# Dietary Supplementation of the Antioxidant Curcumin Halts Systemic LPS-Induced Neuroinflammation-Associated Neurodegeneration and Memory/Synaptic Impairment via the JNK/NF-κB/Akt Signaling Pathway in Adult Rats

Authors: Muhammad Sohail Khan; Tahir Muhammad; Muhammad Ikram; Myeong Ok Kim
Journal: Oxidative Medicine and Cellular Longevity
 (2019)

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## Abstract

Curcumin is a natural polyphenolic compound widely known to have antioxidant, anti-inflammatory, and antiapoptotic properties. In the present study, we explored the neuroprotective effect of curcumin against lipopolysaccharide- (LPS-) induced reactive oxygen species- (ROS-) mediated neuroinflammation, neurodegeneration, and memory deficits in the adult rat hippocampus via regulation of the JNK/NF-κB/Akt signaling pathway. Adult rats were treated intraperitoneally with LPS at a dose of 250 μg/kg for 7 days and curcumin at a dose of 300 mg/kg for 14 days. After 14 days, the rats were sacrificed, and western blotting and ROS and lipid peroxidation assays were performed. For immunohistochemistry and confocal microscopy, the rats were perfused transcardially with 4% paraformaldehyde. In order to verify the JNK-dependent neuroprotective effect of curcumin and to confirm the in vivo results, HT-22 neuronal and BV2 microglial cells were exposed to LPS at a dose of 1 μg/ml, curcumin 100 μg/ml, and SP600125 (a specific JNK inhibitor) 20 μM. Our immunohistochemical, immunofluorescence, and biochemical results revealed that curcumin inhibited LPS-induced oxidative stress by reducing malondialdehyde and 2,7-dichlorofluorescein levels and ameliorating neuroinflammation and neuronal cell death via regulation of the JNK/NF-κB/Akt signaling pathway both in vivo (adult rat hippocampus) and in vitro (HT-22/BV2 cell lines). Moreover, curcumin markedly improved LPS-induced memory impairment in the Morris water maze and Y-maze tasks. Taken together, our results suggest that curcumin may be a potential preventive and therapeutic candidate for LPS-induced ROS-mediated neurotoxicity and memory deficits in an adult rat model.


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## Body

## 1. Introduction

Neuroinflammation is a protective mechanism, which occurs inside the brain and is the primary response to injury. If neuroinflammatory responses are prolonged, however, this can lead to neuronal dysfunction and eventually result in neuronal apoptosis and memory impairments [1, 2]. Increasing evidence indicates that neuroinflammation caused by toxic reactions or disturbances in the homeostasis of antioxidants plays an essential role in the pathogenesis of neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease (AD) [3–5]. Reactive oxygen species (ROS) have been previously identified as potent mediators of neurodegenerative disorders. They have been shown to affect protein, lipids, and nucleic acids, paving the way for cellular dysfunction and neuronal apoptosis. Emerging evidence has suggested that oxidative stress encourages the activation of stress kinases like phosphorylated-c-Jun N-terminal kinase 1 (p-JNK), an important mediator of neuroinflammation and neurodegeneration. Chronic oxidative stress and neuroinflammation are thus key contributors to the advancement of diseases like AD [2, 6].Several lines of investigation have demonstrated that lipopolysaccharide (LPS) and other toxic agents such as ethanol and d-galactose activate numerous ROS-mediated neuroinflammatory and apoptotic signaling pathways that in turn lead to neurodegeneration and memory deficits [7–9]. LPS is a bacterial endotoxin which triggers neuroinflammation and is used as an inflammagen in various animal model studies to evaluate the interaction between inflammation, brain function, and memory impairments. It is well known that LPS treatment leads to an increase in the generation of ROS and cytokine production, ultimately resulting in neuronal cell death and memory impairments [1, 10, 11]. Various mechanisms have been studied and proposed for LPS-induced neuronal damage, the most well-established of which is the increased generation of ROS, subsequently augmenting oxidative stress and neuronal damage. Furthermore, an elevated level of ROS can activate other mediators like phosphorylated-nuclear factor kappa B (p-NF-κB), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), affecting the function of neuronal cells in the hippocampus via neuronal apoptosis and subsequently resulting in memory impairments. Moreover, according to recent studies, LPS given intraperitoneally can activate astrocytes and microglial cells, inducing the processes of astrogliosis and microgliosis, respectively. Activation of both astrocytes and microglia induces neurotoxicity, neuroinflammation, and the production of ROS by stimulating proinflammatory mediators [7, 12–16]. Badshah et al. demonstrated the systemic administration of LPS to adult mice at a dose of 250 μg/kg for 1 week-induced neuronal cell death by increasing the expression of caspase-3 and poly [ADP-ribose] polymerase 1 (PARP-1), triggering the mitochondrial apoptotic pathway [1].Several antioxidants are known for their effectiveness against oxidative stress-mediated neuronal cell death and memory disorders. Most of these have been reported to cross the blood-brain-barrier and reduce the deleterious effects of various toxic molecules such as oxygen and nitrogen free radicals in brain cells. Natural sources of polyphenolic compounds include plants, vegetables, fruits, green tea, olive oil, and red wine [17, 18]. Although no preventative treatments are currently available, studies into neurodegenerative disorders have suggested that lifestyle changes, exercise, and a daily intake of natural polyphenol supplements may help to prevent these diseases. Curcumin, obtained from the rhizomes of the Curcuma longa plant, is a natural yellowish compound which has been used for centuries for the treatment of neuropathological disorders because of its antioxidant and anti-inflammatory properties. Curcumin not only acts as a free radical scavenger but also improves learning and memory deficits. Studies involving curcumin have reported that it has a neuroprotective effect, and it has been shown to counteract AD, depression, and seizures [19–21]. Furthermore, curcumin has also been shown to have a protective role in other conditions such as cancer, pancreatitis, rheumatoid arthritis, liver disease, and pulmonary dysfunction [22, 23]. In addition, curcumin protects the brain from LPS toxicity by inhibiting the production of nitric oxide (NO), prostaglandin E2 (PGE2), ROS, and proinflammatory cytokines [24–26].Although it is known that curcumin has anti-inflammatory and neuroprotective properties, there exists little evidence about its exact underlying mechanism of action, particularly in the context of ROS-mediated neuroinflammation, neurodegeneration, and memory impairments. Therefore, we conducted this study to explore the effects of curcumin against LPS-induced hippocampal microglial activation, neuroinflammation, neurodegeneration, and memory impairments. Our results confirmed that curcumin not only has potent antioxidant, anti-inflammatory, and antiapoptotic properties but also protects against LPS-induced ROS-mediated neuroinflammation, neurodegeneration, and memory impairments. Our results further confirm that natural compounds like curcumin could potentially be used as alternatives to synthetic and semisynthetic drugs for the treatment of neurodegenerative disorders.


## 2. Materials and Methods

### 2.1. Chemicals

The LPS, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), dimethyl sulfoxide (DMSO), and JNK inhibitor (SP600125) used in the experiments were all purchased from Sigma-Aldrich Chemical Co (St. Louis, MO, USA).


### 2.2. Animals

Male Sprague-Dawley rats weighing 250-300 grams were purchased from Samtako Bio (Osan, Republic of Korea). All animals were kept in the university animal house on a 12/12 h light and dark cycle at room temperature and humidity for a week before the start of experimentation to allow acclimatization to the new environment. Animals were allowed to feed ad libitum. Rats were handled carefully according to the guidelines provided by the Animal Ethical Committee of the Gyeongsang National University, South Korea (Approval ID: 125).


### 2.3. Grouping and Treatment of Experimental Animals

Animals were randomly divided into the following three groups (n=15 for each):
(i)
Control (Cont.) group: injected with normal saline for 1 week
(ii)
LPS-treated group: intraperitoneally injected with LPS dissolved in normal saline for 1 week at a dose of 250μg/kg/day, as reported previously [1]
(iii)
LPS+curcumin-treated group: injected with LPS as above and curcumin (300 mg/kg/day for 14 days, as reported [3]) 7 days before and after LPS treatmentCurcumin was dissolved in DMSO, and the final injectable volume was prepared using normal saline. The LPS was dissolved in the same volume of normal saline used for curcumin.


