Circulating exosome-mediated AMPKα-SIRT1 pathway regulates lipid metabolism disorders in calf hepatocytes.

Subclinical ketosis (SCK) in dairy cows is often misdiagnosed because it lacks clinical signs and detection indicators. However, it is highly prevalent and may transform into clinical ketosis if not treated promptly. Due to the negative energy balance, a large amount of fat is mobilized, producing NEFA that exceeds the upper limit of liver processing, which in turn leads to the disturbance of liver lipid metabolism. The silent information regulator 1 (SIRT1) is closely related to hepatic lipid metabolism disorders. Exosomes as signal transmitters, also play a role in the circulatory system. We hypothesize that the circulating exosome-mediated adenosine 5'-monophosphate (AMP)-activated protein kinase alpha (AMPKα)-SIRT1 pathway regulates lipid metabolism disorders in SCK cows. We extracted the exosomes required for the experiment from the peripheral circulating blood of non-ketotic (NK) and SCK cows. We investigated the effect of circulating exosomes on the expression levels of mRNA and protein of the AMPKα-SIRT1 pathway in non-esterified fatty acid (NEFA)-induced dairy cow primary hepatocytes using in vitro cell experiments. The results showed that circulating exosomes increased the expression levels of Lipolysis-related genes and proteins (AMPKα, SIRT1, and PGC-1α) in hepatocytes treated with 1.2 mM NEFA, and inhibited the expression of lipid synthesis-related genes and protein (SREBP-1C). The regulation of exosomes on lipid metabolism disorders caused by 1.2 mM NEFA treatment showed the same trend as for SIRT1-overexpressing adenovirus. The added exosomes could regulate NEFA-induced lipid metabolism in hepatocytes by mediating the AMPKα-SIRT1 pathway, consistent with the effect of transfected SIRT1 adenovirus.

Combination of Zearalenone and Deoxynivalenol Induces Apoptosis by Mitochondrial Pathway in Piglet Sertoli Cells: Role of Endoplasmic Reticulum Stress.

Zearalenone (ZEA) and deoxynivalenol (DON) are widely found in various feeds, which harms livestock's reproductive health. Both mitochondria and endoplasmic reticulum (ER) can regulate cell apoptosis. This study aimed to explore the regulatory mechanism of endoplasmic reticulum stress (ERS) on ZEA- combined with DON-induced mitochondrial pathway apoptosis in piglet Sertoli cells (SCs). The results showed that ZEA + DON damaged the ultrastructure of the cells, induced apoptosis, decreased mitochondrial membrane potential, promoted the expression of cytochrome c (CytC), and decreased the cell survival rate. Furthermore, ZEA + DON increased the relative mRNA and protein expression of Bid, Caspase-3, Drp1, and P53, while that of Bcl-2 and Mfn2 declined. ZEA + DON was added after pretreatment with 4-phenylbutyric acid (4-PBA). The results showed that 4-PBA could alleviate the toxicity of ZEA + DON toward SCs. Compared with the ZEA + DON group, 4-PBA improved the cell survival rate, decreased the apoptosis rate, inhibited CytC expression, and increased mitochondrial membrane potential, and the damage to the cell ultrastructure was alleviated. Moreover, after pretreatment with 4-PBA, the relative mRNA and protein expression of Bid, Caspase-3, Drp1, and P53 were downregulated, while the relative mRNA and protein expression of Bcl-2 and Mfn2 were upregulated. It can be concluded that ERS plays an important part in the apoptosis of SCs co-infected with ZEA-DON through the mitochondrial apoptosis pathway, and intervention in this process can provide a new way to alleviate the reproductive toxicity of mycotoxins.

Zearalenone induces mitochondria-associated endoplasmic reticulum membranes dysfunction in piglet Sertoli cells based on endoplasmic reticulum stress.

