Kelch-like ECH-associated Protein Research Articles

Nodule-specific NRF2-targeted upregulation in patients with KEAP1 mutations and familial nontoxic multinodular goiter.

Kelch-like ECH-associated protein 1 (KEAP1) is associated with nuclear factor erythroid-2 related factor 2 (NRF2) and promotes NRF2 degradation in normal conditions. Genetic abnormality in KEAP1 is a rare disease and presents with familial multinodular goiter. This study assessed the clinical and molecular findings concerning nodular formation in the thyroid gland of patients harboring KEAP1 germline mutations. Next-generation sequencing analysis targeting goiter-associated genes was performed on 39 patients with familial multinodular goiter. The expression of NRF2-targeted genes from surgical thyroid specimens of patients with KEAP1 mutations were analyzed using a whole transcript expression array and immunohistochemistry. We found five probands with pathogenic heterozygous mutations in KEAP1 (p.Q86*, p.L136P, p.V411fs, p.R415C, and p.R483H), which had no meaningful concomitance with mutations of other goiter-associated genes in germline and somatic levels. Their common histopathological features showed multinodular goiters in the entire thyroid gland with few degenerative lesions or complications of malignancy and slow proliferation indicating < 1% at the Ki-67 labeling index. Among 42 NRF2-targeted genes, antioxidant genes were most frequently upregulated (11/12) in the nodule, followed by detoxification genes (6/11). Immunohistochemical analysis showed relatively high expression of glutathione peroxidase 2 and NAD(P)H quinone oxidoreductase 1 (representative NRF2-targeted genes) in the nodules of various patients harboring KEAP1 mutations. KEAP1 germline heterozygous mutations exert excessive NRF2 activity in the thyroid gland and may confer cytoprotective effects even under abundant reactive oxygen species associated with thyroid hormone production, resulting in thyroid hyperplasia with scarce degradation.

Metabolic Rate and Oxidative Stress as a Risk Factors in the Development of Colorectal Cancer

There is growing evidence that the body’s energy expenditures constitute a significant risk factor for the development of most deadly diseases, including cancer. Our aim was to investigate the impact of basal metabolic rate (BMR) on the growth and progression of colorectal cancer (CRC). To do so, we used a unique model consisting of three lines of laboratory mice (Mus musculus) artificially selected for high (HBMR) and low (LBMR) basal metabolic rate and randomly bred individuals (non-selected, NSBMR). The experimental individuals were implanted with human colorectal cancer cells DLD-1. The variation in BMR between the lines allowed for testing the impact of whole-body metabolism on oxidative and antioxidant parameters in the liver throughout the cancerogenesis process. We investigated the dependence between metabolic values, reactive oxygen species (ROS) levels, and Kelch-like ECH-associated protein 1-based E3 ligase complexes (Keap1) gene activity in these animals. We found that the HBMR strain had a higher concentration of oxidative enzymes compared to the LBMR and NSBMR. Furthermore, the growth rate of CRC tumors was associated with alterations in the levels of oxidative stress enzymes and Keap1 expression in animals with a high metabolic rate. Our results indicate that a faster growth and development of CRC line DLD-1 is associated with enzymatic redox imbalance in animals with a high BMR.

A systematic review of the traditional uses, chemistry, and curative aptitude of echinacoside-a phenylethanoid glycoside.

Echinacoside (ECH), a naturally occurring water-soluble phenylethanoid glycoside, is one of the primary bioactive compounds present in several plant species, such as Echinacea, Cistanche, Plantago, Rosa, Buddleja, and Rehmannia. Research has revealed that these plants, rich in ECH, have diverse traditional uses and pharmacological activities, like anti-diabetic, anti-inflammatory, anti-fatigue, anti-allergic, anti-ageing, anti-skin glycation, analgesic, wound healing, and aphrodisiac properties. Among other activities, ophthalmic, haematopoiesis, pulmonary, anti-bacterial, anti-protozoal, anti-fungal, and anti-viral effects of ECH have been reported. Chemically, the compound comprises caffeic acid glycoside containing a trisaccharide that includes two glucose and one rhamnose unit. These units are linked through glycosidic bonds to a caffeic acid and a dihydroxyphenylethanol (hydroxytyrosol) residue, which are connected to the central rhamnose. The biosynthesis of ECH has been reported to start with forming L-phenylalanine and tyrosine precursors via the shikimic acid pathway. The structure-activity relationship of ECH has shown that various functional groups in the structure, particularly phenolic hydroxyl groups, are crucial for antioxidant activities. Similarly, in silico studies have revealed that ECH binds to different receptors, like Kelch-like ECH-associated protein 1 (Keap1), receptor for advanced glycation end products (RAGE), etc., to affect various pharmacological activities. The ECH contents in the reported plants often own these multifaceted properties, highlighting their importance in clinical research. Evident from its therapeutic efficacy, there is a huge potential for a comprehensive understanding of the mechanisms of actions of ECH, which underscores the need for more research in this area. Thus, this review is a compendium of the latest literature to analyse the existing knowledge on ECH, encompassing its distribution, traditional uses, extraction, chemical constituents, biosynthesis, pharmacological activities, structure-activity relationship, and in silico studies, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.

