Kelch-like ECH-associated Protein Research Articles

Therapeutic Effects of Natural Products in the Treatment of Chronic Diseases: The Role in Regulating KEAP1-NRF2 Pathway.

Oxidative stress represents a pivotal mechanism in the pathogenesis of numerous chronic diseases. The Kelch-like ECH-associated protein 1-transcription factor NF-E2 p45-related factor 2 (KEAP1-NRF2) pathway plays a crucial role in maintaining redox homeostasis and regulating a multitude of biological processes such as inflammation, protein homeostasis, and metabolic homeostasis. In this paper, we present the findings of recent studies on the KEAP1-NRF2 pathway, which have revealed that it is aberrantly regulated and induces oxidative stress injury in a variety of diseases such as neurodegenerative diseases, cardiovascular diseases, metabolic diseases, respiratory diseases, digestive diseases, and cancer. Given this evidence, targeting KEAP1-NRF2 represents a highly promising avenue for developing therapeutic strategies for chronic diseases, and thus the development of appropriate therapeutic strategies based on the targeting of the NRF2 pathway has emerged as a significant area of research interest. This paper highlights an overview of current strategies to modulate KEAP1-NRF2, as well as recent advances in the use of natural compounds and traditional Chinese medicine, with a view to providing meaningful guidelines for drug discovery and development targeting KEAP1-NRF2. Additionally, it discusses the challenges associated with harnessing NRF2 as a therapeutic target.

Kurarinone Mitigates LPS-Induced Inflammatory Osteolysis by Inhibiting Osteoclastogenesis Through the Reduction of ROS Levels and Suppression of the PI3K/AKT Signaling Pathway.

Inflammatory bone resorption represents a pathological condition marked by an increase in bone loss, commonly associated with chronic inflammatory conditions such as rheumatoid arthritis and periodontitis. Current therapies primarilyfocuson anti-inflammatory drugs and bisphosphonates; however, these treatments are limited due to side effects, inadequate efficacy, and unpredictable long-term complications. Kurarinone (KR), a bioactive compound isolated from the traditional Chinese herb Sophora flavescens, exhibits a range of biological activities, including anti-inflammatory, anticancer, and cardiovascular protective effects. To address the limitations of existing therapies and enhance drug utilization, this study explores the potential of KR as a therapeutic agent for inflammatory bone resorption and delineates its underlying mechanisms. In vitro experiments reveal that KR notably inhibits osteoclastogenesis and reduces the expression of osteoclastic markers. Additionally, KR decreases the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, while downregulating NADPH oxidase 1 (NOX1) and Kelch-like ECH-associated protein 1 (Keap1) to diminish ROS production. Furthermore, KR activates the nuclear factor erythroid 2-related factor 2 (Nrf2), which enhances the activity of heme oxygenase-1 (HO-1) and catalase (CAT), facilitating the clearance of excess ROS. The compound also hinders osteoclast formation and functionality by inhibiting the PI3K/AKT/GSK-3β signaling pathway. Lentiviral knockdown of CAT can partially reverse these effects of KR. Meanwhile, in vivo experiments indicate that KR effectively mitigates bone loss in an LPS-induced inflammatory bone resorption model. In summary, KR is a promising new star in breaking through the limitations of previous drugs and treating inflammatory bone resorption.

Timosaponin B II as a novel KEAP1-NRF2 inhibitor to alleviate alcoholic liver disease:Receptor structure-based virtual screening and biological evaluation.

Oxidative stress induced by excess ethanol is an important factor in the progression of alcoholic liver disease (ALD). In recent years, inhibiting Kelch-like ECH-associated protein 1 (KEAP1) to activate the antioxidant regulator Nuclear factor erythroid 2-related factor 2 (NRF2) has been considered an effective strategy for treating oxidative stress-related diseases, but its application in ALD remains insufficiently explored. This study aims to discover high-affinity inhibitors targeting the KEAP1 receptor. We conducted virtual screening of a compound library based on a structure-based pharmacophore model, ultimately identifying the candidate compound Timosaponin B II (TBII). Subsequently, we established ALD models in AML-12 cells and C57BL/6 mice, and evaluated the therapeutic effects and mechanisms of TBII on ALD using methods including Immunofluorescence, Western blotting, RT-qPCR, Biochemical assays, and histological staining. Results indicate that TBII significantly improved ethanol-induced liver injury, inhibited the elevation of serum Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Total Cholesterol (T-CHO), and Triglycerides (TG) levels, and reduced lipid droplet accumulation in liver tissues. Furthermore, TBII treatment enhanced the antioxidant capacity of AML-12 cells and mouse liver, increasing Glutathione (GSH) and Superoxide Dismutase (SOD) levels while reducing Malondialdehyde (MDA) and Reactive Oxygen Species (ROS) levels. Mechanistic studies indicated that TBII inhibited the ethanol-induced increase in KEAP1 and reversed the ethanol-induced changes in NRF2 and its downstream targets. In conclusion, this study suggests that TBII may become a potential therapeutic agent for ALD by modulating the KEAP1-NRF2 pathway to alleviate oxidative stress and lipid metabolism abnormalities.

