Keap1 Expression Research Articles

Milk-derived exosomes carrying siRNA-KEAP1 promote diabetic wound healing by improving oxidative stress.

Diabetic wounds are a serious complication of diabetes mellitus (DM) that can lead to persistent infection, amputation, and even death. Prolonged oxidative stress has been widely recognized as a major instigator in the development of diabetic wounds; therefore, oxidative stress is considered a promising therapeutic target. In the present study, Keap1/Nrf2 signaling was confirmed to be activated in streptozotocin (STZ)-induced diabetic mice and methylglyoxal (MGO)-treated human umbilical vein endothelial cells (HUVECs). Knockdown of Keap1 by siRNA reversed the increase in Keap1 levels, promoted the nuclear translocation of Nrf2, and increased the expression of HO-1, an antioxidant protein. To explore therapeutic delivery strategies, milk-derived exosomes (mEXOs) were developed as a novel, efficient, and non-toxic siRNA carrier. SiRNA-Keap1 (siKeap1) was loaded into mEXOs by sonication, and the obtained mEXOs-siKeap1 were found to promote HUVEC proliferation and migration while relieving oxidative stress in MGO-treated HUVECs. Meanwhile, in a mouse model of diabetic wounds, injection of mEXOs-siKeap1 significantly accelerated diabetic wound healing with enhanced collagen formation and neovascularization. Taken together, these data support the development of Keap1 knockdown as a potential therapeutic strategy for diabetic wounds and demonstrated the feasibility of mEXOs as a scalable, biocompatible, and cost-effective siRNA delivery system. The therapeutic effect of siKeap1-loaded mEXOs on diabetic wound healing was assessed. First, we found that the expression of Keap1 was upregulated in the wounds of diabetic mice and in human umbilical vein endothelial cells (HUVECs) pretreated with methylglyoxal (MGO). Next, we extracted exosomes from raw milk by differential centrifugation and loaded siKeap1 into milk-derived exosomes by sonication. The in vitro application of the synthetic complex (mEXOs-siKeap1) was found to increase the nuclear localization of Nrf2 and the expression of the antioxidant protein HO-1, thus reversing oxidative stress. Furthermore, in vivo mEXOs-siKeap1 administration significantly accelerated the healing rate of diabetic wounds (Scheme 1). Scheme 1 Schematic diagram. A Synthesis of mEXOs-siKeap1 complex. B Mechanism of mEXOs-siKeap1 in vitro. C The treatment effect of mEXOs-siKeap1 on an in vivo mouse model of diabetic wounds.

Astragaloside IV mitigates cerebral ischaemia-reperfusion injury via inhibition of P62/Keap1/Nrf2 pathway-mediated ferroptosis

Cerebral ischaemia-reperfusion injury (CIRI) is a critical component of ischaemic stroke pathogenesis. Ferroptosis contributes to and aggravates CIRI, whereas the P62/Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2) pathway exerts neuroprotective effects. Astragaloside IV (AST IV) is the primary active ingredient of Astragalus, an herb with anti-CIRI properties used in traditional Chinese medicine. However, the mechanism of its anti-CIRI action is unclear. This study examined the mechanisms underlying the anti-CIRI action of AST IV using a combination of in vitro and in vivo approaches. We established an erastin-induced ferroptosis model, oxygen and glucose deprivation/reoxygenation (OGD/R)-induced model in SH-SY5Y cells, and middle cerebral artery occlusion-reperfusion (MCAO/R) model using Sprague–Dawley rats. The extent of cell damage and brain damage in rats, ferroptosis indicator changes, and expression of P62, Keap1, and Nrf2 were investigated. AST IV inhibited erastin-induced ferroptosis, attenuated OGD/R-induced cell damage, and ameliorated sensorimotor dysfunction and injury in the MCAO/R model. Further, AST IV promoted Nrf2 activation, inhibited ferroptosis, and reduced cell damage. Notably, these effects were inhibited by ML385, an Nrf2 inhibitor. AST IV increased the P62 and Nrf2 levels and decreased the Keap1 levels. P62 silencing reduced the effects of AST IV on the P62/Keap1/Nrf2 pathway and ferroptosis. Our findings suggest that AST IV mitigates CIRI by inhibiting ferroptosis via activation of the P62/Keap1/Nrf2 pathway. This study provides an important scientific basis and direction for the application and research of AST IV and provides new potential targets and ideas for the study of the pathological mechanism of CIRI.

