The role of Nrf2 signaling in the regulation of antioxidants and detoxifying enzymes after traumatic brain injury in rats and mice

Aim:

To determine whether Nrf2 signaling pathway activation could attenuate oxidative stress and neuronal damage following traumatic brain injury (TBI).

Methods:

Controlled cortical impact (CCI) injury was performed in Sprague-Dawley rats and Nrf2-knockout or control mice. Sulforaphane (SFN), a potent Nrf2 activator, was used to activate Nrf2. Oxidative stress, lesion volume, neuron degeneration, and neurologic dysfunction were determined using biochemical, histopathological and neuroethologic approaches. Protein and mRNA levels of Nrf2 and the antioxidant enzymes heme oxygenase 1 (HO-1) and NAD(P)H:quinine oxidoreductase 1 (NQO1) were assessed using Western blot analysis and RT-PCR.

Results:

Activation of Nrf2 by SFN( 5 mg/kg, ip) induced the nuclear translocation and activation of Nrf2, which resulted in an up-regulation of Nrf2-dependent antioxidant enzymes and a reduction of oxidative damage after TBI. In accordance with these biochemical changes, SFN also significantly reduced neuronal death, contusion volume, and neurological dysfunction after TBI. Furthermore, Nrf2-knockout mice showed more severe oxidative stress and neurologic deficits after TBI and did not benefit from the effects of SFN.

Conclusion:

Nrf2 plays a pivotal role in cell defenses against the oxidative stress of TBI. In addition, pharmacological activation of the Nrf2 signaling pathway by small molecule inducers such as SFN attenuated oxidative stress and neuronal damage following TBI.     

Nrf2 pathway activation contributes to anti-fibrosis effects of ginsenoside Rg1 in a rat model of alcohol- and CCl4-induced hepatic fibrosis

Aim:

To investigate the anti-fibrosis effects of ginsenoside Rg1 on alcohol- and CCl₄-induced hepatic fibrosis in rats and to explore the mechanisms of the effects.

Methods:

Rats were given 6% alcohol in water and injected with CCl₄ (2 mL/kg, sc) twice a week for 8 weeks. Rg1 (10, 20 and 40 mg/kg per day, po) was administered in the last 2 weeks. Hepatic fibrosis was determined by measuring serum biochemical parameters, HE staining, Masson''s trichromic staining, and hydroxyproline and α-SMA immunohistochemical staining of liver tissues. The activities of antioxidant enzymes, lipid peroxidation, and Nrf2 signaling pathway-related proteins (Nrf2, Ho-1 and Nqo1) in liver tissues were analyzed. Cultured hepatic stellate cells (HSCs) of rats were prepared for in vitro studies.

Results:

In the alcohol- and CCl₄-treated rats, Rg1 administration dose-dependently suppressed the marked increases of serum ALT, AST, LDH and ALP levels, inhibited liver inflammation and HSC activation and reduced liver fibrosis scores. Rg1 significantly increased the activities of antioxidant enzymes (SOD, GSH-Px and CAT) and reduced MDA levels in liver tissues. Furthermore, Rg1 significantly increased the expression and nuclear translocation of Nrf2 that regulated the expression of many antioxidant enzymes. Treatment of the cultured HSCs with Rg1 (1 μmol/L) induced Nrf2 translocation, and suppressed CCl₄-induced cell proliferation, reversed CCl_4- induced changes in MDA, GPX, PCIII and HA contents in the supernatant fluid and α-SMA expression in the cells. Knockdown of Nrf2 gene diminished these actions of Rg1 in CCl₄-treated HSCs in vitro.

Conclusion:

Rg1 exerts protective effects in a rat model of alcohol- and CCl₄-induced hepatic fibrosis via promoting the nuclear translocation of Nrf2 and expression of antioxidant enzymes.     

Melatonin Attenuates Memory Impairment Induced by Klotho Gene Deficiency Via Interactive Signaling Between MT2 Receptor,ERK, and Nrf2-Related Antioxidant Potential

Background:

We demonstrated that oxidative stress plays a crucial role in cognitive impairment in klotho mutant mice, a genetic model of aging. Since down-regulation of melatonin due to aging is well documented, we used this genetic model to determine whether the antioxidant property of melatonin affects memory impairment.

