Naringin attenuates Actinobacillus pleuropneumoniae-induced acute lung injury via MAPK/NF-κB and Keap1/Nrf2/HO-1 pathway.

Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia (PCP), which is clinically characterized by acute hemorrhagic, necrotizing pneumonia, and chronic fibrinous pneumonia. Although many measures have been taken to prevent the disease, prevention and control of the disease are becoming increasingly difficult due to the abundance of APP sera, weak vaccine cross-protection, and increasing antibiotic resistance in APP. Therefore, there is an urgent need to develop novel drugs against APP infection to prevent the spread of APP. Naringin (NAR) has been reported to have an excellent therapeutic effect on pulmonary diseases, but its therapeutic effect on lung injury caused by APP is not apparent. Our research has shown that NAR was able to alleviate APP-induced weight loss and quantity of food taken and reduce the number of WBCs and NEs in peripheral blood in mice; pathological tissue sections showed that NAR was able to prevent and control APP-induced pathological lung injury effectively; based on the establishment of an in vivo/in vitro model of APP inflammation, it was found that NAR was able to play an anti-inflammatory role through inhibiting the MAPK/NF-κB signaling pathway and exerting anti-inflammatory effects; additionally, NAR activating the Nrf2 signalling pathway, increasing the secretion of antioxidant enzymes Nqo1, CAT, and SOD1, inhibiting the secretion of oxidative damage factors NOS2 and COX2, and enhancing the antioxidant stress ability, thus playing an antioxidant role. In summary, NAR can relieve severe lung injury caused by APP by reducing excessive inflammatory response and improving antioxidant capacity.

Wine- and stir-frying processing of Cuscutae Semen enhance its ability to alleviate oxidative stress and apoptosis via the Keap 1-Nrf2/HO-1 and PI3K/AKT pathways in H2O2-challenged KGN human granulosa cell line.

BACKGROUND: Cuscutae Semen (CS) has been prescribed in traditional Chinese medicine (TCM) for millennia as an aging inhibitor, an anti-inflammatory agent, a pain reliever, and an aphrodisiac. Its three main forms include crude Cuscutae Semen (CCS), wine-processed CS (WCS), and stir-frying-processed CS (SFCS). Premature ovarian insufficiency (POI) is a globally occurring medical condition. The present work sought a highly efficacious multi-target therapeutic approach against POI with minimal side effects. Finally, it analyzed the relative differences among CCS, WCS and SFCS in terms of their therapeutic efficacy and modes of action against H2O2-challenged KGN human granulosa cell line. METHODS: In this study, ultrahigh-performance liquid chromatography (UPLC)-Q-ExactiveTM Orbitrap-mass spectrometry (MS), oxidative stress indices, reactive oxygen species (ROS), Mitochondrial membrane potential (MMP), real-time PCR, Western blotting, and molecular docking were used to investigate the protective effect of CCS, WCS and SFCS on KGN cells oxidative stress and apoptosis mechanisms. RESULTS: The results confirmed that pretreatment with CCS, WCS and SFCS reduced H2O2-induced oxidative damage, accompanied by declining ROS levels and malondialdehyde (MDA) accumulation in the KGN cells. CCS, WCS and SFCS upregulated the expression of antioxidative levels (GSH, GSH/GSSG ratio, SOD, T-AOC),mitochondrial membrane potential (MMP) and the relative mRNA(Nrf2, Keap1, NQO-1, HO-1, SOD-1, CAT). They inhibited apoptosis by upregulating Bcl-2, downregulating Bax, cleaved caspase-9, and cleaved caspase-3, and lowering the Bax/Bcl-2 ratio. They also exerted antioxidant efficacy by partially activating the PI3K/Akt and Keap1-Nrf2/HO-1 signaling pathways. CONCLUSIONS: The results of the present work demonstrated the inhibitory efficacy of CCS, WCS and SFCS against H2O2-induced oxidative stress and apoptosis in KGN cells and showed that the associated mechanisms included Keap1-Nrf2/HO-1 activation, P-PI3K upregulation, and P-Akt-mediated PI3K-Akt pathway induction.

