Kelch-like ECH-associated Protein Research Articles

Inhibitor of apoptosis stimulating protein of p53 protects against MPP+-induced neurotoxicity of dopaminergic neurons.

Inhibitor of apoptosis stimulating protein of p53 (iASPP) is related to the pathogenesis of several neurological disorders by affecting the oxidative stress and survival of neurons. However, whether iASPP has a role in Parkinson disease (PD) remains to be determined. This work explored the potential regulatory effect of iASPP in an in vitro model of PD based on 1-methyl-4-phenylpyridinium (MPP+)-evoked neurotoxicity of dopaminergic neurons in culture. MN9D neurons were treated with MPP+ at 200 µM in the culture media for 24h to induce neurotoxicity. Overexpression and silencing of iASPP in neurons were achieved by infecting recombinant adenovirus expressing iASPP and sh-iASPP, respectively. Protein expression was examined by immunoblotting. MPP+-evoked neurotoxicity of dopaminergic neurons was determined by cell viability, TUNEL, and flow cytometric assays. The transcriptional activity of nuclear erythroid factor 2-like 2 (Nrf2) was assessed by luciferase reporter assay. Kelch-like ECH-associated protein 1 (Keap1)-knockout neurons were generated by lentiCRISPR/Cas9-Keap1 constructs. Expression levels of iASPP declined in MPP+-stimulated neurons. Overexpression of iASPP in neurons exhibited inhibitory effects on MPP+-evoked apoptosis, α-synuclein accumulation, and oxidative stress, while iASPP-deficient neurons were more sensitive to MPP+-induced neurotoxicity. Overexpression of iASPP led to an enhancing effect on Nrf2 activation in MPP+-stimulated neurons. Mechanism research revealed that iASPP may contribute to the activation of Nrf2 by competing with Nrf2 in binding with Keap1. Notably, the regulatory effect of iASPP on Nrf2 was diminished in Keap1-knockout neurons. The chemical inhibition of Nrf2 or knockdown of Nrf2 abrogated the protective effects of iASPP on MPP+-induced neurotoxicity. To conclude, iASPP protects dopaminergic neurons against MPP+-induced neurotoxicity through modulation of the Keap1/Nrf2 axis. Therefore, iASPP may play a crucial role in mediating the loss of dopaminergic neurons in PD, and targeting the iASPP-Nrf2 axis could be a promising strategy for treating PD.

Molecular docking and molecular dynamics simulation of wheat gluten-derived antioxidant peptides acting through the Keap1-Nrf2 pathway.

In our previous study, we successfully identified five peptides from wheat gluten: Ala-Pro-Ser-Tyr (APSY), Leu-Tyr (LY), Pro-Tyr (PY), Arg-Gly-Gly-Tyr (RGGY) and Tyr-Gln (YQ). Molecular docking and molecular dynamics simulation methods were employed to investigate the interaction between these antioxidant peptides and the Kelch-like ECH-associated protein 1 (Keap1 protein), revealing the molecular mechanism of their non-competitive binding. In addition, the total antioxidant capacity of the five peptides was determined using the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) method. The affinities of APSY, LY, PY, RGGY and YQ were -8.9, -8.3, -8.5, -9.1 and - 7.9 kcal mol-1, respectively. The five peptides effectively bound to Keap1 protein through hydrogen, π-σ, π-alkyl and alkyl interactions. Significant roles were observed for the P1 pocket residue ARG-415 and the P3 pocket residue ALA-556 in the interactions of the Keap1-peptide complexes. Molecular dynamics simulations further elucidated the dynamic process of peptide binding to the Keap1 protein. All five peptides formed stable complexes with Keap1 protein, with van der Waals forces playing crucial roles in these complex systems, indicative of the peptides' strong binding ability to Keap1 protein. The van der Waals forces were -178.74, -123.11, -134.36, -132.59, and -121.44 kJ mol-1 for the Keap1-APSY, Keap1-LY, Keap1-PY, Keap1-RGGY and Keap1-YQ complexes, respectively. These peptides exhibited excellent antioxidant effects. Among them, the YQ peptide exhibited the highest total antioxidant capacity, with an activity value of 1.18 ± 0.06 mmol Trolox equivalent (TE) L-1 at a concentration of 0.10 mg mL-1. The RGGY, PY, LY and APSY peptides followed in descending order, with activity values of 0.91 ± 0.05, 0.72 ± 0.06, 0.62 ± 0.04 and 0.60 ± 0.05 mmol TE L-1, respectively. These results unveiled the molecular mechanism by which the five antioxidant peptides act on active pockets through the Keap1-Nrf2 signaling pathway, providing a theoretical basis for the development of antioxidants. © 2024 Society of Chemical Industry.

