Astaxanthin-mediated Nrf2 activation ameliorates glucocorticoid-induced oxidative stress and mitochondrial dysfunction and impaired bone formation of glucocorticoid-induced osteonecrosis of the femoral head in rats.

BACKGROUND: Osteonecrosis of the femoral head caused by glucocorticoids (GIONFH) is a significant issue resulting from prolonged or excessive clinical glucocorticoid use. Astaxanthin, an orange-red carotenoid present in marine organisms, has been the focus of this study to explore its impact and mechanism on osteoblast apoptosis induced by dexamethasone (Dex) and GIONFH. METHODS: In this experiment, bioinformatic prediction, molecular docking and dynamics simulation, cytotoxicity assay, osteogenic differentiation, qRT-PCR analysis, terminal uridine nickend labeling (TUNEL) assay, determination of intracellular ROS, mitochondrial function assay, immunofluorescence, GIONFH rat model construction, micro-computed tomography (micro-CT) scans were performed. RESULTS: Our research demonstrated that a low dose of astaxanthin was non-toxic to healthy osteoblasts and restored the osteogenic function of Dex-treated osteoblasts by reducing oxidative stress, mitochondrial dysfunction, and apoptosis. Furthermore, astaxanthin rescued the dysfunction in poor bone quality, bone metabolism and angiogenesis of GIONFH rats. The mechanism behind this involves astaxanthin counteracting Dex-induced osteogenic damage by activating the Nrf2 pathway. CONCLUSION: Astaxanthin shields osteoblasts from glucocorticoid-induced oxidative stress and mitochondrial dysfunction via Nrf2 pathway activation, making it a potential therapeutic agent for GIONFH treatment.

Linagliptin ameliorates tacrolimus-induced renal injury: role of Nrf2/HO-1 and HIF-1α/CTGF/PAI-1.

BACKGROUND: Tacrolimus (TAC) is a frequently used immunosuppressive medication in organ transplantation. However, its nephrotoxic impact limits its long-term usage. This study aims to investigate the effect of linagliptin (Lina) on TAC-induced renal injury and its underlying mechanisms. METHODS AND RESULTS: Thirty-two Sprague Dawley rats were treated with TAC (1.5 mg/kg/day, subcutaneously) and/or Lina (5 mg/kg/day, orally) for 4 weeks. Histological examination was conducted, and serum and urinary biomarkers were measured to assess kidney function and integrity. Furthermore, ELISA, Western blot analysis and immunohistochemical assay were employed to determine signaling molecules of oxidative stress, profibrogenic, hypoxic, and apoptotic proteins. Tacrolimus caused renal dysfunction and histological deterioration evidenced by increased serum creatinine, blood urea nitrogen (BUN), urinary cystatin C, and decreased serum albumin as well as elevated tubular injury and interstitial fibrosis scores. Additionally, TAC significantly increased the expression of collagen type-1, alpha-smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1), and transforming growth factor-beta1 (TGF-ß1) renal content. Moreover, TAC decreased the expression of nuclear factor erythroid-2-related factor2 (Nrf2), heme oxygenase 1 (HO-1), and mitochondrial superoxide dismutase (SOD2). In addition, TAC increased protein expression of hypoxia-inducible factor1-alpha (HIF-1α), connective tissue growth factor (CTGF), inducible nitric oxide synthase (iNOS), 8-hydroxy-2-deoxyguanosine (8-OHdG), as well as nitric oxide (NO), 4-hydroxynonenal, caspase-3 and Bax renal contents. Furthermore, TAC decreased Bcl-2 renal contents. The Lina administration markedly attenuated these alterations. CONCLUSION: Lina ameliorated TAC-induced kidney injury through modulation of oxidative stress, hypoxia, and apoptosis related proteins.

Shizukaol C alleviates trimethylamine oxide-induced inflammation through activating Keap1-Nrf2-GSTpi pathway in vascular smooth muscle cell.

