NQO1 Protein Research Articles

Human umbilical cord mesenchymal stem cells protect MC3T3-E1 osteoblasts from dexamethasone-induced apoptosis via induction of the Nrf2-ARE signaling pathway

ObjectiveTo investigate the protective effect human umbilical cord mesenchymal stem cells (hUC-MSCs) have on Dexamethasone (Dex)-induced apoptosis in osteogenesis via the Nrf2-ARE signaling pathway. MethodsGlucocorticoid-induced osteonecrosis of the femoral head (GC-ONFH) was developed in rats through the administration of lipopolysaccharide and methylprednisolone. The incidence of femoral head necrosis, cavity notch, apoptosis of osteoblasts, and bone density were observed by HE staining, TUNEL staining, and Micro-CT. HUC-MSCs were co-cultured with mouse pre-osteoblast MC3T3-E1. The survival rate of osteoblasts was determined by CCK8, and apoptosis and ROS levels of osteoblasts were determined by flow cytometer. The viability of antioxidant enzymes SOD, GSH-Px, and CAT was analyzed by biochemistry. Nrf2 expression levels and those of its downstream proteins and apoptosis-related proteins were analyzed by Western blotting. ResultsIn rats, hUC-MSCs can reduce the rates of empty bone lacuna and osteoblast apoptosis that are induced by glucocorticoids (GCs), while reducing the incidence of GC-ONFH. hUC-MSCs can significantly improve the survival rate and antioxidant SOD, GSH-Px, and CAT activity of MC3T3-E1 cells caused by Dex, and inhibit apoptosis and oxidative stress levels. In addition, hUC-MSCs can up-regulate the expression of osteoblast antioxidant protein Nrf2 and its downstream protein HO-1, NQO-1, GCLC, GCLM, and apoptosis-related protein bcl-2, while also down-regulating the expression of apoptosis-related protein bax, cleaved caspase-3, cleaved caspase-9, and cytochrome C in MC3T3-E1 cells. hUC-MSCs improve the ability of MC3T3-E1 cells to mineralize to osteogenesis. However, the promoting effects of hUC-MSCs were abolished following the blocking of the Nrf2-ARE signaling pathway for osteoblasts. ConclusionThe results reveal that hUC-MSCs can reduce Dex-induced apoptosis in osteoblasts via the Nrf2-ARE signaling pathway.

Genetic variants affecting NQO1 protein levels impact the efficacy of idebenone treatment in Leber hereditary optic neuropathy

Idebenone, the only approved treatment for Leber hereditary optic neuropathy (LHON), promotes recovery of visual function in up to 50% of patients, but we can neither predict nor understand the non-responders. Idebenone is reduced by the cytosolic NAD(P)H oxidoreductase I (NQO1) and directly shuttles electrons to respiratory complex III, bypassing complex I affected in LHON. We show here that two polymorphic variants drastically reduce NQO1 protein levels when homozygous or compound heterozygous. This hampers idebenone reduction. In its oxidized form, idebenone inhibits complex I, decreasing respiratory function in cells. By retrospectively analyzing a large cohort of idebenone-treated LHON patients, classified by their response totherapy, we show that patients with homozygous or compound heterozygous NQO1 variants have the poorest therapy response, particularly if carrying the m.3460G>A/MT-ND1 LHON mutation. These results suggest consideration of patient NQO1 genotype and mitochondrial DNA mutation in the context of idebenone therapy.

Study of the effect of keap1 on oxidative stress in human umbilical cord mesenchymal stem cells