Nano-selenium ameliorates microplastics-induced injury: Histology, antioxidant capacity, immunity and intestinal microbiota of grass carp (Ctenopharyngodon idella)

Microplastics (MPs) are pollutants widely distributed in the aquatic environments and causing various degrees of aquatic toxicity to aquatic organisms, which has attracted global attention in recent years. Nano-selenium (NSe) has been shown to have the potential to mitigate the harmful impacts of toxic substances. However, there is currently no reported evidence regarding the protective influence of NSe against the adverse effects of MPs. The aim of this study is to determine whether NSe could ameliorate the polystyrene (PS)-MPs-induced injury in grass carp (Ctenopharyngodon idella). The individuals of grass carp were assigned into three groups: (1) the control group fed with basal diet, (2) the PS group fed with basal diet and exposed to PS-MPs, and (3) the NSe group fed with diet supplemented with NSe and exposed to PS-MPs. Our results indicated that NSe administration significantly alleviated the histological damage caused by the PS-MPs in the liver and intestine with lower goblet cell count and larger villus height in the intestine, and significantly lower damage score in the liver. Moreover, NSe mitigated PS-MPs-induced oxidative stress through restoring the activities of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA)) except the intestinal CAT activity. Furthermore, NSe supplementation could help fish maintain lower transcriptional level of the immune-related genes (Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88)), inflammation-related genes (major histocompatibility complex class II (MHC-II) and interleukin 8 (IL-8)) and antioxidant enzyme-related genes (nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) and kelch-like ECH-associated protein 1 (Keap-1)) after PS-MPs exposure. Besides, NSe supplementation dramatically helped maintain the intestinal microbial composition, for example, the proportion of Proteobacteria in the grass carp intestine of the NSe group (41 %) was similar to that of the control group (34 %) while 85 % of the PS group. NSe also played a significant protective role in intestinal microbial diversity, effectively resisting the damage on intestinal microbial diversity due to PS-MPs exposure. PS-MPs reduced the beneficial bacteria and increased the pathogenic microorganism like Aeromonas, which was undeniable signs of intestinal dysbiosis. Functional analysis indicated that PS-MPs affected intestinal microbiota functions like inhibition of metabolism, while NSe could significantly alleviate the damage. Our findings suggested that NSe could ameliorate PS-MPs-induced injury, which could contribute to the better understanding of the ecotoxicological effects of MPs on fish and help develop relevant mitigation strategies.

Preparation and characterization of cysteine-rich collagen peptide and its antagonistic effect on microplastic induced damage to HK-2 cells

Cysteine (Cys) plays a significant role in the stability and antioxidant capacity of peptide. This study was to prepare Cys-rich collagen peptide and evaluate its preventive effect on microparticle (MPs)-induced HK-2 cell damage. The results showed that the Cys-rich peptides (AMP-C) were successfully prepared from collagen hydrolysate by plastein reaction in the presence of exogenous Cys. The scavenging activities of 2,2-Diphenyl-1-picrylhydrazyl and hydroxyl radical of AMP-C were significantly higher than those of original peptides without Cys. The fraction with molecular weight < 3 kDa (AMP-CA) significantly improved MPs-induced damage and apoptosis of HK-2 cells. Compared with the same fractions lacking Cys (AMP-A), AMP-CA showed better performance in reducing reactive oxygen species and increasing the total superoxide dismutase and glutathione peroxidase levels, which may be related to its down-regulation of Kelch-like ECH-associated protein 1 (Keap1) expression. In addition, AMP-CA showed strong effects on inhibiting interleukin-6, tumor necrosis factor-α, monocyte chemoattractant protein-1, kidney injury molecule-1, and nuclear factor kappa-B levels. Finally, 330 Cys-containing peptides were isolated from AMP-CA, among which Leucine-Glycine-Asparagine-Glycine-Cysteine-Proline (LGNGCP) showed the lowest binding energy with Keap1 protein. In conclusion, the Cys-rich collagen peptide with higher antioxidant activity was successfully prepared by plastein reaction, which alleviated the HK-2 cells damage induced by MPs through antioxidant and anti-inflammatory. Our findings suggested that Cys-rich collagen peptide might be used as a functional food to mitigate health hazards associated with MPs accumulation.