BACKGROUND: Cardiovascular disease is one of the main causes of global mortality, and there is an urgent need for effective treatment strategies. Gut microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) promotes the development of cardiovascular diseases, and shizukaol C, a natural sesquiterpene isolated from Chloranthus multistachys with various biological activities, might exhibit beneficial role in preventing TMAO-induced vascular inflammation. PURPOSE: The purpose of this study was to investigate the anti-inflammatory effects and the underlying mechanisms of shizukaol C on TMAO-induced vascular inflammation. METHODS: The effect and underlying mechanism of shizukaol C on TMAO-induced adhesion molecules expression, bone marrow-derived macrophages (BMDM) adhesion to VSMC were evaluated by western blot, cell adhesion assay, co-immunoprecipitation, immunofluorescence assay, and quantitative Real-Time PCR, respectively. To verify the role of shizukaol C in vivo, TMAO-induced vascular inflammation model were established using guidewire-induced injury on mice carotid artery. Changes in the intima area and the expression of GSTpi, VCAM-1, CD68 were examined using haematoxylin-eosin staining, and immunofluorescence assay. RESULTS: Our data demonstrated that shizukaol C significantly suppressed TMAO-induced adhesion molecule expression and the bone marrow-derived macrophages (BMDM) adhesion in vascular smooth muscle cells (VSMC). Mechanically, shizukaol C inhibited TMAO-induced c-Jun N-terminal kinase (JNK)-nuclear factor-kappa B (NF-κB)/p65 activation, and the JNK inhibition was dependent on the shizukaol C-mediated glutathione-S-transferase pi (GSTpi) expression. By further molecular docking and protein-binding analysis, we demonstrated that shizukaol C directly binds to Keap1 to induce Nrf2 nuclear translocation and upregulated GSTpi expression. Consistently, our in vivo experiment showed that shizukaol C elevated the expression level of GSTpi in carotid arteries and alleviates TMAO-induced vascular inflammation. CONCLUSION: Shizukaol C exerts anti-inflammatory effects in TMAO-treated VSMC by targeting Keap1 and activating Nrf2-GSTpi signaling and resultantly inhibits the downstream JNK-NF-κB/p65 activation and VSMC adhesion, and alleviates TMAO-induced vascular inflammation in vivo, suggesting that shizukaol C may be a potential drug for treating TMAO-induced vascular diseases.

Total flavonoids of Broussonetia papyrifera alleviate non-alcohol fatty liver disease via regulating Nrf2/AMPK/mTOR signaling pathways.

BACKGROUNDS: Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic liver diseases. The leaves of Broussonetia papyrifera contain a large number of flavonoids, which have a variety of biological functions. METHODS: In vitro experiments, free fatty acids were used to stimulate HepG2 cells. NAFLD model was established in vivo in mice fed with high fat diet (HFD) or intraperitoneally injected with Tyloxapol (Ty). At the same time, Total flavonoids of Broussonetia papyrifera (TFBP) was used to interfere with HepG2 cells or mice. RESULTS: The results showed that TFBP significantly decreased the lipid accumulation induced by oil acid (OA) with palmitic acid (PA) in HepG2 cells. TFBP decreased the total cholesterol (TC), the triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and increased high-density lipoprotein cholesterol (HDLC) in serum. TFBP could also effectively inhibit the generation of reactive oxygen species (ROS) and restrained the level of myeloperoxidase (MPO), and enhance the activity of superoxide dismutase (SOD) to alleviate the injury from oxidative stress in the liver. Additionally, TFBP activated nuclear factor erythroid-2-related factor 2 (Nrf2) pathway to increasing the phosphorylation of AMP-activated protein kinase (AMPK). Meanwhile, protein levels of mTORC signaling pathway were evidently restrained with the treatment of TFBP. CONCLUSION: Our experiments proved that TFBP has the therapeutic effect in NAFLD, and the activation of Nrf2 and AMPK signaling pathways should make sense.

Baicalin inhibits IL-1ß-induced ferroptosis in human osteoarthritis chondrocytes by activating Nrf-2 signaling pathway.

BACKGROUND: Osteoarthritis (OA) is a common degenerative disease involving articular cartilage, in which ferroptosis of chondrocytes plays an important role. Baicalin (BAI) exerts regulatory effects in a wide range of orthopedic diseases including OA, but its effect on ferroptosis of chondrocytes (CHs) is still unclear. The purpose of this study was to determine the effect of BAI on ferroptosis in human OA chondrocytes (OACs), and to explore its possible mechanism. METHODS: CHs were treated with IL-1ß (10 ng/mL) to simulate inflammation in vitro. Immunofluorescence, quantitative RT-PCR, Western blotting and cell viability assay were performed to evaluate the impacts of BAI on Fe2+ level, mitochondrial dysfunction, ferroptosis-related proteins, oxidative stress and cytotoxicity in CHs. Additionally, siRNA was made use of to knock out nuclear factor E2-related factor 2 (Nrf2) to analyze the role played by Nrf2 in BAI-induced CH ferroptosis. RESULTS: BAI eliminated IL-1ß-induced Fe2+ accumulation, changes in mitochondrial membrane potential and ferroptosis-related protein GPX4, SLC7A11, P53 and ACSL4 levels, as well as reactive oxygen species (ROS), lipid peroxidation (LPO) and malondialdehyde (MDA) accumulation in CHs. Besides, BAI reversed IL-1ß-induced decrease of Collagen II and increase of MMP13 in CHs. Meanwhile, BAI attenuated IL-1ß-induced CH toxicity and promoted Nrf2 antioxidant system activation. When Nrf2 was knocked down by siRNA, the effects of BAI on IL-1ß-induced ferroptosis-related proteins and antioxidant stress in CHs were significantly weakened. CONCLUSIONS: This study demonstrates that IL-1ß can induce CH ferroptosis. BAI is able to inhibit IL-1ß-induced CH ferroptosis and ECM degradation, and the specific mechanism may be that it can inhibit IL-1ß-induced CH ferroptosis by activating Nrf2 antioxidant system to attenuate the accumulation of intracellular ROS and lipid ROS.

Mitigative potential of kaempferide against polyethylene microplastics induced testicular damage by activating Nrf-2/Keap-1 pathway.

Polyethylene microplastics (PE-MPs) are one of the environmental contaminants that instigate oxidative stress (OS) in various organs of the body, including testes. Kaempferide (KFD) is a plant-derived natural flavonol with potential neuroprotective, hepatoprotective, anti-cancer, anti-oxidant and anti-inflammatory properties. Therefore, the present study was designed to evaluate the alleviative effects of KFD against PE-MPs-prompted testicular toxicity in rats. Fourty eight adult male albino rats were randomly distributed into 4 groups: control, PE-MPs-administered (1.5 mgkg-1), PE-MPs (1.5 mgkg-1) + KFD (20 mgkg-1) co-treated and KFD (20 mgkg-1) only treated group. PE-MPs intoxication significantly (P < 0.05) lowered the expression of Nrf-2 and anti-oxidant enzymes, while increasing the expression of Keap-1. The activities of anti-oxidants i.e., catalase (CAT), glutathione reductase (GSR), superoxide dismutase (SOD), hemeoxygene-1 (HO-1) and glutathione peroxidase (GPx) were reduced, besides malondialdehyde (MDA) and reactive oxygen species (ROS) contents were increased significantly (P < 0.05) following the PE-MPs exposure. Moreover, PE-MPs exposure significantly (P < 0.05) reduced the sperm motility, viability and count, whereas considerably (P < 0.05) increased the dead sperm number and sperm structural anomalies. Furthermore, PE-MPs remarkably (P < 0.05) decreased steroidogenic enzymes and Bcl-2 expression, while increasing the expression of Caspase-3 and Bax. PE-MPs exposure significantly (P < 0.05) reduced the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone, whereas inflammatory indices were increased. PE-MPs exposure also induced significant histopathological damages in the testes. Nevertheless, KFD supplementation significantly (P < 0.05) abrogated all the damages induced by PE-MPs. The findings of our study demonstrated that KFD could significantly attenuate PE-MPs-instigated OS and testicular toxicity, due to its anti-oxidant, anti-inflammatory, androgenic and anti-apoptotic potential.

A combination of isoliquiritigenin with Artemisia argyi and Ohwia caudata water extracts attenuates oxidative stress, inflammation, and apoptosis by modulating Nrf2/Ho-1 signaling pathways in SD rats with doxorubicin-induced acute cardiotoxicity.