Methods:

First, we examined the effects of melatonin on hippocampal oxidative parameters and the glutathione/oxidized glutathione (GSH/GSSG) ratio and memory dysfunction of klotho mutant mice. Second, we investigated whether a specific melatonin receptor is involved in the melatonin-mediated pharmacological response by application with melatonin receptor antagonists. Third, we examined phospho-extracellular-signal-regulated kinase (ERK) expression, nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, Nrf2 DNA binding activity, and glutamate-cysteine ligase (GCL) mRNA expression. Finally, we examined effects of the ERK inhibitor SL327 in response to antioxidant efficacy and memory enhancement mediated by melatonin.

Results:

Treatment with melatonin resulted in significant attenuations of oxidative damage, a decrease in the GSH/GSSG ratio, and a significant amelioration of memory impairment in this aging model. These effects of melatonin were significantly counteracted by the selective MT2 receptor antagonist 4-P-PDOT. Importantly, 4-P-PDOT or SL327 also counteracted melatonin-mediated attenuation in response to the decreases in phospho-ERK expression, Nrf2 nuclear translocation, Nrf2 DNA-binding activity, and GCL mRNA expression in the hippocampi of klotho mutant mice. SL327 also counteracted the up-regulation of the GSH/GSSG ratio and the memory enhancement mediated by melatonin in klotho mutant mice.

Conclusions:

Melatonin attenuates oxidative stress and the associated memory impairment induced by klotho deficiency via signaling interaction between the MT2 receptor and ERK- and Nrf2-related antioxidant potential.     

Mangiferin activates Nrf2-antioxidant response element signaling without reducing the sensitivity to etoposide of human myeloid leukemia cells in vitro

Aim： Mangiferin is glucosylxanthone extracted from plants of the Anacardiaceae and Gentianaceae families. The aim of this study was to investigate the effects of mangiferin on Nrf2-antioxidant response element （ARE） signaling and the sensitivity to etoposide of human myeloid leukemia cells in vitro. Methods： Human HL-60 myeloid leukemia cells and mononuclear human umbilical cord blood cells （MNCs） were examined. Nrf2 protein was detected using immunofluorescence staining and Western blotting. Binding of Nrf2 to ARE was examined with electrophoretic mobility shift assay. The level of NQ01 was assessed with real-time RT-PCR and Western blotting. DCFH-DA was used to evaluate intracellular ROS level. Cell proliferation and apoptosis were analyzed using MTT and flow cytometry, respectively. Results： Mangiferin （50 pmol/L） significantly increased Nrf2 protein accumulation in HL-60 cells, particularly in the nucleus. Mangiferin also enhanced the binding of Nrf2 to an ARE, significantly up-regulated NQ01 expression and reduced intracellular ROS in HL60 cells. Mangiferin alone dose-dependently inhibited the proliferation of HL-60 cells. Mangiferin （50 mol/L） did not attenuate etoposide-induced cytotoxicity in HL-60 cells, and combined treatment of mangiferin with low concentration of etoposide （0.8 pg/mL） even increased the cell inhibition rate. Nor did mangiferin change the rate of etoposide-induced apoptosis in HL-60 cells. In MNCs, mangiferin significantly relieved oxidative stress, but attenuated etoposide-induced cytotoxicity. Conclusion： Mangiferin is a novel Nrf2 activator that reduces oxidative stress and protects normal cells without reducing the sensitivity to etoposide of HL-60 leukemia Cells in vitro. Mangiferin may be a potential chemotherapy adjuvant.     

Participation of nuclear factor (erythroid 2-related), factor 2 in ameliorating lithocholic acid-induced cholestatic liver injury in mice

BACKGROUND AND PURPOSE

Lithocholic acid (LCA), the most toxic bile acid, induces cholestatic liver injury in rodents. We previously showed that LCA activates the oxidative stress-responsive nuclear factor (erythroid-2 like), factor 2 (Nrf2) in cultured liver cells, triggering adaptive responses that reduce cell injury. In this study, we determined whether Nrf2 protects the liver against LCA-induced toxicity in vivo.

