X-Paste improves wound healing in diabetes via NF-E2-related factor/HO-1 signaling pathway.

BACKGROUND: Diabetic foot ulcers (DFU), as severe complications of diabetes mellitus (DM), significantly compromise patient health and carry risks of amputation and mortality. AIM: To offer new insights into the occurrence and development of DFU, focusing on the therapeutic mechanisms of X-Paste (XP) of wound healing in diabetic mice. METHODS: Employing traditional Chinese medicine ointment preparation methods, XP combines various medicinal ingredients. High-performance liquid chromatography (HPLC) identified XP's main components. Using streptozotocin (STZ)-induced diabetic, we aimed to investigate whether XP participated in the process of diabetic wound healing. RNA-sequencing analyzed gene expression differences between XP-treated and control groups. Molecular docking clarified XP's treatment mechanisms for diabetic wound healing. Human umbilical vein endothelial cells (HUVECs) were used to investigate the effects of Andrographolide (Andro) on cell viability, reactive oxygen species generation, apoptosis, proliferation, and metastasis in vitro following exposure to high glucose (HG), while NF-E2-related factor-2 (Nrf2) knockdown elucidated Andro's molecular mechanisms. RESULTS: XP notably enhanced wound healing in mice, expediting the healing process. RNA-sequencing revealed Nrf2 upregulation in DM tissues following XP treatment. HPLC identified 21 primary XP components, with Andro exhibiting strong Nrf2 binding. Andro mitigated HG-induced HUVECs proliferation, metastasis, angiogenic injury, and inflammation inhibition. Andro alleviates HG-induced HUVECs damage through Nrf2/HO-1 pathway activation, with Nrf2 knockdown reducing Andro's proliferative and endothelial protective effects. CONCLUSION: XP significantly promotes wound healing in STZ-induced diabetic models. As XP's key component, Andro activates the Nrf2/HO-1 signaling pathway, enhancing cell proliferation, tubule formation, and inflammation reduction.

Função do Ventrículo Direito e Estresse Oxidativo Melhoram com a Administração de Hormônios da Tireoide e Suco de Uva em um Modelo de Hipertensão Pulmonar / Right Ventricular Function and Oxidative Stress Improve with the Administration of Thyroid Hormones and Grape Juice in a Pulmonary Hypertension Model

Resumo Fundamento A remodelação adversa dos vasos pulmonares eleva a pressão pulmonar e provoca hipertensão arterial pulmonar (HAP). A HAP resulta em aumento da pós-carga do ventrículo direito (VD), causando hipertrofia ventricular e consequente insuficiência cardíaca. Não existe um tratamento específico para o remodelamento desadaptativo do VD secundário à HAP. Objetivos Este estudo tem como objetivo explorar duas abordagens terapêuticas, o suco de uva (SU) e os hormônios tireoidianos (HT), no tratamento do estresse oxidativo induzido pela HAP e nas alterações funcionais cardíacas. Métodos Parâmetros ecocardiográficos relacionados à resistência dos vasos pulmonares (relação TA/TE), contratilidade do VD (ESPAT) e função diastólica do VD (relação dos picos E/A) foram avaliados. Além disso, foram medidos ROS totais, peroxidação lipídica, enzimas antioxidantes, proteínas de manipulação de cálcio, expressão de proteínas pró-oxidantes e antioxidantes. Valores de p<0,05 foram considerados estatisticamente significativos. Resultados Ambos os tratamentos, com SU e HT, demonstraram uma redução na resistência pulmonar (~22%), além de melhorias na ESPAT (inotropismo ~11%) e na relação TA/TE (~26%) (p<0,05). Não houve alterações entre os grupos na relação do pico de E/A. Embora ROS e TBARS não tenham sido estatisticamente significativos, os tratamentos com SU e HT diminuíram os níveis de xantina oxidase (~49%) e normalizaram a expressão de HSP70 e proteínas de manipulação de cálcio (p<0,05). No entanto, apenas o tratamento com HT melhorou a função diastólica (~50%) e aumentou o imunoconteúdo de NRF2 (~48%) (p<0,05). Conclusões Até onde sabemos, este estudo é pioneiro ao mostrar que o HT administrado em conjunto com o SU promoveu melhorias funcionais e bioquímicas em um modelo de HAP. Além disso, nossos dados sugerem que os tratamentos com SU e HT se mostraram cardioprotetores, sejam combinados ou não, e exibiram seus benefícios ao modular o estresse oxidativo e as proteínas de manipulação do cálcio.

