Nrf2 Inhibitor Research Articles

Acteoside alleviates salsolinol-induced Parkinson's disease by inhibiting ferroptosis via activating Nrf2/SLC7A11/GPX4 pathway

Salsolinol (SAL), i.e.1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroiso-quinoline, is a dopamine metabolite and endogenous neurotoxin that is toxic to dopaminergic neurons, and is involved in the genesis of Parkinson's disease (PD). However, the machinery underlying SAL induces neurotoxicity in PD are still being elucidated. In the present study, we first used RNA sequencing (RNAseq) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to detect differentially expressed genes in SAL-treated SH-SY5Y cells. We found that ferroptosis-related pathway was enriched by SAL, which was validated by in vitro and in vivo SAL models. SAL inducing ferroptosis through downregulating SLC7A11/GPX4 in SH-SY5Y cells, which neurotoxic effect was reversed by ferroptosis inhibitors deferoxamine (DFO) and ferrostatin-1 (Fer-1). Acteoside, a phenylethanoid glycoside of plant origin with neuroprotective effect, attenuates SAL-induced neurotoxicity by inhibiting ferroptosis in in vitro and in vivo PD models through upregulating SLC7A11/GPX4. Mechanistically, acteoside activates Nrf2. Nrf2 inhibitor ML385 abolished acteoside-mediated increased SLC7A11/GPX4 and neuroprotection against SAL in SH-SY5Y cells. Meanwhile, the PI3K inhibitor LY294002 suppressed the acteoside-induced Nrf2 expression and ensued decreased expression of SLC7A11/GPX4 in SAL-treated SH-SY5Y cells. Taken together, these results demonstrate that salsolinol-induced PD through inducing ferroptosis via downregulating SLC7A11/GPX4. Acteoside attenuates SAL-induced PD through inhibiting ferroptosis via activating PI3K/Akt-dependant Nrf2. The present study revealed a novel molecular mechanisms underlining SAL-induced neurotoxicity via induction of ferroptosis in PD, and uncovered a new pharmacological effect against PD through inhibiting ferroptosis. This study highlights SAL-induced ferroptosis -dependent neurotoxicity as a potential therapeutic target in PD.

TWEAK/Fn14 disrupts Th17/Treg balance and aggravates conjunctivitis by inhibiting the Nrf2/HO-1 pathway in allergic conjunctivitis mice.

Allergic conjunctivitis (AC) affects people's daily life and work, especially the health of children. Although there are few relevant studies, Th17/Treg imbalance plays an important role in AC development. The aim of this study was to elucidate the effect of TWEAK/Fn14 on AC and Th17/Treg balance. Ovalbumin induced AC mouse model was utilized to observe the mechanism of TWEAK/Fn14 in vivo. Conjunctivitis was evaluated by hematoxylin-eosin staining, toluidine blue staining and AC clinical score. Flow cytometry was used to measure Th17 and Treg cell ratios. The level of Th17/Treg balance related factors and Nrf2/HO-1 signal was detected by ELISA, WB, qRT-PCR and immunohistochemistry. In the AC state, disruption of Th17/Treg cell balance, increased TWEAK/Fn14 signaling level and conjunctival inflammation were observed. After TWEAK knockdown, Th17 cell differentiation was inhibited, Treg cell differentiation was promoted, and AC symptoms were alleviated in AC mice. Moreover, TWEAK knockdown caused an enhancement of the Nrf2/HO-1 signaling pathway in the AC models. Treatment with Nrf2 inhibitor reversed these changes induced by TWEAK knockdown. Therefore, TWEAK/Fn14 regulated the Nrf2/HO-1 pathway to affect Th17/Treg cell balance and conjunctivitis in AC mouse models. In summary, TWEAK/Fn14 caused Th17/Treg imbalance by inhibiting Nrf2/HO-1 pathway, which might be one potential mechanism of the exacerbation of AC.

Therapeutic potential of hesperidin in diabetes mellitus-induced erectile dysfunction through Nrf2-mediated ferroptosis and oxidative stress.

