Selection, identification and evaluation of optimal reference genes in Chinese sturgeon (Acipenser sinensis) under polypropylene microplastics stress.

Polypropylene microplastics (PP-MPs) are emerging environmental contaminants that have the potential to cause adverse effects on aquatic organisms. Reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) is a valuable tool for assessing the gene expression profiles under PP-MPs stress. To obtain an accurate gene expression profile of tissue inflammation and apoptosis that reflects the molecular mechanisms underlying the impact of PP-MPs on Chinese sturgeon, identifying reliable reference genes is crucial for RT-qPCR analysis. In this study, we constructed an experiment model of Chinese sturgeon exposed to PP-MPs, assessed the pathological injury, metabolic profile responses and oxidative stress in liver, evaluated the reliability of 8 reliable reference genes by 4 commonly used algorithms including GeNorm, NormFinder, BeatKeeper, Delta Ct, and then analyzed the performance of inflammatory response genes in liver, spleen and kidney with the best reference gene. HE staining revealed that the cytoplasm full small vacuoles and nucleus diameter increased were occurred in the liver cell of PP-MPs in treatment groups. Additionally, oxidative and biochemical parameters were significantly changes in the liver of treatment groups. For the reference genes in PP-MPs exposure experiments, this study screening the optimal reference genes including: EF1α and GAPDH for liver and spleen, and GAPDH and RPS18 for kidney. Besides, 2 inflammatory response genes (NLRP3, TNF-α) were chosen to assess the optimal reference genes using the least stable reference gene (TUB) as a control, verified the practicality of the select reference genes in different tissues. We also found that the low concentration of PP-MPs could induce the liver tissue damage and inflammatory response in Chinese sturgeon. Our study initially evaluated the impact of short-time exposure with PP-MPs in Chinese sturgeon and provided 3 sets of validated optimal reference genes in Chinese sturgeon exposure to PP-MPs.

The Effects of Tachykinin1 Gene Products on Prepubertal Dabry's Sturgeon (Acipenser dabrynus) Pituitary Hormone Secretion and Gene Expression.

As an ancient and endangered species unique to the Yangtze River in China, the wild population of the Dabry's sturgeon has become scarce. Due to the long time till the first sexual maturity of Dabry's sturgeon, the population of artificially bred Dabry's sturgeon recovered slowly. As a member of the tachykinin family, TAC1 has been reported to have a variety of functions in mammals such as pain control, smooth muscle contraction and reproductive cycle regulation, but the function of Tac1 in fish has been rarely reported. In this study, we synthesized two tac1 gene products, Substance P (SP) and neurokinin A (NKA), and further verified the effect of two tac1 gene products on the secretion of related hormones in the pituitary of Dabry's Sturgeon by intraperitoneal injection and co-culture of primary cells. Expression studies revealed that the newly cloned tac1 were mainly distributed in the hypothalamus and pituitary tissue of the brain. In prepubertal Dabry's sturgeon, this study showed that the two gonadotropins' mRNA levels in pituitary tissue can be significantly increased by SP and NKA through intraperitoneal injection, and the LH protein level in serum was also increased. Further study showed that both NKA and SP could promote the two gonadotropins' mRNA expression in pituitary cells of Dabry's sturgeon. In addition, we explored the optimal dose and time of SP and NKA on pituitary cells is 24 h and over 10 nM. These results, as a whole, suggested that tac1 gene products play an important role in gonadotropin release and gonadal development in prepubertal Dabry's sturgeon.

Quercetin Ameliorates Deoxynivalenol-Induced Intestinal Injury and Barrier Dysfunction Associated with Inhibiting Necroptosis Signaling Pathway in Weaned Pigs.

