Keap1 Expression Research Articles

Enrichment of polystyrene microplastics induces histological damage, oxidative stress, Keap1-Nrf2 signaling pathway-related gene expression in loach juveniles (Paramisgurnus dabryanus)

Polystyrene microplastics (PS-MPs, particle size<5 mm) cause great harm to aquatic organisms. However, their precise effects are not completely understood. In China, placing plastic film at the pond bottom has become an important loach aquaculture mode. In this mode, MPs will affect loach health. This study investigated the enrichment of PS-MPs and its effects on the growth, liver histomorphology, antioxidant enzymes, and Keap1-Nrf2 signaling pathway-related gene expression in loach juveniles (Paramisgurnus dabryanus). The loach juveniles were raised at the concentration of 1000 μg/L fluorescent polystyrene microplastics (PS-MPs) with particle size of 0.5 µm or 5 µm for seven days, the results showed that fluorescent PS-MPs were found to be enriched in liver, intestine, and gill, and the enrichment amount was higher in liver than in gill and intestine (P < 0.05). Furthermore, the enrichment amount of different-sized PS-MPs was different in liver, gill, and intestine. The loach juveniles were cultured for 21 days in the water of the concentration of 100 or 1000 μg/L PS-MPs with particle size of 0.5 µm or 5 µm, the results showed that the survival rate, weight gain rate, and specific growth rate of loach juveniles were significantly reduced. The histological analysis revealed that PS-MPs caused liver damage. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), and acetylcholinesterase (AChE) were decreased with the extended exposure to PS-MPs. Generally, the expressions of Nrf2 and Keap1 showed the similar change trend. From 7–14 day, the expression trend of oxidative stressed-related genes was not completely consistent with that of Nrf2 gene, but on day 21, the gene expression trend of oxidative stress-related SOD, CAT, and GSH-PX in the downstream of Keap1-Nrf2 signaling pathway was roughly consistent with that of Nrf2 gene. Basically, the change trends of these three gene expression were similar to those of their corresponding enzyme activities. This study provides theoretical basis for the toxicological effects of PS-MPs on freshwater fish.

Importance of Gedunin in Antagonizing Rheumatoid Arthritis via Activating the Nrf2/ARE Signaling.

Objective This study assessed the anti-arthritic effect and protection of Gedunin (GDN) on joint tissues and revealed the possible mechanism in suppressing rheumatoid arthritis (RA). Methods LPS-induced macrophages and TNF-α-stimulated synovial fibroblasts (MH7A) or IL-1β-stimulated primary rheumatoid arthritis synovial fibroblasts (RASFs) were used to evaluate the antiinflammatory effect of GDN. In addition, CIA-induced arthritis was employed here to evaluate the anti-arthritic effect. MTT and BRDU assays were utilized to evaluate the cell viability and proliferation, Q-PCR was conducted to detect the mRNA expression of cytokines, FACS was adopted to monitor ROS production, while western blotting (WB) and siRNA interference were applied in confirming the anti-arthritic effects of GDN via the Nrf2 signaling. Results. In vitro, cell viability was inhibited in macrophages and MH7A cells, but not in RASFs; but the proliferation of RASFs was significantly suppressed in time- and dose-dependent manners. GDN suppressed cytokine levels in LPS-stimulated macrophages and TNF-α-stimulated MH7A cells or RASFs. GDN suppressed ROS expression. Furthermore, GDN treatment notably dose-dependently decreased the mRNA and protein expression of iNOS in LPS-induced macrophages. sip62 interference results showed that GDN cause the less expression of HO-1 and Keap1 and also fail to inhibit cytokines after sip62 interference. In vivo, GDN effectively inhibited paw swelling, arthritis score, and arthritis incidence and cytokines. Conclusions Our study suggested that GDN exhibited strong antagonistic effect on arthritis both in vitro and in vivo via activation of Nrf2 signaling. Our work will provide a promising therapeutic strategy for RA.

Mechanism of Tibetan medicine Ershiwuwei Songshi Pills against liver injury induced by acetaminophen in mice based on Keap1/Nrf2 and TLR4/NF-κB p65 signaling pathways

