Plumbagin protects against hydrogen peroxide-induced neurotoxicity by modulating NF-B and Nrf-2

Abstract
Introduction: Redox signaling initiates pathogenesis of neuronal degenera- tion. Plumbagin is a potential antioxidant with anti-inflammatory, anti-can- cer and radio sensitizing properties. In the present study, we aimed to de- termine the protective role of plumbagin against H2O2-induced neurotoxicity in PC12 cells by determining nuclear factor B (NF-B) and nuclear factor E2-related factor 2 (Nrf-2) pathways.Material and methods: We analyzed oxidative stress by determining re- active oxygen species (ROS) and nitrite levels, and antioxidant enzyme ac- tivities. Nrf-2 and NF-B p65 nuclear localization was determined through immunofluorescence. Further, nuclear levels of p-Nrf-2 and downstream ex- pression of NAD(P)H quinone dehydrogenase 1 (NQO1), heme oxygenase-1 (HO-1) and glutathione-s-transferase (GST) were determined by western blot. Anti-inflammatory activity was analyzed by evaluating NF-B p65, cy- clooxygenase-2 (COX-2) and interleukin (IL-6, IL-8, and MCP-1) expression. Results: The results showed that plumbagin increased (p < 0.01) the cell viability against H2O2-induced cell death in PC12 cells. Plumbagin effectively ameliorated H2O2-induced oxidative stress through reducing oxidative stress (p < 0.01) and activating p-Nrf-2 levels. Further, plumbagin up-regulated an- tioxidant enzyme activities (p < 0.01) against H2O2-induced oxidative stress. Plumbagin showed anti-inflammatory effect by suppressing NF-B p65 ac- tivation and down-regulating NF-B p65 and COX-2 expression. In addition, plumbagin modulated (p < 0.01) inflammatory cytokine expression against H2O2-induced neurotoxic effects.
Conclusions: Together, our results show that plumbagin modulated NF-B and Nrf-2 signaling. Thus, plumbagin might be an effective compound in prevent- ing H2O2-induced neurotoxicity and its associated inflammatory responses.

Introduction
Oxidative stress is a common mediator in the development of neuro- degenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease, multiple sclerosis and Creutzfeldt-Ja- cob disease. Increased generation of reactive oxygen species (ROS), Ca2+ deregulation, loss of mitochondrial permeability transition pore (MPP), and improper clearance of mitochondria are the major factors which ul- timately lead to mitochondrial dysfunction and neuronal oxidative dam-age. These post-mitotic neurons are extremely sensitive to ROS and thus oxidative stress is as- sociated with apoptosis [1, 2]. Thus, an imbalance in protective mechanisms against such oxidativegrown in RPMI-1640 medium supplemented with fetal bovine serum.The cytotoxic dose of H Owas determinedof neuro-toxic effects and degeneration. Endoge-nous antioxidant defense mechanisms are regu- lators of oxidative stress. Decrease in antioxidant capacity in neurotoxicity is well established [3, 4]. Improving antioxidant-mediated defense mecha- nisms is important in modulating oxidative stress and cytoprotection.Plumbagin is a yellow pigmented secondary metabolite isolated from the roots of Plumbag- inaceae, Ancestrocladaceae and Dioncophyllace- ae families. The compound is used in treatment of various diseases from ancient times and is identified with multiple functional properties [5]. Plumbagin is a potential antioxidant with an- ti-inflammatory, anti-cancer and radiosensitizing properties [6–9]. Numerous studies have reported its role in ameliorating redox signaling and there- by preventing oxidative stress associated damage [10, 11]. In this study, we demonstrate the import- ant role of plumbagin in oxidative stress and in- flammatory responses against H2O2-induced neu- rotoxic effects in PC12 cells. In order to evaluate the effect, we identified various oxidative stress markers including ROS, nitrite levels, antioxidantthrough MTT assay. The cells were cultured and cells in log phase were trypsinised and seeded into a 96-well plate. After overnight attachment of the cells, H2O2 at different concentrations (10– 50 µM) was added and incubated for 24 and 48 h. The cells after the respective treatment schedule were treated with DMSO and dissolved formazan crystals were measured at 570 nm [12]. The cell viability was calculated and the IC50 value was determined. Protective effect of plumbagin: After attachment, the cells were pre-treated with plum-bagin for 24 h (5–25 µM) followed by H2O2 treat- ment. The appropriate cytoprotective dose select- ed was used to study the molecular mechanism involved.Increased ROS levels initiate oxidative stress. The ROS levels were determined by DCF-DA as de- scribed previously [13].