Quercetin (Que) is a flavonol compound found in plants, which has a variety of biological activities. Necroptosis, a special form of programmed cell death, plays a vital role in the development of many gastrointestinal diseases. This study aimed to explore whether Que could attenuate the intestinal injury and barrier dysfunction of piglets after deoxynivalenol (DON) exposure through modulating the necroptosis signaling pathway. Firstly, twenty-four weaned piglets were used in a 2 × 2 factorial design and the main factors, including Que (basal diet or diet supplemented with 100 mg/kg Que) and DON exposure (control feed or feed contaminated with 4 mg/kg DON). After feeding for 21 d, piglets were killed for samples. Next, the intestinal porcine epithelial cell line (IPEC-1) was pretreated with or without Que (10 µmol/mL) in the presence or absence of a DON challenge (0.5 µg/mL). Dietary Que increased the body weight, average daily gain, and average daily feed intake (p < 0.05) through the trial. Que supplementation improved the villus height, and enhanced the intestinal barrier function (p < 0.05) indicated by the higher protein expression of occludin and claudin-1 (p < 0.05) in the jejunum of the weaned piglets after DON exposure. Dietary Que also down-regulated the protein abundance of total receptor interacting protein kinase 1 (t-RIP1), phosphorylated RIP1 (p-RIP1), p-RIP3, total mixed lineage kinase domain-like protein (t-MLKL), and p-MLKL (p < 0.05) in piglets after DON exposure. Moreover, Que pretreatment increased the cell viability and decreased the lactate dehydrogenase (LDH) activity (p < 0.05) in the supernatant of IPEC-1 cells after DON challenge. Que treatment also improved the epithelial barrier function indicated by a higher transepithelial electrical resistance (TEER) (p < 0.001), lower fluorescein isothiocyanate-labeled dextran (FD4) flux (p < 0.001), and better distribution of occludin and claudin-1 (p < 0.05) after DON challenge. Additionally, pretreatment with Que also inhibited the protein abundance of t-RIP1, p-RIP1, t-RIP3, p-RIP3, t-MLKL, and p-MLKL (p < 0.05) in IPEC-1 cells after DON challenge. In general, our data suggest that Que can ameliorate DON-induced intestinal injury and barrier dysfunction associated with suppressing the necroptosis signaling pathway.

Effect of plant polysaccharides (Poria cocos and Astragalus polysaccharides) on immune responses and intestinal microbiota of Dabry's sturgeons.

Searching for non-toxic and harmless feed ingredients that can improve growth performance and host immunity has always been the focus of attention in the protected areas for artificially bred Dabry's sturgeons. The present study explored the effect of dietary Poria cocos and Astragalus polysaccharides on the antioxidant status, expression of immune related genes, and composition and putative functions of gut bacterial communities in Dabry's sturgeons for the first time. In this study, Dabry's sturgeons were randomly divided into 3 groups and fed diets of normal, P. cocos polysaccharide-added (200 mg/kg), and Astragalus polysaccharide-added (200 mg/kg) food for 14 days. The results indicated that dietary Astragalus polysaccharide can increase the final body weight of Dabry's sturgeon. Compared with normal breeding individuals, feeding diets containing the P. cocos and Astragalus polysaccharides up-regulated the activity of superoxide dismutase, lysozyme, catalase, and glutathione peroxidase while also decreasing the levels of malondialdehyde. In addition, the Astragalus polysaccharide group had higher gene expression of two inflammatory cytokines, tumor necrosis factor alpha and immunoglobulin M, than the control group. Analysis of intestinal microbiota revealed that the dietary Astragalus polysaccharide improved the richness and diversity of major gut microbiota in Dabry's sturgeons, while the structure in the P. cocos polysaccharide group was clearly distinguished from that of the control group. Our results preliminarily indicated that dietary supplementation of P. cocos and Astragalus polysaccharides may contribute to better performance in growth, development, and inflammatory response for Dabry's sturgeons, and they provide basic guidance for plant polysaccharide additives in artificial breeding of sturgeons.

Effects of short-term water velocity stimulation on the biochemical and transcriptional responses of grass carp (Ctenopharyngodon idellus).