Enhancing radioprotection: exploring the impact of L-carnitine supplementation on the oxidative stress in the liver.

The adverse effects of radiotherapy (RT) primarily occur through oxidative stress, and attempts are being made to mitigate these effects. L-Carnitine (L-Car) involved in physiological functions, possesses antioxidant and tissue-protective properties. The goal of this investigation is to appraise the radioprotective efficacy of L-Car supplementation. The groups were established by dividing thirty-two rats as: control, RT (10Gy), RT + L-Car (200mg/kg/d), L-Car. Upon completion of the experiment, the livers were harvested for histopathological, immunostaining [tumor necrosis factor-alpha (TNF-α), Caspase-3], spectrophotometric [total oxidant status (TOS), total antioxidant status (TAS), oxidative stress index (OSI)], and mRNA expression [(Nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap-1), Heme Oxygenase (HO-1), Transforming growth factor beta 1 (TGF-β1)] analyses. In the damage group, decreased Keap-1, Nrf2, HO-1, and TAS values, along with increased histopathological findings, alanine transferase, aspartate transferase, TNF-α, Caspase-3, TOS, OSI, TGF-β1 levels were found. All findings were improved with L-Car treatment. Considering these findings, it can be inferred that L-Car exhibits tissue-protective effects against organ damage predominantly induced by RT-related oxidative stress. Additionally, it has prevented the development of inflammation, apoptosis, and fibrosis. Therefore, L-Car may be considered as a supplement to reduce complications associated with RT.

Parthenolide ameliorates 3-nitropropionic acid-induced Huntington’s disease-like aberrations via modulating NLRP3 inflammasome, reducing microglial activation and inducing astrocyte shifting

BackgroundHuntington’s disease (HD) is a progressive neurodegenerative disease that causes motor, cognitive, and psychiatric abnormalities, with no satisfying disease-modifying therapy so far. 3-nitropropionic acid (3NP) induces behavioural deficits, together with biochemical and histological alterations in animals’ striata that mimic HD. The role of nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) inflammasome in HD pathogenesis remains largely uncharacterized. Parthenolide (PTL), a naturally occurring nuclear factor kappa B (NF-κB) inhibitor, is also known to inhibit NLRP3 inflammasome. Whether PTL is beneficial in HD has not been established yet.AimThis study evaluated the possible neuroprotective effects of PTL against 3NP-induced behavioural abnormalities, striatal biochemical derangements, and histological aberrations.MethodsMale Wistar rats received PTL (0.5 mg/kg/day, i.p) for 3 weeks and 3NP (10 mg/kg/day, i.p) was administered alongside for the latter 2 weeks to induce HD. Finally, animals were subjected to open-field, Morris water maze and rotarod tests. Rat striata were examined histologically, striatal protein expression levels of glial fibrillary acidic protein (GFAP), cluster of differentiation 45 (CD45) and neuron-specific enolase (NSE) were evaluated immunohistochemically, while those of interleukin (IL)-1β, IL-18, ionized calcium-binding adapter molecule-1 (Iba1) and glutamate were determined by ELISA. Striatal nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein (Keap1), NF-κB, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, S100 calcium-binding protein A10 (S100A10) and complement-3 (C3) were assessed by gene expression analysis.ResultsPTL improved motor, locomotor, cognitive and anxiety-like behaviours, restored neuronal integrity, upregulated Nrf2, and inhibited NLRP3 inflammasome, NF-κB and microglial activation. Additionally, PTL induced astrocyte shifting towards the neuroprotective A2 phenotype.ConclusionPTL exhibits neuroprotection against 3NP-induced HD, that might be ascribed, at least in part, to its modulatory effects on Keap1/Nrf2 and NF-κB/NLRP3 inflammasome signaling.

Quercetin and kaempferol from saffron petals alleviated hydrogen peroxide-induced oxidative damage in B16 cells.

Saffron petals are usually considered as waste after saffron harvest. However, saffron petals contain many important phytochemical components (e.g. quercetin and kaempferol), which may alleviate oxidative damage in human cells. The contents of flavonoids and crocin in different parts of saffron were analyzed. The protective effects of flavonoids from saffron on oxidative damage of B16 cells were investigated. Saffron stigma contained high contents of crocin and picrocrocin, whereas flavonoid content (quercetin, 4.03 ± 0.33 mg g-1 DW; kaempferol, 47.80 ± 0.60 mg g-1 DW) was higher in saffron petals than in other parts. Incubation of B16 cells with quercetin (10-30 μmol L-1) and kaempferol (20-30 μmol L-1) obtained from saffron extracts could significantly increase the total antioxidant capacity (T-AOC) and the activity of NADPH:dehydrogenase quinone-1 (NQO1) to alleviate H2O2-induced oxidative damage. Quercetin was better than kaempferol in increasing NQO1 activity and T-AOC. Quercetin extracted from saffron petals could induce NQO1 expression through regulating kelch-like ECH-associated protein-1/nuclear factor erythroid 2-related factor-2 signaling pathway to protect B16 cells from oxidative damage. The content of kaempferol-3-O-sophoroside and quercetin-3-O-sophoroside was higher in saffron petals than in other parts of saffron. The kaempferol and quercetin obtained from saffron petals could enhance the activity of antioxidant enzyme NQO1 and T-AOC in B16 cells. This indicated that saffron petals, as a potential functional food, may prevent diseases caused by oxidative stress. © 2024 Society of Chemical Industry.

