Dihydroquercetin Ameliorates LPS-induced Neuroinflammation And Memory DeficitⅠ
Apr 20, 2023
ABSTRACT
Dihydroquercetin (DHQ) is a pentahydroxyflavanone that has been used as an important supplement against oxidative stress-related inflammation and neuroinflammation. Neuroinflammation is the activation of the defense mechanism of the central nervous system, upon exposure to stimuli like amyloid β, Lewy bodies, lipopolysaccharide (LPS), and reactive oxygen species. It is an important pathophysiological mediator of several neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and others.
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The objective of the present study is to evaluate the neuroprotective effect of DHQ, a potent antioxidant molecule, against LPS-induced neuroinflammation. On the first day of the experiment (day-1), neuroinflammation was induced through intracerebroventricular injection of LPS (5 μg/5 μl) into each lateral ventricle in the rats. DHQ0.5, 1, and 2 μg/kg were injected into the tail vein in respective groups from day-2 to day-10. Behavioral studies showed that DHQ attenuated the LPS-induced loss in long-term memory and working memory as evaluated by elevated plus maze and Y-maze test, respectively.
Further, the biochemical estimations revealed that DHQ dose-dependently attenuated the LPS-induced decrease in acetylcholine level and increased the acetylcholine-esterase activity in the hippocampal region. DHQ also increased the catalase activity and decreased nitric oxide and lipid peroxidation altered by LPS injection.
DHQ also attenuated interleukin-6 in the brain, which has elevated upon LPS induction. The decrease in IL-6 is attributed to its antioxidant activity. Hence, DHQ could be a potential therapeutic candidate in the management of neuroinflammation and related neurodegenerative disorders.
1 Introduction
Neuroinflammation is the phenomenon of activation of a defense mechanism in the central nervous system against a variety of stimuli (Morales et al., 2015). It is an important pathophysiologic mediator of most neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and others (Lyman et al., 2014; Morales et al., 2015). Various stimuli include amyloid β, Lewy bodies, lipopolysaccharide (LPS), and reactive oxygen species (ROS), which are crucial mediators of neuroinflammation (Lee et al., 2008; Streit et al., 2004).
LPS is an established model for studying the relationship between neuroinflammation and cognitive behavior (Tripathi et al., 2017). LPS is also reported to induce anxiety-like behavior in mice and rat models (Bassi et al., 2012; Savignac et al., 2016). Although the exact pathophysiology of neuroinflammation by LPS is not completely known, one of the mechanisms is a toll-like receptor (TLR)- 4 mediated activation of NFκB, which is a transcription factor for many pro-inflammatory cytokines (Shih et al., 2015). An important mechanism for neuroinflammation by LPS is cellular damage due to reactive oxygen species (ROS) generation and cytokines activation (Hsu and Wen, 2002).
LPS induces the activation of astrocytes and microglia in the CNS and a further increase in the expression of IL-6 (Wang et al., 2015). IL-6 is a soluble mediator with a pleiotropic effect on inflammation, immune response, and hematopoiesis and is considered a marker of neuroinflammation when present in CNS (Beurel and Jope, 2009). LPS causes neurodegeneration and defects in learning and memory (Shaw et al., 2001). It is proven that both muscarinic and nicotinic acetylcholine receptors play a role in learning behavior and memory consolidation via long-term potentiation (LTP) (Hasselmo, 2006).
As per the cholinergic hypothesis, loss of cholinergic function in the CNS is significantly related to memory decline and acetylcholine esterase inhibitors are pivotal drugs for the management of dementia-related symptoms in Alzheimer's disease (Davies, 1985). Neuroinflammation is critically involved in neurodegenerative disorders; however, conventional anti-inflammatory drugs, such as nonsteroidal anti-inflammatory drugs, have produced mixed results, and their toxic effects have not been resolved (Woodling and Andreasson, 2016). Therefore, there is a need to explore newer drugs for the potential treatment of neuroinflammatory disorders.
Various natural products are showing promising results against oxidative stress and neuroinflammation, including carnosine (Caruso et al., 2019), hederagenin (Wang et al., 2020), and resveratrol (Zhang et al., 2013). Dihydroquercetin (DHQ) is a potent antioxidant flavonoid found in onions, French maritime bark, milk thistle, and Douglas fir bark (Weidmann, 2012).
