mRNA Expression Levels Of Nrf2 Research Articles

Protective effects of thymol on deltamethrin-induced toxicity of Channa argus in association with the NF-κB/Nrf2/p53 pathway

This study was aimed to evaluate the ameliorative effects of thymol-supplemented diets on the toxicity damage of Channa argus induced by deltamethrin (DEL). After 28 d, the results are as follows: thymol can attenuate DEL-induced oxidative stress by reversing the decrease of the activities of antioxidant enzyme (SOD, CAT, GSH, GST and GSH-Px) and the increase of the MDA content in the tissues (liver and spleen), and also by reversing the decrease of the mRNA expression levels of Nrf2, Cu/Zn SOD and HO-1, and the increase of the Keap1 mRNA expression level in the tissues (liver and spleen). Thymol can attenuate DEL-induced immunotoxicity of fish by reverse the decrease of the levels of humoral immune parameters (C4, C3, IgM and LYS) in the serum, and by reversing the increase of the expression levels of NF-κB p65, TNF-α, IL-1β, IL-8 and IL-6, and the decrease of the IL-10 expression level in the tissues (liver and spleen). Thymol can attenuate DEL-caused apoptosis reversing the increase of the expression levels of pro-apoptosis (p53, Bax and Apaf-1) and the decrease of the expression level of anti-apoptosis Bcl-2 in the tissues (liver and spleen). Therefore, this study reveals that dietary supplementation of thymol alleviates DEL-caused immunotoxicity, oxidative stress, and apoptosis of C. argus in association with the NF-κB/Nrf2/p53 pathway.

Rhaponticum carthamoides improved energy metabolism and oxidative stress through the SIRT6/Nrf2 pathway to ameliorate myocardial injury

BackgroundRhaponticum carthamoides (Willd.) Iljin (Rha) is a member of the family Compositae that is widely used in folk medicine as a dietary supplement to treat cardiovascular diseases (CVDs), such as senile cardiac insufficiency, and to restore myocardial function after surgery. Sirtuin 6 (SIRT6), an NAD+-dependent class III histone deacetylase, plays a considerable role in the administration of CVDs. However, the specific effects and mechanism of Rha on myocardial injury remain unknown. PurposeThis study aimed to explore the therapeutic potential of Rha against myocardial injury as well as its underlying mechanisms in vivo and in vitro. MethodsA myocardial ischaemia model was established in male SD rats by subcutaneously injecting ISO. The rats were gavaged with Rha (40, 80, 160 mg/kg) or Rho (6 ml/kg) for 14 successive days and then injected subcutaneously with ISO or saline solution on the 13th and 14th days. The positive effects of Rha against myocardial injury in rats were evaluated by ECG assessment, BP measurements, H&E staining, and myocardial enzyme detection. Biochemical indicators of energy metabolism and oxidative stress, such as NAD+/NADH, ATP, and MDA, were analysed by assay kits to assess the effects of Rha. The protein and mRNA expression levels of SIRT6 and Nrf2 in the myocardium were determined by western blotting and real-time PCR. ResultsOur results showed that Rha ameliorated myocardial ischaemia and inhibited energy metabolism disorders (NAD+/NADH ratio, ATP, and LD) and oxidative stress (SOD, ROS, etc.) in rat myocardial tissue and H9c2 cells. In addition, Rha upregulated SIRT6 and Nrf2 expression in myocardial injury. Mechanistic studies then found that SIRT6 knockdown reduced the expression of Nrf2 as well as the effects of Rha on the levels of ATP, LD, and ROS, whereas activation of Nrf2 improved the effects of Rha in cells. In summary, Rha might exert its cardioprotective effects via the SIRT6-mediated Nrf2 signaling pathway. ConclusionThe results suggest that Rha regulates energy metabolism and oxidative stress through the SIRT6/Nrf2 signaling pathway to play a protective role in myocardial injury.

Intervening Effects and Molecular Mechanism of Quercitrin on PCV2-Induced Histone Acetylation, Oxidative Stress and Inflammatory Response in 3D4/2 Cells.