Among erectile dysfunction (ED) caused by metabolic abnormalities, diabetes mellitus-induced ED (DMED) progresses rapidly, manifests with severe symptoms, and shows reduced responsiveness to conventional medications. Hyperglycemia in the corpus cavernosum has been linked to the induction of both ferroptosis and oxidative stress, which are mediated by nuclear factor E2 related factor 2 (Nrf2). Hesperidin (Hes), a flavonoid compound, has been revealed to activate Nrf2 in certain diabetic complications, yet the efficacy of Hes on DMED and the specific mechanism remain unclear. To elucidate the potential mechanism and efficacy of Hes in regulating Nrf2-mediated ferroptosis and oxidative stress in DMED. DMED rats were constructed through the intraperitoneal injection of streptozotocin (STZ), partially supplemented with Hes. In parallel, in vitro research utilized human umbilical vein endothelial cells (HUVECs), with glucose addition to simulating a high glucose (HG) environment, and induced with Hes or ML385 (an Nrf2 inhibitor). Penile tissues and HUVECs were harvested for subsequent analyses. The results of this study indicate that Hes partially reversed the impaired erectile function. The expression of Nrf2, glutathione peroxidase 4 (GPX4), and heme oxygenase-1 (HO-1) in the corpus cavernosum elevated after supplementing with Hes, resulted in an inhibition in ferroptosis and oxidative stress. Moreover, the quantity and function of erectile effector cells were restored, and cavernous fibrosis was ameliorated. In HG-induced HUVECs, Hes ameliorated Nrf2-mediated ferroptosis and oxidative stress, effects which ML385 partially reversed. Hes exerts a therapeutic effect on DMED rats and a regulatory mechanism on the Nrf2-HO-1/GPX4 axis, concurrently revitalizing endothelial and smooth muscle cells, and diminishing fibrosis. Our study provides robust preclinical evidence for employing Hes in treating DMED.

Paeonol attenuates LPS-induced inflammatory injury in mastitis by regulating the MAPK and Nrf2 signaling pathways

Mastitis is an inflammatory udder disease that causes important economic losses in the animal breeding and dairy product industries. While antibiotics have contributed to disease control, their use presents risks due to drug residues in livestock products that can potentially threaten human health. Paeonol, an active ingredient extracted from the dried root bark of paeonia suffruticosa, exhibits various pharmacological effects, including anti-inflammatory and antibacterial properties, but its role in mastitis remains unclear. In this study, we first evaluated the effects of paeonol on LPS-induced mastitis in mice and further explored the potential anti-inflammatory mechanism of paeonol on bovine mammary epithelial cells (BMECs). The results showed that paeonol alleviated LPS-induced mouse mastitis by inhibiting the infiltration of inflammatory cells and the release of inflammatory factors. Cellular assays further demonstrated that paeonol reduced apoptosis and inhibited inflammation and oxidative stress injury by mechanisms involving activation of Nrf2 signaling and inhibition of MAPK signaling in BMECs. Collectively, our results suggest that paeonol holds promise as a therapeutic agent for alleviating LPS-induced mastitis injury while providing novel insights into potential drug targets for mastitis treatment.

Abstract B014: NRF2 regulation of the immune microenvironment in breast cancer

Abstract Immune checkpoint inhibitors (ICIs) have emerged as a significant advance in the treatment of various cancer types, including breast cancer, though their efficacy varies widely depending on the cancer subtype and the stage of the disease. Following their initial approval in metastatic triple-negative breast cancer (TNBC), ICIs have shown promising results in other contexts, including early-stage cancers and ER+ and HER2+ subtypes. However, even in advanced TNBC the objective response rate to monotherapy ICI is only ∼20%. These results highlight the need to better understand determinants of ICI response in breast cancer. A detailed, high-resolution definition of the tumor immune microenvironment (TIME) will be critical for predicting and engineering effective responses to ICIs. In previously published work, we and others found that the antioxidant transcription factor NRF2 is constitutively active in breast cancer and is associated with tumor recurrence, metastasis, and poor prognosis. However, the mechanisms through which NRF2 promotes breast cancer progression remain unclear. In the current study, we explore the role of NRF2 in reshaping the TIME and its impact on breast cancer progression using preclinical models, flow cytometry, and transcriptomic analyses. We find that NRF2 is constitutively activated in a subset of human and mouse breast cancers where it functions to suppress inflammatory gene expression. In models of recurrent and metastatic mammary tumors, NRF2 knockdown results in a marked reduction in tumor growth rates. Furthermore, the NRF2 inhibition significantly alters the composition of innate and adaptive immune cell populations within the tumor microenvironment. Cytokine profiling revealed that these immune alterations are associated with increased levels of inflammatory cytokines in NRF2-deficient tumor cells compared to controls. RNA sequencing of tumor cells further supports the observed shifts in the immune landscape associated with NRF2 deficiency. Collectively, our findings demonstrate that the loss of NRF2 impairs tumor growth in part through remodeling the TIME and suggest that NRF2 may be a promising therapeutic target for sensitizing breast cancers to ICIs. Citation Format: Yasemin Ceyhan Ozdemir, James V. Alvarez. NRF2 regulation of the immune microenvironment in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B014.