It has been found to possess a neuroprotective effect on rat neuronal culture cells that were damaged by oxidative stress (Dok-Go et al., 2003). It has been reported to ameliorate concanavalin-A-induced mouse experimental fulminant hepatitis and increased heme oxygenase-1 (HO-1) expression through mitogen-activated protein kinase/nuclear factor erythroid 2-related 2 (MAPK/Nrf2) antioxidant pathway in RAW264 macrophage cell lines (Zhao et al., 2015).
DHQ has also been shown to inhibit cerebral ischemia-reperfusion injury in rats through suppression of leukocyte infiltration and by inhibiting cyclooxygenase-2 and the expression of inducible nitric oxide synthase (Wang et al., 2006). DHQ has also attenuated the proteasome inhibition-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and caspase-8 and Bid-dependent pathways through antioxidant action (Nam et al., 2015). Therefore, we thought it would be prudent to evaluate the anti-neuroinflammatory activity of DHQ as it possesses potent antioxidant activity.
Therefore, the objective of the present study is to evaluate the potential anti-neuroinflammatory effect of DHQ (0.5, 1, and 2 μg/kg) in the LPS model in rats. Functional analysis of DHQ treatment was done by estimating cerebral blood flow and behavioral tests for working and long-term memory apart from anxiety-like effects using Y-maze and elevated plus maze test, respectively. The level of acetylcholine (ACh) and acetylcholine esterase (AChE) activity was investigated in the hippocampus region to further evaluate learning and memory. The antineuroinflammatory activity of DHQ was evaluated by estimating the expression of IL-6.
2. Materials and methods
2.1. Experimental animals
Inbred albino Wistar male rats of weight (260 20 g) were procured from Central Animal House; Institute of Medical Science (IMS-BHU). The experiments were performed by adopting guidelines (NIH publication number 85–23, revised 2011) and approved by the Institutional Animal Ethical Committee, Banaras Hindu University (BHU; Dean/2015/CAEC/ 1420). Animals were acclimatized for one week in the experimental lab before initiating the experiments.
2.2. Chemicals
Dihydroquercetin (Disto-Pharmaceuticals, India), LPS (E. coli, L3129), bovine serum albumin, and Griess reagent were procured from Sigma-Aldrich (St. Louis, MO, USA). thiobarbituric acid (TBA), NADH, sodium succinate, sodium azide, phenazine methanesulphonate (PMS), and nitro blue tetrazolium (NBT) were purchased from Merck (Darmstadt, Germany). All other chemicals and reagents of high-performance liquid chromatography (HPLC) and analytical grade were procured from local suppliers.
2.3. Experimental procedure
The schematic representation of the experimental schedule has been depicted in Fig. 1. The LPS was injected intracerebroventricular (ICV) using stereotaxic apparatus (Stoelting, USA). Rats were anesthetized with sodium pentobarbitone; i. p. 45 mg/kg (Tripathi et al., 2017). The animal was fixed on the stereotaxic frame, the scalp of the rat was incised, and the bregma was positioned on the scalp of the rat. All coordinates were set from the bregma (0, 0) and drilled 0.8 mm posterior to the bregma, 1.5 mm lateral to the sagittal suture, and 3.8 mm beneath the surface of the brain (Hauss-Wegrzyniak et al., 1998).
LPS was administered to all the groups except sham by using Quintessential Stereotaxic Injector. LPS solution was injected into each of the lateral cerebral ventricles at concentrations of 1 μg/μl, at a rate of 1 μl/min over 5 min with a waiting period of 5 min between the two injections. The Sham group was also exposed to surgery, and only saline (vehicle) was injected. All the sham group results were not significantly different from the control group, so the group was not included further. To limit local infection, after suturing up the incision, we applied betadine (iodine solution) up to day-5, and 1 ml of normal saline was injected intraperitoneally to prevent dehydration in the animals (Jangra et al., 2021).
Rats were closely monitored during the recovery period and kept in a room at 22–26 C. This was considered as day-1, and after 24 h, upon the appearance of neuromotor dysfunction-related symptoms, the drugs were given daily for nine days. All the behavioral tests were performed on day-10 and recorded using ANY-MAZE behavioral tracker version 4.72 (USA). The animals were then immediately killed by decapitation and, hippocampus tissues were microdissected (Paxinos and Charles, 2006). The brain tissues were stored at 80 C until further mechanistic studies.
2.4. Drug administration
DHQ was first dissolved in 10 μl of ethanol, and then the volume was made up using normal saline to obtain a concentration of 1 mg/ml. The concentration of ethanol in the solution injected was less than 0.001% (v/v) and the same concentration excluding DHQ was administered to the vehicle control group. Previously, it has been reported that DHQ ameliorates cerebral ischemia-reperfusion injury in rats through its antioxidative effect and modulation of NFκB activation at the dose of 0.1 and 1.0 μg/kg, intravenous (IV) daily (Wang et al., 2006).
Therefore, we conducted a pilot study to evaluate the DHQ IV dose and finalized three doses of DHQ 0.5 μg/kg, 1 μg/kg, and 2 μg/kg. By following the principle of randomization, grouping was done taking nine animals in each group as follows: (a) Control, (b) LPS (c) LPS þ DHQ 0.5 μg/kg, (d) LPS þ DHQ 1 μg/kg, (e) LPS þ DHQ 2 μg/kg. There was no par se group in the experiment to check the effect of DHQ alone on the rats as the toxic and therapeutic effects of DHQ are well known (Booth and DeEds, 1958; Sunil and Xu, 2019).
2.5. Measurement of cortical blood flow
CBF was recorded using a laser speckle blood flow imaging system (omega zone OZ-2 STD, Tokyo, Japan). Rats were anesthetized, and their skull bones were exposed by a midline scalp incision and placed on the black sponge sheet under the arm stand. The arms stand holds the CCD camera, the lens (ZM10–18, MF12), and the laser unit (780 nm for measurement and 650 nm for positioning).
Raw speckle images were recorded from the skull surface using LSI Software (LSI ver.3.3, Omegawave, Inc., Tokyo, Japan), and average cerebral blood flow was determined by further analysis of images by using LIA Software (LIA ver.3.3, Omegawave, Inc., Tokyo, Japan). The black sheet does not reflect the laser light, and the effect makes the blood flow image clear (Paliwal et al., 2018).
2.6. Behavioral tests
2.6.1. a Y-maze test
Y-maze apparatus helps to assess working memory, general exploratory behavior, and spatial memory. The Y-maze apparatus is made up of three identical arms having dimensions of 32 cm height, 50 cm length, and 16 cm width, which are angled at 120⁰ to each other. Plastic balls of different colors were placed around the arms, and these were not changed every time before the test to maintain novelty for the animals. Soiled animal bedding was spread over the floor of the maze to give a homely atmosphere. In trial 1, the novel arm of the Y-maze was kept closed, and the animals were free to move in the two arms for 15 min.
Trial 2 was performed exactly after 4 h of trial 1, in which animals had free access for 5 min to all three arms. All the entries were recorded using ANY MAZE software. Curiosity behavior was calculated from the total number of entries in all three arms. The percentage of arm entries in novel and known arms is considered indicative of spatial recognition memory. Spontaneous alternation behavior signifies the working memory in which we observe how often an animal repeats its initial choices of arm entries. An arm entry is counted when the animal has his head and front paws inside an arm (Garabadu et al., 2015; Joshi et al., 2014).
2.6.2. b elevated plus maze test
The EPM (Elevated Plus Maze) test was performed to assess anxiety and long-term spatial memory behavior in a fabricated apparatus. The fabricated EPM consisted of two open arms (50 10 cm) and two closed arms (50 10 40 cm) the height of the open arm roof was 50 cm from the floor. Transfer latency (TL) was performed for evaluating long-term spatial memory.
TL was measured as the time taken by the animal to move into one of the enclosed arms with all four legs. The animal was gently pushed into one enclosed arm if the animal is not entering into the enclosed arm within 90 s. In such a case, TL was counted as 90 seconds. On day-10, the trial was performed in which rats were allowed to explore the maze for 2 min. This task was repeated after 24 h to evaluate the retention of memory. Percentage entries and time spent in the open arm were considered as a measure of anxiety (Krishnamurthy et al., 2013).
2.7. Biochemical estimations
2.7.1. a preparation of the samples
The hippocampus tissue homogenate was made, by taking hippocampus tissue in 1 ml of 0.1 M perchloric acid in a Potter–Elvehjem homogenizer with fine circular unidirectional trituration. The homogenate so obtained was taken in a polypropylene tube in which 50 μl of 4 M potassium acetate was added to make its pH 4.0, and later, it was centrifuged for 15 min at 4000 g. The supernatant so obtained was used for the estimation of both Acetylcholine (Ach) and Acetylcholine Esterase (AchE) activity (Muthuraju et al., 2009).
2.7.2. Spectrofluorometric assay of acetylcholine
The amplex red assay kit was used to find the amount of acetylcholine in the homogenate. The reaction was started by adding an Amplex Red reagent/HRP/choline oxidase/AChE working reagent in the homogenate tube. It was incubated for 30 min, and fluorescence was recorded with the help of a microplate reader (BioTek, Synergy H1M, USA) at 530 nm excitation and 590 nm emission wavelengths (Zoukhri and Kublin, 2001).
2.7.3. b estimation of AChE activity
Amplex Red AChE assay kit (Molecular Probes, Inc., USA) was utilized to measure AChE activity. The estimation was done as per the manufacturer's instructions. The reagents were added to the sample and were kept for incubation. After incubation, the fluorescence was determined with the help of a microplate reader (BioTek, Synergy H1M, USA) at 530 nm excitation wavelength and 590 nm emission wavelength
2.7.4. c nitrite level estimation
Nitrite level was estimated as per the method given by Green (Green et al., 1982). 50 μl of the supernatant obtained from brain homogenate was mixed with 5 μl of nicotinamide adenine dinucleotide phosphate (NADPH), 10 μl of flavin adenine dinucleotide (FAD) and 5 μl of nitrate reductase. The mixture was incubated for 1 h at 37 C in the dark. Zinc sulfate was added to precipitate the proteins.
After centrifuging (6000 g), equal volumes of supernatant (100 μl) and Griess reagent (100 μl) (1:1 mixture of 1% sulfanilamide in 3% orthophosphoric acid and 0.1% naphthyl ethylene diamine) were mixed and incubated for 10 min at room temperature in the dark. The plates were then read at 540 nm by UV spectrophotometer, and NOx was calculated by using a sodium nitrite standard curve.
2.7.5. d mitochondrial LPO or malondialdehyde (MDA) formation estimation
Mitochondrial MDA content was measured by following the standard protocol (Ohkawa et al., 1979). The homogenate was boiled along with 10% SDS, KCl (1.15%), acetic acid (20%), and TBA (0.8%). After cooling in running water, it was extracted with n-butanol. The organic layer so obtained by centrifuging was measured at 532 nm. The concentration of MDA was expressed as micromoles of MDA per milligram of protein.
2.7.6. e-assessment of catalase activity
Catalase activity was assayed following the method of Luck (1974). Brain homogenates were centrifuged at 10,000 rpm for 10 min in an Eppendorf microcentrifuge. The supernatant aliquots were mixed with phosphate buffer and hydrogen peroxide The absorbance was measured at 240 nm for 3 min at a 30-s interval. From the decrease in absorbance, the enzyme activity was calculated (Correa et al., 2000).
2.7.7. f estimation of IL-6 using ELISA kit
Cytokine IL-6 was estimated in brain homogenate using a commercially available ELISA kit for rat IL-6 (NOVEX™, Thermo Fischer, USA). The brain samples were suspended in buffer solution (1% Triton X100, aprotinin 200 U/ml, 0.1 mM PMSF, 0.1 mM benzethonium chloride, 1 mM benzamidine, and 1 mM EDTA) and then centrifuged at 14,000 g for 30 min at 4 C and the supernatant was separated for cytokines estimation. The pro-inflammatory cytokine interleukin-6 (IL-6) levels in whole brain samples were quantified using ELISA kits as per the manufacturer's instruction.
2.8. Statistical analysis
All values are expressed as the mean standard deviation (SD). For spatial memory in the EPM task, repeated measures two-way ANOVA followed by Bonferroni post-hoc test was performed for transfer latency between day-9 and day-10. Similarly, two-way ANOVA followed by Bonferroni post-hoc test was performed for spatial recognition memory and curiosity behavior test of the animals in the Y-maze to assess the locomotor activity in both probe and test sessions.
One-way ANOVA followed by post-hoc Student's Newman–Keuls test was performed for the analysis of all other behavioral and biochemical parameters. Graph Pad Prism version 5 (San Diego, CA) has been used for applying the statistics, and groups with p < 0.05 were considered as significantly different.
The mechanism of the Cistanche neuroprotection effect
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To be continued...
Qadir Alam, Sairam Krishnamurthy *
Neurotherapeutics Laboratory, Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (Banaras Hindu University), Varanasi, 221005,
U.P, India
