Protein Expression Levels Of Nrf2 Research Articles

The cold-soaking extract of Chinese yam (Dioscorea opposita Thunb.) protects against erectile dysfunction by ameliorating testicular function in hydrocortisone-induced KDS-Yang rats and in oxidatively damaged TM3 cells

Ethnopharmacological relevanceClinical applications and pharmacological research suggest that Dioscorea opposita Thunb. (Chinese yam), a well-known traditional Chinese medicine which has been used for more than 2000 years to nourish kidney-yang and protect the male reproductive system, might be efficacious for the treatment of erectile dysfunction (ED). Aim of the studyThis study aimed to investigate the active component extract of Chinese yam, determine its effectiveness in hydrocortisone-induced “kidney-yang deficiency syndrome” (KDS-Yang) rats and in oxidatively damaged TM3 cells and explore the underlying mechanism on restoring erectile function. Materials and methodsWe clarified the Chinese yam cold-soaking extract (CYCSE) as the main active extract of Chinese yam by a CCK8 assay and further identified its composition. The KDS-Yang rats were induced by intragastric administration of hydrocortisone. After 10 d of CYCSE intervention, cavernous and testis morphology were stained with hematoxylin and eosin. Inducible nitric oxide synthase (iNOS), cyclic guanosine monophosphate (cGMP), testosterone, 8-hydroxy-2-deoxyguanosine (8-OHdG) and superoxide dismutase (SOD) levels were detected by enzyme-linked immunosorbent assay kits. Leydig cells were performed using immunohistochemistry. Reactive oxygen species were measured using a DCFH-DA fluorescent probe, and testicular collagenous fibers were stained with a Masson kit. Detection of testicular apoptosis was performed by a TUNEL assay. Nrf2 and NQO1 mRNA expression levels were measured by qRT-PCR. The protein expression levels of Nrf2, HO-1, TGF-β1 and SMAD2/3 were analyzed by Western blot. ResultsWe demonstrated in KDS-Yang rats and oxidatively damaged TM3 cells that CYCSE successfully restored erectile function through ameliorating testicular function. Our data suggested that CYCSE can stimulate the NO/cGMP pathway and restore the cavernous morphology to protect against KDS-Yang-induced ED. It also protected testis morphology, increased Leydig cell proliferation and stimulated testosterone secretion. In the damaged testes, excessive increases in 8-OHdG and inhibition of SOD activity were ameliorated, and the Nrf2/HO-1 signaling pathway was enhanced after treatment with CYCSE, indicating that the antioxidant defense system was activated. These findings were also validated in vitro. Additionally, fibrosis of the testes and TM3 cells was reversed by CYCSE through the TGF-β1/SMAD2/3 pathway. ConclusionCYCSE has a therapeutic effect on KDS-Yang-induced ED, and the mechanism includes stimulation of testosterone secretion, resistance to oxidative stress and prevention of fibrosis. These findings provide a new scientific verification for the application of Chinese yam in the treatment of KDS-Yang-induced ED.

Protective effect of Lycium barbarum polysaccharide on ethanol-induced injury in human hepatocyte and its mechanism.

The purpose of this research is to study the effect of Lycium barbarum polysaccharide on ethanol-induced liver injury and its mechanism. The cell survival rate, the apoptosis rate, and the intracellular ROS level was detected by MTT assay, flow cytometry, laser confocal microscopy, and fluorescence spectrophotometry, respectively. The antioxidativeindices were determined by ELISA kits and the protein level was detected by western blot. The result showed Lycium barbarum polysaccharide could protect ethanol-induced cell injury by reducing cell apoptosis and regulating the levels of indicators related to oxidative stress, such as ROS, MDA, SOD, etc. In addition, LBP could increase the nuclear expression of Nrf2 protein and significantly up-regulate the expression levels of Nrf2 protein and its downstream proteins, such as HO-1, NQO1, and GCLC in the cell nucleus. Therefore, Lycium barbarum polysaccharide has a protective effect on ethanol-induced liver cell injury and it plays the role in cell apoptosis pathway and oxidative stress pathway. PRACTICAL APPLICATIONS: Lycium barbarum is a kind of food that can be used as food and medicine in China. The result showed that Lycium barbarum polysaccharide could protect ethanol-induced liver cell injury, which is beneficial to the application of LBP in functional food.

Treatment with catalpol protects against cisplatin-induced renal injury through Nrf2 and NF-κB signaling pathways.

