The Modulatory Influence of Humic Acid on Cognitive Impairment and Neurobehavioral Changes Induced by Colitis in Adult Male Wistar Rats

doi:10.21203/rs.3.rs-4438233/v1

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The Modulatory Influence of Humic Acid on Cognitive Impairment and Neurobehavioral Changes Induced by Colitis in Adult Male Wistar Rats

https://doi.org/10.21203/rs.3.rs-4438233/v1

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Objectives: Ulcerative colitis (UC) is regarded as a chronic inflammatory disorder that involves any part of the colon. It typically presents with symptoms such as abdominal pain, bloody diarrhea, rectal urgency, and comorbid neuropsychiatric manifestations such as anxiety, depression, and cognitive impairment. Different drugs are used in the management of ulcerative colitis, including medicinal plants and other substances from organic sources. This study was conducted to investigate the role of humic acid in attenuating neurobehavioral disorders caused by Dextran sulfate sodium-induced ulcerative colitis in male Wistar rats.

Methods: Twenty male Wistar rats were randomly assigned into groups (n = 5). Group 1[control group]; group 2 [5% Dextran sulfate sodium (DSS) without any additional treatment]; group 3 [5% DSS followed by administration of humic acid (30 mg/kg)]; group 4 [5% DSS followed by administration of sulfasalazine (200 mg/kg)].

Behavioral patterns of the rats were assessed pre-colitis induction, immediately after colitis induction on day 5, and immediately after drug treatment of ulcerative colitis (post-treatment). The disease activity index for colitis was obtained on days 1, 3, 5, and 10 of the experimental duration. Thereafter, the colons were harvested for macroscopic assessment of colitis. Data were analyzed using ANOVA at p < 0.05.

Results:Findings revealed that treatment with humic acid significantly attenuated anxiety, depression-like behavior, and cognitive impairment triggered by colitis, with via modulations on the brain enzymatic antioxidants and neurochemicals.

Conclusion:This study shows that humic acid treatment suppresses colitis-induced changes in neurobehavioral patterns and have neuroprotective effects in addition to its gastroprotective properties.

Brain-gut axis

humic acid

neurobehaviour

antioxidants

ulcerative colitis

Ulcerative colitis (UC), being a chronic condition, is an inflammatory illness that affects the colon and rectum to varying degrees. It was projected that there would be 5 million instances of ulcerative colitis globally in 2023 [1], and the incidence is rising. The etiology of UC is still unknown, but it seems to be associated with a mix of genetic factors and environmental factors, including nutrition and drugs [2, 3]. The levels of sulfate-reducing bacteria tend to be higher in people with ulcerative colitis and have been proposed as the ultimate cause of both ulcerative colitis and Crohn's disease as well [4]. Hence, UC could be linked to the disruption of normal gut microflora [20].

The primary symptoms of ulcerative colitis include weight loss, blood in the stool, and abdominal pain, among others [5]. Secondary symptoms include mental disorders such as anxiety and depressive-like behaviors [6, 7]. Particularly when the illness is active, patients with ulcerative colitis frequently exhibit cognitive impairments such as memory loss, attention problems, and a decline in higher brain function [8]. The most important connections between ulcerative colitis, which is an inflammatory disease, and neurobehavior, which mostly occurs in the brain, have been linked to the brain-gut axis [9]. The brain-gut axis connects the brain's emotional and cognitive regions with the functions of the gut through two-way communication between the enteric neural system and central nervous system [10]. This connection suggests that the gastrointestinal tract is sensitive to anger, anxiety, sadness, and elation.

It is well established that UC is not an uncommon disease, as there are about a hundred thousand cases each year. The frequency and incidence of inflammatory bowel disease (IBD), a disease that affects people all over the world, are rising sharply [11]. Even though in West Africa, including Nigeria, there are no statistics on the occurrence of UC [12], the adoption of Western cuisine and lifestyle poses it to be a major health burden for the country in the future amidst of the skyrocketing cases neurocognitive impairment.

However, there are established methods for managing UC; these include eating a low-fiber diet, especially at the disease's early stages, and the use of drugs such as sulfasalazine and mesalazine, among others [13]. Apart from the high costs of using these drugs, they also have great risks, which may outweigh their advantages. Hence, there is a need for a continuous search for potential drugs or agents that would provide a cure or effective management of symptoms, given the neurocognitive impairment attributed to the progression of this disease.

