Selenium exerts protective effects against heat stress-induced barrier disruption and inflammation response in jejunum of growing pigs.

BACKGROUND: Heat stress (HS) has a negative impact on the intestinal barrier and immune function of pigs. Selenium (Se) may improve intestinal health through affecting selenoproteins. Thus we investigate the protective effect of new organic Se (2-hydroxy-4-methylselenobutanoic acid, HMSeBA) on jejunal damage in growing pigs upon HS and integrate potential roles of corresponding selenoproteins. RESULTS: HS decreased the villus height and increased (P < 0.05) the protein abundance of HSP70, and downregulated (P < 0.05) protein levels of tight junction-related proteins (CLDN-1 and OCLD). HS-induced jejunal damage was associated with the upregulation of four inflammation-related genes and ten selenoprotein-encoding genes, downregulation (P < 0.05) of four selenoprotein-encoding genes and decreased (P < 0.05) the protein abundance of GPX4 and SELENOS. Compared with the HS group, HMSeBA supplementation not only elevated the villus height and the ratio of V/C (P < 0:05), but also reduced (P < 0.05) the protein abundance of HSP70 and MDA content, and increased (P < 0.05) the protein abundance of OCLD. HMSeBA supplementation downregulated the expression of seven inflammation-related genes, changed the expression of 12 selenoprotein-encoding genes in jejunum mucosa affected by HS, and increased the protein abundance of GPX4, TXNRD1 and SELENOS. CONCLUSION: Organic Se supplementation beyond nutritional requirement alleviates the negative effect of HS on the jejunum of growing pigs, and its protective effect is related to the response of corresponding selenoproteins. © 2021 Society of Chemical Industry.

Effects of N-acetyl-l-cysteine on heat stress-induced oxidative stress and inflammation in the hypothalamus of hens.

The purpose of this study was to discuss the effects of N-acetyl-l-cysteine (NAC) on heat stress-induced oxidative stress and inflammation in the hypothalamus of hens in different periods. A total of 120 Hy-Line variety brown laying hens (12 weeks old) were randomly assigned to 4 groups with 6 replicates. The control group (C group) (22 ± 1 °C) received a basal diet, the NAC-treated group (N group) (22 ± 1 °C) received a basal diet with 1000 mg/kg NAC, and 2 heat-stressed groups (36 ± 1 °C for 10 h per day and 22 ± 1 °C for the remaining time) were fed a basal diet (HS group) or a basal diet with 1000 mg/kg NAC (HS + N group) for 21 consecutive days. The influence of NAC on histologic changes, oxidative stress and proinflammatory cytokine production was measured and analysed in hens with heat stress-induced hypothalamic changes. NAC effectively alleviated the hypothalamic morphological changes induced by heat stress. In addition, NAC attenuated the activity of the Nf-κB pathway activated by heat stress and decreased the expression of the proinflammatory cytokines IL-6, IL-18, TNF-α, IKK, and IFN-γ. In addition, NAC treatment regulated the expression of HO-1, GSH, SOD2 and PRDX3 by regulating the activity of Nrf2 at different time points to resist oxidative stress caused by heat exposure. In summary, dietary NAC may be an effective candidate for the treatment and prevention of heat stress-induced hypothalamus injury by preventing Nf-κB activation and controlling the Nrf2 pathway.

Dietary supplementation of acetate-conjugated tryptophan alters feed intake, milk yield and composition, blood profile, physiological variables, and heat shock protein gene expression in heat-stressed dairy cows.

The purpose of this study was to investigate the effects of dietary supplementation of rumen-protected tryptophan (RPT) at four levels on milk yield, milk composition, blood profile, physiological variables, and heat shock protein gene expression in dairy cows under conditions of moderate-severe heat stress (MSHS, THI = 80~89). Sixteen early-lactating dairy cows (body weight = 719 ± 66.4 kg, days in milk = 74.3 ± 7.1, milk yield = 33.55 ± 3.74 kg, means ± SEM) were randomly assigned in a factorial arrangement to one of the four treatments: control group (n = 4, no RPT supplementation), 15 g/d RPT (n = 4), 30 g/d RPT (n = 4), or 60 g/d RPT group per cow (n = 4) supplemented to the TMR. A higher dry matter intake (DMI) and milk yield were found in the 30 g RPT group compared with the other groups, and the 3.5% fat-corrected milk yield, energy-corrected milk yield, milk fat, protein, ß-casein, mono-unsaturated fatty acid, and poly-unsaturated fatty acid contents, and serum glucose content were observed in the 30 g RPT group (p < 0.05). The milk lactose concentration was significantly higher in the 30 g RPT group compared with the control and 60 g RPT groups (p < 0.05). The plasma cortisol level was lower, while the serotonin and melatonin concentrations were higher in the 30 g group compared with the other groups (p < 0.05). Heat shock protein (HSP) 70 expression was downregulated in the control and 15 g RPT groups, whereas the expression of HSP90 and HSPB1 remained unchanged among the groups. In particular, the 30 g RPT group was considered to have an improved DMI, milk yield, and lactose concentration, as well as anti-heat stress effects due to the simulation of serotonin and melatonin during MSHS.