Cisplatin (CP) is one of the most widely used chemotherapy drugs for cancer treatment, but it often leads to nephrotoxicity. It is well known that catalpol exhibits antioxidant and anti-inflammatory functions, thus the present study aimed to investigate the potential protective effects of catalpol on CP-induced kidney injury in rats, in addition to determining the underlying mechanisms. Sprague-Dawley rats were treated with 25, 50 or 100 mg/kg catalpol for two days, injected with 20 mg/kg cisplatin and catalpol on day 3 and sacrificed on day 4. The histological analysis of isolated kidney tissues was performed using hematoxylin and eosin staining, cleaved caspase-3 expression levels were analyzed using western blotting and the expression levels of inflammatory cytokines in the tissues, including tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, IL-10 and inducible nitric oxide synthase (iNOS) were evaluated using ELISAs. Furthermore, the mRNA and protein expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), kelch-like ECH-associated protein 1 (Keap1), NF-κB and inhibitory κB (IκB) were determined using reverse transcription-quantitative PCR and western blotting, respectively. The results revealed that the treatment with catalpol prevented the histopathological injury and renal dysfunction caused by CP. In addition, catalpol significantly suppressed the CP-induced apoptosis of tubular cells, inhibited the CP-induced upregulation of TNF-α, IL-1β, IL-6, IL-8 and iNOS and promoted the production of the anti-inflammatory cytokine IL-10. Additionally, the mRNA and protein expression levels of Nrf2, HO-1 and IκB in the kidney tissues were increased, whereas the expression levels of Keap1 and NF-κB were significantly decreased following the treatment with catalpol. In conclusion, these results suggested that catalpol may inhibit CP-induced renal injury and suppress the associated inflammatory response through activating the Nrf2 and inhibiting the NF-κB signaling pathways, respectively.

Ganoderma Lucidum Polysaccharides Ameliorates Hepatic Steatosis and Oxidative Stress in db/db Mice via Targeting Nuclear Factor E2 (Erythroid-Derived 2)-Related Factor-2/Heme Oxygenase-1 (HO-1) Pathway.

BackgroundType 2 diabetes mellitus (T2DM) and its comorbidities, including obesity, hypertension, and hyperlipidemia, are commonly associated with non-alcoholic fatty liver disease (NAFLD). Ganoderma lucidum polysaccharide (GDLP) is one of the central bioactive components in Ganoderma lucidum with anti-inflammatory, antioxidant, and hepatoprotective properties. However, the effect and mechanisms of GDLP in hepatic steatosis remain largely unknown. In the present study, we aimed to investigate the function of GDLP in hepatic steatosis and the underlying mechanism.Material/MethodsIn this study, male db/db mice were received with a high-fat diet (HFD) to investigate the effect of GDLP in T2DM-induced hepatic steatosis. The biological characteristics of the hepatic steatosis were evaluated through the detection of clinical indicators, including biochemical parameters, histopathology, and related cytokine levels. Additionally, the protein expression levels of Nrf2 (nuclear factor E2 (erythroid-derived 2)-related factor-2) signaling pathway were investigated by using western blotting and immunohistochemical staining.ResultsThe levels of food/water intake, body weight, fasting blood glucose, plasma lipids, urinary biomarkers, hepatic lipid accumulation, and tumor necrosis factor (TNF)-α were observably decreased in GDLP-treated db/db mice. Additionally, administration of GDLP increased the expression of various antioxidases, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), whereas it reduced the level of malonaldehyde (MDA). Furthermore, GDLP was significantly promoted protein expression level of Nrf2 and its downstream target gene HO-1 (heme oxygenase-1) while decreased TNF-α expression.ConclusionsThese results indicate that GDLP against T2DM-induced hepatic steatosis, oxidative stress, and inflammation by improving the Nrf2/HO-1 signaling pathway in db/db mice, suggesting the GDLP may serve as an effective strategy for in fatty liver treatment.

Upregulation of nuclear factor (erythroid-derived 2)-like 2 protein level in the human colorectal adenocarcinoma cell line DLD-1 by a heterocyclic organobismuth(III) compound: Effect of organobismuth(III) compound on NRF2 signaling

An increasing number of metal-based compounds, including arsenic trioxide, auranofin, and cisplatin, have been reported to have antitumor activity. Their beneficial effects are controlled by a transcription factor, nuclear factor (erythroid-derived 2)-like 2 (NRF2). In response to oxidative stress, NRF2 induces the expression of cytoprotective genes. NRF2 protein levels are regulated by Kelch-like ECH-associated protein 1 (KEAP1) via ubiquitination. Bi-chlorodibenzo[c,f][1,5]thiabismocine (compound 3), a bismuth compound, is known for its potent anti-proliferative activity against various cancer cell lines. In the present study, we investigated the effect of compound 3 on NRF2 signaling in the human colorectal adenocarcinoma cell line DLD-1 in terms of cell viability as well as mRNA and protein expression levels of NRF2. Compound 3 upregulated NRF2 protein levels in a time- and concentration-dependent manner, accompanied by a marked increase in heme-oxygenase-1 (HO-1) mRNA and protein levels. We observed that brusatol, an NRF2 inhibitor, as well as small interfering RNA (siRNA)-mediated knockdown of NRF2 in DLD-1 cells suppressed compound 3-induced HO-1 expression. The anticancer activity of compound 3 was enhanced by compounds that downregulate NRF2. These results suggest that compound 3 upregulates HO-1 via NRF2 activation and that the NRF2-HO-1 pathway is the cellular response to compound 3. We also discovered that compound 3 slightly downregulated KEAP1; thus, NRF2 activation may be associated with KEAP1 modification. Collectively, our results indicate that compound 3 simultaneously activates an anti-oxidative stress pathway, such as NRF2 and HO-1, and a pro-cell death signal in DLD-1 cells. Our findings may provide useful information for the development of a potent anticancer organobismuth(III) compound.