[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.

The therapeutic potential of isosakuranetin against perfluorooctane sulfonate instigated cardiac toxicity via modulating Nrf-2/Keap-1 pathway, inflammatory, apoptotic, and histological profile.

Perfluorooctane sulfonate (PFOS) is a pervasive organic toxicant that damages body organs, including heart. Isosakuranetin (ISN) is a plant-based flavonoid that exhibits a broad range of pharmacological potentials. The current investigation was conducted to evaluate the potential role of ISN to counteract PFOS-induced cardiac damage in rats. Twenty-four albino rats (Rattus norvegicus) were distributed into four groups, including control, PFOS (10 mg/kg) intoxicated, PFOS + ISN (10 mg/kg + 20 mg/kg) treated, and ISN (20 mg/kg) alone supplemented group. It was revealed that PFOS intoxication reduced the expressions of Nrf-2 and its antioxidant genes while escalating the expression of Keap-1. Furthermore, PFOS exposure reduced the activities of glutathione reductase (GSR), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), Heme oxygenase-1 (HO-1) and glutathione (GSH) contents while upregulating the levels of reactive oxygen species (ROS) and malondialdehyde (MDA). Besides, PFOS administration upregulated the levels of creatine kinase-MB (CK-MB), troponin I, creatine phosphokinase (CPK), and lactate dehydrogenase (LDH). Moreover, the levels of tumor necrosis factor-alpha (TNF-α), nuclear factor kappa-B (NF-κB), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) were increased after PFOS intoxication. Additionally, PFOS exposure downregulated the expression of Bcl-2 while upregulating the expressions of Bax and Caspase-3. Furthermore, PFOS administration disrupted the normal architecture of cardiac tissues. Nonetheless, ISN treatment remarkably protected the cardiac tissues via regulating aforementioned dysregulations owing to its antioxidative, anti-inflammatory, and antiapoptotic properties.

Dual Deletion of Keap1 and Rbpjκ Genes in Liver Leads to Hepatomegaly and Hypercholesterolemia.

The hepatic deletion of Rbpjκ (RbpjF/F::AlbCre) in the mouse leads to exhibition of the Alagille syndrome phenotype during early postnatal liver development with hyperlipidemia and cholestasis due to attenuated disruption of NOTCH signaling. Given the roles of NRF2 signaling in the regulation of lipid metabolism and bile ductal formation, it was anticipated that these symptoms could be alleviated by enhancing NRF2 signaling in the RbpjF/F::AlbCre mouse by hepatic deletion of Keap1 in compound Keap1F/F::RbpjF/F::AlbCre mice. Unexpectedly, these mice developed higher hepatic and plasma cholesterol levels with more severe cholestatic liver damage during the pre-weaning period than in the RbpjF/F::AlbCre mice. In addition, hypercholesterolemia and hepatic damage were sustained throughout the growth period unlike in the RbpjF/F::AlbCre mouse. These enhanced abnormalities in lipid metabolism appear to be due to NRF2-dependent changes in gene expression related to cholesterol synthetic and subsequent bile acid production pathways. Notably, the hepatic expression of Cyp1A7 and Abcb11 genes involved in bile acid homeostasis was significantly reduced in Keap1F/F::RbpjF/F::AlbCre compared to RbpjF/F::AlbCre mice. The accumulation of liver cholesterol and the weakened capacity for bile excretion during the 3 pre-weaning weeks in the Keap1F/F::RbpjF/F::AlbCre mice may aggravate hepatocellular damage level caused by both excessive cholesterol and residual bile acid toxicity in hepatocytes. These results indicate that a tuned balance of NOTCH and NRF2 signaling is of biological importance for early liver development after birth.

[Antioxidant activity of Euryale ferox seed shell extract and its therapeutic effects on oral ulcer in rats].