BackgroundHucMSCs had shown promising efficacy in treating childhood diseases, but oxidative stress induced by the poor microenvironment at the site of damage resulted in low cell survival after transplantation, thus preventing the cells from maximizing therapeutic efficacy. Therefore, this study aimed to investigate the role and mechanism of keap1 in oxidative stress injury of human umbilical cord mesenchymal stem cells (hucMSCs), and to provide theoretical support for improving the efficacy of stem cell therapy.MethodsThe hucMSCs were treated with hypoxic low-sugar-free serum (GSDH) to mimic the damaged site microenvironment after implantation. Adenoviral overexpression of keap1 gene of hucMSCs was performed in vitro, and cell proliferation ability was detected by CCK8 assay, crystal violet staining assay, and cell cycle assay. Cellular redox level was assessed by Amplex Red, MDA, and GSH/GSSG kit. Mitochondrial morphology was evaluated by mitotracker Red staining. ATP production was estimated by ATP detection kit. The mRNA and protein expression levels were tested by western blotting and RT-qPCR.ResultsGSDH treatment substantially upregulated keap1 expression. Subsequently, we found that overexpression of keap1 notably inhibited cell proliferation and caused cells to stagnate in G1 phase. At the same time, overexpression of keap1 induced the production of large amounts of H2O2 and the accumulation of MDA, but suppressed the GSH/GSSG ratio and the expression of antioxidant proteins NQO1 and SOD1, which caused oxidative stress damage. Overexpression of keap1 induced cells to produce a large number of dysfunctional mitochondria resulting in reduced ATP production. Moreover, Overexpression of keap1 significantly decreased the IKKβ protein level, while upregulating IkB mRNA levels and downregulating P50 mRNA levels.ConclusionsOverexpression of keap1 may induce oxidative stress injury in hucMSCs by down-regulating IKKβ expression and inhibiting NF-κB pathway activation. This implies the importance of keap1 in hucMSCs and it may be a potential gene for genetic modification of hucMSCs.

In silico evaluations of phytochemicals from Withania somnifera exhibiting anticancer activity against NAD[P]H-quinone oxidoreductase.

Cancer is a leading cause of death globally and in the US, prompting research into medicinal plants with anticancer properties. Withania somnifera, or Ashwagandha, is one such plants, known for its diverse pharmacological effects. Withaferin A and Viscosalactone B are two compounds found in Ashwagandha with known anticancer activity. The protein NQO1, overexpressed in various cancers, was the focus of this study. We hypothesize that specific phytochemicals in Withania somnifera can effectively interact with and inhibit the NQO1 protein, thereby exhibiting anticancer properties. This study aims to identify these interactions using in silico approaches. CFDT was performed using the Gaussian 16 program package, followed by QSAR analysis of the compounds in the PASS online web server. The Schrodinger suite was used to carry out ligand and protein preparation, molecular docking, and molecular dynamic simulation to analyse the interaction of these compounds with NQO1 and ADME studies. Protox-II and SWISSADME tools were used to predict the toxicity and blood-brain barrier permeability of the phytochemicals. CDFT and frontier molecular orbital analyses predicted the stability and reactivity of all the selected molecules. QSAR analysis predicted the biological activity and toxicity of the compounds. Withaferin A exhibited the highest glide gscore (-4.953kcal/mol) and demonstrated 6 hydrogen bond interactions with NQO1, suggesting its potential as an anticancer agent. Conceptual density functional theory-based analysis suggested the strong electrophilicity of the ligands, further supporting their potential anticancer activities. Viscosalactone B, another phytochemical from Ashwagandha, also showed interactions involving 6 hydrogen bonds with NQO1, with a glide gscore of (-4.593kcal/mol). Molecular dynamic simulations validated the stability of the Withaferin A-NQO1 complex. ADME-T properties predicted high oral absorption for the selected ligands, indicating that Withaferin A could be a viable orally administered drug.

Quercetin Attenuates Oxidative Stress and Apoptosis in Brain Tissue of APP/PS1 Double Transgenic AD Mice by Regulating Keap1/Nrf2/HO-1 Pathway to Improve Cognitive Impairment.

Objective: The objective of the study is to investigate whether quercetin ameliorates Alzheimer's disease (AD)-like pathology in APP/PS1 double transgenic mice and its hypothesized mechanism, contributing to the comprehension of AD pathogenesis. Methods: A total of 30 APP/PS1 transgenic mice were randomized into model group (APP/PS1), quercetin group (APP/PS1+Q), and donepezil hydrochloride group (APP/PS1+DON). Simultaneously, there were 10 C57 mice of the same age served as a control group. Three months posttreatment, the effects of quercetin on AD mice were evaluated using the Morris water maze (MWM) test, Y maze experiment, immunohistochemistry, immunofluorescence, and western blotting. Results: Results from the water maze and Y maze indicated that quercetin significantly improved cognitive impairment in APP/PS1 transgenic AD mice. Additionally, serum enzyme-linked immunosorbent assay (ELISA) results demonstrated that quercetin elevated MDA, superoxide dismutase (SOD), CAT, GSH, acetylcholine (ACh), and acetylcholinesterase (AChE) levels in AD mice. Hematoxylin-eosin (HE) staining, Nissl staining, and hippocampal tissue thioflavine staining revealed that quercetin reduced neuronal damage and Aβ protein accumulation in AD mice. Western blot validated protein expression in the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 pathway associated with oxidative stress and apoptosis, confirming quercetin's potential molecular mechanism of enhancing AD mouse cognition. Furthermore, western blot findings indicate that quercetin significantly alters protein expression in the Keap1/Nrf2/HO-1 pathway. Moreover, molecular docking analysis suggests that Keap1, NQO1, HO-1, caspase-3, Bcl-2, and Bax proteins in the Keap1/Nrf2/HO-1 pathway may be potential regulatory targets of quercetin. These findings will provide a molecular basis for quercetin's clinical application in AD treatment. Conclusion: Quercetin can improve cognitive impairment and AD-like pathology in APP/PS1 double transgenic mice, potentially related to quercetin's activation of the Keap1/Nrf2/HO-1 pathway and reduction of cell apoptosis.

