PRMT4 Reduced Erastin-Induced Ferroptosis in Nasopharyngeal Carcinoma Cisplatin-Resistant Cells by Nrf2/GPX4 Pathway.

Nasopharyngeal carcinoma (NPC) is one of the common malignant tumors in clinic. In the current study, we aim to investigate the effects of PRMT4 on erastin-induced ferroptosis in NPC by cisplatin resistant. PRMT4 expression in patients with NPC by cisplatin was upregulated. PRMT4 upregulation promoted cell growth of erastin-induced ferroptosis in NPC cisplatin-resistant cells. PRMT4 downregulation reduced cell growth of erastin-induced ferroptosis in NPC cisplatin-resistant cells. PRMT4 promoted tumor volume in mice model of erastin-induced NPC by cisplatin. PRMT4 upregulation reduced erastin-induced ferroptosis in NPC cisplatin-resistant cells by mitochondrial damage. PRMT4 upregulation induced Nrf2 protein expression in model of erastin-induced NPC by cisplatin. Nrf2 reduced the effects of si-PRMT4 on cell growth of erastin-induced ferroptosis in NPC cisplatin-resistant cells. Nrf2 inhibitor reduced the effects of PRMT4 on cell growth of erastin-induced ferroptosis in NPC cisplatin-resistant cells. Nrf2 reduced the effects of si-PRMT4 on erastin-induced ferroptosis in NPC cisplatin-resistant cells by mitochondrial damage. PRMT4 protein interlinked with Nrf2 protein to decrease Nrf2 ubiquitination. Methylation increased PRMT4 DNA stability. Collectively, our data reveal that PRMT4 reduced erastin-induced ferroptosis in NPC cisplatin-resistant cells by Nrf2/GPX4 pathway, suggesting that targeting PRMT4 may present as a potential strategy against the development of NPC.

Buddlejasaponin IVb Alleviates DSS-Induced Ulcerative Colitis through the Nrf2/GPX4 Pathway and Gut Microbiota Modulation.

Buddlejasaponin IVb (BJP-IVb), a triterpenoid saponin derived from Pleurotus ostreatus, is abundant in bioactive constituents. However, its potential therapeutic benefits and underlying mechanism of action against ulcerative colitis (UC) remain elusive. Studies performed using a mouse model of colitis caused by dextran sulfate sodium showed that BJP-IVb helped reduce symptoms of UC, such as weight loss, a higher disease activity index, shorter colon length, and colon damage. It also repaired the intestinal barrier function and suppressed inflammation. BJP-IVb activated the Nrf2/HO-1 antioxidant pathway, inhibited ferroptosis, and affected protein expression and oxidative indicators. The combined use of BJP-IVb and Nrf2 inhibitor ML385 negated the effects of BJP-IVb. In Nrf2-KO mice, the protective effects of BJP-IVb were reversed. BJP-IVb inhibited ferroptosis via the Nrf2/GPX4 axis and alleviated gut microbiota dysbiosis. In conclusion, our research validates the therapeutic and protective efficacy of BJP-IVb on ulcerative colits. Additionally, it offers new perspectives on preventing colitis by focusing on the metabolism of gut microbiota using natural substances.

CircRNA_SLC8A1 alleviates hypertrophic scar progression by mediating the Nrf2-ARE pathway.

BACKGROUND: Hypertrophic scar (HS) is associated with cosmetic defects, mobility, and functional impairments, pruritus, and pain. Previous circRNA microarray analysis identified reduced expression of circRNA_SLC8A1 in HS tissues. Therefore, this study aims to investigate the role of circRNA_SLC8A1 in modulating the abnormal behavior of HS-derived fibroblasts (HSFs) in vitro. METHODS: RT-qPCR and FISH assays were used to assess the differential expression and localization of circRNA_SLC8A1 in normal and HS tissues. Following modulation of circRNA_SLC8A1 expression, CCK-8, flow cytometry, Transwell, and wound healing assays were employed to evaluate the effects of circRNA_SLC8A1 on the biological behaviors of HSFs. The Starbase database, dual-luciferase reporter assays, and Ago2-RIP assays were utilized to predict and validate the interaction between circRNA_SLC8A1 and downstream miRNAs. RESULTS: CircRNA_SLC8A1 was found to be downregulated in HS tissues and was primarily localized in the cytoplasm. Overexpression of circRNA_SLC8A1 reduced cell viability, cell invasion, wound healing, and the expression of Vimentin, N-cadherin, Col I, and Col III, while enhancing apoptosis and E-cadherin expression in HSFs. CircRNA_SLC8A1 activates the Nrf2-ARE pathway by competitively binding to miRNA-27a-3p. miRNA-27a-3p and Nrf2 exhibited high and low expression, respectively in HS tissues, with an inverse correlation between their levels. Overexpression of miRNA-27a-3p counteracted the effects of circRNA_SLC8A1 in HSF proliferation, apoptosis, migration, EMT, collagen deposition, and Nrf2-ARE pathway activity. CONCLUSION: CircRNA_SLC8A1 inhibits the proliferation, migration, EMT, and collagen deposition of HSF through competitive binding with miRNA-27a-3p, thereby activating the Nrf2-ARE pathway. The circRNA_SLC8A1/miRNA-27a-3p/Nrf2-ARE axis may offer a promising molecular target for HS therapy.