Cypermethrin induces cell injury in primary cortical neurons of C57BL/6 mice by inhibiting Nrf2/ARE signaling pathway

To study the role of Nrf2/ARE signaling pathway in cypermethrin-induced oxidative stress and apoptosis of cerebral cortex neurons in C57BL/6 mice. The cortical neurons of C57BL/6 mice were cultured and identified, and a cypermethrin-induced cell injury model was established by treating the cells with 0, 25, 50 and 100 μmol/L of cypermethrin for 48 h. CCK-8 assay was used to analyze the effects of cypermethrin on the cell viability, and the fluorescence probe DCFH-DA was used for detecting intracellular reactive oxygen species (ROS); flow cytometry was performed for determining the apoptosis rate of the cells. The mRNA and protein expression levels of Nrf2 and its downstream genes HO-1 and NQO1 were detected using qPCR and Western blotting. Exposure to cypermethrin at different doses inhibited the viability of the cultured cortical neurons. With the increase of cypermethrin dose, the viability of the neurons decreased progressively, the intracellular ROS and the cell apoptosis rate increased, and the neuronal injury worsened. At the dose of 50 and 100 μmol/L, cypermethrin significantly down-regulated the expressions of HO-1, NQO1 and Nrf2 at both the mRNA and protein levels in the cells (P < 0.01). Cypermethrin exposure shows a dose-dependent neurotoxicity by inhibiting Nrf2/ARE signaling pathway, down-regulating the expression of Nrf2 and its downstream genes HO-1, NQO1 mRNA and protein, and inducing oxidative damage and apoptosis in primary mouse cortical neurons, .

Imatinib inhibits oxidative stress response in spinal cord injury rats by activating Nrf2/HO-1 signaling pathway.

Effect of imatinib on rats with spinal cord injury (SCI) was investigated through the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. Forty-eight Sprague-Dawley rats were randomly divided into sham operation group (n=12), model group (n=12), imatinib group (n=12) and inhibitor group (n=12). The results of immunohistochemistry showed that in comparison with sham operation group, the other three groups had overtly increased positive expression level of Bax and evidently reduced positive expression level of Bcl-2 (P<0.05). Compared with those in model group and inhibitor group, the positive expression level of Bax was distinctly lower, while that of Bcl-2 was notably increased in imatinib group (P<0.05). According to western blot analysis, the protein expression levels of Nrf2 and HO-1 were obviously higher in the other three groups than those in sham operation group (P<0.05), and they were remarkably higher in imatinib group than those in model group and inhibitor group (P<0.05). The results of qPCR assay revealed that the Nrf2 and HO-1 mRNA expression levels were markedly elevated in the other three groups compared with those in sham operation group (P<0.05). Based on ELISA, the other three groups exhibited notably raised content of IL-6, TNF-α, ROS and SOD compared with sham operation group (P<0.05), and imatinib group displayed remarkably decreased content of IL-6, TNF-α and ROS and markedly elevated SOD content in comparison with model group and inhibitor group (P<0.05). The results of TUNEL assay demonstrated that the rate of apoptosis was significantly raised in the other three groups compared with that in sham operation group (P<0.05), and it declined obviously in imatinib group compared with that in model group and inhibitor group (P<0.05). Imatinib inhibits oxidative stress response in SCI rats by activating the Nrf2/HO-1 signaling pathway, thus repressing apoptosis and inflammation.