Chemopreventive effects of atractylenolide-III on mammary tumorigenesis via activation of the Nrf2/ARE pathway through autophagic degradation of Keap1

The incidence of breast cancer is increasing annually, making it a major health threat for women. Chemoprevention using natural, dietary, or synthetic products has emerged as a promising approach to address this growing burden. Atractylenolide-III (AT-III), a sesquiterpenoid present in various medicinal herbs, has demonstrated potential therapeutic effects against several diseases, including tumors, nonalcoholic fatty liver disease, and cerebral ischemic injury. However, its impact on breast cancer chemoprevention remains unexplored. In this study, we used an N-methyl-N-nitrosourea (NMU)-induced rat breast cancer model and 17β-estradiol (E2)-treated MCF-10A cells to evaluate the chemopreventive potential of AT-III on mammary tumorigenesis. AT-III inhibited mammary tumor progression, evidenced by reduced tumor volume and multiplicity, prolonged tumor latency, and the reversal of NMU-induced weight loss. Furthermore, AT-III suppressed NMU-induced inflammation and oxidative stress through the Nrf2/ARE pathway in breast cancer tissues. In vitro, AT-III effectively suppressed E2-induced anchorage-independent growth and cell migration in MCF-10A cells. Nrf2 knockdown attenuated the protective effects of AT-III, highlighting the pivotal role of Nrf2 in AT-III-mediated suppression of tumorigenesis. The mechanism involves the induction of Nrf2 expression by AT-III through the autophagic degradation of Kelch-like ECH-associated protein 1 (Keap1). Overall, the results of this study indicate that AT-III is a promising candidate for breast cancer chemoprevention and provide valuable insights into its molecular interactions and signaling pathways.

Forsythiaside-A improved bile-duct-ligation-induced liver fibrosis in mice: The involvement of alleviating mitochondrial damage and ferroptosis in hepatocytes via activating Nrf2

Liver fibrosis is a key and reversible stage in the progression of many chronic liver diseases to cirrhosis or hepatocellular carcinoma. Forsythiaside-A (FTA), a main compound isolated from Forsythiae Fructus, has an excellent liver protective activity. This study aims to investigate the efficacy of FTA in improving cholestatic liver fibrosis. Bile-duct-ligation (BDL) was conducted to induce liver fibrosis in mice. Hepatic collagen deposition was evaluated by Masson and Sirus red staining. The bile acid spectrum in the liver and serum was analyzed by mass spectrometry. Liver oxidative stress injury and mitochondria damage were observed by using Mito-Tracker Red fluorescence staining, transmission electron microscopy, etc. The level of ferrous iron (Fe2+) and the expression of ferroptosis-associated molecules were detected. The binding between FTA and its target protein was confirmed by Co-immunoprecipitation (Co-IP), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) and surface plasmon resonance (SPR). Our results demonstrated that FTA alleviated BDL-induced liver fibrosis in mice. FTA did not decrease the elevated amount of bile acids in BDL-treated mice, but reduced the bile acid-induced mitochondrial damage, oxidative stress and ferroptosis in hepatocytes, and also induced nuclear factor erythroid 2-related factor-2 (Nrf2) activation. In Nrf2 knock-out mice, the FTA-provided protection against BDL-induced liver fibrosis was disappeared, and FTA's inhibition on mitochondrial damage, oxidative stress and ferroptosis were lowered. Further results displayed that FTA could directly bind to Kelch-like ECH-associated protein-1 (Keap1), thereby activating Nrf2. Moreover, the BDL-induced liver fibrosis was markedly weakened in liver-specific Keap1 knockout mice. Hence, this study suggests that FTA alleviated the BDL-induced liver fibrosis through attenuating mitochondrial damage and ferroptosis in hepatocytes by activating Nrf2 via directly binding to Keap1.

4-Octyl itaconate alleviates dextran sulfate sodium-induced ulcerative colitis in mice via activating the KEAP1-NRF2 pathway.

Ulcerative colitis (UC) is a chronic idiopathic inflammatory bowel disease with a relapsing-remitting course. Although its etiology remains unknown, excessive oxidative stress in colon is a major intermediate factor that can promote the progression of UC. In the present study, we investigated the effect and the underlying mechanisms of 4-Octyl itaconate (OI) on dextran sulfate sodium (DSS)-induced UC in mice. Our work identified that OI alleviated the colitis by reducing the oxidative stress and the apoptosis in colon tissue, then increasing the tight junction proteins expression and in turn enhancing the intestinal barrier function, thereby creating less severe inflammatory responses. Moreover, our results demonstrated that OI reduced the Kelch-like ECH-associated protein 1 (KEAP1) expression and subsequent upregulated nuclear factor E2-related factor (NRF2) expression and its nuclear translocation which in turn induced the expression of glutathione S-transferase (GST) and NAD(P)H: quinone oxidoreductase 1 (NQO1). In addition, ML385, a NRF2 antagonist, can inhibit the protective effects of OI on UC, indicating that the role of OI in this colitis model could be dependent on the activation of KEAP1-NRF2 pathway. Notably, OI co-administration significantly enhanced the therapeutic effects of mesalazine or 1400W on UC. Collectively, itaconate may have a great potential for use in the treatment of IBD.

Long Noncoding RNA MALAT1: Salt-Sensitive Hypertension.

Hypertension stands as the leading global cause of mortality, affecting one billion individuals and serving as a crucial risk indicator for cardiovascular morbidity and mortality. Elevated salt intake triggers inflammation and hypertension by activating antigen-presenting cells (APCs). We found that one of the primary reasons behind this pro-inflammatory response is the epithelial sodium channel (ENaC), responsible for transporting sodium ions into APCs and the activation of NADPH oxidase, leading to increased oxidative stress. Oxidative stress increases lipid peroxidation and the formation of pro-inflammatory isolevuglandins (IsoLG). Long noncoding RNAs (lncRNAs) play a crucial role in regulating gene expression, and MALAT1, broadly expressed across cell types, including blood vessels and inflammatory cells, is also associated with inflammation regulation. In hypertension, the decreased transcriptional activity of nuclear factor erythroid 2-related factor 2 (Nrf2 or Nfe2l2) correlates with heightened oxidative stress in APCs and impaired control of various antioxidant genes. Kelch-like ECH-associated protein 1 (Keap1), an intracellular inhibitor of Nrf2, exhibits elevated levels of hypertension. Sodium, through an increase in Sp1 transcription factor binding at its promoter, upregulates MALAT1 expression. Silencing MALAT1 inhibits sodium-induced Keap1 upregulation, facilitating the nuclear translocation of Nrf2 and subsequent antioxidant gene transcription. Thus, MALAT1, acting via the Keap1-Nrf2 pathway, modulates antioxidant defense in hypertension. This review explores the potential role of the lncRNA MALAT1 in controlling the Keap1-Nrf2-antioxidant defense pathway in salt-induced hypertension. The inhibition of MALAT1 holds therapeutic potential for the progression of salt-induced hypertension and cardiovascular disease (CVD).