Formononetin Exerts Neuroprotection in Parkinson's Disease via the Activation of the Nrf2 Signaling Pathway.

Parkinson's disease (PD) is a prevalent neurodegenerative disease for which no effective treatment currently exists. In this study, we identified formononetin (FMN), a neuroprotective component found in herbal medicines such as Astragalus membranaceus and Glycyrrhiza uralensis, as a potential agent targeting multiple pathways involved in PD. To investigate the anti-PD effects of FMN, we employed Caenorhabditis elegans (C. elegans) PD models, specifically the transgenic strain NL5901 and the MPP(+)-induced strain BZ555, to investigate the effects of FMN on the key pathological features of PD, including dyskinesia, dopamine neuron damage, and reactive oxygen species (ROS) accumulation. The MPP(+)-induced SH-SY5Y cell PD model was utilized to evaluate the effects of FMN on cell viability, ROS accumulation, and mitochondrial dysfunction. The signaling pathway induced by FMN was analyzed using transcriptomic techniques and subsequently validated in vitro. Our results indicate that FMN significantly reduced ROS accumulation and improved both dopaminergic neuron vitality and dyskinesia in the C. elegans PD models. In the cell PD model, FMN significantly reduced ROS accumulation and enhanced mitochondrial membrane potential (MMP) and cell viability. A transcriptomic analysis suggested that the effects of FMN are associated with Nrf2 activation. Furthermore, ML385, a specific Nrf2 inhibitor, blocked the beneficial effects of FMN in vitro, indicating that FMN ameliorates dyskinesia and protects dopaminergic neurons through Nrf2 signaling pathway activation. In addition, the effects of FMN on ameliorating dyskinesia and protecting dopamine neurons were comparable to those of the Nrf2 agonist of sulforaphane (SFN) in vivo. The results of this study confirm that FMN exerts significant anti-PD effects primarily through the Nrf2 signaling pathway. These findings provide crucial insights for the development of anti-PD therapies.

ML385 promotes ferroptosis and radiotherapy sensitivity by inhibiting the NRF2-SLC7A11 pathway in esophageal squamous cell carcinoma

Radiotherapy is important in treating esophageal squamous cell carcinoma (ESCC) comprehensively. Resistance to radiotherapy is a prominent factor contributing to treatment failure in patients with ESCC. The objective of this study was to investigate the impact of ML385, an inhibitor of nuclear factor erythroid 2-related factor 2 (NRF2), on the radiosensitivity of ESCC and elucidate its underlying mechanism. We treated KYSE150 and KYSE510 cells with ML385 and ionising radiation separately or simultaneously, and observed the proliferation, apoptosis, cell cycle and ferroptosis of different conditions by colony formation assay and flow cytometry. Our findings reveal that NRF2 was activated by radiation and translocated from the cytoplasm to the nucleus after radiation. However, ML385 inhibited the expression and cytoplasm-to-nucleus translocation of NRF2. Compared with radiation, ML385 combined with radiation exhibited a significant inhibition on the clone formation ability of ESCC cells, induced apoptosis and promoted G2/M phase arrest. The treatment of ML385 combined with radiation markedly increased ROS and lipid peroxidation levels and decreased glutathione levels compared with the control, thus promoting the occurrence of ferroptosis. In addition, the expression trend of NRF2 was the same as that of proteins related ferroptosis, such as SLC7A11 and GPX4. After overexpression of SLC7A11, we found that significantly restored glutathione levels and alleviated ML385 combined with radiation-induced lipid peroxidation, indicating that ML385 plays a key role in radiotherapy sensitization by inhibiting the NRF2-SLC7A11 pathway. In vivo, ML385 also promoted the killing effect of radiation on xenografted tumours in nude mice. This study identifies NRF2 inhibitor ML385 as a radiosensitizer of ESCC, which highlights the therapeutic potential of the NRF2-SLC7A11 pathway and provides a deeper understanding of the mechanism of ferroptosis in esophageal squamous cell carcinoma.

