Natural Dietary Supplementation of Curcumin Protects Mice Brains against Ethanol-Induced Oxidative Stress-Mediated Neurodegeneration and Memory Impairment via Nrf2/TLR4/RAGE Signaling

Abstract

According to our Western blot and confocal findings, the chronic co-administration of curcumin with ethanol inhibited the suppression of Nrf2/HO-1 in the mice brains, thereby preserving the endogenous antioxidant mechanism of the brains (Figure 2C,D). According to our Western blot and confocal microscopic results, there was an increased expression of GFAP and Iba-1 in the ethanol-treated mice brains, which was significantly rescued with the administration of curcumin (Figure 2). [...]curcumin regulated the expression of these markers. [...]the immunofluorescence results indicated the increased expression of Caspase-3 and PARP-1 in the ethanol-treated mice brains, which were inhibited with chronic administration of curcumin (Figure 4). According to our findings, the synaptic proteins, including PSD-95, synaptophysin, and SNAP-25, were significantly downregulated in the ethanol-treated mice brains compared with the control mice.

1. Introduction

Alcohol is a sedative agent and is pharmacologically similar to other hypnotic drugs, which makes it a potential candidate for abuse worldwide. In Western, European, and North American populations, the percentage of alcoholics among the adult population varies from 2% to 12% [1]. Alcohol and its derivatives promote physiological, behavioral, and cognitive dysfunctions in consumers [2], so worldwide alcohol dependence is considered to be a serious health issue in the modern world [3]. Ethanol has shown strong neurodegenerative consequences in experimental animal brains [4,5]. The neurodegenerative effects are associated with neuroinflammation, apoptotic cell death [6], and synaptic dysfunction [7,8]. Moreover, it has been shown that the consumption of alcohol leads to the generation of free radicals and to the chain reaction of lipid peroxidation that causes damage to the brain and other vital organs [9,10]. To combat the challenges of free radical generation, a potent antioxidant defense mechanism is of crucial importance. In the journey to find a novel, potent, and effective antioxidant system, naturally occurring compounds have always drawn more attention because of their ease of availability, safety, and efficacy. Many natural compounds and their derivatives have shown efficacy in the management of different neurological disorders [11,12,13], and are under consideration and evaluation for these disorders. Curcumin, a compound known to inhibit neuroinflammation, reduces plaque deposition in AD (Alzheimer’s Disease) models and improves vascular dysfunction [14]. It has been shown to antagonize many steps in the inflammatory cascade [15], including the suppression of nuclear factor-B, iNOS (Inducible nitric oxide synthase), and JNK (c-Jun N-terminal kinases) [13,16]. Moreover, it has shown the best antioxidant effects in mice [17]. It has shown effectiveness against the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson’s disease [18] and the MCAO (middle cerebral artery occlusion) animal model of the ischemic brain [19]. Based on the promising therapeutic potentials of naturally occurring curcumin, we hypothesized that chronic curcumin administration may inhibit ethanol-induced neurodegeneration and memory impairment in mice, by regulating reactive oxygen species (ROS), toll-like receptor-4 (TLR4), and Receptor for advanced glycation end products (RAGE)-mediated neuroinflammation, while p-JNK and p-NF-kB triggered the release of cytokines and synaptic dysfunction. The main target of the current study is oxidative stress, regulated by nuclear factor erythroid-2 (Nrf2; an endogenous antioxidant enzyme), and neuroinflammation, initiated by the activation of the innate immune response, mostly tailored by the activation of TLR4 and RAGE, playing a role in recognizing the microbial-associated molecular patterns initiating and modulating the immune response. The activation of the TLR4 signaling promotes the phosphorylation of NF-kB, thereby promoting the inflammatory effects [20]. The signals generated by both the RAGE and TLR4 receptors funnel to the same pathway of neuroinflammation [21]. Compounds counteracting the abnormal ROS generation, inhibition of TLR4/RAGE, and inhibition of the phosphorylation of JNK/NF-kB signaling may render protection to the brain against the mediators of neurodegeneration. Here, we have made an attempt to explore the underlying neuroprotective mechanisms of chronic curcumin administration against ethanol-induced neurodegeneration and memory impairment, by targeting the oxidative stress, neuroinflammation, and apoptotic cell death.

2. Materials and Methods 2.1. Chemicals and Antibodies

The antibodies used in the Western blot and immunofluorescence studies were anti-Nrf2 (sc-722), anti-HO1 (sc-136,961), anti-synaptosomal-associated protein 23 (SNAP-23) (sc-374,215), anti-PSD-95 (sc-71,933), anti-Syntaxin (sc-12,736), anti-tumor necrosis factor-α (TNF-α) (sc-52,746), anti-PARP-1 (sc-8007), TLR4 (sc-16240), Synaptophysin (sc-17750), anti-interleukin (IL)-1β (sc-32,294), anti-Bax (sc-7480), anti-Bcl2 (sc-7382), anti-p-NF-κB (sc-136,548), anti-Iba-1 (sc-32,725), anti-Glial fibrillary acidic protein (GFAP; sc-33,673), and anti-β- actin (sc-47,778) (Santa Cruz Biotechnology, Dallas, TX, USA). In addition, the anti-Cleaved Caspase-3 (#9664) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). For Syntaxin and β-actin, the antibodies were diluted in TBST (1:10000) (Santa Cruz Biotechnology, Dallas, TX, USA). Other primary antibodies were diluted in 1x TBST (1:1000), and Secondary anti-mouse HRP (Horseradish peroxidase) conjugated (Promega Ref# W402) and anti-rabbit HRP conjugated (Promega Ref# W401) were diluted 1:10,000 in 1× TBST and were purchased from Promega, (Fitchburg, WI, USA). TAK242, Resatorvid (CAS 243984-11-4), the specific inhibitor of TLR4. For the confocal microscopic studies, the secondary fluorescent antibodies used were goat anti-mouse (Ref# A11029) and goat anti-rabbit (Ref# 32732) diluted in 1× PBS.