### 2.4. In Vivo ROS Assay

For the determination of the ROS level inside the hippocampus (n=5 per group), we performed the ROS assay. This assay was performed as previously described [12] and was based on the alteration of DCFH-DA to 2,7-dichlorofluorescein (DCF). Briefly, brain homogenates from the hippocampus were diluted with Lock’s buffer at a 1 : 20 ratio, and the final concentration was adjusted to 2.5 mg tissue per 500 μl. The reaction mixture comprised Lock’s buffer (pH 7.4), brain homogenates from hippocampal tissue (0.2 μl), and DCFH-DA (10 μl, 5 mM). The mixture was covered and incubated for 15 min at room temperature. After the incubation, the fluorescent product DCF was measured by a microplate reader (excitation at 484 nm and emission at 530 nm). A blank parallel was used first to evaluate background signal. Results were expressed as pmol DCF formed/min/mg of protein in the tissue homogenate.


### 2.5. In Vitro ROS Assay

The same procedure used in vivo was then used for the quantification of ROS in the BV2 microglial cells. The in vitro ROS assay was also based on the conversation of DCFH-DA to DCF. The microglial BV2 cells used in the in vitro studies were kindly provided as a gift from Dr. I. W. Choi (Inje University, Busan, Republic of Korea). The BV2 microglial cells were seeded in a 75 cm2 Nunc™ EasYFlask™ with a Nunclon™ Delta surface (Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) containing Dulbecco’s modified Eagle medium (DMEM) (Life Technologies, Carlsbad, CA, US) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin) at 37°C in humidified air containing 5% CO2. The cells were grown, counted, and further subcultured in 35 mm Petri dishes (Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) in DMEM supplemented with 10% FBS and 1% antibiotics (penicillin-streptomycin) at 37°C in humidified air containing 5% CO2. When the cells reached 70-80% confluence, they were treated with LPS (1 μg/ml) as reported previously [1], LPS (1 μg/ml)+curcumin (100 μg/ml) as reported previously [6], and LPS+SP600125 (20 μM) as reported previously [7] for 24 hours. For ROS analysis, cells were exposed to DCFH-DA (50 μM) at 37°C for 30 min. The absorbance for ROS-positive cells was measured at 484/530 nm.


### 2.6. In Vivo Lipid Peroxidation (LPO) Assay

To evaluate oxidative stress, we performed the LPO assay. An LPO Assay Kit (CAS 4091-99-0, Santa Cruz Biotechnology, Dallas, TX, USA) was used for evaluating the level of free malondialdehyde (MDA) in the rat hippocampal tissue, as performed previously [12]. The LPO assay was performed as instructed by the manufacturer. In brief, the hippocampal tissues were homogenized on ice in 300 μl of the MDA lysis buffer with 3 μl BHT and centrifuged (13,000×g, 10 min). A total of 10 mg of protein was precipitated by homogenizing the sample in 150 μl dH2O+3 μl BHT, adding 1 vol of 2 N perchloric acid, vertexing, and then centrifuging to remove the precipitated protein. The final volume (200 μl) of the supernatant from each sample was then introduced into a 96-well plate, and the absorbance was measured using a microplate reader at 532 nm. The total MDA content was expressed as nmol/mg of protein in the tissue homogenate.


### 2.7. In Vitro LPO Assay

The LPO assay was also used for evaluating oxidative stress in BV2 cells using the LPO assay kit (BioVision, San Francisco, CA, USA; Cat#739-100). BV2 cells were grown and treated in the same way as previously mentioned in the methodology of the in vitro ROS assay. LPO analysis was performed as mentioned for in vivo experiments.


### 2.8. Protein Extraction

When the in vivo treatment was completed, all animals were sacrificed under anesthesia, and the brains were carefully removed to avoid any damage. The hippocampal tissues were carefully separated, kept in liquid nitrogen, and stored at -80°C for a further biochemical experimental work. When required, the hippocampal tissues were homogenized in pre-prep extraction solution (iNtRON Biotechnology, Inc., Sungnam, South Korea) and centrifuged at 13,000 rpm at 4°C for 25 min. After homogenization, the supernatant was collected and stored at −80°C.


### 2.9. Quantitative Analysis of Proteins by Western Blotting

Before western blotting, the optical densities (OD) of proteins were analyzed using the Bio-Rad protein assay kit (Bio-Rad Laboratories, CA, USA), as described previously [14]. Equal amounts of protein, approximately 20–30 μg, were used for electrophoresis using 4–12% Bolt™ Mini Gels (Life Technologies, Carlsbad, CA, US). Proteins were transferred to PVDF (polyvinylidene fluoride) membranes (Sigma-Aldrich Chemical Co.). The membranes were treated with 5% skim milk (w/v) for 90 min, washed with TBST (3 times, 10 min each), and treated with primary antibodies (anti-caspase-3, anti-TNF-α, anti-IL-1β, anti-p-JNK, anti-p-NF-κB65, anti-Bcl-2-associated X protein (Bax), anti-B-cell lymphoma 2 (Bcl-2), anti-cytochrome c (Cyt c), anti-PARP-1, anti-phosphorylated-glycogen synthase kinase 3 (p-GSK3β) (Ser 9), anti-phosphorylated protein kinase B (p-Akt) (Ser 473), anti-synaptosomal associated protein 25 (SNAP-25), anti-postsynaptic density protein 95 (PSD95), and anti-β-actin, all from Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibodies for 1 h. The membranes were washed with TBST (3 times, 10 min each), treated with a chemiluminescence system (Atto Corporation Tokyo, Japan), and protein bands were then detected on an X-ray film. The protein bands were analyzed by ImageJ software (version 1.50, NIH, https://imagej.nih.gov/ij/, Bethesda, MD, USA) which was used to quantify the integrated density.


### 2.10. Morphological Analysis and Sample Preparation

When the drug treatment and behavioral studies were completed, the animals were perfused transcardially with 4% ice-cold paraformaldehyde, as previously described [7, 12]. In short, the brain was fixed in 4% paraformaldehyde for 72 h and then transferred to 20% sucrose and stored for another 72 h. Following this, the brains were washed with fresh PBS and immediately frozen in O.C.T. Compound (Sakura Finetek USA, Inc., Torrance, CA, USA). Upon solidification of the blocks, 14 μm coronal sections of the hippocampus were cut using a CM 3050C cryostat (Leica, Germany). The sections were thaw-mounted on ProbeOn Plus™ charged slides (Thermo Fisher Scientific, Nunc a/S, Roskilde, Denmark).


### 2.11. Immunofluorescence Staining

Immunofluorescence staining was performed following a previously described protocol [12, 14]. In brief, brain tissue slides were dried overnight, washed twice with PBS (0.01 M) for 10 min, and blocked with blocking serum (2% normal goat serum and 0.3% Triton X-100 in PBS) for 1 hour. The slides were then incubated with the aforementioned primary antibodies (Santa Cruz Biotechnology) overnight at 4°C. The next day, the slides were treated with secondary TRITC/FITC-labelled antibodies (1,100) (Santa Cruz Biotechnology) for 2 h. Slides were washed with PBS (twice for 5 min) and mounted with 4′,6′-diamidino-2-phenylindole (DAPI) and Prolong Antifade Reagent (Molecular Probe, Eugene, OR, USA). Finally, the stained slides were examined under a confocal laser-scanning FluoView FV 1000 MPE microscope (Olympus, Tokyo, Japan).


### 2.12. Fluoro-Jade B (FJB) Staining

Briefly, the tissue slides were dried overnight in a drying chamber and washed with PBS (0.01 M) twice for 5 min. Slides were dipped in a solution containing 1% sodium hydroxide and 80% ethanol for 5 min. Then the slides were kept in 70% alcohol followed by keeping in distilled water for 2 min. Following that, the tissue slides were treated with a solution containing 0.1% acetic acid and 0.06% FJB for 20 min. Slides were washed and left to dry for 10 min, mounted with DPX mounting medium, and coverslips were applied. Slides were examined under a confocal laser-scanning FluoView FV 1000 MPE microscope (Olympus, Tokyo, Japan), and images were taken. Results were analyzed with ImageJ software, and quantification of the immunohistofluorescence and FJB images was performed according to our recently published protocol [7].


### 2.13. TUNEL Staining

TUNEL (terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick-end labeling) staining is used to detect dead neurons and evaluate the extent of neuronal apoptosis. The kit used in this assay was purchased from Sigma-Aldrich Chemical Co. (Cat. No. 11684809910, St. Louis, MO, USA), and the staining was performed according to the manufacturer’s recommendations.


### 2.14. Hematoxylin and Eosin (H&E) Staining

H&E staining is used to analyze cell morphology. In brief, slides from each group (n=5) were first dipped in tap water for a short time and transferred to staining solution. The slides were then transferred to the hematoxylin solution for 8-10 min. Next, the sides were washed with running water for 10-15 min and transferred to the eosin solution for 30 sec. Following this, the slides were then dehydrated with a graded series of alcohol. Finally, all the slides were mounted with mounting medium (Thermo Fisher Scientific, MA, USA) and coverslips applied. Images of the slides were taken using a simple microscope.


