Gut microbiota contributes to bisphenol A-induced maternal intestinal and placental apoptosis, oxidative stress, and fetal growth restriction in pregnant ewe model by regulating gut-placental axis.

BACKGROUND: Bisphenol A (BPA) is an environmental contaminant with endocrine-disrupting properties that induce fetal growth restriction (FGR). Previous studies on pregnant ewes revealed that BPA exposure causes placental apoptosis and oxidative stress (OS) and decreases placental efficiency, consequently leading to FGR. Nonetheless, the response of gut microbiota to BPA exposure and its role in aggravating BPA-mediated apoptosis, autophagy, mitochondrial dysfunction, endoplasmic reticulum stress (ERS), and OS of the maternal placenta and intestine are unclear in an ovine model of gestation. RESULTS: Two pregnant ewe groups (n = 8/group) were given either a subcutaneous (sc) injection of corn oil (CON group) or BPA (5 mg/kg/day) dissolved in corn oil (BPA group) once daily, from day 40 to day 110 of gestation. The maternal colonic digesta and the ileum and placental tissue samples were collected to measure the biomarkers of autophagy, apoptosis, mitochondrial dysfunction, ERS, and OS. To investigate the link between gut microbiota and the BPA-induced FGR in pregnant ewes, gut microbiota transplantation (GMT) was conducted in two pregnant mice groups (n = 10/group) from day 0 to day 18 of gestation after removing their intestinal microbiota by antibiotics. The results indicated that BPA aggravates apoptosis, ERS and autophagy, mitochondrial function injury of the placenta and ileum, and gut microbiota dysbiosis in pregnant ewes. GMT indicated that BPA-induced ERS, autophagy, and apoptosis in the ileum and placenta are attributed to gut microbiota dysbiosis resulting from BPA exposure. CONCLUSIONS: Our findings indicate the underlying role of gut microbiota dysbiosis and gut-placental axis behind the BPA-mediated maternal intestinal and placental apoptosis, OS, and FGR. The findings further provide novel insights into modulating the balance of gut microbiota through medication or probiotics, functioning via the gut-placental axis, to alleviate gut-derived placental impairment or FGR. Video Abstract.

Dietary rumen-protected L-arginine or N-carbamylglutamate enhances placental amino acid transport and suppresses angiogenesis and steroid anabolism in underfed pregnant ewes.

This study aimed to investigate the effects of dietary supplementation of underfed Hu ewes from d 35 to 110 of gestation with either rumen-protected L-arginine (RP-Arg) or N-carbamylglutamate (NCG) on placental amino acid (AA) transport, angiogenic gene expression, and steroid anabolism. On d 35 of gestation, 32 Hu ewes carrying twin fetuses were randomly divided into four treatment groups, each consisting of eight ewes, and were fed the following diets: A diet providing 100% of NRC's nutrient requirements for pregnant ewes (CON); A diet providing 50% of NRC's nutrient requirements for pregnant ewes (RES); RES diet plus 5 g/d NCG (RES + NCG); or RES diet plus 20 g/d RP-Arg (RES + ARG). On the d 110 of pregnancy, blood samples were taken from the mother, and samples were collected from type A cotyledons (COT; the fetal portions of the placenta). The levels of 17ß-estradiol and progesterone in the maternal serum and both the capillary area density (CAD) and capillary surface density (CSD) in type A COT were decreased in response to Arg or NCG supplementation when compared to the RES group. The concentrations of arginine, leucine, putrescine and spermidine in type A COT were higher (P < 0.05) in the RES + ARG or RES + NCG group than in the RES group. The mRNA expression levels of inducible nitric oxide synthase (iNOS) and solute carrier family 15, member 1 (SLC15A1) were increased (P < 0.05) while those of progesterone receptor (PGR) and fibroblast growth factor 2 (FGF2) were decreased in type A COT by supplementation with either NCG or RP-Arg compared to the RES group. The results suggest that providing underfed pregnant ewes from d 35 to 110 of gestation with a diet supplemented with NCG or RP-Arg improves placental AA transport, and reduces the expression of angiogenic growth factor genes and steroid anabolism, leading to better fetal development.

Research progress in physiological effects of resistant substances of Urtica dioica L. on animal performance and feed conversion.

Several members of family Urticaceae are mainly found in the temperate and subtropical zones of the Northern Hemisphere and are important medicinal plants. Among them, Urtica dioica L. (Urticaceae) is an annual or perennial herb that has been used for feeding and medicinal purposes since long time and is the most exploited species of Urticaceae. Recently, it has received attention to be used as animal feed, as its fresh leaves fed to animals in moderate, dried, and other forms. This review details the advantages of U. dioica as an alternative feed in terms of germplasm specificity, nutritional composition, and feed application status. Its roots, stems, leaves, and seeds are rich in active ingredients. It has also been found to have anticancer effects through antioxidant action and promotion of apoptosis of cancer cells. In shady conditions, U. dioica is highly adaptable while under stressful conditions of drought; it also reduces light absorption and ensures carbon assimilation through light energy conversion efficiency. Therefore, it can be added to animal diets as a suitable feed to reduce costs and improve economic efficiency. This paper investigates the feasibility of using U. dioica as a feed and systematically presents the progress of research and exploitation of U. dioica.