2.2. Animals Grouping and Drugs Administration

Male mice (C57BL/6N, n = 60, mice 25 ± 3 g, eight old weeks) were acclimatized to the animal house environment for one week, at 12/12 h light/dark cycles at room temperature. The study was approved and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Division of Applied Life Science, Gyeongsang National University, South Korea (approval ID: 125). Efforts were made to minimize the number of animals used, as well as their suffering. The mice were randomly divided into three groups. (1) The control group mice were treated with an equal volume of vehicle intraperitoneally (physiological saline 0.1 mL/100 g/day) for six weeks, and the mice were freely allowed water and food ad-libitum. (2) The ethanol group mice were treated with an intraperitoneal injection of ethanol for six weeks (5 g/kg i.p., for six weeks, daily). (3) The ethanol and curcumin group mice were treated with curcumin 50 mg/kg for six weeks, daily. The doses of ethanol and curcumin were purely selected on the bases of previously published papers [22]. After the completion of the treatment and behavioral analysis, the mice were sacrificed.

2.3. Tissues Collections for Molecular and Morphological Analysis

For biochemical studies, the mice (8–10/group) were anesthetized, sacrificed, and the brain sections were separated. Next, the brain tissue was homogenized in a protein extraction solution (PRO-PREP^TM), according to the instructions (iNtRON Biotechnology, Inc., Sungnam, South Korea). After homogenization, the samples were centrifuged at 13,000 r.p.m. at 4 °C for 25 min. The supernatants were collected and stored at −80 °C.

For the morphological studies, the mice (seven to eight per group) were anesthetized and perfused transcardially with saline at a flow rate of 10 mL/min for 3 min, followed by perfusion with a 4% paraformaldehyde solution for 8 min using a peristaltic pump, as provided [23]. The brains were removed and fixed in 4% cold neutral buffer paraformaldehyde for 48 h, and cryoprotected by immersing into a 30% sucrose phosphate buffer for 48 h at 4 °C [23]. After that, the whole brain was frozen in an OCT (optimal cutting temperature compound) compound (Sakura, Torrance, CA, USA), and 14 µm sections were made in the coronal planes using a microtome (Leica cryostat CM 3050S, Nussloch, Germany). The sections were mounted on the probe-on plus charged slides (Fisher, Pittsburgh, PA, USA), and were stored at −70 °C for further analyses.

2.4. In Vitro Cell Culturing, Drug Treatment, Nuclear Factor-2 Erythroid-2 (Nrf2) Gene Silencing by Small Interfering RNA (siRNA) and Western Blot Analysis

The mouse hippocampal HT22 and murine BV2 microglial cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with FBS (Fetal bovine serum) (10%) and penicillin/streptomycin (1%) in a 5% CO2 incubator at 37 °C. After attaining a confluency of 70%, the cells were pretreated for 1 h with ethanol (100 µM), followed by curcumin (2 μM) or Nrf2 siRNA for 24 h, or TAK242 (TLR4 specific inhibitor). The Nrf2 gene was knocked down with Nrf2 siRNA at a concentration of 10 μM per transfection for 36 h, as directed (SC: 37049, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The transfection was conducted with lipofectamine™2000 reagent (Invitrogen, Waltham, MA, USA) when the cells culture reached to 75–80%. The control group cells were treated with 0.01% Dimethyl sulfoxide (DMSO).

2.5. Western Blot Analysis

Western blot was performed as described previously, with some modifications [24,25]. The proteins were loaded and separated by SDS–PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P^SQ, Transfer membrane, Merck Millipore, Burlington, MA, USA). The immunoreaction was carried out for 16 h at 4 °C using an appropriate ratio of the primary antibodies. After that, the membranes were washed with 1× TBST three times for 10 min, and reacted with a horseradish peroxidase-conjugated secondary antibody for 2 h, as appropriate. The expression of the respective proteins was detected using an ECL (Enhanced chemiluminescence) -detection reagent, according to the manufacturer’s instructions. The expressions of the different proteins were obtained on X-ray films and were scanned, and the optical densities of the bands were analyzed by densitometry, using the computer-based ImageJ software (version 1.50, NIH, https://imagej.nih.gov/ij/, Bethesda, MD, USA).

2.6. Immunofluorescence Staining

The fluorescence assay was performed as mentioned previously [26,27]. The slides were dried overnight at room temperature, washed with PBS (0.01 mM) for 8–10 min (two times), treated with proteinase K for 5 min, rinsed with PBS, and blocked with normal serum (2% goat/rabbit, as appropriate) in PBS, added with 0.1% Triton X-100. After that, the slides were incubated with primary antibodies overnight at 4 °C. The slides were then incubated with tetramethylrhodamine isothiocyanate–fluorescein isothiocyanate (FITC)-labeled secondary antibodies (antirabbit and antimouse, as appropriate), at room temperature for 95 min. The slides were covered using the fluorescent mounting medium. Images were taken using a confocal laser-microscope (FluoView FV 1000 MPE, Olympus, Tokyo, Japan). Integrated density was used for the quantification of the staining intensity and for the amount in the immunofluorescent microscopic image. ImageJ software ([email protected], https://imagej.nih.gov/ij/) was used to quantify the integrated density, which represents the sum of the pixel values in an image.