Zearalenone (ZEA) is an estrogen-like mycotoxin, which mainly led to reproductive toxicity. The study aimed to investigate the molecular mechanism of ZEA-induced dysfunction of mitochondria-associated endoplasmic reticulum membranes (MAM) in piglet Sertoli cells (SCs) via the endoplasmic reticulum stress (ERS) pathway. In this study, SCs were used as a research object that was exposed to ZEA, and ERS inhibitor 4-Phenylbutyrate acid (4-PBA) was used as a reference. The results showed that ZEA damaged cell viability and increased Ca2+ levels; damaged the structure of MAM; up-regulated the relative mRNA and protein expression of glucose-regulated protein 75 (Grp75) and mitochondrial Rho-GTPase 1 (Miro1), while inositol 1,4,5-trisphosphate receptor (IP3R), voltage-dependent anion channel 1 (VDAC1), mitofusin2 (Mfn2) and phosphofurin acidic cluster protein 2 (PACS2) were down-regulated. After a 3 h 4-PBA-pretreatment, ZEA was added for mixed culture. The results of 4-PBA pretreatment showed that inhibition of ERS reduced the cytotoxicity of ZEA against piglet SCs. Compared with the ZEA group, inhibition of ERS increased cell viability and decreased Ca2+ levels; restored the structural damage of MAM; down-regulated the relative mRNA and protein expression of Grp75 and Miro1; and up-regulated the relative mRNA and protein expression of IP3R, VDAC1, Mfn2, and PACS2. In conclusion, ZEA can induce MAM dysfunction in piglet SCs via the ERS pathway, whereas ER can regulate mitochondria through MAM.

Protective effects of Pudilan Tablets against osteoarthritis in mice induced by monosodium iodoacetate.

Osteoarthritis (OA) is a complicated disorder that is the most prevalent chronic degenerative joint disease nowadays. Pudilan Tablets (PDL) is a prominent traditional Chinese medicine formula used in clinical settings to treat chronic inflammatory illnesses. However, there is currently minimal fundamental research on PDL in the therapy of joint diseases. As a result, this study looked at the anti-inflammatory and anti-OA properties of PDL in vitro and in vivo, as well as the mechanism of PDL in the treatment of OA. We investigated the anti-OA properties of PDL in OA mice that were generated by monosodium iodoacetate (MIA). All animals were administered PDL (2 g/kg or 4 g/kg) or the positive control drug, indomethacin (150 mg/kg), once daily for a total of 28 days starting on the day of MIA injection. The CCK-8 assay was used to test the vitality of PDL-treated RAW264.7 cells in vitro. RAW264.7 cells that had been activated with lipopolysaccharide (LPS) were used to assess the anti-inflammatory properties of PDL. In the MIA-induced OA model mice, PDL reduced pain, decreased OA-induced cartilage damages and degradation, decreased production of pro-inflammatory cytokines in serum, and suppressed IL-1ß, IL-6, and TNF-α mRNA expression levels in tibiofemoral joint. In RAW264.7 cells, PDL treatment prevented LPS-induced activation of the ERK/Akt signaling pathway and significantly decreased the levels of inflammatory cytokines, such as IL-1ß, IL-6, and TNF-α. In conclusion, these results suggest that PDL is involved in combating the development and progression of OA, exerts a powerful anti-inflammatory effect on the knee joint, and may be a promising candidate for the treatment of OA.

Zearalenone Induces Apoptosis in Porcine Endometrial Stromal Cells through JNK Signaling Pathway Based on Endoplasmic Reticulum Stress.

Zearalenone (ZEA) is an estrogen-like mycotoxin characterized mainly by reproductive toxicity, to which pigs are particularly sensitive. The aim of this study was to investigate the molecular mechanism of ZEA-induced apoptosis in porcine endometrial stromal cells (ESCs) by activating the JNK signaling pathway through endoplasmic reticulum stress (ERS). In this study, ESCs were exposed to ZEA, with the ERS inhibitor sodium 4-Phenylbutyrate (4-PBA) as a reference. The results showed that ZEA could damage cell structures, induce endoplasmic reticulum swelling and fragmentation, and decreased the ratio of live cells to dead cells significantly. In addition, ZEA could increase reactive oxygen species and Ca2+ levels; upregulate the expression of GRP78, CHOP, PERK, ASK1 and JNK; activate JNK phosphorylation and its high expression in the nucleus; upregulate the expression Caspase 3 and Caspase 9; and increase the Bax/Bcl-2 ratio, resulting in increased apoptosis. After 3 h of 4-PBA-pretreatment, ZEA was added for mixed culture, which showed that the inhibition of ERS could reduce the cytotoxicity of ZEA toward ESCs. Compared with the ZEA group, ERS inhibition increased cell viability; downregulated the expression of GRP78, CHOP, PERK, ASK1 and JNK; and decreased the nuclear level of p-JNK. The Bax/Bcl-2 ratio and the expression of Caspase 3 and Caspase 9 were downregulated, significantly alleviating apoptosis. These results demonstrate that ZEA can alter the morphology of ESCs, destroy their ultrastructure, and activate the JNK signaling via the ERS pathway, leading to apoptosis.