Till today, plants and other organic products continue to serve as therapeutic agents to cure human diseases [14], especially in the rural regions of sub-Saharan countries where there are limitations to accessing medical facilities and abject poverty, which makes the dwellers unable to afford orthodox medicine. Hence, there is a need for a conventional way to treat illnesses such as ulcerative colitis with readily available nutraceuticals and novel bioactive compounds, such as humic acid.

Humic acid is obtained from humic substances, organic ubiquitous components arising in the process called humification, which includes chemical and microbiological oxidation [15]. The ethno-pharmacological potency of humic acid has been reported, along with its gastrointestinal health-improving function [16] With this growing body of knowledge, little is known about its effects on ulcerative colitis, and its effects on colitis-induced neuropsychiatric disorders have not been scientifically established. Hence, this research was conducted to assess the alterations in the behavioral patterns attributed to ulcerative colitis and the modulatory effect(s) of humic acid on ulcerative colitis and its consequent neurobehavioral symptoms.

Experimental animal

We obtained twenty (20) male Wistar rats (10–12 weeks old) weighing 160 ± 10 g from the University of Medical Sciences, Ondo, Ondo State, animal house. The animals were kept in ventilated plastic cages cushioned with wood shavings and housed in the animal facilities of the University of Medical Sciences, Ondo, Ondo State, Nigeria. The animals were monitored under standard laboratory conditions: at 35.5–37.0°C and a 12-hour light–12-hour darkness cycle. Rats were fed with standardized pellets and clean water. Proper hygiene was also observed in their housing in accordance with the public health service policy on human care and the use of laboratory animals, approved by the Institute of Laboratory Animal Resources, National Research Council (2011).

Experimental design

The male Wistar rats were assigned into 4 groups (n = 5) randomly as follows: Group 1 received distilled water (2 ml/kg) throughout the whole experimental process. Group 2 (negative control) was fed water containing 5% Dextran Sulfate Sodium (DSS) orally for five days according to their body weights, then given distilled water for another five days. Group 3 was fed water containing DSS (5%) for five days, then treated with humic acid (30 mg/kg) for five days. Group 4 was fed water containing (5%) for five days, then treated with sulfasalazine (200 mg/kg) for five days.

Extraction and purification of humic acid

Humic acid was extracted by weighing 1 kg of each soil sample into conical flasks. To each soil sample, 0.1 M NaOH was added and shaken for approximately 30 minutes using a shaker. After 24 hours, the aqueous solution underwent centrifugation at 4000 rpm for 15 minutes to remove suspended particles and was then filtered. The soil residue underwent washing with 500 ml of 0.1 M NaOH, shaken for 15 minutes, and was subjected to centrifugation at 4000 rpm. The process was repeated until the supernatant became clear. The supernatant was acidified to pH 2 with 0.1 M HCl to induce the precipitation of humic acids and allowed to stand overnight. The supernatant containing fulvic acid was separated from the precipitate by centrifugation. The humic acid was then redissolved in 0.1 M NaOH and centrifuged to completely remove clay impurities. The mixture was acidified with 0.1 M HCl to precipitate humic acid, which was subsequently washed with distilled water to remove other impurities. The precipitated humic acids were finally dialyzed in a dialysis bag against distilled water until chloride ions were no longer detected with the AgNO3 solution. The dialyzed humic acids were dissolved in ethanol to separate the insoluble humic acid from the Hymatomelanic acid (soluble), which was present in the mixture, and centrifuged to remove the insoluble humic acid. The purified humic acid was dried in a controlled oven at 40 degrees Celsius and stored for future use.

Induction of colitis

Colitis was induced using 5% (weight per volume) dextran sodium sulfate obtained from Sigma-Aldrich (United States). DSS was added to the drinking water for 5 days. Each drinking solution was administered to the rats in a bottle containing an equal number of moles of the compounds under study. Sulfasalazine was purchased from an accredited pharmaceutical store in Ondo State, Nigeria. Both humic acid and sulfasalazine were solubilized in distilled water as vehicles for oral administration. Humic acid (30 mg/kg) was administered to the rats orally based on their total body weight. Sulfasalazine was administered at a dose of 200 mg/kg via oral gavage.