Construction of a lung adenocarcinoma prognostic model based on KEAP1/NRF2/HO‑1 mutation‑mediated upregulated genes and bioinformatic analysis.

Lung adenocarcinoma (LUAD) is a prevalent malignant tumor of the respiratory tract. The Kelch like ECH associated protein 1 (KEAP1)/nuclear factor erythroid 2-related factor 2 (NRF2)/heme oxygenase 1 (HO-1) axis serves a pivotal role in the occurrence and progression of LUAD. The present study aimed to identify specific genes regulated by mutations of the KEAP1/NRF2/HO-1 axis and to investigate their prognostic potential in LUAD, as well as their association with the tumor microenvironment. Immunohistochemistry was performed to assess the expression levels of KEAP1, NRF2 and HO-1 in LUAD tissues and to evaluate their association with clinical pathology. Sequencing data and clinical information were obtained from The Cancer Genome Atlas (TCGA)-LUAD and Gene Expression Omnibus (GSE68465) databases, whilst mutation information was sourced from the cBio Cancer Genomics Portal website. The R package 'limma' and Venn diagram were utilized to identify upregulated differentially expressed genes. Subsequently, a prognostic model was constructed using univariate Cox regression analysis and 101 machine learning methods. A nomogram of the prognostic model was generated to assess its efficacy in evaluating survival among patients with LUAD. The 'ImmuCellAI' and 'oncoPredict' R packages were used to compare and evaluate differences in immune cell infiltration and immunotherapy between high- and low-risk groups, as well as the sensitivity of LUAD to chemotherapy drugs. Compared with the group with negative expression, the results revealed that the group with positive expression of NRF2 and HO-1 exhibited advanced tumor, lymph node and clinical stages and a worse prognosis. A predictive model incorporating four genes (kynureninase, serpin family B member 5, insulin like 4 and γ-aminobutyric acid type A receptor subunit α3) was constructed based on KEAP1/NRF2/HO-1 mutation-mediated upregulated genes (KNHMUGs). Risk score was an independent prognostic factor for patients with LUAD (hazard ratio, 1.038; 95% confidence interval, 1.034-1.043; P<0.001). A nomogram was developed to predict the prognosis of patients with LUAD, which was validated as a reliable prognostic tool. The low-risk group exhibited higher immune cell infiltration, including CD4+ T, CD8+ T, natural killer (NK) and NKT cells, compared with the high-risk group. In addition, it demonstrated increased expression levels of immune checkpoint inhibitory genes such as cytotoxic T-lymphocyte associated protein 4, T cell immunoreceptor with Ig and ITIM domains, hepatitis A virus cellular receptor 2 and B and T lymphocyte associated protein. Moreover, it displayed enhanced sensitivity to immunotherapy. Drug sensitivity analysis revealed that the high-risk group exhibited increased sensitivity towards vinblastine, docetaxel and cisplatin, whereas the low-risk group showed increased sensitivity to BMS_754807, SB505124_1194 and JQ1_2172. In conclusion, a KNHMUGs-based gene signature was constructed in the present study, which holds promise as a biomarker for evaluating patient prognosis and guiding treatment by effectively assessing immunotherapy response and chemotherapy sensitivity in patients with LUAD.

Unleashing the Power of Covalent Drugs for Protein Degradation.

Targeted protein degradation (TPD) has emerged as a significant therapeutic approach for a variety of diseases, including cancer. Advances in TPD techniques, such as molecular glue (MG) and lysosome-dependent strategies, have shown substantial progress since the inception of the first PROTAC in 2001. The PROTAC methodology represents the forefront of TPD technology, with ongoing evaluation in more than 20 clinical trials for the treatment of diverse medical conditions. Two prominent PROTACs, ARV-471 and ARV-110, are currently undergoing phase III and II clinical trials, respectively. Traditional PROTACs are encountering obstacles such as limited binding affinity and a restricted range of E3 ligase ligands for facilitating the protein of interest (POI) degradation. Covalent medicines offer the potential to enhance PROTAC efficacy by enabling the targeting of previously considered "undruggable" shallow binding sites. Strategic alterations allow PROTAC to establish covalent connections with particular target proteins, including Kirsten rat sarcoma viral oncogene homolog (KRAS), Bruton's tyrosine kinase (BTK), epidermal growth factor receptor (EGFR), as well as E3 ligases such as DDB1 and CUL4 associated factor 16 (DCAF16) and Kelch-like ECH-associated protein 1 (Keap1). The concept of covalent degradation has also been utilized in various new forms of degraders, including covalent molecule glue (MG), in-cell click-formed proteolysis targeting chimera (CLIPTAC), HaloPROTAC, lysosome-targeting chimera (LYTAC) and GlueTAC. This review focuses on recent advancements in covalent degraders beyond covalent PROTACs and examines obstacles and future directions pertinent to this field.

