Chrysophanol attenuates cognitive impairment, neuroinflammation, and oxidative stress by TLR4/ NFκB -Nrf2/HO-1 signaling in ethanol induced neurodegeneration

doi:10.21203/rs.3.rs-5222505/v1

Download PDF

Research Article

Chrysophanol attenuates cognitive impairment, neuroinflammation, and oxidative stress by TLR4/ NFκB -Nrf2/HO-1 signaling in ethanol induced neurodegeneration

https://doi.org/10.21203/rs.3.rs-5222505/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Ethanol-induced neurodegeneration refers to the progressive loss of structure and function of neurons caused by chronic ethanol consumption. According to the World Health Organization (WHO), over 2.3 billion people globally consume alcohol. This contributes to a significant number of alcohol-related brain damage. This study evaluated the effect of chrysophanol in ethanol-induced neurodegeneration. Mice were administered with 10mg/kg i.p chrysophanol, 30 minutes after administration of 2g/kg i.p injection of ethanol for 11 days. Y-maze, Morris water maze (MWM), and novel object recognition (NOR) test were carried out to analyze learning and memory impairment. Analysis of antioxidant levels, histopathological examinations, measurement of COX-2 & NLRP3 using ELISA, and gene expression analysis of TLR4, NFκB, IL-1β, TNF-α, Caspase-3 and Nrf-2, HO-1, and in hippocampus and cortex using RT-PCR as well as DNA damage by comet assay were carried out. Chrysophanol has shown remarkable impact in reversing cognitive decline and spatial memory. It effectively boosted antioxidant levels such as GSH, GST, and CAT, while simultaneously reducing the levels of MDA and NO. The histopathological analysis also showed improvement in overall morphology and survival of neurons. Chrysophanol treatment effectively showed an increase in the expression of HO-1 and Nrf-2 with a decrease in TLR4, NFκB, IL-1β, TNF-α, and Caspase-3 expression confirmed through RT-PCR. Production of inflammatory cytokines, and apoptotic gene expression was successfully reversed after chrysophanol treatment. COX-2 & NLRP3 levels were decreased and improvement in DNA damage were observed after chrysophanol treatment. In conclusion, chrysophanol demonstrated remarkable neuroprotective activity against ethanol-induced neurodegeneration.

Ethanol-induced neurodegeneration refers to the damage and loss of neurons in the brain caused by the chronic consumption of ethanol, the active component of alcoholic beverages. This neurodegenerative process is a significant contributor to the cognitive and behavioral deficits observed in alcohol use disorder (AUD) (Bird et al., 2020, Holloway et al., 2023) Neurodegenerative disorders are a leading cause of disability and mortality worldwide. While specific data on ethanol-induced cases is scarce, alcohol-related neurodegeneration is a significant contributor to this burden, particularly in regions with high alcohol consumption rates. The prevalence of ethanol-induced neurodegeneration is difficult to quantify due to overlapping factors, such as varying definitions of neurodegeneration, differences in alcohol consumption patterns, and the multifactorial nature of neurodegenerative diseases (Crews and Nixon, 2009). As of 2024, alcohol consumption is responsible for approximately 2.3 million deaths annually worldwide, accounting for about 4.7% of all global deaths (WHO). These Figures reflect a significant public health burden, with most alcohol-related deaths occurring among men. Alcohol contributes to a range of health issues, including noncommunicable diseases (such as cardiovascular diseases and cancer), injuries (such as those from traffic accidents and violence), and infectious diseases (such as HIV and tuberculosis, due to their impact on the immune system). The highest alcohol-related mortality rates are found in the European and African regions, with a significant portion of deaths occurring in young adults aged 20 to 39 years. These statistics underscore the need for continued global efforts to address the harmful effects of alcohol consumption, particularly in regions with high mortality rates (Qin and Crews, 2012). Chronic ethanol consumption is widely acknowledged as a significant risk factor for neurodegenerative diseases, resulting in pronounced cognitive deficits, neuronal loss, and brain atrophy (Oscar-Berman and Marinkovic, 2003, Ye et al., 2020). The underlying mechanisms of ethanol-induced neurodegeneration are complex and encompass multiple cellular processes, including oxidative stress, inflammation, and apoptosis. A pivotal pathway involved in these processes is the NF-κB signaling pathway (Lanzillotta et al., 2015).. This pathway plays a critical role in regulating the expression of genes associated with inflammation, cell survival, and apoptosis, thereby serving as a key mediator of ethanol's neurotoxic effects. Ethanol exposure has been demonstrated to activate the NF-κB pathway in several brain regions, including the hippocampus and cortex, which are especially susceptible to damage caused by ethanol (Angelis et al., 2021, Shafaroodi et al., 2012). The activation of NF-κB by ethanol occurs through several mechanisms. Ethanol metabolism generates ROS, contributing to oxidative stress (Alfonso-Loeches et al., 2014) (Bustamante-Barrientos et al., 2023, Wang et al., 2020). ROS are potent activators of the NF-κB signaling pathway. Increased ROS levels resulting from ethanol exposure can cause the phosphorylation and subsequent degradation of IκB proteins, thereby activating NF-κB. A study by (Reddy, 2023) demonstrated that chronic ethanol exposure increases ROS production in the hippocampus, leading to the activation of the NF-κB pathway and subsequent neuroinflammation.

NF-κB regulates the expression of both pro-apoptotic and anti-apoptotic genes (upregulation of pro-apoptotic factors such as Bax and the downregulation of anti-apoptotic factors such as Bcl-2) tipping the balance towards cell death. Studies have reported that chronic ethanol exposure leads to increased activation of caspase-3, a key executor of apoptosis, in an NF-κB-dependent manner, contributing to neuronal apoptosis in the hippocampus and cortex (Waku and Kobayashi, 2021). Mitochondria are essential for cellular energy production and are key regulators of ROS levels. However, chronic exposure to ethanol impairs mitochondrial function, resulting in elevated ROS production and increased oxidative stress. Ethanol decreases the activity of key antioxidant enzymes, and reduction in these enzyme's activity impairs the brain's capacity to neutralize ROS and mitigate oxidative damage (Rowley et al., 2015). Key transcription factor that regulates the expression of antioxidant proteins. Chronic ethanol exposure impairs the Nrf2 signaling pathway, reducing its capacity to activate antioxidant defenses and thereby increasing vulnerability to oxidative stress. (Mattson and Camandola, 2001).