MiR-663-Containing Exosomes Secreted by Bone Marrow Mesenchymal Stem Cells Ameliorate Cardiomyocyte Oxidative Damage

This study assesses the role of miR-663 in the oxidative damage in myocardial cells through regulating BMSC from exosome. BMSC from rats was cultivated and transfected with miR-663 mimics to measure miR-663 level, BMSC proliferation and apoptosis and cTnT level. Exosome in supernatant was collected. The myocardial cells were assigned into control set, damage set and exo-miR-663-BMSC set followed by analysis of cell proliferative and apoptotic activity, miR-663 level, ROS, MDA, SOD and GSH-Px content as well as the expression of Nrf2, keap1 and HO-1. BMSC proliferation was prompted and apoptosis was restrained by miR-663 mimics and BMSC was prompted to be differentiated into myocardial cells. The target gene of miR-663 was keap1. Exo-miR-663-BMSC set showed increased myocardial cell proliferation and decreased apoptosis, reduced ROS and MDA as well as increased SOD and GSH-Px level along with downregulation of keap1 and upregulated of Nrf2 and HO-1. In addition, the recovery of heart injury caused by IRI was significantly prompted by exo-miR-663-BMSC. In conclusion, exo-miR-663 BMSC is capable to ameliorate heart injury induced by IRI.

Identification and antioxidative mechanism of novel mitochondria-targeted MFG-E8 polypeptides in virtual screening and in vitro study

Milk fat globule-EGF factor VIII (MFG-E8) has been identified as an important source of bioactive peptides, which may exert a pivotal role in regulating biologic redox equilibrium. However, the composition of MFG-E8 polypeptides and their mechanisms on mitigating sarcopenia remain unknown. The aim of this study was to identify the composition of MFG-E8 polypeptides and its effects against oxidative stress in dexamethasone-induced L6 cell injury. Simulated digestion in vitro and liquid chromatography-tandem mass spectrometry were used in this investigation. A total of 95 peptides were identified during complete simulated digestion; among them, the contents of 21 peptides were analyzed, having been determined to exceed 1%. Molecular docking assay found that IDLG, KDPG, YYR, and YYK exhibited high binding affinity with keap1. MTT, dichlorodihydrofluorescein diacetate, mito- and lyso-tracker, and transmission electron microscope assay demonstrated that IDLG and KDPG can alleviate oxidative stress-injured L6 cell vitality, mitochondria activity, vacuolation, and function decrease, and increased autophagy, thereby improving mitochondrial homeostasis. From a molecular perspective, IDLG and KDPG can decrease the expression of keap1 and increase the expression of Nrf2, HO-1, and PGC-1α. Therefore, MFG-E8-derived IDLG and KDPG could be potential polypeptides countering oxidative stress in the treatment of sarcopenia, via the keap1/Nrf2/HO-1 signaling pathway.

2,4-di-tert-butylphenol exposure impairs osteogenic differentiation

2,4-di-tert-butylphenol (2,4-DTBP) is a synthetic antioxidant used in polyethylene crosspolymer (PEX) water distribution pipes and food-related plastics. 2,4-DTBP can leach from plastic materials and has been found in breast milk, cord blood, and placental tissue, giving rise to the concern that this compound may interfere with fetal development. The objective of this study is to assess the impacts of 2,4-DTBP on cellular differentiation. Human induced pluripotent stem (HiPS) cells were differentiated into osteoblasts or myoblasts over 40 days, and analyzed for markers of somite, dermomyotome, sclerotome, myoblast, and osteoblast development. When cultured as stem cells, 2,4-DTBP did not alter cell viability and expression of markers (NANOG, OCT4). However, upon differentiation into somite-like cells, 2,4-DTBP had reduced levels of MEOX1 and TBX6 transcripts, while NANOG and OCT4 were in turn upregulated in a dose-dependent manner. At the sclerotome-like stage, PAX9 mRNA decreased by 2-fold in the 0.5 μM and 1.0 μM 2,4-DTBP exposure groups. After 40 days of differentiation into an osteoblast-like lineage, exposure to 2,4-DTBP significantly reduced expression of the osteogenesis transcripts RUNX2 and OSX in a dose-dependent manner. Further, Alizarin Red staining of calcium deposits was decreased in the 0.5 μM and 1.0 μM treatment groups. In contrast, myogenesis was not affected by 2,4-DTBP exposure. Interestingly, KEAP1 expression was significantly increased in the sclerotomal-like cells, but decreased in the dermomytomal-like cells, which may suggest a mechanism of action. Overall, this study shows that 2,4-DTBP can delay key processes during sclerotome and osteoblast development, leading to a potential for bone developmental issues in exposed individuals.