Ohwia caudata (Thunb.) H. Ohashi (Leguminosae) also called as "Evergreen shrub" and Artemisia argyi H.Lév. and Vaniot (Compositae) also named as "Chinese mugwort" those two-leaf extracts frequently used as herbal medicine, especially in south east Asia and eastern Asia. Anthracyclines such as doxorubicin (DOX) are commonly used as effective chemotherapeutic drugs in anticancer therapy around the world. However, chemotherapy-induced cardiotoxicity, dilated cardiomyopathy, and congestive heart failure are seen in patients who receive DOX therapy, with the mechanisms underlying DOX-induced cardiac toxicity remaining unclear. Mitochondrial dysfunction, oxidative stress, inflammatory response, and cardiomyocytes have been shown to play crucial roles in DOX-induced cardiotoxicity. Isoliquiritigenin (ISL, 10 mg/kg) is a bioactive flavonoid compound with protective effects against inflammation, neurodegeneration, cancer, and diabetes. Here, in this study, our aim is to find out the Artemisia argyi (AA) and Ohwia caudata (OC) leaf extract combination with Isoliquiritigenin in potentiating and complementing effect against chemo drug side effect to ameliorate cardiac damage and improve the cardiac function. In this study, we showed that a combination of low (AA 300 mg/kg; OC 100 mg/kg) and high-dose(AA 600 mg/kg; OC 300 mg/kg) AA and OC water extract with ISL activated the cell survival-related AKT/PI3K signaling pathway in DOX-treated cardiac tissue leading to the upregulation of the antioxidant markers SOD, HO-1, and Keap-1 and regulated mitochondrial dysfunction through the Nrf2 signaling pathway. Moreover, the water extract of AA and OC with ISL inhibited the inflammatory response genes IL-6 and IL-1ß, possibly through the NFκB/AKT/PI3K/p38α/NRLP3 signaling pathways. The water extract of AA and OC with ISL could be a potential herbal drug treatment for cardiac hypertrophy, inflammatory disease, and apoptosis, which can lead to sudden heart failure.

Panax quinquefolius saponin inhibits vascular smooth muscle cell calcification via activation of nuclear factor-erythroid 2-related factor 2.

BACKGROUND: Panax quinquefolius saponin (PQS) is the main active component of Panax quinquefolius. Emerging evidence suggests that PQS exerts beneficial effects against cardiovascular diseases. However, the role and mechanism of PQS in vascular calcification are not unclear. The present study investigated the effects of PQS on the calcification of vascular smooth muscle cell (VSMCs). METHODS: The present study used calcification medium containing 3 mM inorganic phosphate (Pi) to induce rat VSMCs calcification. We investigated the effects of PQS on VSMCs calcification using alizarin red staining and alkaline phosphatase (ALP) activity assays. The intracellular reactive oxygen species (ROS) levels and the transcriptional activity of nuclear factor-erythroid 2-related factor 2 (Nrf2) were determined. The mRNA and protein expression levels of Nrf2, the antioxidant gene heme oxygenase-1 (HO-1), osteogenic markers, including runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2), and Kelch-like ECH-associated protein 1 (Keap1) were also measured. RESULTS: Treatment with Pi significantly increased intracellular calcium deposition and ALP activity, which were suppressed by PQS in a concentration-dependent manner. During VSMCs calcification, PQS inhibited the mRNA and protein expression of Runx2 and BMP2. PQS treatment reduced intracellular ROS production and significantly upregulated Nrf2 transcriptional activity and the expression of Nrf2 and its target antioxidant gene HO-1. PQS suppressed the Pi-induced protein expression of Keap1, which is an endogenous inhibitor of Nrf2. Keap1 siRNA treatment induced Nrf2 expression and downregulated Runx2 expression in the presence of Pi and PQS. CONCLUSION: Taken together, these findings suggest that PQS could effectively inhibit VSMCs calcification by ameliorating oxidative stress and regulating osteogenic genes via the promotion of Nrf2 expression.

Natural Products as Modulators of Nrf2 Signaling Pathway in Neuroprotection.

Neurodegenerative diseases (NDs) affect the West due to the increase in life expectancy. Nervous cells accumulate oxidative damage, which is one of the factors that triggers and accelerates neurodegeneration. However, cells have mechanisms that scavenge reactive oxygen species (ROS) and alleviate oxidative stress (OS). Many of these endogenous antioxidant systems are regulated at the gene expression level by the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). In the presence of prooxidant conditions, Nrf2 translocates to the nucleus and induces the transcription of genes containing ARE (antioxidant response element). In recent years, there has been an increase in the study of the Nrf2 pathway and the natural products that positively regulate it to reduce oxidative damage to the nervous system, both in in vitro models with neurons and microglia subjected to stress factors and in vivo models using mainly murine models. Quercetin, curcumin, anthocyanins, tea polyphenols, and other less studied phenolic compounds such as kaempferol, hesperetin, and icariin can also modulate Nrf2 by regulating several Nrf2 upstream activators. Another group of phytochemical compounds that upregulate this pathway are terpenoids, including monoterpenes (aucubin, catapol), diterpenes (ginkgolides), triterpenes (ginsenosides), and carotenoids (astaxanthin, lycopene). This review aims to update the knowledge on the influence of secondary metabolites of health interest on the activation of the Nrf2 pathway and their potential as treatments for NDs.