2.7. ROS Assay

To analyze the effects of curcumin against ethanol-induced oxidative stress, we conducted a ROS assay, as described previously [28,29]. The assay is based on the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate (CAS 4091-99-0, Santa Cruz Biotechnology, Dallas, TX, USA) to 2′ 7′ dichlorofluorescein (DCF). The brain homogenates were diluted with cold Lock’s buffer at 1:20, to yield the final concentration of 2.5 mg tissue/500 μL. The reaction mixture of Lock’s buffer (1 mL; pH ± 7.4), 0.2 mL of homogenate, and 10 mL of DCFH-DA (dichlorodihydrofluorescein diacetate) (5 mM) was incubated at room temperature for 15 min, to convert the DCFH-DA to the fluorescent product DCF. The conversion of DCFH-DA to the DCF was analyzed using a spectrofluorimeter (Promega, Fitchburg, WI, USA), with excitation at 484 nm and emission at 530 nm. For the background fluorescence (conversion of DCFH-DA in the absence of homogenate), parallel blanks were measured. The quantitative expression of ROS has been shown with a histogram.

2.8. Determination of Lipid Peroxidation

Lipid peroxidation (LPO) is used to assess oxidative stress. Free malondialdehyde (MDA), which is an indicator of LPO, was analyzed in the tissue homogenates, by using a lipid peroxidation (MDA) colorimetric/fluorometric assay kit (BioVision, San Francesco, CA, USA, Cat#739-100), according to the manufacturer’s instructions.

2.9. Morris Water Maze Test

The behavioral analysis was done by the MWM (Morris Water Maze) and Y-maze test (n = 12 mice/group), as performed previously [30,31]. The apparatus used for the analysis was made of a circular tank (100 cm in diameter, 40 cm in height), containing water (23 ± 1 °C) to a depth of 15.5 cm. The water was made opaque by adding a non-toxic white color. A platform made of white plastic was kept 1 cm below the water surface, at one quadrant. For five consecutive days, the mice were trained consecutively. The latency to escape from the water (searching the hidden platform) was calculated for each trial. On the sixth day, the probe test was conducted for the evaluation of the memory effects. The probe test was conducted by removing the platform and allowing the mice to explore the tank freely for one hour. The time spent by the mice in the target quadrant and the number of crossings over the position of the platform were recorded. The time spent in the target quadrant was taken to show the degree of memory changes. The data were recorded using video-tracking software (SMART Panlab, Harvard Apparatus, Holliston, MA, USA).

2.10. Y-Maze Test

The apparatus used for the evaluation of the Y-maze was made of black-painted wood, and the dimensions of the arms were 50 cm long and 20 cm height and 10 cm width, as already used by our group [32] and others [33]. The mice were allowed to move freely in the Y-maze, and for that, the mice were kept in the center of the apparatus for 8 min, three times. The arm entries were visualized and observed carefully. The successive entry of the mice into the arms was defined as spontaneous alternations. The alternation behaviors were considered as (successive triplet sets (entries into three different arms consecutively)/total number of arm entries (2)) × 100. A higher percentage (%) of spontaneous alternations was considered to be an indicator of the improved cognitive performance of the experimental mice, and vice versa.

2.11. Fluoro-Jade B Staining

The Fluoro-Jade B staining was done as described previously [28], and according to the instructions provided (Burlington, MA, USA, Cat #AG310, Lot #2159662). The slides were dried at room temperature for 24 h. Furthermore, the slides were dipped in a solution of 1% sodium hydroxide and 80% ethanol, for five min. After that, the slides were kept in 70% ethanol for 2 min and in D-water for 2 min, transferred into a potassium permanganate solution (0.06%) for 10 min, rinsed with D-water, and kept in 0.1% acetic acid solution and 0.01% Fluoro-Jade B solution for 20 min, washed with D-water, and dried for 10 min. The sections were covered using a DPX mounting medium, and the images were taken using a confocal laser microscope (FV 1000, Olympus, Tokyo, Japan). Integrated density was used for the quantification of the staining intensity and for the amount in the immunofluorescent microscopic image. ImageJ software ([email protected]., https://imagej.nih.gov/ij/) was used to quantify the integrated density, which represents the sum of the pixel values in an image.

2.12. Nissl Staining

Nissl staining was performed according to the previously used methods [34], so as to visualize the histological changes in the brain. The slides were washed with 0.01 M BPS, twice for 15 min, and stained with 0.5% cresyl violet solution (containing a few drops of glacial acetic acid) for 12 to 15 min. Then, the sections were washed with D-water and dehydrated in ethanols (70%, 95%, and 100%), and xylene was dropped on the slides and covered by using the non-fluorescent mounting medium. The neurodegeneration was visualized by light microscope, and the densities of the cells were counted by using ImageJ software.

2.13. Data Analysis and Statistics

The densities of the bands were analyzed by densitometry using the ImageJ software. We performed one-way ANOVA (Analysis of variance) with Tukey’s post-hoc test for comparisons among the different experimental groups. The data are presented as the “mean (SD)” of 7–10 mice per group, and are representative of three independent experiments. The calculations and graphs were generated by using Prism 6 software (GraphPad Software, San Diego, CA, USA). Ω is significantly different from the vehicle-treated, Φ is significantly different from the ethanol-treated group. Significance = Ω, p < 0.05, Φ, p < 0.05, and Ψ, p < 0.05.

3. Results 3.1. Chronic Administration of Curcumin Inhibits Ethanol-Induced Oxidative Stress in Mice Brains and In-Vitro HT22 Cells

Oxidative stress has been a significant player in ethanol [35] and other neurotoxins-induced neurodegenerative disorders [36,37]. To find the potential effects of curcumin against ethanol-induced oxidative stress, we performed ROS and LPO assays. According to our findings, curcumin inhibits the elevated level of LPO and ROS (Figure 1A,B). Similarly, the other main antioxidant genes, Nrf2/HO-1, were also analyzed in the experimental groups. According to our Western blot and confocal findings, the chronic co-administration of curcumin with ethanol inhibited the suppression of Nrf2/HO-1 in the mice brains, thereby preserving the endogenous antioxidant mechanism of the brains (Figure 2C,D). Furthermore, we observed a reduced expression of Nrf2 and its target genes HO-1, in the ethanol (100 µM) exposed HT22 cells, which was markedly reversed by curcumin (2 µM), thereby upregulating the expression of Nrf2 and HO-1 in the cells. Interestingly, curcumin could not upregulate the expression of Nrf2/HO-1 in the ethanol-treated cell lines, where the Nrf2 genes were knocked down by Nrf2 siRNA (Figure 1E), indicating that curcumin abrogated the elevated ROS, by upregulating the Nrf2 genes, and the downstream targets of Nrf2.