Korean red ginseng water extract produces antidepressant-like effects through involving monoamines and brain-derived neurotrophic factor in rats

BackgroundGinseng Radix (Panax ginseng Meyer, Araliaceae) has been used medicinally to treat the brain and nervous system problems worldwide. Recent studies have revealed physiological effects that could potentially benefit cognitive performance or mood. The present study aimed to investigate the antidepressant effects of Korean red ginseng water extract (KGE) and its active component in an unpredictable chronic mild stress (UCMS)-induced animal model and elucidate the underlying mechanisms. MethodsThe antidepressant potential of the UCMS model was evaluated using the sucrose preference test and open field tests. The behavioral findings were further corroborated by the assessment of neurotransmitters and their metabolites from the prefrontal cortex and hippocampus of rats. Three doses of KGE (50, 100, and 200 mg/kg) were orally administered during the experiment. Furthermore, the mechanism underlying the antidepressant-like action of KGE was examined by measuring the levels of brain-derived neurotrophic factor (BDNF)/CREB, nuclear factor erythroid 2-related factor 2 (Nrf2), and Kelch-like ECH-associated protein 1 (Keap1) proteins in the prefrontal cortex of UCMS-exposed rats. ResultsKGE treatment normalized UCMS-induced depression-related behaviors. Neurotransmitter studies conducted after completing behavioral experiments demonstrated that KGE caused a reduction in the ratio of serotonin and dopamine, indicating a decrease in serotonin and dopamine turnover. Moreover, the expression of BDNF, Nrf2, Keap1 and AKT were markedly increased by KGE in the prefrontal cortex of depressed rats. ConclusionOur results provide evidence that KGE and its constituents exert antidepressant effects that mediate the dopaminergic and serotonergic systems and expression of BDNF protein in an animal model.

Effects of the juçara fruit (Euterpe edulis Martius) pulp and lyophilized extract on NRF2, KEAP1, SOD1, and GPX2 expression in human colorectal cancer cell lines

We investigated the effects of the juçara fruit (Euterpe edulis Martius) pulp and lyophilized extract on the expression of cytoprotective genes nuclear factor erythroid 2 (NF-E2)-related factor 2 (NRF2), kelch-like ECH-associated protein 1 (KEAP1), superoxide dismutase (SOD1), and glutathione peroxidase (GPX2) in human colorectal cancer cell lines (HT-29 and Caco-2). Cells were cultured for 24 h in Dulbecco's Modified Eagle's Medium containing juçara fruit pulp (5, 10, or 50 mg/mL) or lyophilized extract (0.05, 0.1, or 0.5 mg/mL), and gene expression was quantified using real-time quantitative reverse transcription polymerase chain reaction. All studied genes showed significant variation in gene expression among different concentrations of pulp or lyophilized extract. Overall, the expression of the selected genes decreased in both cell lines following exposure to the pulp or lyophilized extract in a dose-dependent manner for most of the concentrations studied. In summary, our study showed that the compounds in juçara fruit inhibited the expression of cytoprotective genes associated with the antioxidant response and that, although not cytotoxic at the concentrations studied, they could potentially block the activation of the NRF2/KEAP1 pathway.

Koumine ameliorates concanavalin A-induced autoimmune hepatitis in mice: involvement of the Nrf2, NF-κB pathways, and gut microbiota

Gelsemiumelegans(Gardner. & Chapm.) Benth. has long been considered a traditional Chinese medicine effective against rheumatoid pain, cancer, cirrhosis, and skin diseases. Koumine (KM), the most abundant alkaloid in G.elegans Benth., demonstrates a variety of biological effects, including antitumor, analgesic, anxiolytic, anti-inflammatory, antidepressant, antioxidant, immunoregulatory, and hepatoprotective effects. Furthermore, the relatively low toxicity of KM makes it a promising drug candidate. This study aimed to investigate the protective effects of KM and its possible mechanisms using a concanavalin A (Con A)-induced autoimmune hepatitis (AIH) model in mice. Mice were orally administered different doses of KM for 14 d before Con A tail vein injections. The effects of KM on serum biochemical markers and liver histopathology were then evaluated 12h after Con A exposure. The Nrf2 and NF-κB signaling pathways and alterations in gut microbiota were determined using western blotting, immunohistochemistry, and 16S rRNA sequencing to explore the underlying mechanisms of KM exposure. KM pretreatment dose-dependently decreased serum liver injury markers (Alanine aminotransferase, and aspartate aminotransferase) and cytokine levels (Tumor necrosis factor-α and interleukin-6), as well as the liver pathological damage triggered by Con A. Furthermore, the results of the multi-technique analysis indicated that KM activated the Nrf2 pathway, upregulated the expression of anti-oxidation factors HO-1 and Nrf2, and downregulated the expression of Keap1. Moreover, the NF-κB signaling pathway was inhibited. Interestingly, pre-treatment with KM also significantly improved the composition of the gut microbiota probably because it increases the richness of probiotics. Our findings suggest that KM pretreatment could attenuate Con A-induced AIH, the Nrf2 and NF-κB signaling pathways, and that gut microbiota are involved in the process of the hepatoprotective effect. This study provides a theoretical basis for the development of KM as an effective agent against AIH.

High-sulfated derivative of polysaccharide from Ulva pertusa improves Adriamycin-induced nephrotic syndrome by suppressing oxidative stress.

Nephrotic syndrome (NS) is characterized by proteinuria, hyperlipidemia, and hypoalbuminemia. Ulva pertusa, a green seaweed, is a nutritional supplement. In this study, the high-sulfated derivative of Ulva pertusa polysaccharide (HU) was prepared by combining U pertusa polysaccharide with chlorosulfonic acid. The NS rat model was established by tail vein single injection of Adriamycin (6.0 mg kg-1). Normal rats were used as the control group. NS rat models were treated with HU or U (173 mg kg-1 day-1). After treatment for 6 weeks, we assessed urine protein, renal function, and blood lipids, and observed morphology and histologic injury of the kidney and glomerular microstructure. Furthermore, we detected antioxidant enzyme activity and expression level of the Keap1/Nrf2 signaling pathway to explore the potential mechanism of HU. Results showed that HU not only alleviated hyperlipidemia and hypoalbuminemia, but also reduced urine protein by inhibiting podocyte detachment, thickening of the glomerular basement membrane, and expression of kidney fibrosis markers (collagens I and IV). In addition, HU enhanced antioxidant enzyme activity (GSH-Px, CAT, SOD) in both serum and the kidney, which may be due to upregulating the expression of Nrf2 and downregulating the expression of Keap1. In conclusion, HU appears to be effective in attenuating NS in rats through suppressing oxidative stress by regulating the Keap1/Nrf2 signaling pathway.

Icariside II suppresses ferroptosis to protect against MPP+-Induced Parkinson's disease through Keap1/Nrf2/GPX4 signaling.

Parkinson's disease (PD) is recognized as a degenerative and debilitating neurodegenerative disorder. The novel protective role of icariside II (ICS II) as a plant-derived flavonoid compound in neurodegenerative diseases has aroused much attention. Herein, the definite impacts of ICS II on the process of PD and the relevant action mechanism were studied. Human neuroblastoma SK-N-SH cells were challenged with 1-methyl-4-phenylpyridinium ion (MPP+) to construct the PD cell model. MTT assay and flow cytometry analysis, respectively, appraised cell viability and apoptosis. Caspase 3 Activity Assay examined caspase 3 activity. Corresponding kits examined oxidative stress levels. BODIPY 581/591 C11 assay evaluated lipid reactive oxygen species. Iron Assay Kit assessed iron content. Western blot tested the expression of apoptosis-, ferroptosis- and Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) signaling-associated proteins. Molecular docking verified the binding of ICS II with Keap1. The existing experimental results unveiled that ICS II elevated the viability whereas reduced the apoptosis, oxidative stress, and ferroptosis in MPP+-treated SK-N-SH cells in a concentration-dependent manner. Furthermore, ICS II declined Keap1 expression while raised Nrf2, heme oxygenase 1, and GPX4 expression. In addition, ICS II had a strong binding with Keap1 and Nrf2 inhibitor ML385 partially abolished the suppressive role of ICS II in MPP+-triggered apoptosis, oxidative stress, and ferroptosis in SK-N-SH cells. To summarize, ICS II might inhibit apoptosis, oxidative stress, and ferroptosis in the MPP+-stimulated PD cell model, which might be due to the activation of Keap1/Nrf2/GPX4 signaling.

