Nobiletin Protects against Systemic Inflammation-Stimulated

Apr 17, 2019 - Nobiletin Protects against Systemic Inflammation-Stimulated Memory Impairment via MAPK and NF-κB Signaling Pathways ...
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Bioactive Constituents, Metabolites, and Functions

Nobiletin Protects Against Systemic Inflammation-Stimulated Memory Impairment via MAPK and NF-#B Signaling Pathways Guoyuan Qi, Yashi Mi, Rong Fan, Runnan Li, Zhigang Liu, and Xuebo Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00133 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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Nobiletin Protects Against Systemic Inflammation-Stimulated Memory Impairment via MAPK and NF-κB Signaling Pathways Guoyuan Qi, †, 1 Yashi Mi, †, 1 Rong Fan, 2 Runnan Li, 3 Zhigang Liu, 1, * Xuebo Liu 1, *

1Laboratory

of Functional Chemistry and Nutrition of Food, College of Food Science and

Engineering, Northwest A&F University, Yangling, Shaanxi 712100, China

2Department

of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA

3 Department

*

of Animal and Food Science, University of Kentucky, Lexington, KY 40506, USA

Correspondence: Dr. Zhigang Liu, College of Food Science and Engineering, Northwest A&F

University, Xinong Rd. 22, Yangling 712100, China. E-mail: [email protected] *

Correspondence to: Dr. Xuebo Liu, College of Food Science and Engineering, Northwest A&F

University, 22. Xi-nong Road, Yangling 712100, China. Tel: +86-029-87092325; Fax: +86-029-87092325; E-mail: [email protected] † These authors contributed equally.

ABBREVIATION CNS, Central nervous system; AD, Alzheimer disease; NOB, Nobiletin; LPS, Lipopolysaccharide; PD, Parkinson’s disease; HD, ROS, Reactive oxygen species; Huntington’s

disease;

COX-2,

Cyclooxygenase-2;

SNAP-25,

Synaptosomal

associated protein 25; IHC, Immunohistochemical; IBA-1, Ionized calcium-binding

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adapter molecule 1; IL-1β, Interleukin-1β; IL-6, Interleukin-6;MMP, Mitochondrial membrane potential; PSD-95, Postsynaptic density protein 95; TNF-α, Tumor necrosis factor-α.

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ABSTRACT

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Neuroinflammation is intensively demonstrated to be related with various

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neurodegenerative diseases including Parkinson disease (PD), amyotrophic lateral

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sclerosis (ALS), and Alzheimer disease (AD). As a natural polymethoxylated flavone,

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Nobiletin (NOB) is reported to alleviate oxidative stress, insulin resistance, and

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obesity. In this study, we evaluated the protection effects of NOB on

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neuroinflammation and memory deficit. 3-month mice were administrated with NOB

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by oral gavage every day for 6 weeks (100 mg/kg/day), subsequently mice were

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injected intraperitoneally with lipopolysaccharide (LPS) for 7 days. Results of

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behavioral test revealed that NOB dramatically ameliorated LPS-triggered memory

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deficit regarding synaptic dysfunctions and neuronal loss. Also, NOB suppressed the

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microglial activation and pro-inflammatory cytokines secretion, such as COX-2,

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IL-1 TNF- and iNOS Similarly, upon LPS stimulation, pre-treatment NOB

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diminished the secretion of the pro-inflammatory cytokines in BV-2 microglia cells

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by exposure to LPS via modulating MAPKs, PI3K/AKT, and NF-B signaling

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pathways. In addition, NOB alleviated LPS-amplified redox imbalance, disturbance of

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mitochondrial membrane potential (MMP) and dampen the expression of protein

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related to mitochondrial respiration. The present study provide compelling evidences

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that NOB decreased LPS-stimulated neuroinflammation and memory impairment

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through maintaining cellular oxidative balance and blocking NF-κB transcriptional

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pathway, illustrating that the nutritional compound NOB may serve as potential

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approach to alleviate neuroinflammation-related diseases.

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KEYWORDS:

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Neuroinflammation, NF-κB/MAPKs/PI3K/Akt signaling pathways, Mitochondrial

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function.

Nobiletin,

Lipopolysaccharide,

27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

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Memory

impairment,

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INTROUDUCTION

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Neuroinflammation is regard as one of the principal causes of several

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neurodegenerative diseases, such as Parkinson’s disease (PD), Huntington’s diseases

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(HD), and Alzheimer’s disease (AD)

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inflammation within the brain induces greater loss of neurons and influences

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cognition and neuronal plasticity 4. Particularly, microglia plays a crucial part in both

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the inflammatory immune response and degenerative processes 5. Noteworthy ,

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aberrant activation of microglia leads to the overproduction of various

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pro-inflammatory mediator , such as IL-6, IL-1β, and TNF-α, thereby triggering

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neuronal damage and the progression of neurodegenerative disease 6.