The present study investigated the mechanism of the Tibetan medicine Ershiwuwei Songshi Pills(ESP) against the liver injury induced by acetaminophen(APAP) in mice based on the kelch-like ECH-associated protein 1(Keap1)/nuclear transcription factor E2 related factor 2(Nrf2) and Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) p65 signaling pathways. Kunming mice were randomly divided into a blank control group, a model group, an N-acetyl-L-cysteine(NAC) group, and high-(400 mg·kg~(-1)), medium-(200 mg·kg~(-1)), and low-dose(100 mg·kg~(-1)) ESP groups. After 14 days of continuous administration, except for those in the control group, the mice were intraperitoneally injected with 200 mg·kg~(-1) APAP. After 12 h, the serum and liver tissues of mice were collected. Hematoxylin-eosin(HE) staining was performed on pathological sections of the liver, and the levels of aspartate aminotransferase(AST) and alanine aminotransferase(ALT) in the serum and the levels of glutathione(GSH), malondialdehyde(MDA), superoxide dismutase(SOD), catalase(CAT), myeloperoxidase(MPO), and total antioxidant capacity(T-AOC) in liver tissue homogenate were detected to observe and analyze the protective effect of ESP on APAP-induced liver injury in mice. The serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1β), and interleukin-6(IL-6) were determined by enzyme-linked immunosorbent assay(ELISA). The protein expression of Nrf2, Keap1, TLR4, and NF-κB p65 in the liver was determined by Western blot. Quantitative real-time was used to determine the mRNA expression of glutamate-cysteine ligase catalytic subunit(GCLC), glutamate-cysteine ligase regulatory subunit(GCLM), heme oxygenase-1(HO-1), and NAD(P)H dehydrogenase quinone 1(NQO-1) in the liver to explore the mechanism of ESP in improving APAP-induced liver damage in mice. As revealed by results, compared with the model group, the ESP groups showed improved liver pathological damage, decreased ALT and AST levels in the serum and MDA and MPO content in the liver, increased GSH, SOD, CAT, and T-AOC in the liver, reduced TNF-α and IL-6 levels in the serum, down-regulated expression of Keap1 in the liver cytoplasm and NF-κB p65 in the liver nucleus, up-regulated expression of Nrf2 in the liver nucleus, insignificant change in TLR4 expression, and elevated relative mRNA expression levels of antioxidant genes GCLC, GCLM, HO-1, and NQO-1. ESP can reduce the oxidative damage and inflammation caused by APAP, and the mechanism may be related to the Keap1/Nrf2 signaling pathway and the signal transduction factors on the TLR4/NF-κB p65 pathway.

Mechanism of Tibetan medicine Ershiwuwei Shanhu Pills on scopolamine-induced learning and memory impairment in mice based on Keap1/Nrf2/HO-1 signaling pathway

This study aims to investigate the mechanism of the Tibetan medicine Ershiwuwei Shanhu Pills(ESP) in improving scopolamine-induced learning and memory impairment in mice based on Keap1/Nrf2/HO-1 signaling pathway. ICR mice were randomized into blank group, model group, low-dose(200 mg·kg~(-1)), medium-dose(400 mg·kg~(-1)), and high-dose(800 mg·kg~(-1)) ESP groups, and donepezil hydrochloride group. The learning and memory impairment was induced in mice by intraperitoneal injection of scopola-mine. The learning and memory abilities of mice were detected by Morris water maze test, and the damage of hippocampal neurons and cortical neurons was detected based on Nissl staining. The expression of neuron specific nuclear protein(NeuN) in hippocampus and cortex of mice was determined by immunofluorescence assay, and the content of acetylcholine(Ach) and the activity of acetylcholines-terase(AchE) in hippocampus of mice by kits. Moreover, the content of superoxide dismutase(SOD), malondialdehyde(MDA), catalase(CAT), and total antioxidant capacity(T-AOC) in serum of mice was detected. The content of Kelch-like ECH-associated protein 1(Keap1), nuclear factor erythroid 2-related factor 2(Nrf2), and heme oxygenase 1(HO-1) in hippocampus was determined by Western blot. The results showed that there were significant differences in the trajectory map of mice among different groups in the behavioral experiment. Moreover, the latency of ESP groups decreased significantly compared with that in the model group. The hippocampal neurons in the high-dose ESP group were significantly more than those in the model group and the cortical neurons in the high-dose and medium-dose ESP groups were significantly more than those in the model group. The expression of NeuN in the model group was significantly decreased compared with that in the blank group, and the expression in the ESP groups was significantly higher than that in the model group. The AchE activity and MDA level were significantly decreased, and Ach content and levels of SOD, CAT, and T-AOC in the ESP groups were significantly increased in the ESP groups compared with those in the model group. The expression of Keap1 in the model group was significantly increased compared with that in the blank group, and the Keap1 expression increased insignificantly in ESP groups compared with that in the model group. The expression of Nrf2 and HO-1 was significantly lower in the model group than in the blank group, and the expression was significantly higher in the medium-dose ESP group than in the model group. In conclusion, ESP protected mice against the scopolamine-induced learning and memory impairment by regulating the Keap1/Nrf2/HO-1 signaling pathway.

Alloimperatorin activates apoptosis, ferroptosis, and oxeiptosis to inhibit the growth and invasion of breast cancer cells in vitro.