Nrf2 Activation as a Therapeutic Target for Flavonoids in Aging-Related Osteoporosis.

Biological aging is a substantial change that leads to different diseases, including osteoporosis (OP), a condition involved in loss of bone density, deterioration of bone structure, and increased fracture risk. In old people, there is a natural decline in bone mineral density (BMD), exacerbated by hormonal changes, particularly during menopause, and it continues in the early postmenopausal years. During this transition time, hormonal alterations are linked to elevated oxidative stress (OS) and decreased antioxidant defenses, leading to a significant increase in OP. Aging is significantly associated with an abnormal ratio of oxidant/antioxidant and modified nuclear factor erythroid-derived two related factor2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway. OS adversely affects bone health by promoting osteoclastic (bone resorbing) activity and impairing osteoblastic (bone-forming cells). Nrf2 is critical in controlling OS and various cellular processes. The expression of Nrf2 is linked to multiple age-related diseases, including OP, and Nrf2 deficiency leads to unbalanced bone formation/resorption and a consequent decline in bone mass. Various drugs are available for treating OP; however, long-term uses of these medicines are implicated in diverse illnesses such as cancer, cardiovascular, and stroke. At the same time, multiple categories of natural products, in particular flavonoids, were proposed as safe alternatives with antioxidant activity and substantial anti-osteoporotic effects.

Regulation of TGF-β1, PI3K/PIP3/Akt, Nrf-2/Keap-1 and NF-κB signaling pathways to avert bifenthrin induced hepatic injury: A palliative role of daidzein.

Bifenthrin (BFN) is a noxious insecticide which is reported to damage various body organs. Daidzein (DZN) is a natural flavone with excellent pharmacological properties. This research was conducted to evaluate the alleviative strength of DZN to counteract BFN prompted liver toxicity in male albino rats. Thirty-two rats were divided into 4 groups i.e., the control, BFN (7 mg /kg), BFN (7 mg/kg) + DZN (20 mg/kg) and DZN (20 mg/kg) alone group. The biochemical assessment was performed by using qRT PCR as well as standard ELISA protocols. The findings are validated by applying pharmacodynamic techniques including molecular simulation. It was observed that BFN reduced the gene expressions of phosphoinositide 3-kinase (PI3K), phosphatidylinositol-3, 4, 5-triphosphate (PIP3), Protein kinase B (Akt), nuclear factor erythroid 2-related factor 2 (Nrf-2) while promoting the gene expressions of Kelch-like ECH-associated protein 1 (Keap-1). Moreover, BFN notably reduced the activities of glutathione reductase (GSR), heme-oxygenase-1 (HO-1), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) while elevating the levels of reactive oxygen species (ROS) and malondialdehyde (MDA). BFN promoted the levels of matrix metallopeptidase 2 (MMP-2), Procollagen III N-terminal Pro-peptide (PIIINP), alkaline phosphatase (ALP), transforming growth factor-beta-1 (TGF-β1), aspartate aminotransferase (AST), tissue inhibitor of matrix metalloproteinases 1 (TIMP1), and alanine aminotransferase (ALT). The levels of nuclear factor- kappa B (NF-κB), interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) were increased following the BFN intoxication. BFN enhanced the expressions of cysteine-aspartic acid protease-3 (Caspase-3) and Bcl-2-associated X protein (Bax) while suppressing the gene expression of B-cell lymphoma-2 (Bcl-2). Moreover, BFN disrupted the normal histology of liver tissues. Nonetheless, DZN treatment remarkably alleviated hepatic damages owing to its antioxidative, anti-apoptotic as well as anti-inflammatory abilities. However, DZN supplementation remarkably safeguarded which is further confirmed by in-silico assessment.