Since 2011, ecological operation trials of the Three Gorges Reservoir (TGR) have been continuously conducted to improve the spawning quantity of the four major Chinese carp species below the Gezhouba Dam. In particular, exploring the effects of short-term water velocity stimulation on ovarian development in grass carp (Ctenopharyngodon idellus) is essential to understand the response of natural reproduction to ecological flows. We performed ovary histology analysis and biochemical assays among individuals with or without stimulation by running water. Although there were no obvious effects on the ovarian development characteristics of grass carp under short-term water velocity stimulation, estradiol, progesterone, follicle-stimulating hormone (FSH), and triiodothyronine (T3) concentrations were elevated. Then, we further explored the ovarian development of grass carp under short-term water velocity stimulation by RNA sequencing of ovarian tissues. In total, 221 and 741 genes were up- or downregulated under short-term water velocity stimulation, respectively, compared to the control group. The majority of differentially expressed genes (DEGs) were enriched in pathways including ABC transporters, cytokine-cytokine receptor interaction, ECM-receptor interaction, and steroid hormone biosynthesis. Important genes including gpr4, vtg1, C-type lectin, hsd17b1, cyp19a1a, cyp17a1, and rdh12 that are involved in ovarian development were regulated. Our results provide new insights and reveal potential regulatory genes and pathways involved in the ovarian development of grass carp under short-term water velocity stimulation, which may be beneficial when devising further ecological regulation strategies.

Effects of DON on Mitochondrial Function, Endoplasmic Reticulum Stress, and Endoplasmic Reticulum Mitochondria Contact Sites in the Jejunum of Piglets.

Recent research has emphasized the significance of investigating the interplay between organelles, with endoplasmic reticulum mitochondria contact sites (ERMCSs) being recognized as critical signaling hubs between organelles. The objective of the current study was to assess the impact of deoxynivalenol (DON) on jejunal mitochondria, ER, and ERMCSs. Twelve piglets (35 d, 10.22 ± 0.35 kg) were randomized into two groups: control group, basal diet; the DON group, basal diet + 1.5 mg/kg DON. The findings revealed that DON decreased growth performance, induced jejunal oxidative stress, and impaired jejunal barrier function. DON was also found to induce mitochondrial dysfunction, trigger endoplasmic reticulum stress (ERS) in the piglets' jejunum, and activate mitochondrial and ER apoptosis pathways by upregulating apoptosis-related proteins (Caspase-8, Caspase-12, Bax, and CHOP). To investigate the involvement of ERMCSs in DON-induced intestinal injury, we measured the protein levels of ERMCS proteins, such as mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), and glucose-regulated protein 75 (GRP75) and Pearson's correlation coefficient of ERMCS proteins and ERMCS ultrastructure. Our finding showed that DON upregulated the protein level of Mfn2 and GRP75 and increased the percentage of mitochondria with ERMCSs/total mitochondria, the length of ERMCSs compared to the perimeter of mitochondria, and the Pearson's correlation coefficient of voltage-dependent anion-selective channel protein 1 (VDAC1) and inositol 1,4,5-triphosphate receptors (IP3Rs) in piglets' jejunum. Furthermore, DON shortened the distance between mitochondria and ER at ERMCSs. These findings suggested that DON impaired mitochondrial function, triggered ERS, and increased ERMCSs, indicating that the increased ERMCSs could be related to mitochondrial dysfunction and ERS involved in the intestinal injury of piglets induced by DON.

Necroptosis Mediates Muscle Protein Degradation in a Cachexia Model of Weanling Pig with Lipopolysaccharide Challenge.

Necroptosis, an actively researched form of programmed cell death closely related to the inflammatory response, is important in a variety of disorders and diseases. However, the relationship between necroptosis and muscle protein degradation in cachexia is rarely reported. This study aimed to elucidate whether necroptosis played a crucial role in muscle protein degradation in a cachexia model of weaned piglets induced by lipopolysaccharide (LPS). In Experiment 1, the piglets were intraperitoneally injected with LPS to construct the cachexia model, and sacrificed at different time points after LPS injection (1, 2, 4, 8, 12, and 24 h). In Experiment 2, necrostatin-1 (Nec-1), a necroptosis blocker, was pretreated in piglets before the injection of LPS to inhibit the occurrence of necroptosis. Blood and longissimus dorsi muscle samples were collected for further analysis. In the piglet model with LPS-induced cachexia, the morphological and ultrastructural damage, and the release of pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 were dynamically elicited in longissimus dorsi muscle. Further, protein concentration and protein/DNA ratio were dynamically decreased, and protein degradation signaling pathway, containing serine/threonine kinase (Akt), Forkhead box O (FOXO), muscular atrophy F-box (MAFbx), and muscle ring finger protein 1 (MuRF1), was dynamically activated in piglets after LPS challenge. Moreover, mRNA and protein expression of necroptosis signals including receptor-interacting protein kinase (RIP)1, RIP3, and mixed lineage kinase domain-like pseudokinase (MLKL), were time-independently upregulated. Subsequently, when Nec-1 was used to inhibit necroptosis, the morphological damage, the increase in expression of pro-inflammatory cytokines, the reduction in protein content and protein/DNA ratio, and the activation of the protein degradation signaling pathway were alleviated. These results provide the first evidence that necroptosis mediates muscle protein degradation in cachexia by LPS challenge.