Abstract Background Adverse remodeling of lung vessels elevates pulmonary pressure and provokes pulmonary arterial hypertension (PAH). PAH results in increased right ventricle (RV) afterload, causing ventricular hypertrophy and the onset of heart failure. There is no specific treatment for maladaptive RV remodeling secondary to PAH. Objectives This study aims to explore two therapeutic approaches, grape juice (GJ) and thyroid hormones (TH), on PAH-induced oxidative stress and cardiac functional changes. Methods Parameters of echocardiography related to lung vessel resistance (AT/ET ratio), RV contractility (TAPSE), and RV diastolic function (E/A peaks ratio) were evaluated. Also, total ROS, lipid peroxidation, antioxidant enzymes, calcium handling proteins, pro-oxidant and antioxidant protein expression were measured. Values of p<0.05 were considered statistically significant. Results Both GJ and TH treatments demonstrated reductions in pulmonary resistance (~22%) and improvements in TAPSE (inotropism ~11%) and AT/ET ratio (~26%) (p<0.05). There were no changes amongst groups regarding the E/A peak ratio. Although ROS and TBARS were not statistically significant, GJ and TH treatments decreased xanthine oxidase (~49%) levels and normalized HSP70 and calcium handling protein expression (p<0.05). However, only TH treatment ameliorated diastolic function (~50%) and augmented NRF2 immunocontent (~48%) (p<0.05). Conclusions To the best of our knowledge, this study stands as a pioneer in showing that TH administered together with GJ promoted functional and biochemical improvements in a PAH model. Moreover, our data suggest that GJ and TH treatments were cardioprotective, combined or not, and exhibited their beneficial effects by modulating oxidative stress and calcium-handling proteins.

The mechanism of NRF2 regulating cell proliferation and mesenchymal transformation in pulmonary hypertension.

Pulmonary hypertension (PH) is a fatal disease with no existing curative drugs. NF-E2-related factor 2 (NRF2) a pivotal molecular in cellular protection, was investigated in PH models to elucidate its role in regulating abnormal phenotypes in pulmonary artery cells. We examined the expression of NRF2 in PH models and explored the role of NRF2 in regulating abnormal phenotypes in pulmonary artery cells. We determined the expression level of NRF2 in lung tissues of PH model decreased significantly. We found that NRF2 was reduced in rat pulmonary artery endothelial cells (rPAEC) under hypoxia, while it was overexpressed in rat pulmonary artery smooth muscle cells (rPASMC) under hypoxia. Next, the results showed that knockdown NRF2 in rPAEC promoted endothelial-mesenchymal transformation and upregulated reactive oxygen species level. After the rPASMC was treated with siRNA or activator, we found that NRF2 could accelerate cell migration by affecting MMP2/3/7, and promote cell proliferation by regulating PDGFR/ERK1/2 and mTOR/P70S6K pathways. Therefore, the study has shown that the clinical application of NRF2 activator in the treatment of pulmonary hypertension may cause side effects of promoting the proliferation and migration of rPASMC. Attention should be paid to the combination of NRF2 activators.

Sulforaphane upregulates the mRNA expression of NRF2 and NQO1 in non-dialysis patients with chronic kidney disease.

Sulforaphane (SFN), found in cruciferous vegetables, is a known activator of NRF2 (master regulator of cellular antioxidant responses). Patients with chronic kidney disease (CKD) present an imbalance in the redox state, presenting reduced expression of NRF2 and increased expression of NF-κB. Therefore, this study aimed to evaluate the effects of SFN on the mRNA expression of NRF2, NF-κB and markers of oxidative stress in patients with CKD. Here, we observed a significant increase in the mRNA expression of NRF2 (p = 0.02) and NQO1 (p = 0.04) in the group that received 400 µg/day of SFN for 1 month. Furthermore, we observed an improvement in the levels of phosphate (p = 0.02), glucose (p = 0.05) and triglycerides (p = 0.02) also in this group. On the other hand, plasma levels of LDL-c (p = 0.04) and total cholesterol (p = 0.03) increased in the placebo group during the study period. In conclusion, 400 µg/day of SFN for one month improves the antioxidant system and serum glucose and phosphate levels in non-dialysis CKD patients.

A mixture of extracts from natural ingredients reduces the neurotoxic polarization of microglia via modulating NF-κB/NF-E2-related factor 2 activation.

Neurotoxic microglia-provoked neuroinflammation is implicated in cognitive decline in Alzheimer's disease (AD). Supplementation with Ginkgo biloba, phosphatidylserine, Curcuma longa, and propolis is reported to improve the cognitive functions of elderly people; however, the underlying mechanisms of this combination of natural ingredients are unknown. We investigated the effects of a mixture of extracts from propolis, Coffea arabica, Gotu kola, phosphatidylserine, Ginkgo biloba, and Curcuma longa (mixture) on microglia polarization after exposure to amyloid ß1-42 (Aß1-42, 1 µM) and lipopolysaccharide from Porphyromonas gingivalis (PgLPS, 1 µg/mL), using MG6 and BV2 microglial cells. Exposure to Aß1-42 and PgLPS (AL) raised the mRNA expression of IL-1ß, TNF-α, and IL-6, nuclear translocation of p65 NF-κB in MG6 cells and BV2 cells, and mitochondrial reactive oxygen species (ROS) production in MG6 cells. The mixture dramatically suppressed the mRNA expression of IL-1ß, TNF-α, and IL-6, but significantly promoted that of IL-10, TGFß1, and BDNF in AL-exposed MG6 and BV2 cells. Furthermore, the mixture significantly suppressed the nuclear translocation of p65 NF-κB but significantly promoted that of NF-E2-related factor 2 (Nrf2) in AL-exposed MG6 and BV2 cells. Furthermore, the mixture significantly ameliorated mitochondrial ROS production but increased mitochondrial membrane potential in MG6 cells. These observations strongly suggest that the mixture demotes the neuropathic polarization of microglia by modulating NF-κB/Nrf2 activation and improving mitochondrial functions. This study supplies the potential mechanisms of the efficacy of a combination of natural ingredients that can be applied in the prevention of cognitive decline in AD and aging by targeting microglia-mediated neuroinflammation.

Supplementation of Clostridium butyricum Alleviates Vascular Inflammation in Diabetic Mice.

BACKGRUOUND: Gut microbiota is closely related to the occurrence and development of diabetes and affects the prognosis of diabetic complications, and the underlying mechanisms are only partially understood. We aimed to explore the possible link between the gut microbiota and vascular inflammation of diabetic mice. METHODS: The db/db diabetic and wild-type (WT) mice were used in this study. We profiled gut microbiota and examined the and vascular function in both db/db group and WT group. Gut microbiota was analyzed by 16s rRNA sequencing. Vascular function was examined by ultrasonographic hemodynamics and histological staining. Clostridium butyricum (CB) was orally administered to diabetic mice by intragastric gavage every 2 days for 2 consecutive months. Reactive oxygen species (ROS) and expression of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were detected by fluorescence microscopy. The mRNA expression of inflammatory cytokines was tested by quantitative polymerase chain reaction. RESULTS: Compared with WT mice, CB abundance was significantly decreased in the gut of db/db mice, together with compromised vascular function and activated inflammation in the arterial tissue. Meanwhile, ROS in the vascular tissue of db/db mice was also significantly increased. Oral administration of CB restored the protective microbiota, and protected the vascular function in the db/db mice via activating the Nrf2/HO-1 pathway. CONCLUSION: This study identified the potential link between decreased CB abundance in gut microbiota and vascular inflammation in diabetes. Therapeutic delivery of CB by gut transplantation alleviates the vascular lesions of diabetes mellitus by activating the Nrf2/HO-1 pathway.

PHPB ameliorates memory deficits and reduces oxidative injury in Alzheimer's disease mouse model by activating Nrf2 signaling pathway.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is the most common cause of dementia in elderly people and substantially affects patient quality of life. Oxidative stress is considered a key factor in the development of AD. Nrf2 plays a vital role in maintaining redox homeostasis and regulating neuroinflammatory responses in AD. Previous studies show that potassium 2-(1-hydroxypentyl)-benzoate (PHPB) exerts neuroprotective effects against cognitive impairment in a variety of dementia animal models such as APP/PS1 transgenic mice. In this study we investigated whether PHPB ameriorated the progression of AD by reducing oxidative stress (OS) damage. Both 5- and 13-month-old APP/PS1 mice were administered PHPB (100 mg·kg-1·d-1, i.g.) for 10 weeks. After the cognition assessment, the mice were euthanized, and the left hemisphere of the brain was harvested for analyses. We showed that 5-month-old APP/PS1 mice already exhibited impaired performance in the step-down test, and knockdown of Nrf2 gene only slightly increased the impairment, while knockdown of Nrf2 gene in 13-month-old APP/PS1 mice resulted in greatly worse performance. PHPB administration significantly ameliorated the cognition impairments and enhanced antioxidative capacity in APP/PS1 mice. In addition, PHPB administration significantly increased the p-AKT/AKT and p-GSK3ß/GSK3ß ratios and the expression levels of Nrf2, HO-1 and NQO-1 in APP/PS1 mice, but these changes were abolished by knockdown of Nrf2 gene. In SK-N-SH APPwt cells and primary mouse neurons, PHPB (10 µM) significantly increased the p-AKT/AKT and p-GSK3ß/GSK3ß ratios and the level of Nrf2, which were blocked by knockdown of Nrf2 gene. In summary, this study demonstrates that PHPB exerts a protective effect via the Akt/GSK3ß/Nrf2 pathway and it might be a promising neuroprotective agent for the treatment of AD.

Grape Seed Proanthocyanidins Protect Pancreatic ß Cells Against Ferroptosis via the Nrf2 Pathway in Type 2 Diabetes.

Pancreatic ß cell damage is the primary contributor to type 2 diabetes mellitus (T2DM); however, the underlying mechanism remains nebulous. This study explored the role of ferroptosis in pancreatic ß cell damage and the protective effects of grape seed proanthocyanidin extract (GSPE). In T2DM model rats, the blood glucose, water intake, urine volume, HbA1c, and homeostasis model assessment-insulin resistance were significantly increased, while the body weight and the insulin level were significantly decreased, indicating the successful establishment of the T2DM model. MIN6 mouse insulinoma ß cells were cultured in high glucose and sodium palmitate conditions to obtain a glycolipid damage model, which was administered with GSPE, ferrostatin-1 (Fer-1), or nuclear factor erythroid 2-related factor 2 (Nrf2) small interfering (si) RNA. GSPE and Fer-1 treatment significantly improved pancreatic ß-cell dysfunction and protected against cell death. Both treatments increased the superoxide dismutase and glutathione activity, reduced the malondialdehyde and reactive oxygen species levels, and improved iron metabolism. Furthermore, the treatments reversed the expression of ferroptosis markers cysteine/glutamate transporter (XCT) and glutathione peroxidase 4 (GPX4) caused by glycolipid toxicity. GSPE treatments activated the expression of Nrf2 and related proteins. These effects were reversed when co-transfected with si-Nrf2. GSPE inhibits ferroptosis by activating the Nrf2 signaling pathway, thus reducing ß-cell damage and dysfunction in T2DM. Therefore, GSPE is a potential treatment strategy against T2DM.

Radiotherapy induces an increase in serum antioxidant capacity reflecting tumor response.

Background and purpose: Radiotherapy (RT) is a mainstay component of treatment for patients with head and neck squamous cell carcinoma (HNSCC), but responses vary. As RT relies upon oxidative damage, antioxidant expression in response to RT-induced reactive oxygen species (ROS) could compromise treatment response. We aimed to examine local and systemic antioxidant responses to increased RT-induced ROS in relation to treatment success. Materials and methods: Nuclear factor erythroid 2-related factor 2 (NRF2), the main antioxidant transcription factor, was immunofluorescently stained in FaDu cells and in tumor biopsies of patients with oral cavity/oropharynx HNSCC before and after five fractions of RT. Besides, total antioxidant capacity (TAC) was analyzed in HNSCC tumor cells in vitro and in serum of HNSCC patients before, during, and after RT. Results: Data revealed an increase in NRF2 expression and TAC in head and neck cancer cells in vitro over the course of 5 daily fractions of 2 Gy. In accordance, also in patients' tumors NRF2 expression increased, which was associated with increased serum TAC during RT. Increasing serum TAC was related to impaired local tumor control. Conclusion: Radiation induced NRF2 expression and upregulated TAC, which may compromise the effect of RT-induced ROS. Changes in serum TAC during RT could serve as a novel predictor of treatment outcome in HNSCC patients.Medical Ethics Review Committee (CMO) approval - CMO number: 2007/104.

Neferine inhibits the progression of diabetic nephropathy by modulating the miR-17-5p/nuclear factor E2-related factor 2 axis.

OBJECTIVE: To investigate the effect of Neferine (Nef) on diabetic nephropathy (DN) and to explore the mechanism of Nef in DN based on miRNA regulation theory. METHODS: A DN mouse model was constructed and treated with Nef. Serum creatinine (Crea), blood urea (UREA) and urinary albumin were measured in mice by kits, and renal histopathological changes and fibrosis were observed by hematoxylin-eosin staining and Masson staining. Renal tissue superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) activities were measured by enzyme-linked immunosorbent assay (ELISA). Western blotting was used to detect the expression of nuclear factor E2-related factor 2 (Nrf2)/ heme oxygenase 1 (HO-1) signaling pathway-related proteins in kidney tissues. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-17-5p in kidney tissues. Subsequently, a DN in vitro model was constructed by high glucose culture of human mesangial cells (HMCs), cells were transfected with miR-17-5p mimic and/or treated with Nef, and we used qRT-PCR to detect cellular miR-17 expression, flow cytometry to detect apoptosis, ELISAs to detect cellular SOD, MDA, and GSH-Px activities, Western blots to detect Nrf2/HO-1 signaling pathway-related protein expression, and dual luciferase reporter gene assays to verify the targeting relationship between Nrf2 and miR-17-5p. RESULTS: Administration of Nef significantly reduced the levels of blood glucose, Crea, and UREA and the expression of miR-17-5p, improved renal histopathology and fibrosis, significantly reduced MDA levels, elevated SOD and GSH-Px activities, and activated Nrf2 expression in kidney tissues from mice with DN. Nrf2 is a post-transcriptional target of miR-17-5p. In HMCs transfected with miR-17-5p mimics, the mRNA and protein levels of Nrf2 were significantly suppressed. Furthermore, miR-17-5p overexpression and Nef intervention resulted in a significant increase in high glucose-induced apoptosis and MDA levels in HMCs and a significant decrease in the protein expression of HO-1 and Nrf2. CONCLUSION: Collectively, these results indicate that Nef has an ameliorative effect on DN, and the mechanism may be through the miR-17-5p/Nrf2 pathway.

Mechanism of BMAL1 attenuating H2O2-induced cardiomyocyte injury / 天津医药

Objective To investigate the effect of BMAL1 on H2O2-induced cardiomyocyte injury through NRF2-regulated ROS/NLRP3 inflammasome pathway.Methods H9c2 cells and H9c2 cells with stable over-expressed BMAL1 were cultured and divided into the control group,the H2O2 group,the BMAL1-OE group,the BMAL1-OE+H2O2 group,the BMAL1-OE+ML385 group and the BMAL1-OE+ML385+H2O2 group.All groups were pre-intervened with corresponding inhibitors,and then treated with 0.2 mmol/L H2O2,except for the control group and the BMAL1-OE group.After the intervention,CCK-8 assay was used to measure cell viability,fluorescent probe DCFH-DA was used to measure ROS generation and Western blot assay was used to detect BMAL1,NRF2 and NLRP3 protein expressions.ELISA was used to determine IL-1β release.Results Compared with the control group,the cell viability was decreased,ROS generation was increased,BMAL1 and NRF2 protein expressions were decreased,NLRP3 expression and IL-1β release were increased in the H2O2 group(P＜0.05).Compared with the H2O2 group,the cell viability was increased,ROS generation was decreased,BMAL1-OE and NRF2 protein expressions were increased,NLRP3 expression and IL-1β release were decreased in the BMAL1-OE+H2O2 group(P＜0.05).Compared with the BMAL1-OE+H2O2 group,the cell viability was decreased,ROS generation was increased,NLRP3 expression and IL-1β release were increased in the BMAL1-OE+ML385+H2O2 group(P＜0.05).Conclusion BMAL1 attenuates H2O2-induced H9c2 cardiomyocyte injury,and its mechanism may be related to the regulation of ROS/NLRP3 inflammasome pathway through NRF2.

The mechanism of Wumei pill on ulcerative colitis in mice based on Nrf2/ARE antioxidant stress pathway / 天津医药

Objective To explore the mechanism of Wumei pill on ulcerative colitis(UC)in mice based on the anti oxidative stress pathway of nuclear factor erythroid 2-related factor 2(Nrf2)/antioxidant response element(ARE).Methods Seventy SPF male C57BL/6 mice were randomly divided into the control group,the UC group,the mesalazine group(MES group,0.82 g/kg MES),the low dose Wumei pill group(WMW-L group,5 g/kg crude drug),the middle dose Wumei pill group(WMW-M group,10 g/kg crude drug),the high dose Wumei pill group(WMW-H group,20 g/kg crude drug)and the high dose Wumei pills+Nrf2 inhibitor ML-385 group(WMW-H+ML-385 group,Wumei pills crude drug 20 g/kg+20 mg/kg ML-385),with 10 rats in each group.The disease activity index(DAI)score and colonic mucosa injury score were performed in mice after the last administration.Pathological changes of colonic mucosa in mice were observed by HE staining.The levels of interleukin(IL)-1β,tumor necrosis factor-α(TNF-α)and IL-6 in serum and colon tissue of mice were measured by enzyme-linked immunosorbent assay(ELISA).The content of malondialdehyde(MDA)in serum and colon tissue of mice was determined by thiobarbituric acid colorimetry(TBA).The activity of superoxide dismutase(SOD)in serum and colon tissue of mice was measured by xanthine oxidase method.The activity of glutathione peroxidase(GSH-px)in serum and colon tissue of mice was determined by direct method with dithiodinitrobenzoic acid(DTNB).The positive expression of Nrf2 in colon tissue of mice was observed by immunohistochemistry.The expression of heme oxygenase-1(HO-1)and NAD(P)H:quinone oxidoreductase-1(NQO1)proteins in colon tissue of mice were detected by Western blot assay.Results Compared with the control group,the DAI score,colonic mucosa injury score,colonic histopathology score,levels of IL-1β,TNF-α,IL-6 and MDA in serum and colonic tissue,and expression levels of Nrf2,HO-1 and NQO1 protein in colonic tissue of mice were increased in the UC group,levels of SOD and GSH-px in serum and colon tissue decreased(P＜0.05),the colon mucosa of mice was seriously damaged.Compared with the UC group,changes of corresponding indexes were contrary to the above in the MES group,the WMW-M group and the WMW-H group.However,the expression levels of Nrf2,HO-1 and NQO1 proteins in colon tissue were increased(P＜0.05),and the damage of colon mucosa in mice was alleviated.Changes of the above indexes were dose-dependent in the WMW-L group,the WMW-M group and the WMW-H group.There were no significant differences in the above indexes between the WMW-H group and the MES group.ML-385 attenuated the improvement effect of high dose Wumei pill on colon mucosa injury.Conclusion Wumei pill may alleviate the colon mucosal damage of UC mice by activating Nrf2/ARE antioxidant stress pathway.

