Upregulation Of Cytoprotective Genes Research Articles

Exploring the Conformational Landscape of the Neh4 and Neh5 Domains of Nrf2 Using Two Different Force Fields and Circular Dichroism.

The nuclear factor erythroid 2-related factor 2 (Nrf2)-ARE transcriptional response pathway plays a critical role in protecting the cell from oxidative stresses via the upregulation of cytoprotective genes. Aberrant activation of Nrf2 in cancer cells can confer this cytoprotectivity, thereby reducing the efficacy of both chemotherapeutics and radiotherapies. Key to this antioxidant pathway is the interaction between Nrf2 and CREB binding protein (CBP), mediated by the Neh4 and Neh5 domains of Nrf2. Disruption of this interaction via small-molecule therapeutics could negate the effects of aberrant Nrf2 upregulation. Due to the disordered nature of these domains, there remains no three-dimensional structure of Neh4 or Neh5, making structure-based drug design a challenge. Here, we performed 48 μs of unbiased molecular dynamics (MD) simulations with the Amber99SB*-ILDNP and CHARMM36m force fields and circular dichroism (CD) spectropolarimetry experiments to elucidate the free-state structures of these domains; no previous data regarding their conformational landscapes exists. There are two main findings: First, we find Neh5 to be markedly more disordered than Neh4, which has nine residues in the middle of the domain showing α-helical propensity, thus pointing to Neh4 and Neh5 having different binding mechanisms. Second, the two force fields show strong differences for the glutamic acid-rich Neh5 peptide but are in reasonable agreement for Neh4, which has no glutamic acid. The CHARMM36m force field agrees more closely with the CD results.

Evaluation of anti-inflammatory activity of Justicia secunda Vahl leaf extract using in vitro and in vivo inflammation models

BackgroundJusticia secunda Vahl. is a medicinal plant used in ethnomedical practice as therapy to manage inflammation. Therefore, this study was designed to evaluate the anti-inflammatory activity of methanol extract of J. secunda leaves (MEJSL) using in vitro and in vivo inflammation models.MethodsSeventy-percent MEJSL was prepared following standard procedure. In vitro anti-inflammatory assays were performed using heat-induced bovine serum albumin (BSA) denaturation and erythrocyte membrane stabilization assays. Carrageenan and formaldehyde induced inflammation in rat models were used to evaluate the anti-inflammatory activity of MEJSL in vivo. Diclofenac sodium was used as a reference drug. In addition, liver and kidney function assays and hematological analysis were carried out.ResultsData revealed that varying concentrations of MEJSL significantly (P < 0.05) inhibited heat-induced BSA denaturation and stabilized erythrocyte membrane against hypotonicity-induced hemolysis when compared with diclofenac sodium in a concentration-dependent manner. In vivo study showed that 10 mg/kg body weight (b.w.) diclofenac sodium, 100 and 300 mg/kg b.w. MEJSL suppressed carrageenan-induced paw edema at the sixth hour by 71.14%, 83.08%, and 89.05%, respectively. Furthermore, 10 mg/kg b.w. diclofenac sodium, 100 and 300 mg/kg b.w. MEJSL inhibited formaldehyde-induced paw edema by 72.53%, 74.73%, and 76.48%, respectively. Animals treated with varying doses of MEJSL had reduced plasma aspartate aminotransferase and alanine aminotransferase activities; urea and creatinine concentrations; and modulated hematological parameters when compared with the untreated control group.ConclusionsFindings from this study showed that MEJSL exhibited substantial anti-inflammatory actions in the in vitro and in vivo models. It also indicated that MEJSL anti-inflammatory mechanisms of action could be through interference with phase 2 inflammatory stressors, upregulation of cytoprotective genes, stabilization of inflammatory cell membranes and immunomodulatory activity.

Preclinical evaluation of EPHX2 inhibition as a novel treatment for inflammatory bowel disease.