Sequences analysis and pituitary actions of tachykinins in Chinese sturgeon (Acipenser sinensis).

Tachykinins belong to a large, evolutionarily conserved family of brain/gut peptides that are involved in a variety of physiological functions in mammals, such as reproductive regulation. However, little information was available about tachykinins in ancient fish lineage. In the present study, we firstly identified three tachykinin genes (named tac1, tac3 and tac4) and three neurokinin receptors (named nk1r, nk2r and nk3r) from Chinese sturgeon brain and pituitary. Sequence analysis showed that tac1 encoded substance P (SP) and neurokinin A (NKA), tac3 encoded neurokinin B (NKB) and NKB-related peptide (NKBRP), and tac4 encoded hemokin 1 (HK-1) and hemokin 2 (HK-2), respectively. The luciferase reporter assay results showed that NK1R preferentially selected asSP, NK2R preferentially selected asNKA, and NK3R preferentially selected asNKB. Tissue expression analysis showed that the three tac genes were highly detected in the telencephalon and hypothalamus, whereas nkr genes were widely expressed in peripheral tissues. Spatio-temporal expression analysis showed that all three tac genes were highly expressed in unknown sex individuals. Intraperitoneal injection experiments showed that both asSP and asNKB could stimulate luteinizing hormone (LH) release in Chinese sturgeon serum. At the transcriptional level, asSP and asNKB could significantly reduce pituitary follicle-stimulating hormone beta (fshß) mRNA expression, but induce pituitary growth hormone (gh) mRNA expression. In addition, estradiol (E2) could stimulate tac3 mRNA expression in hypothalamus. Taken together, this study provided information on the tachykinin family in Chinese sturgeon and demonstrates that asNKB and asSP could be involved in reproductive and growth regulation in pituitary.

Protocatechuic acid and quercetin attenuate ETEC-caused IPEC-1 cell inflammation and injury associated with inhibition of necroptosis and pyroptosis signaling pathways.

BACKGROUND: Necroptosis and pyroptosis are newly identified forms of programmed cell death, which play a vital role in development of many gastrointestinal disorders. Although plant polyphenols have been reported to protect intestinal health, it is still unclear whether there is a beneficial role of plant polyphenols in modulating necroptosis and pyroptosis in intestinal porcine epithelial cell line (IPEC-1) infected with enterotoxigenic Escherichia coli (ETEC) K88. This research was conducted to explore whether plant polyphenols including protocatechuic acid (PCA) and quercetin (Que), attenuated inflammation and injury of IPEC-1 caused by ETEC K88 through regulating necroptosis and pyroptosis signaling pathways. METHODS: IPEC-1 cells were treated with PCA (40 µmol/L) or Que (10 µmol/L) in the presence or absence of ETEC K88. RESULTS: PCA and Que decreased ETEC K88 adhesion and endotoxin level (P < 0.05) in cell supernatant. PCA and Que increased cell number (P < 0.001) and decreased lactate dehydrogenases (LDH) activity (P < 0.05) in cell supernatant after ETEC infection. PCA and Que improved transepithelial electrical resistance (TEER) (P < 0.001) and reduced fluorescein isothiocyanate-labeled dextran (FD4) flux (P < 0.001), and enhanced membrane protein abundance of occludin, claudin-1 and ZO-1 (P < 0.05), and rescued distribution of these tight junction proteins (P < 0.05) after ETEC infection. PCA and Que also declined cell necrosis ratio (P < 0.05). PCA and Que reduced mRNA abundance and concentration of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-8 (P < 0.001), and down-regulated gene expression of toll-like receptors 4 (TLR4) and its downstream signals (P < 0.001) after ETEC infection. PCA and Que down-regulated protein abundance of total receptor interacting protein kinase 1 (t-RIP1), phosphorylated-RIP1 (p-RIP1), p-RIP1/t-RIP1, t-RIP3, p-RIP3, mixed lineage kinase domain-like protein (MLKL), p-MLKL, dynamin- related protein 1 (DRP1), phosphoglycerate mutase 5 (PGAM5) and high mobility group box 1 (HMGB1) (P < 0.05) after ETEC infection. Moreover, PCA and Que reduced protein abundance of nod-like receptor protein 3 (NLRP3), nod-like receptors family CARD domain-containing protein 4 (NLRC4), apoptosis-associated speck-like protein containing a CARD (ASC), gasdermin D (GSDMD) and caspase-1 (P < 0.05) after ETEC infection. CONCLUSIONS: In general, our data suggest that PCA and Que are capable of attenuating ETEC-caused intestinal inflammation and damage via inhibiting necroptosis and pyroptosis signaling pathways.