Comparative Efficacy of Low-Carbohydrate and Ketogenic Diets on Diabetic Retinopathy and Oxidative Stress in High-Fat Diet-Induced Diabetic Rats.

This study examined the effect of a low-carbohydrate diet (LCD) and a low-carbohydrate ketogenic diet (LCKD) on diabetic retinopathy in high-fat diet-induced diabetes mellitus in rats and studied the mechanisms of action. Rats were divided into four groups: the Control group, which was fed a normal diet for 16 weeks; the HFD group, which was fed a high-fat diet (HFD) for the first 8 weeks and then switched to a normal diet for 8 weeks; the HFD+LCD group, fed a HFD for 8 weeks followed by an LCD for 8 weeks, and the HFD+LCKD group, which was fed a HFD for 8 weeks followed by an LCKD for 8 more weeks. Both the LCD and the LCKD effectively reduced the final body and total fat weights and decreased fasting serum levels of glucose, insulin, hemoglobin A1 (HbA1C), triglycerides, cholesterol, and LDL-c. They also reduced the levels of malondialdehyde (MDA), tumor necrosis factor-α, vascular endothelial factor, caspapse-3, and bax. In the HFD rats, we found increased serum levels of β-Hydroxybutyrate and upregulated expression of Bcl2, glutathione, superoxide dismutase, and hemeoxygenase-1. Moreover, the LCD and LCKD significantly reduced mRNA levels of Kelch-like ECH-associated protein 1 (Keap1) and enhanced mRNA and nuclear concentrations of nuclear factor erythroid factor 2 (Nrf2). All these effects were associated with improved layers of the retina in the HFD - LCD and HFD + LCKD rats but not in HFD animals. The impact of the LCKD was always more profound on all measured parameters and on improving the structure of the retina compared to the LCD. In conclusion, the LCKD is superior to the LCD in preventing diabetic retinopathy in HFD-fed rats. Mechanistically, our results suggest that the hypoglycemic and hypolipidemic conditions and the Nrf2-dependent antioxidant and anti-inflammatory effects may be involved in the preventative effects of the LCD and LCKD.

The NRF2 inducer CDDO-2P-Im provokes a reduction in amyloid β levels in Alzheimer's disease model mice.

Alzheimer's disease (AD) is the most common etiology of dementia. The transcription factor NF-E2-related factor 2 (NRF2) induces the expression of genes encoding phase II detoxification and antioxidant genes. NRF2 is regulated by Kelch-like ECH-associated protein 1 (KEAP1), and the KEAP1-NRF2 system is the key regulatory system involved in cytoprotection. To examine whether pharmacological induction of NRF2 expression alleviates AD phenotypes in vivo, we employed two AD mouse models, i.e., App NL-G-F/NL-G-F (AppNLGF) and APPV717I::TAUP301L (APP/TAU) mice. As the synthetic oleanane triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11-dien-28-oyl)] (CDDO)-4(-pyridin-2-yl)-imidazole (CDDO-2P-Im) exhibits strong NRF2-inducing activity, we treated AD model mice with CDDO-2P-Im. We found that Aβ42 levels were markedly greater in the brains of AppNLGF mice than in those of APP/TAU mice. CDDO-2P-Im treatment significantly decreased Aβ42 levels, but not Aβ40 levels, in APP/TAU mice. Consequently, CDDO-2P-Im also decreased the ratio of Aβ42/Aβ40, a vital marker of amyloid plaque formation. LC-MS/MS analyses revealed that CDDO-2P-Im was delivered to the brains of the APP/TAU mice. CDDO-2P-Im induced the expression of detoxification and antioxidant gene targets of NRF2 and elevated reduced glutathione (GSH) levels in the mouse brain. These results support the notion that CDDO-2P-Im ameliorates AD-related pathologic changes.

KEAP1-mutant atypical meningioma: illustrative case.