Multi-Omics Reveals the Impact of Exogenous Short-Chain Fatty Acid Infusion on Rumen Homeostasis: Insights into Crosstalk between the Microbiome and the Epithelium in a Goat Model.

Emerging data have underscored the significance of exogenous supplementation of butyrate in the regulation of rumen development and homeostasis. However, the effects of other short-chain fatty acids (SCFAs), such as acetate or propionate, has received comparatively less attention, and the consequences of extensive exogenous SCFA infusion remain largely unknown. In our study, we conducted a comprehensive investigation by infusion of three SCFAs to examine their respective roles in regulating the rumen microbiome, metabolism, and epithelium homeostasis. Data demonstrated that the infusion of sodium acetate (SA) increased rumen index while also promoting SCFA production and absorption through the upregulation of SCFA synthetic enzymes and the mRNA expression of SLC9A1 gene. Moreover, both SA and sodium propionate infusion resulted in an enhanced total antioxidant capacity, an increased concentration of occludin, and higher abundances of specific rumen bacteria, such as "Candidatus Saccharimonas," Christensenellaceae R-7, Butyrivibrio, Rikenellaceae RC9 gut, and Alloprevotella. In addition, sodium butyrate (SB) infusion exhibited positive effects by increasing the width of rumen papilla and the thickness of the stratum basale. SB infusion further enhanced antioxidant capacity and barrier function facilitated by cross talk with Monoglobus and Incertae Sedis. Furthermore, metabolome and transcriptome data revealed distinct metabolic patterns in rumen contents and epithelium, with a particular impact on amino acid and fatty acid metabolism processes. In conclusion, our data provided novel insights into the regulator effects of extensive infusion of the three major SCFAs on rumen fermentation patterns, antioxidant capacity, rumen barrier function, and rumen papilla development, all achieved without inducing rumen epithelial inflammation. IMPORTANCE The consequences of massive exogenous supplementation of SCFAs on rumen microbial fermentation and rumen epithelium health remain an area that requires further exploration. In our study, we sought to investigate the specific impact of administering high doses of exogenous acetate, propionate, and butyrate on rumen homeostasis, with a particular focus on understanding the interaction between the rumen microbiome and epithelium. Importantly, our findings indicated that the massive infusion of these SCFAs did not induce rumen inflammation. Instead, we observed enhancements in antioxidant capacity, strengthening of rumen barrier function, and promotion of rumen papilla development, which were facilitated through interactions with specific rumen bacteria. By addressing existing knowledge gaps and offering critical insights into the regulation of rumen health through SCFA supplementation, our study holds significant implications for enhancing the well-being and productivity of ruminant animals.

Homeostatic crosstalk among gut microbiome, hypothalamic and hepatic circadian clock oscillations, immunity and metabolism in response to different light-dark cycles: A multiomics study.

The accelerated pace of life at present time has resulted in tremendous alterations in living patterns. Changes in diet and eating patterns, in particular, coupled with irregular light-dark (LD) cycles will further induce circadian misalignment and lead to disease. Emerging data has highlighted the regulatory effects of diet and eating patterns on the host-microbe interactions with the circadian clock (CC), immunity, and metabolism. Herein, we studied how LD cycles regulate the homeostatic crosstalk among the gut microbiome (GM), hypothalamic and hepatic CC oscillations, and immunity and metabolism using multiomics approaches. Our data demonstrated that central CC oscillations lost rhythmicity under irregular LD cycles, but LD cycles had minimal effects on diurnal expression of peripheral CC genes in the liver including Bmal1. We further demonstrated that the GM could regulate hepatic circadian rhythms under irregular LD cycles, the candidate bacteria including Limosilactobacillus, Actinomyces, Veillonella, Prevotella, Campylobacter, Faecalibacterium, Kingella, and Clostridia vadinBB60 et al. A comparative transcriptomic study of innate immune genes indicated that different LD cycles had varying effects on immune functions, while irregular LD cycles had greater impacts on hepatic innate immune functions than those in the hypothalamus. Extreme LD cycle alterations (LD0/24 and LD24/0) had worse impacts than slight alterations (LD8/16 and LD16/8), and led to gut dysbiosis in mice receiving antibiotics. Metabolome data also demonstrated that hepatic tryptophan metabolism mediated the homeostatic crosstalk among GM-liver-brain axis in response to different LD cycles. These research findings highlighted that GM could regulate immune and metabolic disorders induced by circadian dysregulation. Further, the data provided potential targets for developing probiotics for individuals with circadian disruption such as shift workers.

Dietary L-Arginine or N-Carbamylglutamate Alleviates Colonic Barrier Injury, Oxidative Stress, and Inflammation by Modulation of Intestinal Microbiota in Intrauterine Growth-Retarded Suckling Lambs.