Abstract A016: NRF2 activation promotes a fitness disadvantage in normal urothelium and drives a basal-like phenotype

Abstract Introduction: Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays a key role in protecting cells from oxidative stress. NRF2 is negatively controlled by Kelch-like ECH-associated protein 1 (KEAP1), which facilitates NRF2 ubiquitination and proteosomal degradation under homeostatic conditions. NRF2 activating mutations occur at a frequency of 5.9% in muscle-invasive bladder cancer (MIBC). However, the role of NRF2 in MIBC has not been fully understood. The aim of this study is to clarify how NRF2 affect to the normal epithelial cells and the tumor cells in bladder. Methods: We used genetically engineered mice (GEM) with a Cre inducible Nrf2 activating mutation (LSL-Nrf2-E79Q). To evaluate how Nrf2 affects the tumorigenesis process in vivo, the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) was administered to Upk3a-CreERT2; LSL-Nrf2-E79Q/+ (UN) mice. We also established normal urothelial organoids and BBN tumor derived organoids from wild-type (WT) and Krt5-CreERT2; LSL-Nrf2-E79Q/+ (K5N) mice for ex vivo study. To induce overexpression of NRF2, we utilized KI696 (KEAP1-NRF2 inhibitor) as a pharmacological agent and performed NAD/NADH assay to quantify the reductive stress. Additionally, we performed single cell RNA sequencing (scRNA-seq) with WT, UN and K5N mice bladder to identify the effect of Nrf2E79Q on the normal epithelial cells. Results: In BBN induced tumor model in vivo, UN mice did not differ in time to tumor development from the control WT mice. Furthermore, UN tumors had lost the post-recombination allele of LSL-Nrf2-E79Q in genomic PCR, meaning cells expressing Nrf2E79Q are selected against during the process. In ex vivo study, KI696 induced reductive stress in both WT normal urothelial organoids and BBN tumor derived organoids, but inhibited cell proliferation only in normal urothelial organoids. In K5N organoids, Nrf2E79Q activation enhanced relative mRNA expression of a number of basal related genes. In addition to that, vehicle organoids in differentiation media showed phenotypic differentiation, whereas those treated with tamoxifen did not demonstrate phenotypic signs of differentiation suggesting Nrf2 activation promotes a basal-like phenotype. Furthermore, we annotated three clusters in mice bladder epithelial cells as luminal, intermediate and basal using scRNA-seq and looked at their proportion among WT, UN and K5N mice. We found that the percentage of luminal cells was decreased and intermediate and basal cells increased in both UN and K5N mice compared to WT mice. This indicated that Nrf2E79Q promotes disadvantage in luminal cells in UN mice, whereas it contributes to the increase in intermediate and basal cells in K5N mice. Conclusion: These results suggested that Nrf2E79Q promotes a fitness disadvantage in normal mice urothelium. Furthermore, Nrf2E79Q inhibits differentiation of K5N organoids and drives a basal-like phenotype in GEM models. Further investigation is needed to clarify the mechanism of NRF2 activating mutation contributing the tumorigenesis in bladder. Citation Format: Akihiro Hamada, Yuki Kita, Di Wu, Mi Zhou, Sean Clark-Garvey, Andrew Gdowski, Michael Sturdivant, Wolfgang Beckabir, Elliott Drew Toomer, Emily Kounlavong, Lucia Lim, Kathryn Gessner, Jeffrey Damrauer, William Kim. NRF2 activation promotes a fitness disadvantage in normal urothelium and drives a basal-like phenotype [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A016.

Antioxidative effect of wheat-grain moxibustion on cyclophosphamide-induced liver injury in mice based on Nrf2-Keap1 signaling pathway