While genetic testing of tumors is commonly used to inform the selection of systemic therapies, there is limited evidence for the application of radiotherapy for brain cancer. Recent studies have shown that Kelch-like ECH-associated protein 1 (KEAP1), a key regulator of cellular responses to oxidative and electrophilic stress, is associated with radioresistance in multiple cancer types. Several studies have reported the clinical significance of KEAP1 mutation in brain metastasis; however, the effect of KEAP1 mutations on radioresponse in meningioma has never been reported. The authors present the case of a 40-year-old female with a KEAP1 mutation-positive atypical meningioma that was initially treated with resection followed by intensity-modulated radiation therapy (IMRT). Recurrence was observed at 15 months, requiring reoperation and adjuvant stereotactic radiosurgery (SRS). An excellent treatment response was observed at 7 months post-SRS with an improvement in reported symptoms, although bevacizumab was required for the resolution of radiation necrosis observed 2 months post-SRS. To the authors' knowledge, this is the first report of KEAP1-mutant meningioma, including its clinical course after comprehensive management. Notably, treatment included multimodal radiotherapy with IMRT followed by SRS. SRS led to an excellent treatment response at the 7-month follow-up. However, radiation necrosis developed after both radiotherapy treatments, suggesting that radiological modification can be beneficial in patients with KEAP1 mutations. https://thejns.org/doi/10.3171/CASE24387.

Maintaining KEAP1 levels in retinal pigment epithelial cells preserves their viability during prolonged exposure to artificial blue light

Exposure to artificial blue light, one of the most energetic forms of visible light, can increase oxidative stress in retinal cells, potentially enhancing the risk of macular degeneration. Retinal pigment epithelial (RPE) cells play a crucial role in this process; the loss of RPE cells is the primary pathway through which retinal degeneration occurs. In RPE cells, Kelch-like ECH-associated protein 1 (KEAP1) is located in both the nucleus and cytosol, where it binds to nuclear factor erythroid 2-related factor 2 (NRF2) and p62 (sequestosome-1), respectively. Blue light exposure activates the NRF2-heme oxygenase 1 (HMOX1) axis through both canonical and noncanonical p62 pathways thereby reducing oxidative damage, and initiates autophagy, which helps remove damaged proteins. These protective responses may support the survival of RPE cells. However, extended exposure to blue light drastically decreases the viability of RPE cells. This exposure diminishes the ability of KEAP1 to bind to p62 and reduces the level of KEAP1. Inhibition of autophagy does not prevent KEAP1 degradation, the NRF2-HMOX1 axis, or blue-light-induced cytotoxicity. However, proteasome inhibitor along with a transient increase in the amount of KEAP1 in RPE cells, partially restores the p62-KEAP1 complex and reduces blue-light-induced cytotoxicity. In vivo studies confirmed the downregulation of KEAP1 in damaged RPE cells. Mice subjected to periodic blue light exposure exhibited significant atrophy in the outer retina, particularly in the peripheral areas. Additionally, there was a significant decrease in c-wave electroretinography and pupillary light reflex, indicating functional impairments in both visual and nonvisual physiological processes. These data underscore the essential role of KEAP1 in managing oxidative defense and autophagy pathways triggered by blue light exposure in RPE cells.

Preparation and characterization of novel antioxidant peptides from protein hydrolysate of Ophiocordyceps gracilis

Ophiocordyceps gracilis is a type of fungus commonly utilized in both health food and Chinese medicine. Herein, we prepared antioxidant hydrolysate from O. gracilis, and isolated and purified the prepared hydrolysate to obtain O. gracilis peptides-4-3-1 (OGP-4-3-1) grade with the highest scavenging activity on DPPH, ABTS and hydroxyl radicals. Three new peptides with high antioxidant capacity were identified as LFNHF, PLDRHPF and YPFLRPL, respectively. The three peptides can reduce the proportion of senescent cells conveying positive expression of senescence-associated-β-galactosidase (SA-β-Gal) and increase survival pace of H2O2-induced human hepatocellular carcinomas (HepG2 cells), and the protective effects on cellular against senescence might be related to their increase in the activities of antioxidant enzymes, as well as the decrease in the concentration of malondialdehyde (MDA) and reactive oxygen species (ROS). The molecular docking results indicated that they could form durable hydrophobic and hydrogen bonds with critical amino acid residues of the Kelch-like ECH-associated protein 1 (Keap1), which could potentially modulate pathway of nuclear factor erythroid 2-related factor 2 (Nrf2) in conjunction with Keap1. In conclusion, the current research offers a theoretical foundation for the further development of the anti-aging effect of OGP, which has a broad application prospect in the fields of functional food and cosmetics.