Triptolide induces hepatotoxicity by promoting ferroptosis through Nrf2 degradation

BackgroundTriptolide (TP), a principal active substance from Tripterygium wilfordii, exhibits various pharmacological effects. However, its potential hepatotoxicity has always been a significant concern in clinical applications.PurposeThis research aimed to explore the involvement of ferroptosis in TP-mediated hepatic injury and the underlying mechanisms.MethodsIn this study, in vitro and in vivo experiments were involved. Hepatocyte damage caused by TP was evaluated using MTT assays, liver enzyme measurement and H&E staining technique. Ferroptosis was assessed by measuring iron level, lipid peroxide, glutathione (GSH), mitochondrial morphology and the key protein/mRNA expression implicated in ferroptosis. To verify the contribution of ferroptosis to TP-induced liver damage, the ferroptosis inhibitor Ferrostatin-1 (Fer-1) and a plasmid for overexpressing glutathione peroxidase 4 (GPX4) were employed. Subsequently, nuclear factor erythroid 2-related factor 2 (Nrf2) knockout mice and Nrf2 overexpression plasmid were utilized to investigate the underlying mechanisms. Nontargeted lipidomics was used to analyze lipid metabolism in mouse liver. Moreover, the cellular thermal shift assay (CETSA), cycloheximide (CHX) and MG132 treatments, and immunoprecipitation (IP) assays were applied to validate the binding of TP to Nrf2 and their interactions.ResultsTP triggered ferroptosis in hepatocytes, as indicated by iron accumulation and lipid peroxidation. Ferroptosis was responsible for TP-induced hepatic injury. During the process of TP-induced liver damage, the Nrf2 signaling pathway was significantly suppressed. Notably, the deletion of Nrf2 in mice aggravated the extent of liver injury and ferroptosis associated with TP, whereas enhancing Nrf2 expression in cells significantly reduced TP-induced ferroptosis. Additionally, dysregulation of lipid metabolism was associated with TP-induced liver injury. TP may directly bind to Nrf2 and enhance its degradation through the ubiquitin–proteasome pathway, thereby inhibiting or reducing Nrf2 expression.ConclusionIn summary, the suppression of Nrf2 by TP facilitated the occurrence of ferroptosis, resulting in liver damage.Graphical abstract

Lycium barbarum polysaccharide alleviates ferroptosis in Sertoli cells through NRF2/SLC7A11/GPX4 pathway and ameliorates DEHP-induced male reproductive damage in mice

Di-(2-ethylhexyl)phthalate (DEHP) is a common plasticizer that has been shown to significantly negatively affect male reproductive health. On the other hand, Lycium barbarum polysaccharide (LBP) has been shown to improve reproductive function. Therefore, we hypothesized that LBP may ameliorate DEHP-induced male reproductive damage. Herein, we found that LBP could alleviate DEHP-induced testicular damage and sperm abnormalities. Furthermore, histomorphological analysis of mice testis revealed that LBP primarily ameliorated the DEHP-induced male reproductive damage by targeting Sertoli cells. Moreover, the detection of the function-related genes of Sertoli cells confirmed this finding. The serum of mice in the Control, DEHP, and DEHP+LBP groups was analyzed using non-targeted metabolomics to further elucidate the mechanism of action of LBP in improving DEHP-induced male reproductive damage. According to the results, the differential metabolites were mainly enriched in the glutamate metabolism pathway, implying that LBP may alleviate the ferroptosis-related DEHP-induced testicular injury. Related ferroptosis markers were also found in mice testis. These findings collectively suggest that LBP may ameliorate DEHP-induced testicular injury via alleviating ferroptosis in Sertoli cells. To clarify the specific mechanism, we constructed a cell model in vitro by treating TM4 cells (the Sertoli cell line) with LBP and MEHP (the in vivo DEHP metabolite). Our findings revealed that LBP can improve the function of DEHP-affected Sertoli cells. Furthermore, the analysis of lipid peroxidation, Fe2+ content, and ferroptosis-related protein expressions demonstrated that LBP could ameliorate MEHP-induced ferroptosis in TM4 cells. To clarify the specific mechanism, glutamate metabolism-related proteins involved in the ferroptosis pathway were detected. According to the results, there were significant changes in the expression of NRF2, SLC7A11 and GPX4 proteins, which are involved in the ferroptosis glutamate metabolism pathway. Furthermore, supplementation of NRF2, SLC7A11, and GPX4 inhibitors (ML385, Erastin, and RSL3, respectively) blocked the therapeutic effect of LBP in alleviating MEHP-induced ferroptosis in TM4 cells, implying that LBP could also ameliorate MEHP-induced ferroptosis via the NRF2/SLC7A11/GPX4 pathway. In summary, these findings show that LBP can alleviate DEHP/MEHP-induced ferroptosis through the NRF2/SLC7A11/GPX4 pathway, ameliorating Sertoli cell dysfunction and improving the DEHP-induced male reproductive damage. Therefore, the clinical administration of LBP could be an effective strategy for preventing DEHP-induced male reproductive injury.