11S Glycinin Up-Regulated NLRP-3-Induced Pyroptosis by Triggering Reactive Oxygen Species in Porcine Intestinal Epithelial Cells.

11S glycinin is a major soybean antigenic protein, which induces human and animal allergies. It has been reported to induce intestinal porcine epithelial (IPEC-J2) cell apoptosis, but the role of pyroptosis in 11S glycinin allergies remains unknown. In this study, IPEC-J2 cells were used as an in vitro physiological model to explore the mechanism of 11S glycinin-induced pyroptosis. The cells were incubated with 0, 1, 5, and 10 mg·ml-1 11S glycinin for 24 h. Our results revealed that 11S glycinin significantly inhibited cell proliferation, induced DNA damage, generated active oxygen, decreased mitochondrial membrane potential, and increased the NOD-like receptor protein 3 (NLRP-3) expression of IPEC-J2 cells in a dose-dependent manner. Further, IPEC-J2 cells were transfected with designed sh-NLRP-3 lentivirus to silence NLRP-3. The results showed that 11S glycinin up-regulated the silenced NLRP-3 gene and increased the expression levels of apoptosis-related spot-like protein (ASC), caspase-1, the cleaved gasdermin D, and interleukin-1ß. The IPEC-J2 cells showed pyrolysis morphology. Moreover, we revealed that N-acetyl-L-cysteine can significantly inhibit the production of reactive oxygen species and reduce the expression levels of NLRP-3 and the cleaved gasdermin D. Taken together, 11S glycinin up-regulated NLRP-3-induced pyroptosis by triggering reactive oxygen species in IPEC-J2 cells.

Rutin Supplementation Reduces Oxidative Stress, Inflammation and Apoptosis of Mammary Gland in Sheep During the Transition Period.

Rutin, a common dietary flavonoid, exhibits remarkable pharmacological activities such as antioxidant and anti-inflammatory functions. Metabolic stress in mammals during the transition period affects mammary gland health. The aim of this experiment was to evaluate the protective effect of rutin supplementing against metabolic stress in the mammary glands of sheep during the transition period, particularly after parturition. Transition Hu sheep (2-3 years old with 62.90 ± 2.80 kg) were randomly divided into three groups, the control group was fed a diet without rutin, while rutin (50 and 100 mg/kg body weight/day) was administered to the two treatment groups (-28 day to +28 day relative to parturition). Serum and blood samples were collected from jugular vein on days -14, -7, +1, +2, +7, +14, +21, +28 relative to parturition. Mammary tissue biopsy samples of four sheep from the treatment group were harvested on day +28 postpartum. Compared to that in the control group, rutin supplementation resulted in lower ß-hydroxybutyrate (BHBA) while increasing the concentrations of non-esterified fatty acids (NEFA) and globulin after lactation. Furthermore, rutin treatment led to lower hydrogen peroxide (H2O2) and malonaldehyde (MDA) levels, resulting in increased catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and total antioxidant potential (T-AOC). Compared to that in the control group, rutin inhibits the mRNA expression of inflammatory markers such as tumor necrosis factor-α (TNF-α). In addition, rutin markedly downregulated the ratio of phosphorylated NF-κB p65 (p-p65) to total NF-κB p65 (p65). Meanwhile, rutin supplementation resulted in high mRNA abundance of the nuclear factor erythroid 2-like 2 (NFE2L2, formerly NRF2) and its target gene, heme oxygenase-1 (HO-1), which plays critical roles in maintaining the redox balance of the mammary gland. Furthermore, rutin treatment lowered the levels of various downstream apoptotic markers, including Bax, caspase3 and caspase9, while upregulating anti-apoptotic Bcl-2 protein. These data indicate the positive effect of rutin against inflammation, oxidative stress status, and anti-apoptotic activity in the mammary gland. The mechanism underlying these responses merits further study.