Our previous studies have revealed that dietary N-carbamylglutamate (NCG) and L-arginine (Arg) supplementation improves redox status and suppresses apoptosis in the colon of suckling Hu lambs with intrauterine growth retardation (IUGR). However, no studies have reported the function of Arg or NCG in the colonic microbial communities, barrier function, and inflammation in IUGR-suckling lambs. This work aimed to further investigate how dietary Arg or NCG influences the microbiota, barrier function, and inflammation in the colon of IUGR lambs. Forty-eight newborn Hu lambs of 7 d old were assigned to four treatment groups (n = 12 per group; six male, six female) as follows: CON (normal birth weight, 4.25 ± 0.14 kg), IUGR (3.01 ± 0.12 kg), IUGR + Arg (2.99 ± 0.13 kg), and IUGR + NCG (3.03 ± 0.11 kg). A total of 1% Arg or 0.1% NCG was supplemented in a basal diet of milk replacer, respectively. Lambs were fed the milk replacer for 21 d until 28 d after birth. Compared to the non-supplemented IUGR lambs, the transepithelial electrical resistance (TER) was higher, while fluorescein isothiocyanate dextran 4 kDa (FD4) was lower in the colon of the NCG- or Arg-supplemented IUGR lambs (p < 0.05). The IUGR lambs exhibited higher (p < 0.05) colonic interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)-α, reactive oxygen species (ROS), and malondialdehyde (MDA) levels than the CON lambs; the detrimental effects of IUGR on colonic proinflammatory cytokine concentrations and redox status were counteracted by dietary Arg or NCG supplementation. Both IUGR + Arg and IUGR + NCG lambs exhibited an elevated protein and mRNA expression of Occludin, Claudin-1, and zonula occludens-1 (ZO-1) compared to the IUGR lambs (p < 0.05). Additionally, the lipopolysaccharide (LPS) concentration was decreased while the levels of acetate, butyrate, and propionate were increased in IUGR + Arg and IUGR + NCG lambs compared to the IUGR lambs (p < 0.05). The relative abundance of Clostridium, Lactobacillus, and Streptococcus was lower in the colonic mucosa of the IUGR lambs than in the CON lambs (p < 0.05) but was restored upon the dietary supplementation of Arg or NCG to the IUGR lambs (p < 0.05). Both Arg and NCG can alleviate colonic barrier injury, oxidative stress (OS), and inflammation by the modulation of colonic microbiota in IUGR-suckling lambs. This work contributes to improving knowledge about the crosstalk among gut microbiota, immunity, OS, and barrier function and emphasizes the potential of Arg or NCG in health enhancement as feed additives in the early life nutrition of ruminants.

Dietary N-carbamylglutamate and L-arginine supplementation improves redox status and suppresses apoptosis in the colon of intrauterine growth-retarded suckling lambs.

Previous studies have revealed that dietary N-carbamylglutamate (NCG) or L-arginine (Arg) improves small intestinal integrity and immune function in suckling Hu lambs that have experienced intrauterine growth retardation (IUGR). Whether these nutrients alter redox status and apoptosis in the colon of IUGR lambs is still unknown. This study, therefore, aimed at investigating whether dietary supplementation of Arg or NCG alters colonic redox status, apoptosis and endoplasmic reticulum (ER) stress and the underlying mechanism of these alterations in IUGR suckling Hu lambs. Forty-eight 7-d old Hu lambs, including 12 with normal birth weight (4.25 ± 0.14 kg) and 36 with IUGR (3.01 ± 0.12 kg), were assigned to 4 treatment groups (n = 12 each; 6 males and 6 females) for 3 weeks. The treatment groups were control (CON), IUGR, IUGR + Arg and IUGR + NCG. Relative to IUGR lambs, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) content, as well as proliferation index, were higher (P < 0.05) whereas reactive oxygen species (ROS), malondialdehyde (MDA) levels and apoptotic cell numbers were lower (P < 0.05) in colonic tissue for both IUGR + Arg and NCG lambs. Both mRNA and protein levels of C/EBP homologous protein 10 (CHOP10), B-cell lymphoma/leukaemia 2 (Bcl-2) -associated X protein (Bax), apoptosis antigen 1 (Fas), activating transcription factor 6 (ATF6), caspase 3, and glucose-regulated protein 78 (GRP78) were lower (P < 0.05) while glutathione peroxidase 1 (GPx1), Bcl-2 and catalase (CAT) levels were higher (P < 0.05) in colonic tissue for IUGR + Arg and IUGR + NCG lambs compared with IUGR lambs. Based on our results, dietary NCG or Arg supplementation can improve colonic redox status and suppress apoptosis via death receptor-dependent, mitochondrial and ER stress pathways in IUGR suckling lambs.

Effects of the Gut Microbiota and Barrier Function on Melatonin Efficacy in Alleviating Liver Injury.

