Gentiopicroside alleviates acute myocardial infarction injury in rats by disrupting Nrf2/NLRP3 signaling.

The objective of the present investigation was to assess the protective impact of gentiopicroside (GPS) on acute myocardial infarction (AMI) through the modulation of NF-E2-related factor 2 (Nrf2)/nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3) signaling. H9c2 cells were subjected to varying concentrations of GPS, and subsequently, the cells and Sprague-Dawley (SD) rats were segregated into control, model, GPS, t-BHQ (an Nrf2 activator), and GPS + ML385 (an Nrf2 inhibitor) groups. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were analyzed. Reactive oxygen species (ROS) and cell apoptosis were assessed, while Nrf2 and the expression of the NLRP3 inflammatory body signal pathway were evaluated using western blot and immunofluorescence techniques. The infarct area and pathological changes were also examined. Treatment with varying doses of GPS resulted in increased viability of H9c2 cells. Notably, the model group exhibited significantly elevated levels of cell apoptosis, MDA, and ROS compared to the control group, while SOD and Nrf2 levels were significantly reduced. Furthermore, the expression of NLRP3, cleaved caspase-1, interleukin (IL)-1ß, and IL-18 were found to be augmented. Following the implementation of GPS in cells and animals, there was a notable reduction in MDA and ROS levels, a decrease in the rate of cellular apoptosis, and a mitigation of inflammation scores. In addition, there was an increase in the expression of SOD and Nrf2. However, the protective effects of GPS were negated when co-administered with ML385. GPS exhibits therapeutic properties in AMI rats by activating Nrf2 expression, thereby reducing the NLRP3 inflammatory body and alleviating the inflammatory response and oxidative stress of myocardial cells. GPS may hold promise as a potential drug for the treatment of AMI.

The Gut Microbial Metabolite Pyrogallol Is a More Potent Inducer of Nrf2-Associated Gene Expression Than Its Parent Compound Green Tea (-)-Epigallocatechin Gallate.

(-)-Epigallocatechin gallate (EGCG) has been associated with multiple beneficial effects. However, EGCG is known to be degraded by the gut microbiota. The present study investigated the hypothesis that microbial metabolism would create major catechol-moiety-containing microbial metabolites with different ability from EGCG to induce nuclear factor-erythroid 2-related factor 2 (Nrf2)-mediated gene expression. A reporter gene bioassay, label-free quantitative proteomics and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) were combined to investigate the regulation of Nrf2-related gene expression after exposure of U2OS reporter gene or Hepa1c1c7 cells in vitro to EGCG or to its major microbial catechol-moiety-containing metabolites: (-)-epigallocatechin (EGC), gallic acid (GA) and pyrogallol (PG). Results show that PG was a more potent inducer of Nrf2-mediated gene expression than EGCG, with a 5% benchmark dose (BMD5) of 0.35 µM as compared to 2.45 µM for EGCG in the reporter gene assay. EGC and GA were unable to induce Nrf2-mediated gene expression up to the highest concentration tested (75 µM). Bioinformatical analysis of the proteomics data indicated that Nrf2 induction by PG relates to glutathione metabolism, drug and/or xenobiotics metabolism and the pentose phosphate pathway. Taken together, our findings demonstrate that the microbial metabolite PG is a more potent inducer of Nrf2-associated gene expression than its parent compound EGCG.

Involvement of the Nrf2-Keap1 signaling pathway in protection against thallium-induced oxidative stress and mitochondrial dysfunction in primary hippocampal neurons.

Thallium ion (Tl+) and its neurotoxic products are widely known to cause severe neurological complications. However, the exact mechanism of action remains unknown, with limited therapeutic options available. This study aims to examine the toxic effects of Thallium (I) Nitrate (TlNO3) on primary hippocampal neurons of E17-E18 Wistar rat embryos, and the potential neuroprotective role of Nrf2- Keap1 signaling pathway against thallium-induced oxidative stress and mitochondrial dysfunction. TlNO3 induces a significant increase in reactive oxygen species levels and mitochondrial dysfunction in primary hippocampal neurons. Furthermore, the Nrf2-Keap1 signaling pathway played a protective role against TlNO3-induced hippocampal neuronal cytotoxicity. Moreover, mitochondrial fusion protein Mitofusin 2 (Mfn2) levels significantly decreased in hippocampal neurons when exposed to TlNO3, indicating that Mfn2 protein levels are linked to TlNO3-induced neurotoxicity. t-BHQ, a Nrf2 and phase II detoxification enzyme inducer, counteracted the oxidative damage in hippocampal neurons by activating the Nrf2-Keap1 signaling pathway after TlNO3 exposure; the activated Nrf2-Keap1 pathway could then maintain Mfn2 function by regulating Mfn2 protein expression. Thus, Nrf2-Keap1 pathway activation plays a protective role in Tl+-induced brain damage, and specific agonists have been identified to have great potential for treating thallium poisoning.