Inhibition of the Nrf2-TrxR Axis Sensitizes the Drug-Resistant Chronic Myelogenous Leukemia Cell Line K562/G01 to Imatinib Treatments.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is involved in tumor drug resistance, but its role in imatinib resistance of chronic myeloid leukemia (CML) remains elusive. We aimed to investigate the effects of Nrf2 on drug sensitivity, thioredoxin reductase (TrxR) expression, reactive oxygen species (ROS) production, and apoptosis induction in imatinib-resistant CML K562/G01 cells and explored their potential mechanisms. Stable K562/G01 cells with knockdown of Nrf2 were established by infection of siRNA-expressing lentivirus. The mRNA and protein expression levels of Nrf2 and TrxR were determined by real-time quantitative polymerase chain reaction and western blot, respectively. ROS generation and apoptosis were assayed by flow cytometry, while drug sensitivity was measured by the Cell Counting Kit-8 assay. Imatinib-resistant K562/G01 cells had higher levels of Nrf2 expression than the parental K562 cells at both mRNA and protein levels. Expression levels of Nrf2 and TrxR were positively correlated in K562/G01 cells. Knockdown of Nrf2 in K562/G01 cells enhanced the intracellular ROS level, suppressed cell proliferation, and increased apoptosis in response to imatinib treatments. Nrf2 expression contributes to the imatinib resistance of K562/G01 cells and is positively correlated with TrxR expression. Targeted inhibition of the Nrf2-TrxR axis represents a potential therapeutic approach for imatinib-resistant CML.

Hydrogen gas protects against delayed encephalopathy after acute carbon monoxide poisoning in a rat model

ABSTRACTObjective: The protective effects of 2%-4% hydrogen gas in delayed encephalopathy after acute carbon monoxide poisoning (DEACMP) have been previously reported. This study aimed to assess the neuroprotective effects of high concentration hydrogen (HCH) on DEACMP.Methods: A total of 36 male Sprague-Dawley rats were divided into 3 groups. In the DEACMP group, rats were exposed to CO to induce CO poisoning; in the HCH group, the animals were exposed to 67% H2 and 33% O2 at 3,000 mL/min for 90 min immediately after CO poisoning. Neurological function was evaluated at 1 and 9 days after poisoning. Then, the contents of malondialdehyde, 3-nitrotyrosine and 8-hydroxy-2-deoxyguanosine, as well as superoxide dismutase activity in the serum, cortex and hippocampus were detected by ELISA. Additionally, the mRNA and protein expression levels of Nrf2 and downstream genes were detected by RT-PCR and Western blotting, respectively.Results: Our results showed that CO poisoning significantly impaired neurological function which was improved over time, and HCH markedly attenuated neurological impairment following CO poisoning. In addition, CO poisoning resulted in increased levels of malondialdehyde, 3-nitrotyrosine and 8-hydroxy-2-deoxyguanosine and markedly reduced superoxide dismutase activity at 1 and 9 days, which were significantly inhibited by HCH at 9 days. Finally, CO poisoning increased the mRNA and protein levels of Nrf2 and downstream genes, and HCH further induced the anti-oxidative capability.Conclusion: These findings indicate the neuroprotective effects of HCH on DEACMP, which are related to the activation of Nrf2 signaling pathway.

Toxic effects of glyphosate on intestinal morphology, antioxidant capacity and barrier function in weaned piglets

At present, the public is paying more attention to the adverse effects of pesticides on human and animal health and the environment. Glyphosate is a broad-spectrum pesticide that is widely used in agricultural production. In this manuscript, the effects of diets containing glyphosate on intestinal morphology, intestinal immune factors, intestinal antioxidant capacity and the mRNA expression associated with the Nrf2 signaling pathway were investigated in weaned piglets. Twenty-eight healthy female hybrid weaned piglets (Duroc × Landrace × Yorkshire) were randomly selected with an average weight of 12.24 ± 0.61 kg. Weaned piglets were randomly assigned into 4 treatment groups and fed a basal diet supplemented with 0, 10, 20, and 40 mg/kg glyphosate for a 35-day feeding trial. We found that glyphosate had no effect on intestinal morphology. In the duodenum, glyphosate increased the activities of CAT and SOD (linear, P < 0.05) and increased the levels of MDA (linear and quadratic, P < 0.05). In the duodenum, glyphosate remarkably increased the relative mRNA expression levels of Nrf2 (linear and quadratic, P < 0.05) and NQO1 (linear and quadratic, P < 0.05) and reduced the relative mRNA expression levels of GPx1, HO-1 and GCLM (linear and quadratic, P < 0.05). In the jejunum, glyphosate remarkably increased the relative mRNA expression levels of Nrf2 (linear and quadratic, P < 0.05) and decreased the relative mRNA expression levels of GCLM (linear and quadratic, P < 0.05). Glyphosate increased the mRNA expression levels of IL-6 in the duodenum (linear and quadratic, P < 0.05) and the mRNA expression levels of IL-6 in the jejunum (linear, P < 0.05). Glyphosate increased the mRNA expression of NF-κB in the jejunum (linear, P = 0.05). Additionally, the results demonstrated that glyphosate linearly decreased the ZO-1 mRNA expression levels in the jejunum and the mRNA expression of claudin-1 in the duodenum (P < 0.05). In the duodenum, glyphosate increased the protein expression levels of Nrf2 (linear, P = 0.025). Overall, glyphosate exposure may result in oxidative stress in the intestines of piglets, which can be alleviated by enhancing the activities of antioxidant enzymes and self-detoxification.