Environmental cadmium (Cd) exposure has been associated with severe liver injury. In contrast, melatonin (Mel) is a candidate drug therapy for Cd-induced liver injury due to its diverse hepatoprotective activities. However, the precise molecular mechanism by which Mel alleviates the Cd-induced liver injury, as well as the Mel-gut microbiota interaction in liver health, remains unknown. In this study, mice were given oral gavage CdCl2 and Mel for 10 weeks before the collection of liver tissues and colonic contents. The role of the gut microbiota in Mel's efficacy in alleviating the Cd-induced liver injury was evaluated by the gut microbiota depletion technique in the presence of antibiotic treatment and gut microbiota transplantation (GMT). Our results revealed that the oral administration of Mel supplementation mitigated liver inflammation, endoplasmic reticulum (ER) stress and mitophagy, improved the oxidation of fatty acids, and counteracted intestinal microbial dysbiosis in mice suffering from liver injury. It was interesting to find that neither Mel nor Cd administration induced any changes in the liver of antibiotic-treated mice. By adopting the GMT approach where gut microbiota collected from mice in the control (CON), Cd, or Mel + Cd treatment groups was colonized in mice, it was found that gut microbiota was involved in Cd-induced liver injury. Therefore, the gut microbiota is involved in the Mel-mediated mitigation of ER stress, liver inflammation and mitophagy, and the improved oxidation of fatty acids in mice suffering from Cd-induced liver injury.

Dietary N-carbamylglutamate or L-arginine improves fetal intestinal amino acid profiles during intrauterine growth restriction in undernourished ewes.

Our previous studies demonstrated that prenatal in utero growth restriction impairs postnatal intestinal function. Thus, improving postpartal intestinal absorption capacity and growth by manipulating the maternal diet prepartum is of importance. This work was conducted to determine whether supplementation of N-carbamylglutamate (NCG) or rumen-protected L-arginine (RP-Arg) increased fetal intestinal amino acid (AA) profiles in intrauterine growth retardation (IUGR) fetuses. On d 35 of gestation, Hu ewes (n = 32) carrying twin fetuses were randomized into 4 groups (8 ewes and 16 fetuses in each group), where diets were as follows: 100% of nutrient requirements recommended by National Research Council (NRC, 2007) (CON); 50% of nutrient requirements recommended by NRC (2007) (RES); RES + RP-Arg (20 g/d), (RES + ARG); and RES + NCG (5 g/d), (RES + NCG). On d 110 of gestation, both fetal and maternal tissues were collected and weighed. Compared with RES, solute carrier family 1, member 5 (SLC1A5) was upregulated (P < 0.05) within fetal jejunum, duodenum and ileum when supplementing NCG and RP-Arg. Relative to RES, RP-Arg or NCG supplementation to RES resulted in upregulation (P < 0.05) of peptide transporter 1 protein abundance within the fetal ileum. NCG or RP-Arg supplementation to RES also upregulated phosphorylated mechanistic target of rapamycin (pmTOR)-to-mTOR ratio in the fetal ileum induced by IUGR (P < 0.05). As a result, during IUGR, supplementation of Arg or NCG affected intestinal AA profiles in the fetus in part through controlling mTOR signal transduction as well as AA and peptide transport. Future studies should be conducted to understand the role (if any) of the placenta on the improvement of growth and AA profiles independent of the fetal intestine. This would help demonstrate the relative contribution of intestinal uptake in fetal life.

Dietary rumen-protected L-arginine or N-carbamylglutamate attenuated fetal hepatic inflammation in undernourished ewes suffering from intrauterine growth restriction.

This study aimed to explore whether dietary rumen-protected L-arginine (RP-Arg) or N-carbamylglutamate (NCG) supplementation to feed-restricted pregnant ewes counteracts fetal hepatic inflammation and innate immune dysfunction associated with intrauterine growth retardation (IUGR) in ovine fetuses. On d 35 of pregnancy, twin-bearing Hu ewes (n = 32) were randomly assigned to 4 treatment groups (8 ewes and 16 fetuses per group) and fed diets containing 100% of the NRC requirements (CON), 50% of the NRC requirements (RES), RES + RP-Arg (20 g/d) (RESA), or RES + NCG (5 g/d) (RESN). At 08:00 on d 110 of gestation, fetal blood and liver tissue samples were collected. The levels of triglyceride, free fatty acid, cholesterol and ß-hydroxybutyrate in the fetal blood of RESA and RESN groups were lower (P < 0.05) than those of the RES group, but were higher (P < 0.05) than those of the CON group. The interleukin (IL)-6 and IL-1 levels in fetal blood and liver tissue as well as the myeloid differentiation primary response 88 (MyD88), transforming growth factor ß (TGFß), and nuclear factor kappa B (NF-κB) mRNA levels in the fetal liver were decreased (P < 0.05) by the NCG or RP-Arg supplementation compared to the RES treatment. Similarly, the toll-like receptor (TLR)-4, MyD88, TGFß, and p-c-Jun N-terminal kinase (JNK) protein levels in the fetal liver were reduced (P < 0.05) in the NCG and RP-Arg -supplemented groups compared to the RES group. These results showed that dietary supplementation of RP-Arg or NCG to underfed pregnant ewes could protect against IUGR fetal hepatic inflammation via improving lipid metabolism, down-regulating the TLR-4 and the inflammatory JNK and NF-κB signaling pathways, and decreasing cytokine production in ovine fetal blood and liver tissue.

Biosorption of Copper in Swine Manure Using Aspergillus and Yeast: Characterization and Its Microbial Diversity Study.