Alantolactone inhibits oesophageal adenocarcinoma cells through nuclear factor erythroid 2-related factor 2-mediated reactive oxygen species increment.

BACKGROUND: Oesophageal adenocarcinoma (EAC) is a highly lethal cancer associated with a rapidly rising incidence and a poor prognosis. Alantolactone, a sesquiterpene lactone isolated from inula helenium, has anti-inflammatory, antimicrobial, neuroprotective activities, and anticancer properties. OBJECTIVE: In the present study, the anticancer effects of alantolactone on the human EAC cells were investigated in vitro and in vivo. METHODS AND FINDINGS: After treated with alantolactone, the cell viability of KYAE-1, KYAE-2, OE19, and OE33 cells reduced significantly compared with that of the control cells. Alantolactone induced apoptosis of the EAC cell lines by inhibiting the protein expression levels of nuclear factor erythroid2-related factor 2 (Nrf2). Furthermore, the apoptosis-inducing effect of alantolactone was enhanced by Nrf2 knockdown while reduced by overexpression of Nrf2. Antioxidant α-tocopherol and glutathione can protect EAC cell lines against alantolactone. A xenograft nude mice model showed that alantolactone can inhibit EAC growth in vivo. CONCLUSIONS: Alantolactone inhibits oesophageal adenocarcinoma cells through Nrf2-mediated reactive oxygen species (ROS) increment. Alantolactone maybe a potential therapeutical candidate for treating EAC.

Rosmarinic acid mitigates chlorpyrifos-induced oxidative stress, inflammation, and kidney injury in rats by modulating SIRT1 and Nrf2/HO-1 signaling.

Chlorpyrifos (CPF) is a widely used broad-spectrum pesticide with multi-organ toxic effects. Oxidative stress was found to play a role in the deleterious effects of CPF, including nephrotoxicity. This study investigated the protective effect of the antioxidant polyphenol rosmarinic acid (RA) against CPF-induced kidney injury, with an emphasis on oxidative injury, inflammation, SIRT1, and Nrf2/HO-1 signaling. Rats received 10 mg/kg CPF and 25, 50, and 100 mg/kg RA orally for 28 days, and the samples were collected for analysis. CPF increased serum urea and creatinine and kidney Kim-1 and caused several histopathological alterations. ROS, MDA, NO, NF-κB p65, TNF-α, and IL-1ß were elevated in the kidney of CPF-intoxicated rats. RA ameliorated kidney function markers, prevented tissue injury, suppressed ROS, MDA, and NO, and downregulated NF-κB p65, TNF-α, and IL-1ß in CPF-intoxicated rats in a dose-dependent manner. RA decreased Bax, caspase-3, oxidative DNA damage, and Keap1, boosted antioxidant enzymes and Bcl-2, and upregulated Nrf2, HO-1, and SIRT1 in CPF-administered rats. Molecular docking simulation revealed the binding affinity of RA toward NF-κB, Keap1, HO-1, and SIRT1. In conclusion, RA prevented CPF nephrotoxicity by attenuating oxidative stress, inflammation, and apoptosis and upregulating SIRT1 and Nrf2/HO-1 signaling.

The synthesis of novel thioderivative chalcones and their influence on NF-κB, STAT3 and NRF2 signaling pathways in colorectal cancer cells.

This study aimed to synthesize new thioderivative chalcones and analyze their impact on the NF-κB, STAT3, EGFR and Nrf2 signaling pathways in colorectal cancer cells. Among the studied compounds, derivatives 4 and 5 decreased the activation of NF-κB and the expression of the target gene COX-2. In the case of STAT3, we observed the inhibition of activation of this signaling pathway after influencing derivative 4. Increased activation of the Nrf2 signaling pathway was demonstrated for derivatives 5 and 7 in DLD-1 and HCT116 cells. The results of this study indicated that new chalcone derivatives, especially compounds 4, 5, and-to some degree-7, possess potential applications in the prevention of colorectal cancer.