Mangiferin Ameliorates CCl4-Triggered Acute Liver Injury by Inhibiting Inflammatory Response and Oxidative Stress: Involving the Nrf2-ARE Pathway.

Acute liver injury (ALI) is characterized by inflammation and oxidative stress (OS). Although mangiferin (MGF) has antioxidant and anti-inflammatory effects, its role in ALI remains unclear. Accordingly, we investigated the MGF molecular mechanism in carbon tetrachloride (CCl4)-induced ALI in vivo and in vitro. The CCl4 was utilized to induce ALI in mice. In vivo, the therapeutic effects of MGF on CCl4-induced liver injury were evaluated through biochemical assays and histomorphological analysis. Additionally, immunohistochemistry, immunofluorescence, ELISA and Western blotting were further applied to explore the mechanism. In vitro, The CCK-8 assay and flow cytometry were employed to investigate the protective effects of MGF against CCl4-induced toxicity in HepG2 cells, while mitochondrial reactive oxygen species levels and Western blotting were used to explore the biological effects and molecular mechanisms. MGF treatment resulted in a reduction in serum levels of AST and ALT, diminished concentrations of TNF-α, IL-6, and IL-1β in liver tissue, and concurrently decreased cellular apoptosis. Furthermore, MGF pretreatment enhanced the activity of SOD and GSH while concurrently diminishing the MDA production. This study further demonstrated the upregulation of Nrf2, NQO1, and HO-1 protein expression levels, as well as the downregulation of p-p65 protein expression levels. In vitro investigations revealed that the mitigation of CCl4-induced inflammation and OS by MGF was mediated via the Nrf2- antioxidant response element (ARE) pathway, which was disrupted by ML385 in HepG2 cells. CCl4 can induce liver injury, while treatment with MGF mitigates ALI by inhibiting oxidative stress, inflammation, and apoptosis. The protective mechanism of MGF is mediated by the Nrf2-ARE pathway activation.

Therapeutic effect of phycocyanin on chronic obstructive pulmonary disease in mice

IntroductionThe prevention and treatment of chronic obstructive pulmonary disease (COPD) is closely tied to antioxidation and anti-inflammation. Phycocyanin (PC) has numerous pharmacological effects, such as antioxidation and anti-inflammation. However, it remains unclear whether PC can play a therapeutic role in COPD. ObjectiveAs inflammation and oxidative stress can aggravate COPD, this study is to explore the effect of PC on COPD mice and its mechanisms. MethodsThe COPD mice model was established by exposing them to lipopolysaccharide (LPS) and cigarette smoke (CS); PC was administrated in a concentration of 50 mg/kg for 30 days. On the last day, lung function was measured, and bronchoalveolar lavage fluid (BALF) was obtained and classified for cells. Lung tissue pathological change was analyzed, and organ indices statistics were measured. Based on molecular docking, the mechanism was explored with Western blotting, immunohistochemical, and immunofluorescence in vivo and in vitro. ResultsPC significantly ameliorated the pulmonary function of COPD mice and reduced inflammation of the lung (p < 0.05), and hematoxylin and eosin (H&E) staining showed PC depressed lung inflammatory cell accumulation and emphysema. Periodic acid Schiff (PAS) and Masson staining revealed that PC retarded goblet cells metaplasia and collagen deposition (p < 0.05). In addition, in vivo PC regulated Heme oxygenase 1 (HO-1) (p < 0.05) and NAD(P)H dehydrogenase quinone 1 (NQO1) level (p < 0.01) in the lung, as well as NOX2 level in pulmonary macrophages. Molecular docking results indicate that phycocyanobilin (PCB) in PC had a good binding site in Keap1 and NOX2 proteins; the phycocyanobilin-bound phycocyanin peptide (PCB-PC-peptide) was obtained for further studies. In vitro, PCB-PC-peptide could depress the phospho-NF-E2-related factor 2 (p-Nrf2) and NQO1 protein expression in RAW264.7 cells induced by cigarette smoke extract (CSE) (p < 0.05). ConclusionPC exerts beneficial effects on COPD via anti-inflammatory and antioxidative stress, which may be achieved through PCB.