Lycopene attenuates zearalenone-induced oxidative damage of piglet sertoli cells through the nuclear factor erythroid-2 related factor 2 signaling pathway.

Zearalenone (ZEA) has an estrogenic effect and often causes reproductive damage. Pigs are particularly sensitive to it. Lycopene (LYC) is a type of fat-soluble natural carotenoid that has antioxidant, anti-inflammatory, anti-cancer, anti-cardiovascular and detoxifying effects. In this study, piglet sertoli cells (SCs) were used as research objects to investigate the mechanism of ZEA induced damage to piglet SCs and to evaluate the protective effect of LYC on ZEA induced toxic damage to piglet SCs. The results showed that ZEA damaged the cell structure and inhibited the expression of nuclear factor erythroid-2 related factor 2 (Nrf2) in the nucleus, which down-regulated the relative mRNA expression of heme oxygenase 1 (HO-1) and glutathione peroxidase 1 (GPX1) and decreased the activity of HO-1, glutathione peroxidase (GSH-Px) and total superoxide dismutase (T-SOD), resulting in an increase in malondialdehyde (MDA) and reactive oxygen species (ROS) content. ZEA downregulated the relative mRNA and protein expression of bcl-2 in piglet SCs, promoted cell apoptosis, and upregulated the relative mRNA and protein expression of LC3, beclin-1, and bax. After 3 h LYC-pretreatment, ZEA was added for mixed culture. The results of pretreatment with LYC showed that LYC could alleviate the cytotoxicity of ZEA to porlets SCs. Compared with ZEA group, improved the cell survival rate, promoted the expression of Nrf2 in the nucleus, upregulated the relative mRNA expression of HO-1 and GPX1, increased the activity of antioxidant enzymes, and reduced the levels of MDA and ROS. Moreover, after pretreatment with LYC, the mRNA expression of bcl-2 was upregulated, the apoptosis rate was decreased, the relative mRNA and protein expressions of LC3, beclin-1 and bax were downregulated, and autophagy was alleviated. In conclusion, LYC alleviated the oxidative damage of SCs caused by ZEA by promoting the expression of Nrf2 pathway and decreased autophagy and apoptosis.

N-acetylcysteine ameliorate cytotoxic injury in piglets sertoli cells induced by zearalenone and deoxynivalenol.

Zearalenone (ZEA) and Deoxynivalenol (DON) are two mycotoxins highly detected in agricultural products and feed. Both mycotoxins produce reproductive toxicity and pose a serious threat to human and animal health, among which pigs are the most sensitive animals. Sertoli cells (SCs) play an important role in spermatogenesis; however, the combined toxicity of ZEA and DON and the screening of effective protective agents remains to be determined. By studying the effects of N-acetylcysteine (NAC) on the cells exposed to 20 µM of ZEA and 0.6 µM of DON, we explored the protective mechanism of NAC (4 mM) on the cytotoxic injury of piglets SCs induced by both mycotoxins. The results showed that the combination of ZEA and DON destroy organelles and SCs structures, NAC significantly alleviates the damage caused by ZEA and DON. NAC also significantly increased the expression and distribution of zonula occludens 1 (ZO-1), decreased the relative mRNA and protein expression levels of Bax, Bid, caspase-3, and caspase-9, and increased Bcl-2 expression level and inhibited the decrease of mitochondrial membrane potential. Further, NAC also eases the cell cycle arrest and oxidative stress caused by ZEA and DON. In summary, our results show that NAC could alleviate SCs injury via reducing the oxidative damage and apoptosis caused by ZEA and DON.

Deoxynivalenol Induces Caspase-8-Mediated Apoptosis through the Mitochondrial Pathway in Hippocampal Nerve Cells of Piglet.