Epoxyeicosatrienoic acids (EETs) are signaling lipids produced by cytochrome P450 epoxygenation of arachidonic acid, which are metabolized by EPHX2 (epoxide hydrolase 2, alias soluble epoxide hydrolase or sEH). EETs have pleiotropic effects, including anti-inflammatory activity. Using a Connectivity Map (CMAP) approach, we identified an inverse-correlation between an exemplar EPHX2 inhibitor (EPHX2i) compound response and an inflammatory bowel disease patient-derived signature. To validate the gene-disease link, we tested a pre-clinical tool EPHX2i (GSK1910364) in a mouse disease model, where it showed improved outcomes comparable to or better than the positive control Cyclosporin A. Up-regulation of cytoprotective genes and down-regulation of proinflammatory cytokine production were observed in colon samples obtained from EPHX2i-treated mice. Follow-up immunohistochemistry analysis verified the presence of EPHX2 protein in infiltrated immune cells from Crohn’s patient tissue biopsies. We further demonstrated that GSK2256294, a clinical EPHX2i, reduced the production of IL2, IL12p70, IL10 and TNFα in both ulcerative colitis and Crohn's disease patient-derived explant cultures. Interestingly, GSK2256294 reduced IL4 and IFNγ in ulcerative colitis, and IL1β in Crohn's disease specifically, suggesting potential differential effects of GSK2256294 in these two diseases. Taken together, these findings suggest a novel therapeutic use of EPHX2 inhibition for IBD.

Deletion of Nrf2 enhances susceptibility to eosinophilic sinonasal inflammation in a murine model of rhinosinusitis.

Oxidative stress exacerbates lower airway diseases including asthma and chronic obstructive pulmonary disease (COPD); however, its role in upper airway (sinonasal) chronic inflammatory disorders is less clear. Nuclear erythroid 2 p45-related factor (Nrf2) is an endogenous mechanism that upon activation invokes an antioxidant response pathway via nuclear translocation and upregulation of cytoprotective genes. We sought to determine whether deletion of Nrf2 enhances susceptibility to allergic sinonasal inflammation in vivo. Nrf2-/- mice were subjected to the ovalbumin (Ova)-induced murine model of rhinosinusitis and indices of sinonasal inflammation and epithelial barrier dysfunction were assessed. We show that deletion of Nrf2 results in enhances indices of allergen-induced sinonasal inflammation including aggravated eosinophil accumulation and goblet cell hyperplasia. An exaggerated increase in epithelial derived inflammatory cytokines including interleukin 33 (IL-33) and thymic stromal lymphopoietin (TSLP) was observed in the nasal lavage fluid and sinonasal mucosal tissue of Nrf2-/- mice. Furthermore, Nrf2-/- mice showed heightened Ova-induced barrier dysfunction as measured by serum albumin accumulation in nasal lavage fluid of mice. These data show that the endogenous Nrf2 pathway limits Ova-induced sinonasal inflammation, epithelial derived inflammatory cytokine production, and epithelial barrier dysfunction in vivo and identify a potential therapeutic target in the management of allergic sinonasal inflammatory disorders. This is the first study to our knowledge which shows that Nrf2 regulates allergic inflammation in the sinonasal cavity in vivo.

The Nrf2 activator, tBHQ, exacerbates immediate hypersensitivity response to food allergen (HYP7P.315)

Abstract Nuclear erythroid 2 related factor 2 (Nrf2) is a transcription factor activated by cell stress, including oxidative and electrophilic stimuli, resulting in the upregulation of cytoprotective genes. Nrf2 can be activated experimentally by a number of different pharmacological agents, including tert-butylhydroquinone (tBHQ), which is also used commercially as a food additive. We recently showed that activation of Nrf2 by tBHQ promotes Th2 differentiation and inhibits Th1 differentiation in isolated CD4+ T cells. Given that tBHQ is a food additive and promotes Th2 differentiation, the purpose of the present studies was to determine the effect of tBHQ in a mouse model of food allergy. Mice were administered a diet either with or without tBHQ (0.001%) for two weeks prior to sensitization with ovalbumin. Notably, this dose of tBHQ is lower than that normally present in standard rodent chow (0.0016%). The mice were sensitized weekly transdermally for 4 weeks. Sensitization to ovalbumin caused an increase in ovalbumin-specific IgE in plasma, which was greater in animals exposed to tBHQ. After sensitization, the mice were orally challenged with ovalbumin, causing mild/moderate immediate hypersensitivity, which was exacerbated by tBHQ. Overall, these studies suggest that low doses of the food additive, tBHQ, increase IgE response to food allergen and exacerbate clinical signs of immediate hypersensitivity. (This work was funded by NIH grant: ES018885.)