Carveol Promotes Nrf2 Contribution in Depressive Disorders through an Anti-inflammatory Mechanism.

Major depressive disorder (MDD) is a progressive deteriorating mental state with a feeling of worthlessness and frequent mood swings. Several studies reported the favorable effects of natural drug substances on MMD associated oxidative stress and neuroinflammation. The present study is attempted to examine whether carveol could affect lipopolysaccharide- (LPS-) induced depression, and if so, how nuclear factor E2-related factor (Nrf2) contributed to the neuroprotective effects of carveol mechanistically. Two experimental cohorts were used using the SD rats: first to evaluate the promising dose of carveol (whether 20 mg/kg or 50 mg/kg) and secondly to determine the effect of carveol on Nrf2-mediated antidepression. Significant neuronal alterations were noticed in the cortex and hippocampus regions in the LPS-treated group, accompanied by elevated inflammatory cytokine levels such as tumor necrosis factor-alpha (TNF-α), cyclooxygenase (COX-2), and c-Jun N-terminal kinase (p-JNK). Moreover, amassing of free radicals exacerbated lipid peroxidase (LPO) and oxidative stress with a limited antioxidant capacity. Carveol (20 mg/kg) significantly ameliorated these detrimental effects by promoting the antioxidant Nrf2 gene and protein, which critically regulate the downstream antioxidant and anti-inflammatory pathway. To further elaborate our hypothesis, we employed all-trans retinoic acid (ATRA), an Nrf2 inhibitor, and we found that ATRA exaggerated LPS-induced depressive-like effects associated with elevated neuroinflammatory markers. Our results demonstrated that carveol (20 mg/kg) could activate the endogenous antioxidant Nrf2, which regulates the downstream antioxidant signaling pathway, eventually leading to amelioration of LPS-induced neuroinflammation and neurodegeneration.

Protective effect of rosuvastatin pretreatment against acute myocardial injury by regulating Nrf2, Bcl-2/Bax, iNOS, and TNF-α expressions affecting oxidative/nitrosative stress and inflammation.

Cardiovascular disorders are the leading cause of death globally. Rosuvastatin is a member of statins (inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase) with many pleiotropic properties. This study investigated cardioprotective effects of rosuvastatin in isoprenaline-induced myocardial injury. Male rats were given rosuvastatin (1, 5, or 10 mg/kg, oral) daily for 1 week and on seventh and eighth day isoprenaline (150 mg/kg, subcutaneous) was given to induce cardiac injury. On ninth day, rats were euthanized and different samples were harvested for analysis. Isoprenaline administration resulted in increased cardiac mass, increased cardiac injury marker levels (cTnI, CK-MB, ALT, and AST), increased lipid/protein oxidation, and increased cardiac nitrite levels. It also decreased superoxide dismutase, CAT, GST, and glutathione reductase activities, and total antioxidant activity. Isoprenaline also increased TNF-α and IL-6 levels. Decreased mRNA expression of Nrf2 and Bcl-2 along with increased mRNA expression of Bax, eNOS and iNOS genes was observed in isoprenaline treated animals. Histopathological evaluations of rosuvastatin pre-treated groups showed reduction of myocardial necrosis. Pretreatment with rosuvastatin (5 and 10 mg/kg) reduced many of these pathological changes. The current study showed that rosuvastatin significantly reduces myocardial injury induced by isoprenaline.

Sulforaphane improves cognitive dysfunction after surgery and anesthesia in mice: The role of Keap1-Nrf2 signaling.