New insights into idebenone therapy in relation to NQO1

Idebenone is the only approved treatment for Leber's hereditary optic neuropathy (LHON).It promotes recovery of visual function in up to 50% of patients, but, based on the current knowledge, we can neither predict nor understand the non‐responders. Idebenone is reduced by the cytosolic NAD(P)H oxidoreductase I (NQO1) and directly shuttles electrons to respiratory complex III, bypassing complex I affected in LHON.We explored the possibility that genetic variant in the NQO1 enzyme can impact on idebenone response. We found that two polymorphic variants drastically reduce NQO1 protein levels, when homozygous or in compound heterozygote combination affecting idebenone reduction. In its oxidized form idebenone inhibits complex I, decreasing respiratory function in cells. We also retrospectively analysed a large cohort of LHON treated patients, classified by their response to therapy, demonstrating that patients homozygous or compound heterozygote for the NQO1 variants are characterized by the worst therapy response, particularly if carrying the m.3460G&gt;A/MT‐ND1 LHON mutation.In summary, these results support the concept that the response to idebenone therapy may be influenced by the NQO1 genotype and the different mitochondrial DNA mutations.

Fermented Syzygium samarangense leaves mitigate oxidative stress-triggered hepatotoxicity through Nrf2 activation and epigenetic regulation

The liver, which is a vital organ for metabolism and detoxification, is susceptible to damage caused by oxidative stress. Excessive consumption of acetaminophen (APAP), a commonly used analgesic ingredient, can cause liver injury due to oxidative stress. The aim of this study is to examine the protective effects and mechanisms of fermented Syzygium samarangense (wax apple) leaves (FWAL) against damage caused by APAP in mouse AML-12 hepatocytes. Ethanolic and water extracts of FWAL (FWAL-EE and FWAL-WE) were prepared, and both contained significant phenolic compounds such as gallic acid, EGCG, ellagic acid, myricitrin, and myricetin, with potent DPPH scavenging activity. FWAL-EE demonstrated superior effectiveness in lowering the death ratio, LDH leakage, and oxidative stress in APAP-treated AML-12 cells. The protective effects of FWAL-EE included the downregulation of inflammatory markers TNF-α and cox-2 mRNA, as well as the upregulation of Nrf2, HO-1, and NQO1 proteins in APAP-treated hepatocytes. Additionally, FWAL-EE reduced the expression of epigenetic enzymes HDACs 1, 2, 3, and 4, and DNMTs 1 and 3b while causing an increase in unmethylated DNA levels in the Nrf2 promoter region. Furthermore, EGCG, a prominent active compound in FWAL, protected AML-12 hepatocytes from APAP-induced damage by activating Nrf2 and NQO1, while also inhibiting epigenetic DNMTs and HDACs. These results suggested that FWAL-EE may effectively prevent APAP-induced liver damage by enhancing the activation of the Nrf2 antioxidant system through epigenetic regulation. FWAL-EE has potential as a nutraceutical for liver protection.

Anti-oxidant effects of herbal residue from Shengxuebao mixture on heat-stressed New Zealand rabbits