Dietary copper supplementation in the feed of piglets generally exceeds 250-800 mg/kg, where a higher quantity (>250 mg/kg) can promote growth and improve feed conversion. Despite the reported positive effects, 90% of copper is excreted and can accumulate and pollute the soil. Data indicate that fungi have a biosorptive capacity for copper. Thus, the objectives of the present experiment were to study the effects of adding different strains of fungi on the biosorptive capacity for copper in swine manure and to evaluate potential effects on microbiota profiles. Aspergillus niger (AN), Aspergillus oryzae (AO), and Saccharomyces cerevisiae (SC) were selected, and each added 0.4% into swine manure, which contain 250 mg/kg of copper. The incubations lasted for 29 days, and biosorption parameters were analyzed on the 8th (D8), 15th (D15), 22nd (D22), and 29th (D29) day. Results showed that after biosorption, temperature was 18.47-18.77°C; pH was 6.33-6.91; and content of aflatoxin B1, ochratoxin A, and deoxynivalenol were low. In addition, residual copper concentration with AN was the lowest on D15, D22, and D29. The copper biosorption rate was also highest with AN, averaging 84.85% on D29. Biosorption values for AO reached 81.12% and for SC were lower than 80%. Illumina sequencing of 16S and ITS rRNA gene revealed that fungal treatments reduced the diversity and richness of fungal abundance, but had no effect on bacterial abundance. Unknown_Marinilabiliaceae, Proteiniphilum, Tissierella, and Curvibacter were the dominant bacteria, while Aspergillus and Trichoderma were the dominant fungi. However, the added strain of S. cerevisiae was observed to be lower than the dominant fungi, which contained less than 0.05%. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment predicted via PICRUSt2 that there were bacterial genes potentially related to various aspects of metabolism and environmental information processing. Overall, data indicated that Aspergillus can provide microbial materials for adsorption of copper.

Dietary N-carbamylglutamate or l-arginine supplementation improves hepatic energy status and mitochondrial function and inhibits the AMP-activated protein kinase-peroxisome proliferator-activated receptor γ coactivator-1α-transcription factor A pathway in intrauterine-growth-retarded suckling lambs.

The objective of this study was to investigate the effects of dietary administration of l-arginine (Arg) or N-carbamylglutamate (NCG) on hepatic energy status and mitochondrial functions in suckling Hu lambs with intrauterine growth retardation (IUGR). Forty-eight newborn Hu lambs of 7 d old were allocated into 4 treatment groups of 12 lambs each, in triplicate with 4 lambs per replicate (2 males and 2 females) as follows: CON (lambs of normal birth weight, 4.25 ± 0.14 kg), IUGR (3.01 ± 0.12 kg), IUGR + 1% Arg (2.99 ± 0.13 kg), or IUGR + 0.1% NCG (3.03 ± 0.11 kg). The experiment lasted for 21 d, until d 28 after birth, and all lambs were fed milk replacer as a basal diet. Compared with IUGR lambs, NCG or Arg administration increased (P < 0.05) the adenosine triphosphate (ATP) level and the activities of complexes I/III/IV, isocitrate dehydrogenase and citrate synthase in the liver. Compared with CON lambs, the relative mRNA levels of adenosine monophosphate-activated protein kinase α1 (AMPKα1), peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α) and transcription factor A (TFAM) were increased (P < 0.05) in the liver of IUGR lambs, but were decreased (P < 0.05) in the liver of NCG- or Arg-treated lambs compared with those in the IUGR lambs. Compared with IUGR lambs, NCG or Arg administration decreased (P < 0.05) the total AMPKα (tAMPKα)-to-phosphorylated AMPKα (pAMPKα) ratio and the protein expression of PGC1α and TFAM. The results suggested that dietary Arg or NCG supplements improved hepatic energy status and mitochondrial function and inhibited the AMPK-PGC1α-TFAM pathway in IUGR suckling lambs.

Melatonin ameliorates ochratoxin A induced liver inflammation, oxidative stress and mitophagy in mice involving in intestinal microbiota and restoring the intestinal barrier function.

The mycotoxin ochratoxin A (OTA) is a widespread contaminant in human and animal food products. Previous studies in rats revealed that melatonin (Mel) exhibits a preventive effect against OTA-induced oxidative stress in liver. However, it remains unknown whether gut microbiota respond to Mel and, if so, whether it can prevent OTA-induced inflammation and mitophagy in the liver. In the present study, mice received an oral gavage of Mel and OTA for 3 weeks before harvesting colonic digesta and liver tissue for analyses. In another study, the role of intestinal microbiota on the effects of Mel on OTA-induced liver inflammation and mitophagy was assessed through clearance of intestinal microbiota with antibiotics followed by gut microbiota transplantation (GMT). Oral Mel supplementation ameliorated mitophagy in the liver and reversed gut microbiota dysbiosis. Intriguingly, in antibiotic-treated mice, Mel and OTA failed to induce mitophagy in the liver. Using the GMT approach in which mice were colonised with intestinal microbiota from control-, OTA-, or Mel + OTA-treated mice led us to elucidated the involvement of intestinal microbiota in liver inflammation and mitophagy induced by OTA. The findings suggested that intestinal microbiota play some role in the Mel-induced amelioration of liver inflammation and mitophagy induced by OTA.

l-Arginine Inhibits Apoptosis of Ovine Intestinal Epithelial Cells through the l-Arginine-Nitric Oxide Pathway.