### 2.15. Cresyl Violet Staining

Cresyl violet/Nissl staining is used for the examination and determination of neuronal cell death. First, slides comprising 14μm brain sections were washed with PBS (0.01 M) for 15 min and stained with a solution of Cresyl violet (0.5%) containing a few drops of glacial acetic acid for 10-15 min. Slides were washed with distilled water, dehydrated with a graded series of alcohol (70, 95, and 100%), retained in xylene; nonfluorescent mounting medium was used, and coverslips were applied. Images were taken using a fluorescent light microscope, and quantification of the immunohistochemical images was performed.


### 2.16. Morris Water Maze (MWM) and Y-Maze Tests

For the behavioral studies, a MWM test was conducted on the rats (n=10/group), as described previously [12]. The MWM comprises a circular tank (100 cm in diameter, 40 cm in height) containing water (23±1°C) filled to a depth of 15.5 cm. White ink was added to the water to make it look opaque. A transparent escape platform (10 cm in diameter, 14.4 cm in height) was kept at the midpoint of one quadrant, hidden 1 cm below the water level. Rats were trained for 5 days before the start of the study using a single hidden platform in one quadrant with three quadrants of rotational starting. The escape latency (the time taken to look for and locate the hidden platform) was calculated for every trial. After 24 h of the 5th day, a probe test was then performed for the evaluation of memory consolidation. The platform was removed, and the rats were allowed to swim freely for 60 sec. Then, the length of time spent in the target quadrant and the number of times the rat crossed over the platform location (the platform remained hidden during the training) were recorded. The total time spent by a rat in the target quadrant was considered to be a measure of the degree of memory consolidation. SMART video-tracking software (Panlab Harvard Apparatus, Holliston, MA, USA) was used to record the movement of the rats. The Y-maze test was performed as described previously with necessary changes [14].


### 2.17. In Vitro Cell Culturing and Treatment for Western Blotting and Confocal Microscopy

The HT-22 neuronal cells used in the in vitro studies were kindly provided by Prof. Koh (Gyeongsang National University, South Korea). The HT-22 cells were seeded in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics in a humidified 5% CO2 incubator at 37°C. The HT-22 cell were incubated with LPS (1 μg/ml), LPS (1 μg/ml)+Cur (100 μg/ml) and LPS (1 μg/ml)+SP600125 (20 μM) for 24 h.


### 2.18. Statistical Analyses

Differences between the control, LPS, and LPS+curcumin groups were analyzed using the one-way analysis of variance (ANOVA) and Student’st-test. All data are expressed as the mean±SEM for the three independent experiments and were analyzed using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, US). A P value < 0.05 was considered statistically significant. For the in vivo study, the ∗ symbol denotes a significant difference between the control and LPS groups and the Ώ symbol denotes a significant difference between the LPS and curcumin groups. Likewise, for the in vitro study, the ∗ symbol denotes a significant difference between the control and LPS groups, the Ώ symbol denotes a significant difference between the LPS and curcumin groups, and the # symbol denotes a significant difference between the LPS and JNK inhibitor SP600125 groups.


## 2.1. Chemicals

The LPS, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), dimethyl sulfoxide (DMSO), and JNK inhibitor (SP600125) used in the experiments were all purchased from Sigma-Aldrich Chemical Co (St. Louis, MO, USA).


## 2.2. Animals

Male Sprague-Dawley rats weighing 250-300 grams were purchased from Samtako Bio (Osan, Republic of Korea). All animals were kept in the university animal house on a 12/12 h light and dark cycle at room temperature and humidity for a week before the start of experimentation to allow acclimatization to the new environment. Animals were allowed to feed ad libitum. Rats were handled carefully according to the guidelines provided by the Animal Ethical Committee of the Gyeongsang National University, South Korea (Approval ID: 125).


## 2.3. Grouping and Treatment of Experimental Animals

Animals were randomly divided into the following three groups (n=15 for each):
(i)
Control (Cont.) group: injected with normal saline for 1 week
(ii)
LPS-treated group: intraperitoneally injected with LPS dissolved in normal saline for 1 week at a dose of 250μg/kg/day, as reported previously [1]
(iii)
LPS+curcumin-treated group: injected with LPS as above and curcumin (300 mg/kg/day for 14 days, as reported [3]) 7 days before and after LPS treatmentCurcumin was dissolved in DMSO, and the final injectable volume was prepared using normal saline. The LPS was dissolved in the same volume of normal saline used for curcumin.


## 2.4. In Vivo ROS Assay

For the determination of the ROS level inside the hippocampus (n=5 per group), we performed the ROS assay. This assay was performed as previously described [12] and was based on the alteration of DCFH-DA to 2,7-dichlorofluorescein (DCF). Briefly, brain homogenates from the hippocampus were diluted with Lock’s buffer at a 1 : 20 ratio, and the final concentration was adjusted to 2.5 mg tissue per 500 μl. The reaction mixture comprised Lock’s buffer (pH 7.4), brain homogenates from hippocampal tissue (0.2 μl), and DCFH-DA (10 μl, 5 mM). The mixture was covered and incubated for 15 min at room temperature. After the incubation, the fluorescent product DCF was measured by a microplate reader (excitation at 484 nm and emission at 530 nm). A blank parallel was used first to evaluate background signal. Results were expressed as pmol DCF formed/min/mg of protein in the tissue homogenate.


## 2.5. In Vitro ROS Assay

The same procedure used in vivo was then used for the quantification of ROS in the BV2 microglial cells. The in vitro ROS assay was also based on the conversation of DCFH-DA to DCF. The microglial BV2 cells used in the in vitro studies were kindly provided as a gift from Dr. I. W. Choi (Inje University, Busan, Republic of Korea). The BV2 microglial cells were seeded in a 75 cm2 Nunc™ EasYFlask™ with a Nunclon™ Delta surface (Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) containing Dulbecco’s modified Eagle medium (DMEM) (Life Technologies, Carlsbad, CA, US) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin) at 37°C in humidified air containing 5% CO2. The cells were grown, counted, and further subcultured in 35 mm Petri dishes (Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) in DMEM supplemented with 10% FBS and 1% antibiotics (penicillin-streptomycin) at 37°C in humidified air containing 5% CO2. When the cells reached 70-80% confluence, they were treated with LPS (1 μg/ml) as reported previously [1], LPS (1 μg/ml)+curcumin (100 μg/ml) as reported previously [6], and LPS+SP600125 (20 μM) as reported previously [7] for 24 hours. For ROS analysis, cells were exposed to DCFH-DA (50 μM) at 37°C for 30 min. The absorbance for ROS-positive cells was measured at 484/530 nm.


## 2.6. In Vivo Lipid Peroxidation (LPO) Assay

To evaluate oxidative stress, we performed the LPO assay. An LPO Assay Kit (CAS 4091-99-0, Santa Cruz Biotechnology, Dallas, TX, USA) was used for evaluating the level of free malondialdehyde (MDA) in the rat hippocampal tissue, as performed previously [12]. The LPO assay was performed as instructed by the manufacturer. In brief, the hippocampal tissues were homogenized on ice in 300 μl of the MDA lysis buffer with 3 μl BHT and centrifuged (13,000×g, 10 min). A total of 10 mg of protein was precipitated by homogenizing the sample in 150 μl dH2O+3 μl BHT, adding 1 vol of 2 N perchloric acid, vertexing, and then centrifuging to remove the precipitated protein. The final volume (200 μl) of the supernatant from each sample was then introduced into a 96-well plate, and the absorbance was measured using a microplate reader at 532 nm. The total MDA content was expressed as nmol/mg of protein in the tissue homogenate.


## 2.7. In Vitro LPO Assay

The LPO assay was also used for evaluating oxidative stress in BV2 cells using the LPO assay kit (BioVision, San Francisco, CA, USA; Cat#739-100). BV2 cells were grown and treated in the same way as previously mentioned in the methodology of the in vitro ROS assay. LPO analysis was performed as mentioned for in vivo experiments.


## 2.8. Protein Extraction

When the in vivo treatment was completed, all animals were sacrificed under anesthesia, and the brains were carefully removed to avoid any damage. The hippocampal tissues were carefully separated, kept in liquid nitrogen, and stored at -80°C for a further biochemical experimental work. When required, the hippocampal tissues were homogenized in pre-prep extraction solution (iNtRON Biotechnology, Inc., Sungnam, South Korea) and centrifuged at 13,000 rpm at 4°C for 25 min. After homogenization, the supernatant was collected and stored at −80°C.