Multi-tissue transcriptomic analysis reveals that L-methionine supplementation maintains the physiological homeostasis of broiler chickens than D-methionine under acute heat stress.

The objective of this study was to compare the effects of supplementation with two methionine isoforms, L-methionine (L-Met) or D-methionine (D-Met), on transcriptome expression in broiler chickens under acute heat stress. A total of 240 one-day-old chicks were randomly assigned to one of four treatments in a 2 × 2 factorial arrangement: thermo-neutral vs. acute heat-stress and L-Met vs. D-Met supplementation. On day 14, the heat-stressed group was exposed to 32°C for 5 h, while the others remained at 25°C. Six chicks were randomly selected per treatment and total RNA was isolated from whole blood, ileum, and liver tissues. Two RNA samples from each tissue of each treatment group were randomly selected and pooled in equal amounts. A total of 1.87 billion raw reads obtained from 36 samples (four treatments × three tissues × three composited replicates) were mapped to the reference genome build (Gallus_gallus-5.0) and used to identify differentially expressed genes (DEGs) using DESeq2. Functional enrichment of DEGs was tested using DAVID. Comparing the two isoforms of supplemented methionine, two, three, and ten genes were differentially expressed (> 1 or < -1 log2 fold change) in whole blood, ileum, and liver, respectively. A total of 38, 71, and 16 genes were differentially expressed in response to the interaction between heat stress and Met isoforms in the blood, ileum, and liver, respectively. Three-tissue-specific DEGs were functionally enriched for regulation of cholesterol homeostasis and metabolism, glucose metabolism, and vascular patterning. Chicks fed with L-Met had lower immune (e.g., IL4I1 and SERPINI1) and intestinal angiogenic responses (e.g., FLT1 and FGD5), and stable glucose and lipid metabolism (e.g., PCK1 and LDLR) under heat stress conditions. In conclusion, unlike D-Met, L-Met supplementation seems to help maintain physiological homeostasis and enhances cellular defense systems against external stresses like high environmental temperature.

Dietary enzymatically-treated Artemisia annua L. supplementation could alleviate oxidative injury and improve reproductive performance of sows reared under high ambient temperature.

The medicinal plant Artemisia annua L. is well known for its antimalarial compound artemisinin and the antioxidant capacity of its active ingredients. However, low bioavailability of Artemisia annua L. limits its therapeutic potential, fermentation of Artemisia annua L. can improve its bioavailability. This study was aimed to investigate the effects of dietary supplementation of enzymatically-treated Artemisia annua L. (EA) on reproductive performance, antioxidant status, milk composition of heat-stressed sows and intestinal barrier integrity of their preweaning offspring. 135 multiparous sows of average parity 4.65 (Landrace × large white) at day 85 of pregnancy were randomly distributed into 3 treatments. Sows in the control group were housed at control rooms (temperature: 27.12 ± 0.18 °C, temperature-humidity index (THI): 70.90 ± 0.80) and fed the basal diet. Sows in the HS, HS + EA groups were fed the basal diet supplemented with 0 or 1.0 g/kg EA respectively, and reared at heat stress rooms (temperature: 30.11 ± 0.16 °C, THI: 72.70 ± 0.60). Heat stress increased the malondialdehyde (MDA) content, reduced the activities of total antioxidant capacity (T-AOC) and total superoxide dismutase (T-SOD) of sows and piglets, and seriously compromised the antioxidant capacity of the sows and the intestinal integrity of their offspring. However, dietary supplementation of 1.0 g/kg EA reduced the MDA content, increased the activities of T-SOD and T-AOC in serum, colostrum, and milk of heat-stressed sows, and increased colostrum yield and 14-d milk fat content. EA supplementation also increased piglet weaning weight and the activities of T-SOD and T-AOC in serum. In addition, the abundances of intestinal tight junction proteins claudin-1 and occludin were up-regulated in piglets in EA-supplemented group. In conclusion, dietary EA supplementation at 1.0 g/kg can alleviate the oxidative stress in heat-stressed sows, improve the antioxidant capacity in both sows and their offspring, and promote the intestinal barrier integrity in their offspring. EA may be a potent dietary supplement that ameliorates oxidative stress in livestock production by improving the antioxidant capacity.