PC12 cells were pre-treat- ed with plumbagin (24 h), after which cells were washed with PBS and treated with H O . For de-status, nuclear localization of transcription factors 2 2NF-B p65 and p-Nrf-2, expression of redox regu- lators and inflammatory proteins such as nuclear factor B (NF-B) p65, cyclooxygenase-2 (COX-2), p-Nrf-2, NAD(P)H quinone dehydrogenase 1 (NQO1), glutathione-s-transferase (GST) and heme oxy- genase-1 (HO-1). Further, anti-inflammatory re- sponses were evaluated through pro-inflammato- ry cytokine levels.RPMI-1640, Fetal Calf Serum, Antibiotic and An- timycolytic solution, DCF-DA and interleukins (IL-6, IL-8 and MCP-1) were purchased from Sigma-Al- drich, China. Primary antibodies were: Nrf-2 (Anti- Nrf2 (phospho S40) antibody, Abcam ab76026), GST (Anti-Glutathione S-Transferase antibody, Abcam ab53942), NQO1 (Anti-NQO1 antibody Abcam (ab34173), HO-1 (Abcam-ab13248), NF-B p65 (Cell Signaling Technology-Phospho-NF-B p65 (Ser536)), COX-2 (Abcam-ab15191). Second- ary antibodies were purchased from Cell Signaling Technology, Beverly, USA.Cell culturePC12, pheochromocytoma derived from rat ad- renal medulla was procured from American Type Culture Collection (ATCC-CRL-1721). The cells weretermining individual effects, the cells were treated with H2O2 and plumbagin as a separate group. Af- ter the respective treatment schedule, ROS levels were determined spectrophotometrically (480 nm and 520 nm).Nitrite estimationThe nitrite levels were determined using the Nitrite/Nitrate Assay Kit, Sigma-Aldrich (23479).The specific activity of the antioxidant en- zymes was determined using: Superoxide Dis- mutase Activity Colorimetric Assay Kit (ab65354); Catalase Specific Activity Assay Kit (ab118184), GST Activity Assay Kit (Fluorometric) (ab65325), GPx activity Kit ab102530.

The specific activity was calculated and results were expressed as U/mg of protein.After the respective treatment schedule, nucle- ar and whole cell extracts were isolated and used for protein expression through western blot anal- ysis. 30 µg of protein were separated on precast 12% SDS-PAGE gels and transferred to NC mem- brane. After blocking, primary antibodies (p-Nrf-2,GST, NQO1, HO-1, NF-B p65, COX-2) were addedand incubated overnight. Following TBST wash, appropriate secondary antibodies were added and bands were visualized by the enhanced chemilu- minescence (ECL) system. Image J software was used for densitometric analysis of western blots.The cells were grown on cover slips and coated with lysine. The cells were allowed to attach to the cover slips and treatment was carried out. Plum- bagin was administered for 24 h followed by H2O2 for 3 h. After PBS wash, cells were treated with pri- mary antibody (1 : 50) overnight at 4°C followed by secondary antibody (1 : 2000). Immunofluores- cence was carried out for 2 different antibodies, NF-B-p65 and p-Nrf-2. The images were acquired and nuclear localization was analyzed through Lumi Vision Imager software.Interleukin expressionFollowing treatment with plumbagin and H2O2, the supernatant was determined for interleukin expression (IL-6, IL-8, and MCP-1); R&D Systems China Co., Ltd.28 µM. Plumbagin pre-treatment significantly im- proved the cell viability at a concentration ranging from 10 to 25 µM, from which the lowest concen- tration with the maximum effect was chosen for further studies. Thus, preliminary studies revealed the significant cytoprotective role of plumbagin. Similar neurotoxicity studies have demonstrated that H2O2 has a prime role in inducing cell death through apoptotic induction in rat cerebral cortex neurons [14, 15].