Role of ROS in attenuation of hypoxia-reoxygenation injury in rat cardiomyocytes by pinacidil postconditioning: relationship with Nrf2-ARE signaling pathway / 中华麻醉学杂志

Objective:To evaluate the role of reactive oxygen species (ROS) in attenuation of hypoxia-reoxygenation (H/R) injury in rat cardiomyocytes by pinacidil postconditioning and the relationship with nuclear factor erythrid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway.Methods:Adult rat cardiomyocytes were isolated and cultured and then divided into 4 groups ( n=20 each) by a random number table method: control group (group C), H/R group, pinacidil postconditioning group (group P) and reactive oxygen scavenger N-(2-mercaptopropionyl)-glycine(MPG)+ pinacidil postconditioning group (group MPG+ P). Group C was continuously exposed to 95%O 2+ 5%CO 2 in an incubator at 37 ℃ for 105 min. The cells were exposed to 5%CO 2+ 1%O 2+ 94%N 2 in an incubator at 37 ℃ for 45 min followed by reoxygenation for 60 min to prepare H/R injury model. The cells were exposed to hypoxia for 45 min and then treated with pinacidil 50 μmol/L for 5 min followed by reoxygenation for 60 min in group P. The cells were exposed to hypoxia for 45 min, treated with MPG 2 mmol/L for 10 min, and then treated with pinacidil for 5 min followed by reoxygenation for 60 min in group MPG+ P. The content of Ca 2+ and activity of Nrf2 in cardiomyocytes were measured at the end of reoxygenation. The ultrastructure of cardiomyocytes was observed, and mitochondrial ultrastructure was evaluated using mitochondrial Flameng score. The expression of Nrf2, superoxide dismutase (SOD1), quinone oxidoreductase 1 (NQO1), and heme oxygenase 1 (HO-1) protein and mRNA was detected using Western blot and real-time polymerase chain reaction. Results:Compared with group C, the Ca 2+ content, Nrf2 activity and mitochondrial Flameng score were significantly increased, the expression of Nrf2, SOD1, NQO1 and HO-1 protein and mRNA was down-regulated ( P<0.05), and the damage to the ultrastructure of cardiomyocytes was aggravated in group H/R. Compared with H/R group, the Ca 2+ content and mitochondrial Flameng score were significantly decreased, the Nrf2 activity was increased, the expression of Nrf2, SOD1, NQO1 and HO-1 protein and mRNA was up-regulated ( P<0.05), and the damage to the ultrastructure of cardiomyocytes was attenuated in P group. Compared with P group, the Ca 2+ content and mitochondrial Flameng score were significantly increased, the Nrf2 activity was decreased, the expression of Nrf2, SOD1, NQO1 and HO-1 protein and mRNA was down-regulated ( P<0.05), and the damage to the ultrastructure of cardiomyocytes was aggravated in MPG+ P group. Conclusions:ROS is involved in attenuation of H/R injury by pinacidil postconditioning, which is associated with activation of the Nrf2-ARE signaling pathway in rat cardiomyocytes.

Role of Nrf2/HO-1 signaling pathway in human umbilical cord mesenchymal stem cells-derived exosomes-induced reduction of renal ischemia-reperfusion injury in mice / 中华麻醉学杂志

Objective:To evaluate the role of nuclear factor E2-related factor 2 (Nrf2)/heme oxidase-1 (HO-1) in reduction of renal ischemia-reperfusion (I/R) injury by the human umbilical cord mesenchymal stem cells (hucMSCs)-derived exosomes (hucMSCs-exo) in mice.Methods:The hucMSCs were cultured, and exosomes were extracted and identified by transmission electron microscopy, nanoparticle tracking analysis and Western blot. Thirty-six male SPF-grade C57BL/6 mice, weighing 20-25 g, were used. Thirty mice were selected and divided into 5 groups ( n=6 each) by a random number table method: sham operation group (Sham group), sham operation + Nrf2 inhibitor ML385 group (Sham + ML385 group), renal I/R group (I/R group), renal I/R + exosome group (I/R+ EXO group), and renal I/R + exosome + Nrf2 inhibitor ML385 group (I/R+ EXO+ ML385 group). A model of renal I/R injury was prepared by clamping the bilateral renal pedicles for 45 min followed by perfusion in anesthetized animals. ML385 30 mg/kg was intraperitoneally injected at 45 min before preparing the model in Sham+ ML385 group and I/R+ EXO+ ML385 group, and hucMSCs-exo 100 μg was injected via the tail vein at 15 min before reperfusion in I/R+ EXO group and I/R+ EXO+ ML385 group. Serum blood urea nitrogen (BUN) and creatinine (Cr) concentrations were detected at 24 h of reperfusion. The renal tissues were obtained for examination of the pathological changes and for determination of contents of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and malondialdehyde (MDA), superoxide dismutase (SOD) activity and reactive oxygen species (ROS) levels and expression of Nrf2 and HO-1 protein and mRNA (by Western blot and quantitative real-time polymerase chain reaction). The left 6 mice were allocated to sham operation group (Sham-IM group, n=3) and renal I/R group (I/R-IM group, n=3) by a random number table method for VISQUE in living imaging observation. Results:The exosomes showed a typical cup-shaped morphology with a transmission electron microscope, the nanoparticles tracked and analyzed the average diameter of the exosome, with an average diameter of 96.7 nm, and the positive expression of surface markers CD9, CD63 and TSG101 was detected using Western blot. The renal fluorescence intensity value was significantly increased in I/R-IM group as compared with Sham-IM group ( P<0.05). Compared with Sham group, the serum BUN and Cr concentrations were significantly increased, the contents of IL-6, TNF-α and MDA and ROS levels were increased, the activity of SOD was decreased, the expression of Nrf2 and HO-1 protein and mRNA was down-regulated ( P<0.05), and the pathological changes of renal tissues were aggravated in I/R group, and no significant change was found in serum BUN and Cr concentrations in Sham+ ML385 group ( P>0.05). Compared with I/R group, the serum BUN and Cr concentrations were significantly decreased, the contents of IL-6, TNF-α and MDA and ROS levels were decreased, the activity of SOD was increased, the expression of Nrf2 and HO-1 protein and mRNA was up-regulated ( P<0.05), and the pathological changes of renal tissues were significantly attenuated in I/R+ EXO group. Compared with I/R+ EXO group, the serum BUN and Cr concentrations were significantly increased, the contents of IL-6, TNF-α and MDA and ROS levels were increased, the activity of SOD was decreased, the expression of Nrf2 and HO-1 protein and mRNA was down-regulated ( P<0.05), and the pathological changes of renal tissues were aggravated in I/R+ EXO+ ML385 group. Conclusions:The mechanism by which hucMSCs-exo reduces renal I/R injury may be related to activation of the Nrf2/HO-1 signaling pathway in mice.