Deoxynivalenol (DON) is a common trichothecene mycotoxin found worldwide. DON has broad toxicity towards animals and humans. However, the mechanism of DON-induced neurotoxicity in vitro has not been fully understood. This study investigated the hypothesis that DON toxicity in neurons occurs via the mitochondrial apoptotic pathway. Using piglet hippocampal nerve cells (PHNCs), we evaluated the effects of different concentrations of DON on typical indicators of apoptosis. The obtained results demonstrated that DON treatment inhibited PHNC proliferation and led to morphological, biochemical, and transcriptional changes consistent with apoptosis, including decreased mitochondrial membrane potential, mitochondrial release of cytochrome C (CYCS) and apoptosis inducing factor (AIF), and increased abundance of active cleaved-caspase-9 and cleaved-caspase-3. Increasing concentrations of DON led to decreased B-cell lymphoma-2 (Bcl-2) expression and increased expression of BCL2-associated X (Bax) and B-cell lymphoma-2 homology 3 interacting domain death agonist (Bid), which in turn increased transcriptional activity of the transcription factors AIF and P53 (a tumor suppressor gene, promotes apoptosis). The addition of a caspase-8 inhibitor abrogated these effects. These results reveal that DON induces apoptosis in PHNCs via the mitochondrial apoptosis pathway, and caspase-8 is shown to play an important role during apoptosis regulation.

Tea polyphenols attenuate liver inflammation by modulating obesity-related genes and down-regulating COX-2 and iNOS expression in high fat-fed dogs.

BACKGROUND: Tea polyphenols (TPs) attenuate obesity related liver inflammation; however, the anti-obesity effects and anti-inflammatory mechanisms are not clearly understood. This study aimed to determine whether the anti-obesity and anti-inflammatory TPs mechanisms associated with cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression levels, and obesity-related gene response in dogs. RESULTS: Dogs fed TPs displayed significantly decreased (p < 0.01) mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1ß), and interleukin-6 (IL-6) compared to dogs that consumed high-fat diet (HFD) alone. TPs significantly (p < 0.01) inhibited COX-2 and iNOS expression level, and decreased liver fat content and degeneration. CONCLUSION: These results suggested that TPs act as a therapeutic agent for obesity, liver inflammation, and fat degeneration via COX-2 and iNOS inhibition, with TNF-α, IL-1ß, and IL-6 involvement.

Non-esterified Fatty Acid Induce Dairy Cow Hepatocytes Apoptosis via the Mitochondria-Mediated ROS-JNK/ERK Signaling Pathway.

Elevated plasma non-esterified fatty acid (NEFA) levels and hepatocytes damage are characteristics of ketosis in dairy cows. Oxidative stress is associated with the pathogenesis of NEFA-induced liver damage. However, the exact mechanism by which oxidative stress mediates NEFA-induced hepatocytes apoptosis and liver injury remains poorly understood. The results of the present study demonstrated that NEFA contribute to reactive oxygen species (ROS) generation, resulting in an imbalance between oxidative and antioxidant species, transcriptional activation of p53, transcriptional inhibition of nuclear factor E2-related factor 2 (Nrf2), loss of mitochondria membrane potential (MMP) and release of apoptosis-inducing factor (AIF) and cytochrome c (cyt c) into the cytosol, leading to hepatocytes apoptosis. Besides, NEFA triggered apoptosis in dairy cow hepatocytes via the regulation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), Bcl-2-associated X protein (Bax), B-cell lymphoma gene 2 (Bcl-2), caspase 9 and poly (ADP-ribose) polymerase (PARP). Pretreatment with the inhibitor SP600125 or PD98059 or the antioxidant N-acetylcysteine (NAC) revealed that NEFA-ROS-JNK/ERK-mediated mitochondrial signaling pathway plays a crucial role in NEFA-induced hepatocytes apoptosis. Moreover, the results suggested that the transcription factors p53 and Nrf2 function downstream of this NEFA-ROS-JNK/ERK pathway and are involved in NEFA-induced hepatocytes apoptosis. In conclusion, these findings indicate that the NEFA-ROS-JNK/ERK-mediated mitochondrial pathway plays an important role in NEFA-induced dairy cow hepatocytes apoptosis and strongly suggests that the inhibitors SP600125 and PD98059 and the antioxidant NAC may be developed as therapeutics to prevent hyperlipidemia-induced apoptotic damage in ketotic dairy cows.

The role and regulatory mechanism of autophagy in hippocampal nerve cells of piglet damaged by deoxynivalenol.