Evaluation of Nrf2 with Exposure to Nanoparticles.

Transcription factor Nrf2, nuclear factor (erythroid-derived 2)-like 2, is considered a master regulator of redox homeostasis and plays a central role in antioxidant and anti-inflammatory defence. It has been largely reported that oxidative stress is implicated in nanoparticle-induced toxicity with the involvement of Nrf2. Several basic methods for Nrf2 evaluation with exposure to nanoparticles are described in this chapter including real-time reverse transcription-polymerase chain reaction (RT-PCR), western blotting, immunofluorescence staining, electrophoretic mobility shift assay, DNase I footprinting, dimethylsulfate footprinting, protein pulse-chase analysis, and tert-butylhydroquinone treatment.

t-BHQ Protects Against Oxidative Damage and Maintains the Antioxidant Response in Malnourished Rats.

OBJECTIVE: Tert-butylhydroquinone (t-BHQ) protective effect against oxidative damage in thymus from malnourished pops-rats was evaluated. METHODS: Malnutrition in pops-rats was induced during the lactation period and first-, second-, and third-degree malnourished rats were studied (MN1, MN2, and MN3). To determine t-BHQ protective effect, lipid peroxidation (LPx) was assessed, as well as the carbonyl content. The reduced glutathione and glutathione disulfide content were determined and antioxidant enzyme activities were measured. RESULTS: Oxidative protein damage, LPx, and Nuclear Factor-κB (NF-κB) content, increased in the MN2 and MN3 compared to well-nourished rats, associated with lower protein content and antioxidant activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase. Tert-butylhydroquinone treatment induced a protective effect against lipids and proteins oxidative damage, as well as decrease in NF-κB in MN rats and restored the antioxidant mechanisms, mostly GPx and SOD. No differences were found between male and female animals. CONCLUSIONS: Results show that higher body weight deficit leads to increased oxidative damage and probably inflammation, attributable to alterations in antioxidant mechanisms. These effects were reversed by the t-BHQ-treatment, which restores the antioxidant response. Our findings suggest that t-BHQ could be an interesting pharmacological intervention, but it needs to be studied further.

Effect of tert-butylhydroquinone on DJ-1∕Nrf2 pathway during renal ischemia-reperfusion in diabet-ic rats / 中华麻醉学杂志

Objective To evaluate the effect of tert-butylhydroquinone ( t-BHQ) on DJ-1∕nuclear factor erythroid 2-related factor 2 (Nrf2) pathway during renal ischemia-reperfusion (I∕R) in diabetic rats. Methods Forty SPF healthy adult male Sprague-Dawley rats, weighing 200-220 g, were divided into 4 groups (n＝10 each) using a random number table method: control group (group C), diabetes mellitus group (group D), diabetes mellitus plus renal I∕R group (I∕R group) and t-BHQ group (group T). Diabe-tes mellitus was induced by intraperitoneal streptozotocin 60 mg∕kg and confirmed by fasting blood glucose level＞16. 7 mmol∕L 72 h later. t-BHQ 50 mg∕kg was intraperitoneally injected in 3 times at an interval of 8 h starting from 24 h before surgery in group T, while the equal volume of normal saline was given instead in D and I∕R groups. Blood samples were collected from the apex of the heart at 24 h of reperfusion for deter-mination of serum creatinine (Cr), cystatin C (Cys C) and β2-microglobulin (β2-MG) concentrations. The rats were then sacrificed, and kidneys were removed for determination of pathological changes of kidneys (with a light microscope) and for detection of the expression of DJ-1, Nrf2 and heme oxygenase-1 (HO-1) in renal tissues (by Western blot). Results Compared with group C, the concentrations of serum Cr, Cys C and β2-MG and pathological scores were significantly increased, and the expression of DJ-1, Nrf2 and HO-1 was up-regulated in D, I∕R and T groups ( P＜0. 05). Compared with group D and group I∕R, the concentrations of serum Cr, Cys C and β2-MG and pathological scores were significantly decreased, and the expression of DJ-1, Nrf2 and HO-1 was up-regulated in group T ( P＜0. 05). Conclusion t-BHQ can at- tenuate renal I∕R injury by activating DJ-1∕Nrf2 pathway in diabetic rats.