Chrysophanol is a natural compound found in various plants, particularly in the rhizomes of Rheum palmatum (Chinese rhubarb) and other members of the Polygonaceae family (Zhang et al., 2014, Singh et al., 2017). Chrysophanol is an anthraquinone derivative with a specific chemical structure that contributes to its biological activities. Chrysophanol exhibits strong antioxidant properties, reduces inflammation, antimicrobial properties, and neuroprotective properties (Malik et al., 2010, Prateeksha et al., 2019, Chen et al., 2020). This study aimed to explore the neuroprotective effect of chrysophanol in ethanol-induced degeneration and its underlying mechanisms. Behavioral tests were carried out to evaluate cognitive improvement and various biochemical and histological tests were performed to rule out the beneficial effect of chrysophanol in ethanol-induced neurodegeneration.

Animals

The male BALB/c mice (N = 80) approximately 25 to 30 g, were procured from the NIH in Islamabad for this experiment. The mice were acclimated for one week. They were contained at the Quaid-i-Azam University animal facility in plastic cages with sawdust bedding, provided sterile water, and given rodent food ad libitum. The mice were kept in an optimal environment with a 12-hour light-dark cycle and a temperature of 23 ± 2°C. All in vivo and behavioral assessments were carried out during the light cycle. The methodology was approved by the bioethical committee of QAU (Approval No. #BEC-FBS-QAU2023-561).

Study design

This experiment involved the random division of mice into four separate groups. The study design can be found in the accompanying Table 1 and Fig. 1.

Table 1

Study design for ethanol-induced neurodegeneration
Group	Treatment received
vehicle control	In the vehicle control group, mice were injected with normal saline at a dose of 10 ml/kg i.p daily for 11 days.
Ethanol group	Mice received i.p injections of ethanol at a dose of 2g/kg once daily for 11 days
Donepezil group	Mice in this group were administered donepezil (3mg/kg) as positive control i.p 30 minutes after the i.p injection of ethanol for 11 days.
Chrysophanol group	Mice were injected chrysophanol (10mg/kg) i.p 30 minutes following the i.p injection of ethanol for 11 days.

Behavioral assessment for memory

Y-maze Test

A wooden Y-shaped apparatus measuring 20 cm in height, 10 cm in width, and 50 cm in length is used for evaluating the behavior of mice. Mice were placed in the apparatus and the trial was carried out in three sessions. Each session comprised of 8 minutes and then mice were sent back to their home cages. Spontaneous alteration behavior was evaluated by analyzing the spontaneous uninterrupted entry of mice into the arms. Percent alteration behavior was calculated by using the below-mentioned equation.

Percent Alteration

[three successive sessions x (successive entry of mice into three different arms)/ total arm entries-2] x 100

Morris Water Maze Test

This behavioral test is used to analyzed cognitive deficits and memory impairment in mice. The setup consists of a circular pool 50cm in height and 120cm in diameter, divided into four quadrants with one designated as the target quadrant. The probe is placed in the target quadrant 1cm below the water surface, maintained at a temperature of 25°C. The platform position remains constant throughout the experiment.(Rehman et al., 2021, Vorhees and Williams, 2006).

Training and acclimatization phase

The mice underwent a 3-day training session. On the first day, the mice were placed in the pool environment for 5 minutes to acclimatize. On the second day, the mice were positioned in a quadrant with a visible platform, allowing them to learn to stay in the quadrant. On the third day, the mice were trained with a hidden platform placed 1cm below the water's surface.

Probe trial

During days 9, 10, and 11, a probe trial was conducted to evaluate the impact of ethanol on learning and reference memory. The time spent in the platform quadrant and the number of crossings over the platform position were measured for each animal.

Final test

On day 12, mice were tested for behavioral assessment and escape latency, time spent in the platform quadrant, and no crossing over in each quadrant was measured.

Novel Object Recognition Test

The experiment was conducted in three phases: habituation, familiarization, and test. The apparatus used was a wooden square chamber measuring 40cm x 40cm x 40cm. During the habituation phase, mice were placed in the chamber for five minutes without any objects to get used to the environment. After 24 hours, the mice were placed in the chamber again, this time with two identical objects, for the familiarization phase. In the test phase, the mice were placed in the chamber with one familiar object and one novel object to see if they could recognize the novel object. All the behaviors were recorded using a top-mounted camera for later analysis. The time spent exploring each object was recorded and analyzed for each group. If the mice spent more time exploring the novel object than the familiar object, it indicated that they were able to discriminate between the two. The discrimination index (DI) and recognition index (RI) were calculated based on these observations.

DI = (Total time spent with novel object − Total time spent with familiar object) ÷ (Total time spent with novel object + Total time spent with familiar object)

RI = (Total time spent with novel object) ÷ (Total time spent with novel object t + Total time spent with familiar object)

Sampling

At the end of the experiment, mice were euthanized using xylazine and ketamine i.p. Blood was drawn using intracardiac injection and stored in Eppendorf. Collected blood was centrifuged at 4000rpm for 10 minutes to collect serum. The serum was stored at − 80°C for later testing. After that mice were dissected, and the brain was collected. Desired brain regions such as the hippocampus and cortex were separated from the whole brain. For this purpose, the brain was placed on an iced box, and using sharp blades hippocampus and cortex were precisely separated. Some of the samples were fixed in buffered formalin solution for histological staining and the remaining brain samples were cryopreserved at − 80°C.

Some samples were fixed in 10% buffered formalin for histology, while the remaining brain samples were stored at − 80°C to prepare homogenate. Brain homogenate was prepared by taking an equal amount of tissue (0.1 g) and diluting it with 1 mL phosphate buffer saline (PBS; pH = 7.4) in a 2-mL round-bottom Eppendorf. After homogenization at 5800 rpm for 3–6 min, homogenates were centrifuged at 4°C at 4000 rpm for 10 min to obtain supernatant. The supernatant was collected and used for antioxidant assays.

Antioxidant assay

Antioxidant assays were performed to evaluate the level of GSH, GST, CAT, MDA, and NO in the hippocampus and cortex. The cryopreserved brain samples were thawed and then an equal amount of tissue (0.1gm) was diluted with 1mL phosphate buffer saline in a round bottom Eppendorf. The mixture was homogenized at 5800 rpm for 3–4 minutes and then centrifuged at 4°C at 4000 rpm for 10 minutes to collect supernatant.