Moxibustion Regulates the BRG1/Nrf2/HO-1 Pathway by Inhibiting MicroRNA-222-3p to Prevent Oxidative Stress in Intestinal Epithelial Cells in Ulcerative Colitis and Colitis-Associated Colorectal Cancer.

Oxidative stress is crucial in ulcerative colitis (UC) and colitis-associated colorectal cancer (CAC). Intestinal epithelial cells (IECs) are an important component of the intestinal barrier. In previous studies, we have demonstrated that suppressing microRNA-222-3p (miR-222-3p) can protect against oxidative stress in IECs, which ameliorates colonic injuries in UC mice and prevents the conversion of UC to CAC. In this case, we hope to explore whether moxibustion can alleviate UC and CAC by inhibiting miR-222-3p based on mouse models of UC and CAC. After herb-partitioned moxibustion (HPM) intervention, the disease activity index (DAI) and colon macroscopic damage index (CMDI) were significantly reduced in UC mice, and the number and volume of intestinal tumors were decreased considerably in CAC mice. Meanwhile, we found that HPM suppressed miR-222-3p expression and upregulated the mRNA and protein expression of Brahma-related gene 1 (BRG1), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), while inhibiting Kelch-like ECH-associated protein 1 (Keap1) expression in IECs of UC and CAC mice. With changes in reactive oxygen species (ROS), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and inflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor (TNF)-α), we verified that HPM protects against oxidative stress and inflammation in IECs of UC and CAC mice. The effect of HPM was inhibited in miR-222-3p overexpression mice, further demonstrating that the protective effect of HPM on UC and CAC mice was through inhibiting miR-222-3p. In summary, HPM regulates the BRG1/Nrf2/HO-1 pathway by inhibiting miR-222-3p to attenuate oxidative stress in IECs in UC and CAC.

The Nrf2-HO-1 system and inflammaging.

Nrf2 is a master transcriptional regulator of a number of genes involved in the adaptive response to oxidative stress. Among the genes upregulated by Nrf2, heme oxygenase-1 (HO-1) has received significant attention, given that the products of HO-1-induced heme catabolism have well established antioxidant and anti-inflammatory properties. This is evidenced in numerous models of inflammatory and autoimmune disease whereby induction of HO-1 expression or administration of tolerable amounts of HO-1 reaction products can ameliorate disease symptoms. Unsurprisingly, Nrf2 and HO-1 are now considered viable drug targets for a number of conditions. In recent years, the term 'inflammaging' has been used to describe the low-grade chronic inflammation observed in aging/aged cells. Increased oxidative stress is also a key factor associated with aging and there is convincing evidence that Nrf2, not only declines with age, but that Nrf2 and HO-1 can reduce cellular senescence and the senescence-associated secretory phenotype (SASP) which is now considered an underlying driver of age-related inflammatory disease. In this review, we describe the role of oxidative stress in 'inflammaging' and highlight the potential anti-aging properties of the Nrf2-HO-1 system. We also highlight established and newly emerging Nrf2 activators and their therapeutic application in age-related disease.

GAS5 promotes glucose metabolism reprogramming and resistance to ferroptosis of endothelial progenitor cells through the miR-495-3p/SIX1 and IGF2BP2/NRF2 dual-regulatory pathways in coronary heart disease.