Neurobehavioural studies

Behavioral tests included the open field test, Y-maze test, and elevated-plus maze tests (EPM). The behavioral assessments were conducted in three phases: before inducing colitis (pre-induction), immediately after inducing colitis on day 5 (induction), and immediately after drug treatment on day 10 (post-treatment). All behavioral tests were manually scored by at least two independent, trained observers from the live video recordings. The observers were unaware of the research design and drug treatment. Following each rat's assessment in the OFT, EPM, and Y-maze, the arena was cleaned with 70% alcohol to eliminate olfactory bias, and the area was allowed to dry before introducing a fresh animal.

Open field test

The open-field box utilized was a rectangular space with a firm floor measuring 36 cm by 36 cm by 26 cm, painted white wood. Permanent red markers at the bottom separated the floor into 16 equal squares. The animals were acclimatized in the test laboratory for a minimum of 45 minutes before the open-field test. Subsequently, rats were individually placed in the center of the field, and every five minutes, the frequency of line crossings, rearing, and grooming were recorded for each rat.

Y- maze tests

The Y-maze was utilized to assess short-term spatial memory. Constructed of wood, the Y-shaped maze featured arms measuring 40 cm in length, 30 cm in height, and 10 cm in width. The animals were acclimated in the silent laboratory for at least 45 minutes before the Y-maze test. Each rat was placed in the Y-maze for 5 minutes to evaluate the number of arm entries and the percentage of spontaneous alternation, which were parameters of interest in this study.

Elevated-plus maze (EPM) tests

Anxiety-like behavior in rats was assessed using the Elevated Plus Maze (EPM). The apparatus comprised two open arms (50 cm × 10 cm) intersected with two closed arms of the same dimensions, featuring walls 30 cm high. The maze was elevated 60 cm above the floor. Each rat was placed in the center of the elevated plus maze, facing an open arm, and allowed 5 minutes to explore the apparatus freely. The time spent by each rat in the open arms was measured as an indicator of anxiety-like behavior.

Disease activity index (DAI)

The Disease Activity Index (DAI) was assessed on day 1, 3, and 5 of DSS treatment, as well as on day 10 equivalent to post-drug treatment period. Parameters used for assessment included stool consistency, diarrhea, and bleeding scores, which were obtained from observations. Consistent with the methodology described by Cooper et al.,[18] the DAI was calculated using the formula: DAI = (body weight drop + stool consistency + rectal hemorrhage) / 3. The humane endpoint was defined as DAI = 3, while a diagnosis of ulcerative colitis (UC) was established when DAI was ≥ 1.5.

Preparation of brain tissues homogenates for biochemical assays

After completion of the behavioral tests, rats were euthanized by cervical dislocation.

The brain was carefully dissected out, weighed, washed with normal saline, homogenized in ice-cold sodium phosphate buffer (0.1 M, pH 7.4) and centrifuged at 5000 revolution per minute for 15 min at 4°C. The supernatant was stored at − 20°C for biochemical assays.

Determination of catalase (CAT) activity

Catalase (CAT) activity assay was done using the method of Sinha [66], where hydrogen peroxide (H2O2) disappearance was continuously monitored using two cuvettes at 60 and 180 s intervals at 570 nm spectrophotometrically through its absorbance. The CAT activity was expressed as lmoles of H2O2 decomposed per minutes per mg protein.

Estimation of brain superoxide dismutase (SOD) activity

Measurement of superoxide dismutase (SOD) activity was done using the method described by Misra and Fridovich [65]. This method is based on the inhibition of superoxide dependent adrenaline auto-oxidation in a spectrophotometer adjusted at 480 nm. SOD activity was reported as units per mg of protein.

Estimation of brain level of malondialdehyde (MDA)

The levels of MDA (malondialdehyde) in the striatus and hippocampus were determined using a previously established method [61]. This method relies on the observation that lipid peroxidation generates unstable lipid peroxides, which decompose into various compounds including reactive carbonyl compounds. Specifically, polyunsaturated fatty acid peroxides produce MDA upon decomposition. MDA forms a 1:2 adduct with thiobarbituric acid (TBA), resulting in a pink-colored product when heated under acidic conditions, with maximum absorbance at 532 nm.