Effects of Kisspeptin on the reproductive function in the Dabry's sturgeon (Acipenser dabrynus).

Kisspeptin, a kind of neuropeptide, is involved in various physiological processes such as tumor metastasis inhibition and reproductive regulation due to its ability to interact with Kisspeptin receptor-Kissr. In teleost, Kisspeptin/Kissr system stimulates the hypothalamus-pituitary-gonadal axis (HPG axis), which is crucial for the reproductive regulation. Compared to one Kisspeptin protein Kiss1 was existed in mammals, two Kisspeptin were identified in sturgeon species, including Kiss1 and Kiss2, with specific receptors of Kissr1 and Kissr2, respectively. However, few reports described the effects of the two isoforms of Kisspeptin on the reproductive regulation in sturgeon. The core peptides of Kiss1 and Kiss2 (Kiss1-10 and Kiss2-10) of Dabry's sturgeon were successfully synthesized to explore the functional influence of Kisspeptin on the sturgeon HPG axis in the present study. The present findings suggested that intraperitoneal injection of Kiss1-10 and Kiss2-10 could significantly up-regulate the mRNA expression of Gnrh、Fsh and Lh in the hypothalamus and pituitary and the content of Lh protein in the serum. Assays of Kisspeptin-treated cells demonstrated that Kiss1-10 and Kiss2-10 can significantly promote the expression of Gnrh in hypothalamus cells and Lh and Fsh in pituitary cells of Dabry's sturgeon, indicating their direct-acting effect on pituitary cells and regulatory function on the reproductive development of sturgeon. This study described the reproductive function of the Kisspeptin in the Dabry's sturgeon for the first time, and provided supportive reference for the development of high-efficiency ripening technologies of artificially breeding sturgeon.

Neuropeptides and hormones in hypothalamus-pituitary axis of Chinese sturgeon (Acipenser sinensis).

The hypothalamus and pituitary serve as important neuroendocrine center, which is able to secrete a variety of neuropeptides and hormones to participate in the regulation of reproduction, growth, stress and feeding in fish. Chinese sturgeon is a basal vertebrate lineage fish with a special evolutionary status, but the information on its neuroendocrine system is relatively scarce. Using the transcriptome data on the hypothalamus-pituitary axis of Chinese sturgeon as reference, we found out 46 hypothalamus neuropeptide genes, which were involved in regulation of reproduction, growth, stress and feeding. The results of sequence alignment showed that the neuroendocrine system of Chinese sturgeon evolves slowly, which confirms that Chinese sturgeon is a species with a slow phenotypic evolution rate. In addition, we also isolated six pituitary hormones genes from Chinese sturgeon, including reproductive hormones: follicle-stimulating homone (FSH) and luteinizing hormone (LH), growth-related hormones: growth hormone (GH)/prolactin (PRL)/somatolactin (SL), and stress-related hormone gene: proopiomelanocortin (POMC). Similar to teleost, immunostaining localization analysis in Chinese sturgeon pituitary showed that LH and FSH were located in the pituitary proximal pars distalis, SL was located in the pituitary rostral pars distalis, and POMC was located in the pituitary pars intermedia and pituitary rostral pars distalis. This study will give a contribution to enrich our information on the neuroendocrine system in Chinese sturgeon.