Breast cancer is the most common malignant tumour in women. Our research on alloimperatorin from Angelica dahurica showed that alloimperatorin inhibited breast cancer cell viability in a concentration- and time-dependent manner; it also showed that apoptosis and ferroptosis inhibitors significantly weakened the antisurvival effect of alloimperatorin. Alloimperatorin clearly induced breast cancer cell apoptosis and increased the activities of caspase-3, caspase-8, caspase-9, and poly (ADP-ribose) polymerase; it also caused significant mitochondrial shrinkage, promoted the accumulation of Fe2+, reactive oxygen species, and malondialdehyde, and significantly reduced mRNA and protein expression levels of SLC7A11 and GPX4, indicating that alloimperatorin induces ferroptosis. In addition, alloimperatorin significantly promoted Kelch-like ECH-associated protein 1 (Keap1) expression; although it did not affect the expression of PGAM5 (mitochondrial serine/threonine protein phosphatase) and apoptosis-inducing factor mitochondria associated 1 (AIFM1), it significantly reduced the phosphorylation level of AIFM1. After downregulating the expression of Keap1, PGAM5, or AIFM1, the inhibitory effect of alloimperatorin on cell viability was significantly weakened, indicating that alloimperatorin regulates the Keap1/PGAM5/AIFM1 pathway to promote oxeiptosis. Alloimperatorin significantly inhibited the invasion of breast cancer cells, while Keap1 siRNA or GPX4 overexpression vectors significantly enhanced cell invasion and effectively reversed the anti-invasive effect of alloimperatorin. Therefore, alloimperatorin induces breast cancer cell apoptosis, ferroptosis, and oxeiptosis, thereby inhibiting cell growth and invasion.

Ruscogenin Alleviates Myocardial Ischemia-Induced Ferroptosis through the Activation of BCAT1/BCAT2

Ruscogenin (RUS), a natural steroidal sapogenin, exerts various biological activities. However, its effectiveness for preventing myocardial ischemia (MI) and its molecular mechanisms need further clarification. The model of MI mice and oxygen-glucose deprivation-induced cardiomyocytes injury was performed. RUS significantly alleviated MI, as evidenced by decreased infarct size, ameliorated biochemical indicators and cardiac pathological features, and markedly inhibited ferroptosis by means of the up-regulation of GPX4 and down-regulation of ACSL4 and FLC. Simultaneously, RUS notably mitigated cell injury and oxidative stress, and ameliorated ferroptosis in vitro. Subsequently, HPLC-Q-TOF/MS-based metabolomics identified BCAT1/BCAT2 as possible regulatory enzymes responsible for the cardioprotection of RUS. Importantly, RUS treatment significantly increased the expression of BCAT1 and BCAT2 in MI. Furthermore, we found that BCAT1 or BCAT2 siRNA significantly decreased cell viability, promoted ferroptosis, and increased Keap1 expression, and induced Nrf2 and HO-1 degradation in cardiomyocytes. Conversely, cardiac overexpression of BCAT1 or BCAT2 in MI mice activated the Keap1/Nrf2/HO-1 pathway. Moreover, RUS significantly activated the Keap1/Nrf2/HO-1 pathway in MI, whereas BCAT1 or BCAT2 siRNA partially weakened the protective effects of RUS, suggesting that RUS might suppress myocardial injury through BCAT1 and BCAT2. Overall, this study demonstrated that BCAT1/BCAT2 could alleviate MI-induced ferroptosis through the activation of the Keap1/Nrf2/HO-1 pathway and RUS exerted cardioprotective effects via BCAT1/BCAT2.

Extracts of Dracocephalum tanguticum Maxim Ameliorate Acute Alcoholic Liver Disease via Regulating Transcription Factors in Mice.