Natural product-derived pharmacological modulators of Nrf2/ARE pathway for chronic diseases

Covering: 2000 to 2013. Oxidative stress is the central component of chronic diseases. The nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) pathway is vital in the up-regulation of cytoprotective genes and enzymes in response to oxidative stress and treatment with certain dietary phytochemicals. Herein, we classify bioactive compounds derived from natural products that are Nrf2/ARE pathway activators and recapitulate the molecular mechanisms for inducing Nrf2 to provide favorable effects in experimental models of chronic diseases. Moreover, pharmacological inhibition of Nrf2 signalling has emerged as promising strategy against multi-drug resistance thereby improving the treatment efficacy. We have also enlisted natural product-derived inhibitors of Nrf2/ARE pathway.

Concentration-dependent effects of the soy phytoestrogen genistein on the proteome of cultured cardiomyocytes

The soy-derived phytoestrogen genistein (GEN) has received attention for its potential benefits on the cardiovascular system by providing direct protection to cardiomyocytes against pathophysiological stresses. Here, we employed a proteomic approach to study the concentration-dependent effects of GEN treatments on cardiomyocytes. Cultured HL-1 cardiomyocytes were treated with low (1μM) and high (50μM) concentrations of GEN. Proteins were pre-fractionated by sequential hydrophilic/hydrophobic extraction and both protein fractions from each treatment group were separated by 2D gel electrophoresis (2DE). Overall, approximately 2,700 spots were visualized on the 2D gels. Thirty-nine and 99 spots changed in volume relative to controls (p<0.05) following the low- and high-concentration GEN treatments, respectively. From these spots, 25 and 62 protein species were identified by ESI-MS/MS and Mascot database searching, respectively. Identified proteins were further categorized according to their functions and possible links to cardioprotection were discussed. MetaCore gene ontology analysis suggested that 1μM GEN significantly impacted the anti-apoptosis process, and that both the low and high concentrations of GEN influenced the glucose catabolic process and regulation of ATPase activity. This proteomics study provides the first global insight into the molecular events triggered by GEN treatment in cardiomyocytes.

Abstract P317: Acute Exercise Stress and ROS Signaling: Activation of Nrf2/ARE-Dependent Antioxidant Defense Mechanisms in the Mouse Myocardium

Background: Cellular defense mechanisms are crucial for maintaining intracellular redox state and mitigating free radical accumulation with aging. Nuclear Erythroid 2 p45 related factor-2 (Nrf2) regulates basal and inducible expression of numerous cytoprotective/antioxidant genes. We hypothesize that acute exercise will induce ROS, which triggers Nrf2/ARE signaling and promotes myocardial defense mechanisms. Methods: Age-matched wild-type (WT) and Nrf2−/− (KO) mice at 2 and &gt;20 months were subjected to acute exercise stress (AES) and then we assessed the activation of Nrf2/ARE-dependent transcriptional mechanisms in the heart. Myocardial ROS was measured by electron paramagnetic resonance (EPR) analysis. Results: Under basal conditions, total ROS and GSH levels were identical at 2 months, whereas they were significantly impaired in Nrf2-KO when compared to Wt myocardium at ∼20 months indicating that Nrf2-deficiency is coupled with blemished redox potential. Upon AES, the young WT and KO mice exhibited oxidative stress (OS), but the WT were able compensate for the stress by increasing Nrf2 nuclear translocation and subsequent upregulation of cytoprotective genes. However, the aged (WT &amp; KO) mice developed OS in response to AES. The degree of OS was several fold higher in the aged Nrf2-KO mice when compared with WT, suggesting an important age dependent function for Nrf2 in the myocardium. Western blot analysis revealed significant down regulation of major antioxidants (GCS, Nqo1, Ho1, catalase, G6pd and Gsr) in KO mice, while WT mice exhibited compensatory antioxidant response to the AES. Gene expression (qPCR) analysis revealed profound upregulation of major antioxidants in WT, but there was no such response occurred in KO mice after AES, suggesting Nrf2 independent mechanisms are inadequate to protect the myocardium. Conclusions: Acute exercise induces ROS and thereby activates Nrf2 in the myocardium. However, disruption of Nrf2 increases susceptibility of the myocardium to OS induced damage. Thus Nrf2 signaling might be a potential therapeutic target to protect the heart from ROS and/or age dependent ischemia/reperfusion (I/R) injury and myocardial infarction (MI).