Ginseng Extract Ameliorates the Negative Physiological Effects of Heat Stress by Supporting Heat Shock Response and Improving Intestinal Barrier Integrity: Evidence from Studies with Heat-Stressed Caco-2 Cells, C. elegans and Growing Broilers.

Climatic changes and heat stress have become a great challenge in the livestock industry, negatively affecting, in particular, poultry feed intake and intestinal barrier malfunction. Recently, phytogenic feed additives were applied to reduce heat stress effects on animal farming. Here, we investigated the effects of ginseng extract using various in vitro and in vivo experiments. Quantitative real-time PCR, transepithelial electrical resistance measurements and survival assays under heat stress conditions were carried out in various model systems, including Caco-2 cells, Caenorhabditis elegans and jejunum samples of broilers. Under heat stress conditions, ginseng treatment lowered the expression of HSPA1A (Caco-2) and the heat shock protein genes hsp-1 and hsp-16.2 (both in C. elegans), while all three of the tested genes encoding tight junction proteins, CLDN3, OCLN and CLDN1 (Caco-2), were upregulated. In addition, we observed prolonged survival under heat stress in Caenorhabditis elegans, and a better performance of growing ginseng-fed broilers by the increased gene expression of selected heat shock and tight junction proteins. The presence of ginseng extract resulted in a reduced decrease in transepithelial resistance under heat shock conditions. Finally, LC-MS analysis was performed to quantitate the most prominent ginsenosides in the extract used for this study, being Re, Rg1, Rc, Rb2 and Rd. In conclusion, ginseng extract was found to be a suitable feed additive in animal nutrition to reduce the negative physiological effects caused by heat stress.

Dietary taurine supplementation decreases fat synthesis by suppressing the liver X receptor α pathway and alleviates lipid accumulation in the liver of chronic heat-stressed broilers.

BACKGROUND: Chronic heat stress can enhance fat synthesis and result in lipid accumulation in the liver of broilers. To investigate the effects and molecular mechanisms of dietary taurine supplementation on fat synthesis and lipid accumulation in the liver of chronic heat-stressed broilers, 144 28 day-old chickens (Arbor Acres) were randomly distributed to normal control (NC, 22 °C, basal diet), heat stress (HS, consistent 32 °C, basal diet), or heat stress plus taurine (HS + T, consistent 32 °C, basal diet +5.00 g kg-1 taurine) groups for a 14-day feeding trial. RESULTS: Compared with those of the HS group, dietary taurine supplementation significantly decreased the level of very-low-density lipoprotein and the activity of aspartate aminotransferase in plasma and the relative weight of liver in the HS + T group. In addition, dietary taurine supplementation also significantly decreased the levels of triglyceride, acyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), and suppressed the mRNA expression levels of liver X receptor α (LXRα), sterol response element-binding protein 1c, ACC and FAS in the liver of chronic heat-stressed broilers. Meanwhile, dietary taurine supplementation effectively alleviated lipid accumulation in the liver of broilers exposed to chronic heat stress. CONCLUSION: Chronic heat stress significantly increased fat synthesis and resulted in excess lipid deposition in the liver of broilers. Dietary taurine supplementation can effectively decrease fat synthesis by suppressing the LXRα pathway and alleviate lipid accumulation in the liver of chronic heat-stressed broilers. © 2019 Society of Chemical Industry.

Mechanism underlying berberine's effects on HSP70/TNFα under heat stress: Correlation with the TATA boxes.