Deoxynivalenol (DON), a type B trichothecene mycotoxin mainly affects the health status of pigs and reduced their growth. This study aimed to determine the effects of PI3K/Akt/mTOR pathway on DON-induced autophagy of piglet hippocampal nerve cells (PHNCs), and the relationship between autophagy and apoptosis. The effects of DON on autophagy of PHNCs were examined by cell morphology, cell viability, apoptosis rate, electron microscopy, transient transfection of GFP-LC3 plasmid, immunofluorescence and expression of autophagy-related genes and proteins. The relationship between autophagy and cell apoptosis was analyzed by western blotting, CCK-8 and flow cytometry. The results indicated that, DON inhibited the proliferation of PHNCs and significantly changed cell morphology, and induced apoptosis and autophagy. The expression levels of LC3 protein and gene increased, while the expression levels of PI3K/Akt/mTOR pathway-related genes and proteins decreased, when the concentration of DON increased. Activation of autophagy significantly increased cell viability, reduced apoptosis rate, inhibits autophagy significantly, reduced cell activity and increased apoptosis rate. This data demonstrated that DON exerts certain toxic effect on PHNCs, induced apoptosis and autophagy. PI3K/Akt/mTOR signaling pathway plays a negative regulatory role in DON-induced autophagy of PHNCs. At the same time, autophagy plays a protective role in DON-induced PHNCs injury.

Green tea polyphenols decrease weight gain, ameliorate alteration of gut microbiota, and mitigate intestinal inflammation in canines with high-fat-diet-induced obesity.

Green tea polyphenols (GTPs) exhibit beneficial effects towards obesity and intestinal inflammation; however, the mechanisms and association with gut microbiota are unclear. We examined the role of the gut microbiota of GTPs treatment for obesity and inflammation. Canines were fed either a normal diet or high-fat diet with low (0.48% g/kg), medium (0.96% g/kg), or high (1.92% g/kg), doses of GTPs for 18 weeks. GTPs decreased the relative abundance of Bacteroidetes and Fusobacteria and increased the relative abundance of Firmicutes as revealed by 16S rRNA gene sequencing analysis. The relative proportion of Acidaminococcus, Anaerobiospirillum, Anaerovibrio, Bacteroides, Blautia, Catenibactetium, Citrobacter, Clostridium, Collinsella, and Escherichia were significantly associated with GTPs-induced weight loss. GTPs significantly (P<.01) decreased expression levels of inflammatory cytokines, including TNF-α, IL-6, and IL-1ß, and inhibited induction of the TLR4 signaling pathway compared with high-fat diet. We show that the therapeutic effects of GTPs correspond with changes in gut microbiota and intestinal inflammation, which may be related to the anti-inflammatory and anti-obesity mechanisms of GTPs.

Autophagy protects PC12 cells against deoxynivalenol toxicity via the Class III PI3K/beclin 1/Bcl-2 pathway.

Deoxynivalenol (DON) is a major mycotoxin from the trichothecene family of mycotoxins produced by Fusarium fungi. It can cause a variety of adverse effects on human and farm animal health. Here, we determined the effect of DON on the Class III phosphatidylinositol 3-kinase (PIK3C3)/beclin 1/B cell lymphoma-2 (Bcl-2) pathway in PC12 cells and the relationship between autophagy and apoptosis. The effects of DON were evaluated based on the apoptosis ratio; the typical indicators of autophagy, including cellular morphology, acridine orange- and monodansylcadaverine-labeled vacuoles, green fluorescent protein-microtubule associated protein 1 light chain 3 (LC3) localization, and LC3 immunofluorescence; and the expression of key autophagy-related genes and proteins, that is, PIK3C3, beclin 1, Bcl-2, LC3, and p62. The relationship between autophagy and apoptosis was analyzed by western blot analysis and flow cytometry. DON-induced PC12 cell morphological changes and autophagy significantly. PIK3C3, beclin 1, and LC3 increased in tandem with the DON concentration used; Bcl-2 and p62 expression decreased as DON concentrations increased. Moreover, the PIK3C3/beclin 1/Bcl-2 signaling pathway played a role in DON-induced autophagy. Our findings suggest that DON can induce autophagy by activating the PIK3C3/beclin 1/Bcl-2 signaling pathway and that autophagy may play a positive role in reducing DON-induced apoptosis.

Deoxynivalenol Induces Intestinal Damage and Inflammatory Response through the Nuclear Factor-κB Signaling Pathway in Piglets.

Deoxynivalenol (DON) is highly toxic to animals and humans, but pigs are most sensitive to it. The porcine mucosal injury related mechanism of DON is not yet fully clarified. Here, we investigated DON-induced injury in the intestinal tissues of piglet. Thirty weanling piglets [(Duroc × Landrace) × Yorkshire] were randomly divided into three groups according to single factor experimental design (10 piglets each group). Piglets were fed a basal diet in the control group, while low and high dose groups were fed a DON diet (1300 and 2200 µg/kg, respectively) for 60 days. Scanning electron microscopy results indicated that the ultrastructure of intestinal epithelial cells in the DON-treated group was damaged. The distribution and optical density (OD) values of zonula occludens 1 (ZO-1) protein in the intestinal tissues of DON-treated groups were decreased. At higher DON dosage, interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α mRNA levels were elevated in the intestinal tissues. The mRNA and protein levels of NF-κB p65, IκB-α, IKKα/ß, iNOS, and COX-2 in the small intestinal mucosa were abnormally altered with an increase in DON concentration. These results indicate that DON can persuade intestinal damage and inflammatory responses in piglets via the nuclear factor-κB signaling pathway.

ß-Conglycinin-Induced Intestinal Porcine Epithelial Cell Damage via the Nuclear Factor κB/Mitogen-Activated Protein Kinase Signaling Pathway.

Soybean allergy is a serious health risk to humans and animals; ß-conglycinin is the primary antigenic protein in soybean. Intestinal porcine epithelial (IPEC-J2) cells were used as an in vitro physiological model of the intestinal epithelium to study the effects of different concentrations of soybean antigen protein ß-conglycinin to identify the involved signaling pathways. The cells were divided into eight groups and either untreated or treated with different concentrations of ß-conglycinin, pyrrolidine dithiocarbamate (PDTC), Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), SP600125, and SB202190 either alone or in combination. The cells were incubated with 1, 5, and 10 mg·mL-1 ß-conglycinin or 5 mg·mL-1 ß-conglycinin and 1 µmol·L-1 nuclear factor κB (NF-κB) inhibitor (PDTC), inducible nitric oxide synthase inhibitor (l-NAME), c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and p38 inhibitor (SB202190) for 24 h, separately; controls were left untreated. The mRNA, protein, and phosphorylation levels of NF-κB, p38, and JNK were higher in the treated groups than in the control group. ß-Conglycinin decreased tight junction distribution, destroyed the cytoskeleton of IPEC-J2 cells, and caused cell death. After the addition of the inhibitors, ß-conglycinin-induced IPEC-J2 cell damage was significantly reduced. ß-Conglycinin caused damage to IPEC-J2 cells via the mitogen-activated protein kinase/NF-κB signaling pathway. The results of this study are crucial for exploring the mechanisms underlying allergic reactions caused by soybean antigen proteins.

Uric acid transporters BCRP and MRP4 involved in chickens uric acid excretion.

BACKGROUND: Breast cancer resistance protein (BCRP) and multidrug resistance protein 4 (MRP4) are involved in uric acid excretion in humans and mice. Despite evidence suggesting that renal proximal tubular epithelial cells participate in uric acid excretion in chickens, the roles of BCRP and MRP4 therein remain unclear. This study evaluated the relationship between BCRP and MRP4 expression and renal function in chickens. RESULTS: Sixty laying hens were randomly divided into four treatment groups: a control group (NC) fed a basal diet; a sulfonamide-treated group (SD) fed the basal diet and supplemented with sulfamonomethoxine sodium via drinking water (8 mg/L); a fish meal group (FM) fed the basal diet supplemented with 16% fishmeal; and a uric acid injection group (IU) fed the basal diet and intraperitoneally injected with uric acid (250 mg/kg body weight). The results showed that serum uric acid, creatinine, and blood urea nitrogen levels were significantly higher in the SD and IU, but not FM, than in the NC groups. Renal tubular epithelial cells in the SD and IU groups were damaged. Liver BCRP and MRP4 mRNA and protein levels were significantly decreased in the SD and IU groups, but slightly increased in the FM group. In the SD group, BCRP and MRP4 were significantly increased in the ileum and slightly increased in the kidney. In the FM group, BCRP and MRP4 were significantly increased in the kidney and slightly increased in the ileum. In the IU group, BCRP and MRP4 were significantly increased in the kidney and ileum. BCRP and MRP4 expression in the jejunum was not affected by the treatments. CONCLUSION: Together, these results demonstrate that BCRP and MRP4 are involved in renal and intestinal uric acid excretion in chickens and that BCRP is positively related to MRP4 expression. Further, impairment of renal function results in an increase in serum uric acid as well as a compensatory increase in BCRP and MRP4 in the ileum; however, under normal renal function, renal BCRP and MRP4 are the main regulators of uric acid excretion.