3.2. Chronic Administration of Curcumin Attenuates Ethanol-Induced Astrocytes and Microglia Activation in Ethanol-Treated Mouse Brains and In-Vitro Microglial Cells

Previous literature has shown that there is activated microglia and astrocytes with ethanol intoxication [4], which may promote neurological disorders such as AD and dementia [38]. TLR4, a receptor for innate immune response, has been shown to be upregulated with ethanol; another receptor is RAGE, which has been shown to be playing a similar role in the inflammatory signaling. Our findings demonstrate that there was a significant reduction in the activation of both of the receptors in the curcumin-treated group. The Glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor 1 (Iba-1) are assigned markers for the activities of astrocytes and microglia, respectively [28]. According to our Western blot and confocal microscopic results, there was an increased expression of GFAP and Iba-1 in the ethanol-treated mice brains, which was significantly rescued with the administration of curcumin (Figure 2). Furthermore, the in vitro findings also showed an enhanced expression of TLR4 and Iba-1 in the ethanol treated BV-2 microglial cells, which were significantly reduced in the curcumin and TAK242 (a specific inhibitor of TLR4) treated cells.

3.3. Chronic Administration of Curcumin Regulated Ethanol-Induced Inflammatory Markers in Mouse Brains

Previous studies have shown that ethanol intoxication is responsible for the activation of stress-markers and inducing inflammatory cytokines [39]. To explore whether these markers may be inhibited with the administration of curcumin, we analyzed the expression of stress and inflammatory markers, such as p-JNK, p-NF-κB, cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and tissue necrosis factor-α (TNF-α), in the mice brains. As shown here, treatment with ethanol elevated the expression of p-JNK, p-Nf-κB, IL-1β, COX-2, and TNF-α. However, the mice that were co-administered the curcumin and ethanol had a lower expression of these markers compared with the ethanol-treated group, supporting the hypothesis that curcumin inhibits the expression of inflammatory cytokines, thereby rendering protection to mice brains against ethanol-induced neurodegeneration. The Western blot results were further supported by the confocal microscopic analysis, which showed that curcumin inhibits the effects of ethanol against the activation of p-JNK in mice brains (Figure 3).

3.4. Chronic Administration of Curcumin Rescued Apoptotic Cell Death and Neurodegeneration

Studies have shown that there is an increased expression of apoptotic markers in ethanol-treated mice brains, which leads to apoptotic cell death and neuronal loss [4,40]. Keeping in mind the role of these apoptotic markers in ethanol-induced neurodegeneration, we analyzed the expression of apoptotic markers, including Bax and Bcl-2, Cleaved Caspase-3, and PARP-1 in mice brains. Per our Western blot results, there was an increased expression of pro-apoptotic markers Caspase-3, Bax, and PARP-1, and a decreased expression of Bcl-2 (anti-apoptotic marker) in the ethanol-treated mice brains. However, curcumin regulated the expression of these markers. Moreover, the immunofluorescence results indicated the increased expression of Caspase-3 and PARP-1 in the ethanol-treated mice brains, which were inhibited with chronic administration of curcumin (Figure 4).

3.5. Chronic Administration of Curcumin Rescued the Neuronal Cell Loss, as Assessed by Fluoro-Jade B and Nissl staining

Overall, the neuroprotective effects of curcumin were further confirmed by visualizing the morphology of hippocampal neurons, by using Nissl and FJB staining on the mice brains, as these staining protocols have extensively been used to visualize the morphology of neurons in different experimental settings [41,42]. According to FJB staining, there were increased FJB stained neurons in the ethanol-treated group, which was significantly inhibited with the administration of curcumin, as shown (Figure 5A). Similarly, in the Nissl staining, there was a decrease in the Nissl stained neurons in the ethanol injected group, which was significantly preserved with the administration of curcumin, as shown (Figure 5B), thereby confirming the hypothesis that curcumin plays a rescuing role against ethanol-induced neurodegeneration and memory impairment.

3.6. Chronic Administration of Curcumin Reversed Synaptic Dysfunction and Memory Impairment in the Ethanol-Treated Mouse Brain

Previous studies have shown that ethanol intoxication contributes to the loss of synaptic protein in animal models [39,43]. To explore the effects of curcumin on synaptic marker ethanol-treated mouse brains, we evaluated the expression of synaptic proteins via Western blot and confocal microscopy. According to our findings, the synaptic proteins, including PSD-95, synaptophysin, and SNAP-25, were significantly downregulated in the ethanol-treated mice brains compared with the control mice. Interestingly, curcumin significantly inhibited these effects. The confocal microscopic results of PSD-95 showed significant fluorescence in the curcumin- and ethanol-treated group compared with the ethanol alone group, showing that curcumin preserves the synaptic markers by regulating oxidative stress, neuroinflammation, and apoptotic cell death in mouse brains (Figure 6). Previously, it has been shown that chronic ethanol intoxication causes abnormalities in motor functions and impaired spatial learning and memory [39]. To show whether curcumin could rescue ethanol-induced memory impairment, we performed the MWM and Y-maze tests. First, we recorded the learning abilities of the mice (n = 12 mice/group) with the MWM test. We found that ethanol-treated mice had an enhanced latency to reach to the platform, and the mice that had received curcumin (50 mg/kg, i.p., six weeks) showed a decreased escape latency (Figure 6). Twenty-four hours after the fifth day of training, the platform was removed and the mice were allowed to swim freely. We found that the ethanol-treated mice spent less time in the target quadrant and showed less platform crossings, highlighting that ethanol induces memory impairments. Interestingly, curcumin improved the ethanol-induced memory impairments by increasing the total time spent in the target quadrant, and the number of crossings of the platform (Figure 6F,G). After that, we checked the spontaneous alternation behaviors (showing the spatial working memory or short-term memory) of mice (n = 12 mice/group) in the Y-maze test. In the ethanol-treated mice, there was a reduction in the spontaneous alternation behaviors compared with the saline-treated mice, showing a cognition decline in the ethanol treated mice. Interestingly, curcumin significantly increased the spontaneous alternation behaviors in the ethanol-treated mice compared with the ethanol-treated group (Figure 6H). Collectively, our findings showed that the co-administration of curcumin with ethanol significantly prevents cognitive dysfunctions in mice.