Heat stress can stimulate an increase in body temperature, which is correlated with increased expression of heat shock protein 70 (HSP70) and tumor necrosis factor α (TNFα). The exact mechanism underlying the HSP70 and TNFα induction is unclear. Berberine (BBR) can significantly inhibit the temperature rise caused by heat stress, but the mechanism responsible for the BBR effect on HSP70 and TNFα signaling has not been investigated. The aim of the present study was to explore the relationship between the expression of HSP70 and TNFα and the effects of BBR under heat conditions, using in vivo and in vitro models. The expression levels of HSP70 and TNFα were determined using RT-PCR and Western blotting analyses. The results showed that the levels of HSP70 and TNFα were up-regulated under heat conditions (40 °C). HSP70 acted as a chaperone to maintain TNFα homeostasis with rising the temperature, but knockdown of HSP70 could not down-regulate the level of TNFα. Furthermore, TNFα could not influence the expression of HSP70 under normal and heat conditions. BBR targeted both HSP70 and TNFα by suppressing their gene transcription, thereby decreasing body temperature under heat conditions. In conclusion, BBR has a potential to be developed as a therapeutic strategy for suppressing the thermal effects in hot environments.

Dietary enzymatically treated Artemisia annua L. supplementation alleviates liver oxidative injury of broilers reared under high ambient temperature.

Heat stress induced by high ambient temperature is a major concern in commercial broiler production. To evaluate the effects of dietary enzymatically treated Artemisia annua L. (EA) supplementation on growth performance and liver oxidative injury of broilers reared under heat stress, a total of 320 22-day-old male broilers were randomly allotted into five groups with eight replicates of eight birds each. Broilers in the control group were housed at 22 ± 1 °C and fed the basal diet. Broilers in the HS, HS-EA1, HS-EA2, and HS-EA3 groups were fed basal diet supplemented with 0, 0.75, 1.00, and 1.25 g/kg EA, respectively, and reared under cyclic high temperature (34 ± 1 °C for 8 h/day and 22 ± 1 °C for 16 h/day). Broilers fed EA diets had higher final body weight, average daily body weight gain, and average daily feed intake, as well as liver concentration of reduced glutathione, activities of antioxidant enzymes, abilities to inhibit hydroxyl radical and superoxide radical (HS-EA2 and HS-EA3), and lower liver concentrations of reactive oxygen metabolites, malondialdehyde, and protein carbonyl (HS-EA1, HS-EA2, and HS-EA3) than HS group (P < 0.05). EA treatment downregulated the mRNA levels of heat shock proteins 70 and 90, upregulated the mRNA levels of nuclear factor erythroid 2-related factor 2 (HS-EA1, HS-EA2, and HS-EA3) and heme oxygenase 1 (HS-EA2 and HS-EA3) in liver of heat-treated broilers (P < 0.05). In conclusion, EA alleviated heat stress-induced growth depression and liver oxidative injury in broilers, possibly through improving the antioxidant capacity and regulating the pertinent mRNA expression. The appropriate inclusion level of EA in broiler diet is 1.00-1.25 g/kg.

Epidermal growth factor receptor expression and signaling are essential in glutamine's cytoprotective mechanism in heat-stressed intestinal epithelial-6 cells.

Epidermal growth factor receptor (EGFR) expression and signaling can induce cellular protection after intestinal inflammation. L-Glutamine (GLN) is known to prevent apoptosis after intestinal injury by activating MAPK and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. However, the role of EGFR expression and signaling in GLN-mediated cellular protection in intestinal epithelial-6 (IEC-6) cells after heat stress (HS) is unknown. To address the role of EGFR in GLN-mediated protection, IEC-6 cells were treated with GLN in the presence or absence of EGFR small interfering RNA, the EGFR tyrosine kinase inhibitor AG1478, the ERK1/2 inhibitor PD98059, the p38MAPK inhibitor SB203580, or the PI3-K/Akt inhibitor LY294002 under basal and HS conditions. GLN-mediated cell survival was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Phosphorylated and/or total levels of EGFR, cleaved caspase-3, poly(ADP-ribose) polymerase-1, ERK1/2, p38MAPK, and Akt were assessed by Western blotting. We showed that HS induced a decrease in total, cytoplasmic, and nuclear EGFR levels in IEC-6 cells, which was prevented by GLN supplementation, leading to attenuated apoptosis via EGFR small interfering RNA. Furthermore, the protective effect of GLN was lessened by AG1478, PD98059, and LY294002 but was not affected by SB203580. AG1478 attenuated GLN-mediated increases in ERK1/2 and decreases in p38MAPK phosphorylation. However, AG1478 had no effect on GLN-mediated augmentations in Akt phosphorylation. In summary, EGFR expression was important in the protective mechanism of GLN, as well as GLN-mediated activation of EGFR tyrosine kinase activity. GLN-mediated EGFR signaling activated ERK1/2 and decreased p38MAPK signaling. However, GLN-mediated Akt phosphorylation after HS seems to be independent of EGFR signaling.