Ethyl Acetate Extract of Cichorium glandulosum Activates the P21/Nrf2/HO-1 Pathway to Alleviate Oxidative Stress in a Mouse Model of Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is a significant global health concern, primarily resulting from chronic alcohol consumption, with oxidative stress as a key driver. The ethyl acetate extract of Cichorium glandulosum (CGE) exhibits antioxidant and hepatoprotective properties, but its detailed mechanism of action against ALD remains unclear. This study investigates the effects and mechanisms of CGE in alleviating alcohol-induced oxidative stress and liver injury. Ultra-Performance Liquid Chromatography coupled with Quadrupole-Orbitrap Mass Spectrometry (UPLC-Q-Orbitrap-MS) was used to identify CGE components. A C57BL/6J mouse model of ALD was established via daily oral ethanol (56%) for six weeks, with CGE treatment at low (100 mg/kg) and high doses (200 mg/kg). Silibinin (100 mg/kg) served as a positive control. Liver function markers, oxidative stress indicators, and inflammatory markers were assessed. Transcriptomic and network pharmacology analyses identified key genes and pathways, validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. UPLC-Q-Orbitrap-MS identified 81 CGE compounds, mainly including terpenoids, flavonoids, and phenylpropanoids. CGE significantly ameliorated liver injury by reducing alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels and enhancing antioxidative markers such as total antioxidant capacity (T-AOC) and total superoxide dismutase (T-SOD) while lowering hepatic malondialdehyde (MDA) levels. Inflammation was mitigated through reduced levels of Tumor Necrosis Factor Alpha (TNF-α), Interleukin-1 Beta (IL-1β), and C-X-C Motif Chemokine Ligand 10 (CXCL-10). Transcriptomic and network pharmacology analysis revealed seven key antioxidant-related genes, including HMOX1, RSAD2, BCL6, CDKN1A, THBD, SLC2A4, and TGFβ3, validated by RT-qPCR. CGE activated the P21/Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)/Heme Oxygenase-1 (HO-1) signaling axis, increasing P21, Nrf2, and HO-1 protein levels while suppressing Kelch-like ECH-associated Protein 1 (Keap1) expression. CGE mitigates oxidative stress and liver injury by activating the P21/Nrf2/HO-1 pathway and regulating antioxidant genes. Its hepatoprotective effects and multi-target mechanisms highlight CGE's potential as a promising therapeutic candidate for ALD treatment.

NPA7: A Dual Receptor Activating Peptide That Inhibits Cardiac Oxidative Stress.

Cardiomyocyte oxidative stress significantly contributes to the progression of hypertension-induced heart failure, highlighting the need for targeted therapies. We developed a novel peptide, NPA7, that coactivates the GC-A (guanylyl cyclase A)/cGMP and MasR (Mas receptor)/cAMP pathway. This study aimed to test NPA7's ability to inhibit oxidative stress by modulating the p62-KEAP1 (Kelch-like ECH-associated protein 1)-NRF2 (nuclear factor erythroid 2-related factor 2) pathway in human cardiomyocytes (HCMs) and a rat model of hypertension. Oxidative stress was induced in HCMs using H2O2 with PBS or NPA7 treatment. Intracellular reactive oxygen species levels were assessed via dihydroethidium staining. Western blotting analysis measured p62, KEAP1, and NRF2 protein levels, while GSH/GSSG ratios and antioxidant gene expression were analyzed. HCMs were transfected with siRNA targeting GC-A, MasR, or p62 before NPA7 and H2O2 treatment. In vivo, spontaneously hypertensive rats received saline or NPA7, with normotensive WKY rats as control and cardiac oxidative stress, KEAP1 protein levels, NOX2, and p67 mRNA levels were measured. NPA7 reduced H2O2-induced reactive oxygen species levels and increased GSH/GSSG ratio in HCMs. Silencing GC-A and MasR reversed NPA7's effects. NPA7 activated the KEAP1-NRF2 pathway, enhancing NRF2's antioxidant target gene expression. In p62 knockdown HCMs, NPA7-induced KEAP1 degradation and NRF2 activation were diminished. Reactive oxygen species levels were elevated in spontaneously hypertensive rat versus WKY hearts however, NPA7 treatment reduced myocardial reactive oxygen species, suppressed KEAP1 protein, and decreased NOX2 and p67 mRNA levels. NPA7 exhibits antioxidant properties in HCMs and spontaneously hypertensive rat hearts by targeting GC-A and MasR through the p62-KEAP1-NRF2 pathway, supporting a novel therapeutic approach against cardiovascular disease-related oxidative stress.

An Azomethine Derivative, 1-(4-nitrophenyl)-N-phenylmethanimine (BCS2) Ameliorated 7,12-dimethylbenz(a)anthracene-induced Mammary Carcinoma through Nrf2-Keap1-HO-1 Pathway.