1-3.

In compared to the initial injury,

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As an endotoxin from the outer membrane of Gram-negative bacteria,

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lipopolysaccharide (LPS) is reported to trigger immune response 7. Cumulative

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evidence suggests that systemic administration of LPS elevates several inflammatory

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mediators that initiate neuroinflammation and impair cognitive function 8. LPS is

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mainly recognized by the CD14/TLR-4 receptor that expressed in astrocytes and

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microglia

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activation of microglia will stimulate NF-κB and downstream inflammation signaling

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pathways, such as MAPKs and AKT 9. NF-κB is well-known as a primary

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transcription factor that take part in microglia-mediated inflammation

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Intraperitoneal (i.p.) injection with LPS is reported to induce neuroinflammation and

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memory impairment by targeting kinase cascades and activating transcription factors

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including PI3K/AKT, NF-κB, and MAPKs 3, 4.

3.

Numerous researches demonstrated that LPS-induced excessive

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10.

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Furthermore, ROS production serves as a critical regulator in the etiology of

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neurological disorders 11. It is reported that oxidative stress in microglia-triggered by

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LPS can induce inflammation and contribute to various types of nervous system

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damage

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might serve as promising therapeutic targets to moderate the progression of

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inflammatory-mediated neurodegenerative diseases.

12, 13.

Therefore, neuroinflammation and oxidative stress events in microglia

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NOB is a polymethoxylated flavone which possesses multiple health-promoting

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properties such as anti-obesity 14, anti-allergic 15, and anti-tumor effects 16. Numerous

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researches have confirmed that NOB could alleviate cognitive impairment in several

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mouse model of AD and rescue bulbectomy-induced cholinergic neurodegeneration

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17, 18.

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against LPS-induced migration of NF-κB, excessive ROS generation, and iNOS,

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COX-2 protein expression in RAW264.7 macrophage

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hypothesized

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neuroinflammation by reducing the excessive activation of microglia and oxidative

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stress.

Simultaneously, NOB was also reported to exert anti-inflammatory effect

that

NOB

diminishes

LPS-induced

19.

Accordingly, we

cognitive

deficits

and

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This study aimed at uncovering effects and molecular mechanisms of NOB

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supplementation on LPS-triggered neuroinflammation and cognitive deficits both in

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vivo and in vitro. The present work was also aimed to evaluate the effects of NOB on

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glial activation, inflammatory responses, mitochondrial dysfunction, and redox

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unbalance. This study is significant in providing insights of possible mechanisms of

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NOB in the alleviation of memory deficits and inflammation by targeting PI3K-AKT,

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MAPKs, and NF-KB signaling pathways upon LPS stimulation both in the mouse and

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BV-2 microglial cell models.

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MATERIALS AND METHODS

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Animals and Diet

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C57BL/6J mice (12-week-old, male) were provided by Xi’an Jiaotong University

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(Shaanxi, China). Nobiletin (NOB) (purity of >98%) was purchased from Yuanye

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Biotechnology, Ltd. (Shanghai, China) (Fig. 1A). Mice were random assigned into

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three groups (n=10 each group): Control group, LPS treatment group, and LPS plus

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NOB group under a 12 hr light: 12 hr dark cycle. Control and LPS group mice

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received vehicle (0.3% carboxyl methyl cellulose) by oral gavage, while LPS plus

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NOB group were administration with NOB (100 mg/kg bodyweight/day) mixed with

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the vehicle once daily for 7 weeks. The mice were treated with either vehicle (DMSO)

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or NOB (200 mg/kg body weight) via oral gavage every other day, LPS and LPS plus

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NOB group mice were treated with LPS (0.25 mg/kg bodyweight/day) via i.p.

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injection, while other animals were i.p. injected the same volume of saline alone for 7

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days. 4 h later after injection, we performed behavioral tests during these 7 days (Fig.

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1B). All the animal experimental procedures provided by Xi’an Jiaotong University.

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Y-maze test

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4 h after LPS injection, the Y-maze (Xinruan, Shanghai, China) task was used to 20.

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determine learning and memory capabilities as previously described

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mice in the center of the Y-maze apparatus which contains 3 arms, and the

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overlapping triplet sets was recorded during 8 min.