Lycopene in Feed as Antioxidant and Immuno-Modulator Improves Broiler Chicken's Performance under Heat-Stress Conditions.

Lycopene is a type of carotenoid pigment widely distributed in various plants and fruits, with tomatoes, carrots, and guava being the most abundant sources. Due to its high content of beneficial active components, lycopene has been used in medicine, where it is employed as a dietary additive for cancer therapy, immune modulator, and feed additive to improve livestock productivity. Lycopene is a lipophilic substance that can act as either a prooxidant or a free radical scavenger and is particularly efficient in enhancing broiler performance. Furthermore, lycopene can alleviate heat stress by improving the activity of various antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), as well as increasing the total antioxidant capacity (T-AOC) and nuclear muscle factor erythroid 2-related factor 2 (Nrf2), while simultaneously reducing the levels of malondialdehyde (MDA) and muscle Keap1 expression. In addition, lycopene can improve broiler fertility by enhancing sperm performance and reducing inflammation by modulating the levels of interleukin 1, 2, and 10 (IL-1, IL-2, and IL-10) in cases of infection. In cases of disease by aflatoxin B1 (AFB1), lycopene can modulate interferon-γ (IFN-γ), IL-1, claudin-1 (CLDN-1), and zonula occludens-1 (ZO-1). Furthermore, under the lipopolysaccharide challenge, lycopene can increase the relative weights of immune organ indices such as the bursal, spleen, and thymus.

Possible protective effects of two different doses of cyanocobalamin against methotrexate nephrotoxicity in rats

Abstract 
 Nephrotoxicity is defined as rapid deterioration in kidney functions. It arises from direct exposure to drugs or their metabolites. Methotrexate is a famous chemotherapeutic drug with anti-inflammatory and immunosuppressive properties. A high-dose methotrexate-induced renal dysfunction can be life threatening. Cyanocobalamin, one of the forms of vitamin B12, acts as a coenzyme in the conversion of homocysteine to methionine in the cytosol, and the conversion of methylmalonyl-CoA to succinyl-CoA in the mitochondrion. This study is designed to examine the effect of cyanocobalamin in two different doses each co-administered with methotrexate at 20 mg/kg induced nephrotoxicity in rats through the involvement of Nrf2/keap1 molecular mechanism in this respect. Rats utilized in this study were randomized into 4 groups (ten rats per each group); Group 1- (Control) rats intraperitoneally injected with 0.5ml normal saline once daily for 7 consecutive days. Group 2- Rats intraperitoneally injected with 0.5ml normal saline once daily for 7 consecutive days; and at day 2, a single intraperitoneal dose of methotrexate (20mg/kg) is to be injected. Group 3- Rats intraperitoneally injected with a 0.5mg/kg cyanocobalamin once daily for 7 consecutive days, and at day 2, a single intraperitoneal dose of methotrexate (20mg/kg). Group 4- Rats intraperitoneally injected with a 2mg/kg cyanocobalamin once daily for 7 consecutive days, and at day 2, a single intraperitoneal dose of methotrexate (20mg/kg). Co-administration of cyanocobalamin at doses cyanocobalamin 0.5mg/kg and 2mg/kg with methotrexate showed significant reduction (P<0.05) in malondialdehyde, significant elevation (P<0.05) in reduced glutathione level, significant upregulation in renal Nrf2 expression and significant down regulation in renal keap1 expression each compared to corresponding levels in methotrexate-only treated group. In conclusion this study demonstrated that co-administration of cyanocobalamin at two different doses with MTX resulted in attenuation of its nephrotoxicity by the utilization of selected parameters.
 Keywords: Nephrotoxicity, methotrexate, cyanocobalamin, malondialdehyde, glutathione, Nrf2, Keap1.

