The promoting effect and mechanism of Nrf2 on cell metastasis in cervical cancer.

BACKGROUND: Cervical cancer (CC) has poor prognosis and high mortality rate for its metastasis during the disease progression. Epithelial-mesenchymal transition (EMT) and anoikis are initial and pivotal steps during the metastatic process. Although higher levels of Nrf2 are associated with aggressive tumor behaviors in cervical cancer, the detailed mechanism of Nrf2 in cervical cancer metastasis, especially EMT and anoikis, remains unclear. METHODS: Immunohistochemistry (IHC) was used to examine Nrf2 expression in CC. Wound healing assay and transwell analysis were used to evaluate the migration ability of CC cells. Western blot, qTR-PCR and immunofluorescent staining were used to verify the expression level of Nrf2, the EMT associated markers and anoikis associated proteins. Flow cytometry assays and cell counting were used to detect the apoptosis of cervical cancer cells. The lung and lymph node metastatic mouse model were established for studies in vivo. The interaction between Nrf2 and Snail1 was confirmed by rescue-of-function assay. RESULTS: When compared with cervical cancer patients without lymph node metastasis, Nrf2 was highly expressed in patients with lymph node metastasis. And Nrf2 was proved to enhance the migration ability of HeLa and SiHa cells. In addition, Nrf2 was positively correlated with EMT processes and negatively associated with anoikis in cervical cancer. In vivo, a xenograft assay also showed that Nrf2 facilitated both pulmonary and lymphatic distant metastasis of cervical cancer. Rescue-of-function assay further revealed the mechanism that Nrf2 impacted the metastasis of CC through Snail1. CONCLUSION: Our fundings established Nrf2 plays a crucial role in the metastasis of cervical cancer by enhancing EMT and resistance to anoikis by promoting the expression of Snail1, with potential value as a therapeutic candidate.

Melatonin alleviates particulate matter-induced liver fibrosis by inhibiting ROS-mediated mitophagy and inflammation via Nrf2 activation.

OBJECTIVE: Fine particulate matter (PM2.5) is a source of pollution worldwide, that causes inflammation and liver fibrosis. Melatonin, as the predominant hormone secreted by the pineal gland, can inhibit PM2.5-induced lung injury by activating nuclear factor erythroid 2-related factor 2 (Nrf2) to inhibit ferroptosis. However, the possible role of melatonin in PM2.5-induced liver damage remains unclear. EXPERIMENTAL APPROACH: In vitro, the effects of melatonin on PM2.5-induced oxidative stress and LX-2 cell activation were examined. In vivo, a PM2.5-induced inflammation and liver fibrosis mouse model was used to evaluate the hepatoprotective effect of melatonin. RESULTS: In vitro, melatonin induced the expression of Nrf2 and its downstream genes and inhibited PM2.5-induced reactive oxygen species (ROS) production and mitochondrial damage. Melatonin also ameliorated the PM2.5-induced oxidative stress and fibrogenic marker upregulation. However, the antifibrotic effect of melatonin was abolished in siNrf2-treated LX-2 cells. In vivo, we observed mitochondrial abnormalities and mitochondrial fragmentation, which were accompanied by increased PTEN-induced kinase 1 (PINK1) and Parkin expression, in PM2.5-treated mouse hepatocytes. These changes were partially reversed by melatonin. In addition, melatonin activated the Nrf2 signaling pathway and protected against PM2.5-induced oxidative stress. Furthermore, melatonin alleviated inflammation and liver fibrosis. Moreover, Nrf2-KO mice exhibited more severe inflammation and liver fibrosis after PM2.5 exposure than wild-type mice, and the protective effect of melatonin on PM2.5- treated Nrf2-KO mice was greatly compromised. CONCLUSION: These data suggest that melatonin effectively inhibits PM2.5-induced liver fibrosis by activating Nrf2 and inhibiting ROS-mediated mitophagy and inflammation.

PM2.5 inhibits system Xc- activity to induce ferroptosis by activating the AMPK-Beclin1 pathway in acute lung injury.