4. Discussion

The current study has the following main findings. (i) Curcumin has strong anti-oxidant potentials against ethanol-induced oxidative stress, in vivo mouse brains, and in vitro mouse hippocampal HT22 cells, and the antioxidant effects of curcumin are solely dependent on Nrf2. (ii) Curcumin has rescuing effects against ethanol induced activated astrocytes and microglia, in vivo and in vitro, as revealed by the reduced expression of TLR4/RAGE, GFAP, and Iba-1. (iii) Curcumin may rescue the mice brain from ethanol-induced apoptotic cell death, synaptic dysfunction, and memory impairment in mice brains. Several lines of studies have also reported the neuroprotective effects of curcumin against neuroinflammation and apoptotic cell death [44,45,46]. One study completely focused on oxidative stress mediated neuroinflammation [47]. However, they have not further extended their study to the main antioxidant enzymes, such as Nrf2/HO-1. Curcumin was administered intraperitoneally, and although it is considered a parenteral route of administration, the pharmacokinetics of substances administered intraperitoneally are more similar to those seen after oral administration. In both cases, the primary route of absorption is into the mesenteric vessels, which drain into the portal vein and pass through the liver [48].

Curcumin has long since been reported to be a potent antioxidant, but no studies have yet reported its effects on the endogenous antioxidant mechanisms (Nrf2/HO-1) and TLR4/RAGE mediated neurodegeneration. A wide range of effects are associated with ethanol intoxication, including oxidative stress, apoptotic neurodegeneration, excitotoxicity, and the disruption of cell to cell interactions [40,49]. The production of ROS may cause other serious consequences, such as an altered metabolism, deleterious structural modifications of proteins, DNA mishandlings, and altered mitochondrial homeostasis. Here, we report that ethanol-induced oxidative stress mediated neuroinflammation via Nrf-2/TLR4 signaling. The induction of oxidative stress with the administration of ethanol is in accordance with previous studies [50]. Interestingly, the in vitro findings also support the results, showing antioxidant effects against ethanol induced oxidative stress. For more confirmatory purposes, we used specific Nrf2 siRNA, and the findings showed that curcumin may relieve the elevated ROS level through Nrf2.

The other outcome of our study is that curcumin may inhibit the TLR4/RAGE triggered neuroinflammation in mice brains and in in vitro microglial cells. TLR4, which is mainly expressed in the microglial cells, induces microglial activation and the expression of proinflammatory cytokines, such as TNF-α and p-NF-kB, in response to a variety of stimuli [51]. Besides TLR4, other receptors are also involved in neurodegenerative conditions, such as RAGE. Here, we also evaluated the expression of RAGE in the experimental groups. According to our findings, there was an enhanced expression of TLR4/RAGE and its downstream inflammatory mediators in the ethanol-treated mice brain. Interestingly, these markers were significantly reduced with the administration of curcumin. For more confirmatory purposes, we used a specific TLR4 inhibitor (TAK242). Interestingly, the rescuing effects of curcumin against TLR4/iba-1 in the microglial cells were comparable to TAK 242. GFAP and Iba-1, which are the assigned markers for the activated astrocytes and microglia, were significantly upregulated in the ethanol-treated group, however, there was a significantly lower expression in the curcumin-treated group. The upregulation of GFAP and Iba1 in the ethanol-treated group is in accordance with the previous reports [4]. The inhibition of TLR4 further inhibited the expression of its downstream effectors (Iba-1) in the microglial cells. The upregulation of TLR4, RAGE, and the iba-1 in the ethanol-treated mice brains is in accordance with the previous reported effects of ethanol [52].

The other inducer of the neurodegenerative conditions are as follows: the phosphorylation of MAP kinases, which phosphorylates and translocates the NF-kB to the nucleus, playing a pivotal role in the release of inflammatory mediators [53]. According to our findings, there were enhanced expressions of p-JNK and p-NF-kB, as well as other inflammatory mediators (TNF-α, Cox-2, and IL-1β) in the ethanol treated mice, which is in accordance with previous studies [39]. Another main contributor to the neurodegeneration is apoptotic cell death in neurodegenerative conditions [54], as apoptotic cell death has closely been linked to the oxidative damage [55]. For the evaluation of the effects of curcumin against apoptotic cell death, the expression of proapoptotic (Bax, Caspase-3, and PARP-1) and antiapoptotic (Bcl-2) markers were evaluated; according to our findings, ethanol promoted proapoptotic cell death, which was significantly inhibited with the administration of curcumin. For more confirmatory purposes, we performed Nissl and FJB staining, which showed that curcumin significantly inhibited the neurotoxic effects of ethanol on mice brains. The ultimate consequence of neurodegeneration is synaptic dysfunction and loss of memory, which have been extensively reported with ethanol intoxication [56].

For the evaluation of synaptic dysfunction and memory impairment, we performed behavioral studies in Morris water maze and Y-maze tests, as well as the protein markers related to synaptotoxicity. According to our results, curcumin significantly improved the behavioral alterations and synaptic markers (postsynaptic density protein-95, SNAP-23, and synaptophysin) in the mice brain. The protective effects of curcumin on the behavioral changes and synaptic dysfunction are in accordance with previous studies [43,57].