OBJECTIVE: To investigate the therapeutic effect of Euryale ferox seed shell extract on oral ulcer in rats and its underlying mechanism. METHODS: The contents of polyphenols and flavonoids in Euryale ferox seed shells were determined by Folin-phenol assay and aluminum nitrate colorimetry, respectively. DPPH·, ABTS+·, ·OH and·O2- scavenging experiments were performed to evaluate the antioxidant activities of Euryale ferox seed shell extract in vitro. In a rat model of oral ulcer induced by burning with glacial acetic acid, the therapeutic effect of Euryale ferox seed shell extract was assessed by detecting changes in serum levels of oxidative factors by enzyme-linked immunosorbent assay (ELISA) and observing pathological changes of the ulcerous mucosa using HE staining; the therapeutic mechanism of the extract was explored by detecting the expression levels of Keap1, Nrf2, Nes-Nrf2 and HO-1 proteins in ulcerous mucosa using Western blotting. RESULTS: The ethyl acetate extract of Euryale ferox seed shells contained 306.74±1.04 mg/g polyphenols and 23.43±0.61 mg/g flavonoids and had IC50 values for scavenging DPPH· and ABTS+· free radicals of 3.42 ± 0.97 µg/mL and 3.32 ± 0.90 µg/mL, respectively. In the rat models, the ethyl acetate extract significantly ameliorated oral mucosal ulcer, increased serum CAT level, and decreased serum MDA level. The protein expression levels of Nes-Nrf2 and HO-1 were increased and Keap1 protein expression was lowered significantly in the ulcerous mucosa of the rats after treatment with the extract (P<0.05 or 0.01). CONCLUSION: The therapeutic effect of Euryale ferox seed shell extract on oral ulcers in rats is mediated probably by activation of the Keap1/Nrf2/HO-1 signaling pathway.

Melatonin ameliorates 10-hydroxycamptothecin-induced oxidative stress and apoptosis via autophagy-regulated p62/Keap1/Nrf2 pathway in mouse testicular cells.

10-Hydroxycamptothecin (HCPT) is a widely used clinical anticancer drug but has a significant side effect profile. Melatonin has a beneficial impact on the chemotherapy of different cancer cells and reproductive processes, but the effect and underlying molecular mechanism of melatonin's involvement in the HCPT-induced side effects in cells, especially in the testicular cells, are poorly understood. In this study, we found that melatonin therapy significantly restored HCPT-induced testicular cell damage and did not affect the antitumor effect of HCPT. Further analysis found that melatonin therapy suppressed HCPT-induced DNA damage associated with ataxia-telangiectasia mutated- and Rad3-related and CHK1 phosphorylation levels in the testis. Changes in apoptosis-associated protein levels (Bax, Bcl-2, p53, and Cleaved caspase-3) and in reactive oxygen species-associated proteins (Nrf2 and Keap1) and index (malondialdehyde and glutathione) suggested that melatonin treatment relieved HCPT-induced cell apoptosis and oxidative damage, respectively. Mechanistically, melatonin-activated autophagy proteins (ATG7, Beclin1, and LC3bII/I) may induce p62-dependent autophagy to degrade Keap1, eliciting Nrf2 from Keap1-Nrf2 interaction to promote antioxidant enzyme expression such as HO-1, which would salvage HCPT-induced ROS production and mitochondrial dysfunction. Collectively, this study reveals that melatonin therapy may protect testicular cells from HCPT-induced damage via the activation of autophagy, which alleviates oxidative stress, mitochondrial dysfunction, and cell apoptosis.

Identification of ROS and KEAP1-related genes and verified targets of α-hederin induce cell death for CRC.