Urban airborne fine particulate matter (PM2.5) is a global pollution source that has been strongly related to multiple respiratory diseases involving various types of regulated cell death (RCD). However, the role of ferroptosis, a novel form of RCD, in PM2.5-induced acute lung injury (ALI), has not been elucidated. Herein, we define the role and mechanism of ferroptosis in a PM2.5-induced ALI model. First, we demonstrated that lipid peroxidation and iron accumulation were significantly enhanced in ALI models and were accompanied by activation of the AMP-activated protein kinase (AMPK)-Beclin1 signaling pathway and inhibition of the key subunit SLC7A11 of System Xc-. However, these abnormalities were partially reversed by ferroptosis inhibitors. We further revealed that Beclin1 knockdown or overexpression ameliorated or exacerbated PM2.5-induced ferroptosis, respectively. Mechanistically, we verified that Beclin1 blocks System Xc- activity to trigger ferroptosis by directly binding to SLC7A11. Finally, knockdown of Beclin1 by AAV-shRNA or inhibition of AMPK, an upstream activator of Beclin1, ameliorated PM2.5-induced ferroptosis and ALI. Taken together, our results revealed that ferroptosis plays a novel role in PM2.5-induced ALI and elucidated the specific mechanism involving the AMPK-Beclin1 pathway and System Xc-, which may provide new insight into the toxicological effects of PM2.5 on respiratory problems.

Leonurine inhibits the TXNIP/NLRP3 and NF-κB pathways via Nrf2 activation to alleviate carrageenan-induced pleurisy in mice.

Oxidative stress and inflammation play important roles in pleurisy. Leonurine (Leo) has been confirmed to exert antioxidative and antiinflammatory effects in many preclinical experiments, but these effects have not been studied in pleurisy. The aim of this study was to explore the therapeutic effect and mechanism of Leo in a carrageenan (CAR)-induced pleurisy model. In this study, we found that the increase of reactive oxygen species (ROS), myeloperoxidase (MPO), and malondialdehyde (MDA) and decrease of glutathione (GSH) induced by CAR could be reversed by the treatment of Leo. Leo effectively reduced the levels of proinflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and the percentages of mature macrophages and increased the levels of antiinflammatory cytokines (IL-10). Furthermore, Western blotting revealed that Leo significantly activated the Nrf2 pathway to restrain the thioredoxin-interacting protein/NOD-like receptor protein 3 (TXNIP/NLRP3) and nuclear factor kappa-B (NF-κB) pathways. However, the protective effect of Leo was significantly weakened in Nrf2-deficient mice. These results indicate that Leo confers potent protection against CAR-induced pleurisy by inhibiting the TXNIP/NLRP3 and NF-κB pathways dependent on Nrf2, which may serve as a promising agent for attenuating pleurisy.

Targeting Nrf2 may reverse the drug resistance in ovarian cancer.

BACKGROUND: Acquired resistance to therapeutic drugs has become an important issue in treating ovarian cancer. Studies have shown that the prevalent chemotherapy resistance (cisplatin, paclitaxel etc.) for ovarian cancer occurs partly because of decreased production of reactive oxygen species within the mitochondria of ovarian cancer cells. MAIN BODY: Nuclear erythroid-related factor-2 (Nrf2) mainly controls the regulation of transcription of genes through the Keap1-Nrf2-ARE signaling pathway and protects cells by fighting oxidative stress and defending against harmful substances. This protective effect is reflected in the promotion of tumor cell growth and their resistance to chemotherapy drugs. Therefore, inhibition of the Nrf2 pathway may reverse drug resistance. In this review, we describe the functions of Nrf2 in drug resistance based on Nrf2-associated signaling pathways determined in previous studies. CONCLUSIONS: Further studies on the relevant mechanisms of Nrf2 may help improve the outcomes of ovarian cancer therapy.

Hyperoside relieves particulate matter-induced lung injury by inhibiting AMPK/mTOR-mediated autophagy deregulation.