Antidiabetic, Antihyperlipidemic, Antioxidant, Anti-inflammatory Activities of Ethanolic Seed Extract of Annona reticulata L. in Streptozotocin Induced Diabetic Rats.

Annona reticulata L. (Bullock's heart) is a pantropic tree commonly known as custard apple, which is used therapeutically for a variety of maladies. The present research was carried out to evaluate the possible protective effects of Annona reticulata L. (A. reticulata) ethanolic seed extract on an experimentally induced type 2 diabetes rat model. Male Albino Wistar rats were randomly divided into five groups with six animals in each group viz., control rats in group I, diabetic rats in group II, diabetic rats with 50 and 100 mg/kg/bw of ethanolic seed extract of A. reticulata in groups III and IV, respectively, and diabetic rats with metformin in group V. Treatment was given for 42 consecutive days through oral route by oro-gastric gavage. Administration of A. reticulata seed extract to diabetes rats significantly restored the alterations in the levels of body weight, food and water intake, fasting blood glucose (FBG), insulin levels, insulin sensitivity, HbA1c, HOMA-IR, islet area and insulin positive cells. Furthermore, A. reticulata significantly decreased the levels of triglycerides, cholesterol, LDL, and significantly increased the HDL in diabetic rats. A. reticulata effectively ameliorated the enzymatic (ALT, AST, ALP, GGT) and modification of histopathological changes in diabetic rats. The serum levels of the BUN, creatinine levels, uric acid, urine volume, and urinary protein were significantly declined with a significant elevation in CCr in diabetic rats treated with A. reticulata. MDA and NO levels were significantly reduced with an enhancement in SOD, CAT, and GPx antioxidant enzyme activities in the kidney, liver, and pancreas of diabetic rats treated with A. reticulata. Diabetic rats treated with A. reticulata have shown up-regulation in mRNA expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), NAD(P)H:quinone oxidoreductase 1 (NQO1), Heme oxygenase-1 (HO-1) and protein expression level of Nrf2 with diminution in Keap1 mRNA expression level in pancreas, kidney, and liver. From the outcome of the current results, it can be inferred that seed extract of A. reticulata exhibits a protective effect in diabetic rats through its anti-diabetic, anti-hyperlipidemic, antioxidant and anti-inflammatory effects and could be considered as a promising treatment therapy in the treatment of diabetes mellitus.

Effect of miR-101 on proliferation and oxidative stress-induced apoptosis of breast cancer cells via Nrf2 signaling pathway.

To explore the influence of micro ribonucleic acid (miR)-101 on breast cancer cell proliferation and apoptosis via nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway. All MCF-7 cells were divided into 3 groups, namely control group, miR-101 mimic group (the cells were treated with 50 nmol/L miR-101 mimic), and miR-101 inhibitor group (the cells were treated with 50 nmol/L miR-101 inhibitor). The impact of miR-101 expression level on MCF-7 cell proliferation was evaluated via cell counting kit-8 (CCK-8) and colony formation assays. After the MCF-7 cells in the three groups were treated with 100 nM H2O2 for 12 h, the change in the apoptosis rate was detected via flow cytometry. Moreover, the influence of miR-101 expression level on the Nrf2 signaling pathway was detected via reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. According to the CCK-8 assay results, compared with that in control group, the proliferation rate of cells notably declined at 48, 72, and 96 h in miR-101 mimic group, and the difference was statistically significant (p<0.01), while it was substantially raised in miR-101 inhibitor group, showing a statistically significant difference (p<0.01). Compared that in control group, the cell colony formation rate was remarkably lowered in miR-101 mimic group, and the difference was statistically significant (p<0.01), while it was substantially raised in miR-101 inhibitor group (p<0.01). According to the flow cytometry assay results, compared with that in control group, the apoptosis of MCF-7 cells was markedly enhanced in miR-101 mimic group, showing a statistically significant difference (p<0.01), while it was weakened in miR-101 inhibitor group, with a statistically significant difference (p<0.01). The influence of miR-101 on the expression level of Nrf2 was detected via RT-PCR, and it was found that the messenger RNA (mRNA) expression level of Nrf2 was notably lower in miR-101 mimic group than that in control group (p<0.01), while it was raised in miR-101 inhibitor group. Western blotting results showed that compared with control group, miR-101 mimic group had a substantially lowered protein expression level of Nrf2 in the cell nucleus, with a statistically significant difference (p<0.01), while it was notably raised in miR-101 inhibitor group and the difference was statistically significant (p<0.01), indicating that miR-101 can remarkably lower the nucleoprotein expression level of Nrf2. The results of this study imply that miR-101 can inhibit the expression of Nrf2 to suppress the proliferation of breast cancer cells and enhance their sensitivity to oxidative stress, which provides a theoretical basis for reversal of tumor resistance.