BACKGROUND: In nonruminants, many of the biological roles of l-arginine (Arg) at the intestinal level are mediated through the Arg-nitric oxide (Arg-NO) pathway. Whether the Arg-NO pathway is involved in controlling the immune response and viability in ovine intestinal epithelial cells (IOECs) is unclear. OBJECTIVES: The current study aimed to examine the role of the Arg-NO pathway in apoptosis, antioxidant capacity, and mitochondrial function of IOECs. METHODS: The IOECs were incubated in Arg-free DMEM supplemented with 150 µM Arg (CON) or 300 µM Arg (ARG) alone or with 350 µM Nw-nitro-l-arginine methyl ester hydrochloride (l-NAME) (CON + NAME, ARG + NAME) for 24 h. The reactive oxygen species (ROS) concentration, antioxidant capacity, and cell apoptotic percentage were determined. RESULTS: Arg supplementation decreased (P < 0.05) the ROS concentration (38.9% and 22.7%) and apoptotic cell percentage (57.2% and 54.8%) relative to the CON and CON + NAME groups, respectively. Relative to the CON and ARG treatments, the l-NAME administration decreased (P < 0.05) the mRNA abundance of superoxide dismutase 2 (32% and 21.3%, respectively) and epithelial NO synthase (36% and 29.1%, respectively). Arg supplementation decreased (P < 0.05) the protein abundance of apoptosis antigen 1 (FAS) (52.0% and 43.9%) but increased (P < 0.05) those of nuclear respiratory factor 1 (31.3% and 22.9%) and inducible NO synthase (35.2% and 41.8%) relative to the CON and CON + NAME groups, respectively. CONCLUSIONS: The inhibition of apoptosis in IOECs due to the increased supply of Arg is associated with the mitochondria- and FAS-dependent pathways through the activity of the Arg-NO pathway. The findings help elucidate the role of the Arg-NO pathway in IOEC growth and apoptosis.

Dietary supplementation of l-arginine and N-carbamylglutamate enhances duodenal barrier and mitochondrial functions and suppresses duodenal inflammation and mitophagy in suckling lambs suffering from intrauterine-growth-restriction.

The current work aimed at investigating the effects of the dietary supplementation of N-carbamylglutamate (NCG) or l-arginine (Arg) on the duodenal mitophagy, mitochondrial function, inflammation, and barrier function in suckling lambs suffering from intrauterine-growth-retardation (IUGR). Forty-eight neonate Hu lambs were used in this study: 12 lambs with normal birth weight (NBW: 4.25 ± 0.14 kg) and 36 lambs with IUGR (3.01 ± 0.13 kg). Seven day old lambs were assigned to 4 treatment groups (12 lambs in each group) as follows: control group (CON), IUGR group, IUGR + Arg, and IUGR + NCG. Lambs were fed the experimental diets for 21 days from 7 days to 28 days of age. Compared with IUGR lambs, the Arg or NCG-treated IUGR lambs had a markedly higher duodenal transepithelial electrical resistance (TER) and lower fluorescein isothiocyanate dextran (FD4) (P < 0.05), respectively. The duodenal mitochondrial membrane potential change (ΔΨm), relative mitochondrial DNA (mtDNA) content, adenosine triphosphate (ATP) level, together with the activities of the respiratory complexes I, III, and IV were markedly higher in Arg or NCG-treated IUGR lambs than those in non-supplemented IUGR lambs (P < 0.05). The expressions of the integrity-related proteins (occludin and zonula occludens-1 (ZO-1)), antioxidant- and apoptosis-related proteins (B-cell lymphoma/leukaemia 2 (Bcl2), superoxide dismutase 2 (SOD2), catalase (CAT), and glutathione peroxidase 1 (GPx1)), and the nitric oxide-dependent pathway-related proteins (epithelial NO synthase (eNOS) and inducible NO synthase (iNOS)) were higher in NCG or Arg-supplemented IUGR lambs than those in nontreated IUGR lambs (P < 0.05). The duodenal expressions of the mitophagy-related proteins (microtubule-associated protein light chain 3 (LC3) I, LC3 II, Belin1, PTEN induced putative kinase 1 (PINK1), and Parkin) and the immune function-related proteins (myeloid differentiation factor 88 (MyD88), IL-6, nuclear factor kappa B (p65), toll-like receptor (TLR4) and TNF-α) were reduced (P < 0.05) in NCG or Arg-supplemented IUGR lambs compared with non-supplemented IUGR lambs. These results demonstrated that the dietary supplementation of Arg or NCG enhanced the duodenal barrier function and mitochondrial function, mitigated duodenal inflammation, and suppressed mitophagy in suckling lambs suffering from IUGR.

Dietary Supplementation of L-Arginine and N-Carbamylglutamate Attenuated the Hepatic Inflammatory Response and Apoptosis in Suckling Lambs with Intrauterine Growth Retardation.