Autophagy-mediated cell death plays a critical role in the pathogenesis of PMs-induced lung injury. Hyperoside (Hyp), a flavonoid glycosides, is known to exert protective effects on many diseases by inhibiting autophagic activity. The current study aimed to explore the protective effect and mechanism of Hyp against PMs-induced lung injury in PM2.5 challenged Beas-2b cells in vitro and BALB/C mice in vivo. In vitro, we found that the organic solvent-extractable fraction of SRM1649b (O-PMs) caused more severe cytotoxicity in Beas-2b cells than the water solvent-extractable fraction of SRM1649b (W-PMs). O-PMs treatment dose-dependently upregulated the expression of autophagy markers (beclin-1, p62, atg3 and LC3II) and apoptotic proteins. This cytotoxicity of O-PMs was attenuated by Hyp pretreatment in parallel with downregulation of the expression of autophagy markers, apoptotic proteins, and p-AMPK and upregulation of p-mTOR expression. Notably, the therapeutic effect of Hyp was attenuated by pretreated with AICAR (an AMPK inducer), but enhanced by CC and 3-MA treatment. In vivo, Hyp reduced pathological lung injury and decreased the levels of PMs-induced inflammatory cytokines (TNF-α and IL-6), and the number of total cells in the BALF by inhibiting AMPK/mTOR signaling. Furthermore, cotreatment with AICAR (500 mg/kg) reduced but did not abrogate the pulmonary protective effect of Hyp. These findings indicate that Hyp protects against PMs-induced lung injury by suppressing autophagy deregulation and apoptosis through regulation of the AMPK/mTOR pathway.

Food-Derived Pharmacological Modulators of the Nrf2/ARE Pathway: Their Role in the Treatment of Diseases.

Oxidative stress, which refers to unbalanced accumulation of reactive oxygen species (ROS) levels in cells, has been linked to acute and chronic diseases. Nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) pathway plays a vital role in regulating cytoprotective genes and enzymes in response to oxidative stress. Therefore, pharmacological regulation of Nrf2/ARE pathway is an effective method to treat several diseases that are mainly characterized by oxidative stress and inflammation. Natural products that counteract oxidative stress by modulating Nrf2 have contributed significantly to disease treatment. In this review, we focus on bioactive compounds derived from food that are Nrf2/ARE pathway regulators and describe the molecular mechanisms for regulating Nrf2 to exert favorable effects in experimental models of diseases.

Oridonin attenuates carrageenan-induced pleurisy via activation of the KEAP-1/Nrf2 pathway and inhibition of the TXNIP/NLRP3 and NF-κB pathway in mice.

The classic NLRP3 inflammasome and NF-κB molecular pathways are activated in many inflammatory-related diseases, such as pleurisy. Because oridonin (Ori) has been indicated as a covalent NLRP3 inhibitor with strong anti-inflammasome activity, we herein aimed to assess the effects of Ori in a mouse model of carrageenan (CAR)-induced pleurisy. The results showed that CAR caused hemorrhaging and exudation of lung tissues and the release of inflammatory factors (TNF-α, IL-6 and IL-1ß), effects that were significantly reduced by treatment with Ori. In addition, increased neutrophil infiltration, protein concentrations and volumes were found in the exudates of the CAR group, and these phenomena were suppressed by Ori treatment. Regarding cellular pathways, Ori could alleviate the CAR-activated NF-κB and TXNIP/NLRP3 pathways. Additionally, oxidative stress was shown to be involved in the pathogenesis of pleurisy, but possible mechanisms remain to be explored. Herein, Ori reversed the CAR-induced depletion of GSH and SOD and the CAR-induced increases in ROS, MPO and MDA levels. Furthermore, Ori inhibited NOX-4 levels, initiated the dissociation of KEAP-1 from Nrf2, activated the downstream genes HO-1 and exerted antioxidative effects on CAR-induced pleurisy. In conclusion, Ori conferred protection against CAR-induced pleurisy via Nrf2-dependent antioxidative and NLRP3-dependent anti-inflammatory properties.

Oridonin protects LPS-induced acute lung injury by modulating Nrf2-mediated oxidative stress and Nrf2-independent NLRP3 and NF-κB pathways.