Screening of orthopedic medicines identifies raloxifene hydrochloride as a novel ferroptosis inhibitor for spinal cord injury therapy

Spinal cord injury (SCI) is a severe condition that can lead to irreversible central nervous system damage. Spinal cord injury patients frequently present with coexisting orthopedic conditions, and many of them also have underlying bone and joint diseases. Recent studies have identified ferroptosis as a significant contributor that exacerbates the progression of spinal cord injury. This study conducted a screening in common orthopedic medications, which includes anti-osteoporosis agents and calcium supplements, in order to identify potential ferroptosis inhibitors and investigate their therapeutic effects on spinal cord injury. Among the 8 drugs screened, raloxifene hydrochloride was found to significantly inhibit ferroptosis induced by RSL3 in neural cells. Subsequent studies confirmed its inhibitory effect on ferroptosis both in vitro and in vivo. It was also demonstrated that Nrf2 inhibitor Brusatol could reverse the anti-ferroptotic effect of Raloxifene hydrochloride in neural cells in vitro as well as its therapeutic effect on SCI in vivo, suggesting its inhibitory effect on ferroptosis is through Nrf2. This study identifies a novel ferroptosis inhibitor among orthopedic medicines and also confirms the therapeutic effect of Raloxifene hydrochloride on SCI. The results of the current study may provide reference for the clinical administration of SCI.

ΔNp63α promotes radioresistance in esophageal squamous cell carcinoma through the PLEC-KEAP1-NRF2 feedback loop.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers and is highly prevalent in China, exhibiting resistance to current treatments. ΔNP63α, the main isoform of p63, is frequently amplified in ESCC and contributes to therapeutic resistance, although the molecular mechanisms remain unknown. Here, we report that ΔNP63α is highly expressed in ESCC and is associated with radioresistance by reducing ROS level. Furthermore, ΔNP63α plays a critical role in radioresistance by directly transactivating the expression of PLEC. PLEC competitively interacts with KEAP1, resulting in the release of NRF2 from KEAP1 and its translocation from the cytosol to the nucleus, where it activates gene expression to facilitate ROS elimination. Additionally, radiotherapy-induced ROS also activates ΔNP63α expression via NRF2. Pharmacologic inhibition of NRF2 effectively improves radiosensitivity in nude mice. Collectively, our results strongly suggest that the ΔNp63α/PLEC/NRF2 axis plays a key role in radioresistance in ESCC, indicating that targeting NRF2 is a promising therapeutic approach for ESCC treatment.

Gypenosides alleviates HaCaT keratinocyte hyperproliferation and ameliorates imiquimod-induced psoriasis in mice.

Psoriasis is an autoimmune skin condition characterized by hyperproliferation of keratinocytes and chronic immune responses. Gypenosides (Gyp) exhibits anti-proliferative and anti-inflammatory effects on different diseases. However, its functioning and mechanism of Gyp on psoriasis remains unknown. To explore the effect and mechanism of Gyp on psoriasis. The impact and mechanism of Gyp on psoriasis in vitro and in vivo were probed through cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, reverse transcription quantitative polymerase chain reaction, hematoxylin and eosin staining, enzyme-linked immunosorbent serologic assay, immunofluorescence, and Western Blotting assays. Gyp inhibited cell proliferation and the release of inflammatory cytokinesin interleukin (IL-22)-induced spontaneously transformed human aneuploid immortal keratinocyte cell line (HaCaT). In addition, Gyp demonstrated enhancement in erythema and scaling as well as reductions in the thickness of epidermal layers, release of inflammatory factors, and Ki-67 (a nuclear protein) level in imiquimod (IMQ)-induced mice. Mechanistically, Gyp upregulated nuclear factor erythroid 2-related factor 2 (Nrf-2) expression and diminished the level of p-p65/p65 and p-STAT3/STAT3 in skin tissues from IMQ-induced mice and IL-22-induced HaCaT cells, which were reversed with the application of ML385, an inhibitor of Nrf2. In addition, the administration of ML385 reversed decrease in cell viability and reduced the expressions of IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in IL-22-induced HaCaT cells caused by Gyp. In summary, Gyp reduced excessive cell growth and inflammation in psoriasis by suppressing nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3) through activation of Nrf2.