The procedure involves mixing 0.4 mL of the sample with 1.6 mL of Tris-potassium chloride (Tris-KCl) buffer, followed by the addition of 0.5 mL of 30% trichloroacetic acid (TCA). Then, 0.5 mL of 0.75% TBA is added to the mixture, which is subsequently heated in a water bath for 45 minutes at 80°C. After cooling in ice and centrifugation at 1200 g for 15 minutes, the clear supernatant is collected and its absorbance is measured against a reference blank of distilled water at 532 nm. Finally, the MDA concentration is calculated using a molar extinction coefficient of 1.56 × 105 M − 1 cm − 1 and the result is expressed as nanomoles of MDA per milligram of protein (nmol MDA/mg protein).

Determination of brain nitrite level

To evaluate the impact of treatments involving humic acid and sulfasalazine on nitric oxide (NO) production, nitrite levels were assessed in the homogenates of the striatum and hippocampus using the method outlined by Green et al. [62]. Following centrifugation at 5400 g for 10 minutes, the supernatant of the homogenate was collected. NO production was determined utilizing the Griess reaction. 100 µL of the supernatant was incubated with an equal volume of Griess reagent (consisting of sulfanilamine in 1% H3PO4, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride, 1% H3PO4, and distilled water, in a 1:1:1:1 ratio, sourced from Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 10 minutes. The absorbance was subsequently measured at 550 nm using a UV-spectrophotometer. A standard curve was constructed with various concentrations of NaNO2, ranging from 0.75 to 100 µM, and results were expressed as µmol per mg tissue.

Determination of brain glutamine transferase (GST) concentration

The activity of GST (Glutathione S-Transferase) enzyme was assessed in duplicate following a method described by Habig et al. [60]. 10 µl of each cytosolic fraction was combined with 2.0 ml of potassium phosphate buffer at pH 6.5, supplemented with 1.0 mmol/l of 1-chloro-2,4-dinitrobenzene (CDNB; sourced from Sigma Chemical Co.) and 5.0 mmol/l of GSH (also from Sigma Chemical Co.).

Determination of brain glutathione (GSH) concentration

The activity of Glutathione (GSH) was assessed using the method outlined by Jollow et al. [63]. This method relies on the development of a stable yellow color when 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) is introduced to sulfhydryl compounds. Upon reaction with reduced GSH, DTNB produces a chromophoric product known as 2-nitro-5-thiobenzoic acid, which exhibits a characteristic absorbance at 412 nm. The quantity of reduced GSH in the sample is directly proportional to the absorbance.at this wavelength. The concentration of reduced GSH in the striatum and hippocampus tissues were expressed as µmol/mg/protein.

Measurement of acetylcholinesterase activity in brain

The method used to assess Acetylcholinesterase (AChE) enzyme activity, a marker indicative of cholinergic neurotransmission, for evaluating the cognitive effects of humic acid on colitis-induced behavioral alterations was conducted according to a previously described protocol [64]. The estimation of AChE enzyme activity was performed on the homogenates of the striatum and hippocampus. Aliquots of homogenates (0.4 mL) from each mouse in the respective treatment groups were combined with 2.6 mL of phosphate buffer (0.1 M, pH 7.4) and 0.1 mL of 5,5′-dithio-bis(2-nitrobenzoic acid). The reaction mixture was then incubated at room temperature (25°C) for 5 minutes. Subsequently, 0.1 mL of acetylthiocholine iodide solution was added to the reaction mixture. The absorbance was measured using a spectrophotometer at a wavelength of 412 nm, and changes in absorbance were recorded every 10 minutes over a 120-minute period. The rate of acetylcholinesterase activity was determined by monitoring the increase in color resulting from the reaction of thiocholine with 5,5′-dithio-bis(2-nitrobenzoic acid). The change in absorbance per minute was calculated, and the rate of acetylcholinesterase activity was expressed as µmol/min/mg tissue.

Statistical analysis

This study utilized GraphPad Prism version 9.0.5 (GraphPad Software, San Diego, USA) for the analysis of collected data. The data was presented as mean ± standard error of mean (SEM) with n = 5. An analysis of variance (two-way ANOVA) was employed to examine mean differences, and the Tukey post hoc test was used for multiple comparisons. A significance level of P < 0.05 was considered statistically significant.

Effect of DSS and drug treatments on the disease activity index of colitis

As shown in Fig. 1, the results of the Disease Activity Index (DAI) indicated the progressive effect of DSS on colitis induction, with the maximal effect observed on day 5 of colitis induction. However, treatment with humic acid and sulfasalazine significantly reduced the DAI by day 10 (post-drug treatment period).