BACKGROUND: Oxidative stress and the resulting inflammation are essential pathological processes in acute lung injury (ALI). Nuclear factor erythroid 2-related factor 2 (Nrf2), a vital transcriptional factor, possesses antioxidative potential and has become a primary target to treat many diseases. Oridonin (Ori), isolated from the plant Rabdosia Rrubescens, is a natural substance that possesses antioxidative and anti-inflammatory effects. Our aim was to study whether the anti-inflammatory and antioxidant effects of Ori on LPS-induced ALI were mediated by Nrf2. METHODS: MTT assays, Western blotting analysis, a mouse model, and hematoxylin-eosin (H & E) staining were employed to explore the mechanisms by which Ori exerts a protective effect on LPS-induced lung injury in RAW264.7 cells and in a mouse model. RESULTS: Our results indicated that Ori increased the expression of Nrf2 and its downstream genes (HO-1, GCLM), which was mediated by the activation of Akt and MAPK. Additionally, Ori inhibited LPS-induced activation of the pro-inflammatory pathways NLRP3 inflammasome and NF-κB pathways. These two pathways were also proven to be Nrf2-independent by the use of a Nrf2 inhibitor. In keeping with these findings, Ori alleviated LPS-induced histopathological changes, the enhanced production of myeloperoxidase and malondialdehyde, and the depleted expression of GSH and superoxide dismutase in the lung tissue of mice. Furthermore, the expression of LPS-induced NLRP3 inflammasome and NF-κB pathways was more evident in Nrf2-deficient mice but could still be reversed by Ori. CONCLUSIONS: Our results demonstrated that Ori exerted protective effects on LPS-induced ALI via Nrf2-independent anti-inflammatory and Nrf2-dependent antioxidative activities.

Pterostilbene Reduces Acetaminophen-Induced Liver Injury by Activating the Nrf2 Antioxidative Defense System via the AMPK/Akt/GSK3ß Pathway.

BACKGROUND/AIMS: Pterostilbene (Pts), a natural dimethylated analog of resveratrol from blueberries, exerts antioxidative and anti-apoptotic effects in various diseases. This study aims to investigate the protective effects and mechanism of Pts against acetaminophen (APAP)-induced hepatotoxicity in vivo. METHODS: C57BL/6 mice were treated with APAP or APAP+Pts. HepG2 cells were used to further explore the underlying mechanisms in vitro. The effects of Pts on hepatotoxicity were measured by commercial kits, Hematoxylin and Eosin (H&E) straining, TUNEL assay, Western blot analysis, and Flow cytometry assay. RESULTS: In vivo, Pts treatment effectively protected against APAP-induced severe liver injury by decreasing the lethality rate, the serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, liver histological injury, liver malondialdehyde (MDA) formation and myeloperoxidase (MPO) levels and by increasing liver glutathione (GSH) and superoxide dismutase (SOD) levels. Moreover, in Pts-treated mice, the nuclear factor-erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway was activated; however, APAP-induced c-Jun NH2-terminal kinase (JNK) activation, mitochondrial Bcl-2 Associated X Protein (Bax) translocation, apoptosis-inducing factor (AIF) levels and cytochrome c release were attenuated. In vitro, Pts was found to reverse hydrogen peroxide (H2O2) -induced cytotoxicity, reactive oxygen species (ROS) production and apoptosis that depended on Nrf2 activation. Moreover, Pts induced a dose-dependent increase in the phosphorylation of AMP-activated protein kinase (AMPK), serine/threonine kinase (Akt), and glycogen synthase kinase 3ß (GSK3ß) in HepG2 cells. Moreover, Pts protect against APAP or H2O2-induced toxicity were effectively attenuated or abolished in HepG2 Nrf2-/- cells and Nrf2-/- mice. CONCLUSION: Our data suggest that Pts protects against APAP-induced toxicity by activating Nrf2 via the AMPK/Akt/GSK3ß pathway.

Esculentoside A Attenuates Allergic Airway Inflammation via Activation of the Nrf-2 Pathway.