Discovery of novel 5-phenyl-1H-pyrrole-2-carboxylic acids as Keap1-Nrf2 inhibitors for acute lung injury treatment

Oxidative stress is intricately linked to acute lung injury (ALI) and cerebral ischemic/reperfusion (I/R) injury. The Keap1 (Kelch-like ECH-Associating protein 1)-Nrf2 (nuclear factor erythroid 2-related factor 2)-ARE (antioxidant response element) signaling pathway, recognized as a crucial regulatory mechanism in oxidative stress, holds immense potential for the treatment of both diseases. In our laboratory, we initially screened a compound library and identified compound 3, which exhibited a dissociation constant of 5090 nM for Keap1. To enhance its binding affinity, we developed a novel 5-phenyl-1H-pyrrole-2-carboxylic acid Keap1-Nrf2 inhibitor through scaffold hopping from compound 3. Structure-activity relationship studies identified compound 19 as the most potent, with a KD2 of 42.2 nM against Keap1. Furthermore, compound 19 showed significant protection against LPS-induced injury in BEAS-2B cells and promoted Nrf2 nuclear translocation. Subsequently, we investigated its therapeutic effects in mouse models of ALI injury. Compound 19 effectively alleviated symptoms at doses of 15 mg/kg for ALI injury. Additionally, it facilitated Nrf2 translocation to the nucleus, increased Nrf2 levels, and upregulated the expression of HO-1 and NQO1 in affected tissues.

Effect of Agkistrodon acutus venom (AAVC-I) on apoptosis through modulation of the Keap1/Nrf2 pathway in HSC-3 oral squamous cell carcinoma cells.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral and maxillofacial regions. Patients with OSCC exhibit a poor response to conventional chemoradiotherapies, which are associated with severe side effects. Therefore, it is essential to identify an effective therapeutic method to treat patients with OSCC. An anti-tumor compound, Agkistrodon acutus venom component I (AAVC-I), purified from Agkistrodon acutus venom, has demonstrated anticancer activity both in vitro and in vivo. However, the mechanism of AAVC-I's anticancer activity in cancer cells has yet to be established. This study aimed to investigate the mechanism of AAVC-I-induced apoptosis in HSC-3 OSCC cells and explore its regulatory effect on oxidative stress. Survival rates of human OSCC cell HSC-3 were detected by Cell Counting Kit-8 (CCK-8). The reactive oxygen species (ROS) level was analyzed by flow cytometry and fluorescence microscopy. The mitochondrial membrane potential was analyzed by cytometry and fluorescent microplate reader. Apoptosis of HSC-3 cells was analyzed using flow cytometry. The oxidative stress level was evaluated using glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) kits. In addition, the target proteins were analyzed by western blot. AAVC-I reduced HSC-3 cells' survival rates in a dose-dependent manner with a 50% inhibiting concentration (IC50) of 8.86 µg/mL. It induced apoptosis of HSC-3 cells and the expression of cleaved caspase-3, cleaved caspase-9, and Cyt-c increased significantly, whereas the expression level of Bcl-2 decreased in AAVC-I-treated HSC-3 cells. Thus, AAVC-I caused apoptosis of HSC-3 via the activation of the intrinsic apoptotic pathway. In addition, AAVC-I reduced the mitochondrial membrane potential in HSC-3, enhanced intracellular ROS, and increased intracellular oxidative stress levels in comparison to that of untreated control cells. Furthermore, AAVC-I increased the expression of Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 (Keap1/Nrf2) levels. These findings demonstrate the inhibitory effects and associated mechanisms of AAVC-I on the HSC-3 OSCC cell line. This insight could be valuable for investigating AAVC-I as a potential therapeutic option for patients with OSCC.

Lutein protects senescent ciliary muscle against oxidative stress through the Keap1/Nrf2/ARE pathway

BackgroundAging-induced decline in ciliary muscle function is an important factor in visual accommodative deficits in elderly adults. With this study, we provide an innovative investigation of the interaction between ciliary muscle aging and oxidative stress. MethodsTricolor guinea pigs were used for the experiments in vivo and primary guinea pig ciliary smooth muscle cells were used for the experiments in vitro. ResultsWe enriched for genes associated with muscle-aging-lutein relationship using bioinformatics, including Nuclear factor-erythroid 2-related factor-2 (Nrf2), Glutathione Peroxidase (GPx) gene family, Superoxide Dismutase (SOD) gene family, NAD(P)H: Quinone Oxidoreductase 1 (NQO1) and Heme Oxygenase-1 (HO-1). After gavage to aged guinea pigs, lutein reduced Reactive Oxygen Species (ROS) and P21 levels in senescent ciliary muscle; lutein decreased refractive error and restored accommodation of the eye. In addition, lutein increased GPx, SOD, and Catalase (CAT) levels in serum; lutein increased GPx and CAT levels in ciliary bodies. Lutein regulated the expression of proteins such as Nrf2, Kelch-like ECH-associated protein 1 (Keap1), and downstream proteins in senescent ciliary bodies. Similarly, guinea pig ciliary muscle cell senescence was associated with oxidative stress. In vitro, 100 μM lutein reversed the damage caused by 800 μM H2O2; it reduced Senescence-Associated β-galactosidase (SA-β-Gal) and ROS activites, cell apoptosis and cell migration. Also, lutein increased the expression of smooth muscle contractile proteins. Lutein also increased the expression of Nrf2, GPx2, NQO1 and HO-1, decreased the expression of Keap1. A reduction in Nrf2 activity led to a reduction in the ability of lutein to activate antioxidant enzymes in the cells, thus reducing its inhibitory effect on cell senescence. Conclusionlutein improved resistance to oxidative stress in senescent ciliary muscle in vivo and in vitro by regulating the Keap1/Nrf2/Antioxidant Response Element pathway. We have innovatively demonstrated the molecular pharmacological mechanism by which lutein reverse age-related ciliary muscle systolic and diastolic deficits.