Protective effect of lactobacillus plantarum on alcoholic liver injury and regulating of keap-Nrf2-ARE signaling pathway in zebrafish larvae.

This research investigated the protective effect of lactobacillus plantarum against alcohol-induced liver injury and the regulatory mechanism of Keap-Nrf2-ARE signal pathway in zebrafish. Firstly, a zebrafish alcoholic liver injury model was established using1.0mM of ethanol concentration, then two forms of lactobacillus plantarum treatment were designed to perform repair, including a lactobacillus plantarum thallus suspension (LPS) and a lactobacillus plantarum thallus breaking solution (LPBS). After 24h of alcohol injury, lactobacillus plantarum concentrations of 0, 1.0×105, 1.0×106, 1.0×107 and 1.5×107 cfu/mL were added to protect zebrafish larvae. Then with the treatment of lactobacillus plantarum after 48h, activities of alanine transaminase (ALT), aspartate transaminase (AST), superoxide dismutase (SOD) and malondialdehyde (MDA) in zebrafish tissue homogenate were respectively determined. Keap-Nrf2-ARE signal pathway related gene expression conditions were also analyzed, including nuclear factor (erythroid-derived 2)-like 2(Nrf2), Kelch like ECH associated protein 1(Keap1), catalase(CAT), hemooxygenase1(HO1) and Glutathione S-Transferase Kappa 1(gstk1). Results showed that: in comparison with the control group, the LPBS with dosage of 1.0×107 cfu/mL remarkably improved the activities of SOD, CAT, HO1and gstk1 in zebrafish larvae liver (P<0.05), resulting in significant increase of the protein expression level of Nrf2 (225.78%) and suppression of Keap1 gene expression (73.67%)(P<0.01). As confirmed by the results, lactobacillus plantarum activated the Keap-Nrf2-ARE signal pathway from the level of transcription, the up-regulation of the expression quantity of Nrf2 protected the organism from oxidative stress and maximally reduced liver injury.

Protective effect of cycloartenyl ferulate on lipopolysaccharide induced endothelial cell apoptosis and its mechanism

Objective To investigate the effect of cycloartenyl ferulate (CF) on lipopolysaccharide (LPS)-induced apoptosis in human umbilical vein endothelial cells (HUVEC), and to explore its relative mechanism. Methods Human umbilical vein endothelial cells were cultured in vitro, and the experiment was divided into the normal group, CF group, LPS group, and LPS+CF groups at different concentrations. After the corresponding treatment of cells in each group, the cell viability and apoptosis of each group were tested by the cell counter Kit-8 (CCK-8) assay and TdT-mediated dUTP nick end labeling (TUNEL) assay. The protein expression of Bax, Bcl-2, caspase-3 and the effect of CF on the protein expression of Nrf2/HO-1 pathway were determined by Western blot. One-way ANOVA were performed in multigroup mean comparison, LSD-t test was used to compare the mean values of two samples, and P<0.05 was considered statistically significant. Results Compared with the LPS group, the survival rate of HUVECs cells in the CF group was significantly increased with different doses, and the survival rate increased significantly in the 320 μmol/L CF group [(69.85±1.2)% vs ( 100.2±1.824)%, P< 0.01]. TUNEL staining showed that compared with the LPS group, the number of apoptotic positive cells in the 320 μmol/L CF group was significantly reduced [(27.33 ± 3.40) vs (11.67 ± 2.04), P<0.01]. Compared with the LPS group, Bcl-2 protein expression level was significantly increased in the CF group at different doses (P<0.01), caspase-3 and Bax protein expression were significantly decreased (P<0.01). Nrf2 protein expression level increased significantly (P<0.01), and HO-1 protein level increased significantly (P<0.01). Conclusion CF can alleviate LPS-induced apoptosis of HUVEC, which may be related to the increase of bcl-2 expression, the decrease of caspase-3 and Bax protein expression and the activation of Nrf2/ HO-1 signaling pathway. Key words: Cycloartenyl ferulate; Lipopolysaccharide; Vascular endothelial cells; Apoptosis; Nrf2/HO-1 signaling pathway

Protective effect of polysaccharides of sea cucumber Acaudina leucoprocta on hydrogen peroxide-induced oxidative injury in RAW264.7 cells

The aim of this experiment was to investigate the protective effects of polysaccharides of sea cucumber Acaudina leucoprocta (ALP) against hydrogen peroxide (H2O2) induced oxidative injury in RAW264.7 cells. Analysis of monosaccharide composition and structure of one fraction from ALP (ALPN) were analyzed by High Performance Liquid Chromatography (HPLC) and Fourier Transform Infrared Spectoscopy (FT-IR). The results showed that ALPN contain sulfate groups, which is sulfated polysaccharides. The results from MTT assay indicated that ALPN could markedly increase viability of cells compared with RAW264.7 cells exposed to H2O2. Moreover, ALPN significantly increased the levels of catalase (CAT), glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD), decreased the production of malondialdehyde (MDA) and lactate dehydrogenase (LDH) in RAW264.7 cells. The data from RT-PCR showed that ALPN (300 μg/mL) could increase the gene expression levels of SOD1 and GPX1. ALPN could also observably increase the protein expression level of Nrf2 and decrease the protein expression level of Keap1 with western blot. Collectively, this study suggested that polysaccharides of sea cucumber Acaudina leucoprocta (ALP) could effectively protect RAW264.7 cells against H2O2-induced oxidative injury. This protection mechanism may be related to activation of the Nrf2/Keap1 signaling pathway.