Syringic acid ameliorates testicular oxidative stress via the conservation of endogenous antioxidant markers and inhibition of the activated Nrf2-Keap1-NQO1-HO1 signaling in methyl cellosolve-administered rats

IntroductionSyringic acid (SYRAC) isolated from Ban Lan Gen or Isatis root, a famous Chinese traditional medicine is known to possess antiendotoxic effect. Methyl cellosolve (MCE) on the other hand, is an important constituent of car brake fluids, pesticides, inks, paints, and liquid soaps, rendering humans inevitable to its exposure. MethodsThis study therefore investigated the effect of SYRAC treatments on MCE-induced testicular oxidative stress in rats. Six (6) groups of five (5) rats each were involved in this study that lasted for 30 days. The first group was the control that contained rats served food and water throughout, the second group were administered 100 mg/kg body weight of MCE everyday for 30 days, the third, fourth, and fifth group were treated same way as rats in second group but were treated with 25, 50, and 75 mg/kg body weight of SYRAC respectively for 30 days, while rats in the sixth group were administered 75 mg/kg body weight of SYRAC only. ResultsAt the end of administrations, RTW, testicular GSH level, and activities of GPx, GST, CAT, and SOD were significantly decreased, while MDA, total mRNA expressions of HO-1, NQO1, Keap1, and Nrf2 were significantly increased by MCE compared with control. Compared with MCE only, treatments with SYRAC significantly increased the testicular levels of NO and GSH, as well as activities of GPx, GST, SOD, and CAT, while MDA, mRNA expressions of Keap1, NQO1, Nrf2, and HO-1 were significantly decreased. ConclusionConclusively, MCE-induced testicular oxidative stress was recorded. SYRAC demonstrated a gonadoprotective effect by maintaining the normal levels of the endogenous antioxidants and inhibiting MECE-induced Nrf2 activation and ARE-dependent genes in rats.

Hepatopancreas-Intestinal Health in Grass Carp (Ctenopharyngodon idella) Fed with Hydrolyzable Tannin or Rapeseed Meal.

This study evaluated the effect of dietary rapeseed meal (RM) and hydrolyzable tannin on grass carp (Ctenopharyngodon idella) and determined the possible role of tannin on health when RM was added to the diet. Eight diets were formulated. Four were semipurified-diets with 0, 0.75, 1.25, and 1.75% hydrolyzable tannin (T0, T1, T2, and T3), and the other four were practical diets containing 0, 30, 50, and 70% RM (R0, R30, R50, and R70), which had similar tannin contents as semipurified-diets. After the 56 d feeding trial, the antioxidative enzymes and relative biochemical indexes showed a similar tendency in practical and semipurified groups. In hepatopancreas, superoxide dismutase (SOD) and catalase (CAT) activities increased with RM and tannin levels, respectively, while glutathione (GSH) content and glutathione peroxidase (GPx) activity increased. Malondialdehyde (MDA) content increased and decreased in T3 and R70, respectively. In the intestine, MDA content and SOD activity increased with RM and tannin levels, while GSH content and GPx activity decreased. The expression levels of interleukin 8 (IL-8) and interleukin 10 (IL-10) were upregulated with RM and tannin levels, and the Kelch-like ECH-associated protein 1 (Keap1) expression was upregulated in T3, whereas it was downregulated in R50. This study demonstrated that 50% of RM and 0.75% of tannin induced oxidative stress, injured hepatic antioxidant ability, and resulted in intestinal inflammation in grass carp. Therefore, the tannin in rapeseed meal cannot be neglected in aquatic feeding.

Effect of substitution of taurine for methionine and additional taurine supplementation on the performance and antioxidative capacity of laying hens

Taurine (TAU), a sulfur-containing amino acid that synthesized from methionine and cystine, plays vital roles in maintenance of redox balance. The effect of substitution of TAU for methionine was evaluated in vivo and in vitro. The effects of replacing methionine with TAU and additional TAU supplementation on the performance and antioxidant capacity of laying hens were evaluated. The in vitro cultured chicken primary hepatocytes and intestinal epithelial cells were further employed. Two hubdred eighty-eight 40-wk-old Isa brown laying hens were divided into 4 groups and subjected one to the following treatments: fed with basal diet with 0.17% crystallized DL-Met (CON), the control diet and replace 25% (21% total Met, 21TAU) or 50% (42% total Met, 42TAU) of crystallized DL-Met with taurine, the control diet supplemented with 0.1% taurine (0.1% TAU). The laying rate, feed intake, egg weight, and feed efficiency were not influenced (P > 0.05) by TAU replacement or additional TAU supplementation. In the liver, 0.1% TAU decreased SOD but increased GSH-Px activity (P < 0.01). In duodenum, 42TAU decreased SOD activity (P < 0.05) while 0.1% TAU decreased GSH level and SOD activity (P < 0.05). In the hepatocytes, TAU treatment decreased (P < 0.05) the MDA and GSH contents, whereas increased SOD and GSH-Px activities (P < 0.05). Meanwhile, TAU treatment decreased (P < 0.05) the protein expression of Nrf2 while increase Keap1 expression. The mRNA expression of Nrf2, SOD1, SOD2, CAT, and GCLC were increased (P < 0.05) and GSR were decreased (P < 0.05) by 0.1% TAU. In the intestinal epithelial cells, TAU treatment decreased (P < 0.05) SOD activity, increased (P < 0.05) CAT activity, and decreased (P < 0.05) the mRNA and protein expression of Nrf2. In summary, partial substitution methionine for taurine (21-42%) has no influence on egg performance of hens. Taurine enhances the antioxidative capacity in hepatocyte but not in the enterocytes and if taurine could offer an improved effect on antioxidant capacity needs to be verified under oxidative stress-challenged conditions.