GSH level

Following the described protocol, the GSH level was evaluated in the hippocampus and cortex (Habig et al., 1974). This is a conjugation reaction between the free thiol of GSH and DTNB that generates a yellowish chromophore. 40ul of DTNB solution was added in each well of the plate followed by the addition of 6.6uL of sample and 153 uL of phosphate buffer to maintain the pH at 8. Absorbance was recorded at the wavelength of 412nm using the spectrophotometer.

GST level

GST level was evaluated in the hippocampus and cortex regions of the brain by following previously reported protocol (Habig et al., 1974). This is based on a conjugation reaction between GST CDNB. Each well-contained 10uL of 1mM CDNB, 10uL of 5mM of reduced glutathione, 10uL of sample, and 270uL of buffer solution. The absorbance was recorded at 340nm wavelength. using a plate reader.

Catalase level

Catalase activity was analyzed in the hippocampus and cortex by using the prescribed protocol (Bouguiyoud et al., 2022). This reaction follows the decomposition of H2O2 by catalase. Absorbance was taken on 240nm of wavelength by using a microplate reader. Absorbance is directly proportional to the H2O2 level in tissue.

MDA and NO level

MDA and NO levels were analyzed by using the reported protocols respectively (Ohkawa et al., 1979, Tsikas, 2007). To measure MDA levels, precisely mix 20µl of ferric chloride with 200µl of supernatant and 200µl of ascorbic acid. Incubate the mixture at 37°C for exactly 30 minutes, then add 500µl of TCA and TBA. Measure the absorbance at 535nm. Estimate NO levels using the Griess reagent method in tissue homogenate and record the absorbance at 540nm.

Histological testing

The formalin-fixed parts of the brain were embedded in the paraffin. A 5 µm thick section was carefully obtained using a rotary microtome and then meticulously stained with hematoxylin & eosin. The slides were meticulously examined under an optical light microscope to assess any neurodegenerative changes in the hippocampus CA1, CA3, and DG, and to determine the extent of surviving neurons in the hippocampus and cortex. The precisely stained sections were rigorously evaluated for any signs of neuronal injury and inflammation using a precise four-point scale ranging from 0 to 5, where 0 indicates no damage, 1–2 indicates mild damage, 3–4 indicates moderate damage, and 5 indicates severe damage.

mRNA Expression in Hippocampus and Cortex by RT-PCR

The hippocampus and cortex of mice were isolated, and total RNA was extricated by HiPure Total RNA Kit by Magen Biotechnology (Catalogue No. IVD4121).by following the manufacturer’s specifications to determine mRNA expression. The concentration of isolated RNA was measured using a nano-drop light spectrophotometer. After analyzing the purity and adjusting the concentration to 2000ng, RNA was converted to DNA (cDNA) using Thermo Fisher cDNA kit (HRP013 100T) (ZOKEYO, China). The reaction was carried out using 0.5ul of cDNA with a total 10ul reaction mixture using SYBR Select Master Mix (CatNo. 4472903, ZOKEYO, China) on Step One Plus Real-time PCR system for relative quantification of each sample (Applied Biosystems, USA). Then, 2 − ΔΔCT was calculated after expression analysis. The primers for GAPDH, TLR4, NF-κB, IL-1β, TNF-α, Nrf2, HO-1, Caspase 3 are presented in Table 2.

Table 2

Detail of primers used in RT-PCR for mRNA expression.
S.No	Gene	Forward Primer (5’-3’)		Reference
1	GADPH	ACTCCACTCAC GGCAAATTCA	TCTCGCTCCT GGAAGATGGT	(Rehman et al., 2022)
2	TLR4	ATGGCATGGC TTACACCACC	GAGGCCAATT TTGTCTCCACA	(Dong et al., 2022)
3	NF-κB	GCCAGACACA GATGATCGCC	GTTTCGGGTA GGCACAGCAA	(Dong et al., 2022)
4	IL-1β	TGGACCTTCCA GGATGAGGACA	GTTCATCTCG GAGCCTGTAGTG	(Javaid et al., 2023)
5	TNF-α	CCACCACGCT CTTCTGTCTAC	AGGGTCTGGG CCATAGAACT	(Javaid et al., 2023)
6	Nrf2	CACATCCAG A CAGACACCAGT	CTACAAATGGG AATGTCTCTGC	(Alvi et al., 2021)
7	HO-1	CGTGCAGAG A ATTCTGAGTTC	AGACGCTTTAC GTAGTGCTG	(Alvi et al., 2021)
8	Caspase 3	ACAGTGGAAC TGACGATGATATG	TCCCTTGAATTT CTCCAGGAATAG	(Huang et al., 2021)

Estimation of COX-2 and NLRP3 levels by ELISA

The COX-2 and NLRP3 levels were determined using the hippocampus and cortex using an ELISA kit according to the manufacturer’s specifications (Catalog No. 30205Ra, purchased from Nanjing Pars Biochem CO., LTD.) and Elisa kit of rat (Catalog No A5652 purchased from Shanghai MLBIO Biotechnology C. LTD. China) respectively.

The SCGE comet analysis was employed to assess DNA damage in the hippocampus and cortex regions of the brain. This involved a series of precise steps. Initially, slides were meticulously cleaned by burning them in methanol to eliminate any machine oil and dust particles. Subsequently, three-quarters of the sterile slides were submerged in a 1% NMPA agarose solution at a controlled temperature of 25 ˚C. A homogenate of separated brain parts was prepared with 1 ml of cold lysing solution. The homogenate, combined with low melting point agarose (LMPA), was layered onto the NMPA-coated slides. After covering the slides with a coverslip and allowing them to sit on ice for 10–12 minutes, the coverslips were removed, and the slides were treated with LMPA. This process was repeated three times, followed by dipping the slides in lysing solution for 10 minutes and then placing them in the freezer for 2 hours. Following electrophoresis, the slides were meticulously observed and analyzed for DNA damage. Ethidium bromide (1%) was used to stain the slides, which were then examined under a fluorescent microscope. The degree of DNA damage was precisely measured and analyzed using Image J Comet software.