Demyelination in cuprizone mice is ameliorated by calycosin mediated through astrocyte Nrf2 signaling pathway

Oxidative stress plays a pivotal role in multiple sclerosis (MS), triggering demyelination predominantly through excessive peroxide production and the depletion of antioxidants. The accumulation of oxidative damage can be caused by dysregulation of astrocytes, which are the brain's main regulators of oxidative homeostasis. Calycosin, an essential bioactive component extracted from Astragalus, is recognized for its neuroprotective properties. Although recent research has highlighted calycosin's neuroprotective capabilities, its role in demyelinating conditions like MS remains unclear. In this work, we examined the possible molecular mechanism of calycosin's neuroprotective effect on cuprizone (CPZ)-induced demylination in mice. According to our research, calycosin successfully reduced demyelination and behavioral dysfuction in CPZ mice. Calycosin also decreased the production of oxidative stress and enhanced the expression of antioxidants in CPZ mice and in astrocytes induced by hydrogen peroxide (H2O2). Furthermore, both in vivo and in vitro experiments demonstrated that calycosin promoted the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) along with the upregulation of heme oxygenase 1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), and superoxide dismutase (SOD). Importantly, the application of all-trans retinoic acid (ATRA), a specific inhibitor of Nrf2, effectively reversed the myelin-protective and antioxidant effects conferred by calycosin. This study suggested that calycosin might exert neuroprotection by inhibiting oxidative stress and reducing demyelination via the activation of astrocyte Nrf2 signaling. These findings indicated that calycosin might be a potential candidate for treating MS.

Nuclear factor erythroid 2-related factor 2 alleviates lung endothelial cells injury by inhibition of ferroptosis.

In recent years, the survival rate of preterm infants has significantly improved due to the application of pulmonary surfactant (PS) and advancements in lung-protective mechanical ventilation strategies. However, this has been accompanied by an increased incidence of complications, particularly lung diseases triggered by elevated reactive oxygen species (ROS) induced by hyperoxia. The primary mechanism of hyperoxic lung injury (HLI) involves the excessive production of ROS within cells and the aggregation of inflammatory cells. Currently, no effective prevention or treatment methods are available. Ferroptosis, a newly identified form of cell death, is closely linked to ROS accumulation and is likely involved in HLI. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates both HLI and ferroptosis, and targeting Nrf2 to inhibit ferroptosis may represent a key therapeutic approach for treating HLI. This study aimed to investigate the involvement of ferroptosis in HLI and to elucidate the regulatory role of Nrf2. We employed the human pulmonary microvascular endothelial cell (HPMEC) model of hyperoxia exposure and corresponding intervention groups. Mitochondrial morphological alterations within HPMECs exposed to hyperoxia and various control groups were examined using transmission electron microscopy (TEM). Cell viability was assessed via the Cell Counting Kit-8 (CCK-8) assay, whereas intracellular ROS levels were quantified using the dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Furthermore, the expression levels of GPX4 and Nrf2 were analyzed through quantitative polymerase chain reaction (qPCR) and western blot techniques. Relative to the control group, the HPMECs subjected to hyperoxic conditions exhibited diminished viability, heightened ROS levels, decreased GPX4 expression, and increased Nrf2 expression. These cells also demonstrated mitochondrial morphological alterations characteristic of ferroptosis, including reduced mitochondrial cristae and shrinkage. The application of a ferroptosis inhibitor mitigated cellular damage, lipid peroxidation, and the morphological manifestations of mitochondrial ferroptosis, whereas Nrf2 inhibitor ML385 reversed this effect. Ferroptosis appears to contribute to the pathogenesis of HLI, with Nrf2 serving a protective function by mitigating ferroptosis.

Berberine safeguards sepsis-triggered acute gastric damage and inhibits pyroptosis in gastric epithelial cells via suppressing the ubiquitination and degradation of Nrf2.

Berberine (BBR), a widely recognized traditional Chinese medicine, has attracted considerable attention for its promising anti-inflammatory effects. The activation of nuclear factor erythroid 2-related factor 2 (Nrf2) effectively safeguards against organ damage stemming from sepsis-induced oxidative stress and inflammatory responses. This study examined the potential of BBR in alleviating sepsis-induced acute gastric injury, with a particular focus on elucidating whether its mechanism of action involves the activation of the Nrf2 signaling pathway. Following intraperitoneal injection of BBR, mice were subjected to the cecal ligation and puncture (CLP) method to induce sepsis. In vitro experiments involved pre-treating the normal gastric epithelial cells (GES-1) with BBR, followed by treatment with lipopolysaccharide (LPS). Functional assays were then performed to assess cell proliferation and apoptosis. To validate the role of Nrf2 in pyroptosis and inflammation, siRNA targeting Nrf2 (si-Nrf2) was transfected into LPS-treated GES-1 cells. Additionally, mice were administered the Nrf2 inhibitor ML385 to confirm the protective effects of BBR in vivo. BBR displayed a dose-dependent effect in mitigating gastric tissue damage, suppressing the release of inflammatory cytokines, and reducing the expression of NLRP3, ASC, and GSDMD-N. In vitro, BBR fostered GES-1 cell proliferation, hindered apoptosis, and suppressed the levels of TNF-α, IL-18, IL-1β, NLRP3, ASC, and GSDMD-N. Further analysis revealed that knocking down Nrf2 reversed BBR's inhibitory effect on pyroptosis in LPS-treated GES-1 cells. Through binding to Keap1, BBR efficiently prevented the ubiquitination and degradation of Nrf2, ultimately promoting its nuclear translocation. In vivo experiments confirmed that ML385 reversed the protective effect of BBR on pyroptosis and inflammation. Our research reveals that BBR interacts with Keap1 to activate the Keap1/Nrf2 signaling pathway in gastric epithelial cells, thereby suppressing pyroptosis and inflammation in sepsis-induced acute gastric injury.

Kaempferol alleviates myocardial ischemia injury by reducing oxidative stress via the HDAC3-mediated Nrf2 signaling pathway

IntroductionKaempferol (KAE) is a flavonoid found in various plants. Recent studies showed that high dietary intake of KAE was associated with a lower risk of myocardial infarction; however, the cardioprotective mechanism of KAE remains unknown. ObjectivesTo determine the effect of KAE on cardiac injury in isoproterenol (ISO)-induced rats and cobalt chloride (CoCl2)-treated cardiomyocytes, and the underlying mechanisms. MethodsMale rats were pretreated with different doses of KAE for 14 days, and then injected with ISO to induce myocardial ischemia injury. We also established a model of myocardial cell injury using rat H9c2 cardiomyocytes stimulated with CoCl2. ResultsWe found that KAE pretreatment significantly alleviated myocardial injury and improved cardiac function in ISO-injected rats. In addition, KAE reduced oxidative stress in rats with myocardial ischemia by decreasing malondialdehyde concentration and increasing superoxide dismutase activity, and protection of the myocardial mitochondrial structure. KAE also attenuated CoCl2-induced injuryof H9c2 cardiomyocytes via suppression ofoxidative stress. With regard to the mechanism, we found that KAE down-regulated HDAC3 expression and up-regulated Nrf2 expression in ISO-induced rats and CoCl2-stimulated cardiomyocytes. Incubation of cardiomyocytes with HDAC3-selective inhibitor RGFP966 augmented the protective effect of KAE and reduced oxidative stress. By contrast, HDAC3 overexpression by adenovirus attenuated the effect of KAE on oxidative stress compared with KAE treatment group. HDAC3 also regulated Nrf2 expression in the cardiomyocytes with RGFP966 or an adenovirus overexpressing HDAC3; but Nrf2 inhibition reduced the effect of KAE on ROS generation in CoCl2-induced cardiomyocytes. Immunoprecipitation assay showed that HDAC3 interacted with Nrf2 in cardiomyocytes. Further studies found that KAE increased the acetylation level of Nrf2, while HDAC3 overexpression decreased the acetylation of Nrf2 compared with KAE treatment group. ConclusionOur data show that KAE ameliorates cardiac injury by reducing oxidative stress via the HDAC3-mediated Nrf2 signaling pathway in cardiomyocytes.

GLP-1 receptor agonist liraglutide alleviates kidney injury by regulating nuclear translocation of NRF2 in diabetic nephropathy.

Diabetic nephropathy (DN) is a severe renal disorder that arises as a complication of diabetes. Liraglutide, an analogue of a glucagon-like peptide 1 (GLP-1) receptor agonist, has been shown to decrease diabetes-caused renal damage. Nevertheless, the complete understanding of the roles and mechanism remains unclear. In our study, diabetic rat models were created through a single intraperitoneal injection of streptozotocin (STZ). The level of fasting blood glucose, 24-h urine protein, serum creatinine (Scr) and blood urea nitrogen (BUN) were assessed. Periodic acid-Schiff (PAS) staining was applied to examine the pathological changes in renal tissues. Reactive oxygen species (ROS) formation was measured via dichloro-dihydro-fluorescein diacetate (DCFH-DA) probes. Western blot was conducted to examine the levels of oxidative stress-related and extracellular matrix (ECM)-associated proteins. The nuclear translocation of NRF2 was investigated through immunofluorescence and Western blot assays. We demonstrated that liraglutide attenuated DN-induced oxidative stress and ECM deposition in vitro and in vivo. Liraglutide exerted a reno-protective effect by promoting nuclear translocation of NRF2 in mesangial cells. ML385, an NRF2 inhibitor, counteracted the beneficial impact of liraglutide.

Unveiling the structural characteristics and anti-inflammatory potential of a novel polysaccharide from sweet potato peels

Sweet potato tubers generate a significant amount of peel waste during starch production, leading to resource inefficiency and environmental pollution. This research focused on the isolation/purification of a novel polysaccharide from sweet potato peel, named as sweet potato polysaccharide (SPP-W). The SPP-W has a molecular weight of 5.764 kDa and consist of glucose monosaccharides. The backbone of SPP-W was composed of →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→, and →3,4)-β-D-Glcp-(1 → . The branched chain was α-D-Glcp-(1→ linked to the O-6 position of the sugar residue→4,6)-α-D-Glcp-(1→ and the O-3 position of the sugar residue→3,4)- β-D-Glcp-(1→ as confirmed by methylation and NMR analysis. SPP-W was found helpful in reducing the excessive NO, up-regulating IL-10, down-regulating the release of IL-6, IL-1β and TNF-α with LPS-induced RAW264.7 cell inflammation model. Furthermore, SPP-W effectively mitigates oxidative stress by reducing ROS levels, preventing nuclear damage, and preserving mitochondrial membrane potential in the context of inflammation. Immunoassays demonstrated that the clearance of endogenous ROS and the improvement of inflammatory conditions were attributable to the activation of the Nrf2 signaling pathway and inhibition of the NF-κB signaling pathway by SPP-W. Conclusively, SPP-W holds promise as a potential active ingredient for anti-inflammatory functional foods, offering unique and beneficial properties for further research and development.

Etomidate Inhibits Hepatic Ischemia-Reperfusion Injury Depending on the Activation of Nrf2-HO-1 Signaling Pathway.

Hepatic ischemia-reperfusion (I/R) injury (HIRI) is recognized as a local aseptic inflammatory response driven by innate immunity and is considered a leading cause of early organ dysfunction and failure following liver transplantation. Etomidate (Eto), an anesthetic drug known for its ability to inhibit inflammatory response and apoptosis, was the focus of our investigation. In this study, we conducted hepatic I/R surgery invivo on C57 mice, analyzing liver damage through histopathology. Additionally, primary hepatocytes isolated from mice were cultured and subjected to hypoxia/reoxygenation (H/R) insult in vitro, with cell activity assessed using the CCK8 assay and immunofluorescence staining employed to analyze liver inflammatory cell infiltration and apoptosis. Results showed that Eto effectively inhibited liver injury, inflammatory response, and apoptosis induced by HIRI surgery, with the greatest effect observed at an Eto concentration of 10 mg/kg. Furthermore, Eto also showed the ability to inhibit H/R-induced cell damage, inflammatory activation, and apoptosis in primary hepatocytes. Further mechanistic studies revealed that Eto could promote the activation of the Nrf2-HO-1 signaling pathway, and the protective effect of Eto on HIRI was nullified when the Nrf2 inhibitor ML385 was utilized. This study highlights the potential of Eto to protect against HIRI by promoting the Nrf2-HO-1 signaling axis.

NRF2 Modulators of Plant Origin and Their Ability to Overcome Multidrug Resistance in Cancers.

Cancer is one of the most common causes of death in the world. Despite the fact that there are many types of therapies available, cancer treatment remains a major challenge. The main reason for the ineffectiveness of chemotherapy is the acquisition of multidrug resistance (MDR) by cancer cells. One of the factors responsible for the acquisition of MDR is the NRF2 transcription factor, which regulates the expression of proteins such as HO-1, NQO1, MRP1, MRP2, and GST. In normal cells, NRF2 is the first line of defense against oxidative stress, thereby preventing carcinogenesis. Still, its hyperactivation in cancer cells causes them to acquire MDR, which significantly reduces or eliminates the effectiveness of chemotherapy. Considering the important role NRF2 plays in the acquisition of MDR, its modulators and, above all, inhibitors are being sought after, including among compounds of plant origin. NRF2 inhibition may prove to be a key element of anticancer therapy. This review summarizes the current state of knowledge about plant NRF2 inhibitors and presents the effects of their use in overcoming MDR in cancer.