EXPERIMENTAL APPROACH

Nrf2 disrupted (Nrf2^−/−) and wild-type mice were treated with LCA (125 mg·kg⁻¹ body weight) to induce liver injury. Levels of mRNA, protein and function of important Nrf2 target genes coupled with liver histology and injury biomarkers of mice were examined.

KEY RESULTS

In 4 day LCA treatments, we observed a significantly higher hepatic induction of Nrf2 target, cytoprotective genes including thioredoxin reductase 1, glutamate cysteine ligase subunits, glutathione S-transferases, haeme oxygenase-1 and multidrug resistance-associated proteins 3 and 4 in the wild type as compared with the Nrf2^−/− mice. Moreover, basal and LCA-induced hepatic glutathione and activities of glutathione S-transferases and thioredoxin reductases were higher in wild-type than in Nrf2^−/− mice. This reduced production of cytoprotective genes against LCA toxicity rendered Nrf2^−/− mice more susceptible to severe liver damage with the presence of multifocal liver necrosis, inflamed bile ducts and elevation of lipid peroxidation and liver injury biomarkers, such as alanine aminotransferase and alkaline phosphatase.

CONCLUSIONS AND IMPLICATIONS

Nrf2 plays a crucial cytoprotective role against LCA-induced liver injury by orchestrating adaptive responses. The pharmacological potential of targeting liver Nrf2 in the management of cholestatic liver diseases is proposed.     

Epigallocatechin gallate reduces human monocyte mobility and adhesion in vitro

Background and purpose:

Monocytes/macrophages are an important population of immune inflammatory cells that have diverse effector functions in which their mobility and adhesion play a very relevant role. Epigallocatechin gallate (EGCG), a major component of green tea, has been reported to have anti-allergic and anti-inflammatory activities, but its effects on monocytes remain to be determined. Here we investigated the effects of EGCG on the migration and adhesion of monocytes.

Experimental approach:

We used a human monocyte cell line (THP-1) to analyse the effects of treatment with EGCG under non-cytotoxic conditions on the expression levels of the monocyte chemotactic protein-1 (MCP-1) and of the MCP-1 receptor (CCR2) and on the activation of β1 integrin. A functional validation was carried out by evaluating the inhibitory effect of EGCG on monocyte adhesiveness and migration in vitro.

Key results:

Treatment of THP-1 cells with EGCG decreased MCP-1 and CCR2 gene expression, together with MCP-1 secretion and CCR2 expression at the cell surface. EGCG also inhibited β1 integrin activation. The effects on these molecular targets were in agreement with the EGCG-induced inhibition of THP-1 migration in response to MCP-1 and adhesion to fibronectin.

Conclusions and implications:

Under our experimental conditions, EGCG treatment inhibited the migration and adhesion of monocytes. These inhibitory effects of EGCG on monocyte function should be considered as a promising new anti-inflammatory response with a potential therapeutic role in the treatment of inflammation-dependent diseases.     

Native profiles of alpha(1A)-adrenoceptor phenotypes in rabbit prostate

Background and purpose:

α₁-Adrenoceptors in the rabbit prostate have been studied because of their controversial pharmacological profiles in functional and radioligand binding studies. The purpose of the present study is to determine the native profiles of α₁-adrenoceptor phenotypes and to clarify their relationship.

Experimental approach:

Binding experiments with [³H]-silodosin and [³H]-prazosin were performed using intact tissue segments and crude membrane preparations of rabbit prostate and the results were compared with α₁-adrenoceptor-mediated prostate contraction.

Key results:

[³H]-Silodosin at subnanomolar concentrations bound specifically to intact tissue segments of rabbit prostate. However, [³H]-prazosin at the same range of concentrations failed to bind to α₁-adrenoceptors of intact segments. Binding sites of [³H]-silodosin in intact segments were composed of α_1L phenotype with low affinities for prazosin (pKi=7.1), 5-methyurapidil and N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethamine hydrochloride (RS-17053), and α_1A-like phenotype with moderate affinity for prazosin (pKi=8.8) but high affinity for 5-methyurapidil and RS-17053. In contrast, both radioligands bound to a single population of α₁-adrenoceptors in the membrane preparations at the same density with a subnanomolar affinity, showing a typical profile of ‘classical'' α_1A-adrenoceptors (pKi for prazosin=9.8). The pharmacological profile of α₁-adrenoceptor-mediated prostate contraction was in accord with the α_1L phenotype observed by intact segment binding approach.