In this study, we analyzed and verified differentially expressed genes (DEGs) in ROS and KEAP1 crosstalk in oncogenic signatures using GEO data sets (GSE4107 and GSE41328). Multiple pathway enrichment analyses were finished based on DEGs. The genetic signature for colorectal adenocarcinoma (COAD) was identified by using the Cox regression analysis. Kaplan-Meier survival and receiver operating characteristic curve analysis were used to explore the prognosis value of specific genes in COAD. The potential immune signatures and drug sensitivity prediction were also analyzed. Promising small-molecule agents were identified and predicted targets of α-hederin in SuperPred were validated by molecular docking. Also, expression levels of genes and Western blot analysis were conducted. In total, 48 genes were identified as DEGs, and the hub genes such as COL1A1, CXCL12, COL1A2, FN1, CAV1, TIMP3, and IGFBP7 were identified. The ROS and KEAP1-associated gene signatures comprised of hub key genes were developed for predicting the prognosis and evaluating the immune cell responses and immune infiltration in COAD. α-hederin, a potential anti-colorectal cancer (CRC) agent, was found to enhance the sensitivity of HCT116 cells, regulate CAV1 and COL1A1, and decrease KEAP1, Nrf2, and HO-1 expression significantly. KEAP1-related genes could be an essential mediator of ROS in CRC, and KEAP1-associated genes were effective in predicting prognosis and evaluating individualized CRC treatment. Therefore, α-hederin may be an effective chemosensitizer for CRC treatments in clinical settings.

Echinatin attenuates acute lung injury and inflammatory responses via TAK1-MAPK/NF-κB and Keap1-Nrf2-HO-1 signaling pathways in macrophages.

Echinatin is an active ingredient in licorice, a traditional Chinese medicine used in the treatment of inflammatory disorders. However, the protective effect and underlying mechanism of echinatin against acute lung injury (ALI) is still unclear. Herein, we aimed to explore echinatin-mediated anti-inflammatory effects on lipopolysaccharide (LPS)-stimulated ALI and its molecular mechanisms in macrophages. In vitro, echinatin markedly decreased the levels of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated murine MH-S alveolar macrophages and RAW264.7 macrophages by suppressing inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) expression. Furthermore, echinatin reduced LPS-induced mRNA expression and release of interleukin-1ß (IL-1ß) and IL-6 in RAW264.7 cells. Western blotting and CETSA showed that echinatin repressed LPS-induced activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) pathways through targeting transforming growth factor-beta-activated kinase 1 (TAK1). Furthermore, echinatin directly interacted with Kelch-like ECH-associated protein 1 (Keap1) and activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway to enhance heme oxygenase-1 (HO-1) expression. In vivo, echinatin ameliorated LPS-induced lung inflammatory injury, and reduced production of IL-1ß and IL-6. These findings demonstrated that echinatin exerted anti-inflammatory effects in vitro and in vivo, via blocking the TAK1-MAPK/NF-κB pathway and activating the Keap1-Nrf2-HO-1 pathway.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Deuterium-Depleted Water in Cancer Therapy: A Systematic Review of Clinical and Experimental Trials.

Chemotherapy exhibits numerous side effects in anti-tumour therapy. The clinical experiments indicated that deuterium-depleted water (DDW) monotherapy or in combination with chemotherapy was beneficial in inhibiting cancer development. To further understand the potential mechanism of DDW in cancer therapy, we performed a systematic review. The data from experiments published over the past 15 years were included. PubMed, Cochrane and Web of Science (January 2008 to November 2023) were systemically searched. Fifteen studies qualified for review, including fourteen in vivo and in vitro trials and one interventional trial. The results showed that DDW alone or in combination with chemotherapy effectively inhibited cancer progression in most experiments. The combination treatment enhances the therapeutic effect on cancer compared with chemotherapeutic monotherapy. The inhibitory role of DDW in tumours is through regulating the reactive oxygen species (ROS)-related genes in Kelch-like ECH-associated protein 1 (Keap 1) and Nuclear erythroid 2-related factor 2 (Nrf2) signalling pathways, further controlling ROS production. An abnormal amount of ROS can inhibit the tumour progression. More extensive randomized controlled trials should be conducted to evaluate the accurate effect of DDW in Keap1-Nrf2 signalling pathways.

FGFR2-triggered autophagy and activation of Nrf-2 reduce breast cancer cell response to anti-ER drugs.