Targeting specific cell signaling transduction pathways by dietary and medicinal phytochemicals in cancer chemoprevention

Natural phytochemicals derived from dietary sources or medicinal plants have gained significant recognition in the potential management of several human clinical conditions. Much research has also been geared towards the evaluation of plant extracts as effective prophylactic agents since they can act on specific and/or multiple molecular and cellular targets. Plants have been an abundant source of highly effective phytochemicals which offer great potential in the fight against cancer by inhibiting the process of carcinogenesis through the upregulation of cytoprotective genes that encode for carcinogen detoxifying enzymes and antioxidant enzymes. The mechanistic insight into chemoprevention further includes induction of cell cycle arrest and apoptosis or inhibition of signal transduction pathways mainly the mitogen-activated protein kinases (MAPK), protein kinases C (PKC), phosphoinositide 3-kinase (PI3K), glycogen synthase kinase (GSK) which lead to abnormal cyclooxygenase-2 (COX-2), activator protein-1 (AP-1), nuclear factor-kappaB (NF-κB) and c-myc expression. Effectiveness of chemopreventive agents reflects their ability to counteract certain upstream signals that leads to genotoxic damage, redox imbalances and other forms of cellular stress. Targeting malfunctioning molecules along the disrupted signal transduction pathway in cancer represent a rational strategy in chemoprevention. NF-κB and AP-1 provide mechanistic links between inflammation and cancer, and moreover regulate tumor angiogenesis and invasiveness, indicating that signaling pathways that mediate their activation provide attractive targets for new chemotherapeutic approaches. Thus cell signaling cascades and their interacting factors have become important targets of chemoprevention and phenolic phytochemicals and plant extracts seem to be promising in this endeavor.

MHC CLASS I ALLOCHIMERIC MOLECULE MODULATES ALLOANTIBODY RESPONSE AND UPREGULATES EXPRESSION OF PROTECTIVE GENES: INFLUENCE ON DEVELOPMENT OF GRAFT ACCOMMODATION

P1028 Aims: Specific transplantation tolerance can be induced in rat recipients treated during peri-transplant period with MHC class I antigens in which 4 donor a.a. are substituted onto host MHC class I sequences. The tolerance characterized with upregulation of cyto-protective and anti-apoptotic genes and accumulation of IgG type alloantibodies in the vessel endothelium. This study was designated to investigate possible influence of this therapy on development of graft accommodation through passive transfer of alloantibodies to a new cohort of recipients. Methods: 1 ml of serum from tolerant ACI rats after allochimeric protein therapy was injected through the portal vein to lightly irradiated (130 Rad) ACI recipients of WF hearts. Grafts were evaluated at 100 days. Results: Passive transfer of sera from rats conditioned with allochimeric therapy, but not from naive rats, to lightly irradiated syngeneic ACI recipients of WF hearts resulted in indefinite survival of cardiac allografts (MST 135+21.9, n=13). Histopathology examination revealed intact myocardial architecture, absent or minimal intramiocardial inflammation, and no evidence of neointimal hyperplasia. The degree of neointimal proliferation and TVS within each vessel was quantitated by the neointimal index (NI). Serum conditioned hosts demonstrated NI of 6.5 with 32% of vessels effected. In contrast, control group receiving low dose radiation in conjunction with naive ACI serum had NI of 39 with 70% of vessels affected. Upregulation of genes for Th2-type cytokines (IL-10 and IL-4) was found in tolerated grafts in comparison to control. FACScan analysis of serum from passive transferred tolerated ACI rats revealed low level of IgM alloantibodies. IgG production was markedly abrogated but noticeably higher than in naive control, IgG isotype analysis showed upregulation for IgG1 and IgG2a alloantibodies. Immunohistology staining of tolerant grafts with anti-IgM and IgG antibodies showed deposits of IgG granules around of internal intima when in control hearts after high doses of CsA such patterns were not revealed. To detect level of apoptotic cells within myocardium of long term survived tolerant grafts in compare to chronic rejected tissue in situ hybridization was performed (TUNEL). Analysis showed decreased number of apoptotic cells in tolerant hosts as compared to controls (7.5+1.2; 13.4+ 2.5, per 5-6 fields, respectively). Conclusions: Allochimeric MHC class I therapy orchestrates the development of donor-specific alloantibodies than can be transferred to new recipient and might cause allograft vascular accommodation, characterized by inhibition of apoptotic cell death, upregulation of cyto-protective genes and IgG antibody binding to vascular endothelium.