Statistical analysis

Data were presented as mean ± SD. The IBM® Statistical Package for Social Sciences (SPSS) version 25 was used for One-way and two-way ANOVA (as per requirements of data) with the least significant difference (LSD) test for multiple comparisons, was used for data analysis. A p-value < 0.05 was used to assign the statistical significance. All Graphs were created using GraphPad Prism version 9.5.0 software.

Y-maze

In this study, Y-maze tests were conducted to evaluate the impact of chrysophanol on ethanol-induced cognitive decline in mice (Fig. 2). Results of the y-maze test have shown that ethanol-treated mice have demonstrated cognitive deficit by the lowest percent of spontaneous alteration compared to vehicle control (p < 0.05). Comparing the results of the chrysophanol group with vehicle control has shown least significant variation. However, chrysophanol has improved mice's spontaneous alteration behavior, signifying cognitive improvement compared to the ethanol group (p < 0.05). The Chrysophanol group has shown comparable results with the donepezil group.

Morris Water Maze

In the probe trial, three parameters were taken for the assessment i.e. time spent in the target quadrant, escape latency, and number of crossing over through platform positions (Fig. 3). The results of MWZ showed that the ethanol-treated mice had increased cognitive deficits, as evidenced by their longer latency times in the MWM test (p < 0.05) compared to the vehicle control group (p < 0.05). The Chrysophanol group has the least significant differences in terms of escape latency compared to the vehicle control group. Comparing the results of the chrysophanol group with the ethanol group, a highly significant difference was observed (p < 0.05). Treatment with chrysophanol significantly improved cognitive deficits, as demonstrated by a decrease in latency period (p < 0.05) and a reduction in the number of crossings over the platform quadrant (p < 0.05) compared to the ethanol group. Additionally, the time spent in the platform quadrant was significantly lower in the ethanol-treated mice compared to the vehicle control group (p < 0.05), but chrysophanol treatment resulted in an improvement in the time spent in the platform quadrant compared to the ethanol group (p < 0.05). Chrysophanol treatment significantly increased the mice's staying time in the platform quadrant compared to the ethanol group (p < 0.05).

Novel Object Recognition

The results of novel object recognition (NOR) have shown that (Fig. 4) sniffing for new objects was maximum for the vehicle control group and mice in the ethanol group had the lowest time spent sniffing the new object (p < 0.05). However, ethanol-treated mice spent more time sniffing old objects in contrast to the vehicle control group (p < 0.05). This indicates the cognitive deficit in ethanol-treated mice. Treatment with chrysophanol improved this cognitive deficit in mice as results showed increased sniffing time of new objects in the chrysophanol group and decreased sniffing time with old objects as compared to the ethanol group. The Chrysophanol group has the least significant differences with the vehicle control group. The discrimination index was calculated for each group. Results showed that ethanol-treated mice were unable to discriminate between old and new objects. Chrysophanol group has increased DI which means mice in this group were able to discriminate between new and old objects. The recognition index was maximum for vehicle control and chrysophanol group as compared to the ethanol group indicating the cognitive ability of mice to recognize old and new objects.

Chrysophanol improved the antioxidant and oxidative stress markers in ethanol-induced neurodegeneration

Free radicals formed by ROS can be inactivated by an antioxidant defense system before they cause any cellular damage. Results indicated a notable reduction in GSH, GST, and catalase levels (Fig. 5) in the ethanol group with a significant increase in oxidant production such as NO and MDA as compared to the vehicle control group (p < 0.05). moreover, the chrysophanol group was found to improve the antioxidant defense system by upgrading the level of GSH, GST, and CAT. In addition to that chrysophol group successfully reduced the level of MDA and NO (Fig. 6) in the hippocampus and cortex. These observations ascertain the effective role of chrysophanol in preventing cellular damage and neurodegeneration caused by ethanol.

Effect of chrysophanol on histology of hippocampus and cortex

The effects of chrysophanol on ethanol-induced brain tissue injury were investigated. H&E staining was utilized to identify and analyze pathological changes in the hippocampus DG region, CA1, CA3, and cortex. Results revealed that ethanol administration is associated with neurodegeneration, vacuolation, and swelling (Fig. 7) as compared to the vehicle control group. Administration of chrysophanol was effectively found to alleviate neuropathological changes and reduce neuroinflammation as compared to the ethanol group. Graph has shown the scoring of neuron damage and inflammation in each group. It showed that maximum neuronal damage and inflammation were seen in DG, CA1, CA3, and cortex of ethanol-treated mice as compared to vehicle control. The Chrysophanol group has shown better recovery, less inflammation, and neuronal damage as compared to the ethanol group. Moreover, chrysophanol has shown results comparable to those of the vehicle control group.

The mRNA expression of TLR4, NFκB, IL-1β, TNF-α, Caspase-3 Nrf-2 and HO-1, in hippocampus and cortex

The mRNA expression of TLR4, NFκB, IL-1β, TNF-α, Caspase-3 and Nrf2, HO-1 in the hippocampus and cortex was determined using quantitative PCR. Results have shown that the ethanol group had increased mRNA expression of TLR4, NFκB, IL-1β, TNF-α, and Caspase-3 (Fig. 8) in the hippocampus and cortex compared to vehicle control. Ethanol administration is associated with increased expression of these proteins. In contrast to the ethanol group, chrysophanol administration reduced the expression of TLR4, NFκB, IL-1β, TNF-α, and Caspase-3 in the hippocampus and cortex. Comparing the mRNA expression of Nrf-2, HO-1 in the ethanol group with vehicle control, a significant reduction has been observed in the ethanol group (Fig. 9). Chrysophanol improved the Nrf2 and HO-1 expression as compared to the ethanol group. Comparing the results of chrysophanol with vehicle control, no significant difference was observed. It is observed that chrysophanol has a highly significant effect on the protein expression of TLR4, NFκB, IL-1β, TNF-α, Caspase-3, Nrf-2 and HO-1 in the hippocampus and cortex.

Effect of chrysophanol on COX-2 and NLRP3 expression by ELISA

Expression of COX-2 and NLRP3 in the hippocampus and cortex was determined using the ELISA technique. ELISA results (Fig. 10) have indicated that the ethanol group has significantly increased expression of COX-2 and NLRP3 in the hippocampus and cortex as compared to the vehicle control group (p < 0.05). However, comparing the results of chrysophanol with the ethanol group, a highly significant reduction was obtained in the chrysophanol group. (p < 0.05). The results of the chrysophanol group were not significantly different from the vehicle control group and the donepezil group. (p < 0.05).