BACKGROUND: The role of airway inflammation and inflammation-induced oxidative stress in the pathogenesis and progression of chronic inflammatory airway diseases has received increasing attention in recent years. We investigated the potential anti-inflammatory and antioxidative effects of esculentoside A (EsA), a saponin isolated from the Chinese herb Phytolacca esculenta, in comparison to dexamethasone, a potent corticosteroid, in a murine model of allergic asthma. METHODS: EsA was added to cultures of A549 cells at different concentrations or for different lengths of time, and nuclear factor erythroid 2-related factor 2 (Nrf-2) translocation and heme oxygenase 1 expression were monitored. Mice treated with or without EsA and Nrf-2 siRNA were sensitized and challenged with ovalbumin (OVA) and developed airway inflammation and oxidative lung damage. The Th2-type cytokine levels and inflammatory cells in bronchoalveolar lavage fluid (BALF) and the serum immunoglobulin production and adhesion molecule expression in the lung tissues were measured. The activities of related antioxidases and glutathione were measured using assay kits. RESULTS: EsA enhanced nuclear Nrf-2 translocation in both A549 cells and the lungs of OVA-challenged mice. Airway inflammation induced by OVA was reduced. Additionally, EsA increased mRNA expression of antioxidant enzymes regulated by Nrf-2, leading to a reduction in Th2 cytokines and the expression of adhesion molecule mRNA in the BALF and lung tissues. Inhibition of Nrf-2 by siRNA abrogated the regulatory effects of EsA on inflammation and oxidant stress. CONCLUSIONS: This is the first study to illustrate that EsA acts as a novel Nrf-2 activator, which modulates the oxidative stress pathway to improve lung injury and ameliorate the development of airway inflammation.

Differential requirement of RasGRP1 for γδ T cell development and activation.

γδ T (γδT) cells belong to a distinct T cell lineage that performs immune functions different from αß T (αßT) cells. Previous studies established that Erk1/2 MAPKs are critical for positive selection of αßT cells. Additional evidence suggests that increased Erk1/2 activity promotes γδT cell generation. RasGRP1, a guanine nucleotide-releasing factor for Ras, plays an important role in positive selection of αßT cells by activating the Ras-Erk1/2 pathway. In this article, we demonstrate that RasGRP1 is critical for TCR-induced Erk1/2 activation in γδT cells, but it exerts different roles for γδT cell generation and activation. Deficiency of RasGRP1 does not obviously affect γδT cell numbers in the thymus, but it leads to increased γδT cells, particularly CD4(-)CD8(+) γδT cells, in the peripheral lymphoid organs. The virtually unhindered γδT cell development in the RasGRP1(-/-) thymus proved to be cell intrinsic, whereas the increase in CD8(+) γδT cells is caused by non-cell-intrinsic mechanisms. Our data provide genetic evidence that decreased Erk1/2 activation in the absence of RasGRP1 is compatible with γδT cell generation. Although RasGRP1 is dispensable for γδT cell generation, RasGRP1-deficient γδT cells are defective in proliferation following TCR stimulation. Additionally, RasGRP1-deficient γδT cells are impaired to produce IL-17 but not IFNγ. Together, these observations revealed that RasGRP1 plays differential roles for γδ and αß T cell development but is critical for γδT cell proliferation and production of IL-17.

Aging-related NOX4-Nrf2 redox imbalance increases susceptibility to cisplatin-induced acute kidney injury by regulating mitophagy.

BACKGROUND: During aging, excessive ROS production in the kidneys leads to redox imbalance, which contributes to oxidative damage and impaired organ homeostasis. However, whether and how aging-related NOX4-Nrf2 redox imbalance increases susceptibility to cisplatin-induced acute kidney injury remain largely unknown. METHODS: In this study, we used cisplatin-challenged aging mouse models and senescent HK-2 cells to investigate the effects and mechanisms of aging on susceptibility to cisplatin-induced acute kidney injury. RESULTS: In vivo, we found that cisplatin stimulation caused more severe renal damage, oxidative stress, mitochondrial dysfunction and mitophagy impairment in aging mice than in young mice. Moreover, Nrf2 deficiency aggravated cisplatin-induced acute kidney injury by exacerbating NOX4-Nrf2 redox imbalance and defective mitophagy. In vitro experiments on D-gal-treated human renal tubular epithelial cells (HK-2) demonstrated that senescent renal epithelial cells exhibited increased susceptibility to cisplatin-induced apoptosis, NOX4-Nrf2 redox imbalance-mediated oxidative stress and defective mitophagy. Mechanistically, we found that knockdown of Nrf2 in HK2 cells resulted in increased ROS and aggravated mitophagy impairment, whereas these effects were reversed in NOX4-knockdown cells. CONCLUSION: The present study indicates that NOX4-Nrf2 redox imbalance is critical for mitophagy deficiency in aged renal tubular epithelial cells and is a therapeutic target for alleviating cisplatin-induced acute kidney injury in elderly patients.