Next, we studied the detailed molecular mechanism of plumbagin-induced cel- lular protective mechanisms. Plumbagin prevent- ed H2O2-induced oxidative stress by reducing the generation of ROS levels and nitrite levels. Further, plumbagin prevented redox signaling through in- creasing the activation of Nrf-2 and the expres- sion of antioxidant defense system proteins such as HO-1, NQO1, and GST. Plumbagin increased the overall antioxidant status by enhancing the activ- ities of glutathione levels and various enzymic an- tioxidants: SOD, CAT, GST, GPx. Nrf-2 (nuclear fac- tor erythroid 2 (NF-E2)-related factor 2) belongs to the Cap ’n’ Collar (CNC) family of proteins involved in cytoprotective defense and survival. In normal conditions, it is associated with Keap-1 (Kelch-like erythroid cell-derived protein with CNC homolo-proteins including COX-2 and inducible nitric ox- ide synthase. In the present study, H2O2 induced NF-B p65 nuclear translocation and expression with subsequent up-regulation of the inflamma- tory protein COX-2. However, plumbagin pre-treat- ment suppressed the inflammation by preventing NF-B p65 translocation. Further, expression of COX-2 and pro-inflammatory cytokines such as IL-6, IL-8 and MCP-1 was down-regulated. The immunomodulatory effect of plumbagin by sup- pressing NF-B signaling was previously reported in ConA-induced inflammation in lymphocytes [6]. Plumbagin prevented cellular invasion by activa- tion of the NF-B pathway and tumor necrosis factor (TNF)- mediated apoptosis [5]. Anti-in- flammatory effects of plumbagin were demon- strated in lipopolysaccharides-induced inflam- mation through suppression of NF-B and MAPK signaling in Raw 264.7 cells [22].In conclusion, in the present study we demon- strated that plumbagin acts as a neuroprotectant by regulating mechanisms involving both redox signaling and inflammation.

Thus, plumbagin might potentially target early activation of ROS and inflammatory proteins, thereby preventing H O -induced neuronal toxicity and damage and upon oxidative insult Nrf-2 localizes into the nucleus, thereby escaping Keap-1 mediated ubiq- uitination. Inside the nucleus, it associates with small Maf proteins, binds to the antioxidant re- sponse element (ARE) and induces transcription of NAD(P)H-quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1), glutamate-cysteine ligase (GCL) and glutathione-S-transferases (GSTs) [16, 17]. In the present study, we found that H2O2 pre- vented the nuclear translocation of p-Nrf-2 and its expression. However, plumbagin significantly prevented these events by inducing nuclear trans- location of p-Nrf-2 and increased its expression. In addition, expression of its downstream target proteins such as HO-1, NQO1 and GST was sig- nificantly increased during plumbagin pre-treat- ment. Previous in vivo and in-vitro studies on plumbagin mediated effects on cerebral ischemia demonstrated the protective role by inducing Nrf-2/ARE signaling and thereby reduced the ox- idative insult [18]. Thus, plumbagin offers neuro- protection by increasing the antioxidant defense mechanisms.Down-regulation of Nrf-2 activates inflamma- tory signaling [19]; thus oxidative stress and in- flammatory activation are interrelated. In neuro- degenerative diseases, activation and sustained inflammatory responses lead to dysfunction of neurons, resulting in disease progression [20, 21]. NF-B p65 activation induces pro-inflamma- tory signaling, which results in nuclear transloca- tion and activation of various downstream G6PDi-1 target.