Determination of DNA damage by comet assay

Comet analysis was carried out to check of extent of ethanol-induced DNA damage and the effect of chrysophanol treatment in reducing DNA damage in the hippocampus and cortex (Fig. 11). Results indicated that the ethanol group has shown maximum DNA damage in the hippocampus. The ethanol group exhibited maximum tail length and % DNA in the tail in the hippocampus and cortex as compared to the vehicle control group. The Chrysophanol group has shown significant improvement in preventing DNA damage. Chrysophanol treatment significantly reduced the tail length (p < 0.05) mitigating the ethanol-induced DNA damage in the hippocampus and cortex. % DNA in the tail was also significantly reduced after chrysophanol treatment as compared to the ethanol group (p < 0.05).

Ethanol-induced neurodegeneration is a growing concern in terms of chronic alcohol use which has been demonstrated to cause extensive damage to CNS. Alcohol use disorder remains a serious public health issue worldwide. Researchers have elucidated extensive mechanisms of alcohol-related neurodegeneration. From the generation of ROS to the activation of an inflammatory cascade in the CNS, alcohol acts to exert its neurotoxic effect on the body. Alcohol contributes to long-term cognitive deficits and neurodegenerative conditions such as alcohol-related dementias (Crews and Nixon, 2009). Various studies have examined the mechanisms behind alcohol-induced neurodegeneration, focusing on OS, apoptosis, and inflammation. Chronic ethanol consumption leads to significant neuronal loss and cognitive impairment, mediated by increased oxidative damage and neuroinflammation (Oscar-Berman and Marinkovic, 2003). Alcohol-induced neurodegeneration impacts various brain regions, leading to deficits in cognitive and motor functions (Crews et al., 2017). Literature has reported the negative impact of chronic ethanol use on memory and cognitive functions (Charlton and Perry, 2022). Findings of previous studies indicate that chronic ethanol consumption impairs various cognitive domains, including working memory, spatial navigation, and attention, due to neurochemical and structural brain changes (Stragier et al., 2015).

Our study demonstrates that chrysophanol protects the brain against alcohol-related neurodegenerations. The neuroprotective actions of chrysophanol are based on its ability to reduce oxidative stress and prevent the formation of ROS, hinder the activation of inflammatory pathways, and prevent apoptosis. Observations made on behavioral and tissue levels showed the positive effect of chrysophanol in mitigating alcohol-induced neurodegeneration. Our study investigates how chronic ethanol exposure leads to cognitive decline, focusing on memory impairments and executive dysfunction. Various mechanisms such as hippocampal damage, synaptic loss, and alterations in neurotransmitter systems, contribute to deficits in learning and memory. Key findings include deficits in memory consolidation and retrieval, along with impaired synaptic plasticity and neurodegeneration in the hippocampus. Long-term effects of chronic ethanol exposure on cognitive function, particularly memory, and learning have been established. Sustained alcohol use leads to remarkable cognitive impairments, including deficits in episodic memory and spatial learning, which are associated with structural and functional alterations in the brain. The behavioral results of this study including NOR Y-MAZE and MWZ tests analyzed cognitive deficit and memory impairment in mice. Results have shown remarkable recovery in chrysophanol-treated mice's cognitive decline and spatial memory. The formation of ROS is one of the major factors contributing to ethanol-induced neurodegeneration (Afzal et al., 2023). ROS plays a central role in ethanol-induced neurodegeneration. The research demonstrates that chronic ethanol exposure leads to elevated ROS levels, which contribute to neuronal damage through oxidative stress, inflammation, and apoptosis. Various mechanisms by which ROS mediate neurodegeneration and potential interventions to mitigate these effects. Key mechanisms include oxidative stress, activation of glial cells, and disruption of neurovascular integrity (Karoly et al., 2021). Chrysophanol has a remarkable impact in preventing damage related to ROS. Results of this study have shown that chrysophanol meticulously enhanced the antioxidant defense system and reduced the level of oxidant production. Results of our study show that chrysophanol significantly reduces ROS production and enhances antioxidant enzyme activity, suggesting its potential as a protective agent against oxidative damage. These findings follow the reports of Zhao and coworkers who explored the defensive effects of chrysophanol against oxidative stress, focusing on its role in reducing oxidant production (Zhao et al., 2018). The results show that chrysophanol reduces ROS production and modulates oxidative stress pathways, highlighting its capacity as an anti-inflammatory and antioxidant agent as supported by the previous findings.

Chrysophanol was also found to activate the Nrf2 pathway and increase the production of antioxidant enzymes such as GSH, GST, and CAT to strengthen the antioxidant defense system. Chrysophanol also decreases the production of oxidants such as MDA and NO to prevent further damage This study explores how chrysophanol activates the Nrf2 pathway, leading to enhanced cellular defense against oxidative stress. Our results show that chrysophanol induces Nrf2 translocation to the nucleus, increasing the expression of antioxidant enzymes and reducing oxidative damage in cellular models. Findings indicate that chrysophanol activates Nrf2, which in turn upregulates the expression of antioxidant and detoxifying enzymes, thereby mitigating oxidative stress and protecting neuronal cells from damage. The study provides evidence that chrysophanol enhances Nrf2 expression and its downstream targets, contributing to reduced cellular damage and improved antioxidant defense. These results are strengthened by the previously published studies by various researchers (Wen et al., 2021) (Ma et al., 2021).

Alcohol consumption has detrimental effects on the histology of both the hippocampus and cortex of the brain typically involved in cognitive and behavioral functions reported by researchers (Mira et al., 2020) (Moreira-Júnior et al., 2023). (Goldowitz et al., 2014). Alcohol consumption leads to neurotoxic effects in the hippocampus and cortex including shrinkage of the hippocampus and cortical thinning, contributing to cognitive deficits and impaired brain function. (Zahr and Pfefferbaum, 2017). (Geil et al., 2014).

Our result showed that chrysophanol protects the number of survival neurons in different regions of the brain (cortex and hippocampus). H&E staining of brain regions of ethanol treated group has shown extensive neuronal loss in the hippocampus, particularly in the CA1 and CA3 dentate gyrus and cortex. Chrysophanol treatment was successfully able to reverse this neuronal damage with reduced inflammation in these brain regions suggesting the amelioration of neuroinflammation.

Ethanol consumption activates the microglia in the hippocampus and causes the activation of the NFkB pathway by activating TLRs. Chronic ethanol exposure leads to microglial activation in the hippocampus, which in turn triggers the NF-κB, signaling through the activation of TLRs (Li et al., 2019).This activation leads to downstream activation of NF-κB and the production of pro-inflammatory cytokines contributing to neuroinflammation and neurodegeneration (Vetreno and Crews, 2015). Ethanol is also associated with reduced Nrf2 activity and contributes to neuronal stress. (Chen et al., 2013). The decreased Nrf2 activity leads to impaired antioxidant defenses, resulting in increased oxidative stress and neuronal damage(Shanmugam et al., 2019, Chen et al., 2013). Restoring Nrf2 activity through pharmacological intervention can reduce ethanol-induced neuronal stress and protect against cognitive decline. HO-1 is an enzyme regulated by NRF-2 that plays a role in the breakdown of heme into biliverdin, free iron, and carbon monoxide, which have antioxidant and anti-inflammatory properties (Young et al., 2003, Moon et al., 2013). Ethanol consumption also causes the activation of Caspase-3 leads to apoptotic cell death in neurons, contributing to the loss of neuronal populations in the hippocampus and cortex.

Our experimental Results of PCR for mRNA expression of TLR4, NFκB, IL-1β, TNF-α, Caspase-3 Nrf2 and HO-1 in the hippocampus and cortex have shown the effective role of chrysophanol in reducing the effects of these inflammatory and apoptotic gene expressions. COX-2 contributes to neuroinflammation through the formation of prostaglandins and ROS, while the NLRP3 inflammasome promotes the activation of pro-inflammatory cytokines like IL-1β. The interplay between these molecules amplifies the inflammatory response, leading to sustained neuroinflammation, oxidative stress, and ultimately, neuronal death. ELISA Results of Chrysophanol-treated groups have been shown to reduce the level of COX-2 and NLRP3 levels and mitigate inflammation.

Results of the comet assay showed that ethanol had increased tail length with increased percent of DNA in the tail. It may be caused by oxidative stress, impaired apoptotic process, and impaired DNA repair. Ethanol exposure leads to increased levels of inflammatory cytokines and markers of DNA damage in both the hippocampus and cortex, suggesting a link between inflammation and DNA damage. Moreover, the chrysophanol-treated group effectively managed to reduce tail length and was involved in the DNA repair process. Overall, this study demonstrates that chrysophanol has remarkable neuroprotective properties and is found effective in reversing ethanol-induced neurodegeneration. Chrysophanol has shown potential in mitigating DNA damage in the hippocampus through its antioxidant properties, enhancement of DNA repair mechanisms, and overall neuroprotective effects. These findings also follow previously reported data by (Crews and Nixon, 2009). This study highlights the ability of chrysophanol to reduce oxidative stress, improve DNA repair, and protect against neuronal damage, making it a promising candidate against ethanol-induced neurodegeneration.

Ethanol-induced neurodegeneration is a remarkable concern, as chronic alcohol consumption leads to substantial damage to the brain. Chrysophanol treatment leads to improved neurodegeneration and cognitive recovery in ethanol-exposed animals, highlighting its potential for reversing neurodegenerative changes. These findings underscore chrysophanol's potential as a therapeutic agent for alleviating ethanol-induced neurodegeneration and improving neurological health (figure 12).

MWM: Morris Water Maze, NOR: Novel object recognition, NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells, TNF-α: Tumor necrosis factor-alpha, IL: Interleukins, GST: Glutathione S-transferase, GSH: Reduced glutathione, CAT: Catalase MDA: Malonaldehyde, NO: Nitric oxide, ROS: Reactive oxygen species, H&E: Hematoxylin and eosin

Data availability

The data analyzed in this study can be made available by the corresponding author upon request.

Ethics Statement

All research procedures were carefully carried out following the guidelines of Quaid-i-Azam University for animal care and use. The bioethical committee thoroughly approved the methodology, ensuring the ethical treatment of the animals involved in the study (Approval No. #BEC-FBS-QAU2023-561).

Authorship contributions

JZK: Conceptualization, Investigation, Methodology, Software use, Visualization, and Writing-original draft. SRZ: Conceptualization, Investigation, Methodology, Software use, Visualization, and Writing-original draft. Fawad Ali Shah: Data curation, Formal analysis, Validation, and Visualization. Muhammad Khalid Tipu: Resources, data curation, revising the final manuscript version, conceptualizing and designing the study, statistical analysis of data, investigation, supervision, revising, and approval of the final manuscript version.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This work was supported by the Prince Sattam bin Abdulaziz University (Grant No. PSAU/2024/R/1445).