Protective effect of esculentoside A on lipopolysaccharide-induced acute lung injury in mice.

BACKGROUND: Esculentoside A (EsA) is a saponin isolated from the Chinese herb Phytolacca esculenta. In our study, we sought to investigate the protective effects of EsA on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. MATERIALS AND METHODS: To determine the effects of EsA on the reduction of histopathologic changes in mice with ALI, inflammatory cell count in bronchoalveolar lavage fluid (BALF) and lung wet-to-dry weight ratio were measured in LPS-challenged mice, and lung histopathologic changes observed via paraffin section were assessed. Next, cytokine production induced by LPS in BALF was measured by enzyme-linked immunosorbent assay. To further study the mechanism of EsA protective effects on ALI, IκBa, p38, and extracellular signal receptor-activated kinase pathways were investigated in lung tissue of mice with ALI. RESULTS: In the present investigation, EsA showed marked effects by reducing inflammatory infiltration, thickening of the alveolar wall, and pulmonary congestion. Levels of tumor necrosis factor α and interleukin 6 elevated by LPS were significantly decreased in BALF in EsA-pretreated ALI model. Furthermore, EsA significantly suppressed phosphorylation of IκBa, p38, and extracellular signal receptor-activated kinase. CONCLUSIONS: Taken together, our results suggest that EsA suppressed inflammatory responses in LPS-induced ALI through inhibition of the nuclear factor kappa B and mitogen activated protein kinase signaling pathways. EsA may be a promising potential preventive agent for ALI treatment.

Daphnetin ameliorates PM2.5-induced airway inflammation by inhibiting NLRP3 inflammasome-mediated pyroptosis in CS-exposed mice.

Epidemiologic studies have shown that fine particulate matter 2.5 (PM2.5) exaggerates airway inflammation associated with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Daphnetin (Daph) is a natural compound with a variety of biological activities. At present, there are limited data on whether Daph can protect against cigarette smoke (CS)-induced chronic obstructive pulmonary disease (COPD) and PM2.5-CS-induced AECOPD. Therefore, this study systematically evaluated the effects of Daph on CS-induced COPD and PM2.5-CS-induced AECOPD and determined its mechanism of action. First, in vitro studies showed that PM2.5 exacerbated cytotoxicity and NLRP3 inflammasome-mediated pyroptosis induced by low-dose cigarette smoke extracts (CSE). However, the effect was reversed by si-NLRP3 and MCC950. Similar results were obtained in PM2.5-CS-induced AECOPD mice. The results of the mechanistic studies suggested that blocking NLRP3 abolished PM2.5 combined with cigarette induced cytotoxicity, lung damage, NLRP3 inflammasome activation and pyroptosis in vitro and in vivo. Second, Daph suppressed the expression of NLRP3 inflammasome and pyroptosis in BEAS-2B cells. Third, Daph significantly protected against CS-induced COPD and PM2.5-CS-induced AECOPD by inhibiting the NLRP3 inflammasome and pyroptosis in mice. Our findings identified the NLRP3 inflammasome as a critical contributor to PM2.5-CS-induced airway inflammation, and Daph as a negative regulator of NLRP3-mediated pyroptosis, which has implications for the pathophysiology of AECOPD.

PM2.5 increases susceptibility to acute exacerbation of COPD via NOX4/Nrf2 redox imbalance-mediated mitophagy.