Glycine Alleviated Intestinal Injury by Inhibiting Ferroptosis in Piglets Challenged with Diquat.

The purpose of this research was to examine the impact of glycine on intestinal injury caused by oxidative stress in piglets. A 2 × 2 factorial experiment with diets (basic diet vs. 1% glycine diet) and oxidative stress (saline vs. diquat) was conducted on 32 weanling piglets. On day 21, all piglets received an injection of either saline or diquat. After 7 days, all pigs were slaughtered and intestinal samples were collected. Dietary glycine supplementation improved intestinal mucosal morphology, increased the activities of disaccharidases and enhanced intestinal mucosal antioxidant capacity, while regulating the expression of ferroptosis mediators in the piglets under oxidative stress. These findings suggested that dietary glycine supplementation improved the morphology and function of the intestinal mucosa, which was involved in regulating antioxidant capacity and ferroptosis.

Lycopene alleviates multiple-mycotoxin-induced toxicity by inhibiting mitochondrial damage and ferroptosis in the mouse jejunum.

Multiple mycotoxins contamination in foods and feeds threatens human and animal health after they accumulate in the food chain, producing various toxic effects. The common mycotoxins contaimination in feeds are zearalenone (ZEN), deoxynivalenol (DON), and aflatoxin B1 (AFB1), but the effects of their co-exposure on the jejunum are not well understood. Lycopene (LYC) has been reported to have antioxidant activity that alleviates jejunal damage. In this study, we investigated the possible role of LYC as a treatment to mitigate the combined effects of ZEN, DON, and AFB1 on the jejunum of mice. Eighty male specific-pathogen-free ICR mice were randomly allocated to treatments with LYC (10 mg kg-1) and/or ZEN + DON + AFB1 (10 mg kg-1 ZEN, 1 mg kg-1 DON, and 0.5 mg kg-1 AFB1). The results indicated that LYC alleviated ZEN + DON + AFB1-induced jejunal injury by ameliorating the jejunal structural injury and increasing the villus height/crypt depth ratio and the levels of tight junction proteins (zonula occludens 1 [ZO1], occludin1 and claudin1) in the mouse jejunum. LYC also inhibited the oxidative stress induced by co-exposure to ZEN, DON, and AFB1 via reducing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and enhancing the total antioxidant capacity (T-AOC). LYC also alleviated jejunal mitochondrial damage in the ZEN + DON + AFB1-affected mice, evident as an increase in mitochondrial fission 1 (Fis1) transcription and a reduction in mitochondrial mitofusin 1 (Mfn1) and Mfn2 transcription. Co-exposure to ZEN, DON, and AFB1 also significantly increased the transcription of ferroptosis-related genes (transferrin receptor 1 (Tfr1), ferritin heavy chain 1 [Fth1], solute carrier family 3 member 2 [Slc3a2], and glutathione peroxidase 4 [Gpx4]), TFR1 and Fe2+ concentration. Notably, LYC potentially alleviated ZEN + DON + AFB1-induced jejunal ferroptosis. These results demonstrate that LYC alleviates ZEN + DON + AFB1-induced jejunal toxicity by inhibiting oxidative stress-mediated ferroptosis and mitochondrial damage in mice.

Necroptosis Contributes to LPS-Induced Activation of the Hypothalamic-Pituitary-Adrenal Axis in a Piglet Model.