Sodium Danshensu Cream Promotes the Healing of Pressure Ulcers in Mice through the Nrf2/HO-1 and NF-κB Pathways.

On the basis of the mice pressure ulcers (PU) model, the protective effect and potential mechanism of sodium Danshensu (SDSS) cream against PU were investigated. The mice were randomly divided into three groups: the negative control group (cream without 0.5 g SDSS), the SDSS group (cream containing 0.5 g SDSS), and the positive group (0.5 g Hirudoid®). After 7 and 14 days of ointment application, the wound-healing rate of the SDSS and positive groups was significantly higher than that of the control group (p &lt; 0.05). The results of hematoxylin-eosin staining also indicated that SDSS has the potential to promote the healing of PU. In addition, the serum IL-6, IL-1β, TNF-α, and MDA levels decreased significantly (p &lt; 0.01) after 14 days of SDSS treatment, while the SOD, CAT, and GSH-Px activities increased significantly (p &lt; 0.01). In addition, SDSS cream was able to significantly increase the expression of Nrf2, HO-1, GCLM, NQO1, NF-κB p65, NF-κB p50, IKKα, and IKKβ while decreasing the expression of Keap1 and IκBαin the Nrf2/HO-1 and NF-κB pathways. Our research will provide a foundation for the future clinical prevention and treatment of PU with SDSS cream.

Effect of dietary oregano oil on growth performance, disease resistance, intestinal morphology, immunity, and microbiota of hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂)

The study aimed to investigate the effects of oregano oil (OO) supplement in the diet on the growth performance, disease resistance, immunity, and intestinal health of hybrid grouper. Six isonitrogenous and isolipidic diets were fed to the grouper with an initial weight of 19.10 ± 0.12 g for 8 weeks, of which the dietary OO levels were 0 (Control), 0.1 (OO-0.1), 0.3 (OO-0.3), 0.6 (OO-0.6), 0.9 (OO-0.9), or 1.2 (OO-1.2) g kg-1. The results showed that there were no significant differences in growth performance among the six groups, while the OO supplement could improve the disease resistance ability of grouper. Our findings have demonstrated that the OO supplement, especially 0.3 g kg-1, could improve the intestinal morphology via increasing the villus number and muscle thickness and elevate digestive enzyme activities such as those of amylase (AMS) and lipase. The 0.3-g kg-1 OO supplement also can activate the antioxidant defense system, reduce nuclear factor erythroid 2-related factor 2-like 2 (Nrf2)/Kelch-like-ECH-associated protein 1 (Keap1) expression, and then enhance the immune ability via acid phosphatase (ACP), lysozyme (LYS), complement C3 (C3), and tight junction-associated signaling pathways in hybrid grouper. Meanwhile, the relative abundance of Firmicutes and Bacteroidetes in the OO-0.3 group was significantly higher than that of Control group (P&amp;lt; 0.05), while the relative abundance of Proteobacteria showed a basically opposite trend (P&amp;lt; 0.05). These findings suggested that dietary OO supplementation could significantly improve intestinal health and induce the antioxidant defense system and immunity, which means that OO could be used as a functional feed additive to protect the intestine, and the appropriate addition level of OO should be 0.3 g kg-1.

Mesenchymal Stem Cells Promote Intestinal Mucosal Repair by Positively Regulating the Nrf2/Keap1/ARE Signaling Pathway in Acute Experimental Colitis.