Conclusions and implications:

Three distinct phenotypes (α_1L and α_1A-like phenotypes in the intact segments and a classical α_1A phenotype in the membranes) with different affinities for prazosin were detected in rabbit prostate. It appears that the three phenotypes are phenotypic subtypes of α_1A-adrenoceptors, but are not genetically different subtypes.     

The anti-fibrotic effects of epigallocatechin-3-gallate in bile duct-ligated cholestatic rats and human hepatic stellate LX-2 cells are mediated by the PI3K/Akt/Smad pathway

Aim:

(−)-Epigallocatechin-3-gallate (EGCG) is one of the most abundant polyphenols in green tea with strong antioxidant activity and various therapeutic effects. In this study, we investigated the anti-fibrotic effects of EGCG and underlying mechanisms in bile duct-ligated (BDL) rats and a liver fibrosis model in vitro.

Methods:

BDL rats were treated with EGCG (25 mg·kg⁻¹·d⁻¹, po) for 14 d, and then the serum, bile and liver samples were collected. Liver fibrosis was assessed by serum, urine and bile biochemistry analyses and morphological studies of liver tissues. TGF-β1-stimulated human hepatic stellate LX-2 cells were used as a liver fibrosis model in vitro. The expression of liver fibrogenic genes and signaling proteins in the PI3K/Akt/Smad pathway was examined using Western blotting and/or real-time PCR.

Results:

In BDL rats, EGCG treatment significantly ameliorates liver necrosis, inflammation and fibrosis, and suppressed expression of the genes associated with liver inflammation and fibrogenesis, including TNF-α, IL-1β, TGF-β1, MMP-9, α-SMA, and COL1A1. In LX-2 cells, application of EGCG (10, 25 μmol/L) dose-dependently suppressed TGF-β1-stimulated expression of COL1A1, MMP-2, MMP-9, TGF-β1, TIMP1, and α-SMA. Furthermore, EGCG significantly suppressed the phosphorylation of Smad2/3 and Akt in the livers of BDL rats and in TGF-β1-stimulated LX-2 cells. Application of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a specific inhibitor of PI3K, produced similar effects as EGCG did in TGF-β1-stimulated LX-2 cells, but co-application of EGCG and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 did not produce additive effects.

Conclusion:

EGCG exerts anti-fibrotic effects in BDL rats and TGF-β1-stimulated LX-2 cells in vitro via inhibiting the PI3K/Akt/Smad pathway.     

The ecto-nucleotidase NTPDase1 differentially regulates P2Y1 and P2Y2 receptor-dependent vasorelaxation

Background and purpose:

Extracellular nucleotides produce vasodilatation through endothelial P2 receptor activation. As these autacoids are actively metabolized by the ecto-nucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), we studied the effects of this cell surface enzyme on nucleotide-dependent vasodilatation.

Experimental approach:

Vascular NTPDase expression and activity were evaluated by immunohistochemistry and histochemistry. The vascular effects of nucleotides were tested in vivo by monitoring mean arterial pressure, and in vitro comparing reactivity of aortic rings using wild-type and Entpd1^−/− (lacking NTPDase1) mice.

Key results:

The absence of NTPDase1 in Entpd1^−/− mice led to a dramatic drop in endothelial nucleotidase activity. This deficit was associated with an exacerbated decrease in blood pressure after nucleotide injection. Following ATP injection, mean arterial pressure was decreased in Entpd1^+/+ and Entpd1^−/− mice by 5.0 and 17%, respectively, and by 0.1 and 19% after UTP injection (10 nmole·kg⁻¹ both). In vitro, the concentration-response curves of relaxation to ADP and ATP were shifted to the left, revealing a facilitation of endothelial P2Y1 and P2Y2 receptor activation in Entpd1^−/− mice. EC₅₀ values in Entpd1^+/+ versus Entpd1^−/− aortic rings were 14 µM versus 0.35 µM for ADP, and 29 µM versus 1 µM for ATP. In Entpd1^−/− aortas, P2Y1 receptors were more extensively desensitized than P2Y2 receptors. Relaxations to the non-hydrolysable analogues ADPβS (P2Y1) and ATPγS (P2Y2) were equivalent in both genotypes confirming the normal functionality of these P2Y receptors in mutant mice.