ShRNA-interfered of Nrf2 reveals a critical role for Keap1-Nrf2 signaling pathway during effects of Zearalenone induced oxidative stress in IPEC-J2 cells.

This study aims to verify the protective effect of the Kelch-like ECH-associated protein1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways by studying the effect of plasmids containing Nrf2-small hairpin RNA (shRNA) interference down-regulation of Nrf2 on zearalenone (ZEA) -induced intestinal porcine epithelial cells (IPEC-J2) oxidative stress. We constructed an IPEC-J2 model that interferes with Nrf2 expression, set blank (Control), negative control group (Sh-control), positive control group (Sh-Nrf2), and added 10, 20, and 40 μmol/L ZEA experimental group (Sh-Nrf2+ZEA10, Sh-Nrf2+ZEA20, and Sh-Nrf2+ZEA40). The study results showed that, compared with the Sh-Nrf2 group, ZEA significantly increased the apoptosis rate of IPEC-J2 in a time- and dose-dependent manner. Compared with the Sh-Nrf2 group, the activities of T-SOD and GSH-PX and relative expressions of Keap1 at mRNA and protein level in the Sh-Nrf2+ZEA20 and Sh-Nrf2+ZEA40 groups were significantly reduced, the MDA level, and the fluorescence intensity around and within the nucleus of ROS and Nrf2, and the relative expressions of Nrf2, Nqo1, and Ho1 at mRNA and protein level significantly increased. These results further prove that interfering with the expression of Nrf2 in IPEC-J2 cells affected the activation of the Keap1-Nrf2 signaling pathway and reduced the ability of cells to resist ZEA-induced oxidative stress. Therefore, the Keap1-Nrf2 signaling pathway had an important protective effect in ZEA-induced intestinal oxidative stress.

Research on the Anti-Fatigue Effects and Mechanisms of Arecoline in Sleep-Deprived Mice.

This research aims to estimate the anti-fatigue effects of arecoline and explore its underlying mechanisms using a murine model of central fatigue precipitated by sleep deprivation (SD). Seventy-two male C57BL/6 mice were randomly assigned to six groups: a control group, an SD-induced fatigue model group, a group that received Rhodiola Rosea capsules (2.5 mg/kg), and three arecoline groups, which were administered at low, medium, and high doses (10, 20, and 40 mg/kg, respectively). Following 28 days of continuous administrations, the effects of arecoline on mouse fatigue-related behaviors were assessed by behavioral tests, including grip strength, rotarod performance, and weight-bearing swimming endurance. The release levels of the related biochemical markers were measured by enzyme-linked immunosorbent assays (ELISAs). Western blotting was employed to quantify the expression levels of nuclear factor erythroid 2-related factor (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), heme oxygenase 1 (HO-1), sequestosome-1 (p62), and NADPH quinone oxidoreductase 1 (NQO1) in the gastrocnemius muscle. Arecoline administration notably enhanced grip strength, delayed the onset of fatigue as evidenced by extended latencies in rotarod tests, and increased the duration of weight-bearing swimming in mice. In the elevated plus maze, arecoline obviously decreased both the number of entries and the total distance traveled in the open arms. Arecoline markedly decreased the contents of creatine kinase, blood urea nitrogen, lactate dehydrogenase, triglycerides, and cholesterol in the serum, while it elevated the levels of total testosterone, lactate dehydrogenase, and immunoglobulin G. Furthermore, it significantly increased the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase in the gastrocnemius muscle, reduced malondialdehyde levels, augmented hippocampal SOD and CAT activity, and elevated glycogen stores in both liver and muscle tissues. Neurotransmitter levels showed significant increases, cytokine levels were markedly reduced, and the expressions of Nrf2, Keap1, NQO1, p62, and HO-1 in brain tissues were significantly upregulated. This study demonstrates that arecoline has anti-fatigue activity, and the specific mechanisms are associated with elevating glucose and lipid metabolism levels, relieving oxidative stress damage, inhibiting neuroinflammatory response, and regulating neurotransmitter levels and the Keap1/Nrf2/HO-1 signaling pathway. The research provides a new direction for arecoline's potential in preventing and improving fatigue.