Porcine circovirus type 2 (PCV2) is the main pathogen causing porcine circovirus-associated diseases (PCVD/PCVADs), and infection of the host induces immunosuppression. Since quercitrin (QUE) has anti-inflammatory and antiviral activity, it is worth exploiting in animal diseases. In this study, the interventional effects and the molecular mechanism of QUE on PCV2-induced oxidative stress and inflammatory responses in 3D4/2 cells and the modulation of histone acetylation modifications were investigated. The ROS production was measured by DCFH-DA fluorescent probes. HAT and HDAC enzyme activity were determined by ELISA. Histone acetylation, oxidative stress and inflammation-related gene expression levels were measured by q-PCR. Histone H3 and H4 (AcH3 and AcH4) acetylation, oxidative stress and inflammation-related protein expression levels were measured by Western blot. The results showed that QUE treatment at different concentrations on PCV2-infected 3D4/2 cells was able to attenuate the production of ROS. Moreover, QUE treatment could also intervene in oxidative stress and decrease the enzyme activity of HAT and the mRNA expression level of HAT1, while it increased the enzyme activity of HDAC and HDAC1 mRNA expression levels and downregulated histone H3 and H4 (AcH3 and AcH4) acetylation modification levels. In addition, QUE treatment even downregulated the mRNA expression levels of IL-6, IL-8, IκB, AKT and p38, but upregulated the mRNA expression levels of IL-10, SOD, GPx1, p65, Keap1, Nrf2, HO-1 and NQO1. As to protein expression, QUE treatment downregulated the levels of iNOS, p-p65 and IL-8 as well as the phosphorylation expression of IκB and p38, while it upregulated the levels of HO-1 and NQO1. It was shown that QUE at 25, 50 or 100 μmol/L regulated p38MAPK and PI3K/AKT signaling pathways by downregulating cellular histone acetylation modification levels while inhibiting the NF-κB inflammatory signaling pathway and activating the Nrf2/HO-1 antioxidant signaling pathway, thus regulating the production of inflammatory and antioxidant factors and exerting both anti-inflammatory and antioxidant effects.

Sagittaria sagittifolia polysaccharides regulates Nrf2/HO-1 to relieve liver injury caused by multiple heavy metals in vivo and in vitro

This study explored whether Sagittaria sagittifolia polysaccharides(SSP) activates the nuclear factor erythroid-2-related factor2(Nrf2)/heme oxygenase-1(HO-1) signaling pathway to protect against liver damage jointly induced by multiple heavy metals. First, based on the proportion of dietary intake of six heavy metals in rice available in Beijing market, a heavy metal mixture was prepared for inducing mouse liver injury and HepG2 cell injury. Forty male Kunming mice were divided into five groups: control group, model group, glutathione positive control group, and low-and high-dose SSP groups, with eight mice in each group. After 30 days of intragastric administration, the liver injury in mice was observed by HE staining. In the in vitro experiment, MTT assay was conducted to detect the effects of SSP at 0.25, 0.5, 1, and 2 mg·mL~(-1) on HepG2 cell survival at different time points. The content of alanine transaminase(ALT) and aspartate aminotransferase(AST) in the 48-h cell culture fluid was measured using micro-plate cultivation method, followed by the detection of the change in reactive oxygen species(ROS) content by flow cytometry. The mRNA expression levels of Nrf2 and HO-1 in cells were determined by RT-PCR, and their protein expression by Western blot. HE staining results showed that compared with the model group, the SSP administration groups exhibited significantly alleviated inflammatory cell infiltration and fatty infiltration in the liver, with better outcomes observed in the high-dose SSP group. In the in vitro MTT assay, compared with the model group, SSP at four concentrations all significantly increased the cell survival rate, decreased the ALT, AST, and ROS content(P<0.05), and down-regulated Nrf2 and HO-1 mRNA and protein expression(P<0.05). SSP significantly improves inflammatory infiltration in the liver tissue of mice exposed to a variety of heavy metals and corrects the liver fat degeneration, which may be related to its regulation of the Nrf2/HO-1 signaling pathway, reduction of ROS, and alleviation of oxidative damage.

Pulsed-Ultrasound Irradiation Induces the Production of Itaconate and Attenuates Inflammatory Responses in Macrophages.

BackgroundItaconate is a key metabolite in the innate immune system and exerts strong anti-inflammatory effects in macrophages. For the production of itaconate in macrophages, immune-responsive gene 1 (IRG1) is an imperative enzyme, and activating the IRG1-itaconate pathway is reported to alleviate inflammatory diseases by upregulating nuclear factor-erythroid 2-related factor 2 (NRF2). However, there are very few reports on strategies to increase itaconate production. Ultrasound therapy is a widely used intervention for anti-inflammatory and soft-tissue regeneration purposes. Here we show the effect of ultrasound irradiation on the production of itaconate in macrophages.MethodsMurine bone marrow-derived macrophages (BMDMs) were exposed to pulsed ultrasound (3.0 W/cm2) for 5 minutes. Three hours after irradiation, the intracellular levels of metabolites and mRNA expression levels of Irg1 and Nrf2 were measured using CE/MS and qPCR, respectively. To evaluate macrophage inflammation status, 3 h after irradiation, the cells were stimulated with 100 ng/mL lipopolysaccharide (LPS) for 1.5 h and the mRNA expression levels of pro-inflammatory factors (Il-1β, Il-6, and Tnf-α) were measured. Student’s t-test, one-way ANOVA and Tukey’s multiple comparison test were used for statistical processing, and the significance level was set to less than 5%.ResultsUltrasound irradiation significantly increased the intracellular itaconate level and the expression levels of Irg1 and Nrf2 in BMDMs. Upregulation of Il-1β, Il-6, and Tnf-α by LPS was significantly suppressed in BMDMs treated with ultrasound. Ultrasound irradiation did not affect cell viability and apoptosis.ConclusionUltrasound irradiation induces the production of itaconate by upregulating Irg1 expression and attenuates inflammatory responses in macrophages via Nrf2. These results suggest that ultrasound is a potentially useful method to increase itaconate production in macrophages.