To observe the protective effect of wheat-grain moxibustion on cyclophosphamide (CTX)-induced liver injury in mice, and explore its mechanism based on the nuclear factor E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) signaling pathway. Twenty-four male CD-1 (ICR) mice were randomly divided into a blank group, a model group, and a moxibustion group, with 8 mice in each group. The mice in the model group and the moxibustion group were intraperitoneally injected with CTX (80 mg/kg) to induce liver injury. The mice in the moxibustion group were treated with wheat-grain moxibustion at "Guanyuan" (CV 4) and bilateral "Zusanli" (ST 36) and "Sanyinjiao" (SP 6), with each acupoint being treated by 3 cones, approximately 30 seconds per cone, once daily for 7 days. After intervention, the general condition of the mice was observed; the liver mass was measured and the liver index was calculated; HE staining was used to observe the morphology of the liver, and the liver tissue pathological score was assessed; ELISA was used to detect the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH) and the levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) in the liver; Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of Nrf2, Keap1, and quinione acceptor oxidoreductase 1 (NQO1) in the liver. Compared with the blank group, the mice in the model group showed sluggishness, unsteady gait, and decreased body weight; liver index was increased (P<0.01); liver cells were loosely arranged, with a small number of cell swollen and exhibiting balloon-like changes; liver tissue pathological score was increased (P<0.05); the serum levels of AST, ALT, GLDH, and level of MDA in the liver were increased (P<0.05), and the levels of SOD and GSH-Px in the liver were decreased (P<0.05); protein and mRNA expression of Nrf2 and NQO1 in the liver was decreased (P<0.01), protein and mRNA expression of Keap1 in the liver was increased (P<0.01). Compared with the model group, the mice in the moxibustion group showed improvement in general condition; liver index was decreased (P<0.01); liver cell structure was relatively intact and clear, and liver tissue pathological score was decreased (P<0.05); the serum levels of AST, ALT, GLDH, and level of MDA in the liver were decreased (P<0.05), and the levels of SOD and GSH-Px in the liver were increased (P<0.05, P<0.01); protein and mRNA expression of Nrf2 and NQO1 in the liver was increased (P<0.05), protein and mRNA expression of Keap1 in the liver was decreased (P<0.05). The wheat-grain moxibustion may alleviate CTX-induced liver injury by activating the Nrf2-Keap1 signaling pathway and enhancing the expression of antioxidative enzyme system in the body.

Silybin prevented avermectin-induced cardiotoxicity in carp by modulating oxidative stress, inflammation, endoplasmic reticulum stress, mitochondrial apoptosis, and autophagy

Avermectin is one of the widely used anthelmintics in aquaculture and exhibits substantial toxicity to aquatic organisms. Silybin is extensively used for its anti-inflammatory, antioxidant and anti-apoptotic biological properties. Heart is essential for the survival of fish and plays a vital role in pumping blood oxygen and nutrients. Residual avermectin in water poses harm to carp. However, there is still insufficient research on whether silybin can mitigate the toxicity of avermectin to carp heart tissues. In this research, we established a model involving carp subjected to acute avermectin exposure and administered diets containing silybin to explore the potential protective effects of silybin against avermectin-induced cardiotoxicity. The results revealed that avermectin induced oxidative stress, inflammation, endoplasmic reticulum (ER) stress, mitochondrial pathway apoptosis and autophagy in the cardiac tissues of carp. Compared with the avermectin group, silybin significantly reduced ROS accumulation in cardiac tissues, restored antioxidant enzyme activity, inhibited mRNA transcript levels of pro-inflammatory-related factors, and attenuated ER stress, mitochondrial pathway apoptosis and autophagy. Protein-protein interaction (PPI) analysis demonstrated that silybin mitigated avermectin-induced cardiac oxidative stress, inflammation, ER stress, mitochondrial pathway apoptosis and autophagy. Silybin exerted anti-inflammatory effects through the Nuclear Factor kappa B (NF-κB) pathway, antioxidant effects through the Nuclear factor erythroid 2-related factor 2 (Nrf2) - Kelch-like ECH-associated protein 1 (Keap1) pathway, alleviated cardiac ER stress through the Glucose-regulated protein 78 (GRP78)/Activating Transcription Factor 6 (ATF6)/C/EBP homologous protein (CHOP) axis, suppressed apoptosis through the mitochondrial pathway, and inhibited excessive autophagy initiation through the PTEN-induced putative kinase 1 (PINK1)/Parkin RBR E3 ubiquitin protein ligase (PARKIN) signaling pathway. This study provided evidence supporting the protective effect of silybin against avermectin-induced cardiotoxicity in carp, highlighting its potential as a dietary additive to protect fish from adverse effects caused by avermectin exposure.

Adenosine A2A receptor antagonist SCH58261 improves the cognitive function in Alzheimer's disease model mice through activation of Nrf2 via an autophagy-dependent pathway.

Adenosine, an important endogenous neuromodulator, contributes to a broad set of several neurodegenerative diseases. The adenosine A2A receptor (A2AR) is the most involved in neuropathological effects and plays an important role in the pathogenesis of Alzheimer's disease (AD). However, the effect of A2AR antagonist and the underlying mechanism in AD model mice remains unclear. The amyloid beta (Aβ)1-42-induced mice AD models were used in this study. Several behavioral experiments were performed to evaluate the improvement of AD mice treat with A2AR receptor antagonist. For mechanism analysis, autophagy-related proteins, Kelch-like ECH-associated protein1(Keap1)- nuclear factor erythroid-derived factor 2-related factor (Nrf2) pathway activation and synaptic function were studied by using western blot, immunofluorescence (IF), immunohistochemistry (IHC), transmission electron microscope (TEM), real-time quantitative (PCR) and patch clamp. Pharmacological blockade of adenosine A2AR by SCH58261 (SCH) ameliorated cognitive deficits and decreased expression levels of several AD biomarkers, including Aβ and hyperphosphorylation of Tau. Moreover, SCH activated the Nrf2 pathway through autophagy mediated Keap1 degradation, resulting in the improvement of neurons autophagy dysfunction, synaptic plasticity, and synaptic transmission. Our data clarified that the SCH58261 (an antagonist of adenosine A2A receptor) could increase the level of autophagy, promote the ability of anti-oxidative stress by the activation of Keap1-Nrf2 pathway, and improve the synaptic function in Aβ1-42-induced Alzheimer's disease mice or cell model, which provided a potential therapeutic strategy for AD. A2AR antagonism represents a promising strategy for the anti-AD agents development through autophagy-dependent pathway.

Roquin-2 promotes oxidative stress-induced cell death by ubiquitination-dependent degradation of TAK1

Reactive oxygen species (ROS) are highly reactive and their accumulation causes oxidative damage to cells. Cells maintain survival upon mild oxidative stress with anti-oxidative systems, such as the kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) system. On the other hand, upon severe oxidative stress, cells undergo regulated cell death, including apoptosis, for eliminating damaged cells. To execute efficient cell death, cells need to turn off the anti-oxidant systems, while triggering cell death. However, it remains unknown how cells orchestrate these two conflicting systems under excessive oxidative stress.Herein, we show that when cells are exposed to excessive oxidative damage, an E3 ubiquitin ligase Roquin-2 (also known as RC3H2) plays a key role in switching cell fate from survival to death by terminating activation of transforming growth factor-β-activated kinase 1 (TAK1), a positive regulator for Nrf2 activation. Roquin-2 interacted with TAK1 via four cysteine residues in TAK1 (C96, C302, C486, and C500) that are susceptible to oxidative stress and participate in oligomer formation via disulfide bonds, promoting K48-linked polyubiquitination and degradation of TAK1. Nrf2 was inactivated upon lethal oxidative stress in wild-type mouse embryonic fibroblast (MEF) cells, whereas it sustained activation and conferred resistance to Roquin-2 deficient cells, which was reversed by pharmacological or genetic inhibition of TAK1. These data demonstrate that in response to excessive ROS exposure, Roquin-2 promotes ubiquitination and degradation of TAK1 to suppress Nrf2 activation, and thereby contributes to an efficient cell death, providing insight into the pathogenesis of oxidative stress-related diseases, including cancer.

Anti-aging mechanism and effect of treatment with raw and wine-steamed Polygonatum sibiricum on D-galactose-induced aging in mice by inhibiting oxidative stress and modulating gut microbiota.

Polygonatum sibiricum (PS) is a traditional Chinese medicine (TCM) first recorded in Mingyi Bielu. The book documents that PS can nourish five internal organs, be taken for a long time, relax the body and prolong lifespan. Presently, PS is widely used in TCM to prevent premature graying of hair. Based on TCM theory and clinical trials, the wine steaming processed product from PS provides a better effect. However, no published study has elucidated the anti-aging mechanism. The study aim was to investigate the anti-aging mechanism of PS and its wine steaming processed product in mice, specifically focusing on the effect of D-galactose (D-gal) surrounding the intestinal flora and the Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2-antioxidant response elements (Keap1/Nrf2/ARE) pathway. The chemical components in Raw PS (RPS) and Wine-steamed PS (WPS) were identified by ultra-performance liquid chromatography-hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS). An aging model using Kunming mice was established through intraperitoneally injected D-gal. Concentrations of RPS and WPS at 5, 10, or 15g/kg/day levels were administered intragastrically, respectively. The body weight, liver and spleen indexes, superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and malondialdehyde (MDA) activities in serum and brain tissue were recorded. Hematoxylin and eosin (HE) stained brain tissue was histopathologically examined. The expressions of Keap1, Nrf2 and heme oxygenase 1 (HO-1) in the brain tissue at the mRNA and protein levels were respectively detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot (WB). Moreover, an Illumina Hiseq platform was used for 16S ribosomal RNA (16S rRNA) high-throughput sequencing to evaluate the proportions of intestinal flora in aging mice. The proportions of saccharides, flavonoids, and triterpene acids were different between RPS and WPS. In the aging model mice, WPS outperformed RPS in improving body weight and mental state by increasing the spleen index, SOD and GSH-PX activities, decreasing the liver index and MDA activities, and restoring the histopathological morphology in D-gal-induced aging mice. At the mRNA levels, RPS and WPS significantly reduced the expression of Keap1 and increased the expressions of Nrf2 and HO-1. The trend in protein expressions was similar to that of the mRNA results, and WPS had a stronger effect than RPS. Fecal microbiota analysis showed that RPS and WPS restored intestinal microbiota proportions to normal levels. The results demonstrated that PS and its WPS had a positive effect in relieving oxidative stress in aging mice. WPS outperformed RPS, which might be related to the activation of the Keap1/Nrf2/ARE pathway and regulation of intestinal flora.

Lead induced cerebellar toxicology of developmental Japanese quail (Coturnix japonica) via oxidative stress-based Nrf2/Keap1 pathway inhibition and glutathione-mediated apoptosis signaling activation

Lead (Pb) is a heavy metal that has been recognized as a neurotoxin, meaning it can cause harmful effects on the nervous system. However, the neurotoxicology of Pb to birds still needs further study. In this study, we examined the neurotoxic effects of Pb exposure on avian cerebellum by using an animal model-Japanese quail (Coturnix japonica). The one-week old male chicks were exposed to 50, 200 and 500 mg/kg Pb of environmental relevance in the feed for five weeks. The results showed Pb caused cerebellar microstructural damages charactered by deformation of neuroglia cells, granule cells and Purkinje cells with Nissl body changes. Moreover, cerebellar neurotransmission was disturbed by Pb with increasing acetylcholine (ACh) and decreasing acetylcholinesterase (AChE), dopamine (DA), γ-Aminobutyric Acid (GABA) and Na+/K+ ATPase. Meanwhile, cerebellar oxidative stress was caused by Pb exposure represented by increasing reactive oxygen species (ROS) and malondialdehyde (MDA) as well as decreasing catalase (CAT), glutathione peroxidase (GPX), glutathione (GSH) and superoxide dismutase (SOD). Moreover, RNA-Seq analysis showed that molecular signaling pathways in the cerebellum were disrupted by Pb exposure. In particular, the disruption of nuclear factor erythroid-2-related factor 2 (Nfr2)/kelch-like ECH-associated protein 1 (Keap1) pathway and glutathione metabolism pathway indicated increasing cell apoptosis and functional disorder in the cerebellum. The present study revealed that Pb induced cerebellar toxicology through structural injury, oxidative stress, neurotransmission interference and abnormal apoptosis.

Mechanism of tetramethylpyrazine combined with neural stem cells transplantation on cerebral ischemia-reperfusion rats

This study aimed to investigate the intervention effect of tetramethylpyrazine(TMP) combined with transplantation of neural stem cells(NSCs) on middle cerebral artery occlusion(MCAO) rat model and to explore the mechanism of TMP combined with NSCs transplantation on ischemic stroke based on the regulation of stem cell biological behavior. MCAO rats were randomly divided into a model group, a TMP group, an NSCs transplantation group, and a TMP combined with NSCs transplantation group according to neurological function scores. A sham group was set up at the same time. The neurological function score was used to evaluate the improvement of neurological function in MCAO rats after TMP combined with NSCs transplantation. The proliferation, migration, and differentiation of NSCs were evaluated by BrdU, BrdU/DCX, BrdU/NeuN, and BrdU/GFAP immunofluorescence labeling. The protein expression of stromal cell-derived factor 1(SDF-1), C-X-C motif chemokine receptor 4(CXCR4), as well as oxidative stress pathway proteins nuclear factor erythroid 2-related factor 2(Nrf2), Kelch-like ECH-associated protein 1(KEAP1), heme oxygenase 1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1) was detected by Western blot to study the migration mechanism of TMP combined with NSCs. The results showed that TMP combined with NSCs transplantation significantly improved the neurological function score in MCAO rats. Immunofluorescence staining showed a significant increase in the number of BrdU~+, BrdU~+/DCX~+, BrdU~+/NeuN~+, and BrdU~+/GFAP~+ cells in the TMP, NSCs transplantation, and combined treatment groups, with the combined treatment group showing the most significant increase. Further Western blot analysis revealed significantly elevated expression of CXCR4 protein in the TMP, NSCs transplantation, and combined treatment groups, along with up-regulated protein expression of Nrf2, HO-1, and NQO1, and decreased KEAP1 protein expression. This study showed that both TMP and NSCs transplantation can promote the recovery of neurological function by promoting the proliferation, migration, and differentiation of NSCs, and the effect of TMP combined with NSCs transplantation is superior. The mechanism of action may be related to the activation of the Nrf2/HO-1/CXCR4 pathway.

Kaempferol sophoroside glucoside mitigates acetaminophen-induced hepatotoxicity: Role of Nrf2/NF-κB and JNK/ASK-1 signaling pathways

APAP (Acetaminophen)-induced hepatic injury is a major public health threat that requires continuous searching for new effective therapeutics. KSG (Kaempferol-3-sophoroside-7-glucoside) is a kaempferol derivative that was separated from plant species belonging to different genera. This study explored the protective effects of KSG on ALI (acute liver injury) caused by APAP overdose in mice and elucidated its possible mechanisms. The results showed that KSG pretreatment alleviated APAP-induced hepatic damage as it reduced hepatic pathological lesions as well as the serum parameters of liver injury. Moreover, KSG opposed APAP-associated oxidative stress and augmented hepatic antioxidants. KSG suppressed the inflammatory response as it decreased the genetic and protein expression as well as the levels of inflammatory cytokines. Meanwhile, KSG enhanced the mRNA expression and level of anti-inflammatory cytokine, IL-10 (interleukin-10). KSG repressed the activation of NF-κB (nuclear-factor kappa-B), besides it promoted the activation of Nrf2 signaling. Additionally, KSG markedly hindered the elevation of ASK-1 (apoptosis-signal regulating-kinase-1) and JNK (c-Jun-N-terminal kinase). Furthermore, KSG suppressed APAP-induced apoptosis as it decreased the level and expression of Bax (BCL2-associated X-protein), and caspase-3 concurrent with an enhancement of anti-apoptotic protein, Bcl2 in the liver. More thoroughly, Computational studies reveal indispensable binding affinities between KSG and Keap1 (Kelch-like ECH-associated protein-1), ASK1 (apoptosis signal-regulating kinase-1), and JNK1 (c-Jun N-terminal protein kinase-1) with distinctive tendencies for selective inhibition. Taken together, our data showed the hepatoprotective capacity of KSG against APAP-produced ALI via modulation of Nrf2/NF-κB and JNK/ASK-1/caspase-3 signaling. Henceforth, KSG could be a promising hepatoprotective candidate for ALI.

Molecular docking, dynamics, and preclinical experimental studies identify Morin hydrate as a potent PPAR-γ and NRF-2 transcription factor agonist that mitigates colon inflammation

Introduction: Peroxisome proliferator-activated receptor gamma (PPAR-γ) plays a vital role in regulating intestine inflammation. PPAR-γ is highly expressed in the adipose tissue and the colon. The nuclear erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein1 (Keap1) system protects cells from oxidative stress and inflammation. Therefore, PPAR-γ and Nrf2 ligands offer a drug-targeting system that can be exploited to treat inflammatory bowel diseases (IBDs), especially ulcerative colitis. IBD prevalence is on the rise globally. Current mainstream therapies for IBD offer protection but with many side effects; therefore, up to 30% of IBD patients turn to alternate therapy using bioactive phytochemicals. Morin hydrate, a naturally occurring bioflavonoid in many fruits, stems, and leaves of the Moraceae family plant, possesses potent anti-inflammatory properties. The docking studies reveal that it is a potent activator of the PPAR-γ and Nrf2 transcription factors. Therefore, in this study, we investigated the role of morin hydrate modulating colon epithelial PPAR-γ transcription in colon inflammation. Aim: Therefore, this study aims to investigate morin hydrate’s anti-inflammatory properties using both in vivo and in vitro models of colon inflammation. Methodology: C57BL/6J black mice were administered 2% dextran sodium sulfate (DSS) morin hydrate administered at concentrations of 25 &amp; 50mg/kg body for in vivo studies. The DAI, colon length, MPO, and histology changes were determined. Proinflammatory cytokines (IL-1β, IL-6, TNF-α, &amp; IL17A) were measured using ELISA and mRNA using real-time PCR. PPAR-γ and Nrf2 expression and phosphoNF-κB/NF-κB expression were estimated. HT-29 cells were cultured in DMEM media and were stimulated using TNF-α (1ng/ml). Different concentrations of morin hydrate were treated to HT-29 cells to determine a safe dose range for further in vitro experiments by estimating cell toxicity assay. CXCL-1, IL-8, and mRNA levels were determined using real-time PCR after morin hydrate co-treatment. Morin hydrate effect on PPAR-γ and Nrf2 promoter activity was determined using full-length promoter transfection studies. Results: Morin treatment significantly ( p&lt;0.01) decreased DAI, MPO level, and restored colon length dose-dependently in the treated group. Histological scoring for colonic inflammation was significantly ( p&lt;0.001) improved upon morin treatment. Morin treatment also inhibited the proinflammatory cytokines (IL-1β, IL-6, TNF-α, &amp; IL17A) significantly ( p&lt;0.01) both at protein and mRNA levels. Morin significantly inhibited COX-2 and iNOS both at protein and mRNA levels and also decreased tissue nitrite levels. Morin also activated PPAR-γ protein expression NRF2 nuclear translocation and inhibited NF-κB expression in DSS-administered mice. Morin significantly decreased antioxidant surrogate markers superoxide dismutase (SOD) and catalase activity. Morin significantly ( p&lt;0.01) decreased proinflammatory cytokine (CXCL-1 &amp; IL-8) mRNA expression when HT-29 cells were challenged with TNF-α. Morin also increased PPAR-γ and Nrf2 promoter activity. Conclusion: Morin hydrate is a potent PPAR-γ and Nrf2 transcription factor activator that mitigates colon inflammation through its anti-inflammatory and antioxidant activities. This is supported from UAE University grant, fund number 12R006 and 12R169. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Procyanidin A1 and its digestive products alleviate acrylamide-induced IPEC-J2 cell damage through regulating Keap1/Nrf2 pathway

Our previous study has revealed that procyanidin A1 (A1) and its simulated digestive product (D-A1) can alleviate acrylamide (ACR)-induced intestine cell damage. However, the underlying mechanism remains unknown. In this study, we elucidated the molecular mechanism for A1 and D-A1 to alleviate ACR-stimulated IPEC-J2 cell damage. ACR slightly activated nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and its target genes, but this activation could not reduce intestine cell damage. A1 and D-A1 could alleviate ACR-induced cell damage, but the effect was abrogated in cells transiently transfected with Nrf2 small interfering RNA (siRNA). Further investigation confirmed that A1 and D-A1 interacted with Kelch-like ECH-associated protein 1 (Keap1), which boosted the stabilization of Nrf2, subsequently promoted the translocation of Nrf2 into the nucleus, and further increased the expression of antioxidant proteins, thereby inhibiting glutathione (GSH) consumption, maintaining redox balance and eventually alleviating ACR-induced cell damage. Importantly, there was no difference between A1 and D-A1 treated groups, indicating that A1 can tolerate gastrointestinal digestion and may be a potential compound to limit the toxicity of ACR.

The Role of Endothelial Cell Specific Nrf2 on Skeletal Muscle Mitochondrial Function