## 2.9. Quantitative Analysis of Proteins by Western Blotting

Before western blotting, the optical densities (OD) of proteins were analyzed using the Bio-Rad protein assay kit (Bio-Rad Laboratories, CA, USA), as described previously [14]. Equal amounts of protein, approximately 20–30 μg, were used for electrophoresis using 4–12% Bolt™ Mini Gels (Life Technologies, Carlsbad, CA, US). Proteins were transferred to PVDF (polyvinylidene fluoride) membranes (Sigma-Aldrich Chemical Co.). The membranes were treated with 5% skim milk (w/v) for 90 min, washed with TBST (3 times, 10 min each), and treated with primary antibodies (anti-caspase-3, anti-TNF-α, anti-IL-1β, anti-p-JNK, anti-p-NF-κB65, anti-Bcl-2-associated X protein (Bax), anti-B-cell lymphoma 2 (Bcl-2), anti-cytochrome c (Cyt c), anti-PARP-1, anti-phosphorylated-glycogen synthase kinase 3 (p-GSK3β) (Ser 9), anti-phosphorylated protein kinase B (p-Akt) (Ser 473), anti-synaptosomal associated protein 25 (SNAP-25), anti-postsynaptic density protein 95 (PSD95), and anti-β-actin, all from Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibodies for 1 h. The membranes were washed with TBST (3 times, 10 min each), treated with a chemiluminescence system (Atto Corporation Tokyo, Japan), and protein bands were then detected on an X-ray film. The protein bands were analyzed by ImageJ software (version 1.50, NIH, https://imagej.nih.gov/ij/, Bethesda, MD, USA) which was used to quantify the integrated density.


## 2.10. Morphological Analysis and Sample Preparation

When the drug treatment and behavioral studies were completed, the animals were perfused transcardially with 4% ice-cold paraformaldehyde, as previously described [7, 12]. In short, the brain was fixed in 4% paraformaldehyde for 72 h and then transferred to 20% sucrose and stored for another 72 h. Following this, the brains were washed with fresh PBS and immediately frozen in O.C.T. Compound (Sakura Finetek USA, Inc., Torrance, CA, USA). Upon solidification of the blocks, 14 μm coronal sections of the hippocampus were cut using a CM 3050C cryostat (Leica, Germany). The sections were thaw-mounted on ProbeOn Plus™ charged slides (Thermo Fisher Scientific, Nunc a/S, Roskilde, Denmark).


## 2.11. Immunofluorescence Staining

Immunofluorescence staining was performed following a previously described protocol [12, 14]. In brief, brain tissue slides were dried overnight, washed twice with PBS (0.01 M) for 10 min, and blocked with blocking serum (2% normal goat serum and 0.3% Triton X-100 in PBS) for 1 hour. The slides were then incubated with the aforementioned primary antibodies (Santa Cruz Biotechnology) overnight at 4°C. The next day, the slides were treated with secondary TRITC/FITC-labelled antibodies (1,100) (Santa Cruz Biotechnology) for 2 h. Slides were washed with PBS (twice for 5 min) and mounted with 4′,6′-diamidino-2-phenylindole (DAPI) and Prolong Antifade Reagent (Molecular Probe, Eugene, OR, USA). Finally, the stained slides were examined under a confocal laser-scanning FluoView FV 1000 MPE microscope (Olympus, Tokyo, Japan).


## 2.12. Fluoro-Jade B (FJB) Staining

Briefly, the tissue slides were dried overnight in a drying chamber and washed with PBS (0.01 M) twice for 5 min. Slides were dipped in a solution containing 1% sodium hydroxide and 80% ethanol for 5 min. Then the slides were kept in 70% alcohol followed by keeping in distilled water for 2 min. Following that, the tissue slides were treated with a solution containing 0.1% acetic acid and 0.06% FJB for 20 min. Slides were washed and left to dry for 10 min, mounted with DPX mounting medium, and coverslips were applied. Slides were examined under a confocal laser-scanning FluoView FV 1000 MPE microscope (Olympus, Tokyo, Japan), and images were taken. Results were analyzed with ImageJ software, and quantification of the immunohistofluorescence and FJB images was performed according to our recently published protocol [7].


## 2.13. TUNEL Staining

TUNEL (terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick-end labeling) staining is used to detect dead neurons and evaluate the extent of neuronal apoptosis. The kit used in this assay was purchased from Sigma-Aldrich Chemical Co. (Cat. No. 11684809910, St. Louis, MO, USA), and the staining was performed according to the manufacturer’s recommendations.


## 2.14. Hematoxylin and Eosin (H&E) Staining

H&E staining is used to analyze cell morphology. In brief, slides from each group (n=5) were first dipped in tap water for a short time and transferred to staining solution. The slides were then transferred to the hematoxylin solution for 8-10 min. Next, the sides were washed with running water for 10-15 min and transferred to the eosin solution for 30 sec. Following this, the slides were then dehydrated with a graded series of alcohol. Finally, all the slides were mounted with mounting medium (Thermo Fisher Scientific, MA, USA) and coverslips applied. Images of the slides were taken using a simple microscope.


## 2.15. Cresyl Violet Staining

Cresyl violet/Nissl staining is used for the examination and determination of neuronal cell death. First, slides comprising 14μm brain sections were washed with PBS (0.01 M) for 15 min and stained with a solution of Cresyl violet (0.5%) containing a few drops of glacial acetic acid for 10-15 min. Slides were washed with distilled water, dehydrated with a graded series of alcohol (70, 95, and 100%), retained in xylene; nonfluorescent mounting medium was used, and coverslips were applied. Images were taken using a fluorescent light microscope, and quantification of the immunohistochemical images was performed.


## 2.16. Morris Water Maze (MWM) and Y-Maze Tests

For the behavioral studies, a MWM test was conducted on the rats (n=10/group), as described previously [12]. The MWM comprises a circular tank (100 cm in diameter, 40 cm in height) containing water (23±1°C) filled to a depth of 15.5 cm. White ink was added to the water to make it look opaque. A transparent escape platform (10 cm in diameter, 14.4 cm in height) was kept at the midpoint of one quadrant, hidden 1 cm below the water level. Rats were trained for 5 days before the start of the study using a single hidden platform in one quadrant with three quadrants of rotational starting. The escape latency (the time taken to look for and locate the hidden platform) was calculated for every trial. After 24 h of the 5th day, a probe test was then performed for the evaluation of memory consolidation. The platform was removed, and the rats were allowed to swim freely for 60 sec. Then, the length of time spent in the target quadrant and the number of times the rat crossed over the platform location (the platform remained hidden during the training) were recorded. The total time spent by a rat in the target quadrant was considered to be a measure of the degree of memory consolidation. SMART video-tracking software (Panlab Harvard Apparatus, Holliston, MA, USA) was used to record the movement of the rats. The Y-maze test was performed as described previously with necessary changes [14].


## 2.17. In Vitro Cell Culturing and Treatment for Western Blotting and Confocal Microscopy

The HT-22 neuronal cells used in the in vitro studies were kindly provided by Prof. Koh (Gyeongsang National University, South Korea). The HT-22 cells were seeded in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics in a humidified 5% CO2 incubator at 37°C. The HT-22 cell were incubated with LPS (1 μg/ml), LPS (1 μg/ml)+Cur (100 μg/ml) and LPS (1 μg/ml)+SP600125 (20 μM) for 24 h.


## 2.18. Statistical Analyses

Differences between the control, LPS, and LPS+curcumin groups were analyzed using the one-way analysis of variance (ANOVA) and Student’st-test. All data are expressed as the mean±SEM for the three independent experiments and were analyzed using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, US). A P value < 0.05 was considered statistically significant. For the in vivo study, the ∗ symbol denotes a significant difference between the control and LPS groups and the Ώ symbol denotes a significant difference between the LPS and curcumin groups. Likewise, for the in vitro study, the ∗ symbol denotes a significant difference between the control and LPS groups, the Ώ symbol denotes a significant difference between the LPS and curcumin groups, and the # symbol denotes a significant difference between the LPS and JNK inhibitor SP600125 groups.