We aimed to explore the potential along with mechanism of lncRNA growth arrest-specific 5 (GAS5) in modulating glucose metabolism and ferroptosis of endothelial progenitor cells (EPCs) in coronary heart disease (CHD). CCK-8, flow cytometry, EdU, colony formation, scratch test as well as transwell assays were implemented to assess cell biological behaviors. Glucose uptake testing, lactic acid production assay, and detection of extracellular acidification rate (EACR) together with oxygen consumption rate (OCR) were used to assess glucose metabolism. Iron, GSH and MDA detection were used to measure ferroptosis. Besides, a series of mechanical experiments were implemented to clarify the modulatory relationship between GAS5 and nuclear factor erythroid 2-related factor 2 (NRF2) as well as sine oculis homeobox 1 (SIX1). We found that GAS5 was down-regulated in CHD patients relative to healthy controls. GAS5 depletion repressed EPCs proliferation, migration along with invasion while elevated cell apoptosis. GAS5 promoted the reprogramming of glucose metabolism and inhibited ferroptosis in EPCs. GAS5 affected glycometabolic reprogramming and ferroptosis resistance through regulating SIX1 and NRF2. On the one hand, GAS5 promoted NRF2 mRNA stability through IGF2BP2. On the other hand, GAS5 regulated the miR-495-3p/SIX1 axis in EPCs. To sum up, GAS5 promotes glucose metabolism reprogramming and resistance to ferroptosis of EPCs through the miR-495-3p/SIX1 and IGF2BP2/NRF2 dual-regulatory pathways in CHD.

Continuous Theta Burst Stimulation Inhibits Oxidative Stress-Induced Inflammation and Autophagy in Hippocampal Neurons by Activating Glutathione Synthesis Pathway, Improving Cognitive Impairment in Sleep-Deprived Mice.

Sleep deprivation (SD) has been reported to have a negative impact on cognitive function. Continuous theta burst stimulation (cTBS) shows certain effects in improving sleep and neurological diseases, and its molecular or cellular role in SD-induced cognition impairment still need further exploration. In this study, C57BL/6 mice were subjected to 48 h of SD and cTBS treatment, and cTBS treatment significantly improved SD-triggered impairment of spatial learning and memory abilities in mice. Additionally, cTBS reduced malondialdehyde levels, increased superoxide dismutase activities, and inhibited the production of inflammatory cytokines, alleviating oxidative stress and inflammation levels in hippocampal tissues of SD model mice. cTBS decreased LC3II/LC3I ratio, Beclin1 protein levels, and LC3B puncta intensity, and elevated p62 protein levels to suppress excessive autophagy in hippocampal tissues of SD-stimulated mice. Then, we proved that inhibiting oxidative stress alleviated inflammation, autophagy, and death of hippocampal neuron cells through an in vitro cellular model for oxidative stress, and cTBS treatment promoted the production of glutathione (GSH), the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the mRNA expression of GSH synthesis-related genes to enhance antioxidant capacity in hippocampal tissues of SD mice. An Nrf2 inhibitor ML385 or a GSH synthesis inhibitor BSO reversed the alleviating effects of cTBS treatment on oxidative stress-associated damage of hippocampal tissues and cognitive impairment in SD model mice. Altogether, our study demonstrated that cTBS mitigates oxidative stress-associated inflammation and autophagy through activating the Nrf2-mediated GSH synthesis pathway, improving cognitive impairment in SD mice.

Heat shock protein 22 alleviates doxorubicin-induced kidney injury by suppressing oxidative stress and apoptosis.

This study investigated the effects of heat shock protein 22 (HSP22) against doxorubicin (DOX)-induced kidney injury. Mice were randomly assigned to four groups: CON, ad-HSP22, DOX, and ad-HSP22 + DOX. Adeno-associated virus carrying the HSP22 gene (ad-HSP22) was administered via tail vein injection for four weeks, followed by intraperitoneal simulation with DOX (20 mg/kg) for another five days. Upon euthanasia, ELISA, histological staining (H&E, IHC, DHE, and TUNEL), and western blot analyses were employed to assess relevant markers. Serum biomarkers of kidney injury, SCr, and BUN, were upregulated after DOX administration but normalized with HSP22 overexpression. Pathological changes induced by DOX were also reversed by HSP22 overexpression in H&E, IHC, DHE, and TUNEL stains. DOX-induced upregulation of NOX-2 and NOX-4 and downregulation of SOD-1 and SOD-2 were reversed by HSP22 overexpression. Similarly, DOX-induced increases in Bax and decrease in Bcl-2 were attenuated by HSP22 overexpression. The study further demonstrated that the Nrf2/HO-1 signaling pathway was activated by HSP22 overexpression. In vitro experiments corroborated the findings from in vivo experiments. In conclusion, HSP22 alleviates DOX-induced kidney injury by suppressing oxidative stress and apoptosis, primarily through the activation of the Nrf2/HO-1 signaling pathway. These results suggest HSP22 as a potential therapeutic target for DOX-induced kidney injury.