Increased mRNA Expression Levels of Nrf2 and TSPO Genes as a Markers of Brain Injury-induced by Aflatoxicosis and Ameliorated by Arginine Administration in Male Rabbits

L-protective arginine's function in adult male rabbits administered Aflatoxin B1 and l-arginine is the focus of this investigation. Four equal groups of 20 intact male rabbits were proposed: control group receiving distal water, (G1) receiving Aflatoxin B1 (oral dose of 4μg / animal.), (G2) receiving l-arginine (300 mg/animal orally) and (G3) receiving both Aflatoxin B1 and l-arginine. As compared to other groups, the Aflatoxin group (G1) had a large increase in reverse transcription (mRNA) of Nrf2 and Tspo genes (G2 and G3). In addition, we found that the arginine group had lower amounts of Nrf2 and TSPO mRNA than the Aflatoxin group (G1). According to these findings, Arginine protects the brain from Aflatoxin B1-induced oxidative damage.

The Protective Effect of Trichilia catigua A. Juss. on DEHP-Induced Reproductive System Damage in Male Mice.

The present study aimed to explore the protective effect and molecular mechanisms of Trichilia catigua A. Juss. extract (TCE) against di (2-ethylhexyl) phthalate (DEHP)-induced damage to the reproductive system of mice. Acute toxicity tests revealed that the maximum tolerated dose (MTD) in mice was up to 2.7 g kg−1. After induction with DEHP, TCE (L-TCE, M-TCE, H-TCE) was orally administered to mice for 28 days. Differences in indicators among groups showed that TCE significantly improved the anogenital distance and the organ indexes of the epididymides and testes. It also significantly reduced varicocele and interstitial cell lesions compared to the model group. H-TCE reduced the sperm abnormality rate, increased the levels of sex hormones, Na+K+ and Mg2+, Ca2+-ATPase enzyme activity, antioxidant enzyme vitality, coupled with a significant decrease in LH and MDA contents. The levels of testicular marker enzymes ACP and LDH were significantly augmented by both M-TCE and H-TCE. Further studies claimed that DEHP induction reduced the mRNA expression levels of Nrf2, SOD2, SOD3, CDC25C CDK1, CYP11A1, 3β-HSD, 5ɑ-R, AR, SF1, and CYP17A1, increased the level of Keap1, while TCE reversed the expression levels of these genes. Meanwhile, IHC results demonstrated a significant change in the expression activity of the relevant proteins compared to the control group. The results suggest that M-TCE and H-TCE enabled the recovery of DEHP-induced reproductive system damage in male mice by improving testicular histopathology, repairing testicular function, and reducing oxidative stress damage. The oxidation-related Keap1-Nrf2 pathway, SODs enzyme, the cell cycle control-related CDC25C-CDK1 pathway, and the steroidogenic-related pathway may contribute to this protective effects of TCE.

6-Paradol alleviates Diclofenac-induced acute kidney injury via autophagy enhancement-mediated by AMPK/AKT/mTOR and NLRP3 inflammasome pathways

Diclofenac (DIC)-induced acute kidney injury (AKI) causes high morbidity and mortality. With the absence of satisfactory treatment, we investigated the protective effects of 6-Paradol (PDL) against DIC-induced AKI, with focus on renal autophagy and NLRP3 inflammasome pathways . PDL has anti-inflammatory, antioxidant and AMPK-activation properties. PDL was administered to DIC-challenged rats. Nephrotoxicity, oxidative stress, inflammatory, and autophagy markers and histopathological examinations were evaluated. Compared to DIC, PDL restored serum nephrotoxicity, renal oxidative stress and pro-inflammatory markers. PDL almost restored renal architecture, upregulated renal Nrf2 pathway via enhancing Nrf2 mRNA expression and HO-1 levels. PDL suppressed renal NF-κB mRNA expression, and NLRP3 inflammasome pathway expression. Moreover, PDL enhanced renal autophagy through upregulating LC3B, AMPK and SIRT-1, and suppressed mTOR, p-AKT mRNA expressions and phosphorylated-p62 levels. Our study confirmed that autophagy suppression mediates DIC-induced AKI via AMPK/mTOR/AKT and NLRP3-inflammasome pathways. Also, PDL’s nephroprotective effects could provide a promising therapeutic approach against DIC-induced AKI.

Spenceria ramalana Trimen total polyphenols modulate the inflammatory response and intestinal flora in DSS-induced ulcerative colitis in C57BL/6 mice

Ethnopharmacological relevanceSpenceria ramalana Trimen is a perennial herb of the genus Spenceria in the family Rosaceae, the roots of which can be used as medicine. It is mainly distributed in Tibet autonomous region, Yunnan province, and Sichuan province of P. R. China at an altitude of 2950∼5000 m. Spenceria ramalana Trimen has been documented to have laxative, antidiarrheal, and balancing effects. Plant polyphenols with significant anti-inflammatory and antioxidant effects appear to have therapeutic potential for ulcerative colitis. However, the mechanism of SRT total polyphenols on ulcerative colitis (UC) are still unclear. Aim of the studyTo investigate the pharmacological effects and the potential mechanisms of Spenceria ramalana Trimen total polyphenols on dextran sodium sulfate (DSS)-induced UC in C57BL/6 mice. MethodsThe components of Spenceria ramalana Trimen total polyphenols were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and the therapeutic targets of the bioactive components and UC related targets were collected by searching the database to obtain their intersection targets. The “drug-component-target” network and protein interaction network were constructed for screening the key components and potential targets. DSS-induced UC model in C57BL/6 mice was constructed for the experimental verification of Spenceria ramalana Trimen total polyphenols through systematic evaluation, histopathological analysis, expression levels of inflammatory cytokines, and gut microbiota analysis. ResultsA total of 84 Spenceria ramalana Trimen polyphenolic components were detected, including 25 core components. 138 UC related targets were collected and 22 core targets were included. Enrichment analysis showed that the intersection targets were involved in various inflammation-related pathways and functions. Animal experiments revealed that Spenceria ramalana Trimen total polyphenols reduced the DAI score and histological index score, and significantly inhibited the shortening of the colon length. Spenceria ramalana Trimen total polyphenols significantly decreased the contents of TNF-α, IL-6, TGF-β, and the mRNA expression levels of TNF-α, IL-6, IL-1β, TGF-β were also significantly down-regulated in colon tissues. Oxidative stress-related indicators were not significantly regulated by Spenceria ramalana Trimen total polyphenols, and the mRNA expression levels of Nrf2 and HO-1 were inhibited compared with the model group. Gut microbiota analysis showed that Spenceria ramalana Trimen total polyphenols could alleviate the DSS-induced intestinal flora dysbiosis. ConclusionSpenceria ramalana Trimen total polyphenols could significantly alleviate DSS-induced UC, and the potential mechanism might be related to the regulation of the expression levels of TNF signaling pathway-related factors and the restoration of gut microbiota homeostasis.

JFNE-A isolated from Jing-Fang n-butanol extract attenuates lipopolysaccharide-induced acute lung injury by inhibiting oxidative stress and the NF-κB signaling pathway via promotion of autophagy

BackgroundJing-Fang powder consists of Jingjie (Nepeta tenuifolia Benth, (Lamiaceae)). and Fangfeng (Saposhnikovia divaricata (Turcz.) Schischk, (Apiaceae)) Previous studies have revealed that the Jing-Fang powder n-butanol extract (JFNE) has anti-acute lung injury (ALI) and anti-inflammatory properties; however, the active ingredient and mechanism remain unknown. PurposeIn the present study, we investigated the anti-inflammatory effect of a bioactive fraction obtained from JFNE(JFNE-A) on lipopolysaccharide (LPS)-induced ALI in mice and explored the underlying mechanism. Study designThe anti-acute lung injury effect and mechanism of JFNE-A was investigated by prophylactic administration of JFNE-A in mice with LPS-induced acute lung injury. MethodsThe expression levels of myeloperoxidase(MPO) in lung tissues of mice and interleukin(IL)-6, tumor necrosis factor(TNF)-α, IL-1β, IL-5, interferon (IFN)-γ, monocyte chemotactic protein (MCP)-1, macrophage colony stimulating factor (M-CSF), macrophage inflammatory protein (MIP)-1α, and MIP-1β in bronchi alveolar lavage fluid (BALF) were detected by reagent kit and the histological changes were examined by hematoxylin and eosin (H & E) for general histopathological conditions under a light microscope. In addition, the ultrastructure of the cells in lung tissues were observed and photographed under a transmission electron microscope. The expression levels of protein were detected via Western blotting and the mRNA expression of relative genes were determined of via reverse transcriptase polymerase chain reaction (RT-PCR). What's more, we also further clarified the potential targets of JFNE-A through network pharmacology analysis, which could be utilized in ALI treatment. ResultsOur results showed that pretreatment with JFNE-A for 7 days significantly reduced the lung pathological injury score, alleviated pulmonary edema, and decreased the lung tissue MPO level. Mechanistically, JFNE-A dramatically downregulated the protein levels of IL-6, TNF-α, IL-1β, M-CSF, and IFN-γ in BALF and mRNA expression levels of IL-6, TNF-α, IL-1β, and IFN-γ in lung tissues. JFNE-A also significantly lowered the protein levels of iNOS and phosphorylated NF-κB (p65) and mRNA expression levels of iNOS, Rela, CHUK, and NF-κB1, and also elevated the protein expression levels of Nrf2, HO-1, and SOD1 and the mRNA expression levels of Nrf2, Hmox1, and Keap-1 in the lungs. Moreover, JFNE-A significantly decreased the protein expression of p62 and increased the ratio of LC3II/LC3I. It also upregulated the mRNA expression levels of Atg5 and Beclin-1, whereas it reduced the mRNA expression level of SQSTM1 and increased autophagosome structures. ConclusionOverall, treatment with JFNE-A ameliorated LPS-induced ALI in mice by suppressing the NF-κB signaling pathways and promoting Nrf2 signaling pathways by accelerating autophagy.

Expression of LIAS and NRF2 in PBMCs from patients with silicosis and their correlation with silicosis

Objective: To investigate the expression of lipoic acid synthase gene (LIAS) and nuclear factor-erythroid 2-related factor 2 gene (NRF2) in peripheral blood mononuclear cells (PBMCs) from patients with silicosis and their correlation with silicosis. Methods: A total of 45 healthy controls and 107 patients with silicosis were randomly selected in this study in May 2019. PBMCs were isolated from peripheral blood and NRF2 protein expression was detected by immunofluorescence. The mRNA levels of LIAS and NRF2 in PBMCs were determined by real-time PCR. The dose-response relationship beween LIAS and NRF2 mRNA expression levels and their association with silicosis were analyzed by restricted cubic spline (RCS) and logistic regression. Results: Compared with the control group, the number of monocytes in the case group was significantly increased, and the forced expiratory volume in the first second (FEV(1.0)) decreased, the difference was statistically significant (P<0.05) . The positive expression rate of NRF2 in PBMCs of silicosis patients in stage Ⅰ group was significantly higher than that in the control group, and the positive expression rate of NRF2 in silicosis patients in stageⅡ and Ⅲ groups was lower than that in silicosis patients in control group and stage Ⅰ group (P<0.01) . Results of RCS showed that there was a linear dose-response relationship between LIAS and NRF2 mRNA expression (overall correlation test, χ(2)=213.710, P<0.01; non-linear test, χ(2)=1.340, P=0.511) . There was a positive correlation between mRNA expression of LIAS and that of NRF2 (r=0.651, P<0.01) . The results of multivariate analysis showed that LIAS and NRF2 were increased the risk of incidence in silicosis patients with stageⅠ (OR=11.184, 4.332, P<0.05) and NRF2 was the protective factor in silicosis patients with stage Ⅱ and Ⅲ (OR=0.225, 0.208, P<0.05) after adjusting for potential confounding factors including age, education level, BMI and smoking. Conclusion: There is a linear dose-response relationship between the expression of LIAS and NRF2 mRNA in PBMCs of silicosis patients, LIAS and NRF2 are involved in the pathogenesis of silicosis.

Chlorogenic acid supplementation alleviates dextran sulfate sodium (DSS)-induced colitis via inhibiting inflammatory responses and oxidative stress, improving gut barrier integrity and Nrf-2/HO-1 pathway

• CGA supplementation can enhance antioxidant capacity in DSS-induced colitis mice. • CGA supplementation can activate Nrf-2/HO-1 pathway in DSS-induced colitis mice. • CGA supplementation can improve gut barrier integrity in DSS-induced colitis mice. Chlorogenic acid (CGA) is an important source of phenolic acids with multiple health functions such as antioxidation, anti-inflammation, and free radical scavenging effects. This study aimed to investigate whether the nuclear factor erythroid 2-related factor 2 (Nrf-2)/HO-1 pathway played a role in CGA ameliorating DSS-induced colitis in mice. 500 mg/kg CGA (high chlorogenic acid, HCGA) and 250 mg/kg CGA (low chlorogenic acid, LCGA) supplementation could alleviate the symptoms of colitis and the related mechanisms. qPCR results indicated that HCGA treatment dramatically upregulated mRNA expression levels of anti-inflammatory cytokine, antioxidative enzymes, occluding, and Nrf-2/HO-1 pathway downstream targets. Western blot results proved that HCGA supplementation significantly upregulated HO-1 level. Besides, Spearman’s correlation analysis showed that the colon mRNA expression levels of antioxidative enzymes had a positive correlation with Nrf-2/HO-1 pathway downstream targets. Collectively, CGA supplementation could effectively ameliorate DSS-induced colitis by activating the Nrf-2/HO-1 pathway to suppress oxidative stress and inflammatory responses, and promoting gut barrier.

Honokiol alleviates LPS-induced acute lung injury by inhibiting NLRP3 inflammasome-mediated pyroptosis via Nrf2 activation in vitro and in vivo

BackgroundHonokiol (HKL) has been reported to ameliorate lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, its potential mechanism of its protective effects remains unclear. In this study, the protective mechanism of HKL on LPS-induced ALI was explored in vivo and in vitro.MethodsIn vivo, the SD rats were intratracheally instilled with LPS (5 mg/kg) to establish an acute lung injury model and then treated with HKL (1.25/2.5/5 mg/kg) or ML385 (30 mg/kg) intraperitoneally. In vitro, the human bronchial epithelial cell line (BEAS-2B) was stimulated with LPS and ATP to induce pyroptosis and treated with HKL (12.5/25/50 μM). Small interfering RNA (siRNA) technique was used to knockdown Nrf2 in BEAS-2B cells. The protein and mRNA expression levels of Nrf2, HO-1, NLRP3, ASC, CASP1, and GSDMD in cells and lung tissues were detected by western blot and real time-PCR. The expression levels of interleukin (IL)-1β, IL-18, MPO, MDA, and SOD in bronchoalveolar lavage fluid (BALF) and supernatant were determined by ELISA. The degree of pathological injury of lung tissue was evaluated by H&E staining.ResultsThe results showed that HKL could alleviate oxidative stress and inflammatory responses by regulating the levels of MPO, MDA, SOD, IL-1β, IL-18 in supernatant. And it could also inhibit the expression levels of NLRP3, ASC, CASP1, GSDMD via activation of Nrf2 in BEAS-2B cells. Further studies revealed that HKL could attenuate the pathological injury in LPS-induced ALI rats, and the molecular mechanism was consistent with the results in vitro.ConclusionsOur study demonstrated that HKL could alleviate LPS-induced ALI by reducing the oxidative stress and inhibiting NLRP3 inflammasome-mediated pyroptosis, which was partly dependent on the Nrf2 activation.Graphical

The Protective Effect of Sulforaphane against Oxidative Stress through Activation of NRF2/ARE Pathway in Human Granulosa Cells.

Objective Sulforaphane (SFN) is a natural free radical scavenger that can reduce oxidative stress (OS) through mediating nuclear factor (erythroid-derived 2)-like 2 (NF-E2-related factor 2 or NRF2)/antioxidant response element (ARE) signaling pathway and the downstream antioxidant enzymes. Here, we intended to study the role of SFN in OS- induced human granulosa cells (GCs) by investigating the intracellular levels of reactive oxygen species (ROS), cell death, and NRF2-ARE pathway. Materials and Methods This experimental study was conducted on GCs of 12 healthy women who had normal menstrual cycles with no history of polycystic ovary syndrome (PCOS), endometriosis, menstrual disorders, hyperprolactinemia, or hormonal therapy. After isolation of GCs, the MTT assay was performed to explore GCs viability after treatment with SFN in the presence or absence of H2O2. Flow cytometry was utilized to determine the intracellular ROS production and the apoptosis rate. Evaluation of the mRNA and protein expression levels of NRF2 and phase II enzymes including superoxide dismutase (SOD) and catalase (CAT) was performed by quantitative real-time polymerase chain reaction (PCR) and western blotting. Finally, the data were analyzed by SPSS software using One-way ANOVA and the suitable post-hoc test. Significance level was considered as P<0.05. Results Pretreatment of GCs with SFN attenuated intracellular ROS production and apoptosis rate in the H2O2-exposed cells. Moreover, SFN treatment increased the mRNA expression level of NRF2, SOD, and CAT. Higher expression of NRF2 and SOD was also observed at the protein level. ConclusionOur study demonstrated that SFN protects human GCs against H2O2induced-OS by reducing the intracellular ROS production and the following apoptosis through a mechanism by which NRF2 increases the antioxidant enzymes such as SOD and CAT. This result may have a potential application in assisted reproduction cycles by improving the quality of GCs and the embedded oocyte, especially in PCOS patients.

Protective Effect of Chitosan Oligosaccharide against Hydrogen Peroxide-Mediated Oxidative Damage and Cell Apoptosis via Activating Nrf2/ARE Signaling Pathway.

Chitosan oligosaccharide (COS), hydrolyzed and deacetylated from chitosan, has been reported to possess varieties of biological activities. Alzheimer's disease (AD) is a multifactorial progressive neurodegenerative disorder characterized by cognitive decline and memory loss, where oxidative stress was reported to be an overwhelming cause of the occurrence of AD. We have previously reported that COS could significantly decrease cell death, ROS generation, and lipid peroxidation, though the potential mechanism was yet to be determined. This study was designed to investigate the neuroprotective effect of COS against hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis in neuronal SH-SY5Y cells. Our results indicated that COS could dose-dependently scavenge H2O2 in the cell-free systems. Accordingly, COS markedly decreased H2O2-induced cell apoptosis and intracellular ROS generation, while increased antioxidant capacity in SH-SY5Y cells. Further, COS significantly reduced the expression of Bax and upregulated Bcl-2. The mRNA and protein expression levels of nuclear Nrf2, heme oxygenase 1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO1) were significantly increased upon COS treatment. Moreover, Nrf2-siRNA evidently reversed the promotive effect of COS on expression levels of HO-1 and NQO1, and ARE-driven transcriptional activity as determined by double-luciferase reporter gene assay. Besides, COS reversed H2O2-mediated increased phosphorylation of ERK1/2 and p38 MAPK. In conclusion, our findings indicate that COS could protect SH-SY5Y cells from oxidative damage and apoptosis via regulating Nrf2/ARE signaling pathway, which may provide new applications for the prevention and treatment of AD.

The Keap1/Nrf2-ARE signaling pathway is involved in atrazine induced dopaminergic neurons degeneration via microglia activation

ObjectiveTo investigate the mechanisms of ATR-induced dopaminergic toxicity by microglia activation and the response of the Keap1/ Nrf2- ARE signaling pathway. MethodsWistar rats were treated with 50, 100 and 200 mg/kg ATR and BV-2 microglia cells were treated with 50, 100 μM ATR or 100 ng/mL LPS, respectively. Rats behavioral responses and histopathological changes were monitored. Immunohistochemical and immunofluorescence analysis detected Iba-1 and TH+ cells in rats. Keap1/Nrf2-ARE signaling-related proteins and inflammatory factors from BV-2 cells and rats were detected using ELISA, Western blot and Real-time PCR. ResultsAfter ATR treatment, the grip strength of Wistar rats was significantly decreased, and anxiety were clearly observed. TH+ neurons were reduced, however, the number of microglia cells and Iba-1 levels were increased clearly in SN. The release of ROS, TNF-α and IL-Iβ were increased, and levels of SOD and GSH-Px were significantly decreased. Keap1 mRNA expression and protein levels were decreased, while nuclear Nrf2 mRNA expression and protein levels were both increased in vivo and in vitro. ConclusionATR could significantly activate microglia and exacerbate neurotoxicity and neuroinflammation, leading to accelerate dopaminergic neuron cell death by inhibiting Keap1/Nrf2-ARE signaling pathway.

Effect of peroxisome proliferator-activated receptor-γ coactivator-1α on liver injury induced by periodontitis in rats.

To investigate the effect of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) on liver injury induced by periodontitis in rats. Twenty-four male Wistar rats were randomly divided into two groups: control group and periodontitis group, twelve per group. In periodontitis group, the periodontitis models were established for the maxillary first molars in rats by means of "wire ligation+vaccinationwith Porphyromonas gingivalis", the control group was inoculated with the equal volume of 2% sodium carboxymethyl cellulose in the same position, for 6 weeks. The probing depth, tooth mobility and sulcus bleeding index were detected. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of liver tissues in rats. The quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were used to detect the gene and protein expression levels of PGC-1α, nuclear factor erythroid 2-related factor 2 (Nrf2) and mitochondrial transcription factor A (TFAM) in liver tissues of rats. The probing depth, tooth mobility and sulcus bleeding index in periodontitis group were significantly higher than that in control group. HE staining showed in periodontitis group, hepatic cords ranged disorderly and there were vacuoles in cells and inflammatory cells infiltrated in liver tissues of rats, and there was no obvious abnormality in control group. The qRT-PCR results showed that the mRNA expression levels of Pgc-1α, Nrf2 and Tfam in liver tissues of rats in periodontitis group were lower obviously than that in control group. IHC results showed that the protein expression level of PGC-1α in liver tissues of rats in periodontitis group was decreased significantly than that in control group. PGC-1α may be involved in the process of periodontitis-induced liver injury in rats.