Anesthesia and surgery are likely causing cognitive dysfunction in patients, especially the elderly. However, the underlying pathogenic mechanisms largely remain unclear. Accumulating evidence suggest that signaling between Kelch-like erythroid cell-derived protein with CNC homology (ECH)-associated protein 1 (Keap1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) plays an important role in the pathogenesis and treatment of brain dysfunction, while sulforaphane (SFN), a natural compound acting as an Nrf2 agonist, can improve brain function. In the present study, we used 9-month-old mice to perform tibial fracture surgery under isoflurane general anesthesia. Hierarchical cluster analysis of Morris water maze test (MWMT) analysis was performed to classify mice into post-operative cognitive dysfunction (POCD) versus non-POCD phenotypes. Expression levels of Keap1 and Nrf2 were significantly decreased in the medial prefrontal cortex (mPFC), hippocampus and liver, but not in the nucleus accumbens, muscle and gut of POCD mice compared to control and non-POCD mice. Interestingly, both pretreatment and posttreatment with SFN significantly improved the abnormal behaviors of mice in the MWMT, in parallel with the up-regulated levels of Keap1-Nrf2 signaling in the mPFC, hippocampus and liver. In conclusion, these results suggest that decreased Keap1-Nrf2 signaling in the mPFC, hippocampus and liver may contribute to the onset of POCD, and that SFN exerts facilitating effects on POCD symptoms by increasing Keap1-Nrf2 signaling.

Role of ferroptosis in promoting cardiotoxicity induced by Imatinib Mesylate via down-regulating Nrf2 pathways in vitro and in vivo.

Imatinib Mesylate (IMA) has been widely used to treat with chronic myeloid leukemia (CML). However, cardiotoxicity associated with IMA is included among the therapeutic strategies. The present study was aimed to discover whether ferroptosis, a programmed iron-dependent cell death, is involved in IMA-induced cardiotoxicity. In vivo, mouse model was established after treated with 25 mg/kg, 50 mg/kg and 100 mg/kg IMA. Serum CK, LDH, AST activities were determined. Cardiac tissues were examined by H&E and Oil Red O staining. MDA was measured to assess production of lipid peroxide. Tissue iron and GSH content were measured. In vitro, cell viability, mitochondria membrane potential, generation of reactive oxygen species (ROS) and cellular iron levels were performed to explore the mechanism of IMA. The in vivo results revealed that IMA treatment significantly increased serum CK, LDH and AST. H&E staining showed that IMA caused cardiac structural injuries. The dose-dependent decrease of GSH and increase of tissue iron and MDA were observed in IMA-treated groups. Oil Red O staining suggested obvious cardiac lipid accumulation after treated with IMA. In H9c2 cardiomyocytes, IMA significantly inhibited cell proliferation in a dose-dependent manner. Mitochondria membrane potential assay showed that IMA destroyed the mitochondrial function. Additionally, IMA increased the cellular ROS and iron levels. Furthermore, IMA down-regulated the expression of Nrf2 and up-regulated the expression of P53 and TfR. These results provided compelling evidence that ferroptosis participates in IMA-induced cardiotoxicity. Ferroptosis could be regarded as a target to protect against cardiotoxicity in IMA-exposed patients.

Repeated radon exposure induced lung damage via oxidative stress-mediated mitophagy in human bronchial epithelial cells and mice.

This study aimed to investigate the potential molecular mechanism underlying radon-induced lung damage. Our results showed that long-term radon exposure induced mitochondrial damage and redox imbalance in BEAS-2B cells and a time-dependent lung pathological injury in mice. The activation of Nrf-2 and its down-stream antioxidants, and the gene expression of the indicated markers at different stages of autophagy were found to be induced with the increasing of radon exposure time. Changes in the gene expression of PINK-1, Parkin, and p62 induced by radon showed differences in mechanisms of mitophagy activation and profiles of autophagic flux between BEAS-2B cells and mice. Our findings not only demonstrated that long-term radon exposure induced damages to bronchial epithelial cells and the mice lung through increasing oxidative stress, decreasing mitochondrial function and activating mitophagy with different profiles of autophagic flux, but also revealed Nrf-2 as a central regulator of mitochondrial homeostasis and lung damage.

Licochalcone A Induces Cholangiocarcinoma Cell Death Via Suppression of Nrf2 and NF-κB Signaling Pathways.

OBJECTIVE: To investigate the anti-tumor effect of licochalcone A (LCA) on proliferation and migration in cholangiocarcinoma (CCA) cells and to elucidate their underlying mechanisms. METHODS: Human CCA cells, KKU-100, KKU-213, KKU-214, KKU-156, and KKU-452 were used to study effect of LCA on proliferation and migration by a cytotoxicity assay, wound healing assay. Reactive oxygen species levels were evaluated using DHE-fluorescent probes. Proteins associated with cancer survival and progression were analyzed by immune blotting assay. RESULTS: LCA suppressed proliferation and induced cell death in CCA cells including KKU-100, KKU-213, KKU-214, KKU-156, and KKU-452. The CCAs cells were suppressed in association with LCA-induced accumulation of intracellular reactive oxygen species (ROS). Increased formation of ROS was causally related with suppression of Nrf2 and its down-stream antioxidant and cytoprotective enzymes. These effects may lead to the expression of Bax and release of cytochrome c and ensuring cell death.  Interestingly, LCA could also inhibit cell migration and cell cycle arrest at low concentrations. These effects were associated with down-regulation of NF-kB, STAT3 and their down-stream proteins, cyclin D1, VEGF, and ICAM-1. CONCLUSIONS: These results suggest that LCA has potential therapeutic activity in suppression of CCA cells.