Effect of treatment with Humic acid on the neurobehavioral patterns observed in open field test (OFT) of DSS-induced colitis

As presented in Figs. 2a, b, and c, DSS-induced colitis significantly (p < 0.05) decreased locomotion activity, as evidenced by the reduction in the frequency of line crossing, grooming, and rearing compared with the control group. However, treatment with humic acid resulted in a significant (p < 0.05) increase in the frequency of line crosses and rearing in the Open Field Test (OFT) compared with the DSS-exposed and untreated rats.

Effect of treatment with Humic acid on the neurobehavioral patterns observed in Y-maze test of rats induced with colitis

As shown in Figs. 3a and b, our data indicated a significant (p < 0.05) decrease in the percentage of spontaneous alternation and arm entries in the Y-maze test, a measure of spatial working memory function, in DSS-exposed rats compared to the control group. On the other hand, treatment with humic acid significantly (p < 0.05) increased the percentage of spontaneous alternation when compared with the DSS-only group.

Effect of treatment with Humic acid on the neurobehavioral patterns observed in elevated plus maze test of rats induced with colitis

As shown in Fig. 4a and b, our data indicated a significant (p < 0.05) decrease in the time spent in the open arm and a decrease in the time spent in the closed arm in the elevated plus maze (EPM) test, a measure of anxiety-related behavior, with DSS-treated rats compared to the control group. On the other hand, treatment with humic acid significantly(p < 0.05) increased the time spent in the open arm and decreased the time spent in the closed arm among the treated groups compared with the DSS-only group.

Effect of treatment with Humic acid on the brain oxidative and inflammatory markers of rats induced with colitis

As shown in Fig. 5a to f, our data indicated a significant (p < 0.05) increase in the striatal and hippocampal MDA and nitrite levels; markers of lipid peroxidation and neuroinflammation respectively, also antioxidant levels (Catalase, GSH, GST, SOD) significantly (p < 0.05) decreased with DSS-treated rats compared to the control group. On the other hand, treatment with humic acid significantly(p < 0.05) increased the also antioxidant levels (Catalase, GSH, GST, SOD) compared with the DSS-only group.

Effect of treatment with Humic acid on the brain acetylcholinesterase activity of rats induced with colitis

As shown in Fig. 6 our data indicated a significant (p < 0.05) increase in striatal and hippocampal acetylcholinesterase activity in DSS-treated rats compared to the control group. On the other hand, treatment with humic acid significantly(p < 0.05) reduced acetylcholinesterase activity compared with the DSS-only group.

The incidence of ulcerative colitis (UC), a chronic inflammatory bowel disease (IBD) affecting the colon and rectum, is rising quickly in developing nations [2]. Ulcerative colitis has shown comorbidity with psychiatric symptoms such as depression, anxiety, and cognitive impairment, which might be due to the severity of ulcerative colitis and its impairing effects on daily life activities [19]. Even though much research has been conducted on the connection between higher brain functions and ulcerative colitis in human patients, there is still much to be done using animal models in order to substantiate these interactions.

In this present study, after inducing ulcerative colitis with DSS, animals exhibited similar symptoms to IBD, including weight loss, mucosal ulceration, and inflammation [21]. Upon examination, the colons of rats in different groups corroborated the inflammation caused by DSS treatment and the evident ulceration and megacolon and shortened colon in the DSS only group describe the inflammatory status. This is consistent with the previous studies where DSS treatment was used to induce colitis [19, 22, 23]. The results of the significant increase in the disease activity index of this studye as presented brought great insights that the severity of colitis progresses with time and when untreated, could have impacted greatly not only on the gastrointestinal membrane architecture but also on mental functions.

The findings of this study show that DSS-induced colitis greatly impaired spatial working memory with lesser percentage of spontaneous alternation as compared to the control group in the Y-Maze test. Memory impairment is often due to progression of untreated anxiety and depression [47]. The manifestation of the decline in the cognitive function in ulcerative colitis-induced rats as evident in this study aligns with the work of Elbaz et al [24] which reported that acetic induced colitis triggered neurocognitive impairment in rat model as well. According to a functional MRI study, there was a strong correlation between memory loss and decreased hippocampal activity. In patients with UC, executive function and attention network dysfunction are linked to higher posterior cingulate activity and strength of functional connectivity [8, 25]. These changes in brain structure and functions have been linked to gut microbiome dysbiosis [26].