Afzal, S., Abdul Manap, A. S., Attiq, A., Albokhadaim, I., Kandeel, M. & Alhojaily, S. M. (2023). From imbalance to impairment: the central role of reactive oxygen species in oxidative stress-induced disorders and therapeutic exploration. Front. pharmacol., 14, 1269581.
Alfonso-Loeches, S., Ureña-Peralta, J. R., Morillo-Bargues, M. J., Oliver-De La Cruz, J. & Guerri, C. (2014). Role of mitochondria ROS generation in ethanol-induced NLRP3 inflammasome activation and cell death in astroglial cells. Frontiers in cellular neuroscience, 8, 216.
Angelis, D., Savani, R. & Chalak, L. (2021). Nitric oxide and the brain. Part 1: Mechanisms of regulation, transport and effects on the developing brain. Pediatr. Res., 89, 738-745.
Bird, C. W., Barber, M. J., Post, H. R., Jacquez, B., Chavez, G. J., Faturos, N. G. & Valenzuela, C. F. (2020). Neonatal ethanol exposure triggers apoptosis in the murine retrosplenial cortex: Role of inhibition of NMDA receptor-driven action potential firing. Neuropharmacology, 162, 107837.
Bouguiyoud, N., Roullet, F., Bronchti, G., Frasnelli, J. & Al Aïn, S. (2022). Anxiety and depression assessments in a mouse model of congenital blindness. Frontiers in Neuroscience, 15, 807434.
Bustamante-Barrientos, F. A., Luque-Campos, N., Araya, M. J., Lara-Barba, E., de Solminihac, J., Pradenas, C., Molina, L., Herrera-Luna, Y., Utreras-Mendoza, Y. & Elizondo-Vega, R. (2023). Mitochondrial dysfunction in neurodegenerative disorders: Potential therapeutic application of mitochondrial transfer to central nervous system-residing cells. Journal of Translational Medicine, 21, 613.
Charlton, A. J. & Perry, C. J. (2022). The effect of chronic alcohol on cognitive decline: do variations in methodology impact study outcome? An overview of research from the past 5 years. Frontiers in Neuroscience, 16, 836827.
Chen, R.-r., Liu, J., Chen, Z., Cai, W.-j., Li, X.-f. & Lu, C.-l. (2020). Anthraquinones extract from Morinda angustifolia Roxb. Root alleviates hepatic injury induced by carbon tetrachloride through inhibition of hepatic oxidative stress. Evidence‐Based Complementary and Alternative Medicine, 2020, 9861571.
Chen, X., Liu, J. & Chen, S.-y. (2013). Over-expression of Nrf2 diminishes ethanol-induced oxidative stress and apoptosis in neural crest cells by inducing an antioxidant response. Reproductive Toxicology, 42, 102-109.
Crews, F. T., Lawrimore, C. J., Walter, T. J. & Coleman Jr, L. G. (2017). The role of neuroimmune signaling in alcoholism. Neuropharmacology, 122, 56-73.
Crews, F. T. & Nixon, K. (2009). Mechanisms of neurodegeneration and regeneration in alcoholism. Alcohol & Alcoholism, 44, 115-127.
Geil, C. R., Hayes, D. M., McClain, J. A., Liput, D. J., Marshall, S. A., Chen, K. Y. & Nixon, K. (2014). Alcohol and adult hippocampal neurogenesis: promiscuous drug, wanton effects. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 54, 103-113.
Goldowitz, D., Lussier, A. A., Boyle, J. K., Wong, K., Lattimer, S. L., Dubose, C., Lu, L., Kobor, M. S. & Hamre, K. M. (2014). Molecular pathways underpinning ethanol-induced neurodegeneration. Frontiers in Genetics, 5, 203.
Habig, W. H., Pabst, M. J. & Jakoby, W. B. (1974). Glutathione S-transferases: the first enzymatic step in mercapturic acid formation. Journal of biological Chemistry, 249, 7130-7139.
Holloway, K. N., Douglas, J. C., Rafferty, T. M., Majewska, A. K., Kane, C. J. & Drew, P. D. (2023). Ethanol-induced cerebellar transcriptomic changes in a postnatal model of fetal alcohol spectrum disorders: Focus on disease onset. Frontiers in Neuroscience, 17, 1154637.
Karoly, H. C., Skrzynski, C. J., Moe, E. N., Bryan, A. D. & Hutchison, K. E. (2021). Exploring relationships between alcohol consumption, inflammation, and brain structure in a heavy drinking sample. Alcoholism: Clinical and Experimental Research, 45, 2256-2270.
Lanzillotta, A., Porrini, V., Bellucci, A., Benarese, M., Branca, C., Parrella, E., Spano, P. F. & Pizzi, M. (2015). NF-κB in innate neuroprotection and age-related neurodegenerative diseases. Frontiers in neurology, 6, 98.
Li, Q., Liu, D., Pan, F., Ho, C. S. & Ho, R. C. (2019). Ethanol exposure induces microglia activation and neuroinflammation through TLR4 activation and SENP6 modulation in the adolescent rat hippocampus. Neural Plasticity, 2019, 1648736.
Ma, S., Xu, H., Huang, W., Gao, Y., Zhou, H., Li, X. & Zhang, W. (2021). Chrysophanol relieves cisplatin-induced nephrotoxicity via concomitant inhibition of oxidative stress, apoptosis, and inflammation. Frontiers in Physiology, 12, 706359.
Malik, S., Sharma, N., Sharma, U. K., Singh, N. P., Bhushan, S., Sharma, M., Sinha, A. K. & Ahuja, P. S. (2010). Qualitative and quantitative analysis of anthraquinone derivatives in rhizomes of tissue culture-raised Rheum emodi Wall. plants. Journal of plant physiology, 167, 749-756.
Mattson, M. P. & Camandola, S. (2001). NF-κB in neuronal plasticity and neurodegenerative disorders. The Journal of clinical investigation, 107, 247-254.
Mira, R. G., Lira, M., Tapia-Rojas, C., Rebolledo, D. L., Quintanilla, R. A. & Cerpa, W. (2020). Effect of alcohol on hippocampal-dependent plasticity and behavior: role of glutamatergic synaptic transmission. Frontiers in behavioral Neuroscience, 13, 288.
Moon, Y., Kwon, Y. & Yu, S. (2013). How does ethanol induce apoptotic cell death of SK-N-SH neuroblastoma cells. Neural Regen. Res., 8, 1853.
Moreira-Júnior, R. E., Guimarães, M. A. d. F., Etcheverria da Silva, M., Maioli, T. U., Faria, A. M. C. & Brunialti-Godard, A. L. (2023). Animal model for high consumption and preference of ethanol and its interplay with high sugar and butter diet, behavior, and neuroimmune system. Frontiers in Nutrition, 10, 1141655.
Ohkawa, H., Ohishi, N. & Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical biochemistry, 95, 351-358.
Oscar-Berman, M. & Marinkovic, K. (2003). Alcoholism and the brain: an overview. Alcohol research & health, 27, 125.
Prateeksha, Yusuf, M. A., Singh, B. N., Sudheer, S., Kharwar, R. N., Siddiqui, S., Abdel-Azeem, A. M., Fernandes Fraceto, L., Dashora, K. & Gupta, V. K. (2019). Chrysophanol: a natural anthraquinone with multifaceted biotherapeutic potential. Biomolecules, 9, 68.
Qin, L. & Crews, F. T. (2012). NADPH oxidase and reactive oxygen species contribute to alcohol-induced microglial activation and neurodegeneration. Journal of neuroinflammation, 9, 1-19.
Reddy, V. P. (2023). Oxidative stress in health and disease. Biomedicines, 11, 2925.
Rehman, M. U., Ali, N., Jamal, M., Kousar, R., Ishaq, M., Awan, A. A., Hussain, I., Sherkheli, M. A. & ul Haq, R. (2021). Comparison of acute and chronic effects of Bacopa monnieri, Ginkgo biloba, and Lavandula angustifolia and their mixture on learning and memory in mice. Phytother. Res., 35, 2703-2710.
Rowley, S., Liang, L.-P., Fulton, R., Shimizu, T., Day, B. & Patel, M. (2015). Mitochondrial respiration deficits driven by reactive oxygen species in experimental temporal lobe epilepsy. Neurobiology of disease, 75, 151-158.
Shafaroodi, H., Moezi, L., Fakhrzad, A., Hassanipour, M., Rezayat, M. & Dehpour, A. R. (2012). The involvement of nitric oxide in the anti-seizure effect of acute atorvastatin treatment in mice. Neurol. Res., 34, 847-853.
Shanmugam, S., Patel, D., Wolpert, J. M., Keshvani, C., Liu, X., Bergeson, S. E., Kidambi, S., Mahimainathan, L., Henderson, G. I. & Narasimhan, M. (2019). Ethanol impairs NRF2/antioxidant and growth signaling in the intact placenta in vivo and in human trophoblasts. Biomolecules, 9, 669.
Singh, B. N., Upreti, D. K., Gupta, V. K., Dai, X.-F. & Jiang, Y. (2017). Endolichenic fungi: a hidden reservoir of next generation biopharmaceuticals. Trends in biotechnology, 35, 808-813.
Stragier, E., Martin, V., Davenas, E., Poilbout, C., Mongeau, R., Corradetti, R. & Lanfumey, L. (2015). Brain plasticity and cognitive functions after ethanol consumption in C57BL/6J mice. Translational psychiatry, 5, e696-e696.
Tsikas, D. (2007). Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction: appraisal of the Griess reaction in the L-arginine/nitric oxide area of research. Journal of Chromatography B, 851, 51-70.
Vetreno, R. P. & Crews, F. T. (2015). Binge ethanol exposure during adolescence leads to a persistent loss of neurogenesis in the dorsal and ventral hippocampus that is associated with impaired adult cognitive functioning. Frontiers in neuroscience, 9, 35.
Vorhees, C. V. & Williams, M. T. (2006). Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat. Protoc., 1, 848-858.
Waku, T. & Kobayashi, A. (2021). Pathophysiological potentials of NRF3-regulated transcriptional axes in protein and lipid homeostasis. International Journal of Molecular Sciences, 22, 12686.
Wang, W., Zhao, F., Ma, X., Perry, G. & Zhu, X. (2020). Mitochondria dysfunction in the pathogenesis of Alzheimer’s disease: Recent advances. Molecular neurodegeneration, 15, 1-22.
Wen, Q., Lau, N., Weng, H., Ye, P., Du, S., Li, C., Lv, J. & Li, H. (2021). Chrysophanol exerts anti-inflammatory activity by targeting histone deacetylase 3 through the high mobility group protein 1-nuclear transcription factor-kappa B signaling pathway in vivo and in vitro. Frontiers in bioengineering and biotechnology, 8, 623866.
Ye, T., Li, X., Zhou, P., Ye, S., Gao, H., Hua, R., Ma, J., Wang, Y. & Cai, B. (2020). Chrysophanol improves memory ability of d-galactose and Aβ 25–35 treated rat correlating with inhibiting tau hyperphosphorylation and the CaM–CaMKIV signal pathway in hippocampus. 3 Biotech, 10, 1-8.
Young, C., Klocke, B., Tenkova, T., Choi, J., Labruyere, J., Qin, Y., Holtzman, D., Roth, K. & Olney, J. (2003). Ethanol-induced neuronal apoptosis in vivo requires BAX in the developing mouse brain. Cell Death & Differentiation, 10, 1148-1155.
Zahr, N. M. & Pfefferbaum, A. (2017). Alcohol’s effects on the brain: neuroimaging results in humans and animal models. Alcohol research: current reviews, 38, 183.
Zhang, J., Yan, C., Wang, S., Hou, Y., Xue, G. & Zhang, L. (2014). Chrysophanol attenuates lead exposure-induced injury to hippocampal neurons in neonatal mice. Neural Regeneration Research, 9, 924-930.
Zhao, Y., Huang, Y., Fang, Y., Zhao, H., Shi, W., Li, J., Duan, Y., Sun, Y., Gao, L. & Luo, Y. (2018). Chrysophanol attenuates nitrosative/oxidative stress injury in a mouse model of focal cerebral ischemia/reperfusion. Journal of Pharmacological Sciences, 138, 16-22.