The Nrf2 Pathway Alleviates Overloading Force-Induced TMJ Degeneration by Downregulating Oxidative Stress Reactions.

Objective: Oxidative stress is involved in the mechanisms associated with temporomandibular joint (TMJ) diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial oxidative stress marker, but the specific mechanisms of its regulation in the early stages of mandibular condylar cartilage (MCC) degeneration remain unclear. This study aimed to explore the regulatory role of Nrf2 and its related oxidative stress signaling pathway in the early stage of MCC degeneration. Materials and Methods: Overloading force-induced MCC degeneration was performed in wild-type and Nrf2 knockout mice, as well as in mice after treatment with the Nrf2 activator cardamonin. Changes in MCC degeneration and the expression of oxidative stress markers in the corresponding situations were observed. Results: Nrf2 and NADPH oxidase 2 (NOX2) expression were elevated during early MCC degeneration induced by an overloading force. MCC degeneration was aggravated when Nrf2 was knocked out, accompanied by increased NOX2 and superoxide dismutase 2 (SOD2) expression. The MCC degeneration process was alleviated after cardamonin treatment, with activation of the Nrf2 pathway and decreased NOX2 and SOD2 expression. Conclusion: Early MCC degeneration is accompanied by mild oxidative stress progression. Activated Nrf2 and related pathways could alleviate the degeneration of MCC.

High Expression of NRF2 and Low Expression of KEAP1 Predict Worse Survival in Patients With Operable Triple-Negative Breast Cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer. Currently, no effective treatment options for this condition exist. Nuclear factor erythroid 2-related factor 2 (NRF2), encoded by nuclear factor erythroid-derived 2-like 2 (NFE2L2) gene and its endogenous inhibitor, Kelch-like ECH-associated protein 1 (KEAP1), both participate in cellular defense mechanisms against oxidative stress and contribute to chemoresistance and tumor progression in numerous types of cancers. This study aimed to evaluate the expression patterns of NRF2 and KEAP1 and their prognostic value in operable TNBC. METHODS: Tissue microarrays were prepared using tumor tissues collected from 203 patients with TNBC who underwent surgery. Immunohistochemical staining analyses of NRF2 and KEAP1 were performed. The expression of each immunomarker was categorized into two groups (low or high) based on the median H-score. We analyzed the association between the expression of each immunomarker and clinicopathological information to predict survival. A total of 225 TNBC samples from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset were used to validate our results. RESULTS: NRF2 immunoreactivity was detected in the nucleus and was associated with histologic grade and Ki-67 index, whereas KEAP1 immunoreactivity was detected in the cytoplasm and was associated with the Ki-67 index. Survival analyses showed that NRF2 and KEAP1 expressions were independent prognostic factors for overall survival (OS) (hazard ratio [HR], 2.45 and 0.30; p = 0.015 and 0.016, respectively) and disease-free survival (HR, 2.27 and 0.42; p = 0.019 and 0.022, respectively). NFE2L2 mRNA expression was an independent prognostic factor for OS (HR, 0.59; p = 0.009) in the METABRIC dataset. CONCLUSION: High NRF2 and low KEAP1 expressions independently predicted poor survival in patients with operable TNBC. Further investigations are warranted to examine the possible therapeutic benefits of targeting the KEAP1-NRF2 pathway for TNBC treatment.

Effect of miR-27b-3p and Nrf2 in human retinal pigment epithelial cell induced by high-glucose.