Antifatigue effects of Hechong (Tylorrhynchus heterochaetus) through modulation of Nrf2/ARE- mediated antioxidant signaling pathway

This study was to evaluate the antifatigue effect of T. heterochaetus and explore the underlying mechanism of action. T. heterochaetus extract was treated to mice for 28 days. On the 28th day, after weight loaded swimming test. The levels of antioxidant enzymes and levels of pro- and anti-inflammatory cytokines in the liver and muscles of exercised mice were evaluated. mRNA and protein expression levels of Nrf2, SOD, HO-1, and Keap-1 were evaluated using RT-PCR and western blot analysis. The low (2.70 mg/0.5 ml/20 g) and medium (5.41 mg/0.5 ml/20 g) dose enhanced the activities of antioxidant enzymes like SOD, CAT and GPx in the liver and skeletal muscle thereby enhancing the antifatigue effect. The low and medium doses showed good anti-inflammatory effects by evaluating the levels of pro and anti-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-10 both in the liver and skeletal muscle. Furthermore, RT-PCR and western blot analysis showed increased expression of HO-1, SOD, Nrf2, and decreased expression of Keap-1 gene and proteins in liver and skeletal muscle of T. heterochaetus treated group mice. The current results indicate that T. heterochaetus exert the antifatigue effect through attenuating oxidative stress injury and inflammatory responses through the Nrf2/ARE-mediated signaling pathway.

Caffeine‐induced neurotoxicity mediated by Nrf2 pathway in PC12 cells and zebrafish larvae

Caffeine is one of the most widely used psychostimulants in the world and possesses central excitative, anti-depressive, and neuroprotective properties. However, excessive ingestion or abuse of caffeine can lead to intoxication. Many toxic effects are attributed to oxidative damage, and nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical intracellular regulator of the oxidative stress response. Here, we investigated the neurotoxicity of caffeine in rat pheochromocytoma PC12 cells and zebrafish larvae. It was found that caffeine inhibited the viability of PC12 cells in a dose- and time-dependent manner. Furthermore, it induced PC12 cell apoptosis and elevated reactive oxygen species (ROS) production. Quantitative polymerase chain reaction (qPCR) and western blotting revealed that caffeine also inhibited the expression levels of Nrf2 mRNA and protein and its target genes (e.g., NADPH quinone oxidoreductase 1 [NQO1]). Furthermore, Nrf2 silencing attenuated the toxic effects of caffeine. In addition, zebrafish larvae were treated with different doses of caffeine. Behavioral experiments showed that a low dose of caffeine (0.05 to 0.3 mM) increased the average distance of movement and promoted excitation. Survivorship curves showed that caffeine (0.2 to 1.5 mM) caused lethality. Finally, qPCR revealed that a higher dose of caffeine inhibited mRNA levels in the Nrf2 pathway. Based on these results, this study identified for the first time that overuse of caffeine can induce neurotoxicity by inhibiting the Nrf2 pathway. These results will provide a new perspective for studies on caffeine toxicity.

Ginkgo biloba extract (EGb 761) mitigates methotrexate-induced testicular insult in rats: Targeting oxidative stress, energy deficit and spermatogenesis

Methotrexate (MTX) is commonly used as a therapeutic agent in the treatment of malignancies and autoimmune disorders. Risk of subsequent infertility following MTX administration has been reported as a significant side effect due to testicular toxicity. The aim of the study was to evaluate the modulatory effects of Ginkgo biloba (standardized extract, EGb 761) on MTX-induced testicular oxidative stress, energy deficits and spermatogenic status in rats. All groups received intraperitoneal injection of MTX (0.5 mg/kg) twice weekly for four weeks except the control group that received the corresponding vehicles. Other groups received oral EGb761, at doses 25, 50 or 100 mg/kg/day, for four weeks, concurrently with MTX. Blood and semen sampling followed by testis dissection were performed 24 h after last EGb 761 treatment. Semen was examined for sperm progressive motility, percent of normal spermatozoa and sperm cell concentration as well as seminal plasma essential and non-essential amino acids. Serum LH, FSH and testosterone were detected as well as testicular MDA, GSH, GSSG, TNF-α, IL-1β, IL-6, NF-κB and the nuclear, cytoplasmic and mRNA expression levels of Nrf-2 besides the testicular cell energy; AMP, ADP and ATP. Histopathological studies of interstitial fibrosis and the severity of germ cell degeneration were also conducted. MTX induced significant decline in sperm quality along with decreased essential and non-essential amino acids in seminal plasma. MTX reduced serum FSH, LH and testosterone as well as testicular ATP, GSH and Nrf2, while increased levels of testicular ADP, AMP, MDA, GSSG and TNF-α. Results were confirmed by prominent interstitial fibrosis and severe germ cell degeneration in MTX group. Concurrent treatment with EGb 761 alleviated MTX-induced testicular insult evidenced by amelioration of oxidative stress biomarkers, energy functions, seminal sperms abnormalities and spermatogenesis status. The present study suggests a beneficial role of EGb 761 in MTX-induced testicular injury and subsequent distortion of spermatogenesis.