The aim of this study is the evaluation of an Azomethine derivative, BCS2, for its antioxidant and anti-tumor activities against mammary carcinoma through the Nrf2- Keap1-HO-1 pathway. The global prevalence of breast cancer is rising at an alarming rate. The facilitation of abnormal cell proliferation in mammary carcinoma occurs due to the disruption of signaling pathways that balance pro- and antioxidant status, thereby producing oxidative stress that disrupts genomic stability. Therefore, introducing a potent antioxidant molecule with antitumor activity is of paramount importance for treating breast cancer. Synthesis, characterization, and in-vitro, in-vivo, and in-silico evaluation of an Azomethine derivative, BCS2, for its antioxidant and anti-tumor activities against chemical carcinogen- induced mammary carcinogenesis in Sprague-Dawley rats. An azomethine derivative, 1-(4-nitrophenyl)-N-phenylmethanimine (BCS2), was synthesized and characterized based on its spectral data. The cytotoxic potential was observed on breast cancer cells, MCF-7, MDA-MB-231, and MDA-MB-468. The in vivo chemotherapeutic potential of BCS2 was established on 7,12-dimethylbenz(a)anthracene (DMBA) induced breast cancer in Sprague-Dawley (SD) rats. The effect of BCS2 on kelch-like ECH-associated protein- 1 (Keap1), Nrf2, heme oxygenase-1 (HO-1), mitogen-activated protein kinase (MAPK), and nuclear factor kappa-light-chain-enhancer of activated-B (NF-κB) was evaluated through ELISA and qPCR techniques. Furthermore, the binding potential and stability of BCS2 with Keap-1, HO-1, and MAPK were predicted using in silico molecular docking and dynamics studies. Additionally, drug-likeness properties of BCS2 were evaluated using in silico ADMET tools. BCS2 showed remarkable cytotoxic activity on MCF-7 cells followed by MDA-MB- 231 and MDA-MB-468 cells having an IC50 of 2.368μM, 4.843μM and 6.472μM respectively, without affecting normal breast cells, MCF-10A. In the DMBA-induced animal model, BCS2 showed potent antitumor potential and showed protective action on endogenous-enzymatic and non-enzymatic antioxidants in cancer-bearing animals. Marked improvement in cellular architecture and ultrastructure of breast/tumor tissues excised from experimental animals was noted through histopathological and field emission scanning electron microscopy (FESEM) analyses. Significant upregulation of antioxidant proteins, Keap1 and HO-1, and downregulation of inflammatory proteins, MAPK, and NF-κB was observed after BCS2 treatment. The in silico computational studies predicted the potent binding of BCS2 with the active pockets of Keap1, HO-1, and MAPK proteins that validated the biological findings. The study revealed BCS2's potent antioxidant and antitumor potential against mammary carcinoma through the Nrf2-Keap1-HO-1 signaling pathway.

Resveratrol enhances post-injury muscle regeneration by regulating antioxidant and mitochondrial biogenesis.

Resveratrol (RES), a natural polyphenolic compound, has shown promise in enhancing skeletal muscle regeneration and metabolic function. This study aims to explore the impact of RES on muscle regeneration after injury through the regulation of antioxidant capacity and mitochondrial biogenesis. RES treatment significantly increased the ratio of tibialis anterior muscle mass to body weight, alongside reduced fasting glucose levels. Following cardiotoxin-induced injury, RES treatment improved muscle regeneration, characterized by greater formation of new fibers and better structural repair compared to the control. Notably, gene expression analyses demonstrated that RES-treated mice exhibited elevated levels of myogenic markers, such as paired box 7 (Pax7), myogenic factor 5 (Myf5), myoblast determination protein (MyoD), and Myogenin (MyoG). Concurrently, yes-associated protein (YAP) increased, underscoring its role in regulating satellite cell activity. Transcriptomic analysis identified enriched pathways related to muscle regeneration and mitochondrial biogenesis, with increased expression of mitochondrial transcription factors and higher mitochondrial DNA content in RES-treated mice. Furthermore, RES enhanced antioxidant capacity via the Kelch-like ECH-associated protein 1 (KEAP-1)/nuclear factor erythroid 2-related factor 2 (NRF2)/heme oxygenase-1 (HO-1) signaling pathway, as indicated by elevated activities of total antioxidant capacity, Glutathione peroxidase (GSH-PX), and superoxidase dismutase (SOD). Collectively, these findings suggest that RES not only promotes muscle regeneration but also supports mitochondrial function and antioxidant defenses, positioning it as a food-derived pharmaceutical for targeting muscle repair after injury.

Heat stress induces oxidative stress and activates the KEAP1-NFE2L2-ARE pathway in reproduction-related cells.

Heat stress negatively affects the reproductive function of in animals and humans. Although a relationship between heat and oxidative stress has been suggested, the underlying mechanism has not been sufficiently examined in reproduction-related cells. Therefore, we aimed to investigate whether heat stress induces oxidative stress using a variety of reproduction-related cells including bovine placental and cumulus-granulosa cells, human cell lines derived from cervical and endometrial cancers, and fibroblasts derived from endometrium. Quantitative polymerase chain reaction analysis showed that the expression levels of representative heat and oxidative stress-related genes were significantly increased in cells cultured at high temperatures compared with those in cells cultured at basal temperatures. Moreover, luciferase reporter assays showed that the reporter activity of the heat shock element and antioxidant responsive element (ARE) was increased in cells cultured at high temperatures compared with that in cells cultured at basal temperatures. Furthermore, the stability of nuclear factor erythroid 2 like 2 (NFE2L2), a master regulator of the cellular stress response, increased under high temperatures. Point mutations in Kelch-like ECH-associated protein 1 (KEAP1) cysteine residues reduced the luciferase activity. Our results suggest that heat stress induces oxidative stress and that the KEAP1-NFE2L2-ARE pathway may play a protective role in reproduction-related cells against heat stress.