5. Conclusions

Altogether, our findings support the hypothesis that the chronic administration of curcumin may protect mice brains against the detrimental effects of ethanol, by serving as a strong antioxidant, anti-inflammatory, and antiapoptotic agent in vivo mice brains and in vitro cells.

[Image omitted. See PDF.]

[Image omitted. See PDF.]

[Image omitted. See PDF.]

[Image omitted. See PDF.]

[Image omitted. See PDF.]

[Image omitted. See PDF.]

Author Contributions

M.I designed and conducted the experiments, and wrote manuscript; K.S., and M.S.K. conducted the experiments; A.K., T.M., M.G.J., and S.U.R. conducted the statistical analysis, reviewed and edited the manuscripts; M.O.K. supervised and organized the final version of the manuscript. All of the authors reviewed and approved the paper.

Funding

This research was supported by the Brain Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science-ICT (2016M3C7A1904391).

Conflicts of Interest

The authors declare no competing financial interests.

References

1. Kohnke, M.D. Approach to the genetics of alcoholism: A review based on pathophysiology. Biochem. Pharmacol. 2008, 75, 160-177.

2. Lee, J.W.; Lee, Y.K.; Lee, B.J.; Nam, S.Y.; Lee, S.I.; Kim, Y.H.; Kim, K.H.; Oh, K.W.; Hong, J.T. Inhibitory effect of ethanol extract of Magnolia officinalis and 4-O-methylhonokiol on memory impairment and neuronal toxicity induced by β-amyloid. Pharmacol. Biochem. Behav. 2010, 95, 31-40.

3. Badshah, H.; Kim, T.H.; Kim, M.J.; Ahmad, A.; Ali, T.; Yoon, G.H.; Naseer, M.I.; Kim, M.O. Apomorphine attenuates ethanol-induced neurodegeneration in the adult rat cortex. Neurochem. Int. 2014, 74, 8-15.

4. Saito, M.; Chakraborty, G.; Hui, M.; Masiello, K.; Saito, M. Ethanol-induced neurodegeneration and glial activation in the developing brain. Brain Sci. 2016, 6.

5. Naseer, M.I.; Ullah, I.; Narasimhan, M.L.; Lee, H.Y.; Bressan, R.A.; Yoon, G.H.; Yun, D.J.; Kim, M.O. Neuroprotective effect of osmotin against ethanol-induced apoptotic neurodegeneration in the developing rat brain. Cell Death Dis. 2014, 5, e1150.

6. von Haefen, C.; Sifringer, M.; Menk, M.; Spies, C.D. Ethanol enhances susceptibility to apoptotic cell death via down-regulation of autophagy-related proteins. Alcohol. Clin. Exp. Res. 2011, 35, 1381-1391.

7. Menetski, J.; Mistry, S.; Lu, M.; Mudgett, J.S.; Ransohoff, R.M.; Demartino, J.A.; Macintyre, D.E.; Abbadie, C. Mice overexpressing chemokine ligand 2 (CCL2) in astrocytes display enhanced nociceptive responses. Neuroscience 2007, 149, 706-714.

8. Lucas, S.M.; Rothwell, N.J.; Gibson, R.M. The role of inflammation in CNS injury and disease. Br. J. Pharmacol. 2006, 147 (Suppl. 1), S232-S240.

9. Manoharan, J.P.S.; Subramaniyan, A.V.D. Neuroprotective Effects of pterois volitans venom against alcohol induced oxidative dysfunction in Rats. J. Environ. Anal. Toxicol. 2015, 5.

10. Ojala, J.; Sutinen, E. The role of interleukin-18, oxidative stress and metabolic syndrome in Alzheimer's disease. J. Clin. Med. 2017, 6, 55.

11. Bent, S. Herbal medicine in the United States: Review of efficacy, safety, and regulation: Grand rounds at University of California, San Francisco Medical Center. J. Gen. Intern. Med. 2008, 23, 854-859.

12. Jun, Y.L.; Bae, C.H.; Kim, D.; Koo, S.; Kim, S. Korean Red Ginseng protects dopaminergic neurons by suppressing the cleavage of p35 to p25 in a Parkinson's disease mouse model. J. Ginseng Res. 2015, 39, 148-154.

13. Begum, A.N.; Jones, M.R.; Lim, G.P.; Morihara, T.; Kim, P.; Heath, D.D.; Rock, C.L.; Pruitt, M.A.; Yang, F.; Hudspeth, B.; et al. Curcumin structure-function, bioavailability, and efficacy in models of neuroinflammation and Alzheimer's disease. J. Pharmacol. Exp. Ther. 2008, 326, 196-208.

14. Sompamit, K.; Kukongviriyapan, U.; Nakmareong, S.; Pannangpetch, P.; Kukongviriyapan, V. Curcumin improves vascular function and alleviates oxidative stress in non-lethal lipopolysaccharide-induced endotoxaemia in mice. Eur. J. Pharmacol. 2009, 616, 192-199.

15. Ng, Q.; Soh, A.; Loke, W.; Venkatanarayanan, N.; Lim, D.; Yeo, W.-S. A meta-analysis of the clinical use of curcumin for irritable bowel syndrome (IBS). J. Clin. Med. 2018, 7, 298.

16. Weber, W.M.; Hunsaker, L.A.; Gonzales, A.M.; Heynekamp, J.J.; Orlando, R.A.; Deck, L.M.; Vander Jagt, D.L. TPA-induced up-regulation of activator protein-1 can be inhibited or enhanced by analogs of the natural product curcumin. Biochem. Pharmacol. 2006, 72, 928-940.