BACKGROUND: Genetic abnormalities in the FGFR signalling occur in 40% of breast cancer (BCa) patients resistant to anti-ER therapy, which emphasizes the potential of FGFR-targeting strategies. Recent findings indicate that not only mutated FGFR is a driver of tumour progression but co-mutational landscapes and other markers should be also investigated. Autophagy has been recognized as one of the major mechanisms underlying the role of tumour microenvironment in promotion of cancer cell survival, and resistance to anti-ER drugs. The selective autophagy receptor p62/SQSTM1 promotes Nrf-2 activation by Keap1/Nrf-2 complex dissociation. Herein, we have analysed whether the negative effect of FGFR2 on BCa cell response to anti-ER treatment involves the autophagy process and/or p62/Keap1/Nrf-2 axis. METHODS: The activity of autophagy in ER-positive MCF7 and T47D BCa cell lines was determined by analysis of expression level of autophagy markers (p62 and LC3B) and monitoring of autophagosomes' maturation. Western blot, qPCR and proximity ligation assay were used to determine the Keap1/Nrf-2 interaction and Nrf-2 activation. Analysis of 3D cell growth in Matrigel® was used to assess BCa cell response to applied treatments. In silico gene expression analysis was performed to determine FGFR2/Nrf-2 prognostic value. RESULTS: We have found that FGFR2 signalling induced autophagy in AMPKα/ULK1-dependent manner. FGFR2 activity promoted dissociation of Keap1/Nrf-2 complex and activation of Nrf-2. Both, FGFR2-dependent autophagy and activation of Nrf-2 were found to counteract the effect of anti-ER drugs on BCa cell growth. Moreover, in silico analysis showed that high expression of NFE2L2 (gene encoding Nrf-2) combined with high FGFR2 expression was associated with poor relapse-free survival (RFS) of ER+ BCa patients. CONCLUSIONS: This study revealed the unknown role of FGFR2 signalling in activation of autophagy and regulation of the p62/Keap1/Nrf-2 interdependence, which has a negative impact on the response of ER+ BCa cells to anti-ER therapies. The data from in silico analyses suggest that expression of Nrf-2 could act as a marker indicating potential benefits of implementation of anti-FGFR therapy in patients with ER+ BCa, in particular, when used in combination with anti-ER drugs.

Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways.

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

Cholesterol accumulation impairs HIF-1α-dependent immunometabolic reprogramming of LPS-stimulated macrophages by upregulating the NRF2 pathway.

Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis. Yet, how lipid loading modulates Mφ inflammatory responses remains unclear. We endeavored to gain mechanistic insights into how pre-loading with free cholesterol modulates Mφ metabolism upon LPS-induced TLR4 signaling. We found that activities of prolyl hydroxylases (PHDs) and factor inhibiting HIF (FIH) are higher in cholesterol loaded Mφs post-LPS stimulation, resulting in impaired HIF-1α stability, transactivation capacity and glycolysis. In RAW264.7 cells expressing mutated HIF-1α proteins resistant to PHDs and FIH activities, cholesterol loading failed to suppress HIF-1α function. Cholesterol accumulation induced oxidative stress that enhanced NRF2 protein stability and triggered a NRF2-mediated antioxidative response prior to and in conjunction with LPS stimulation. LPS stimulation increased NRF2 mRNA and protein expression, but it did not enhance NRF2 protein stability further. NRF2 deficiency in Mφs alleviated the inhibitory effects of cholesterol loading on HIF-1α function. Mutated KEAP1 proteins defective in redox sensing expressed in RAW264.7 cells partially reversed the effects of cholesterol loading on NRF2 activation. Collectively, we showed that cholesterol accumulation in Mφs induces oxidative stress and NRF2 stabilization, which when combined with LPS-induced NRF2 expression leads to enhanced NRF2-mediated transcription that ultimately impairs HIF-1α-dependent glycolytic and inflammatory responses.

Keap1/Nrf2 signaling pathway participating in the progression of epilepsy via regulation of oxidative stress and ferroptosis in neurons.