The manifestations of anxiety and depression as presented in the EPM and OFT tests respectively, showed locomotive decline and time spent in the open and closed arms of the elevated plus maze respectively. Decline in locomotory parameters such as grooming, rearing and line crossing signals depressive-like behaviour and reduction in time spent in the open arm of EPM pointed to the manifestation of anxiety. These results are in line with the previous study as reported by Zhou et al. and Lee et al [27, 50] in the mouse model, with similar trends in various studies in the human model [6, 28, 29]. Significant neuronal inflammation and oxidative stress was observed in the hippocampus of rodents afflicted with colitis induced by DSS. These factors are closely linked to the emergence of behaviors resembling depression [56].

The pathogenesis of most neuropsychiatry disorders including depression and memory impairment have been linked to oxidative stress, neuroinflammation and neurotransmitter imbalance [59]. Our findings as shown in Fig. 6. revealed significant increases in markers of lipid peroxidation (MDA) and neuroinflammation (nitrite levels) in the striatum and hippocampus of rats with colitis induced by dextran sulfate sodium (DSS). Additionally, we observed a notable decrease in major enzymatic antioxidants measured, including catalase, glutathione (GSH), glutathione S-transferase (GST), and superoxide dismutase (SOD), in the DSS-treated group compared to the control group. These findings are consistent with previous studies demonstrating the detrimental effects of colitis on oxidative stress and neuroinflammation in the brain[49, 51].

However, treatment with humic acid significantly reversed these alterations. We observed a significant increase in antioxidant enzyme levels in the humic acid-treated group compared to the DSS-only group. This suggests that humic acid possesses potent antioxidant properties, which may help mitigate oxidative stress and neuroinflammation associated with colitis-induced brain dysfunction [45, 52].

Furthermore, our study investigated the effect of humic acid treatment on acetylcholinesterase (AChE) activity in the striatum and hippocampus. AChE is an enzyme involved in the breakdown of acetylcholine, a neurotransmitter critical for cognitive function. Our results demonstrated a significant increase in AChE activity in DSS-treated rats compared to the control group, indicating impaired cholinergic neurotransmission. However, treatment with humic significantly reduced AChE activity, suggesting a protective effect against colitis-induced cognitive dysfunction [53].

The pathogenic mechanisms of inflammatory bowel diseases are multifaceted and associated with oxidative stress, unbalanced gut microbiota, and aberrant immune response [48]. Connection between ulcerative colitis and neurobehavioural functions has been established to be via the Brain -gut axis (BGA) [10]. This communication is central to the pathophysiology and comorbidity of anxiety, depression and cognitive impairment [30]. According to Kasarello et al. [31], BGA is a bidirectional communication channel that connects the peripheral intestinal processes and immune activation, intestinal permeability, gut enteric reflex, and entero-endocrine signaling with the cognitive center and emotional base of the brain.

The gut microbiota acts as the center for the brain gut axis. Evidence suggests that the gut microbiota has a great effect on the gut brain axis, interacting not only locally with enteric nervous system and intestinal cells but also directly with the central nervous system through metabolic and neuroendocrine pathways [32, 33]. Studies conducted have also shown that the microbiome affects anxiety-like behaviors and stress reactivity [34]. Disruption of the normal microbiome community of the gut could pose a great threat to mental health as most neurotransmitters, such as serotonin, dopamine and acetylcholine are produced in the gut as by-products of the metabolism of gut microbes [35, 36]. Due to the relevance of this neurotransmitters in the pathophysiology of anxiety, depression, memory impairment and other neuropsychiatric disorders. It is believed that upon disruption of the gut microbiomes, the level of these neurotransmitters could be altered as well, hence, the development of anxiety, depression and cognitive impairment [37, 38].

Evidence from our study show the reversal of the anxiety, depressive-like and cognitive impairment in DSS-induced colitis rats by humic acid treatment. The experimental colitis in rats caused by acetic acid was stopped using donepezil by significantly alleviating the microscopic and macroscopic changes associated with colitis and its associated cognitive impairment by regulating inflammatory/oxidative/apoptotic pathways [24]. A crude alkaloidal extract of Picralima nitida seeds; as a result of its anti-inflammatory role, was reported to have attenuated the intestinal oxidative and inflammatory damages in an animal model of colitis as well [39]. Long term treatment with anti-tumor necrotic factor and immunomodulators also showed significant improvement in both anxiety and depressive symptoms in ulcerative colitis patients [40].