AIM: To determine whether the microRNA-27b-3p (miR-27b-3p)/NF-E2-related factor 2 (Nrf2) pathway plays a role in human retinal pigment epithelial (hRPE) cell response to high glucose, how miR-27b-3p and Nrf2 expression are regulated, and whether this pathway could be specifically targeted. METHODS: hRPE cells were cultured in normal glucose or high glucose for 1, 3, or 6d before measuring cellular proliferation rates using cell counting kit-8 and reactive oxygen species (ROS) levels using a dihydroethidium kit. miR-27b-3p, Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) mRNA and protein levels were analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunocytofluorescence (ICF), respectively. Western blot analyses were performed to determine nuclear and total Nrf2 protein levels. Nrf2, NQO1, and HO-1 expression levels by RT-qPCR, ICF, or Western blot were further tested after miR-27b-3p overexpression or inhibitor lentiviral transfection. Finally, the expression level of those target genes was analyzed after treating hRPE cells with pyridoxamine. RESULTS: Persistent exposure to high glucose gradually suppressed hRPE Nrf2, NQO1, and HO-1 mRNA and protein levels and increased miR-27b-3p mRNA levels. High glucose also promoted ROS release and inhibited cellular proliferation. Nrf2, NQO1, and HO-1 mRNA levels decreased after miR-27b-3p overexpression and, conversely, both mRNA and protein levels increased after expressing a miR-27b-3p inhibitor. After treating hRPE cells exposed to high glucose with pyridoxamine, ROS levels tended to decreased, proliferation rate increased, Nrf2, NQO1, and HO-1 mRNA and protein levels were upregulated, and miR-27b-3p mRNA levels were suppressed. CONCLUSION: Nrf2 is a downstream target of miR-27b-3p. Furthermore, the miR-27b-3p inhibitor pyridoxamine can alleviate high glucose injury by regulating the miR-27b-3p/Nrf2 axis.

Unraveling the protective role of Nrf2 in molluscs: Insights into mitochondrial and apoptosis pathways in the defense against Bap-induced oxidative stress.

Benzopyrene (Bap) is a major constituent of petroleum pollutants commonly found in aquatic environments, and its mutagenic and carcinogenic properties have adverse effects on aquatic organisms' development, growth, and reproduction. The antioxidant defense system element, NF-E2-related factor 2 (Nrf2), has been linked to the oxidative stress response in marine invertebrates exposed to toxic substances. In a previous study, a novel Nrf2 homologue, McNrf2, was identified in mussel Mytilus coruscus, a significant model marine molluscs in ecotoxicology studies. McNrf2 showed the potential to trigger an antioxidant defense against oxidative stress induced by Bap. Here, we employed an Nrf2 overexpression and inhibition model using SFN and ML385 as Nrf2 inducer and inhibitor, respectively. Next, immunofluorescence technique was used to evaluate the nuclear activation of Nrf2 induced by Bap-mediated oxidative stress. Transmission electron microscopy revealed that overexpression of Nrf2 could maintain the quantity and structural integrity of mitochondria, while flow cytometry analysis showed that Nrf2 could alleviate Bap-induced cellular apoptosis. These findings suggest that Nrf2 can protect molluscs from Bap-induced oxidative stress through the mitochondria and apoptosis pathways, providing a novel perspective on Nrf2's antioxidant function.

An ERK5-NRF2 Axis Mediates Senescence-Associated Stemness and Atherosclerosis.

BACKGROUND: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis. METHODS: A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow-derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis. RESULTS: We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow-derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)-induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors. CONCLUSIONS: We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.

Possible Involvement of Dermal Fibroblasts in Modulating Nrf2 Signaling in Epidermal Keratinocytes.

Epidermal keratinocytes protect themselves by cooperating with neighboring cells against internal and external stresses, which leads not only to the maintenance of cell homeostasis but also to the prevention of skin aging. Although it is known that nuclear factor (NF)-E2-related factor 2 (Nrf2) signaling plays a pivotal role in ameliorating oxidative stress and inflammatory responses under stress situations, it is unclear whether Nrf2 signaling in keratinocytes cooperates with neighboring cells such as dermal fibroblasts. Thus, this study was conducted to examine the influence of dermal fibroblasts on Nrf2 signaling in epidermal keratinocytes using a co-culture system. The results show that expression levels of Nrf2-regulated antioxidant factors, such as glutathione and heme oxygenase-1, in HaCaT keratinocytes (HaCaT KCs) are up-regulated in the presence of normal human dermal fibroblasts (NHDFs). In addition, the secretion of pro-inflammatory molecules, including interleukin-1α (IL-1α) and prostaglandin E2 (PGE2), is suppressed in co-cultures of NHDFs and UVB-irradiated HaCaT KCs. Interestingly, the localization of Nrf2 protein in HaCaT KCs was immediately translocated from the cytoplasm to the nucleus after the co-culture with NHDFs. These results suggest the possibility that Nrf2 signaling in keratinocytes is regulated in cooperation with dermal fibroblasts.