A Mechanism Study on the Antioxidant Pathway of Keap1-Nrf2-ARE Inhibiting Ferroptosis in Dopaminergic Neurons.

The pathology of Parkinson's disease (PD) indicates that iron deposition exists in dopaminergic neurons, which may be related to the death of cellular lipid iron peroxide. The extracellular autophagy adaptor SQSTM1(p62) of dopamine (DA) neurons can activate the intracellular Keap1-Nrf2-ARE signaling pathway to inhibit ferroptosis, which has a protective effect on DA neurons. The objective of this study was to investigate the protective mechanism of the Keap1- Nrf2-ARE antioxidant pathway against iron death in dopaminergic neurons. The experiment was divided into a control group (Control group), 1-methyl-4- phenylpyridiniumion control group (MPP+ Control group), p62 overexpression group (MPP+OVp62), and p62 overexpression no-load group (MPP+ OV-P62-NC). The inhibitors brusatol and ZnPP inhibited the activation of NF-E2-related factor 2(Nrf2) and Heme oxygenase-1(HO-1), respectively, and were divided into brusatol group (MPP+OV-p62+brusatol) and ZnPP group (MPP+OV-p62+ZnPP). RT-qPCR was used to detect transfection efficiency, and Cell Counting Kit-8 (CCK8) was used to detect cell activity. FerroOrange, 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA), and Liperfluo probes were used to detect intracellular iron, reactive oxygen species (ROS), and lipid peroxidation (LPO) levels. Western Blotting detected the levels of Nrf2, HO-1, Kelch-like ECH-associated protein1 (Keap1), and their downstream Glutathione peroxidase 4(GPX4) and Acyl-CoA synthetase long-chain family member 4(ACSL4). The levels of LGlutathione (GSH) and Malondialdehyde (MDA) were detected by GSH and MDA kits, and the activation of Keap1-Nrf2-ARE pathway was verified at the cellular level to have an antioxidant protective effect on iron death in dopaminergic neurons. (1) The results of RT-qPCR showed that compared with the control group, the expression of the p62 gene was significantly increased in the MPP+OV-p62 groups (P = 0.039), and the p62 gene was significantly increased in the brusatol and ZnPP groups, indicating successful transfection (P =0.002; P=0.008). (2) The immunofluorescence probe flow results showed that compared to the normal control group, the contents of three kinds of probes in MPP+ model group were significantly increased (P =0.001; P ＜0.001; P＜0.001), and the contents of three kinds of probes in MPP+OV-p62 group were decreased compared to the MPP+ model group (P =0.004). The results indicated that the levels of iron, ROS, and LPO were increased in the MPP+ group and decreased in the MPP+OV-p62 group. (3) Compared with the control group, the expressions of Nrf2, HO-1, and GPX4 in the MPP+OV-p62 group were increased (P =0.007; P =0.004; P=0.010), and the expressions of Keap1 and ACSL4 in MPP+p62 overexpression group were decreased (P =0.017; P =0.005). Compared with the MPP+ control group, Nrf2 and GPX4 were increased in the MPP+OV-p62 group, and ACSL4 was decreased in the MPP+OVp62 group (P =0.041; P ＜0.001; P ＜0.001). The results of the GSH and MDA kit showed that compared with the normal control group, the content of GSH in the MPP+ control group was decreased (P < 0.01), and the content of MDA was increased (P < 0.01). Compared with the MPP+ model group, GSH content was increased (P = 0.003), and MDA content was decreased in the MPP+OV-p62 group (P < 0.001). Nrf2, HO-1, and GPX4 increased in the MPP+p62 overexpression group but decreased in the brusatol group and ZnPP group (P < 0.001). Based on the transfection of P62 plasmid, it was found that P62 plasmid can inhibit the lipid peroxidation of iron death in dopaminergic nerve cells by activating the Nrf2 signaling pathway, thus playing a protective role in dopaminergic nerve cells.

Discovery of a novel pyrimidine derivative for treatment of acute lung injury through reducing oxidative stress and inflammatory response.

Acute lung injury (ALI) is a multifactorial respiratory disease characterized by uncontrolled inflammatory response and has high morbidity and mortality. There is currently a lack of effective drugs for ALI treatment. In this study, through nitric oxide (NO) release inhibition and cytotoxicity screening from the in-house compound library, hit compound 6 was discovered. Using 2,4,5-trichloropyrimidine as raw material, 27 new molecules were rapidly synthesized as modified products of compound 6 through nucleophilic substitution reaction and Buchwald-Hartwig reaction. Further activity evaluation and structure-activity relationship study confirmed that compound 32 was a low-toxicity, highly efficient lead compound. Action mechanism studies indicated that compound 32 can significantly reduce the inflammatory response induced by lipopolysaccharide (LPS) in RAW264.7 cells, manifested by the down-regulation of the levels of cytokines, reactive oxygen species (ROS), and the protein expression of Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) and Kelch-like ECH-associated protein-1/nuclear factor-erythroid 2-related factor 2/heme oxygenase-1 (Keap1-NRF2-HO-1). An in vivo anti-inflammatory study showed that it can reduce the severity of lung injury in the ALI model, accompanied by a reduction in the levels of inflammatory factors and related protein expression levels.