## 3. Results

### 3.1. Curcumin Ameliorated LPS-Induced Increases in ROS Generation, Oxidative Stress, and P-JNK Level in the Adult Rat Hippocampus and in LPS-Treated BV2 Cell

Recently, it has been suggested that curcumin has strong antioxidant properties and can reduce the ROS burden. It is also well known that JNK is a crucial stress kinase and is highly expressed during increased intracellular ROS generation [7, 20]. Therefore, we analyzed the expression of p-JNK through western blotting, confocal microscopy, and immunohistochemistry. Our results showed that treatment with LPS significantly increased the expression of p-JNK in the adult rat hippocampus. On the other hand, treatment with 300 mg/kg/i.p. curcumin for 2 weeks significantly reduced the expression of p-JNK, providing evidence that curcumin has potent antioxidant properties (Figures 1(f), 1(g), and 1(j)). Furthermore, to investigate if curcumin could inhibit p-JNK activation in a similar way to the JNK inhibitor SP600125, we exposed BV2 microglial cells to 1 μg/ml LPS, 100 μg/ml curcumin, and 20 μM SP600125. The in vitro immunoblot and confocal microscopy results confirmed that LPS treatment significantly increased the expression of p-JNK in BV2 cells. Curcumin significantly reduced this increase similar to SP600125 treatment (Figures 1(h) and 1(i)). Studies using animal models have shown that LPS treatment induces the generation of ROS [21–23]. We therefore carried out ROS and LPO assays on rats treated with saline, LPS, and LPS+curcumin. We found that treatment with LPS enhanced ROS generation and oxidative stress in the hippocampus of adult rats, and curcumin ameliorated both of these in the LPS+curcumin-treated group (Figures 1(b) and 1(c)). We further investigated whether curcumin could reduce ROS generation and oxidative stress in a similar way to SP600125 by performing ROS and LPO assays on BV2 cell lines. The in vitro results demonstrated that LPS treatment significantly increased the levels of DCF and MDA (markers of ROS and oxidative stress) in the BV2 cells. Curcumin treatment significantly reduced the levels of DCF and MDA in a similar way to that of the JNK inhibitor SP600125 (Figures 1(d) and 1(e).Figure 1
Curcumin mitigated the LPS-induced increase in ROS and oxidative stress and JNK activation in the adult rat hippocampus and in BV2 cells. (a) Presenting study plan for the current research work. Rats were divided into three groups: (1) control (2), LPS (3), and LPS+curcumin (b–e), representative histograms showing the ROS/LPO assays both in vivo and in vitro. Five animals were kept per group, and each experiment was repeated 3 times; i.e., (n=5)/(n=3). (f) Identifying the western blot results of p-JNK in the hippocampus of control, LPS, and LPS+curcumin. (g) Indicating the confocal results of p-JNK in the hippocampus of the adult rat. (h) Showing the western blot results of p-JNK in BV2 microglial cells. (i) Showing the confocal microscopy results of p-JNK in BV2 cells. (j) Immunohistochemistry results of p-JNK in the CA1 region of adult rat hippocampus. In each case of western blot assay, the same immunoblot was probed using β-actin as a loading control. 15 animals were kept per group; i.e., n=15. Eight animals per group for western blot (n=8), while 5 animals per group were used for confocal microscopy (n=5). (n) Showing numbers of animal per group. Sigma Gel software was used to quantify the western blot results whereas ImageJ software was used to analyze the immunofluorescence results of p-JNK. Green color (FITC) and blue color (DAPI) represents the confocal microscopy results of p-JNK in the hippocampus while red color indicates immunofluorescent reactivity of p-JNK in BV2 microglial cells. Magnifications: 20x. Scalebar=50μm. ∗P<0.05 shows the significant difference between the control and LPS groups while the Ώ symbol shows the significance between the LPS and LPS+curcumin groups. In the in vitro study, the # symbol represents the significant difference between the LPS and LPS+SP600125 groups.
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### 3.2. Curcumin Attenuated LPS-Induced Proinflammatory Cytokine Production and Restored the P-Akt/P-GSK3β Survival Pathway

Recent studies have demonstrated that LPS treatment induces microglial activation and cytokine production both in vivo and in vitro, playing a significant role in neuroinflammation-induced neurodegenerative disorders. In addition, the inhibitory effects of curcumin on the release of proinflammatory cytokines are well documented [24, 25]. Therefore, to determine the inhibitory role of curcumin against LPS-induced neuroinflammation in adult rats, we evaluated the expression of p-NF-κB (a transcription factor), TNF-α and IL-1β (proinflammatory cytokines), and GFAP and Iba1 (markers of reactive microglia and astrocytes) through western blotting. We found that LPS treatment significantly increased the expression of p-NF-κB, TNF-α, IL-1β, GFAP, and Iba1. Curcumin treatment inhibited this overexpression of p-NF-κB, TNF-α, IL-1β, GFAP, and Iba-1 in the hippocampal tissue of LPS-treated adult rats (Figure 2(a)). Confocal microscopy also showed an increased immunoreactivity of p-NF-κB and TNF-α in the hippocampus of LPS-treated adult rats. Curcumin treatment significantly reduced the immunoreactivity of TNF-α and p-NF-κB, suggesting that it prevents cytokine production in LPS-treated rats (Figures 2(b) and 2(c)). To investigate if curcumin prevents neuroinflammation via a JNK-dependent mechanism, we exposed BV2 microglial cells to LPS at a dose of 1 μg/ml for 24 h. We found that curcumin (100 μg/ml) and the JNK inhibitor SP600125 (20 μM) analogously attenuated the LPS-induced elevated level of proinflammatory cytokines, providing evidence that curcumin may inhibit cytokine production via a JNK-dependent mechanism (Figures 2(d)–2(g)).Figure 2
Curcumin treatment suppressed neuroinflammatory cytokines both in vivo/in vitro while restoring the survival pathway in adult rat hippocampus. (a) Western blot analysis of activated microglia (GFAP, Iba1), transcription factor (p-NF-κB), and inflammatory cytokines (TNF-α, IL-1β) in adult rats. (b, c) Indicating the immunofluorescent results of p-NF-κB (transcription factor) and TNF-α (inflammatory cytokines) in the CA1 region of an adult rat hippocampus. (d) Showing the western blot results of inflammatory cytokines in LPS-exposed BV2 microglial cells. (e–g) Representing the confocal results of inflammatory markers (TNF-α, IL-1β) and transcription factor (p-NF-κB) in BV2 cells. (h–j) Signifying the western blot and confocal results of survival proteins in an adult rat hippocampus. n=5 for confocal microscopy, whilen=8 for western blot. Green: FITC, blue: DAPI, red: TRITC; n=3 experiments. Magnification: 20x. Scalebar=30/50μm. One-way ANOVA followed by Student’s t-test was used to determine the mean±SEM, whereas statistical analysis was evaluated by using GraphPad Prism 5 software. Sigma Gel software was used to quantify the western blot results whereas ImageJ software was used to analyze the immunofluorescence results. β-Actin was used as a loading control in western blot analysis.
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(j)Mounting studies have reported that LPS treatment inhibited the phosphorylation of Akt and GSK3-β in rat models of Parkinson’s disease [26]. Our western blot and immunofluorescent results indicated that LPS treatment disrupts the survival pathway, demonstrated by a decrease in the expression and phosphorylation of Akt and GSK3-β proteins compared to saline-treated adult rats. Treatment with curcumin at a dose of 300 mg/kg/day not only restored this survival pathway but also significantly increased the level of p-Akt and p-GSK3β in the adult rat hippocampus (Figures 2(h)–2(j)).


