAP also induced the formation of LC3-GFP autophagic puncta in bovine hepatocytes. We also knocked down the NOD1 with siRNA. NOD1 silencing suppressed the autophagy and inflammation-related genes and proteins. The application of the autophagy inhibitor increased the expression of inflammatory molecules and alleviated autophagy-associated molecules. Taken together, these findings suggest that NOD1 is a key player for regulating both ATG16L1 and RIPK2-ULK1 directed autophagy during inflammation in response to iE-DAP in bovine hepatocytes.
Assuntos
Autofagia/efeitos dos fármacos , Ácido Diaminopimélico/análogos & derivados , Hepatócitos/metabolismo , Inflamação/patologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Animais , Autofagossomos/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Bovinos , Sobrevivência Celular/fisiologia , Células Cultivadas , Ácido Diaminopimélico/farmacologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismoRESUMO
Glutamine (GLN) has many types of biological activity in rats, including anti-inflammatory, antioxidative stress, and anti-apoptosis effects. However, little is known about the effects of GLN on bovine mammary epithelial cells (BMEC). γ-d-Glutamyl-meso-diaminopimelic acid (iE-DAP) is a cell wall peptidoglycan component of gram-negative bacteria that can be recognized by the intracellular receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and can cause bovine mastitis. The goal of the present study was to investigate whether GLN protects BMEC from iE-DAP-induced inflammation, oxidative stress, and apoptosis. We cultured BMEC in a GLN-free medium for 24 h and then separated them into 4 groups: cells treated with 1× PBS for 26 or 32 h (control); cells stimulated by 10 µg/mL iE-DAP for 2 or 8 h (2- or 8-h iE-DAP); cells pretreated with 8 or 4 mM GLN for 24 h followed by 2 or 8 h of 1× PBS treatment (8 or 4 mM GLN); and cells pretreated with 8 or 4 mM GLN for 24 h followed by 2 or 8 h of iE-DAP treatment (DG). In the 2-h iE-DAP group, when levels of inflammation peaked, iE-DAP treatment increased both the mRNA and protein expression of NOD1, inhibitor of nuclear factor-κB (NFKBIA, IκB), and nuclear factor-κB subunit p65 (RELA, NF-κB p65), as well as the mRNA expression of IL6 and IL8 and levels of IL-6 and tumor necrosis factor-α in cell culture supernatants. In contrast, 8 mM GLN pretreatment inhibited the mRNA and protein expression of inflammatory-related factors by suppressing the NOD1/NF-κB pathway. In the 8-h iE-DAP group, iE-DAP treatment decreased the mRNA and protein expression of extracellular regulated kinase (Erk, ERK) and nuclear factor erythroid 2-associated factor2 (NFE2L2, Nrf2), as well as the mRNA expression of superoxide dismutase 1 (SOD1), catalase (CAT), coenzyme II oxidoreductase 1 (NQO1), and heme oxygenase 1 (HMOX1, HO1). In addition, iE-DAP treatment increased the expression of malondialdehyde in BMEC when oxidative stress levels peaked. Interestingly, 4 mM GLN pretreatment induced the mRNA and protein expression of antioxidative stress-related factors and inhibited the expression of reactive oxygen species in BMEC by promoting the ERK/Nrf2 pathway. Moreover, GLN reduced apoptosis caused by inflammation and oxidative stress in BMEC. This is the first report showing that GLN protects against iE-DAP-induced inflammation and oxidative stress via the NOD1/NF-κB and ERK/Nrf2 pathways in BMEC.