[Effects of penetration needling from "Zhibian" (BL54) to "Shuidao" (ST28) on SIRT1/PGC-1α/Nrf2 signaling pathway in rats with premature ovarian insufficiency]. / "秩边透水道"é’ˆæ³•å¯¹æ—©å‘æ€§åµå·¢åŠŸèƒ½ä¸å ¨å¤§é¼ SIRT1/PGC-1α/Nrf2信号通路的影响.

OBJECTIVES: To observe the effect of penetration needling from "Zhibian" (BL54) to "Shuidao"(ST28) on silencing information regulator 1 (SIRT1) /peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) /nuclear factor E2 related factor 2 (Nrf2) signaling in rats with premature ovarian insufficiency (POI), so as to explore its mechanisms underlying improvement of POI. METHODS: A total of 48 female SD rats were equally and randomly allocated to blank control, POI model, shallow needling and penetration needling (from "Zhibian" ï¼»BL54ï¼½ to "Shuidao" ï¼»ST28ï¼½) groups. The POI model was established by intraperitoneal injection of cyclophosphamide (50 mg·kg-1·d-1 on the 1st day and 8 mg·kg-1·d-1 from the 2nd to 15th day, for a total of 15 days). After successful modeling, for rats of the shallow needling group, a filiform needle was inserted into BL54 to a depth about 5-8 mm, and then retained for 30 min. And for rats of the penetration needling group, a filiform needle was inserted into BL54 area and advanced to the unilateral ST28 to a depth about 12-15 mm, and then retained for 30 min (bilateral acupoints were used at the same time). The treatments were conducted once daily, 6 times a week for 4 weeks. After the interventions, the contents of serum follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and anti-Müllerian hormone (AMH) were detected using ELISA, and the activity of superoxide dismutase (SOD), catalase (CAT) and content of malondialdehyde (MDA) in the ovarian tissue were detected using colorimetry. Histopathological changes of the ovarian tissue were observed after H.E. staining. The immunoactivities and expression levels of SIRT1, PGC-1α, and Nrf2 mRNA and protein in the ovarian tissues were detected using immunohistochemistry, quantitative real-time PCR and Western blot, respectively. RESULTS: After modeling, the rats' estrus cycles were disordered, contents of serum FSH and LH levels significantly increased, and the E2 level markedly decreased compared with those of the blank control group (P<0.01), indicating that the POI model was successfully established. Relevant to the blank control group, the model group had an increase in serum FSH and LH, ovarian MDA contents, and the number of atretic oocytes (P<0.01), and a decrease in serum E2 and AMH contents, ovarian SOD and CAT activities, number of growing oocytes, immunoactivities and expressions of ovarian SIRT1, PGC-1α and Nrf2 protein and mRNA (P<0.01, P<0.05). Following interventions, both the increased levels of serum FSH and LH and ovarian MDA contents, and the number of atretic oocytes, and the decreased levels of E2 and AMH contents, ovarian SOD and CAT activities, number of growing oocytes, immunoactivities and expressions of ovarian SIRT1, PGC-1α and Nrf2 protein and mRNA were reversed by penetration needling of BL54-ST28 (P<0.01, P<0.05), but not by shallow needling, except serum FSH, LH, E2 and AMH contents. The effects of penetration needling were obviously superior to those of shallow needling in up-regulating the levels of serum AMH, ovarian SOD and CAT, number of growing oocytes, and the expressions of ovarian SIRT1, PGC-1α and Nrf2 protein and mRNA (P<0.05, P<0.01), and in down-regulating the level of MDA and the number of atretic oocytes (P<0.05). CONCLUSIONS: Penetration needling stimulation of BL54 to ST28 can increase the number of ovarian growing oocytes and reduce the number of atretic oocytes, regulate the serum hormone levels and relieve the ovarian oxidative stress level in POI rats, which may be associated with its functions in activating ovarian SIRT1/PGC-1α/Nrf2 signaling pathway.

Potential of NRF2 Inhibitors-Retinoic Acid, K67, and ML-385-In Overcoming Doxorubicin Resistance in Promyelocytic Leukemia Cells.