17. Ataie, A.; Sabetkasaei, M.; Haghparast, A.; Moghaddam, A.H.; Kazeminejad, B. Neuroprotective effects of the polyphenolic antioxidant agent, Curcumin, against homocysteine-induced cognitive impairment and oxidative stress in the rat. Pharmacol. Biochem. Behav. 2010, 96, 378-385.

18. Venkatesan, R.; Ji, E.; Kim, S.Y. Phytochemicals that regulate neurodegenerative disease by targeting neurotrophins: A comprehensive review. BioMed Res. Int. 2015, 2015, 814068.

19. Thiyagarajan, M.; Sharma, S.S. Neuroprotective effect of curcumin in middle cerebral artery occlusion induced focal cerebral ischemia in rats. Life Sci. 2004, 74, 969-985.

20. Nakano, Y.; Shimazawa, M.; Ojino, K.; Izawa, H.; Takeuchi, H.; Inoue, Y.; Tsuruma, K.; Hara, H. Toll-like receptor 4 inhibitor protects against retinal ganglion cell damage induced by optic nerve crush in mice. J. Pharmacol. Sci. 2017, 133, 176-183.

21. Frasnelli, S.C.; de Medeiros, M.C.; Bastos Ade, S.; Costa, D.L.; Orrico, S.R.; Rossa Junior, C. Modulation of immune response by RAGE and TLR4 signalling in PBMCs of diabetic and non-diabetic patients. Scand. J. Immunol. 2015, 81, 66-71.

22. Sorrenti, V.; Contarini, G.; Sut, S.; Dall'Acqua, S.; Confortin, F.; Pagetta, A.; Giusti, P.; Zusso, M. Curcumin prevents acute neuroinflammation and long-term memory impairment induced by systemic lipopolysaccharide in mice. Front. Pharmacol. 2018, 9, 183.

23. Jackson-Lewis, V.; Przedborski, S. Protocol for the MPTP mouse model of Parkinson's disease. Nat. Protoc. 2007, 2, 141-151.

24. Badshah, H.; Ullah, I.; Kim, S.E.; Kim, T.H.; Lee, H.Y.; Kim, M.O. Anthocyanins attenuate body weight gain via modulating neuropeptide Y and GABAB1 receptor in rats hypothalamus. Neuropeptides 2013, 47, 347-353.

25. Khan, A.; Ali, T.; Rehman, S.U.; Khan, M.S.; Alam, S.I.; Ikram, M.; Muhammad, T.; Saeed, K.; Badshah, H.; Kim, M.O. Neuroprotective effect of quercetin against the detrimental effects of LPS in the adult mouse brain. Front. Pharmacol. 2018, 9, 1383.

26. Badshah, H.; Kim, T.H.; Kim, M.O. Protective effects of anthocyanins against amyloid β-induced neurotoxicity in vivo and in vitro. Neurochem. Int. 2015, 80, 51-59.

27. Lee, Y.; Chun, H.J.; Lee, K.M.; Jung, Y.S.; Lee, J. Silibinin suppresses astroglial activation in a mouse model of acute Parkinson's disease by modulating the ERK and JNK signaling pathways. Brain Res. 2015, 1627, 233-242.

28. Amin, F.U.; Shah, S.A.; Kim, M.O. Vanillic acid attenuates Aβ1-42-induced oxidative stress and cognitive impairment in mice. Sci. Rep. 2017, 7, 40753.

29. Ikram, M.; Muhammad, T.; Rehman, S.U.; Khan, A.; Jo, M.G.; Ali, T.; Kim, M.O. Hesperetin confers neuroprotection by regulating Nrf2/TLR4/NF-κB signaling in an Aβ mouse model. In Molecular Neurobiology; Springer: Berlin, Germany, 2019.

30. Khan, M.; Shah, S.A.; Kim, M.O. 17β-Estradiol via SIRT1/Acetyl-p53/NF-kB signaling pathway rescued postnatal rat brain against acute ethanol intoxication. Mol. Neurobiol. 2017.

31. Lin, Y.-T.; Wu, Y.-C.; Sun, G.-C.; Ho, C.-Y.; Wong, T.-Y.; Lin, C.-H.; Chen, H.-H.; Yeh, T.-C.; Li, C.-J.; Tseng, C.-J. Effect of resveratrol on reactive oxygen species-induced cognitive impairment in rats with angiotensin II-induced early alzheimer's disease. J. Clin. Med. 2018, 7, 329.

32. Rehman, S.U.; Ahmad, A.; Yoon, G.-H.; Khan, M.; Abid, M.N.; Kim, M.O. Inhibition of c-Jun N-Terminal kinase protects against brain damage and improves learning and memory after traumatic brain injury in adult mice. Cereb. Cortex 2017.

33. Ayaz, M.; Junaid, M.; Ullah, F.; Subhan, F.; Sadiq, A.; Ali, G.; Ovais, M.; Shahid, M.; Ahmad, A.; Wadood, A.; et al. Anti-Alzheimer's studies on β-Sitosterol isolated from Polygonum hydropiper L. Front. Pharmacol. 2017, 8, 697.

34. Muhammad, T.; Ali, T.; Ikram, M.; Khan, A.; Alam, S.I.; Kim, M.O. Melatonin rescue oxidative stress-mediated neuroinflammation/neurodegeneration and memory impairment in scopolamine-induced amnesia mice model. J. Neuroimmune Pharmacol. 2018.

35. Bauer, A.K.; Fitzgerald, M.; Ladzinski, A.T.; Lenhart Sherman, S.; Maddock, B.H.; Norr, Z.M.; Miller, R.R., Jr. Dual behavior of N-acetylcysteine during ethanol-induced oxidative stress in embryonic chick brains. Nutr. Neurosci. 2017, 20, 478-488.

36. Jo, M.G.; Ikram, M.; Jo, M.H.; Yoo, L.; Chung, K.C.; Nah, S.Y.; Hwang, H.; Rhim, H.; Kim, M.O. Gintonin mitigates MPTP-induced loss of nigrostriatal dopaminergic neurons and accumulation of alpha-synuclein via the Nrf2/HO-1 pathway. Mol. Neurobiol. 2018.