Daphnetin ameliorates intervertebral disc degeneration via the Keap1/Nrf2/NF-κB axis in vitro and in vivo.

Intervertebral disc degeneration (IVDD) is the primary cause of low back pain (LBP). Enhanced inflammation and reactive oxygen species (ROS) levels can cause apoptosis, which is one of the initial factors of IVDD. Our previous study showed that daphnetin (DAP) alleviates IVDD; however, the underlying mechanisms remain unknown. An IVDD mouse model was established by lumbar disc puncture to investigate the mechanisms of DAP regulation, and DAP was injected intraperitoneally. Moreover, nucleus pulposus cells (NPCs) were challenged with tumor necrosis factor-alpha (TNF-α)/H2O2 to mimic IVDD. Additionally, NPC apoptosis, ROS, and the expression of proinflammatory cytokines were comprehensively assessed. We found that DAP can reverse H2O2-induced ROS and play an anti-inflammatory role by inhibiting Nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Moreover, we found that DAP inhibits the apoptosis of NPCs induced by H2O2/TNF-α. DAP may regulate ROS production and apoptosis via the Kelch-like ECH-associated protein 1/NF-E2-related factor 2/heme oxygenase-1 (Keap1/Nrf2/HO-1) pathway. These findings were confirmed by in vivo results. The comprehensive nature of our research provides a strong foundation for the potential use of DAP as a therapeutic agent to alleviate IVDD.

5-(3-(N-(Carboxymethyl)naphthalene-2-sulfonamido)phenyl)-1-ethyl-1H-pyrrole-2-carboxylic acid as a Keap1-Nrf2 inhibitor for cerebral ischemia/reperfusion injury treatment.

The Keap1 (Kelch-like ECH-Associating Protein 1)-Nrf2 (Nuclear Factor Erythroid 2-Related Factor 2)-ARE (Antioxidant Response Element) signaling pathway plays a crucial role in the oxidative stress response and has been linked to the development and progression of various diseases. Its influence on cerebral ischemia/reperfusion (I/R) injury has garnered significant attention. In our study, we investigated the effect of compound 2, a non-covalent inhibitor of the Keap1-Nrf2 interaction, which was previously discovered by our research group. Specifically, we used 5-(3-(N-(carboxymethyl)naphthalene-2-sulfonamido)phenyl)-1-ethyl-1H-pyrrole-2-carboxylic acid (compound 2) to assess its therapeutic potential in a cerebral I/R injury model. The results demonstrated that compound 2 had a significant therapeutic effect, promoting the translocation of Nrf2 from the cytoplasm to the nucleus in diseased tissue. Additionally, it increased the production of key antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH).

Scopoletin ameliorates hyperlipidemia and hepatic steatosis via AMPK, Nrf2/HO-1 and NF-κB signaling pathways.

Scopoletin (SC) is one of the important phenolic coumarin constituents derived from many edible plants and fruits, and exerts a wide range of biological activities. In the present study, we investigated the effects of SC on tyloxapol (TY)-induced hyperlipidemia and hepatic steatosis in C57BL/6j mice and free fatty acid (FFA) 0.5mM-stimulated lipid accumulation in human L02 cells. Our results showed that TY injection significantly increased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), triglyceride (TG), total cholesterol (TC), low density lipoprotein (LDL-C) as well as malondialdehyde (MDA) in the livers of the mice (p<0.001, respectively), and decreased serum levels of high density lipoprotein (HDL-C), IL-10 levels as well as superoxide dismutase (SOD) in the livers (p<0.001, respectively). On the other hand, SC pretreatment reversed these changes. SC obviously alleviated TY-induced liver steatosis by upregulating the AMP-activated kinase (AMPK), acetyl-CoA carboxylase (ACC) phosphorylation, and significantly downregulated sterol regulatory element binding protein (SREBP)1c and fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD1), Lipin 1, phospho-hormone-sensitive triglyceride lipase (p-HSL) proteins and SREBP-2, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) mRNA expressions. In the meantime, SC upregulated the expressions of the lipolysis-associated genes LDL receptor (LDLR), adipose triglyceride lipase (ATGL), and HSL. In addition, SC significantly inhibited TNF-α, F4/80, caspase-1 (cas-1), cas-1p10, IL-1β, Kelch-like ECH-associated protein 1 (Keap1) expressions, nuclear factor-kappa B (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2) translocation, and increased heme oxygenase 1 (HO-1) expressions in TY-induced hyperlipidemia and hepatic steatosis mice. The in vivo results were similar to that those in the in vitro experiment, for example, SC markedly lowered TG and TC levels and protected lipid accumulation via AMPK, NF-κB, and Nrf2/HO-1 signaling pathway in FFA-induced L02 cells. These results indicate that SC has protective potential against hyperlipidemia and hepatic steatosis, and the underlying mechanism may be closely associated with AMPK activation and Nrf2/HO-1 and NF-κB inhibition.