Alcoholic liver disease (ALD) caused by excessive drinking is a health and economic concern worldwide. Given the high morbidity, mortality, and the progressive nature of ALD, finding effective interventions is essential. Previous studies have confirmed that edible food plants and their bioactive compounds exert a protective effect against ALD. Dracocephalum tanguticum Maxim (DTM) is one of the important traditional Tibetan medicines in China with the effect of clearing away liver heat, used for the treatment of hepatitis. In this study, the DTM chloroform extract (DtM-C), ethyl acetate extract (DtM-E), and n-butanol extract (DtM-B) were obtained by ethanol extraction combined with fractional extraction. Acute ALD was induced in mice given intragastric ethanol. Serum and liver biochemical markers were detected by ELISA. Liver histological observation, Oil Red O, and Masson’s trichrome staining were performed. Liver injury cells were induced by ethanol. The cell vitality was detected by using MTT colorimetry. The expressions of Nrf2, NF-κB, STAT3, AP-1, CREB, HIF-1α, HO-1, NQO-1, GSTA1, IKB2, and Keap1 were detected by real-time polymerase chain reaction (PCR) to elucidate the mechanism of hepatoprotective effect, and the results were verified by using Western blot. The results of serum liver function indicators (ALT, AST, and ADH), serum hepatic lipid indicators (TC, TG, HDL-C, and LDL-C), and lipid peroxidation indicators (ADH, MDA, SOD, CAT, and GSH-Px) in liver tissue and liver histological observation showed that DtM-E could improve liver function, alleviate fatty degeneration, edema, cell necrosis, and liver fibrosis caused by alcohol. DtM-E also increased the vitality of EtOH-induced liver injury cells, upregulated the mRNA expression of Nrf2, HO-1, NQO-1, and GSTA1, while downregulated the expression of Keap-1, p65, and NF-κB. Western blot results were consistent with PCR. The results suggest that DtM-E has a protective effect against ALD in vitro and in vivo, and its mechanism of action may be related to the activation of Nrf2/Keap-1 and inhibition of the P65/NF-κB signaling pathways.

Hypoxia Induces Oxidative Injury and Apoptosis via Mediating the Nrf-2/Hippo Pathway in Blood Cells of Largemouth Bass (Micropterus salmoides)

Investigating how aquatic animals respond to hypoxia brought about by changes in environmental temperature may be of great significance to avoid oxidative injury and maintain the quality of farmed fish in the background of global warming. Here, we investigated the effects of hypoxia on oxidative injury and environment-sensing pathway in blood cells of Micropterus salmoides. The total blood cell count (TBCC) and Giemsa staining showed that hypoxia could lead to damage of blood cells. Flow cytometry analysis confirmed that the apoptosis rate, Ca2+ level, NO production and ROS of blood cells were significantly increased under hypoxia stress. Environment-sensing pathways, such as Nrf2 pathway showed that hypoxia resulted in significant up-regulation of hiF-1 alpha subunit (Hif-1α), nuclear factor erythroid 2-related factor 2 (Nrf2) and kelch-1ike ECH- associated protein l (Keap1) expression. Meanwhile, the expression of Hippo pathway-related genes such as MOB kinase activator 1 (MOB1), large tumor suppressor homolog 1/2 (Lats1/2), yes-associated protein/transcriptional co-activator with PDZ-binding motif (YAP/TAZ), protein phosphatase 2A (PP2A) were significantly increased in blood cells after hypoxia exposure. In addition, hypoxia stress also increased the expression of catalase (CAT) and glutathione peroxidase (GPx), but decreased the expression of superoxide dismutase (SOD). Consequently, our results suggested that hypoxia could induce oxidative injury and apoptosis via mediating environment-sensing pathway such as Nrf2/Hippo pathway in blood cells of M. salmoides.

Novel Prenylated Indole Alkaloids with Neuroprotection on SH-SY5Y Cells against Oxidative Stress Targeting Keap1-Nrf2.

Oxidative stress has been implicated in the etiology of Parkinson’s disease (PD). Molecules non-covalently binding to the Keap1–Nrf2 complex could be a promising therapeutic approach for PD. Herein, two novel prenylated indole alkaloids asperpenazine (1), and asperpendoline (2) with a scarce skeleton of pyrimido[1,6-a]indole were discovered from the co-cultivated fungi of Aspergillus ochraceus MCCC 3A00521 and Penicillium sp. HUBU 0120. Compound 2 exhibited potential neuroprotective activity on SH-SY5Y cells against oxidative stress. Molecular mechanism research demonstrated that 2 inhibited Keap1 expression, resulting in the translocation of Nrf2 from the cytoplasm to the nucleus, activating the downstream genes expression of HO-1 and NQO1, leading to the reduction in reactive oxygen species (ROS) and the augment of glutathione. Molecular docking and dynamic simulation analyses manifested that 2 interacted with Keap1 (PDB ID: 1X2R) via forming typical hydrogen and hydrophobic bonds with residues and presented less fluctuation of RMSD and RMSF during a natural physiological condition.

6-Gingerol exerts a protective effect against hypoxic injury through the p38/Nrf2/HO-1 and p38/NF-κB pathway in H9c2 cells

Ginger, one of the most widely consumed condiment for various foods and beverages, has many pharmacological effects. 6-gingerol, a naturally occurring phenol, is one of the major pungent constituents of ginger. The purpose of this study was to characterize the effect of 6-gingerol on the p38/Nrf2/HO-1 and p38/NF-κB signaling pathway, as a possible means of combating hypoxia-related oxidative stress. H9c2 cells were chemically induced with CoCl2 to mimic hypoxia-associated cellular damage. Cardiomyocyte injury was assessed by lactate dehydrogenase and creatine kinase. Reactive oxygen species production was assessed by 2′,7′-dichlorodihydrofluorescein diacetate. The antioxidative property of 6-gingerol was measured by estimating the activities of superoxide dismutase, catalase, glutathione and glutathione disulfide. Apoptosis was detected by flow cytometry after Annexin V-FITC-propidium iodide double staining. Western blotting was used to evaluate levels of p-p38, p38, cytoplasm p65, nuclear p65, total p65, nuclear Nrf2, total Nrf2, Keap1, HIF-1α, and HO-1. 6-gingerol was able to counter hypoxia-induced cardiomyocyte injury as evidenced by inhibiting the levels of oxidative stress indexes and increasing the percentage of apoptosis. Furthermore, 6-gingerol was able to down-regulate p-p38/p38, nuclear p65, total p65 and Keap1 expression induced by CoCl2 stimulation and increased cytoplasm p65, nuclear Nrf2, total Nrf2, HO-1, and HIF-1α expression. However, treatment with specific Nrf2 inhibitor blunted the activation of Nrf2 signaling and removed the protective effects of 6-gingerol. These experiments provide evidence that 6-gingerol exerts cytoprotective effects, which may be associated with the regulation of oxidative stress and apoptosis, potentially through activating the Nrf2 pathway and inhibiting the p38/NF-κB pathways.

Impact of Auricularia cornea var. Li polysaccharides on the physicochemical, textual, flavor, and antioxidant properties of set yogurt

This study evaluated quality attributes and in vivo antioxidant activity of Auricularia cornea var. Li polysaccharide (ACP)-fortified set yogurt during 21 days of storage (4 °C). Set yogurt was manufactured using a commercial yogurt culture, and 3% (w/v) ACP was added. Physicochemical (pH, titratable acidity, and water-holding capacity), textural, rheological, microstructural, flavor, and antioxidant properties of set yogurt were investigated. The results showed that the addition of ACP significantly enhanced WHC, viscosity, firmness, and cohesiveness, while inhibiting post-acidification of set yogurt during storage. The yogurt supplemented with ACP showed a larger hysteresis area and higher G′ and G″ values, formed a porous, dense, mesh-like structure and exhibited a unique mushroom flavor. Antioxidant results showed that administration of ACP-fortified yogurt significantly decreased serum alanine aminotransferase and aspartate aminotransferase enzyme activities and malondialdehyde levels, while increasing superoxide dismutase, catalase, phospholipid hydroperoxide glutathione peroxidase, and total antioxidant capacity in the liver and hippocampus of the mice. ACP-fortified yogurt might alleviate hepatic damage and hippocampal neuroinflammation induced by d-galactose. Additionally, ACP-fortified yogurt downregulated the expression of Keap1 and upregulated the expression of Nrf2 and HO-1 in the liver. In conclusion, ACP may be used as an ingredient to produce yogurt with desired properties.

Blockage of ERCC6 Alleviates Spinal Cord Injury Through Weakening Apoptosis, Inflammation, Senescence, and Oxidative Stress.

Objective: Spinal cord injury (SCI) is a devastating disease resulting in lifelong disability, but the molecular mechanism remains unclear. Our study was designed to observe the role of excision repair cross-complementing group 6 (ERCC6) following SCI and to determine the underlying mechanism. Methods: SCI mouse models and LPS-induced microglia cell models were established. ERCC6 expression was blocked by ERCC6-siRNA-carrying lentivirus. Nissl staining was utilized for detecting neuronal damage, and apoptosis was analyzed with TUNEL and Western blotting (apoptotic markers). Immunofluorescence was used for measuring macrophage markers (CD68 and F4/80) and astrocyte and microglia markers (GFAP and Iba-1). Pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) were measured via ELISA. Senescent cells were estimated via SA-β-Gal staining as well as Western blot (senescent markers p21 and p27). Oxidative stress was investigated by detecting the expression of 4-HNE, Nrf2, and Keap1, and intracellular ROS levels. Results: ERCC6 expression was remarkably upregulated both in the spinal cord of SCI mice and LPS-induced microglia cells. ERCC6 deficiency alleviated neuronal damage and apoptosis. Macrophage infiltration and inflammatory response were suppressed by si-ERCC6 treatment. Moreover, ERCC6 blockage ameliorated astrocyte and microglia activation and cell senescence in the damaged spinal cord. Excessive oxidative stress was significantly decreased by ERCC6 knockdown in SCI. Conclusion: Collectively, ERCC6 exerts crucial functions in mediating physiological processes (apoptosis, inflammation, senescence, and oxidative stress), implying that ERCC6 might act as a prospective therapeutic target against SCI.

Diosmetin alleviates cerebral ischemia-reperfusion injury through Keap1-mediated Nrf2/ARE signaling pathway activation and NLRP3 inflammasome inhibition.

Diosmetin was found to exert protective effect on renal and myocardial ischemia-reperfusion (IR) injury. This study aimed to investigate the role of diosmetin in cerebral IR (CIR) injury. PC12 neurons were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to establish CIR injury model in vitro and then incubated with diosmetin, and we found that diosmetin alleviated OGD/R-induced viability inhibition, LDH release, apoptosis, and oxidative stress in PC12 cells. Then our results showed that diosmetin downregulated kelch like ECH-associated protein 1 (Keap1) expression, and upregulated nuclear factor E2-related factor 2 (Nrf2) expression, antioxidant response element (ARE) activity and the mRNA and protein expression of heme oxygenase 1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1). Keap1 overexpression or Nrf2 silencing both attenuated the neuroprotective effect of diosmetin on PC12 cells. Moreover, diosmetin inhibited the levels of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) pyrin domain containing 3 (NLRP3) inflammasome pathway related proteins and inflammatory cytokines interleukin (IL)-1β and IL-18. Additionally, a middle cerebral artery occlusion (MCAO) rat model was established and diosmetin was injected for treatment. Diosmetin alleviated CIR-induced neurological deficits, cerebral infarction, brain edema and histopathological damage, and neuronal apoptosis and oxidative stress in MCAO rats. In conclusion, diosmetin attenuated OGD/R-induced PC12 cell viability inhibition, apoptosis, oxidative stress and inflammation through Keap1-mediated Nrf2/ARE signaling activation and NLRP3 inflammasome inhibition, and alleviated CIR-induced neurological injury in MCAO rat model. Our study may provide a novel therapeutic strategy for CIR injury.

FOXO acts as a positive regulator of CncC and deltamethrin tolerance in the red flour beetle, Tribolium castaneum

Both forkhead box O (FOXO) and nuclear factor erythroid-derived 2-like-2 (Nrf2) are key transcription factors related to stress responses. Whereas limited studies in mammals and Caenorhabditis elegans have revealed the interaction between FoxO/DAF-16 and Nrf2/SKN-1, the role of FOXO in metabolic detoxification and regulation of the Nrf2-Keap1 signaling pathway are poorly understood in insects. Using Tribolium castaneum as a model organism, we found that RNA interference-mediated knockdown of FOXO enhanced deltamethrin-induced lethality by affecting the messenger RNA (mRNA) levels of CYP6BQ cluster genes. We further demonstrated that injection of dsFOXO into the beetle larvae decreased expression of CncC and KEAP1 at both the mRNA and protein level. Notably, dual-luciferase and electrophoretic mobility shift assays both confirmed direct regulation of CncC by FOXO, whereas Keap1 was directly regulated by CncC. FOXO can directly regulate the expression of CncC and indirectly regulate the expression of Keap1 through CncC. The data provide insights into the regulatory mechanisms of the Nrf2-Keap1 signaling pathway in insects.

Exosome-derived miR-200a promotes esophageal cancer cell proliferation and migration via the mediating Keap1 expression.

Previous studies have reported that exosomes bearing certain microRNAs (miRNAs) are related to the physiological functions of different types of cancer cells. Our study aimed to elucidate the role of miR-200a in esophageal squamous cell carcinoma (ESCC). We observed that miR-200a expression is higher in esophageal carcinoma cells, tissues, and exosomes than in normal cells and healthy tissues. We showed that exosome-shuttled miR-200a promotes the proliferation, migration, and invasion of esophageal cells and inhibits apoptosis, thereby leading to the progression of ESCC. We showed that miR-200a exerts its effects through its interaction with Keap1, thus altering the Keap1/Nrf2 signaling pathway. Our results suggest that exosome-shuttled miR-200a might be useful as a biomarker for prognosis in patients with ESCC.

Honeysuckle extract (Lonicera pallasii L.) exerts antioxidant properties and extends the lifespan and healthspan of Drosophila melanogaster.

Honeysuckle Lonicera pallasii (Lonicera caerulea L.) is an excellent source of anthocyanins which have a number of health-promoting properties mainly associated with antioxidant and anti-inflammatory activities. Cyanidin-3-O-glucoside (C3G) is one of the most common anthocyanins naturally found in honeysuckle. The goal of the present study was to investigate antioxidant and anti-aging properties of Lonicera pallasii (Lonicera caerulea L.) extract (LE) and C3G using red blood cells (RBC) and Drosophila melanogaster models. LE and C3G treatment at a concentration of 100μM induced enhancement of median and maximum lifespan up to 8%. LE and C3G supplementation at a concentration of 100μM increased stress resistance up to 10%. The locomotor activity decreased during LE and C3G treatment in 4 and 6weeks up to 52% in females. The integrity of the intestinal barrier was increased by 4% after LE treatment. These effects were accompanied by increased expression of Hif1 (pro-longevity gene) in response to C3G treatment and decreased expression of Keap1 (anti-longevity gene) after C3G and LE supplementation. RNA interference-mediated knockdown of Sirt6 completely abolished the positive effect obtained of LE and C3G supplementation in males which indicates that lifespan-extending effect is associated with Sirt6 activation. The experiments on the various in-vitro models (including radical scavenging activity and oxidative hemolysis of RBC demonstrated antioxidant and membrane-protective activities of LE and C3G. The present study indicates that Lonicera extract can prolong the lifespan and improve the healthspan of Drosophila model through biological and antioxidant activities.

Trilobatin, a Natural Food Additive, Exerts Anti-Type 2 Diabetes Effect Mediated by Nrf2/ARE and IRS-1/GLUT2 Signaling Pathways.

Oxidative stress and aberrant insulin signaling transduction play vital roles in type 2 diabetes mellitus (T2DM). Our previous research has demonstrated that trilobatin (TLB), derived from the leaves of Lithocarpus Polystachyus (Wall.), exhibits a potent antioxidative profile. In the current study, we investigated the anti-T2DM effect of TLB on KK-Ay diabetic mice and further explored the potential mechanisms. Our results showed that TLB significantly reduced the high fasting blood glucose level and insulin resistance and promoted the tolerances to exogenous glucose and insulin in KK-Ay mice. Moreover, TLB reduced the content of reactive oxygen species; enhanced antioxidant enzymes activities, including serum catalase, glutathione peroxidase, and superoxide dismutase; and regulated the abnormal parameters of lipid metabolism, including triglyceride, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, and free fatty acid, as evidenced by enzyme-linked immunosorbent assay. Additionally, TLB markedly ameliorated the pancreatic islet morphology near normal and increased the insulin expression of the islet. Whereafter, TLB promoted Nrf2 that was translocated from cytoplasm to nucleus. Moreover, it increased the protein expressions of HO-1, NQO-1, and GLUT-2, and phosphorylation levels of Akt and GSK-3β Ser 9 and decreased the protein expressions of keap1 and phosphorylation levels of IRS-1Ser 307 and GSK-3β Tyr 216. Taken together, our findings reveal that TLB exhibits an anti-T2DM effect in KK-Ay mice by activating the Nrf2/ARE signaling pathway and regulating insulin signaling transduction pathway, and TLB is promising to be developed into a novel candidate for the treatment of T2DM in clinic due to its favorable druggability.

Dihydrotanshinone I inhibits ovarian tumor growth by activating oxidative stress through Keap1-mediated Nrf2 ubiquitination degradation

Dihydrotanshinone I (DHT), a bioactive compound in Salvia miltiorrhiza, was reported to exhibit cytotoxicity against various malignancies. However, the underlying mechanism on ovarian cancer remains unclear. Here, DHT inhibited cell viability of ovarian cancer HO8910PM, SKOV3, A2780 and ES2 cells. It showed moderate inhibitory effect on ovarian epithelial IOSE80 cells and lower toxicity than chemotherapy drugs. DHT induced apoptosis and G2 cell cycle arrest accompanied by reduced expression of Bcl-2, Caspase-3, and increased Bax. Meanwhile, DHT increased ROS accumulation, decreased mitochondrial membrane potential and activated oxidative stress in HO8910PM and ES2 cells. Mechanistically, DHT inhibited Nrf2 and p62 expression, Nrf2 target genes and enzymes, and Nrf2 nuclear translocation, while increased the expression of Nrf2 inhibitor Keap1. NAC, a ROS scavenger, rescued DHT-induced proliferation inhibition, ROS generation and Nrf2 inhibition. DHT alleviated tBHQ-induced Nrf2 expression and increased its mRNA level. However, the proteasome inhibitor MG132 blocked DHT-induced Nrf2 inhibition, suggesting a post-translational regulation manner. DHT enhanced Nrf2 binding with Keap1, leading to potentiated Nrf2 ubiquitination degradation. Furthermore, Nrf2 and p62 overexpression blocked DHT-induced Nrf2 and p62 inhibition. Consistent with the in vitro results, DHT significantly delayed tumor growth in HO8910PM and ES2 xenograft nude mice, decreased tumor marker HE4 and CA125 levels, reversed the abnormally expressed proteins including Ki67, Nrf2, p62, Keap1, Bcl-2, CyclinB1, Cdc-2, and antioxidant enzymes SOD, CAT in vivo. Serum from DHT-treated mice also inhibited cell growth in vitro. Taken together, DHT exhibits anti-ovarian tumor effect by activating oxidative stress through ubiquitination-mediated Nrf2 degradation. Our findings implicate a potential application of DHT for ovarian cancer therapy.