Rutin protects against lipopolysaccharide-induced mastitis by inhibiting the activation of the NF-κB signaling pathway and attenuating endoplasmic reticulum stress.

Rutin, found widely in traditional Chinese medicine materials, is used to treat eye swelling and pain, hypertension, and hyperlipidemia. In the present study, a mouse mastitis model induced by lipopolysaccharide (LPS) was established to explore rutin's inhibitory mechanism on mastitis via nuclear factor kappa B (NF-κB) inflammatory signaling and the relationship between NF-κB signaling and endoplasmic reticulum (ER) stress. Mice were divided into six groups: Control group, LPS model group, LPS + rutin (25, 50, and 100 mg/kg) and LPS + dexamethasone (DEX) group. DEX, rutin, and PBS (control and LPS groups) were administered 1 h before and 12 h after perfusion of LPS. After LPS stimulation for 24 h, to evaluate rutin's therapeutic effect on mastitis, the mammary tissues of each group were collected to detect histopathological injury, tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, and IL-6 mRNA and protein levels; and glucose-regulated protein, 78 kDa (GRP78) protein levels. The protein and mRNA levels of TNF-α, IL-1ß, and IL-6 in the LPS + rutin group were significantly lower than those in the LPS model group. Similarly, p50/p105, phosphorylated (p)-p65/p65 and p-inhibitor of nuclear factor kappa b kinase subunit beta (p-IKKß)/IKKß ratios in the LPS + rutin group (50 mg/kg) and LPS + rutin group (100 mg/kg) decreased significantly. GRP78 protein expression was significantly higher in LPS + rutin group (100 mg/kg). The structure of mammary tissue became gradually more intact and vacuolization of acini decreased as the rutin concentration increased. The nuclear quantity of p65 in the LPS + rutin group decreased significantly in a rutin dose-dependent manner. Rutin had an anti-inflammatory effect in the LPS-induced mouse mastitis model, manifested by inhibition of NF-κB pathway activation and attenuation of ER stress.

Glucagon attenuates lipid accumulation in cow hepatocytes through AMPK signaling pathway activation.

The ketotic cows displayed hepatic lipid metabolic disorder and high blood concentration of glucagon. Importantly, adenosine monophosphate-activated protein kinase (AMPK) signaling pathway plays an important role in the hepatic lipid homeostasis. Therefore, the aim of this study was to investigate the effect of glucagon on AMPK pathway and its underlying mechanism on lipid metabolism in cow hepatocytes. Cow hepatocytes were cultured and treated with glucagon and AMPK inhibitor (BML-275). The results showed that glucagon significantly promoted the expression of glucagon receptor and increased the phosphorylation level and activity of AMPKα. Activated AMPKα increased the expression level and transcriptional activity of peroxisome proliferator-activated receptor α, which further increased the expression of fatty acid oxidation genes and lipid oxidation. Furthermore, activated AMPKα inhibited the expression level and transcriptional activity of sterol regulatory element binding protein-1c and carbohydrate response element binding protein, which decreased the expression of lipogenic genes, thereby decreasing lipid synthesis. In addition, glucagon also increased the expression of very-low-density lipoprotein (VLDL) assembly to export intracellular triglycerides (TG). Consequently, the content of intracellular TG was significantly decreased in cow hepatocytes. These results indicate that glucagon activates the AMPK signaling pathway to increase lipid oxidation and VLDL assembly and decrease lipid synthesis in cow hepatocytes, thereby reducing liver fat accumulation.