### 3.3. Curcumin Mitigated LPS-Induced Neuronal Apoptosis and Neurodegeneration in the Adult Rat Hippocampus and in HT-22 Neuronal Cells

Recently, interest has been focused on LPS-induced ROS-mediated neuroinflammation-associated neurodegeneration. Badshah et al. demonstrated that LPS could induce neuronal apoptosis by increasing the expression of apoptotic markers like caspase-3 and PARP-1 [1]. Other studies have also shown that LPS induces prolonged neuroinflammatory responses by activating microglial cells, subsequently initiating the mitochondrial apoptotic pathway and neuronal cell death [27–29]. In the present study, we validated the antiapoptotic properties of curcumin against LPS-induced neuroinflammation-associated neurodegeneration. Our immunoblot and immunofluorescence results showed that LPS at a dose of 250 μg/kg for 7 days increased the expression of the apoptotic proteins Bax, caspase-3, Cyt c, and PARP-1 and decreased the expression of the antiapoptotic protein Bcl-2 in the adult rat hippocampus. Treatment with curcumin at a dose of 300 mg/kg for 14 days significantly decreased these apoptotic markers and inhibited neuronal apoptosis (Figures 3(a)–3(d)).Figure 3
Curcumin treatment abrogated neuronal cell apoptosis in LPS-treated adult rats and in HT-22 hippocampal neuronal cells. (a–d) Representative western blot and immunofluorescent analysis of neuronal apoptotic protein markers in an adult rat hippocampus. (e–g) Showing the confocal and western blot results of apoptotic markers in LPS-exposed HT-22 neuronal cells. (h) Representing the FJB results, (i) indicating the tunnel assay results, and (j) specifying the immunohistochemistry results of caspase-3 in the hippocampus of the adult rat. (k, l) Representing the H&E and Nissl results, respectively. Sigma Gel software was used to quantify the western blot results of the related protein bands. Beta-actin was used as a loading control.∗/#/Ώ were used to show the level of significance. ∗P<0.05. One-way ANOVA followed by Student’s t-test was used to determine the mean±SEM whereas statistical analysis was evaluated by using GraphPad Prism 5 software. n=3 means that the number of experiments were repeated 3 times. (n=5 animals/group for confocal and histochemistry); n=3 experiments. Magnification: 20x. Scalebar=30/50μm. The presented data are relative to the control. The number of animal used per group and the level of significance are mentioned in Materials and Methods.
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(l)There exists considerable evidence in the literature regarding the exposure of HT-22 cells to LPS-induced injury. Ji et al. reported that stimulation of hippocampal HT-22 neuronal cells with LPS remarkably upregulated the expression of the apoptotic markers Bax and caspase-3 and downregulated the expression of the antiapoptotic protein Bcl-2 [30]. Similarly, in the current study, we treated HT-22 neuronal cells with LPS in order to evaluate JNK-dependent and inflammation-associated neuronal apoptosis. Our western blot and confocal results demonstrated that LPS treatment at a dose of 1 μg/ml significantly elevated the levels of caspase-3, Cyt c, and PARP-1. Curcumin at a dose of 100 μg/ml and the JNK inhibitor SP600125 at a dose of 20 μM markedly reduced their expression (Figures 3(e)–3(g)).Additionally, to determine the extent of LPS-induced neurodegeneration, we performed FJB, TUNEL, and Nissl staining. We found that LPS treatment increased the number of FJB-positive cells in the hippocampus of adult rats. However, treatment with curcumin reduced the number of FJB-positive cells and inhibited neuronal cell death (Figure3(h)). Next, the Nissl staining results demonstrated that LPS treatment for 7 days at a dose of 250 μg/kg significantly decreased the number of viable neurons. Curcumin treatment for 14 days at a dose of 300 mg/kg prevented neuronal apoptosis and remarkably increased the number of viable neurons (Figure 3(l)). The results of the TUNEL assay also showed that LPS increased the number of dead neurons. Curcumin significantly improved the cell viability of the affected neurons and ameliorated neurodegeneration (Figure 3(i)). To determine the effect of LPS on cell morphology, we conducted H&E staining. The results showed that LPS treatment dysregulated cell morphology, demonstrated by the appearance of shrunken neuronal cells. Curcumin treatment restored cell morphology and improved cell survival (Figure 3(k)).


### 3.4. Curcumin Increased the Expression of Pre- and Postsynaptic Protein Markers and Rescued Memory-Related Deficits in an Adult Rat Model of LPS Challenge

A recent study indicated that neuroinflammation and neurodegeneration in response to ROS can lead to synaptic deficits and memory impairments [31, 32]. To analyze the effect of curcumin against LPS-induced synaptic degeneration, we examined the expression level of the presynaptic protein SNAP25 and the postsynaptic protein PSD95 through western blotting and confocal microscopy. Results showed that LPS treatment, due to its toxic effects, decreased the expression of both SNAP25 and PSD95 compared to the control and curcumin-treated groups (Figures 4(a)–4(c)). Moreover, to evaluate the memory, we performed the MWM and Y-maze tests. The LPS-treated rats exhibited an increased latency time, indicative of learning and memory deficits. Conversely, a reduced latency time, indicative of memory improvements, was exhibited by the curcumin-treated group (Figure 4(d)). A probe test was also performed on the 5th day in which the hidden platform was removed. The LPS-treated rats exhibited fewer platform crossings and a reduced length of time spent in the target quadrant. Curcumin treatment increased the number of platform crossings and the length of time spent in the target quadrant (Figures 4(e) and 4(f)). Next, we performed a Y-maze test to investigate spatial working memory by assessing spontaneous alternation percentage. We found that the LPS-treated rats exhibited a lower alteration percentage than the saline-treated rats, indicating poor working memory. Curcumin significantly increased alteration percentage in LPS-challenged rats, indicating that curcumin can improve memory dysfunctions (Figure 4(g)).Figure 4
Curcumin treatment improves memory impairment and increases the expression level of synaptic proteins. (a–c) Immunoblot and confocal analysis indicating the expression of synaptic proteins including SNAP-25 and PSD95 in the hippocampus of the saline-treated group, LPS-treated group, and LPS+curcumin-treated group (n=8 for western and n=5 for confocal). Each immunoblot was probed with an anti-β-actin antibody as a loading control. Bands were quantified with Sigma gel software. FITC (green) showing the immunoreactivity of the related antibodies while DAPI (blue) showing the nucleus of the cell. The number of experiments was repeated 3 times (n=3). ImageJ software was used for the quantification of confocal analysis. (d) The related histogram representing a total of 5 days of latency of the control, LPS, and LPS+curcumin groups. (e) The histogram represents the number of crossings during the probe test among the control, LPS, and LPS+curcumin groups. (f) Graphical representation showing time spent in the target quadrant by control, LPS, and LPS+curcumin. (g) The representative histogram shows the spontaneous alteration (%) in the Y-maze test. (h) Showing the trajectories of the water maze test.
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## 3.1. Curcumin Ameliorated LPS-Induced Increases in ROS Generation, Oxidative Stress, and P-JNK Level in the Adult Rat Hippocampus and in LPS-Treated BV2 Cell

Recently, it has been suggested that curcumin has strong antioxidant properties and can reduce the ROS burden. It is also well known that JNK is a crucial stress kinase and is highly expressed during increased intracellular ROS generation [7, 20]. Therefore, we analyzed the expression of p-JNK through western blotting, confocal microscopy, and immunohistochemistry. Our results showed that treatment with LPS significantly increased the expression of p-JNK in the adult rat hippocampus. On the other hand, treatment with 300 mg/kg/i.p. curcumin for 2 weeks significantly reduced the expression of p-JNK, providing evidence that curcumin has potent antioxidant properties (Figures 1(f), 1(g), and 1(j)). Furthermore, to investigate if curcumin could inhibit p-JNK activation in a similar way to the JNK inhibitor SP600125, we exposed BV2 microglial cells to 1 μg/ml LPS, 100 μg/ml curcumin, and 20 μM SP600125. The in vitro immunoblot and confocal microscopy results confirmed that LPS treatment significantly increased the expression of p-JNK in BV2 cells. Curcumin significantly reduced this increase similar to SP600125 treatment (Figures 1(h) and 1(i)). Studies using animal models have shown that LPS treatment induces the generation of ROS [21–23]. We therefore carried out ROS and LPO assays on rats treated with saline, LPS, and LPS+curcumin. We found that treatment with LPS enhanced ROS generation and oxidative stress in the hippocampus of adult rats, and curcumin ameliorated both of these in the LPS+curcumin-treated group (Figures 1(b) and 1(c)). We further investigated whether curcumin could reduce ROS generation and oxidative stress in a similar way to SP600125 by performing ROS and LPO assays on BV2 cell lines. The in vitro results demonstrated that LPS treatment significantly increased the levels of DCF and MDA (markers of ROS and oxidative stress) in the BV2 cells. Curcumin treatment significantly reduced the levels of DCF and MDA in a similar way to that of the JNK inhibitor SP600125 (Figures 1(d) and 1(e).Figure 1
Curcumin mitigated the LPS-induced increase in ROS and oxidative stress and JNK activation in the adult rat hippocampus and in BV2 cells. (a) Presenting study plan for the current research work. Rats were divided into three groups: (1) control (2), LPS (3), and LPS+curcumin (b–e), representative histograms showing the ROS/LPO assays both in vivo and in vitro. Five animals were kept per group, and each experiment was repeated 3 times; i.e., (n=5)/(n=3). (f) Identifying the western blot results of p-JNK in the hippocampus of control, LPS, and LPS+curcumin. (g) Indicating the confocal results of p-JNK in the hippocampus of the adult rat. (h) Showing the western blot results of p-JNK in BV2 microglial cells. (i) Showing the confocal microscopy results of p-JNK in BV2 cells. (j) Immunohistochemistry results of p-JNK in the CA1 region of adult rat hippocampus. In each case of western blot assay, the same immunoblot was probed using β-actin as a loading control. 15 animals were kept per group; i.e., n=15. Eight animals per group for western blot (n=8), while 5 animals per group were used for confocal microscopy (n=5). (n) Showing numbers of animal per group. Sigma Gel software was used to quantify the western blot results whereas ImageJ software was used to analyze the immunofluorescence results of p-JNK. Green color (FITC) and blue color (DAPI) represents the confocal microscopy results of p-JNK in the hippocampus while red color indicates immunofluorescent reactivity of p-JNK in BV2 microglial cells. Magnifications: 20x. Scalebar=50μm. ∗P<0.05 shows the significant difference between the control and LPS groups while the Ώ symbol shows the significance between the LPS and LPS+curcumin groups. In the in vitro study, the # symbol represents the significant difference between the LPS and LPS+SP600125 groups.
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## 3.2. Curcumin Attenuated LPS-Induced Proinflammatory Cytokine Production and Restored the P-Akt/P-GSK3β Survival Pathway