Piceatannol inhibits oxidative stress through modification of Nrf2-signaling pathway in testes and attenuates spermatogenesis and steroidogenesis in rats exposed to cadmium during adulthood.

BackgroundCadmium (Cd) is considered a heavy metal and potential pollutant to the environment.PurposeThe purpose of this study was to evaluate the protective potential of piceatannol (PT; 10 mg/kg body weight/day) against cadmium (Cd; 5 mg/kg body weight/day)-induced testicular dysfunction in Wistar rats.Materials and methodsRats were randomly divided into four groups: control, PT, Cd, and Cd + PT.ResultsTreatment with Cd resulted in a significant decrease in body, testicular, and epididymal weights, sperm quantity and quality, steroidogenic marker–enzyme activities, mRNA- and protein-expression levels of SF1, StAR, and P450 side chain–cleaving enzyme, and serum male sex hormonal levels when compared to controls. Testicular malondialdehyde levels were significantly increased, with a significant reduction in enzymatic and nonenzymatic antioxidants in Cd-treated rats compared to control rats. Testicular histomorphometric results supported the biochemical and molecular alterations observed in the study. In addition, significant downregulation in mRNA- and protein-expression levels of cytosolic Nrf2, HO1, γGCS, GPx, and NQO1, as well as significant upregulation in mRNA- and protein-expression levels of Nrf2 and Keap1 in testicular tissue, were noticed in rats administered Cd. PT treatment inCd-treated rats caused marked alleviation in body and organ weights, sperm analysis, steroidogenesis, serum hormonal levels, histomorphometric changes, and oxidative and antioxidative status in testes when compared to Cd alone–treated rats. Further, treatment of rats with PTl showed a marked improvement in mRNA- and protein-expression levels of Nrf2 and its regulated genes and proteins.ConclusionThe present study provides compelling evidence that PT treatment results in significant protection against Cd-induced testicular dysfunctions, such as spermatogenesis, steroidogenesis, and oxidative stress in rats, possibly through modification of the Nrf2–Keap1 signalling pathway.

High glucose promotes prostate cancer cells apoptosis via Nrf2/ARE signaling pathway.

To explore the influences of high glucose on the proliferation and apoptosis of prostate cancer cells and analyze its possible mechanism of action. Human prostate cancer cell line LNCaP was divided into control group, mannitol group, and high glucose group. Then, the proliferation in each group was detected via methyl-thiazolyl-tetrazolium (MTT) assay. Hoechst staining assay was performed to determine the apoptosis level in each group. Western blotting was employed to measure the expression levels of apoptosis-related proteins and nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and γ-glutamylcysteine synthetase (γ-GCS) proteins. The cellular reactive oxygen species (ROS) level was measured through 2,7-dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Enzyme-linked immunosorbent assay (ELISA) was carried out to detect the content of lactate dehydrogenase (LDH) and inflammatory factors. High glucose significantly promoted the proliferation of prostate cancer cells LNCaP (p<0.01) and increased the apoptosis level of cells (p<0.01). In high glucose group, the expression level of Caspase-3 protein was overtly increased (p<0.01), while that of B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X protein (Bax) was significantly decreased (p<0.01). High glucose group had clearly increased the content of ROS (p<0.01), LDH (p<0.01), and interleukin-6 (IL-6) (p<0.01), but decreased the content of IL-10 (p<0.01). High glucose notably lowered the protein expression levels of Nrf2, HO-1, and γ-GCS in the cells (p<0.01). High glucose represses the activation of the Nrf2/anti-oxidation response element (ARE) signaling pathway in prostate cancer cells and increases the content of ROS, IL-6, and the expression of apoptotic proteins in the cells, thus promoting the apoptosis of prostate cancer cells.