Drug resistance is one of the major obstacles to the clinical use of doxorubicin, an extensively used chemotherapeutic drug to treat various cancers, including leukemia. Inhibition of the nuclear factor erythroid 2-related factor 2 (NRF2) seems a promising strategy to reverse chemoresistance in cancer cells. NRF2 is a transcription factor that regulates both antioxidant defense and drug detoxification mechanisms. In this study, we investigated the potential of three inhibitors of NRF2-K67, retinoic acid and ML-385-to overcome doxorubicin resistance in promyelocytic leukemia HL-60 cells. For this purpose, low-dose doxorubicin was used to establish doxorubicin-resistant HL-60/DR cells. The expression of NRF2 and its main repressor, Kelch-like ECH-associated protein 1 (KEAP1), at mRNA and protein levels was examined. HL-60/DR cells overexpressed NRF2 at mRNA and protein levels and down-regulated KEAP1 protein compared to drug-sensitive HL-60 cells. The effects of NRF2 inhibitors on doxorubicin-resistant HL-60/DR cell viability, apoptosis, and intracellular reactive oxygen species (ROS) levels were analyzed. We observed that NRF2 inhibitors significantly sensitized doxorubicin-resistant HL-60/DR cells to doxorubicin, which was associated with increased intracellular ROS levels and the expression of CAS-9, suggesting the participation of the mitochondrial-dependent apoptosis pathway. Furthermore, ML-385 inhibitor was used to study the expression of NRF2-KEAP1 pathway genes. NRF2 gene and protein expression remained unchanged; however, we noted the down-regulation of KEAP1 protein upon ML-385 treatment. Additionally, the expression of NRF2-regulated antioxidant and detoxification genes including SOD2, HMOX2, and GSS was maintained upon ML-385 treatment. In conclusion, our results demonstrated that all the studied inhibitors, namely K67, retinoic acid, and ML-385, increased the efficacy of doxorubicin in doxorubicin-resistant HL-60/DR cells, and suggested a potential strategy of combination therapy using NRF2 inhibitors and doxorubicin in overcoming doxorubicin resistance in leukemia.

Eucalyptus Wood Smoke Extract Elicits a Dose-Dependent Effect in Brain Endothelial Cells.

The frequency, duration, and size of wildfires have been increasing, and the inhalation of wildfire smoke particles poses a significant risk to human health. Epidemiological studies have shown that wildfire smoke exposure is positively associated with cognitive and neurological dysfunctions. However, there is a significant gap in knowledge on how wildfire smoke exposure can affect the blood-brain barrier and cause molecular and cellular changes in the brain. Our study aims to determine the acute effect of smoldering eucalyptus wood smoke extract (WSE) on brain endothelial cells for potential neurotoxicity in vitro. Primary human brain microvascular endothelial cells (HBMEC) and immortalized human brain endothelial cell line (hCMEC/D3) were treated with different doses of WSE for 24 h. WSE treatment resulted in a dose-dependent increase in IL-8 in both HBMEC and hCMEC/D3. RNA-seq analyses showed a dose-dependent upregulation of genes involved in aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (NRF2) pathways and a decrease in tight junction markers in both HBMEC and hCMEC/D3. When comparing untreated controls, RNA-seq analyses showed that HBMEC have a higher expression of tight junction markers compared to hCMEC/D3. In summary, our study found that 24 h WSE treatment increases IL-8 production dose-dependently and decreases tight junction markers in both HBMEC and hCMEC/D3 that may be mediated through the AhR and NRF2 pathways, and HBMEC could be a better in vitro model for studying the effect of wood smoke extract or particles on brain endothelial cells.

Novel Indirect Antioxidant Activity Independent of Nrf2 Exerted by Lactic Acid Bacteria.

In recent years, the health benefits of lactic acid bacteria have garnered attention, but their antioxidant activity remains relatively underexplored. We have been analyzing the antioxidant activities of various dietary phytochemicals by assessing their ability to mitigate oxidative stressor-induced toxicity in zebrafish larvae through pretreatment. In this study, the antioxidant activities of 24 strains of heat-killed lactic acid bacteria from various origins were examined using this zebrafish assay system. The results revealed that all 24 strains possessed antioxidant activity that reduces hydrogen peroxide toxicity. Further detailed analysis using the H61 strain, which exhibited the strongest activity, showed that no direct antioxidant activity was observed in the assay system, suggesting that the detected antioxidant activity was entirely indirect. Moreover, it was found that pretreatment of zebrafish larvae with the H61 strain for more than 6 h was required to exert its antioxidant activity. This duration was similar to that required by dietary antioxidants that activate the Keap1-Nrf2 pathway, suggesting potential involvement of this pathway. However, analysis using Nrf2-knockout zebrafish revealed that the antioxidant activity of strain H61 is independent of Nrf2, indicating that it represents a novel indirect antioxidant activity that does not involve the Keap1-Nrf2 pathway. To further characterize this activity, the ability to mitigate the toxicity of oxidative stressors other than hydrogen peroxide was examined. The results indicated that while the toxicity of tert-butyl hydroperoxide was reduced, unlike with the Keap1-Nrf2 pathway, it was not effective in counteracting the toxicity of paraquat or arsenite, which generate superoxide radicals. In conclusion, we have identified a novel indirect antioxidant activity in lactic acid bacteria.

Immune-response gene 1 deficiency aggravates inflammation-triggered cardiac dysfunction by inducing M1 macrophage polarization and aggravating Ly6Chigh monocyte recruitment.

The immune response gene 1 (IRG1) and its metabolite itaconate are implicated in modulating inflammation and oxidative stress, with potential relevance to sepsis-induced myocardial dysfunction (SIMD). This study investigates their roles in SIMD using both in vivo and in vitro models. Mice were subjected to lipopolysaccharide (LPS)-induced sepsis, and cardiac function was assessed in IRG1 knockout (IRG1-/-) and wild-type mice. Exogenous 4-octyl itaconate (4-OI) supplementation was also examined for its protective effects. In vitro, bone marrow-derived macrophages and RAW264.7 cells were treated with 4-OI following Nuclear factor, erythroid 2 like 2 (NRF2)-small interfering RNA administration to elucidate the underlying mechanisms. Our results indicate that IRG1 deficiency exacerbates myocardial injury during sepsis, while 4-OI administration preserves cardiac function and reduces inflammation. Mechanistic insights reveal that 4-OI activates the NRF2/HO-1 pathway, promoting macrophage polarization and attenuating inflammation. These findings underscore the protective role of the IRG1/itaconate axis in SIMD and suggest a therapeutic potential for 4-OI in modulating macrophage responses.

O-GlcNAcylation promotes malignancy and cisplatin resistance of lung cancer by stabilising NRF2.

BACKGROUND: The transcription factor NRF2 plays a significant role in regulating genes that protect cells from oxidative damage. O-GlcNAc modification, a type of posttranslational modification, is crucial for cellular response to stress. Although the involvement of both NRF2 and O-GlcNAc in maintaining cellular redox balance and promoting cancer malignancy has been demonstrated, the potential mechanisms remain elusive. METHODS: The immunoblotting, luciferase reporter, ROS assay, co-immunoprecipitation, and immunofluorescence was used to detect the effects of global cellular O-GlcNAcylation on NRF2. Mass spectrometry was utilised to map the O-GlcNAcylation sites on NRF2, which was validated by site-specific mutagenesis and O-GlcNAc enzymatic labelling. Human lung cancer samples were employed to verify the association between O-GlcNAc and NRF2. Subsequently, the impact of NRF2 O-GlcNAcylation in lung cancer malignancy and cisplatin resistance were evaluated in vitro and in vivo. RESULTS: NRF2 is O-GlcNAcylated at Ser103 residue, which hinders its binding to KEAP1 and thus enhances its stability, nuclear localisation, and transcription activity. Oxidative stress and cisplatin can elevate the phosphorylation of OGT at Thr444 through the activation of AMPK kinase, leading to enhanced binding of OGT to NRF2 and subsequent elevation of NRF2 O-GlcNAcylation. Both in cellular and xenograft mouse models, O-GlcNAcylation of NRF2 at Ser103 promotes the malignancy of lung cancer. In human lung cancer tissue samples, there was a significant increase in global O-GlcNAcylation, and elevated levels of NRF2 and its O-GlcNAcylation compared to paired adjacent normal tissues. Chemotherapy promotes NRF2 O-GlcNAcylation, which in turn decreases cellular ROS levels and drives lung cancer cell survival. CONCLUSION: Our findings indicate that OGT O-GlcNAcylates NRF2 at Ser103, and this modification plays a role in cellular antioxidant, lung cancer malignancy, and cisplatin resistance.

Sleeve gastrectomy ameliorates renal injury in obesity-combined hyperuricemic nephropathy mice by modulating the AMPK/Nrf2/ABCG2 pathway.

Hyperuricemic nephropathy (HN) is renal injury caused by hyperuricemia (HUA). While sleeve gastrectomy (SG) has shown promise in improving renal injury in patients with obesity-related HN, the mechanisms are not fully understood. This study induced an obesity-combined HN model in male ob/ob mice and measured serum uric acid (SUA), creatinine, and other biochemical indicators 6 weeks post-surgery. Renal histological changes were evaluated through staining techniques, and the study also assessed renal adenosine monophosphate-activated protein kinase (AMPK) and nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation levels and urate transporter ABCG2 expression. In vitro experiments involved Nrf2 knockdown in AMPK-activated HK-2 cells and ChIP to confirm Nrf2 binding to the ABCG2 promoter. Results showed that SG reduced SUA levels, serum creatinine, and blood urea nitrogen, increased p-AMPK, p-Nrf2 protein, and ABCG2 expression, and alleviated renal fibrosis and inflammation. In vitro, Nrf2 knockdown down-regulated ABCG2 expression, and ChIP confirmed Nrf2's role in ABCG2 transcription. The study suggests that SG may improve renal injury in HN mice by modulating the AMPK/Nrf2 pathway and upregulating ABCG2 transcription.

ATF3 as a response factor to regulate Cd-induced reproductive damage by activating the NRF2/HO-1 ferroptosis pathway.

Cadmium (Cd) has garnered significant attention due to reproductive toxicity in inducing ferroptosis. However, the specific mechanisms underlying Cd-induced germ cell ferroptosis remain poorly understood. This study aimed to systematically explore the molecular mechanisms of germ cell ferroptosis by investigating differential changes in transcription factors and proteins in male mice treated orally with CdCl2 (0.5 g/L) reaching postnatal day 60, alongside Leydig cell (TM3) and Sertoli cell (TM4) lines. Results demonstrated that Cd exposure led to increased iron overload and oxidative stress in mouse testes, disrupted intracellular mitochondrial morphology characteristic of ferroptosis. RNA sequencing revealed significant upregulation of Atf3 and Hmox1 in Cd-exposed germ cells, along with increased expression of ATF3 and HO-1. Intervention in ferroptosis or HO-1 effectively rescued cells from Cd-induced mortality by breaking the detrimental cycle between lipid peroxidation and HO-1 activation. Further findings showed that NRF2 and HO-1 expression was notably elevated upon ATF3 overexpression in TM3 and TM4 cells, activating the Keap1-Nrf2 pathway and triggering ferroptosis in testes, whereas NRF2 and HO-1 expression levels were reversed when ATF3 was silenced. This study provides novel insights into ATF3-mediated NRF2/HO-1 signaling in Cd-induced mitochondrial ferroptosis in testes, shedding light on the mechanisms underlying Cd-induced ferroptosis and testicular injury.

Perfluorooctanoic acid (PFOA) induces cardiotoxicity by activating the Keap1/Nrf2 pathway in zebrafish (Danio rerio) embryos.

Perfluorooctanoic acid (PFOA), a perfluoroalkyl compound, is linked to congenital heart diseases, though its underlying mechanisms remain unclear. We hypothesized that PFOA induces cardiac defects through the inhibition of the Keap1/Nrf2 pathway, leading to oxidative damage in cardiomyocytes. In this study, zebrafish embryos exposed to PFOA showed significant cardiac malformations and dysfunction, characterized by excessive reactive oxygen species (ROS), malondialdehyde (MDA) production, decreased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities. Additionally, we observed dysregulation in the expression of key cardiac development genes (vmhc, gata4, nkx2.5, and sox9b). PFOA also reduced the expression of keap1, nrf2, and ho-1. After overexpression of Nrf2, levels of ROS and MDA decreased, while levels of SOD, CAT, and GSH-Px increased. Additionally, cardiomyocyte apoptosis and cardiac malformations were alleviated. These findings have suggested that PFOA induces oxidative stress through Keap1/Nrf2 pathway inhibition, ultimately leading to cardiac defects.

The antioxidant betulinic acid enhances porcine oocyte maturation through Nrf2/Keap1 signaling pathway modulation.

During in vitro maturation, excess levels of reactive oxygen species (ROS) are a major cause of developmental defects in embryos. Betulinic acid (BA) is a naturally produced antioxidant in white birch bark. Recent studies have shown that BA exhibits antioxidant properties in various cells through the activation of antioxidant genes. Therefore, we investigated the effect of BA treatment on porcine oocytes and its underlying mechanism during oocyte maturation. Treatment with 0.1 µM BA significantly increased the proportion of MII oocytes compared with controls, and BA-treated oocytes had significantly higher development rates, trophectoderm cell numbers, and cell survival rates than controls. These results demonstrate that BA treatment improved the developmental competence of oocytes. Following BA treatment, oocytes exhibited reduced ROS levels and elevated glutathione (GSH) levels, accompanied by the enhanced expression of antioxidant genes, compared with control oocytes. To evaluate the antioxidant effects of BA, oocytes were exposed to H2O2, a potent ROS activator. Impaired nuclear maturation, ROS levels, and GSH levels induced in oocytes by H2O2 exposure was restored by BA treatment. As these antioxidant genes are regulated by the Nrf2/Keap1 signaling pathway, which is involved in antioxidant responses, we applied the Nrf2 inhibitor brusatol to investigate the effects of BA on this pathway. The negative effects of brusatol on meiotic maturation and oocyte quality, including levels of ROS, GSH, and antioxidant-related gene expression, were mitigated by BA treatment. Our results suggested that BA plays an effective role as an antioxidant in porcine oocyte maturation through adjusting the Nrf2/Keap1 signaling pathway. This finding provides valuable insights into the mechanisms governing oocyte maturation and embryonic development.

Genetic Variations in the NRF2 Microsatellite Contribute to the Regulation of Bovine Sperm-Borne Antioxidant Capacity.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor protein-coding gene, considered a master regulator of the cellular stress response. The genetic variations of the NRF2 could influence its transcriptional profile and, subsequently, the stress resilience in all cell types, including sperm cells. Therefore, the sperm-borne antioxidants abundance in association with the genetic variation of a GCC microsatellite located at the 5' upstream region of the NRF2 gene was investigated in young (n = 8) and old (n = 8) Holstein bulls' sperm cells at different seasons. The sperm DNA was sequenced using Sanger sequencing, while- the sperm-borne mRNA analysis was carried out using the synthesized cDNA and qPCR. The data were statistically analyzed using GraphPad Prism 10.0.2 software. The results showed that two bulls had a heterozygous genotype of eight and nine GCC repeats, while biallelic of eight, nine, and fifteen repeats were identified in two, ten, and two bulls, respectively. The computational in silico analysis revealed that the NRF2 upstream sequence with 15, 9, and 8 GCC repeats bound with 725, 709, and 707 DNA-binding transcription factor proteins, respectively. Lower quality of sperm DNA was detected in the spring season compared to other seasons and in young bulls compared to old ones, particularly in the summer and autumn seasons. The mRNA expression analysis revealed that the PRDX1 gene was the abundant transcript among the studied sperm-borne antioxidants and was significantly determined in old bulls' spermatozoa. Moreover, two transcripts of the NRF2 gene and antioxidant (SOD1, CAT, GPX1, TXN1, NQO1) genes displayed differential expression patterns between the age groups across seasons in an antioxidant-dependent manner. The bulls with a heterozygous GCC sequence exhibited elevated sperm-borne mRNA levels of NRF2 and PRDX1 transcripts. Taken together, the findings suggest that the NRF2-GCC microsatellite may contribute to the transcription regulation of NRF2 transcripts and their subsequent downstream antioxidants in bovine sperm cells.

ALDH2 alleviates inflammation and facilitates osteogenic differentiation of periodontal ligament stem cells in periodontitis by blocking ferroptosis via activating Nrf2.

This paper elucidated the effects and mechanisms of aldehyde dehydrogenase 2 (ALDH2) on periodontitis. Rat model of periodontitis and periodontal ligament stem cell (PDLSC) model of periodontitis were constructed. PDLSC were transfected by ALDH2 overexpression vectors, and then treated by ML385 (Nrf2 inhibitor), ferrostatin-1 (ferroptosis inhibitor) and FIN56 (ferroptosis inducer), respectively. ALDH2, nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione peroxidase 4 (GPX4) proteins was evaluated by immunohistochemistry and Western blot. Ferroptosis-related factors, including Fe2+ and glutathione (GSH), were assessed by commercial kits. Pro-inflammatory factors (interleukin-6 [IL-6] and tumor necrosis factor-α [TNF-α]) and osteogenic differentiation-related proteins (osteocalcin [OCN] and runt-related transcription factor 2 [RUNX2]) were scrutinized by commercial kits and Western blot. In both periodontal tissues of periodontitis rats and PDLSC model of periodontitis, down-regulated ALDH2, Nrf2, GPX4 and GSH, but elevated Fe2+ level was discovered. ALDH2 overexpression in PDLSC resulted in an increase in Nrf2 expression. In PDLSC model of periodontitis, ALDH2 increased GPX4 and GSH levels, decreased Fe2+, IL-6 and TNF-α levels, and elevated OCN and RUNX2 expression. However, these effects of ALDH2 were counteracted by ML385. Additionally, the suppression of ALDH2 on IL-6 and TNF-α levels and promotion of it on OCN and RUNX2 expression in PDLSC model of periodontitis was further intensified by ferrostatin-1, but reversed by FIN56. ALDH2 may alleviate inflammation and facilitate osteogenic differentiation of PDLSC in periodontitis by hindering ferroptosis via activating Nrf2, suggesting it to be a promising candidate for treating periodontitis.