Recent studies have demonstrated that LPS treatment induces microglial activation and cytokine production both in vivo and in vitro, playing a significant role in neuroinflammation-induced neurodegenerative disorders. In addition, the inhibitory effects of curcumin on the release of proinflammatory cytokines are well documented [24, 25]. Therefore, to determine the inhibitory role of curcumin against LPS-induced neuroinflammation in adult rats, we evaluated the expression of p-NF-κB (a transcription factor), TNF-α and IL-1β (proinflammatory cytokines), and GFAP and Iba1 (markers of reactive microglia and astrocytes) through western blotting. We found that LPS treatment significantly increased the expression of p-NF-κB, TNF-α, IL-1β, GFAP, and Iba1. Curcumin treatment inhibited this overexpression of p-NF-κB, TNF-α, IL-1β, GFAP, and Iba-1 in the hippocampal tissue of LPS-treated adult rats (Figure 2(a)). Confocal microscopy also showed an increased immunoreactivity of p-NF-κB and TNF-α in the hippocampus of LPS-treated adult rats. Curcumin treatment significantly reduced the immunoreactivity of TNF-α and p-NF-κB, suggesting that it prevents cytokine production in LPS-treated rats (Figures 2(b) and 2(c)). To investigate if curcumin prevents neuroinflammation via a JNK-dependent mechanism, we exposed BV2 microglial cells to LPS at a dose of 1 μg/ml for 24 h. We found that curcumin (100 μg/ml) and the JNK inhibitor SP600125 (20 μM) analogously attenuated the LPS-induced elevated level of proinflammatory cytokines, providing evidence that curcumin may inhibit cytokine production via a JNK-dependent mechanism (Figures 2(d)–2(g)).Figure 2
Curcumin treatment suppressed neuroinflammatory cytokines both in vivo/in vitro while restoring the survival pathway in adult rat hippocampus. (a) Western blot analysis of activated microglia (GFAP, Iba1), transcription factor (p-NF-κB), and inflammatory cytokines (TNF-α, IL-1β) in adult rats. (b, c) Indicating the immunofluorescent results of p-NF-κB (transcription factor) and TNF-α (inflammatory cytokines) in the CA1 region of an adult rat hippocampus. (d) Showing the western blot results of inflammatory cytokines in LPS-exposed BV2 microglial cells. (e–g) Representing the confocal results of inflammatory markers (TNF-α, IL-1β) and transcription factor (p-NF-κB) in BV2 cells. (h–j) Signifying the western blot and confocal results of survival proteins in an adult rat hippocampus. n=5 for confocal microscopy, whilen=8 for western blot. Green: FITC, blue: DAPI, red: TRITC; n=3 experiments. Magnification: 20x. Scalebar=30/50μm. One-way ANOVA followed by Student’s t-test was used to determine the mean±SEM, whereas statistical analysis was evaluated by using GraphPad Prism 5 software. Sigma Gel software was used to quantify the western blot results whereas ImageJ software was used to analyze the immunofluorescence results. β-Actin was used as a loading control in western blot analysis.
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(j)Mounting studies have reported that LPS treatment inhibited the phosphorylation of Akt and GSK3-β in rat models of Parkinson’s disease [26]. Our western blot and immunofluorescent results indicated that LPS treatment disrupts the survival pathway, demonstrated by a decrease in the expression and phosphorylation of Akt and GSK3-β proteins compared to saline-treated adult rats. Treatment with curcumin at a dose of 300 mg/kg/day not only restored this survival pathway but also significantly increased the level of p-Akt and p-GSK3β in the adult rat hippocampus (Figures 2(h)–2(j)).


## 3.3. Curcumin Mitigated LPS-Induced Neuronal Apoptosis and Neurodegeneration in the Adult Rat Hippocampus and in HT-22 Neuronal Cells

Recently, interest has been focused on LPS-induced ROS-mediated neuroinflammation-associated neurodegeneration. Badshah et al. demonstrated that LPS could induce neuronal apoptosis by increasing the expression of apoptotic markers like caspase-3 and PARP-1 [1]. Other studies have also shown that LPS induces prolonged neuroinflammatory responses by activating microglial cells, subsequently initiating the mitochondrial apoptotic pathway and neuronal cell death [27–29]. In the present study, we validated the antiapoptotic properties of curcumin against LPS-induced neuroinflammation-associated neurodegeneration. Our immunoblot and immunofluorescence results showed that LPS at a dose of 250 μg/kg for 7 days increased the expression of the apoptotic proteins Bax, caspase-3, Cyt c, and PARP-1 and decreased the expression of the antiapoptotic protein Bcl-2 in the adult rat hippocampus. Treatment with curcumin at a dose of 300 mg/kg for 14 days significantly decreased these apoptotic markers and inhibited neuronal apoptosis (Figures 3(a)–3(d)).Figure 3
Curcumin treatment abrogated neuronal cell apoptosis in LPS-treated adult rats and in HT-22 hippocampal neuronal cells. (a–d) Representative western blot and immunofluorescent analysis of neuronal apoptotic protein markers in an adult rat hippocampus. (e–g) Showing the confocal and western blot results of apoptotic markers in LPS-exposed HT-22 neuronal cells. (h) Representing the FJB results, (i) indicating the tunnel assay results, and (j) specifying the immunohistochemistry results of caspase-3 in the hippocampus of the adult rat. (k, l) Representing the H&E and Nissl results, respectively. Sigma Gel software was used to quantify the western blot results of the related protein bands. Beta-actin was used as a loading control.∗/#/Ώ were used to show the level of significance. ∗P<0.05. One-way ANOVA followed by Student’s t-test was used to determine the mean±SEM whereas statistical analysis was evaluated by using GraphPad Prism 5 software. n=3 means that the number of experiments were repeated 3 times. (n=5 animals/group for confocal and histochemistry); n=3 experiments. Magnification: 20x. Scalebar=30/50μm. The presented data are relative to the control. The number of animal used per group and the level of significance are mentioned in Materials and Methods.
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(l)There exists considerable evidence in the literature regarding the exposure of HT-22 cells to LPS-induced injury. Ji et al. reported that stimulation of hippocampal HT-22 neuronal cells with LPS remarkably upregulated the expression of the apoptotic markers Bax and caspase-3 and downregulated the expression of the antiapoptotic protein Bcl-2 [30]. Similarly, in the current study, we treated HT-22 neuronal cells with LPS in order to evaluate JNK-dependent and inflammation-associated neuronal apoptosis. Our western blot and confocal results demonstrated that LPS treatment at a dose of 1 μg/ml significantly elevated the levels of caspase-3, Cyt c, and PARP-1. Curcumin at a dose of 100 μg/ml and the JNK inhibitor SP600125 at a dose of 20 μM markedly reduced their expression (Figures 3(e)–3(g)).Additionally, to determine the extent of LPS-induced neurodegeneration, we performed FJB, TUNEL, and Nissl staining. We found that LPS treatment increased the number of FJB-positive cells in the hippocampus of adult rats. However, treatment with curcumin reduced the number of FJB-positive cells and inhibited neuronal cell death (Figure3(h)). Next, the Nissl staining results demonstrated that LPS treatment for 7 days at a dose of 250 μg/kg significantly decreased the number of viable neurons. Curcumin treatment for 14 days at a dose of 300 mg/kg prevented neuronal apoptosis and remarkably increased the number of viable neurons (Figure 3(l)). The results of the TUNEL assay also showed that LPS increased the number of dead neurons. Curcumin significantly improved the cell viability of the affected neurons and ameliorated neurodegeneration (Figure 3(i)). To determine the effect of LPS on cell morphology, we conducted H&E staining. The results showed that LPS treatment dysregulated cell morphology, demonstrated by the appearance of shrunken neuronal cells. Curcumin treatment restored cell morphology and improved cell survival (Figure 3(k)).