Dynamic changes in DNA methylation during thermal control establishment affect CREB binding to the brain-derived neurotrophic factor promoter.

Thermal control establishment develops during a critical period by alterations in cellular properties in the frontal hypothalamus. These alterations may be modulated by the epigenetic code that determines the repertoire of transcribed proteins. Here we demonstrate transient changes in the expression of brain-derived neurotrophic factor (Bdnf) during both thermal conditioning and re-exposure of conditioned chicks to heat stress, relative to their age-matched naive counterparts. These changes coincide with changes in CpG methylation pattern in the avian Bdnf promoter region. Reduction in methylation during heat conditioning was observed at a cAMP response element-binding (CREB) site which coincided with both elevation in phospho-CREB levels and its binding to the Bdnf promoter. At the same time, an increase in methylation was observed at two other CpG sites, accompanied by elevation of the DNA methyltransferase 3a (DNMT3a) expression. DNMT3a was also found to bind to the two elevated methyl CpG sites, but not to the CREB binding site. These data suggest that complex and dynamic changes in DNA methylation are involved in the regulation of Bdnf expression during thermotolerance acquisition.

Acclimatory-phase specificity of gene expression during the course of heat acclimation and superimposed hypohydration in the rat hypothalamus.

The induction of the heat-acclimated phenotype involves reprogramming the expression of genes encoding both constitutive and inducible proteins. In this investigation, we studied the global genomic response in the hypothalamus during heat acclimation, with and without combined hypohydration stress. Rats were acclimated for 2 days (STHA) or for 30 days (LTHA) at 34 degrees C. Hypohydration (10% decrease in body weight) was attained by water deprivation. 32P-labeled RNA samples from the hypothalamus were hybridized onto cDNA Atlas array (Clontech no. 1.2) membranes. Clustering and functional analyses of the expression profile of a battery of genes representing various central regulatory functions of body homeostasis demonstrated a biphasic acclimation profile with a transient upregulation of genes encoding ion channels, transporters, and transmitter signaling upon STHA. After LTHA, most genes returned to their preacclimation expression levels. In both STHA and LTHA, genes encoding hormones and neuropeptides, linked with metabolic rate and food intake, were downregulated. This genomic profile, demonstrating an enhanced transcription of genes linked with neuronal excitability during STHA and enhanced metabolic efficiency upon LTHA, is consistent with our previously established integrative acclimation model. The response to hypohydration was characterized by an upregulation of a large number of genes primarily associated with the regulation of ion channels, cell volume, and neuronal excitability. During STHA, the response was transiently desensitized, recovering upon LTHA. We conclude that hypohydration overrides the heat acclimatory status. It is notable that STHA and hypohydration gene profiles are analogous with the physiological profile described in the response to various types of brain injury.

Differences in hypothalamic Fos expressions between two heat stress conditions in conscious mice.

Hyperthermia and dehydration were important physiological phenomena in heat stress. But, the degrees of these phenomena were changed by heat stress conditions, and the distinction between both phenomena is necessary for investigation of response for individual phenomenon. Heat stress at 34 degrees C for 60 min increased rectal temperature, and heat stress at 38.5 degrees C for 60 min further increased rectal temperature and increased osmolality in mice. We investigated the activated region in hypothalamus, which played a role in thermoregulation, fluid regulation and so on, using immunostaining for Fos protein under these conditions in conscious mice. At 34 degrees C, Fos-positive neurons increased in the median preoptic nucleus, lateral preoptic area and anterior hypothalamic area, which were known to be the thermoregulatory center, and the dorsomedial hypothalamic nucleus, which was known to control eating. At 38.5 degrees C, Fos-positive neurons further increased in the regions mentioned above and appeared in the lateral septal nucleus, medial preoptic area, lateral hypothalamic area and zona incerta, which were thought to be involved in thermoregulation and/or fluid regulation, and the paraventricular hypothalamic nucleus, supraoptic nucleus and supraoptic nucleus in the retrochiasmatic part, which were known to be involved in neuroendocrine effector systems. These results support that the activated regions in hypothalamus differed with heat stress conditions, which induced only hyperthermia and both hyperthermia and dehydration.