Humic acid as a complex mixture of organic compounds that is gotten from decaying plants has been shown to have modulated the stimulation of immune system and also inflammation. Even though little is known about the pharmacological actions of humic acid but few emerging studies have shown that humic acid has immune boosting potential. Ahfeethah et al.[41], reported that a daily dose of humic acid treatment on broiler chicken had stimulating and enhancing influence on the cellular and humoral immune systems. The support humic acid has for the immune system has been linked to its ability to bind to biomolecules, assemble sugars in the body to form glycoproteins. These glycoproteins attach to killer and T cells in the immune system, allowing for communication [42].

In a recent study by Huang et al. [43], it was reported that humic acid alleviated DSS-induced colitis as it helped to restore intestinal morphology, alter the community of gut microbiota, reduce the levels of inflammatory cytokines by its modulatory role on the NF-κB-TLR4 pathway, and additionally enhanced the expression of tight junction proteins, which guard against DSS damage to the gut barrier. Modulation of hippocampal oxidative stress marker as well as hippocampal pro-inflammatory mediators by rice bran extract was reported [54]. Treatment with Magnolia officinalis bark extract significantly decreased DSS-induced proliferation of microglia and astrocytes in the hippocampal region, and attenuated the increase in pro-inflammatory cytokines, modulated serotonin level [55].

Şehitoğlu et al., [44] also reported that humic acid is gastroprotective as it alleviates gastric ulcer. Humic acid was also reported to be neuroprotective against oxidative stress as it improved traumatic spinal cord injury in rat a model as well [45]. In this vein, administration of humic substance modulated serotonin and its metabolite, while normalizing dopamine, and noradrenaline in rat’s brain [46].

The possible mechanism(s) by which humic acid can reverse the anxiety, depresssive-like symptoms and memory impairment caused by DSS-induced colitis is/are not far- fetched as it could be attributed to its antioxidant properties, which can help protect cells from damage caused by free radicals and have anti-inflammatory and immunity- boosting effects [57]. The available data also suggest that humic acid may support gastrointestinal health by promoting a healthy balance of gut bacteria and neurochemical modulation [52, 56, 57]. The gut microbiota being positively influenced by humic acid, leading to improved brain health and cognitive functions.

The comorbidity of anxiety, depression and memory impairment and DSS-induced colitis was established in this study. Treatment with humic acid attenuated this neurocognitive damage, this poses humic acid as gastroprotective and may be neuroprotective as well via its modulatory role on gut microbiota, oxidative and inflammatory pathways.

Ethical Approval

Ethical approval was requested from the University of Medical Sciences Research and Ethics Committee on Animal use and care and granted with the approval reference UNIMED-AREC/Apv/2023/033.

Consent for publication

Not applicable.

Availability of data and Material

The relevant data supporting the findings of this study can be found in the article and available upon request. Requests for access to the data should be directed to the corresponding author.

Conflicting interests

There is no conflict of any kind to declare

Funding

No external funding was received.

Authors' contributions

OBO and AGA conceived, designed and supervised the research, OOE co-supervised the experiments, did literature search, analyzed data, and wrote the manuscript, ADO conducted the experiment, AJK helped in the extraction and purification of the humic acid.

Acknowledgment

The authors express their gratitude to the technical personnel of the Animal House and the Department of Physiology, University of Medical Sciences, Ondo, Nigeria.

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The Modulatory Influence of Humic Acid on Cognitive Impairment and Neurobehavioral Changes Induced by Colitis in Adult Male Wistar Rats

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Abstract

Figures

Introduction

Materials and methods

Experimental animal

Experimental design

Extraction and purification of humic acid

Induction of colitis

Neurobehavioural studies

Open field test

Y- maze tests

Elevated-plus maze (EPM) tests

Disease activity index (DAI)

Preparation of brain tissues homogenates for biochemical assays

Determination of catalase (CAT) activity

Estimation of brain superoxide dismutase (SOD) activity

Estimation of brain level of malondialdehyde (MDA)

Determination of brain nitrite level

Determination of brain glutamine transferase (GST) concentration

Determination of brain glutathione (GSH) concentration

Statistical analysis

Results

Effect of DSS and drug treatments on the disease activity index of colitis

DISCUSSION

Conclusion

Declarations

References

Additional Declarations

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