Honokiol alleviates acetaminophen-induced hepatotoxicity via decreasing generation of acetaminophen-protein adducts in liver

AimAcetaminophen (APAP) overdose is the most frequent cause of drug-induced liver damage. Magnolia officinalis is a traditional hepatoprotective Chinese medicine and Honokiol (HO) is the major active constituent. The present study was to investigate the effect of HO on APAP-induced hepatotoxicity and related mechanisms. Main methodsFour groups of mice were subjected to treatment as vehicle, APAP, APAP + HO and APAP + HO + NRF2 inhibitor. The morphological and biochemical assessments were used to evaluate the hepatoprotective effects. The extent of APAP-protein adducts was determined through evaluate the hepatic content 3‑(cystein‑S‑yl)acetaminophen (APAP-Cys), the hydrolysis products of APAP-protein adducts. The activities of CYP2E1, CYP1A2 and CYP3A4 were evaluated by cocktail incubation, and the protein expression levels of NRF2, GCLC, GCLM, GS and GST were evaluated by western blot analysis. Key findingsMorphological and biochemical assessments clearly demonstrated that HO could alleviate APAP-induced liver damage. The hepatoprotective effect of HO was positively associated with the reduction of APAP-protein adducts. Further investigation suggested that HO induced inhibition of CYP 2E1 and CYP2A1 as well as upregulation of GSH co-contributed to the reduction of APAP-protein adducts. Furthermore, HO induced activations of NRF2 and its target enzymes, such as GCLC, GCLM and GST, gave rise to the upregulation of GSH. SignificanceOur results suggested that HO could alleviate APAP-induced liver damage through reducing the generation of APAP-protein adducts, which might be mediated by inhibiting the activity of CYP 2E1 and CYP2A1 as well as enhancing the generation of GSH via NRF2 pathway.

Effect of the Nrf2-ARE signaling pathway on biological characteristics and sensitivity to sunitinib in renal cell carcinoma.

The aim of the present study was to examine the effects of the nuclear factor erythroid-2 related factor 2-antioxidant-responsive element (Nrf2-ARE) signaling pathway on the biological characteristics and sensitivity to targeted therapy in human renal cell carcinoma (RCC) cells. RCC tissues and adjacent tissues were collected and assessed by immunohistochemistry to determine the expression of Nrf2, NAD(P)H dehydrogenase [quinone] 1 (NQO1) and heme oxygenase-1 (HO-1) to analyze the clinicopathological features of RCC. A series of in vitro experiments were conducted to analyze the biological characteristics of Nrf2-ARE signaling in RCC. The renal cancer cell line, 786-0 was used, and cells was divided into a mock group, negative control group and small hairpin (sh)RNA-Nrf2 group. A Cell Counting Kit-8 assay was performed alongside flow cytometry to detect cell viability, cell cycle stage and apoptosis following treatment with sunitinib. The results demonstrated that Nrf2, NQO1 and HO-1 were significantly upregulated in RCC tissues compared with adjacent tissues and were associated with tumor node metastasis stage, Fuhrman classification and lymph node metastasis. Following shRNA-Nrf2 transfection, the 786-0 cells demonstrated a significant decrease in viability, cell invasion and scratch healing rate, and the mRNA and protein expression levels of Nrf2, NQO1, HO-1 and glutathione transferase were significantly decreased, which enhanced the sensitivity to sunitinib, arrested cells in the G0/G1 phase and increased apoptosis. In conclusion, Nrf2-ARE signaling is important for RCC progression, and its inhibition may increase sensitivity to targeted drugs to provide novel developments for RCC treatment.

The Nrf2/HO-1 axis can be a prognostic factor in clear cell renal cell carcinoma.

ObjectiveTo study the protein expression level of Nrf2/HO-1 in clear cell renal cell carcinoma (ccRCC) and adjacent normal tissue and to explore its relationship with clinicopathological characteristics and prognosis in ccRCC patients.Materials and methodsIn total, 152 ccRCC patients with available follow-up and clinical data were enrolled, and sample microarrays were prepared for immunohistochemistry studies. The human ccRCC cell lines 786-O, OS-RC-2, A498, and ACHN were cultured for immunofluorescence. The protein concentrations of five ccRCC patients’ tumor and adjacent normal renal tissues were prepared for Western blotting. Chi-squared tests, Fisher’s exact test, Kaplan–Meier analyses, log-rank tests, and Cox regression were performed for statistical analyses.ResultsThe immunoreactivity results showed that the Nrf2 and HO-1 proteins were found in consistent locations in vitro and were expressed both in ccRCC and adjacent normal tissues. The two proteins were localized in the cytoplasm and nucleus of RCC tumor cells and in adjacent normal tissue cells. The expression levels of Nrf2 and HO-1 were significantly higher in ccRCC tissues than in the adjacent normal tissues. The Nrf2 protein level was found to be significantly correlated with the tumor size. Additionally, higher protein expression levels of Nrf2 and HO-1 were also correlated with worse overall survival outcomes and could potentially be used to predict the prognosis of ccRCC patients.ConclusionOur study provides an important theoretical basis for evaluating the clinical prognosis of ccRCC patients, which implies that the Nrf2/HO-1 axis can be a prognostic factor in ccRCC.