37. Ali, T.; Kim, T.; Rehman, S.U.; Khan, M.S.; Amin, F.U.; Khan, M.; Ikram, M.; Kim, M.O. Natural dietary supplementation of anthocyanins via PI3K/Akt/Nrf2/HO-1 pathways mitigate oxidative stress, neurodegeneration, and memory impairment in a mouse model of alzheimer's disease. Mol. Neurobiol. 2018, 55, 6076-6093.

38. Fernandez-Lizarbe, S.; Pascual, M.; Guerri, C. Critical role of TLR4 response in the activation of microglia induced by ethanol. J. Immunol. 2009, 183, 4733-4744.

39. Shah, S.A.; Yoon, G.H.; Kim, M.O. Protection of the developing brain with anthocyanins against ethanol-induced oxidative stress and neurodegeneration. Mol. Neurobiol. 2015, 51, 1278-1291.

40. Crews, F.T.; Nixon, K. Mechanisms of neurodegeneration and regeneration in alcoholism. Alcohol. Alcohol. 2009, 44, 115-127.

41. Damjanac, M.; Rioux Bilan, A.; Barrier, L.; Pontcharraud, R.; Anne, C.; Hugon, J.; Page, G. Fluoro-Jade B staining as useful tool to identify activated microglia and astrocytes in a mouse transgenic model of Alzheimer's disease. Brain Res. 2007, 1128, 40-49.

42. Ullah, N.; Naseer, M.I.; Ullah, I.; Kim, T.H.; Lee, H.Y.; Kim, M.O. Neuroprotective profile of pyruvate against ethanol-induced neurodegeneration in developing mice brain. Neurol. Sci. 2013, 34, 2137-2143.

43. Muhammad, T.; Ikram, M.; Ullah, R.; Rehman, S.U.; Kim, M.O. Hesperetin, a citrus flavonoid, attenuates lps-induced neuroinflammation, apoptosis and memory impairments by modulating TLR4/NF-κB signaling. Nutrients 2019, 11.

44. Fan, C.; Song, Q.; Wang, P.; Li, Y.; Yang, M.; Yu, S.Y. Neuroprotective effects of curcumin on IL-1β-induced neuronal apoptosis and depression-like behaviors caused by chronic stress in rats. Front. Cell. Neurosci. 2018, 12, 516.

45. Xu, L.; Ding, L.; Su, Y.; Shao, R.; Liu, J.; Huang, Y. Neuroprotective effects of curcumin against rats with focal cerebral ischemia-reperfusion injury. Int. J. Mol. Med. 2019.

46. Cole, G.M.; Teter, B.; Frautschy, S.A. Neuroprotective effects of curcumin. In The Molecular Targets and Therapeutic Uses of Curcumin in Health and Disease; Springer: Boston, MA, USA, 2007; pp. 197-212.

47. Tiwari, V.; Chopra, K. Protective effect of curcumin against chronic alcohol-induced cognitive deficits and neuroinflammation in the adult rat brain. Neuroscience 2013, 244, 147-158.

48. Turner, P.V.; Brabb, T.; Pekow, C.; Vasbinder, M.A. Administration of substances to laboratory animals: Routes of administration and factors to consider. J. Am. Assoc. Lab. Anim. Sci. 2011, 50, 600-613.

49. Goldowitz, D.; Lussier, A.A.; Boyle, J.K.; Wong, K.; Lattimer, S.L.; Dubose, C.; Lu, L.; Kobor, M.S.; Hamre, K.M. Molecular pathways underpinning ethanol-induced neurodegeneration. Front. Genet. 2014, 5, 203.

50. Comporti, M.; Signorini, C.; Leoncini, S.; Gardi, C.; Ciccoli, L.; Giardini, A.; Vecchio, D.; Arezzini, B. Ethanol-induced oxidative stress: Basic knowledge. Genes Nutr. 2010, 5, 101-109.

51. McColl, B.W.; Allan, S.M.; Rothwell, N.J. Systemic infection, inflammation and acute ischemic stroke. Neuroscience 2009, 158, 1049-1061.

52. Crews, F.T.; Vetreno, R.P. Mechanisms of neuroimmune gene induction in alcoholism. Psychopharmacology 2016, 233, 1543-1557.

53. Saha, R.N.; Jana, M.; Pahan, K. MAPK p38 regulates transcriptional activity of NF-κB in primary human astrocytes via acetylation of p65. J. Immunol. 2007, 179, 7101-7109.

54. Radi, E.; Formichi, P.; Battisti, C.; Federico, A. Apoptosis and oxidative stress in neurodegenerative diseases. J. Alzheimers Dis. 2014, 42 (Suppl. 3), S125-S152.

55. Khan, A.; Ikram, M.; Muhammad, T.; Park, J.; Kim, M.O. Caffeine modulates cadmium-induced oxidative stress, neuroinflammation, and cognitive impairments by regulating Nrf-2/HO-1 in vivo and in vitro. J. Clin. Med. 2019, 8, 680.

56. Wang, P.; Luo, Q.; Qiao, H.; Ding, H.; Cao, Y.; Yu, J.; Liu, R.; Zhang, Q.; Zhu, H.; Qu, L. The neuroprotective effects of carvacrol on ethanol-induced hippocampal neurons impairment via the antioxidative and antiapoptotic pathways. Oxid. Med. Cell. Longev. 2017, 2017, 4079425.

57. Wang, X.S.; Zhang, Z.R.; Zhang, M.M.; Sun, M.X.; Wang, W.W.; Xie, C.L. Neuroprotective properties of curcumin in toxin-base animal models of Parkinson's disease: A systematic experiment literatures review. BMC Complement. Altern. Med. 2017, 17, 412.