Conclusions and implications:

NTPDase1 controls endothelial P2Y receptor-dependent relaxation, regulating both agonist level and P2 receptor reactivity.     

The effects of a single nucleotide polymorphism in SLCO1B1 on the pharmacodynamics of pravastatin

AIM

To determine whether the SNP rs4149056 in SLCO1B1 alters the pharmacodynamics of pravastatin.

METHODS

rs4149056 was genotyped in 626 pravastatin-treated participants in the WOSCOPS trial and the response after 1 year of treatment was compared between the different genotypes.

RESULTS

Pravastatin reduced serum LDL cholesterol by 22.2% in TT homozygotes, by 22.2% in TC heterozygotes and by 17.7% in CC homozygotes (TT + TC vs. CC P value 0.33). There were no significant differences in the response of total cholesterol, LDL, HDL, triglycerides or CRP to pravastatin between the genotypes.

CONCLUSION

The rs4149056 SNP did not significantly affect the pharmacodynamics of pravastatin.     

Differential role of sodium channels SCN1A and SCN2A gene polymorphisms with epilepsy and multiple drug resistance in the north Indian population

AIMS

To evaluate sodium channel genes as candidates for epilepsy susceptibility and their role in therapeutic efficacy, we screened coding single-nucleotide polymorphism of SCN1A p. Thr 1067 Ala or c.3184 A→G (rs2298771) and SCN2A p.Arg19Lys or c.56 G→A (rs17183814) in north Indian epilepsy patients.

METHODS

The genotyping was performed in 160 control subjects and 336 patients with epilepsy, of whom 117 were drug resistant and 219 were drug responsive. Therapeutic drug monitoring for phenytoin, carbamazepine, phenobarbital and valproate was also performed in 20% of the patients to confirm compliance.

RESULTS

AG genotype of SCN1A 3184 A→G polymorphism was significantly higher and associated in epilepsy patients [P= 0.005; odds ratio (OR) 1.76, 95% confidence interval (CI) 1.19, 2.61], whereas A variant of SCN2A c.56 G→A was associated with multiple drug resistance in north Indian patients with epilepsy (P= 0.03; OR 1.62, 95% CI 1.03, 2.56).

CONCLUSIONS

Overall, results indicate a differential role of genetic polymorphisms of sodium channels SCN1A and SCN2A in epilepsy susceptibility and drug response.     

Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation

Aim:

To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects.

Methods:

MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 was measured using real-time PCR. Western blot analysis was used to detect the expression of AMPK and eNOS proteins.

Results:

Bezafibrate increased the viability and proliferation of MC3T3-E1 cells in a dose- and time-dependent manner. Bezafibrate (100 μmol/L) significantly enhanced osteoblastic mineralization and expression of the differentiation markers ALP, collagen I and osteocalcin. Bezafibrate (100 μmol/L) increased phosphorylation of AMPK and eNOS, which led to an increase of NO production by 4.08-fold, and upregulating BMP-2 and Runx-2 mRNA expression. These effects could be blocked by AMPK inhibitor compound C (5 μmol/L), or the PPARβ inhibitor GSK0660 (0.5 μmol/L), but not by the PPARα inhibitor MK886 (10 μmol/L). Furthermore, GSK0660, compound C, or N^G-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mmol/L) could reverse the stimulatory effects of bezafibrate (100 μmol/L) on osteoblast proliferation and differentiation, whereas MK886 only inhibited bezafibrate-induced osteoblast proliferation.

Conclusion:

Bezafibrate stimulates proliferation and differentiation of MC3T3-E1 cells, mainly via a PPARβ-dependent mechanism. The drug might be beneficial for osteoporosis by promoting bone formation.