Introduction: Cardiovascular disease (CVD) is a leading cause of death worldwide. Excessive reactive oxygen species (ROS) in the vasculature has been suggested as one of the major players in CVD. In addition, CVDs such as peripheral arterial disease (PAD) have elevated oxidative stress (OS) damage, stemming from an imbalance between antioxidants and ROS in both the vasculature and skeletal muscles. Although a vascular disease, the most common symptom of PAD is leg pain due to skeletal muscle ischemia and OS damage. The Nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is a redox transcription factor and is likely the master regulator of endogenous antioxidants. Modulation of Nrf2 expression may be a potential therapeutic target in CVDs. However, the role of endothelial cell (EC) Nrf2 on skeletal muscle mitochondrial function is currently unknown. Therefore, the purpose of this study was to examine the role of endothelial specific Nrf2 in skeletal muscle mitochondrial function.Hypothesis: We hypothesized that 1) EC-specific Nrf2-knockout (Tie2-Cre-Nrf2 Floxed) would result in blunted skeletal muscle mitochondrial function and 2) EC specific Keap1 knockout (Tie2-Cre-Keap1 Floxed) would improve skeletal muscle mitochondrial function. Methods: Gastrocnemius muscles were harvested from 6-month-old C57BL/6 mice (WT, n = 11), EC specific Nrf2-KO mice (n = 15) and EC specific Keap1-KO mice (n = 7). Muscle fibers were permeabilized and placed in a high-resolution respirometer to assess mitochondrial respiratory complex function (Oroboros Instruments). Results: Complex I state 3 respiration was blunted in Nrf2-KO compared to both Keap1-KO and WT (6.5±1.7 vs 13.6±3.5 vs 18.5±3.3 pmols−1･mg−1, respectively; p&lt;0.05). Complex I+II state 3 respiration was attenuated in Nrf2-KO compared to Keap1-KO (13.63±1.5 vs 27.8±4.2 pmols−1･mg−1; p&lt;0.05). Complex II state 3 respiration was increased in the Keap1-KO compared to both Nrf2-KO and WT (23.9±2.8 vs 7.1±1.1 vs 5.6±0.7 pmols−1･mg−1, respectively; p&lt;0.05) and Nrf2-KO exhibited reduced Complex IV respiration compared to both Keap1-KO and WT (19.5±6.1 vs 57.7±13.2 vs 76.6±12.4 pmols−1･mg−1, respectively; p&lt;0.05).Conclusions: This study provides novel findings showing skeletal muscle mitochondrial dysfunction is present in EC specific Nrf2-KO mice. Yet, EC specific Keap1-KO mice had increased mitochondrial respiration. To our knowledge, this is the first study to demonstrate a substantial impact that EC specific Nrf2 contributes to skeletal muscle mitochondrial function. Our findings provide evidence that the vascular EC Nrf2 pathway can be a potential therapeutic target for skeletal muscle dysfunction in CVDs such as PAD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Muscle p62 stimulates the expression of antioxidant proteins alleviating cancer cachexia

Cancer cachexia is characterized by a significant reduction in body weight predominantly resulting from severe loss of skeletal muscle. Oxidative stress plays an important role in skeletal muscle atrophy during cancer cachexia, and more glycolytic muscles are preferentially affected. Sequestosome1/SQSTM1 (i.e., p62), particularly when phosphorylated at Ser 349 (Ser 351 in mice), competitively binds to the Kelch-like ECH-associated protein 1 (Keap1) activating Nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 then stimulates the transcription of antioxidant/electrophile-responsive elements (ARE/EpRE) in target genes. However, a potential role for p62 in the protection of muscle wasting in cachexia remains to be determined. Here, using the well-established cachexia-inducing model of Lewis Lung Carcinoma (LLC) in mice we demonstrate higher expression of antioxidant proteins (i.e., NQO1, HO-1, GSTM1, CuZnSOD, MnSOD, and EcSOD) in the more oxidative and cachexia resistant soleus muscle than in the more glycolytic and cachexia prone extensor digitorum longus muscle. This was accompanied by higher p62 (total and phosphorylated) and nuclear Nrf2 levels in the soleus, which were paralleled by higher expression of proteins known to either phosphorylate or promote p62 phosphorylation (i.e., NBR1, CK1, PKCδ, and TAK1). Muscle-specific p62 gain-of-function (i.e., in p62 mTg mice) activated Nrf2 nuclear translocation and increased the expression of multiple antioxidant proteins (i.e., CuZnSOD, MnSOD, EcSOD, NQO1, and GSTM1) in glycolytic muscles. Interestingly, skeletal muscle Nrf2 haplodeficiency blunted the increases of most of these proteins (i.e., CuZnSOD, EcSOD, and NQO1) suggesting that muscle p62 stimulates antioxidant protein expression also via additional, yet to be determined mechanisms. Of note, p62 gain-of-function mitigated glycolytic muscle wasting in LLC-affected mice. Collectively, our findings identify skeletal muscle p62 as a potential therapeutic target for cancer cachexia. This research was supported by Grant-in-Aid for JSPS Fellows (20J15551), Grant-in-Aid for Early-Career Scientists (22K17733), Suzuken Memorial Foundation, The Nakatomi Foundation, and The Uehara Memorial Foundation (to M.Y.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Liquid Chromatography/Tandem Mass Spectrometry Analysis of Sophora flavescens Aiton and Protective Effects against Alcohol-Induced Liver Injury and Oxidative Stress in Mice.

In this study, we investigated the hepatoprotective effects of an ethanol extract of Sophora flavescens Aiton (ESF) on an alcohol-induced liver disease mouse model. Alcoholic liver disease (ALD) was caused by the administration of ethanol to male C57/BL6 mice who were given a Lieber-DeCarli liquid diet, including ethanol. The alcoholic fatty liver disease mice were orally administered ESF (100 and 200 mg/kg bw/day) or silymarin (50 mg/kg bw/day), which served as a positive control every day for 16 days. The findings suggest that ESF enhances hepatoprotective benefits by significantly decreasing serum levels of aspartate transaminase (AST) and alanine transaminase (ALT), markers for liver injury. Furthermore, ESF alleviated the accumulation of triglyceride (TG) and total cholesterol (TC), increased serum levels of superoxide dismutase (SOD) and glutathione (GSH), and improved serum alcohol dehydrogenase (ADH) activity in the alcoholic fatty liver disease mice model. Cells and organisms rely on the Kelch-like ECH-associated protein 1- Nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) system as a critical defensive mechanism in response to oxidative stress. Therefore, Nrf2 plays an important role in ALD antioxidant responses, and its level is decreased by increased reactive oxidation stress (ROS) in the liver. ESF increased Nrf2, which was decreased in ethanol-damaged livers. Additionally, four polyphenol compounds were identified through a qualitative analysis of the ESF using LC-MS/MS. This study confirmed ESF's antioxidative and hangover-elimination effects and suggested the possibility of using Sophora flavescens Aiton (SF) to treat ALD.