L-arginine (Arg) is a semiessential amino acid with several physiological functions. N-Carbamylglutamate (NCG) can promote the synthesis of endogenous Arg in mammals. However, the roles of Arg or NCG on hepatic inflammation and apoptosis in suckling lambs suffering from intrauterine growth restriction (IUGR) are still unclear. The current work is aimed at examining the effects of dietary Arg and NCG on inflammatory and hepatocyte apoptosis in IUGR suckling lambs. On day 7 after birth, 48 newborn Hu lambs were selected from a cohort of 432 twin lambs. Normal-birthweight and IUGR Hu lambs were allocated randomly (n = 12/group) to control (CON), IUGR, IUGR+1% Arg, or IUGR+0.1% NCG groups. Lambs were fed for 21 days from 7 to 28 days old. Compared with CON lambs, relative protein 53 (P53), apoptosis antigen 1 (Fas), Bcl-2-associated X protein (Bax), caspase-3, cytochrome C, tumor necrosis factor alpha (TNF-α), nuclear factor kappa-B (NF-κB) p65, and NF-κB pp65 protein levels were higher (P < 0.05) in liver from IUGR lambs, whereas those in liver from IUGR lambs under Arg or NCG treatment were lower than those in IUGR lambs. These findings indicated that supplementing Arg or NCG reduced the contents of proinflammatory cytokines at the same time when the apoptosis-related pathway was being suppressed, thus suppressing the IUGR-induced apoptosis of hepatic cells.

Supply of methionine and arginine alters phosphorylation of mechanistic target of rapamycin (mTOR), circadian clock proteins, and α-s1-casein abundance in bovine mammary epithelial cells.

Methionine (Met) and arginine (Arg) regulate casein protein abundance through alterations in activity of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. A potential role for the circadian clock network on the regulation of protein synthesis, partly via activity of mTORC1, has been highlighted in non-ruminants. The main objective of the study was to determine in ruminant mammary cells alterations in mRNA, protein abundance and phosphorylation status of mTORC1-related upstream targets, circadian clock proteins, and protein kinase AMP-activated catalytic subunit alpha (AMPK) in relation to α-s1-casein protein (CSN1S1) abundance in response to greater supply of Met and Arg alone or in combination. Primary bovine mammary epithelial cells (BMEC) were incubated for 12 h in a 2 × 2 arrangement of treatments with control media (ideal profile of amino acids, IPAA), or media supplemented with increased Met (incMet), Arg (incArg), or both (incMet + incArg). Data were analyzed testing the main effects of Met and Arg and their interaction. Among 7 amino acid (AA) transporters known to be mTORC1 targets, increasing supply of Arg downregulated SLC1A5, SLC3A2, SLC7A1, and SLC7A5, while increasing supply of Met upregulated SLC7A1. mRNA abundance of the cytosolic Arg sensor (CASTOR1) was lower when supply of Arg and Met alone increased. p-TSC2 (TSC complex subunit 2) was greater when the Arg supply was increased, while the phosphoralation ratio of p-AKT (AKT serine/threonine kinase 1):total (t) AKT and p-AMPK:tAMPK were lower. In spite of this, the ratio of p-mTOR:tmTOR nearly doubled with incArg but such response did not prevent a decrease in CSN1S1 abundance. The abundance of period circadian regulator 1 (PER1) protein nearly doubled with all treatments, but only incMet + incArg led to greater clock circadian regulator (CLOCK) protein abundance. Overall, data suggest that a greater supply of Met and Arg could influence CSN1S1 synthesis of BMEC through changes in the mTORC1, circadian clock, and AMPK pathways. Identifying mechanistic relationships between intracellular energy, total AA supply, and these pathways in the context of milk protein synthesis in ruminants merits further research.

N-Carbamylglutamate and l-arginine supplementation improve hepatic antioxidant status in intrauterine growth-retarded suckling lambs.

The influence of dietary supplementation of l-arginine (Arg) or N-carbamylglutamate (NCG) on the hepatic antioxidant status in intrauterine-growth-retarded (IUGR) suckling lambs remains unclear. The current work aimed to investigate the regulatory mechanisms whereby dietary Arg or NCG alter hepatic antioxidant status in suckling lambs suffering from IUGR. Forty-eight newborn Hu lambs of normal birth weight (CON) and IUGR were allocated randomly into four groups of 12 animals each: CON (4.25 ± 0.14 kg), IUGR (3.01 ± 0.12 kg), IUGR + 1% Arg (2.99 ± 0.13 kg), or IUGR + 0.1% NCG (3.03 ± 0.11 kg). All lambs were raised for a period of 21 days from 7 to 28 days after birth. Compared with the IUGR suckling animals, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and reduced glutathione (GSH) content were greater (P < 0.05), and protein carbonyl and malondialdehyde (MDA) levels were reduced (P < 0.05) in the livers of both IUGR + 1% Arg and 0.1% NCG suckling animals. Relative to IUGR suckling lambs, supplementing with Arg or NCG markedly reduced (P < 0.05) reactive oxygen species (ROS) levels, apoptosis, and necrosis in liver. Relative to IUGR suckling lambs, protein and mRNA expression of GSH-Px1, SOD2, catalase (CAT), heme oxygenase-1 (HO-1), inducible nitric oxide (NO) synthase (iNOS), and epithelial NO synthase (eNOS) increased in IUGR animals receiving Arg or NCG (P < 0.05). Both Arg and NCG can protect neonates from IUGR-induced hepatic oxidative damage through promoting the expression of antioxidative enzymes (including SOD, CAT, and GSH-Px), phase II metabolizing enzymes, and activation of the NO pathway.