MiR-24-3p Attenuates Doxorubicin-induced Cardiotoxicity via the Nrf2 Pathway in Mice.

OBJECTIVE: The nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) is associated with doxorubicin (DOX)-induced cardiac injury. It has been reported that microRNA-24-3p (miR-24-3p) may regulate the Keapl by mRNA degradation, whereas Keapl can suppress the activation of Nrf2. However, the role of miR-24-3p in DOX-related cardiotoxicity remains unclear. METHODS: The mice receiving DOX were used as cardiac injury model. In this study, an adenoassociated virus 9 system was used to deliver miR-24-3p or miR-scramble to mice hearts. The echocardiographic and hemodynamic analyses were used to evaluate the effects of miR-24-3p on cardiac function under DOX stimulation. ELISA and RT-PCR were used to detect protein or mRNA expressions associated with cardiac injury, inflammation response, apoptosis and oxidative stress. Western Blot were used for quantitative analysis of the roles of miR-24-3p in regulating Nrf2 expression. H9C2 cells used to verify the role of miR-24-3p in vitro. RESULTS: We found that miR-24-3p mRNA was significantly decreased in DOX-treated mice and cardiomyocytes. Overexpression of miR-24-3p blocked cardiac injury caused by DOX injection, as reflected by the reduction in the levels of cardiac troponin I, creatinine kinase isoenzyme MB and the N-terminal pro brain natriuretic peptide. Furthermore, miR-24-3p reduced oxidative stress and cell loss without affecting the inflammation response. As expected, we found that Nrf2 was upregulated by miR-24-3p supplementation, and that the protective efforts of miR-24-3p supplementation were abolished when Nrf2 was silenced. CONCLUSION: The results from this study suggest that miR-24-3p protects cardiomyocytes against DOX-induced heart injury via activation of the Nrf2 pathway. miR-24-3p supplementation may be a novel strategy to counteract the cardiac side effects of DOX treatment.

Isoliquiritigenin prevents Doxorubicin-induced hepatic damage in rats by upregulating and activating SIRT1.

This study evaluated if the hepatic protective effect of Isoliquiritigenin (ISL) against doxorubicin (DOX)-treated rats involves upregulating sirtuin-1 (SIRT1) signaling. Adult male was divides into 5 groups (n = 6 rats/each) as control (vehicle), ISL (25 mg/kg), DOX (15 mg/kg), DOX + ISL, and DOX + ISL + EX-527 (a SIRT1 inhibitor, 5 mg/kg). ISL and EX-527 were administered 10 days before and after the single treatment of DOX. Also, cultured AML-12 hepatocytes (5 ×104) were treated with 10 µM of ISL for 24 h with or without DOX-treatments (10 µM) and in the presence or absence of EX-527 (5 µM). ISL prevented hepatocyte damage and decreased serum levels of hepatic transaminases, hepatic levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and hepatic mRNA levels of Bax and caspases-3,8, and 9. In the liver of the control and DOX-treated rats, ISL reduced levels of malondialdehyde (MDA) but increased hepatic levels of glutathione (GSH), superoxide dismutase (SOD), and catalase, as well as mRNA levels of Bcl2. In vitro, ISL stimulated cell survival and lowered levels of ROS but increased GSH levels. In vivo and in vitro, in the livers of control and DOX-treated animals, ISL significantly increased the nuclear activity and mRNA levels of SIRT1, enhanced the nuclear levels of Nrf2, and reduced nuclear levels of NF-κB p65. In conclusion, ISL alleviates DOX-induced hepatocyte toxicity by stimulating the Nrf2/antioxidants axis and concomitant suppression of NF-κB, mainly by upregulating/activating SIRT1.