Heat stress can lead to hormonal imbalances, weakened immune system, increased metabolic pressure on the liver, and ultimately higher animal mortality rates. This not only seriously impairs the welfare status of animals, but also causes significant economic losses to the livestock industry. Due to its rich residual bioactive components and good safety characteristics, traditional Chinese medicine (TCM) residue is expected to become a high-quality feed additive with anti-oxidative stress alleviating function. This study focuses on the potential of Shengxuebao mixture herbal residue (SXBR) as an anti-heat stress feed additive. Through the UPLC (ultra performance liquid chromatography) technology, the average residue rate of main active ingredients from SXBR were found to be 25.39%. SXBR were then added into the basal diet of heat stressed New Zealand rabbits at the rates of 5% (SXBRl), 10% (SXBRm) and 20% (SXBRh). Heat stress significantly decreased the weight gain, as well as increased neck and ear temperature, drip loss in meat, inflammation and oxidative stress. Also, the hormone levels were disrupted, with a significant increase in serum levels of CA, COR and INS. After the consumption of SXBR in the basal diet for 3 weeks, the weight of New Zealand rabbits increased significantly, and the SXBRh group restored the redness value of the meat to a similar level as the control group. Furthermore, the serum levels T3 thyroid hormone in the SXBRh group and T4 thyroid hormone in the SXBRm group increased significantly, the SXBRh group showed a significant restoration in inflammation markers (IL-1β, IL-6, and TNF-α) and oxidative stress markers (total antioxidant capacity, HSP-70, MDA, and ROS) levels. Moreover, the real-time fluorescence quantitative PCR analysis found that, the expression levels of antioxidant genes such as Nrf2, HO-1, NQO1, and GPX1 were significantly upregulated in the SXBRh group, and the expression level of the Keap1 gene was significantly downregulated. Additionally, the SXBRm group showed significant upregulation in the expression levels of HO-1 and NQO1 genes. Western blot experiments further confirmed the up-regulation of Nrf2, Ho-1 and NQO1 proteins. This study provides a strategy for the utilization of SXBR and is of great significance for the green recycling of the TCM residues, improving the development of animal husbandry and animal welfare.

The Gastroprotective Effect of Walnut Peptides: Mechanisms and Impact on Ethanol-Induced Acute Gastric Mucosal Injury in Mice

The objective of this research was to explore the protective impact of walnut peptides (WP) against ethanol-induced acute gastric mucosal injury in mice and to investigate the underlying defense mechanisms. Sixty male BALB-c mice were divided into five groups, and they were orally administered distilled water, walnut peptides (200 and 400 mg/kg bw), and omeprazole (20 mg/kg bw) for 24 days. Acute gastric mucosal injury was then induced with 75% ethanol in all groups of mice except the blank control group. Walnut peptides had significant protective and restorative effects on tissue indices of ethanol-induced gastric mucosal damage, with potential gastric anti-ulcer effects. Walnut peptides significantly inhibited the excessive accumulation of alanine aminotransferase (ALT), aspartate transferase (AST), and malondialdehyde (MDA), while promoting the expression of reduced glutathione (GSH), total antioxidant capacity (T-AOC), glutathione disulfide (GSSG), and mouse epidermal growth factor (EGF). Furthermore, the Western blot analysis results revealed that walnut peptides significantly upregulated the expression of HO-1 and NQO1 proteins in the Nrf2 signaling pathway. The defensive impact of walnut peptides on the gastric mucosa may be achieved by mitigating the excessive generation of lipid peroxides and by boosting cellular antioxidant activity.

Effects of terbuthylazine on myocardial oxidative stress and ferroptosis via Nrf2/HO-1 signaling pathway in broilers

Terbuthylazine (TBA) is one of the most commonly used and effective herbicides. However, due to its affinity for soil organic matter and water solubility, TBA can lead to biological health concerns. This study exposed broilers to TBA (0 mg/kg bw, 0.4 mg/kg bw, 4 mg/kg bw) for 28 days. The results showed significant pathological damage in broiler myocardial tissue, such as widening of the interstitial space, rupture of muscle fibers, and deposition of myocardial collagen fibers. In addition, Under the 0.4 mg/kg bw TBA exposure, myocardial oxidative stress was observed in broilers, which was accompanied by the activation of Nrf2/HO-1 pathway and the increased protein and mRNA levels of NQO1, NOX2 and SOD2 antioxidant enzymes. However, Nrf2/HO-1 protein and mRNA levels were reversed at 4 mg/kg bw TBA exposure. Meanwhile, the Nrf2/HO-1 mediated antioxidant defense was impaired. In contrast with the low dose, the protein and gene expression levels of NQO1, NOX2, and SOD2 were reduced in 4 mg/kg bw TBA group. The expression of GPX4 and SLC7A11 was significantly downregulated at both protein and mRNA levels. Beyond that, ACSL4 expression was significantly up-regulated, and the protein result was consistent with the mRNA expression, demonstrating the occurrence of ferroptosis. In general, TBA exposure activated the Nrf2/HO-1 pathway, resulting in ferroptosis. This study links ferroptosis to the Nrf2/HO-1 pathway, providing new insights into the potential role of TBA in myocardial toxicity.