## 3.4. Curcumin Increased the Expression of Pre- and Postsynaptic Protein Markers and Rescued Memory-Related Deficits in an Adult Rat Model of LPS Challenge

A recent study indicated that neuroinflammation and neurodegeneration in response to ROS can lead to synaptic deficits and memory impairments [31, 32]. To analyze the effect of curcumin against LPS-induced synaptic degeneration, we examined the expression level of the presynaptic protein SNAP25 and the postsynaptic protein PSD95 through western blotting and confocal microscopy. Results showed that LPS treatment, due to its toxic effects, decreased the expression of both SNAP25 and PSD95 compared to the control and curcumin-treated groups (Figures 4(a)–4(c)). Moreover, to evaluate the memory, we performed the MWM and Y-maze tests. The LPS-treated rats exhibited an increased latency time, indicative of learning and memory deficits. Conversely, a reduced latency time, indicative of memory improvements, was exhibited by the curcumin-treated group (Figure 4(d)). A probe test was also performed on the 5th day in which the hidden platform was removed. The LPS-treated rats exhibited fewer platform crossings and a reduced length of time spent in the target quadrant. Curcumin treatment increased the number of platform crossings and the length of time spent in the target quadrant (Figures 4(e) and 4(f)). Next, we performed a Y-maze test to investigate spatial working memory by assessing spontaneous alternation percentage. We found that the LPS-treated rats exhibited a lower alteration percentage than the saline-treated rats, indicating poor working memory. Curcumin significantly increased alteration percentage in LPS-challenged rats, indicating that curcumin can improve memory dysfunctions (Figure 4(g)).Figure 4
Curcumin treatment improves memory impairment and increases the expression level of synaptic proteins. (a–c) Immunoblot and confocal analysis indicating the expression of synaptic proteins including SNAP-25 and PSD95 in the hippocampus of the saline-treated group, LPS-treated group, and LPS+curcumin-treated group (n=8 for western and n=5 for confocal). Each immunoblot was probed with an anti-β-actin antibody as a loading control. Bands were quantified with Sigma gel software. FITC (green) showing the immunoreactivity of the related antibodies while DAPI (blue) showing the nucleus of the cell. The number of experiments was repeated 3 times (n=3). ImageJ software was used for the quantification of confocal analysis. (d) The related histogram representing a total of 5 days of latency of the control, LPS, and LPS+curcumin groups. (e) The histogram represents the number of crossings during the probe test among the control, LPS, and LPS+curcumin groups. (f) Graphical representation showing time spent in the target quadrant by control, LPS, and LPS+curcumin. (g) The representative histogram shows the spontaneous alteration (%) in the Y-maze test. (h) Showing the trajectories of the water maze test.
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## 4. Discussion

The aim of the present study was to evaluate the underlying antioxidant neuroprotective mechanism of the dietary polyphenolic compound curcumin in LPS-treated adult rats via regulation of the JNK/NF-κB/Akt signaling pathway. Several studies have demonstrated that LPS triggers ROS generation/oxidative stress and activates the stress kinase JNK, subsequently mediating the pathogenesis of various neurodegenerative disorders [33, 34]. Similarly, in our study, LPS treatment elevated ROS generation and enhanced LPO, demonstrated by increased levels of DCF and MDA, and upregulated the expression of p-JNK in the hippocampus of adult rats and in HT-22/BV2 cells. Dietary curcumin supplementation reduced these elevated levels of ROS/oxidative stress and p-JNK.Many studies have reported that LPS induces JNK activation which mediates neuroinflammatory responses and LPS has been extensively used in models studying neuroinflammation. Recently, other studies have suggested that systemic administration of LPS activates oxidative stress and JNK which initiates downstream neuroinflammatory cascades. Numerous in vivo and in vitro studies have proposed that LPS provokes microgliosis and activates a number of signaling pathways including activation of NF-κB via several mediators including ROS/oxidative stress and p-JNK, which in turn promote the release of proinflammatory cytokines such as TNF-α and IL-1β [4, 35, 36]. In agreement with earlier studies, our rat model of LPS challenge exhibited an increased expression of Iba-1, GFAP, p-NF-κB, TNF-α, and IL-1β. On the other hand, our in vivo and in vitro results demonstrated that curcumin treatment significantly diminished this ROS/JNK-mediated increased expression of NF-κB, Iba-1, GFAP, TNF-α, and IL-1β in a similar manner to the JNK inhibitor SP600125.Several studies have supported the notion that LPS-induced ROS/JNK-mediated neuroinflammation provokes neuronal cell death and downregulates the level of survival proteins such as p-Akt/p-GSK3β via microgliosis and the production of proinflammatory cytokines. With respect to neuroinflammation, it has been suggested that several mechanisms are involved in the secretion of proinflammatory cytokines by astrocytes and microglial cells. One such mechanism is the LPS-induced p-JNK pathway that functions as a link between ROS/oxidative stress and neuroinflammation-mediated apoptotic neurodegeneration [4, 26, 37, 38]. It is well known that dietary curcumin is a strong candidate for the prevention of ROS/oxidative stress-mediated neuroinflammation-related neurodegeneration [39]. Additionally, curcumin can inhibit the dysfunction of mitochondrial activity and suppress the apoptotic proteins Bax, caspase-3, Cyt c, and PARP-1. Curcumin also upregulates the expression of the antiapoptotic protein Bcl-2, which prevents the dysregulation of mitochondrial homeostasis [28, 40–42]. Here, our results also demonstrated that curcumin treatment markedly inhibited neuronal apoptosis in the adult rat hippocampus via the suppression of neuroinflammation, regulation of the survival proteins p-Akt and p-GSKβ, and the reduction of apoptotic protein markers. Additionally, many studies have shown that exposure of HT-22 neuronal cells to LPS activates cell death pathways and mitochondrial dysfunction [30]. Likewise, in the present study, we observed that curcumin treatment prevented neuronal apoptosis via a JNK-dependent mechanism in LPS-treated HT-22 neuronal cells. To further explore the neuroprotective effect of curcumin, we performed TUNEL, FJB, Nissl, and H&E staining. Our results demonstrated that in the adult rat hippocampus, the number of TUNEL- and FJB-positive neuronal cells was remarkably decreased in the LPS-treated group, while the number of viable neurons in the Nissl assay was greatly increased upon curcumin treatment. Moreover, consistent with previous studies, our findings demonstrated that curcumin treatment restored fragmented and shrunken neuronal cells during H&E immunohistochemistry.Studies in animal models have shown that LPS induces detrimental physiological responses such as chronic neuroinflammation, disrupted mitochondrial function, and alteration of the homeostasis of antioxidants/oxidation, in turn resulting in memory disorders [1, 36, 43, 44]. Polyphenolic compounds with antioxidant properties, particularly curcumin, have thus been studied for their effectiveness in reducing oxidative stress/neuroinflammation-associated learning and memory deficits [17, 45, 46]. The results of this study demonstrated that curcumin, a potent scavenger of ROS, improved memory deficits by increasing the expression level of memory-related proteins, detected using western blotting and confocal microscopy. Furthermore, curcumin treatment significantly decreased latency time, increased the number of platform crossings, increased the length of time spent in the target quadrant, and enhanced alteration percentage in the Morris water maze and Y-maze tasks.


## 5. Conclusions

In conclusion, we obtained notable data about the underlying antioxidant neuroprotective mechanism of dietary curcumin in a rat model of LPS-induced neurotoxicity. Our results demonstrated that curcumin regulated the JNK/NF-κB/Akt signaling pathway and consequently ameliorated ROS generation, oxidative stress, and neuroinflammation-associated neurodegeneration. Curcumin also improved memory-associated pre- and postsynaptic markers, as well as cognitive functions, in LPS-treated adult rats (Figure 5). Taken together, these data suggest that dietary curcumin acts as a potent antioxidant and anti-inflammatory agent and could be beneficial as a dietary supplement for the prevention of oxidative stress/neuroinflammation-related neurological disorders.Figure 5
Proposed schematic diagram of LPS-induced ROS-mediated neuroinflammation, neurodegeneration, and memory deficits via dysregulation of the JNK/NF-κB/Akt signaling pathway.



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*Source: 7860650-2019-11-07.xml*