Mesenchymal stem cells (MSCs) are a type of adult pluripotent stem cell that has anti-inflammatory and immunomodulatory effects, and whose conditioned medium (CM) has also been found to be effective. We used MSC and CM enemas to investigate their ameliorative effects in a mouse model of colitis. We employed MSCs, CM, and MSCs + ML385 (an inhibitor of Nrf2) in dextran sodium sulfate (DSS)-induced colitis. Mice were sacrificed on day 8, and the effects of MSC or CM treatment on the levels of inflammation and oxidative stress in colonic epithelial cells were evaluated by histological analyses. MSCs inhibited inflammatory cell infiltration and proinflammatory cytokine expression in the colon. In addition, MSCs reduced extracellular matrix deposition and maintained the mechanical barrier and permeability of colonic epithelial cells. Mechanistically, MSCs activated Nrf2, which then increased HO-1 and NQO-1 levels and downregulated the expression of Keap1 to suppress reactive oxygen species production and MDA generation, accompanied by increases in components of the enzymatic antioxidant system, including SOD, CAT, GSH-Px, and T-AOC. However, after administering an Nrf2 inhibitor (ML385) to block the Nrf2/Keap1/ARE pathway, we failed to observe protective effects of MSCs in mice with colitis. CM alone also produced some of the therapeutic benefits of MSCs but was not as effective as MSCs. Our data confirmed that MSCs and CM can effectively improve intestinal mucosal repair in experimental colitis and that MSCs can improve this condition by activating the Nrf2/Keap1/ARE pathway.

Activation of the Nrf2 Antioxidant Pathway by Longjing Green Tea Polyphenols in Mice Livers

Previous studies have revealed that green tea polyphenol (GTP) could protect against liver injury due to oxidative stress. However, the mechanism underlying the bioactive actions of GTP in the liver has not been systematically evaluated. This study aimed to investigate the effect of GTP on the activation of the nuclear factor erythroid-2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (keap1) pathway, using in silico and in vivo methods. Furthermore, the regulation of Nrf2 downstream target antioxidant response element (ARE) was also evaluated. The high-performance liquid chromatography analysis indicated that GTP includes 9 major compounds, and molecule docking analysis demonstrated that most of these polyphenols have a strong binding affinity with the keap1 Kelch domain, where keap1 binds to the Neh2 domain of Nrf2. Remarkably, the predominant compound of GTP, that is, epigallocatechin gallate, displayed the best binding affinity score, which can fully occupy all 3 polar subpockets of the keap1 Kelch domain. The Nrf2, keap1, and Nrf2 downstream target gene expression levels were changed in the livers compared to the control group. It showed that the Nrf2 expression level was significantly upregulated in GTP-induced mice liver across most treatments, while the keap1 expression level remained unchanged. Subsequently, we observed a significant increasing trend in the expression of the downstream ARE, including antioxidative enzymes, liver phase II enzymes, and liver efflux transporters in mice livers. The present study demonstrated that GTP could activate the Nrf2 signaling pathway by interrupting the Nrf2-keap1 protein–protein interaction

Asiatic acid alleviates LPS-induced acute kidney injury in broilers by inhibiting oxidative stress and ferroptosis via activation of the Nrf2 pathway

Asiatic acid (AA), a triterpenoid compound isolated from Centella asiatica, has anti-inflammatory, antioxidant and anticancer biological characteristics. To explore the effect of AA on LPS-induced acute kidney injury (AKI) in broilers, a total of 60 one-day-old broilers were randomly divided into 6 groups, including the normal group, AKI model group, AKI+AA 15mg/kg group, AKI+AA 30mg/kg group, AKI+AA 60mg/kg group and normal+AA 60mg/kg group. Hematoxylin-eosin staining was used to observe the histopathology in kidney tissue, and the mRNA and protein expressions related to oxidative stress and ferroptosis were tested by qPCR and western blotting respectively. AA mitigated vacuolar degeneration and enlarged glomerular space caused by LPS in kidney tissue. Additionally, AA significantly increased the mRNA levels of Nrf2, HO-1, NQO1, GCLC, GCLM, GPX4, SLC7A11 and FTH1, and decreased the mRNA levels of Keap1 and PTGS2 in LPS-induced AKI. Likewise, AA significantly upregulated the protein expressions of Nrf2, HO-1, NQO1, GPX4, SLC7A11 and FTH1, and downregulated the protein expressions of Keap1 and PTGS2 in LPS-induced AKI. These results suggested that AA alleviated LPS-induced AKI by inhibiting oxidative stress and ferroptosis through targeting regulation of the Nrf2 pathway in broilers.