Multifunctional agents against Alzheimer’s disease based on oxidative stress: Polysubstituted pyrazine derivatives synthesized by multicomponent reactions

As Alzheimer’s disease (AD) is a neurodegenerative disease with a complex pathogenesis, the exploration of multi-target drugs may be an effective strategy for AD treatment. Multifunctional small molecular agents can be obtained by connecting two or more active drugs or privileged pharmacophores by multicomponent reactions (MCRs). In this paper, two series of polysubstituted pyrazine derivatives with multifunctional moieties were designed as anti-AD agents and synthesized by Passerini-3CR and Ugi-4CR. Since the oxidative stress plays an important role in the pathological process of AD, the antioxidant activities of the newly synthesized compounds were first evaluated. Subsequently, selected active compounds were further screened in a series of AD-related bioassays, including Aβ1-42 self-aggregation and deaggregation, BACE-1 inhibition, metal chelation, and protection of SH-SY5Y cells from H2O2-induced oxidative damage. Compound A3B3C1 represented the best one with multifunctional potencies. Mechanism study showed that A3B3C1 acted on Nrf2/ARE signaling pathway, thus increasing the expression of related antioxidant proteins NQO1 and HO-1 to normal cell level. Furthermore, A3B3C1 showed good in vitro human plasma and liver microsome stability, indicating a potential for further development as multifunctional anti-AD agent.

Osmundacetone alleviates cerebral ischemia-reperfusion injury in rats.

Osmundacetone (DHBAc) is an antioxidant compound that has been shown to have neuroprotective and immunomodulatory activities. However, few studies have estimated its effect on cerebral ischemia-reperfusion (I/R) injury. In this study, we investigated the protective effect of DHBAc on the brain tissue of rats with cerebral I/R injury. Rats were respectively given nimodipine (NI), low dose (L-DHBAc) and high dose (H-DHBAc) Osmundacetone, and they were killed under anesthesia after 24 hours of reperfusion. And neurological impairment scores, cerebral infarct size and cerebral pathological changes were respectively detected, and mRNA expression of recombinant kelch like ECH associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2), protein expression levels of caspase3, cleaved caspase3, heme oxygenase-1 (HO-1) and quinone oxidoreductase1 (NQO1) in ischemic brain tissue were measured. Compared with the I/R group, neurological impairment scores of the DHBAc groups were significantly decreased, and their infarct sizes were significantly smaller. DHBAc could improve the pathological status of brain tissue with cerebral I/R injury, including reducing number of inflammatory cells and area of vacuoles and restoring number of normal neurons. Expression levels of Keap1 mRNA and proteins of cleaved caspase3 were significantly decreased in the DHBAc groups than those of the I/R group, while expression levels of Nrf2 mRNA, HO-1 and NQO1 proteins were remarkably increased. The effect of H-DHBAc was similar to those of NI. These results suggest that DHBAc could mitigate damage to brain tissue in rats with cerebral I/R injury.

Afzelin induces immunogenic cell death against lung cancer by targeting NQO2

BackgroundLung cancer is one of the most common malignant cancers worldwide. Previous studies have shown that Afzelin, a flavonoid, possesses anticancer activity. The aim of this study was to explore Afzelin’s effect on lung cancer cells and delineate potential anti-cancer mechanism.MethodsThe effect of Afzelin on cell viability, proliferation, and apoptosis of lung cancer cells i.e., A549 and H1299 cells, was studied. The targets for Afzelin in lung cancer were predicted using SwissTargetPrediction, Next, the GO analysis and pathway enrichment were analyzed using String. For in vitro studies, the overexpression plasmid of NQO2, the identified target of Afzelin, was transfected into Afzelin-treated cells to verify the regulatory role of Afzelin on its target and signaling pathway.ResultsIn in vitro studies, Afzelin markedly inhibited cell viability, proliferation, and raised apoptotic rate of A549 and H1299 cells. In addition, Afzelin activated endoplasmic reticulum (ER) stress and increased ATP, HMGB1, and CRT levels in lung cancer cells, indicating that Afzelin induced immunogenic cell death (ICD). SwissTargetPrediction identified NQO2 as a target of Afzelin. Further, Afzelin markedly inhibited NQO2 protein expression and in turn, overexpression of NQO2 attenuated the effect of Afzelin on A549 and H1299 cells.ConclusionAfzelin inhibits lung cancer progression by targeting NQO2, in turn, activating ER stress and inducing ICD.