Inhibition of MALAT1 facilitates ROS accumulation via the Keap1/HO-1 pathway to enhance photodynamic therapy in secondary hyperparathyroidism.

The prevalence of secondary hyperparathyroidism (SHPT) in advanced chronic kidney disease (CKD) exceeds 80%. Our previous study indicated that photodynamic therapy (PDT) has potential for treating SHPT. Long noncoding RNA (lncRNA) is involved in various oxidative stress and apoptotic processes, but the molecular mechanism remains unreported. In this study, we found that PDT induced apoptosis in SHPT through reactive oxygen species (ROS) accumulation. The expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and heme oxygenase 1 (HO-1) within SHPT was upregulated after PDT. Inhibition of MALAT1 increased PDT-induced ROS, which promoted the apoptosis. Pearson correlation analysis confirmed that there was a positive correlation between MALAT1 and HO-1, and MALAT1 inhibition down-regulated HO-1, whereas concomitant overexpression of HO-1 was able to eliminate the PDT-induced ROS and inhibit apoptosis. The direct binding of MALAT1 to Kelch-like ECH-associated protein 1 (Keap1) protein was confirmed by high-throughput sequencing, RNA pulldown, silver staining and western blotting assays. Si-Keap1 was able to rescue the down-regulation of HO-1 caused by MALAT1 inhibition, restoring the elimination of ROS by HO-1 and attenuating the effect of PDT. In addition, PDT effectively reduced parathyroid hormone (PTH) secretion in SHPT rats, and this effect was further enhanced in combination with MALAT1 inhibitors. Overall, MALAT1 activates downstream HO-1 expression by binding to Keap1, thereby reducing ROS and inhibiting apoptosis, which in turn mediates PDT resistance in SHPT. Inhibition of MALAT1 significantly enhanced the efficacy of PDT, suggesting a potential therapeutic target for improving PDT for SHPT outcomes.

Psoralen and Isopsoralen Activate Nuclear Factor Erythroid 2-Related Factor 2 Through Interaction With Kelch-Like ECH-Associated Protein 1.

As natural furocoumarins, psoralen and its isomer isopsoralen are widely distributed in various fruits including Ficus carica L., vegetables including celery, and medicinal herbs including Psoralea corylifolia L. Although psoralen and isopsoralen have been used as dietary supplements because of their bioactivities such as antibacterial and anti-inflammatory properties; however, the potential mechanisms underlying the antioxidant activities of these two furocoumarins still need to be explored. Hence, the aims of this work were to examine the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) by psoralen and isopsoralen, as well as the binding interaction of Kelch-like ECH-associated protein 1 (Keap1) with these two furocoumarins. Interestingly, both psoralen and isopsoralen induced Nrf2 nuclear translocation in a dose-dependent manner in HEK293T cells. These two furanocoumarins also activated antioxidant response element (ARE)-driven luciferase activity. The mRNA expression of GCLM, HO-1, and NQO1 genes was significantly upregulated by treatment of HEK293T cells with psoralen and isopsoralen, respectively. Similarly, the expression of proteins can be promoted. Both psoralen and isopsoralen were located in the top of the central pocket of the Keap1 Kelch domain, suggesting that they were natural ligands of Keap1. In conclusion, both psoralen and isopsoralen activate Nrf2 through interaction with Keap1, thereby serving as natural antioxidants.

Kelch-like ECH-associated Protein 1/Nuclear Factor Erythroid 2-related Factor 2 Pathway and Its Interplay with Oncogenes in Lung Tumorigenesis.

Nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor regulating cellular redox homeostasis, exhibits a complex role in cancer biology. Genetic mutations in the Kelch-like ECH-associated protein 1 (KEAP1)/NRF2 system, which lead to NRF2 hyperactivation, are found in 20% to 30% of lung cancer cases. This review explores the intricate interplay between NRF2 and key oncogenic pathways in lung cancer, focusing on the interaction of KEAP1/NRF2 system with Kirsten rat sarcoma virus (KRAS), tumor protein P53 (TP53), epidermal growth factor receptor (EGFR), and phosphatidylinositol 3-kinases (PI3K)/AKT signaling. While NRF2 activation alone is insufficient to initiate tumorigenesis, it can significantly impact tumor initiation and progression when combined with oncogenic drivers such as KRAS. The review highlights the context-dependent effects of NRF2, from its protective role against chemical carcinogen-induced tumor initiation to its potential promotion of tumor growth in established cancers. These findings suggest the need for nuanced, stage-specific approaches to targeting the NRF2 pathway in cancer therapy.