Stressors cause activation of the hypothalamic-pituitary-adrenal (HPA) axis and a systemic inflammatory response. As a newly proposed cell death manner in recent years, necroptosis occurs in a variety of tissue damage and inflammation. However, the role of necroptosis in HPA axis activation remains to be elucidated. The aim of this study was to investigate the occurrence of necroptosis and its role in HPA activation in a porcine stress model induced by Escherichia coli lipopolysaccharide (LPS). Several typical stress behaviors like fever, anorexia, shivering and vomiting were observed in piglets after LPS injection. HPA axis was activated as shown by increased plasma cortisol concentration and mRNA expression of pituitary corticotropin-releasing hormone receptor 1 (CRHR1) and adrenal steroidogenic acute regulatory protein (StAR). The mRNA expression of tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß) and IL-6 in the hypothalamus, pituitary gland and adrenal gland was elevated by LPS, accompanied by the activation of necroptosis indicated by higher mRNA expression of necroptosis signals including receptor-interacting protein kinase (RIP) 1, RIP3, and phosphorylated mixed-lineage kinase domain-like protein (MLKL). Furthermore, necrostatin-1 (Nec-1), an inhibitor of necroptosis, inhibited necroptosis indicated by decreased mRNA levels of RIP1, RIP3, MLKL, and phosphoglycerate mutase family member 5 (PGAM5) in the hypothalamus, pituitary gland and adrenal gland. Nec-1 also decreased the mRNA expression of TNF-α and IL-ß and inhibited the activation of the HPA axis indicated by lower plasma cortisol concentration and mRNA expression of adrenal type 2 melanocortin receptor (MC2R) and StAR. These findings suggest that necroptosis is present and contributes to HPA axis activation induced by LPS. These findings provide a potential possibility for necroptosis as an intervention target for alleviating HPA axis activation and stress responses.

Polyphenols Sourced from Ilex latifolia Thunb. Relieve Intestinal Injury via Modulating Ferroptosis in Weanling Piglets under Oxidative Stress.

Polyphenols sourced from Ilex latifolia Thunb. (PIT) contain high levels of phenolic acids, tannic acids, triterpenoids and so on, which play important roles in antioxidant function. This study was conducted to investigate the effects of PIT against intestinal injury in piglets under oxidative stress. Thirty-two weanling piglets were arranged by a 2 × 2 factorial experiment with diets (basal diet vs. PIT diet) and oxidative stress (saline vs. diquat). All piglets were injected with saline or diquat on d 21, respectively. After 7 days, all pigs were slaughtered and intestinal samples were collected. PIT enhanced jejunal villus heights and crypt depth in the piglets under oxidative stress. PIT increased the activities of intestinal mucosal lactase, sucrase and maltase in the challenged piglets. PIT also increased the jejunal ratio of protein to DNA and ileal protein content. PIT increased the jejunal activities of GSH-PX and GSH content and reduced the ileal MDA amounts. Furthermore, PIT regulated the expression of ferroptosis mediators, such as TFR1, HSPB1, SLC7A11 and GPX4. These results indicate that dietary PIT supplementation enhances the histological structure and function of the intestinal mucosa, which is involved in modulating antioxidant capacity and ferroptosis.

Lysine-Specific Demethylase 1 in Energy Metabolism: A Novel Target for Obesity.

Obesity develops from an imbalance of energy homeostasis and is associated with the development of metabolic disorders, including insulin resistance and type 2 diabetes. Identification of the underlying molecular mechanisms and effective therapeutic approaches is highly needed. Lysine-specific demethylase 1 (LSD1), an flavin adenine dinucletide-dependent amine oxidase, is implicated in a wide variety of biological processes, including tumorigenesis, stem cell fate decisions, and embryonic development. Recent studies have suggested a vital role of LSD1 in regulating adaptive thermogenesis, mitochondrial biogenesis, glucose, and lipid metabolism. More recently, LSD1 activity was found to be regulated by nutrients, energy status, and hormonal signals, suggesting that it may act as a novel sensor for nutritional regulation of metabolic health. Here, we first discuss the effects of LSD1 on physiological phenotypes, including body weight, fat mass, body temperature, and glucose homeostasis. We also summarize recent understanding of the physiological roles and underlying mechanisms of LSD1 in controlling metabolic functions of adipose and other tissues. Hopefully, a better understanding of the roles of LSD1 in metabolic regulation may provide new perspectives for the nutritional prevention and treatment of obesity.

Natural Cyclooxygenase-2 Inhibitors Synergize With Butyrate to Augment Chicken Host Defense Peptide Gene Expression.