OBJECTIVE: This study aims to analyze the relationship between the Kelch-like ECH-associated protein 1 (Keap1)/Nuclear factor-erythroid 2-related factor 2 (Nrf2) and Epilepsy (EP), as well as its mechanism of action. METHODS: Thirty Wistar rats were divided into a control group (without treatment), a model group (EP modeling), and an inhibition group (EP modeling + intervention by Keap1/Nrf2 signaling pathway inhibitor ATRA) and subject to Morris water maze experiment. Then, the expression of Oxidative Stress (OS) markers, ferroptosis-associated proteins and Keap1/Nrf2 pathway in rat hippocampus was measured. In addition, rat hippocampal neuronal cell HT22 was purchased and treated accordingly based on the results of grouping, and cell proliferation and apoptosis in the three groups were determined. RESULTS: Compared with rats in the model group, those in the inhibition group showed shorter escape latency and an increased number of platform crossings (p < 0.05). Significant OS and neuron ferroptosis, increased apoptosis rate, elevated Keap1 expression, and decreased Nrf2 expression were observed in the model group compared to the control group (p < 0.05). The inhibition group exhibited notably improved OS and ferroptosis, as well as enhanced neuronal viability (p < 0.05). CONCLUSION: Inhibition of the Keap1/Nrf2 pathway can reverse the OS and neuron viability in EP rats.

Wheat peptide alleviates DSS-induced colitis by activating the Keap1-Nrf2 signaling pathway and maintaining the integrity of the gut barrier.

Inflammatory bowel disease (IBD) is difficult to cure, and formulating a dietary plan is an effective means to prevent and treat this disease. Wheat peptide contains a variety of bioactive peptides with anti-inflammatory and antioxidant functions. The results of this study showed that preventive supplementation with wheat peptide (WP) can significantly alleviate the symptoms of dextran sulfate sodium (DSS)-induced colitis in mice. WP can increase body weight, alleviate colon shortening, and reduce disease activity index (DAI) scores. In addition, WP improved intestinal microbial disorders in mice with colitis. Based on LC-MS, a total of 313 peptides were identified in WP, 4 of which were predicted to be bioactive peptides. The regulatory effects of WP and four bioactive peptides on the Keap1-Nrf2 signaling pathway were verified in Caco-2 cells. In conclusion, this study demonstrated that WP alleviates DSS-induced colitis by helping maintain gut barrier integrity and targeting the Keap1-Nrf2 axis; these results provided a rationale for adding WP to dietary strategies to prevent IBD.

Arginine deprivation enriches lung cancer proteomes with cysteine by inducing arginine-to-cysteine substitutants.

Many types of human cancers suppress the expression of argininosuccinate synthase 1 (ASS1), a rate-limiting enzyme for arginine production. Although dependency on exogenous arginine can be harnessed by arginine-deprivation therapies, the impact of ASS1 suppression on the quality of the tumor proteome is unknown. We therefore interrogated proteomes of cancer patients for arginine codon reassignments (substitutants) and surprisingly identified a strong enrichment for cysteine (R>C) in lung tumors specifically. Most R>C events did not coincide with genetically encoded R>C mutations but were likely products of tRNA misalignments. The expression of R>C substitutants was highly associated with oncogenic kelch-like epichlorohydrin (ECH)-associated protein 1 (KEAP1)-pathway mutations and suppressed by intact-KEAP1 in KEAP1-mutated cancer cells. Finally, functional interrogation indicated a key role for R>C substitutants in cell survival to cisplatin, suggesting that regulatory codon reassignments endow cancer cells with more resilience to stress. Thus, we present a mechanism for enriching lung cancer proteomes with cysteines that may affect therapeutic decisions.

Rosmarinic acid plus deferasirox inhibits ferroptosis to alleviate crush syndrome-related AKI via Nrf2/Keap1 pathway.