Swimming training attenuates doxorubicin induced cardiomyopathy by targeting the mir-17-3p/KEAP1/NRF2 axis

Doxorubicin (DOX), as a first-line anticancer drug, is widely used in the treatment of various cancers. However, its clinical application is restricted due to its severe cardiac toxicity. Previous studies have indicated exercise training can alleviate the DOX-induced cardiotoxicity (DIC), but the underlying mechanism remains unclear. Our research has discovered, post-exercise, an elevated expression level of mir-17-3p, but in DIC its level decreases. Therefore, we further studied the effect of exercise mir-17-3p axis on DIC. In vivo, we simulated DIC mouse model, followed by an intervention using swimming and adenovirus to inhibit mir-17-3p. We found that inhibition of mir-17-3p can weaken the protection of exercise against DIC, presenting as weakened heart function. Besides, the levels of Malondialdehyde and Fe2+ in the cardiac tissue increased, along with diminished glutathione peroxidase 4 and Solute Carrier Family 7 Member 11 levels, and a decline in the concentration of glutathione, causing an increase in ferroptosis. Moreover, in vitro, we used dual-luciferase assay to confirm that Kelch Like ECH Associated Protein 1 (KEAP1) can be a target gene of mir-17-3p. We used Keap1/NFE2 Like BZIP Transcription Factor 2 (NRF2) inhibitor brusatol and Stimulator of Interferon Response CGAMP Interactor 1 (STING) agonist SR-717 to verify the mir-17-3p/KEAP1 axis can affect the Cyclic GMP-AMP Synthase (CGAS)/STING pathway, leading to further ferroptosis in DIC. This manifested as a reduction in ferroptosis. In summary, our research suggests swimming training enhances the levels of mir-17-3p, thereby activating the KEAP1/NRF2 pathway, and weakening the CGAS/STING pathway, improving ferroptosis in DIC.

Effects of oligomeric proanthocyanidins on growth performance, antioxidant capability, immunity, liver and intestinal health of Channa argus

To determine the effects of proanthocyanidins on the growth, antioxidant capability, immunity, intestinal morphology, intestinal microbiota, and liver health of Channa argus (10.12 ± 0.12 g), four experimental diets with various oligomeric proanthocyanidin (OPC) concentrations (0, 200, 400, and 800 mg/kg) were fed to triplicate groups for 8 weeks. The final body weight (FBW), weight gain (WG), feeding rate (FER), protein efficiency ratio (PER), and specific growth rate (SGR) were significantly improved in the 400 and 800 mg/kg OPC-supplemented diets (P < 0.05). The superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione (GSH), catalase (CAT), and glutathione peroxidase (GSH-Px) activities in the liver and intestines were increased significantly, whereas the malondialdehyde (MDA) content exhibited the opposite pattern (P < 0.05). The GST, Cu/Zn superoxide dismutase (Cu/Zn SOD), nuclear factor erythroid 2-related factor 2 (Nrf2), NAD(P)H quinone dehydrogenase 1 (NQO-1), and heme oxygenase 1 (HO-1) mRNA expression levels in the intestines and liver were significantly increased, whereas kelch-like ECH-associated protein 1 (Keap1) expression showed the opposite pattern (P < 0.05). Significant increases were noted in the serum lysozyme (LYS), immunoglobulin M (IgM), complement 3 (C3), and complement 4 (C4) levels, and significant decreases were noted in the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bile acid (TBA), and total cholesterol (TC) levels (P < 0.05). The interferon gamma (IFN-γ), interleukin-8 (IL-8), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) mRNA expression levels in the liver and intestines were significantly decreased, whereas the expression of interleukin-10 (IL-10) showed the opposite trend (P < 0.05). Compared with the control diet, the 400 and 800 mg/kg OPC-supplemented diets markedly improved liver and intestinal morphology. With increasing OPC concentration, the relative abundance of the beneficial bacteria, namely, Firmicutes increased, whereas the opposite trend was observed for the potential pathogenic bacteria, namely, Proteobacteria, Actinobacteria, Chlamydiae, Bacteroidetes, Cyanobacteria, and Acidobacteria. Therefore, dietary OPC supplementation facilitates growth performance and enhances antioxidant capability and immunity, as well as improves liver health, intestinal morphology, and microbiota composition of C. argus, of which the optimal level is 400 mg/kg.