No competing interests reported.

Download PDF

Reviewers agreed at journal
04 Nov, 2024
Reviewers agreed at journal
02 Nov, 2024
Reviewers agreed at journal
29 Oct, 2024
Reviewers agreed at journal
29 Oct, 2024
Reviewers agreed at journal
29 Oct, 2024
Reviewers invited by journal
29 Oct, 2024
Editor assigned by journal
18 Oct, 2024
Submission checks completed at journal
18 Oct, 2024
First submitted to journal
08 Oct, 2024

You are reading this latest preprint version

Chrysophanol attenuates cognitive impairment, neuroinflammation, and oxidative stress by TLR4/ NFκB -Nrf2/HO-1 signaling in ethanol induced neurodegeneration

Status:

Version 1

Abstract

Figures

Introduction

Material & Methods

Animals

Study design

Behavioral assessment for memory

Y-maze Test

Morris Water Maze Test

Training and acclimatization phase

Probe trial

Final test

Novel Object Recognition Test

Sampling

Antioxidant assay

GSH level

GST level

Catalase level

MDA and NO level

Histological testing

mRNA Expression in Hippocampus and Cortex by RT-PCR

Estimation of COX-2 and NLRP3 levels by ELISA

Statistical analysis

Results

Y-maze

Morris Water Maze

Novel Object Recognition

Chrysophanol improved the antioxidant and oxidative stress markers in ethanol-induced neurodegeneration

Effect of chrysophanol on histology of hippocampus and cortex

Effect of chrysophanol on COX-2 and NLRP3 expression by ELISA

Determination of DNA damage by comet assay

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1