BACKGROUND: Myoglobin (Mb) induced death of renal tubular epithelial cells (RTECs) is a major pathological factor in crush syndrome-related acute kidney injury (CS-AKI). It is unclear whether ferroptosis is involved and could be a target for treatment. PURPOSE: This study aimed to evaluate the potential therapeutic effects of combining the natural small molecule rosemarinic acid (RA) and the iron chelator deferasirox (Dfe) on CS-AKI through inhibition of ferroptosis. METHODS: Sequencing data were downloaded from the GEO database, and differential expression analysis was performed using the R software limma package. The CS-AKI mouse model was constructed by squeezing the bilateral thighs of mice for 16 h with 1.5 kg weight. TCMK1 and NRK-52E cells were induced with 200 µM Mb and then treated with RA combined with Dfe (Dfe + RA, both were 10 µM). Functional and pathological changes in mouse kidney were evaluated by glomerular filtration rate (GFR) and HE pathology. Immunofluorescence assay was used to detect Mb levels in kidney tissues. The expression levels of ACSL4, GPX4, Keap1, and Nrf2 were analyzed by WB. RESULTS: We found that AKI mice in the GSE44925 cohort highly expressed the ferroptosis markers ACSL4 and PTGS2. CS-AKI mice showed a rapid decrease in GFR, up-regulation of ACSL4 expression in kidney tissue, and down-regulation of GPX4 expression, indicating activation of the ferroptosis pathway. Mb was found to deposit in renal tubules, and it has been proven to cause ferroptosis in TCMK1 and NRK-52E cells in vitro. We found that Dfe had a strong iron ion scavenging effect and inhibited ACSL4 expression. RA could disrupt the interaction between Keap1 andNrf2, stabilize Nrf2, and promote its nuclear translocation, thereby exerting antioxidant effects. The combination of Dfe and RA effectively reversed Mb induced ferroptosis in RTECs. CONCLUSION: In conclusion, we found that RA combined with Dfe attenuated CS-AKI by inhibiting Mb-induced ferroptosis in RTECs via activating the Nrf2/Keap1 pathway.

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).

[Stellera chamaejasme against multidrug resistance of triple-negative breast cancer MDA-MB-231 cell through Nrf2].

This study aims to investigate the effect and mechanism of Stellera chamaejasme extract(SCL) on multidrug resistance(MDR) in breast cancer. Human triple-negative breast cancer cell line MDA-MB-231 and its adriamycin-resistant cell line MDA-MB-231/ADR were used in the experiment. Cell viability was detected by methyl thiazolyl tetrazolium(MTT) assay, and cell apoptosis was detected by DAPI staining and Annexin-V/Pi double staining. Western blot(WB) was used to detect the expression levels of Keap1, Nrf2, HO-1, Bcl-2, Bax, caspase-9, and caspase-3. Immunofluorescence staining was used to observe the distribution of Nrf2 in the cell, and flow cytometry was used to detect the level of reactive oxygen species(ROS) in the cell. The results showed that the resis-tance factor of SCL was 0.69, and that of adriamycin and paclitaxel was 8.40 and 16.36, respectively. DAPI staining showed that SCL could cause nuclear shrinkage and fragmentation of breast cancer cells. Annexin-V/Pi double staining showed that the average apoptosis rate of the drug-resistant cells was 32.64% and 50.29%, respectively under medium and high doses of SCL. WB results showed that SCL could significantly reduce the expression levels of anti-apoptotic proteins Bcl-2, caspase-9, and caspase-3 and significantly increase the expression level of pro-apoptotic protein Bax. Further studies showed that SCL could significantly promote the expression of Keap1, significantly inhibit the expression of Nrf2 and HO-1, and significantly reduce the expression level of Nrf2 in the nucleus. Correspondingly, flow cytometry showed that the intracellular ROS level was significantly increased. In conclusion, SCL can significantly inhibit the proliferation of MDA-MB-231 multidrug-resistant cells of triple-negative breast cancer and cause cell apoptosis, and the mechanism is related to inhibiting Keap1/Nrf2 signaling pathway, leading to ROS accumulation in drug-resistant cells and increasing the expression of apoptosis-related proteins.