Assuntos
Doenças dos Bovinos/prevenção & controle , Ácido Diaminopimélico/análogos & derivados , Glutamina/uso terapêutico , Inflamação/veterinária , Glândulas Mamárias Animais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Bovinos , Células Cultivadas , Ácido Diaminopimélico/antagonistas & inibidores , Células Epiteliais/metabolismo , Feminino , Heme Oxigenase-1/metabolismo , Inflamação/induzido quimicamente , Inflamação/prevenção & controle , Glândulas Mamárias Animais/citologia , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/farmacologiaRESUMO
BACKGROUND: Long-term high-concentrate (HC) diet feeding increased bacterial endotoxins, which translocated into the mammary glands of dairy goats and induced inflammatory response. γ-d-Glutamyl-meso-diaminopimelic acid (iE-DAP), bacterial peptidoglycan component, triggered inflammatory response through activating nucleotide oligomerization domain protein 1 (NOD1) signaling pathway. While dietary supplemented with sodium butyrate (SB) relieved inflammatory response and improved animal health and production. To investigate the effects and the mechanisms of action of SB on the inflammatory response in the mammary glands of dairy goats fed HC diet, 12 Saanen dairy goats were randomly assigned into HC group and SB regulated (BHC) group. RESULTS: The results showed that SB supplementation attenuated ruminal pH decrease caused by HC diet in dairy goats resulting in a decrease of proinflammatory cytokines and iE-DAP plasma concentration and the mRNA expression of NOD1 and other inflammation-related genes. The protein levels of NOD1, NF-κB p65 and NF-κB pp65 were decreased by the SB supplementation. The expression of histone deacetylase 3 (HDAC3) was also inhibited by the SB supplementation. Meanwhile, the chromatin compaction ratios and DNA methylation levels of NOD1 and receptor-interacting protein 2 (RIP2) of BHC group were upregulated. CONCLUSION: Collectively, the SB supplementation mitigated the inflammatory response in the mammary glands of dairy goats during HC-induced subacute ruminal acidosis (SARA) by inhibiting the activation of the NOD1/NF-κB signaling pathway through the decrease of the iE-DAP concentration in the rumen fluid and plasma and HDAC3 expression. DNA methylation and chromatin remodeling also contributed to the anti-inflammatory effect of SB. © 2020 Society of Chemical Industry.
Assuntos
Ácido Butírico/administração & dosagem , Ácido Diaminopimélico/análogos & derivados , Doenças das Cabras/tratamento farmacológico , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/imunologia , Acidose/tratamento farmacológico , Acidose/imunologia , Acidose/veterinária , Ração Animal/efeitos adversos , Ração Animal/análise , Animais , Ácido Diaminopimélico/efeitos adversos , Ácido Diaminopimélico/análise , Dieta/efeitos adversos , Dieta/veterinária , Suplementos Nutricionais/análise , Feminino , Doenças das Cabras/imunologia , Cabras/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Proteína Adaptadora de Sinalização NOD1/imunologiaRESUMO
The innate immune response plays a crucial role in recovery from infectious diseases by promoting the clearance of pathogens. Sodium butyrate (NaB) is an energy source for cellular processes with the potential to regulate the innate immune response. The present study aimed to evaluate the effect of NaB on the innate immune response in a bovine mammary alveolar cell line (MAC-T) initiated by lipopolysaccharides (LPS). Thus, treatments were conducted as follows: treated with 1× PBS for 24 h (control), pretreated with 1 mM NaB (optimized by cell viability assays and dose-dependent experiment) for 18 h followed by treatment of 1× PBS for 6 h (NaB), pretreated with 1× PBS for 18 h followed by stimulation with LPS (1 µg/mL) for 6 h (LPS), and pretreated with 1 mM NaB for 18 h followed by stimulation with LPS (1 µg/mL) for 6 h (NaB + LPS). Different inhibitors were also used to elucidate the underlying mechanism. Furthermore, cells were treated with NaB and heat-inactivated Escherichia coli to test the effect of NaB on transcription of genes related to the innate immune response triggered by the major causative pathogen of mastitis. Each treatment had 3 replicates and was repeated 3 times. Proinflammatory cytokines, chemokines, and ß-defensins are crucial secretion factors in innate immunity, and transcription of these factors was increased by NaB during challenge with LPS or heat-inactivated E. coli in MAC-T cells. Acetylation of histone H3 protein, which promotes gene expression by affecting the structure of chromatin, was also upregulated by NaB in response to LPS stimulation. P38 mitogen-activated protein kinases (MAPK), JNK, and Erk 1 and 2 are key upstream regulators of the expression of proinflammatory cytokines, chemokines, and ß-defensins, and their activity was enhanced by NaB during LPS stimulation. Furthermore, inhibitors were used to assess the role of MAPK signaling in the effects of NaB. The results showed that inhibitors of p38 MAPK, Erk, and JNK attenuated the NaB-induced upregulation of TNF and ß-defensin 5 (DEFB5) transcription, and that the inhibitor of Erk attenuated the NaB-induced upregulation of IL1B transcription during LPS challenge. Enhanced transcription of CXCL8 by NaB was blocked by the inhibitor of Erk and p38 MAPK during LPS stimulation. Overall, NaB boosted the LPS-induced innate immune response by promoting the expression of proinflammatory cytokines, chemokines, and ß-defensins, which was associated with enhanced MAPK signaling activation and histone H3 acetylation.