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

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. 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. 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. 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.

Evaluation of Schisandra chinensis extract on anti-fatigue activity in mice

Many herbs and plant extracts have traditionally been used to enhance physical endurance and increase vitality. These natural products may contain active compounds such as antioxidants, adaptogens, and neuroprotective agents, which help alleviate fatigue and restore energy, but have not been developed enough as new anti-fatigue nutritional supplements. Schisandra chinensis, also known as Wu Wei Zi, is a medicinal and edible plant native to China. It is rich in various bioactive components and possesses significant pharmacological activities. The current study was designed to investigate the effect of Schisandra chinensis extract (SCE) on fatigue in mice, and further to elucidate its possible underlying mechanism. The results showed that SCE significantly prolonged the swimming time, increased the levels of hepatic glycogen and muscular glycogen, while reducing the levels of lactic acid (LA), blood urea nitrogen (BUN), and malondialdehyde (MDA). Furthermore, it enhanced the activities of glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), and catalase (CAT). Pearson correlation analysis also revealed a strong correlation between the anti-fatigue effects and antioxidant activities of SCE in mice vivo. Western blotting results showed that SCE alleviated fatigue by activating the AMPK/PGC-1α signaling pathway and simultaneously increasing the expression levels of Nrf2, HO-1, and NQO1 proteins in mouse skeletal muscle. Therefore, SCE could be considered as a potential anti-fatigue nutritional supplement in functional foods, beneficial for alleviating exercise-induced fatigue.

Nicotinamide mononucleotide inhibits oxidative stress-induced damage in a SIRT1/NQO-1-dependent manner

Oxidative stress causes endothelial dysfunction, which is associated with vascular cellular aging and is causally related to cardiovascular disease pathogenesis. Preclinical studies indicate that a nicotinamide adenine dinucleotide (NAD+) precursor, nicotinamide mononucleotide (NMN), alleviates oxidative stress in aged vessels, granting vasoprotective effects. However, the associated cellular mechanism remains largely unclear. In this study, we used human umbilical vein endothelial cells (HUVECs) to demonstrate that NMN inhibits oxidative stress-induced damage by activating the sirtuin 1 (SIRT1)/NAD(P)H: quinone oxidoreductase 1 (NQO-1) axis. We found that NMN inhibited H2O2-induced cytotoxicity and senescence-associated protein expression, such as p16 and p21. Furthermore, NMN prevented H2O2-induced actin cytoskeletal disorganization via inhibiting reactive oxygen species (ROS) production. NMN increased NQO-1 mRNA and protein expression that in turn was abrogated by SIRT1 inhibition, suggesting that NMN-inducible NQO-1 was associated with SIRT1 activity. SIRT1 and NQO-1 inhibition attenuated the inhibitory effect of NMN on H2O2-inducible cytotoxicity, senescence-related protein upregulation, and actin cytoskeletal disorganization. Our findings provide new insights into the mechanism by which NMN exerts protective effects against vascular oxidative stress.

Herbal cake-separated moxibustion relieves aspirin induced gastric mucosal injury by activating Nrf2/ARE/HO-1 signaling in rats