Age-Related Mitochondrial Impairment and Renal Injury Is Ameliorated by Sulforaphane via Activation of Transcription Factor NRF2.

Age is one of the major risk factors for the development of chronic pathologies, including kidney diseases. Oxidative stress and mitochondrial dysfunction play a pathogenic role in aging kidney disease. Transcription factor NRF2, a master regulator of redox homeostasis, is altered during aging, but the exact implications of altered NRF2 signaling on age-related renal mitochondrial impairment are not yet clear. Herein, we investigated the role of sulforaphane, a well-known NRF2 activator, on age-related mitochondrial and kidney dysfunction. Young (2–4 month) and aged (20–24 month) male Fischer 344 rats were treated with sulforaphane (15 mg/kg body wt/day) in drinking water for four weeks. We observed significant impairment in renal cortical mitochondrial function along with perturbed redox homeostasis, decreased kidney function and marked impairment in NRF2 signaling in aged Fischer 344 rats. Sulforaphane significantly improved mitochondrial function and ameliorated kidney injury by increasing cortical NRF2 expression and activity and decreasing protein expression of KEAP1, an NRF2 repressor. Sulforaphane treatment did not affect the renal NRF2 expression or activity and mitochondrial function in young rats. Taken together, our results provide novel insights into the protective role of the NRF2 pathway in kidneys during aging and highlight the therapeutic potential of sulforaphane in mitigating kidney dysfunction in elders.

A Network Pharmacology-Based Study on Antidepressant Effect of Salicornia europaea L. Extract with Experimental Support in Chronic Unpredictable Mild Stress Model Mice.

To investigate the pharmacodynamic material basis, mechanism of actions and targeted diseases of Salicornia europaea L. (SE) based on the network pharmacology method, and to verify the antidepressant-like effect of the SE extract by pharmacological experiments. Retrieval tools including Chinese medicine (CM), PubMed, PharmMapper, MAS 3.0 and Cytoscape were used to search the components of SE, predict its targets and related therapeutic diseases, and construct the "Component-Target-Pathway" network of SE for central nervous system (CNS) diseases. Further, protein-protein interaction (PPI) network, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) function annotation of depression-related targets were analyzed to predict the antidepressant mechanism of SE. Chronic unpredictable mild stress (CUMS) model was used to construct a mouse model with depression-like symptoms. And the animals were randomly divided into 6 groups (n=10) including the normal group (nonstressed mice administered with distilled water), the CUMS group (CUMS mice administered with distilled water), the venlafaxine group (CUMS mice administered with venlafaxine 9.38 mg/kg), SE high-, medium-, and low-dose groups (CUMS mice administered with SE 1.8, 1.35 and 0.9 g/kg, respectively). Then some relevant indicators were determined for experimental verification by the forced swim test (FST), the tail suspension test (TST) and open-field test (OFT). Dopamine (DA) concentration in hippocampus and cerebral cortex, IL-2 and corticosterone (CORT) levels in blood, and nuclear factor E2 related factor 2 (Nrf2), kelch-like epichlorohydrin related protein 1 (Keap1), NAD(P) H dehydrogenase [quinone] 1 (NQO1) and heme oxygenase-1 (HO-1) levels in mice were measured by enzyme linked immunosorbent assay (ELISA) and Western blot respectively to explore the possible mechanisms. The "target-disease" network diagram predicted by network pharmacology, showed that the potential target of SE involves a variety of CNS diseases, among which depression accounts for the majority. The experimental results showed that SE (1.8, 1.35 g/kg) significantly decreased the immobility period, compared with the CUMS group in FST and TST in mice after 3-week treatment, while SE exhibited no significant effect on exploratory behavior in OFT in mice. Compared with CUMS group, the SE group (0.9 g/kg) showed significant differences (P<0.05) in DA levels in the hippocampus and cerebral cortex. In addition, compared with CUMS control group, SE (1.8 g/kg) group showed a significant effect on decreasing the activities of CORT (P<0.05), and serum IL-2 level with no statistical significance. Finally, Western blot results showed that compared with the model group, Nrf2, Keap1, NQO1 and HO-1 protein expressions in SE group (1.8 g/kg) were up-regulated (all P<0.01). The SE extract may have an antidepressant effect, which appeared to regulate Nrf2-ARE pathway and increased levels of DA and CORT in the hippocampus and cortex.