The increasing abundance of fine particulate matter (PM2.5) in the environment has increased susceptibility to acute exacerbation of COPD (AECOPD). During PM2.5 exposure, excessive reactive oxygen species (ROS) production triggers a redox imbalance, which contributes to damage to organelles and disruption of homeostasis. At present, there are limited data on whether NOX4/Nrf2 redox imbalance increases susceptibility to acute exacerbation of COPD (AECOPD), and the underlying mechanism is unclear. Therefore, the current study was aimed to evaluate the role of NOX4/Nrf2 redox balance on AECOPD induced by PM2.5-CS-exposure. Here, we report that PM2.5 exacerbates cytotoxicity by enhancing NOX4/Nrf2 redox imbalance-mediated mitophagy. First, exposure to a low-dose of PM2.5 (200 µg/ml) significantly exacerbated oxidative stress and mitochondrial damage by increasing the ROS overproduction, enhancing the excessive NOX4/Nrf2 redox imbalance, decreasing the mitochondrial membrane potential (MMP), and enhancing the mitochondrial fragmentation that were caused by a low-dose of CSE (2.5%). Second, coexposure to PM2.5 and CSE (PM2.5-CSE) induced excessive mitophagy. Third, PM2.5 exacerbated CS-induced COPD, as shown by excessive inflammatory cell infiltration, inflammatory cytokine production and mucus hypersecretion, goblet cell hyperplasia, NOX4/Nrf2 redox imbalance, and mitophagy, these effects triggered excessive ROS production and mitochondrial damage in mice. Mechanistically, PM2.5-CS-induced excessive levels of mitophagy by triggering redox imbalance, leading to greater cytotoxicity and AECOPD; however, reestablishing the NOX4/Nrf2 redox balance via NOX4 blockade or mitochondria-specific ROS inhibitor treatment alleviated this cytotoxicity and ameliorated AECOPD. PM2.5 may exacerbate NOX4/Nrf2 redox imbalance and subsequently enhance mitophagy by increasing the ROS and mito-ROS levels, thereby increasing susceptibility to AECOPD.

Xanthohumol protect against acetaminophen-induced hepatotoxicity via Nrf2 activation through the AMPK/Akt/GSK3ß pathway.

OBJECTIVE: Acetaminophen (APAP) is one of the world's popular and safe painkillers, and overdose can cause severe liver damage and even acute liver failure. The effect and mechanism of the xanthohumol on acetaminophen-induced hepatotoxicity remains unclear. METHODS: The hepatoprotective effects of xanthohumol were studied using APAP-induced HepG2 cells and acute liver injury of mouse, seperately. RESULTS: In vitro, xanthohumol inhibited H2O2- and acetaminophen-induced cytotoxicity and oxidative stress. Xanthohumol up-regulated the expression of Nrf2. Further mechanistic studies showed that xanthohumol triggered Nrf2 activation via the AMPK/Akt/GSK3ß pathway to exert a cytoprotective effect. In vivo, xanthohumol significantly ameliorated acetaminophen-induced mortality, the elevation of ALT and AST, GSH depletion, MDA formation and histopathological changes. Xanthohumol effectively suppressed the phosphorylation and mitochondrial translocation of JNK, mitochondrial translocation of Bax, the activation o cytochrome c, AIF secretion and Caspase-3. In vivo, xanthohumol increased Nrf2 nuclear transcription and AMPK, Akt and GSK3ß phosphorylation in vivo. In addition, whether xanthohumol protected against acetaminophen-induced liver injury in Nrf2 knockout mice has not been illustated. CONCLUSION: Thus, xanthohumol exerted a hepatoprotective effect by inhibiting oxidative stress and mitochondrial dysfunction through the AMPK/Akt/GSK3ß/Nrf2 antioxidant pathway.

UBE2E2 enhances Snail-mediated epithelial-mesenchymal transition and Nrf2-mediated antioxidant activity in ovarian cancer.

Dissemination of ovarian cancer (OvCa) cells can lead to inoperable metastatic lesions in the bowel and omentum, which have a poor prognosis despite surgical and chemotherapeutical options. A better understanding of the mechanisms underlying metastasis is urgently needed. In this study, bioinformatics analyses revealed that UBE2E2, a less-studied ubiquitin (Ub)-conjugating enzyme (E2), was upregulated in OvCa and was associated with poor prognosis. Subsequently, we performed western blot analysis and IHC staining with 88 OvCa and 26 normal ovarian tissue samples, which further confirmed that UBE2E2 protein is highly expressed in OvCa tissue but weakly expressed in normal tissue. Furthermore, the silencing of UBE2E2 blocked OvCa cell migration, epithelial-mesenchymal transition (EMT) and metastasis in vitro, whereas UBE2E2 overexpression exerted the opposite effects. Mechanistically, UBE2E2 promoted p62 accumulation and increased the activity of the Nrf2-antioxidant response element (ARE) system, which ultimately activated the Snail signaling pathway by inhibiting the ubiquitin-mediated degradation of Snail. Additionally, co-IP and immunofluorescence demonstrated that a direct interaction exists between UBE2E2 and Nrf2, and the N-terminal of UBE2E2 (residues 1-52) is required and sufficient for its interaction with Nrf2 protein. Mutations in the active site cysteine (Cys139) impaired both the function and cellular distribution of UBE2E2. More importantly, the deletion of UBE2E2 reduced tumorigenicity and metastasis in xenograft OvCa mouse models. Taken together, our findings reveal the role of the UBE2E2-Nrf2-p62-Snail signaling axis in OvCa and thus provides novel therapeutic targets for the prevention of OvCa metastasis.