To investigate whether herbal cake-separated moxibustion can activate nuclear factor erythroid 2-related factor 2 （Nrf2） / antioxidant responsive element （ARE） /hemeoxygenase-1 （HO-1） signaling pathway to repair aspirin induced gastric mucosal injury （GMI） in rats. SD rats （half male and half female） were randomly divided into blank control, model, moxibustion, inhibitor of HO-1 （inhibitor）, model+inhibitor, moxibustion + inhibitor groups, with 20 rats in each group. The GMI model was established by gavage of aspirin 150 mg/kg（1 mL/100 g）. Herbal cake-separated moxibustion was alternatively applied to bilateral "Zusanli" （ST36） and "Zhongwan" （CV12） and bilateral "Pishu" （BL20） and "Weishu" （BL21） for 30 min, once daily for 8 days. The rats in the three inhibitor groups received intraperitoneal injection of HO-1 inhibitor zinc protoporphyrin （5 mg/kg）. The rats' behavior score, emotional response score, skin hair score, diet score and stool state score were given. The GMI index was calculated according to Guth's methods. Histopathological changes of gastric mucosa were observed by H.E. staining. The activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the gastric mucosal tissue and serum were detected by ELISA. The levels of Nrf2/ARE/HO-1 signaling pathway-related factors Keap1, Nrf2, HO-1, CAT, GST, and NQO1 in the gastric mucosal tissue were detected by quantitative real-time PCR and Western blot, separately. Compared with the blank control group, the behavior score, emotional response score, skin hair score, diet score and stool state score, GMI index, MDA contents of gastric mucosal tissue and serum, expression le-vels of Keap1 mRNA and protein were significantly increased （P<0.01）, while the activity of gastric mucosal and serum SOD, the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins were considerably decreased （P<0.01） in the model group. Compared with the model group, moxibustion obviously reversed the increase of emotional response score, skin hair score, stool state score, GMI index, MDA levels of gastric mucosal tissue and serum, and expression levels of Keap1 mRNA and protein （P<0.01, P<0.05）, and the decrease of activity of SOD of gastric mucosal and serum, and the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. After administration of antagonist of HO-1, the effects of moxibustion were eliminated or weakened pronouncedly in reducing skin hair score, GMI index, contents of gastric mucosal and serum MDA, and expression of Keap1 mRNA and protein, and in up-regulating gastric mucosal and serum SOD, and expression of HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. Herbal-cake separated moxibustion can improve the GMI in rats, which may be associated with its effects in reducing oxidative stress and activating Nrf2/ARE/HO-1 signaling pathway.

Herbal cake-separated moxibustion relieves aspirin induced gastric mucosal injury by activating Nrf2/ARE/HO-1 signaling in rats

To investigate whether herbal cake-separated moxibustion can activate nuclear factor erythroid 2-related factor 2 （Nrf2） / antioxidant responsive element （ARE） /hemeoxygenase-1 （HO-1） signaling pathway to repair aspirin induced gastric mucosal injury （GMI） in rats. SD rats （half male and half female） were randomly divided into blank control, model, moxibustion, inhibitor of HO-1 （inhibitor）, model+inhibitor, moxibustion + inhibitor groups, with 20 rats in each group. The GMI model was established by gavage of aspirin 150 mg/kg（1 mL/100 g）. Herbal cake-separated moxibustion was alternatively applied to bilateral "Zusanli" （ST36） and "Zhongwan" （CV12） and bilateral "Pishu" （BL20） and "Weishu" （BL21） for 30 min, once daily for 8 days. The rats in the three inhibitor groups received intraperitoneal injection of HO-1 inhibitor zinc protoporphyrin （5 mg/kg）. The rats' behavior score, emotional response score, skin hair score, diet score and stool state score were given. The GMI index was calculated according to Guth's methods. Histopathological changes of gastric mucosa were observed by H.E. staining. The activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the gastric mucosal tissue and serum were detected by ELISA. The levels of Nrf2/ARE/HO-1 signaling pathway-related factors Keap1, Nrf2, HO-1, CAT, GST, and NQO1 in the gastric mucosal tissue were detected by quantitative real-time PCR and Western blot, separately. Compared with the blank control group, the behavior score, emotional response score, skin hair score, diet score and stool state score, GMI index, MDA contents of gastric mucosal tissue and serum, expression le-vels of Keap1 mRNA and protein were significantly increased （P<0.01）, while the activity of gastric mucosal and serum SOD, the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins were considerably decreased （P<0.01） in the model group. Compared with the model group, moxibustion obviously reversed the increase of emotional response score, skin hair score, stool state score, GMI index, MDA levels of gastric mucosal tissue and serum, and expression levels of Keap1 mRNA and protein （P<0.01, P<0.05）, and the decrease of activity of SOD of gastric mucosal and serum, and the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. After administration of antagonist of HO-1, the effects of moxibustion were eliminated or weakened pronouncedly in reducing skin hair score, GMI index, contents of gastric mucosal and serum MDA, and expression of Keap1 mRNA and protein, and in up-regulating gastric mucosal and serum SOD, and expression of HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. Herbal-cake separated moxibustion can improve the GMI in rats, which may be associated with its effects in reducing oxidative stress and activating Nrf2/ARE/HO-1 signaling pathway.