N-carbamylglutamate and l-arginine promote intestinal function in suckling lambs with intrauterine growth restriction by regulating antioxidant capacity via a nitric oxide-dependent pathway.

Data indicate that intrauterine growth restriction (IUGR) in newborns can be partly alleviated through the supply of l-arginine (Arg) and N-carbamylglutamate (NCG). The current work aimed to explore whether Arg and NCG promote intestinal function by regulating antioxidant capacity in suckling lambs with IUGR via a nitric oxide (NO)-dependent pathway. Forty eight newly born Hu lambs with normal weights at birth (CON) or suffering from IUGR were randomly divided into 4 groups (n = 12 per group), namely, the CON, IUGR, IUGR + 1% Arg, and IUGR + 0.1% NCG groups. The animals were used for experiments from the age of day 7 to 28. Compared with the lambs in the IUGR group, the lambs in the Arg or NCG group had higher (P < 0.05) final body weights. The plasma insulin, NO, and NO synthase (NOS) concentrations in the IUGR group were higher (P < 0.05) compared with those in IUGR + 1% Arg or IUGR + 0.1% NCG. The jejunal level of the tumor necrosis factor α (TNF-α) in the IUGR lambs was greater (P < 0.05) compared with that in IUGR + 1% Arg or IUGR + 0.1% NCG. The plasma and jejunal total antioxidant capacity (T-AOC) values for the IUGR + 1% Arg or IUGR + 0.1% NCG group were greater (P < 0.05) compared with those for the IUGR group. Compared with the IUGR + 1% Arg or IUGR + 0.1% NCG lambs, the IUGR lambs had lower (P < 0.05) abundance of mRNA and protein abundance of glutathione peroxidase 1 (GPx1), catalase (CAT), superoxide dismutase 2 (SOD2), nuclear factor erythroid 2-related factor 2 (Nrf2), quinone oxidoreductase 1 (NQO1), heme oxygenase (HO-1), zonula occludens-1 (ZO-1), occludin, inducible NOS (iNOS), and epithelial NOS (eNOS). Overall, the data suggest that the Arg or NCG supplementation to suckling lambs with IUGR enhances the intestinal function by regulating the oxidant status via the NO-dependent pathway.

Dietary N-carbamylglutamate and l-arginine supplementation improves intestinal energy status in intrauterine-growth-retarded suckling lambs.

This study explores the roles of l-arginine (Arg) and N-carbamylglutamate (NCG) supplementation in the diet in intestine damage, energy state, as well as the associated protein kinase signaling pathways activated by AMP in intrauterine growth retarded (IUGR) suckling lambs. A total of 48 newborn Hu lambs with a normal birth weight (CON) and those with IUGR were randomly divided into four groups, CON, IUGR, IUGR + 1% Arg, and IUGR + 0.1% NCG, with 12 animals in each group. All animals were fed for 21 days, from day 7-28, following birth. Our results indicated that the IUGR suckling Hu lambs in the Arg or NCG groups were associated with reduced (P < 0.05) plasma diamine oxidase (DAO) and d-lactic acid levels compared with IUGR suckling lambs. In addition, IUGR suckling Hu lambs in the Arg or NCG group were also linked with a higher (P < 0.05) villous height : crypt depth ratio (VCR), as well as villous height in the duodenum relative to those obtained for IUGR suckling Hu lambs. Relative to IUGR suckling Hu lambs, IUGR suckling Hu lambs in the Arg or NCG groups were found to have higher (P < 0.05) ATP, ADP and TAN contents, and AEC levels, and smaller (P < 0.05) AMP : ATP ratios in the duodenum, jejunum and ileum. Moreover, IUGR suckling Hu lambs in the Arg or NCG group were also linked with higher citrate synthase, isocitrate dehydrogenase and alpha-oxoglutarate dehydrogenase complex activities in the duodenum, jejunum and ileum compared with those found for IUGR suckling Hu lambs (P < 0.05), except for the activity of isocitrate dehydrogenase in the ileum. IUGR suckling Hu lambs in the Arg or NCG group were linked with a lower ratio of pAMPKα/tAMPKα and protein expression of Sirt1 and PGC1α in the ileum relative to those of the IUGR suckling Hu lambs (P < 0.05). Taken together, these findings show that supplementation of NCG and Arg in the diet can ameliorate intestinal injury, improve energy status, motivate key enzyme activities in the tricarboxylic acid (TCA) cycle, and also inhibit the AMP-activated protein kinase signaling pathways in IUGR suckling Hu lambs.