Activation of Nrf2 signalling pathway by tectoridin protects against ferroptosis in particulate matter-induced lung injury.

BACKGROUND AND PURPOSE: Our previous research showed that ferroptosis plays a crucial role in the pathophysiology of PM2.5-induced lung injury. The present study aimed to investigate the protective role of the Nrf2 signalling pathway and its bioactive molecule tectoridin in PM2.5-induced lung injury by regulating ferroptosis. EXPERIMENTAL APPROACH: We examined the regulatory effect of Nrf2 on ferroptosis in PM2.5-induced lung injury and Beas-2b cells using Nrf2-knockout (KO) mice and Nrf2 siRNA transfection. The effects and underlying mechanisms of tectoridin on PM2.5-induced lung injury were evaluated in vitro and in vivo. KEY RESULTS: Nrf2 deletion increased iron accumulation and ferroptosis-related protein expression in vivo and vitro, further exacerbating lung injury and cell death in response to PM2.5 exposure. Tectoridin activated Nrf2 target genes and ameliorated cell death caused by PM2.5. In addition, tectoridin prevented lipid peroxidation, iron accumulation and ferroptosis in vitro, but in siNrf2-treated cells, these effects almost disappeared. In addition, tectoridin effectively mitigated PM2.5-induced respiratory system damage, as evaluated by HE, PAS, and inflammatory factors. Tectoridin also augmented the antioxidative Nrf2 signalling pathway and prevented changes in ferroptosis-related morphological and biochemical indicators, including MDA levels, GSH depletion and GPX4 and xCT downregulation, in PM2.5-induced lung injury. However, the effects of tectoridin on ferroptosis and respiratory injury were almost abolished in Nrf2-KO mice. CONCLUSION AND IMPLICATIONS: Our data proposed the protective effect of Nrf2 activation on PM2.5-induced lung injury by inhibiting ferroptosis-mediated lipid peroxidation and highlight the potential of tectoridin as a PM2.5-induced lung injury treatment.

Short-term roxithromycin treatment attenuates airway inflammation via MAPK/NF-κB activation in a mouse model of allergic asthma.

OBJECTIVE: We investigated whether roxithromycin reduces ovalbumin-specific allergic asthma symptoms in mice, and we further investigated the inhibitory mechanism of roxithromycin in ovalbumin-specific allergic asthma. METHODS: Mice were divided into five groups (n = 10 for each): control group, roxithromycin-treated groups (5, 20 and 40 mg/kg) and ovalbumin-challenged group. We measured the recruitment of inflammatory cells into the bronchoalveolar lavage fluid (BALF) or the lung tissues by Kwik-Diff and hematoxylin and eosin (H&E) staining, goblet cell hyperplasia by alcian blue-periodic acid-Schiff (AB-PAS) staining, airway hyperresponsiveness (AHR) by whole-body plethysmograph chamber, cytokine and immunoglobulin E (IgE) levels by ELISA, and the activation of mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-κB) in the lung tissues by Western blotting. RESULTS: Treatment with roxithromycin resulted in fewer inflammatory cells in the BALF and peribronchial areas, and decreased AHR, goblet cell hyperplasia, IgE levels and inflammatory cytokines, as well as MAP kinases and NF-κB activation, which are increased in lung tissues of mice with ovalbumin-induced allergic asthma. CONCLUSIONS: Our data suggest that oral administration of roxithromycin suppresses ovalbumin-induced airway inflammation and AHR by regulating the inflammatory cytokines via MAP kinases/NF-κB pathway in inflammatory cells. Based on these results, we suggest that roxithromycin may be used as a therapeutic agent for allergy-induced asthma.