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Placed the

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Morris water maze test

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The Morris water maze is a circular pool (Xinruan, Shanghai, China), which

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contain four quadrants of equal areas. A platform was placed at one quadrant. The test

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was performed as previously described with minor modifications

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lasted for 5 days. The mice were received four training periods each day. For each

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trial, the escape latency and the swimming route of the mice was recorded by

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SuperMaze software. After the last test trials 24 h, probe trial was proceeding. During

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this trial, the platform was removed and the mouse was placed in the water to swim

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freely 60 s before being removed. The time spent in each quadrant and the number of

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platform crossings was calculated.

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Immunohistochemical staining

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20, 21.

The test trials

The immunohistochemical staining was conducted as previous reported

22, 23.

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Briefly, sections were cut off by using a microtome and dehydrated through xylene.

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The Sections were pre-incubated with 3 % H2O2 for 10 min, and then the goat serum

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was applied 20 min for blocking. Next the sections were then incubated with primary

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antibodies overnight. After washing, the brain sections were incubated secondary

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antibody for 20 min and reacted with HRP-Streptavidin (Zhongshan Golden Bridge,

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Beijing, China) for 20 min at 25 °C, and visualized by the chromogen DAB kit

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(Zhongshan Golden Bridge, Beijing, China). The brain slides were detected by

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microscope (Olympus, Tokyo, Japan) (×200).

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Cell culture

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In current study, we obtained BV-2 cells from Kunming Institute of Zoology

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(Kunming, Yunnan, China), which maintained in RPMI 1640 (Hyclone Co., USA)

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supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 µg/mL

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streptomycin 7. The BV-2 cells were pre-treteate with NOB (0, 25, 50, 100 µM) for 4

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h, and then treated with LPS for 12 h.

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Western blot analysis Cell lysate and brain tissue homogenate was generated with SDS lysis buffer

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24,

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25.

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The

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(NADPHdiaphorase) (SC-20493), complex II (COX2) (sc65239), Actin (I-19), Nrf2

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(SC-722), complex III (cytochrome reductase) (SC-69064), NQO-1 (SC-16464),

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p-p44/42 MAPK (ERK1/2) (9101), iNOS (2982), AKT (4060), Lamin B (SC-6217),

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p-AKT (ser473) (8242), HO-1 (SC-1796), p44/42 MAPK (ERK1/2) (9102), NF-κB

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p65 (8242), p-p38 MAPK(Thr180/Tyr182) (4511), p38 MAPK (9212), IB (9242)

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and p-IB (9246S) (Cell Signaling Technology, Shanghai, China); Complex IV

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(cytochrome oxidase) (AC610) (Beyotime, Jiangsu, China) and HRP-conjugated

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secondary antibodies (Santa Cruz, USA); The blots was quantified by the Quantity

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One (Bio-Rad, CA, USA).

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RT-qPCR

Total proteins diluted by SDS/PAGE were transferred to nitrocellulose membranes. appropriate

antibodies

applied

were

COX-2

(SC-1747),

complex

I

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Total RNA was extracted and purified from brain with RNA extraction kit

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(TaKaRa, Dalian, China) as previously described 2. The RNA purity was measured by

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spectrophotometer (Quawell Technology, San Jose, CA, USA). Reverse transcription

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to cDNA was using the reverse transcription kit (TaKaRa, Dalian, China), and target

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gene were further assessed by RT-PCR on CFX96 real-time system (B io-Rad,

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Hercules, CA). The mRNA expression was normalized to GAPDH. The primers

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information was were listed in Table 1.

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Biochemical analysis

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The supernatant of brain tissues homogenate in lysis buffer was used for the

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biochemical analysis. The protein concentration was determined using BCA protein

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assay kit (Thermo Scientific, USA). TNF-α and IL-1β in serum were measured with

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ELISA kit (Xinle Biology Technology, Shanghai, China).

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Fluorescence staining assays

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JC-1 assay was applied to evaluate mitochondrial membrane potential (MMP) as 26, 27.

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previous described

The BV-2 cells were seeded in 96-well plates overnight.

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Then, 100 μL JC-1 solutions (5 μg/mL) was added into the cells and treated for 1 h at

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37 °C in dark. Fluorescence intensities were detected by a Multimode plate reader

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(Tecan, Switzerland).

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Following treatment, ROS were measured by using DCFH-DA fluorescent dye

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(Beyotime, Jiangsu, China). After washing with PBS, DCFH-DA dye was added into

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the cells. Followed by 30 min incubation at 37 °C, green fluorescence was detected by

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fluorescence microscope (Olympus IX71, Tokyo, Japan) (× 200) and quantified using

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micro plate reader.

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Data analysis

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Data were presented as the means ± SEM. Differences between groups were

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determined by One-way ANOVA using Graphpad Prism software. Differences at p

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