Enhancing the synthesis of microbicidal and immunomodulatory host defense peptides (HDP) is a promising host-directed antimicrobial strategy to combat a growing threat of antimicrobial resistance. Here we investigated the effect of several natural cyclooxygenase-2 (COX-2) inhibitors on chicken HDP gene regulation. Our results indicated that phenolic COX-2 inhibitors such as quercetin, resveratrol, epigallocatechin gallate, anacardic acid, and garcinol enhanced HDP gene expression in chicken HTC macrophage cell line and peripheral blood mononuclear cells (PBMCs). Moreover, these natural COX-2 inhibitors showed a strong synergy with butyrate in augmenting the expressions of multiple HDP genes in HTC cells and PBMCs. Additionally, quercetin and butyrate synergistically promoted the expressions of mucin-2 and claudin-1, two major genes involved in barrier function, while suppressing lipopolysaccharide-triggered interleukin-1ß expression in HTC macrophages. Mechanistically, we revealed that NF-κB, p38 mitogen-activated protein kinase, and cyclic adenosine monophosphate signaling pathways were all involved in the avian ß-defensin 9 gene induction, but histone H4 was not hyperacetylated in response to a combination of butyrate and quercetin. Because of their HDP-inducing, barrier-protective, and antiinflammatory activities, these natural COX-2 inhibitors, when combined with butyrate, may be developed as novel host-directed antimicrobial therapeutics.

Glutamate attenuates lipopolysaccharide induced intestinal barrier injury by regulating corticotropin-releasing factor pathway in weaned pigs.

OBJECTIVE: The purpose of this study was to evaluate the protection of glutamate (GLU) against the impairment in intestinal barrier function induced by lipopolysaccharide (LPS) stress in weaned pigs. METHODS: Twenty-four weaned pigs were divided into four treatments containing: i) non-challenged control, ii) LPS-challenged control, iii) LPS+1.0% GLU, and iv) LPS+2.0% GLU. On day 28, pigs were treated with LPS or saline. Blood samples were collected at 0, 2, and 4 h post-injection. After blood samples collection at 4 h, all pigs were slaughtered, and spleen, mesenteric lymph nodes, liver and intestinal samples were obtained. RESULTS: Dietary GLU supplementation inhibited the LPS-induced oxidative stress in pigs, as demonstrated by reduced malondialdehyde level and increased glutathione level in jejunum. Diets supplemented with GLU enhanced villus height, villus height/crypt depth and claudin-1 expression, attenuated intestinal histology and ultrastructure impairment induced by LPS. Moreover, GLU supplementation reversed intestinal intraepithelial lymphocyte number decrease and mast cell number increase induced by LPS stress. GLU reduced serum cortisol concentration at 4 h after LPS stress and downregulated the mRNA expression of intestinal corticotropin-releasing factor signal (corticotrophin-releasing factor [CRF], CRF receptor 1 [CRFR1], glucocorticoid receptor, tryptase, nerve growth factor, tyrosine kinase receptor A), and prevented mast cell activation. GLU upregulated the mRNA expression of intestinal transforming growth factor ß. CONCLUSION: These findings indicate that GLU attenuates LPS-induced intestinal mucosal barrier injury, which is associated with modulating CRF signaling pathway.

Glycine alleviated diquat-induced hepatic injury via inhibiting ferroptosis in weaned piglets.

OBJECTIVE: The beneficial effects of glycine were tested in piglets with diquat-induced hepatic injury. METHODS: Thirty-two piglets were assigned by a 2×2 factorial experimental design including glycine supplementation and diquat challenge. After 3 weeks of feeding with a basic diet or a 1% glycine supplemented diet, piglets were challenged with diquat or saline. After 1 week later, the piglets were slaughtered and samples were collected. RESULTS: Our results indicated that glycine alleviated diquat induced morphological hepatic injury, decreased the activities of plasma alanine aminotransferase, aspartate aminotransferase and glutamyl transpeptidase in the piglets under diquat challenge, and increased total antioxidant capacity and antioxidative enzyme activity significantly. Adding glycine enhanced the concentrations of hepatic adenosine triphosphate and adenosine diphosphate. Transmission electron microscope observation showed that diquat induced clear hepatocytes ferroptosis and its effect could be alleviated by glycine to a certain degree. Moreover, glycine significantly affected mRNA and protein expression of ferroptosis-related signals in the liver. CONCLUSION: These results demonstrated that glycine attenuated liver damage via inhibiting ferroptosis.