Effects of Nrf2-autophagy pathway in rats of the incisional pain-remifentanil-induced hyperalgesia / 天津医药

Objective To investigate the effect of nuclear factor erythroid 2-related factor 2(Nrf2)-autophagy pathway on the incisional pain-remifentanil-induced hyperalgesia of rat model. Methods Twenty-four male Sprague-Dawley rats were randomly allocated into three groups:saline+incisional pain group(group I),remifentanil+incisional pain group(group RI)and Nrf2 agonist t-BHQ group(group tBHQ),with 8 rats in each group.In group I and RI,normal saline at 0.1 mL/(kg· min) and remifentanilat 1 μg/(kg·min) were respectively infused into caudal vein for 60 min. Rats in group t-BHQ were injected intraperitoneally with Nrf2 agonist t-BHQ(15 mg/kg),the first time at 0.5 h before remifentanil infusion,per 12 h once,4 times in a row,the rest management was the same as group RI.Brennan incision pain model rats were constructed along with the infusions in the three groups. The thermal paw withdraw latency (PWL) and mechanical paw withdraw threshold(PWT)were measured at 24 h before the infusion(T0)and at 2 h(T1),6 h(T2),24 h(T3),48 h(T4)after the infusion. Rats were sacrificed after the tests, then the L4-6segments of signal cord were removed and the expression levels of autophagy-related proteins,microtubule associated protein 1 light chain 3Ⅱ(LC3Ⅱ),Beclin 1,Nrf2 and Nrf2 downstream molecular hemeoxygenase-1 (HO-1) were detected by Western blot assay. Results The PWT and PWL values were decreased significantly with the prolongation of the processing time in the three groups. Compared with group I, PWL and PWT values were decreased at T1-4,the expression levels of LC3Ⅱand Beclin-1 were increased while Nrf2 and HO-1 were decreased at T4in group RI (P<0.05). While compared with group RI, PWL and PWT values were increased, and the expressions of Nrf2 and HO-1 were increased, LC3Ⅱ and Beclin-1 protein were upregulated in group tBHQ (P<0.05).Conclusion The activation of Nrf2-autophagy pathway can improve the incisional pain-remifentanil induced hyperalgesia.

Concerted action of Nrf2-ARE pathway, MRN complex, HMGB1 and inflammatory cytokines - implication in modification of radiation damage.

Whole body exposure to low linear energy transfer (LET) ionizing radiations (IRs) damages vital intracellular bio-molecules leading to multiple cellular and tissue injuries as well as pathophysiologies such as inflammation, immunosuppression etc. Nearly 70% of damage is caused indirectly by radiolysis of intracellular water leading to formation of reactive oxygen species (ROS) and free radicals and producing a state of oxidative stress. The damage is also caused by direct ionization of biomolecules. The type of radiation injuries is dependent on the absorbed radiation dose. Sub-lethal IR dose produces more of DNA base damages, whereas higher doses produce more DNA single strand break (SSBs), and double strand breaks (DSBs). The Nrf2-ARE pathway is an important oxidative stress regulating pathway. The DNA DSBs repair regulated by MRN complex, immunomodulation and inflammation regulated by HMGB1 and various types of cytokines are some of the key pathways which interact with each other in a complex manner and modify the radiation response. Because the majority of radiation damage is via oxidative stress, it is essential to gain in depth understanding of the mechanisms of Nrf2-ARE pathway and understand its interactions with MRN complex, HMGB1 and cytokines to increase our understanding on the radiation responses. Such information is of tremendous help in development of medical radiation countermeasures, radioprotective drugs and therapeutics. Till date no approved and safe countermeasure is available for human use. This study reviews the Nrf2-ARE pathway and its crosstalk with MRN-complex, HMGB1 and cytokines (TNF-a, IL-6, IFN-? etc.). An attempt is also made to review the modification of some of these pathways in presence of selected antioxidant radioprotective compounds or herbal extracts.

Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes.

Isorhamentin is a 3'-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes.