https://orcid.org/0000-0002-1405-1728
Figures
Abstract
This study tested the hypothesis that flavonoids (diosmine, hesperidin and rutin®) play a protective role in the brain and duodenum against 3-Nitropropionic acid (3-NPA) free radical induced inflammation. This was achieved by measuring the levels of GABA, 5-HIAA, dopamine and some inflammation and oxidative stress markers. Male young Wistar rats (weight 60g) received Salmonella thyphimurium ATCC14028 1x106 CFU/g every week, for two consecutive weeks, plus the following treatments: group A, 0.9% NaCl (Control); group B, diosmine (300 mg) + hesperidin (33.3g) + rutin (150 mg/kg); group C, 3-NPA; group D, 3-NPA + mix of flavonoids with 1 ml of rutin® per rat. 3-NPA administration was at 24 mg/kgW by intraperitoneal route and the flavonoids were orally administered at every 48 hours for 15 days. At the moment of euthanasia, the blood was obtained to assess Interleukine-6, glucose, triglycerides and hemoglobin levels. Brain and duodenum were obtained to measure GABA, dopamine, 5-HIAA, lipoperoxidation, reduced glutathione (GSH), total ATPase concentrations and catalase activity using validated methods. The brains, stomach and duodenum were dissected for histological analysis. In group A, there is a decrease in interleukine-6 levels (p = 0.009). In the cortex region of animals in group C, dopamine experienced a significant decrease (p = 0.012). GABA increased (p = 0.001) in cerebellum regions of animals in the groups A, B and C. ATPase activity increased (p = 0.013) in cerebellum regions in animals of groups C and D. Lipoperoxidation diminished (p = 0.043) in cerebellum region of group D. Catalase activity diminished (p = 0.043) in Cortex region of animals in groups B and D, and (p = 0.002) in cerebellum region of animals in groups A and B. Besides, histological changes revealed marked lesions of neuronal cells in experimental animals treated with nitro propionic acid. The protective role of flavonoids and rutin compounds on inhibition of the inflammatory response and correction of the fundamental oxidant/antioxidant imbalance in animals suffering from Huntington diseases are important vistas for further research.
Citation: Calderón Guzmán D, Osnaya Brizuela N, Ortiz Herrera M, Juárez Olguín H, Valenzuela Peraza A, Rojas Ochoa A, et al. (2026) Therapeutic potential of flavonoids and rutin against inflammation-induced on a huntington animal model. PLoS One 21(7): e0339016. https://doi.org/10.1371/journal.pone.0339016
Editor: Shengqian Sun, Yantai Institute of Technology, CHINA
Received: December 23, 2025; Accepted: May 21, 2026; Published: July 16, 2026
Copyright: © 2026 Calderón Guzmán et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: “Yes - all data are fully available without restriction; All relevant data are within the paper.”.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: AKT, Threonine Kinase; Anova, Analysis of variance; ATCC, American Type Culture Collection; ATP, Adenosine Triphosphatase; CNS, Central nervous system; UFC, Colony-forming units; DNA, Deoxyribonucleic Acid; Rutin, Diosmine/hesperidine; DA, Dopamine; ELISA, Enzyme-Linked Immunosorbent Assay; EDTA, Ethylenediaminetetraacetic acid; GABA, γ-Aminobutyric acid; GSH, Glutathione; 5-HIAA, 5-Hydroxyindole acetic acid; 3-NPA, 3-Nitropropionic acid; NF-kB, Factor Nuclear Kappa B; NOGSH, Nitroso-glutathione; KH2PO4, Potassium dihydrogen phosphate; PI3K, Phosphoinositide 3 Kinase; RNS, Reactive nitrogen species; ROS, Reactive oxygen species; RPM, Revolutions per minute; Na+, K+ ATPase, Sodium potassium ATPase; TBARS, Thiobarbaturic acid reactive substances; Tris, Tris-hidroxymethyl aminomethane
Introduction
The sum of the years of life lost because of premature mortality from a disease and the years lived with a disability associated with prevalent cases of the disease in a population include neurological disorders [1]. Neurological disease is one of the leading causes of death worldwide, and some common mechanisms are excitotoxicity and calcium overload, oxidative stress, and neuroinflammation [2]. This situation triggers lethal pathway for reactive oxygen (ROS) and nitrogen species production [3], provoking oxidative stress and release of nitric oxide (NOˉˉ) species. NO not only causes physical changes in the structure of the mitochondria and negative impact on their functions but also leads to DNA damage [4]. 3-nitropropionic acid (3-NPA) is a potent mitochondrial toxin and generally causes damage to mitochondria and DNA.
In Wistar rats, 3-NPA promotes the degeneration of neurons. Its systemic delivery in rats’ heralds Huntington disease model [5], where it works in the basal ganglia using up the store of monoamine neurotransmitters, such as serotonin, norepinephrine, and dopamine [6]. Apart from its toxic potential, NOˉˉ, in the low nanomolar range of 10ˉ8 to 10ˉ6 M could function as a neuromodulator, however any deviation from this amount may trigger fatal consequences on the cells through the formation of nitroso-glutathione (NOGSH) and increase of in oxidative stress [7]. The fact that cellular structural components are susceptible to damage by free radicals [8], more specially the membrane lipids [9], the central nervous system (CNS) must be protected from NOˉˉ imbalance through the maintenance of the right amount of antioxidants. This point is extremely important during development, because this is the time brain metabolism and growth is at peak level [10]. Therefore, it is necessary to regulate energy and glucose homeostasis via the maintenance of dopaminergic system in hypothalamic neurocircuits and higher brain circuits [11].
Apoptosis of macrophages in the body is found during systemic infection, and the activation of kinase pathways leads to balanced pro- and antiapoptotic regulatory factors in the cell. In the intestine, Salmonella mediates macrophagic death by caspase-1 activation, which also releases interleukins. The above situation bolsters inflammation and triggers chemiotactic influx of phagocytic cells such as macrophages, thus carrying the infectious agent to tissues outside the intestine [12]. However, this disseminative channel is not the only pathway Salmonella can spread to systemic level. Some strains can invade intestinal epithelial cells through the “zipper” and “trigger” pathways leading to seriously fatal infections and production of extensive cytopathology that reaches out to many systemic organs [13]. In any case, the overall effectors involved in this dissemination pathways remain poorly clear.
In the context of neurodegenerative diseases, phytonutrient compounds, such as flavonoids, are natural products with preventive potentials. These diseases share the same pattern based on progressive deterioration of dopaminergic (DA) neurons [14]. The flavonoids activate the antiapoptotic pathways, such as P13K/AKT and NF-KB, that are critical intracellular pathways geared towards the regulation of cell survival, growth and proliferation, as well as the protection of the mitochondria from dysfunction and activation of neurotrophic factors. Findings of Burda and Oleszek [15], suggest that flavonoids with hydroxyl group in position C-3 possess elevated antioxidant activity, while the hydroxyl in C-4 possesses anti-free radical activity, and that these compounds have shown antimicrobial activity [16]. The flavonoids as Diosmin and Hesperidin are natural polyphenolic compounds with antioxidant and anti-inflammatory effects, both are biological compounds activity obtained from lime citrus, present in pharmaceutical formula as Daflon® [17]. The intake of Rutin® is recommended for its neuroprotective roles helping to downplay neurodegenerative disorders due to its antioxidant activities and is an-herbal polyphenolic compound [18]. Particularly, rutin® has some protective effects in Huntington´s Disease models [19] although the underlying mechanisms are still unknown.
Plasma membrane phospholipids in brain are in close contact with structural proteins that are embedded in the lipid bilayer [20], from which Na+, K+ ATPase is responsible of keeping the ionic interchange through this bilayer by the stimulation of Na+ and K+ flows [21]. The inhibition of the Na+, K+ ATPase activity induces excitatory amino acid release within the Central Nervous System (CNS) [22].
Taken the above reports and findings as a background, the purpose of the present study is to compare the protective effect of flavonoids (diosmin and hesperidin) in combination with rutin® on the levels of dopamine, GABA, 5-HIAA and on select oxidative stress and anti-inflammatory markers in brain regions, duodenum and stomach of animal model with experimentally induced inflammation and Huntington´s disease.
Materials and methods
Experimental animals
Animals were purchased from certified bioterium of Instituto Politecnico Nacional, Mexico City. The animals were placed in four meshed plastic cages; each containing eight rats and were exposed to 12 h light-dark cycle and natural environmental conditions. Free access to pelleted laboratory rodent feed (Purine 5001) and water was allowed during the experiment. Before the study, the animals were allowed 1–2-week period of acclimatization to the animal house facility conditions with food and water. Animal management and care were conducted according to the National and International guidelines of animal care. This study protocol was approved with the reference number 026/2022.
Chemicals
Thiobarbituric, Glutathione, catalase, ATP, GABA, Dopamine, 5-HIAA and Ortho Pthaldialdehyde were acquired from Sigma-Aldrich, St. Louis, MO, USA. Hydrochloride acid, Sulfuric acid, Nitric acid, Bisulphite, Trichloro acetic acid, Sodium phosphate, Magnesium chloride and Methyl alcohol were purchased from Merck, Darmstad, Germany. Triglycerides and glucose Roche devices were used in the study. Catalase Assay Kit was from Cayman Chemical Company, and Rat IL6 and Interleukin-6 Elisa Kit were obtained from OriGene Technologies Inc.
Experimental model
Thirty young male Wistar rats, 25 days old (60−65 g) were separated into 4 groups and treated as follows: Group A, 0.9% NaCl + Salmonella T (control). Group B, S. typhimurium + Diosmin/hesperidin/Rutin® (1 ml); group C, S. typhimurium + 3-NPA (24 mg/kg); group D, S. typhimurium + Diosmin/hesperidin/Rutin (1 ml) + 3-NPA (24 mg/kg) per rat. Diosmin/hesperidin/Rutin® was orally administered every 48 hours for 15 days. Live culture of Salmonella typhimurium (S. typhimurium) ATCC14028, 1 x 106 colony-forming units/rat (CFU/rat) was given once a week in two doses, and 3-NPA in a single dose at the end (Experimental design). 120 minutes after receiving the drugs, the animals were put under anaesthesia (sodium pentobarbital 50 mg/kg) and euthanized with guillotine to obtain the brain, stomach and duodenum, and then put in saline (NaCl 0.9%) at 4 °C. Eight animals (two rats for each group and 20 slices by tissues) were stained with hematoxylin-eosin to evaluate the histological abnormalities. The blood was assessed to measure Interleukin-6, triglycerides, hemoglobin and glucose. Brain was dissected into cortex, hemispheres and cerebellum. Brain regions, stomach and duodenum were put in 5 volumes of 0.05 M TRIS-HCl, pH 7.4 to evaluate lipoperoxidation (TBARS), total ATPase and catalase. An aliquot was homogenized in 0.1 M perchloric acid (HClO4) (50:50 v / v) to evaluate γ-Aminobutyric acid (GABA), reduced glutathione (GSH), dopamine and 5-hydroxyindole acetic acid (5-HIAA) concentrations.
Inoculation of rats with Salmonella thyphimurium strain ATCC14028
The S. thyphimurium inoculated in the rats were obtained from strain bank (ceparium) of Experimental Bacteriology laboratory of National Institute of Pediatrics, Mexico City. Following its re-identification, an aliquot was taken from a maintenance medium and injected in a culture medium of Salmonella Shigella (SS agar). This was subjected to 18- to 24-hour incubation at an ambient temperature of 37 °C using bacteria incubator, Zhengzhou Nanbei instruments, Henan, China. Subsequently, proven colonies containing S. thyphimurium, after reconfirmation through conventional biochemical tests, were selected and inoculated in TSA (Trypticasein Soya Agar). This was subjected to 18-hour incubation at 37 °C. Using Hyssop, the collection of bacterial biomasses was made. The masses were suspended in PBS buffer, pH = 6.8. Subsequently, with DU 640 spectrometer (Beckman, USA) the masses were adjusted to an AS450nm = 0.175 (equivalent to 3 x 108 UFC / ml) and diluted to a concentration of 1 x 106 UFC / ml [23]. A non-lethal volume (1 ml) was taken and administered to each animal using orogastric tube.
Technique to measure glucose and triglycerides in blood
Glucose and triglycerides were measured using 20 µl of tail-end blood, twice collected after the treatment. The collection was made without anticoagulant. This volume of blood was smeared on a reactive filter paper in Accu-Chek active (Roche Mannheim Germany) equipment. The glucose and triglyceride concentrations were read and reported in mg/dL.
Measurements of Interleukin (IL-6)
The measurement of IL-6 was carried out at the end of treatment. 3 mL of fresh blood was obtained from the heart by cardiac punction after anesthesia. This was centrifuged at 3,500 rpm for 10 min in a clinical centrifuge (HERMLE Labnet, Z 326 K). The plasma obtained was processed with Rat IL6/Interleukin-6 Elisa Kit from OriGene Technologies Inc. The samples were read in triplicate at 450 nm in a microplate reader. Molecular devices Spectra max plus 384 and software SoftMax Pro 6.0 were used, and the concentration was expressed in pg/ml.
Dopamine concentration determination
The determination of dopamine (DA) concentrations was carried out with HCLO4 homoginzed tissue supermatant resulting from a 10-minute centrifugation at 9,000 rpm performed with a microcentrifuge (Hettich Zentrifugen, model Mikro 12–42, Germany). The technique employed was reported by Calderon et al, [24]. A portion of the HClO4 supernatant was mixed with 1.9 ml volume of chemical buffer solution (0.003M octyl-sulphate, 0.035 M KH2PO4, 0.03 M citric acid, 0.001 M ascorbic acid) in a test tube. In total darkness at room temperature, the incubation of the mixture was performed for a 5-minute period. Thereafter, the reading of the samples was carried out using a spectrofluorometer (Perkin Elmer LS 55, England) with an excitation wavelength of 282 nanometers and electromagnetic radiation at a specific wavelength of 315 nanometers The FL Win Lab version 4.00.02 software was used. Using a previously standardize curve, the concentration values of DA was deduced and this was reported in nanomol per gram (nMoles/g) wet tissue.
Measurement of γ-Aminobutyric acid
The determination of the concentration of γ-Aminobutyric acid (GABA) was carried out with the HCLO4 homogenized tissue supernatant previously subjected to a 10-minute centrifugation at a revolution of 9,000 per minute using a microcentrifuge (Hettich Zentrifugen, model Mikro 12–42, Germany). The technique employed in this determination was developed by Hsieh et al, [25]. An aliquot of the HClO4 supernatant and work solution (Buffer of Boric acid 0.1M pH 9.3 + MeOH + Orto-Phthalaldehyde + Mercaptoethanol) was collected and loaded in a test tube. Subsequently, this was subjected to 5-minute incubation at room temperature in total darkness. Following the incubation, GABA concentrations in the samples were fluorometrically read using FL Win Lab version 4.00.02 of Perkin Elmer LS 55 (England) spectrofluorometer with an excitation wavelength of 340 nanometers and electromagnetic radiation at wavelength of 455 nanometers. Using a previously standardized curve, the concentration values of GABA was deduced, and this was reported in nanomole per gram (nMoles/g) wet tissue.
5-HIAA concentration Determination
The levels of 5-HIAA were evaluated using the floating tissues of the brain regions previously mixed with HClO4 (2:1 v/v). The tissues were made to undergo a 10-minute centrifugation at a revolution of 10,000 per minute using a micro centrifuge (Hettich Zentrifugen, model Mikro 12–42, Germany). To process the brain regions, an aliquot of each region was fed to Perkin Elmer LS 55 fluorometer that processed the tissues at an excitation wavelength of 296 nanometers, and an emission of 333 nanometers and FL Win Lab version 4.00.02 software was to determine the concentration values [26]. The values were extrapolated in a standard curve previously standardized and reported in nM/g of wet tissue.
Reduced Glutathione (GSH) concentration Determination
To measure the concentration of GSH, the HCLO4 homogenized tissue supernatant that was previously centrifuged with MIkro 12–42 centrifuge (Germany) during a 5-minute period at a revolution of 9,000 per minute was used applying Hissin and Hilf modified method [27]. In a phosphate buffer [pH 8.0, EDTA 0.2%] with a capacity of 1.8 mL, the supernatant aliquot (20 μL) was mixed with ortho-phthaldehyde (100 mL) and methanol (1 mg/mL). The mixture was put in a test tube and subjected to a 15-minute incubation at room temperature in absolute darkness. Perkin Elmer LS 55, software FL Win Lab 4.00.02 version, that possesses an excitation wavelength of 296 nanometers, and an emission of 333 nanometers was used to read GSH concentration in the centrifuged samples. From a previously standardised curve the concentration of GSH was extrapolated and reported in nanomole per gram (nMoles/g) wet tissue.
Total ATPase Measurement
Adenosine triphosphate enzyme activity was analyzed based on Calderón and colleagues´ method [28]. The analysis was conducted with 1 mg (10%) weight-by-volume (w/v) of the tissues of brain, duodenum and stomach that were previously homogenized in tris-HCl 0.05 M pH 7.4. The 1 mg of each of the tissues were put in a solution containing 3 mM MgCl2, 7 mM KCl, and 100 mM NaCl and subjected to a 15-minute incubation. Following this incubation, 4 mM tris-ATP was added to the solution. With Dubnoff Labconco shaking water bath, a second-round 30-minute incubation of the new solution was carried out at 37 °C. To stop the reaction, 100 µL (10%) weight-by-volume (w/v) of trichloroacetic acid was used. Subsequently, the samples were centrifuged at 100 g for 5 minutes at 4 °C. The measurement of inorganic phosphate (Pi), a product of ATP hydrolysis by ATPase, was carried out in triplicates using one supernatant aliquot as proposed by Fiske and Subarrow [29]. The supernatant absorbance reading was made with BECKMAN DU 640 spectrophotometer at 660 nanometers. The absorbance was expressed as mM Pi/g wet tissue per minute.
Measurement of Catalase
The determination of catalase was made with catalase kit (Cayman Chemical®) using the modified technique of Sinha [30]. Each brain region (cortex, hemispheres, cerebellum/medulla oblongata), stomach and intestine were homogenized in 3 mL of tris-HCl 0.05 M pH 7.4 buffers. From the diluted homogenates, 100 µL was taken. The samples were read in triplicate at 570 nm in a microplate reader. Molecular devices Spectra max plus 384 and software SoftMax Pro 6.0 were used. Catalase activity was expressed in µM/g of wet tissue.
Lipid peroxidation (TBARS) determination technique
The measurement of TBARS was made using the brain tissues homogenized in tris-HCl 0.05 M pH 7.4 buffer solution in accordance with the modified technique of Gutteridge and Halliwell [9]. 1mL of the homogenized brain tissue sample was mixed with 2mL solution containing 1.25 g of thiobarbaturic acid (TBA), trichloroacetic acid (TCA) 40 g, and concentrated hydrochloric acid 6.25 mL that was diluted with deionized water 250 mL. The mixture was put in a Thermomix 1420 and subjected to 30-minute heat. Thereafter, it was cooled for 5 minutes in an ice bath followed by 15-minute centrifugation at 700 g-force (Sorvall RC-5B Dupont). Subsequently, the reading of the floating tissue absorbance was carried out in triplicate at 532 nanometer using BECKMAN DU 640 spectrophotometer. The reactive substance concentration to TBARS was reported as µM of Malondialdehyde/g of wet tissue.
Histological analysis in brain regions, stomach and duodenum
The brain, stomach and duodenal tissues were gentle cleansed in saline solution (0.9% NaCl) following their extraction with the objective of removing all the sticking debris on them. They were immediately subjected to histological analysis consisting of fixing them for 24 hours in a 10% NBF (neutral buffered formalin) solution. This was followed by washing and removing excess parts on the tissue samples and dehydrating them in a graded series of alcohol. Thereafter, the samples were immersed in xylene to remove the water and alcohol, and then paraffin embedded. Subsequently, they were cut to a thickness of 4–6 millimeters and H&E stained (Hematoxyn and Eosin). Next, the H&E-stained tissues were viewed under stereology microscope (Olympus BX51). Software Stereo Investigator 11 (SI 11) was used to quantify the biochemical indicators [31].
Statistical analysis
Descriptive statistic tables containing central measures of tendency and dispersion (mean + /- SD) were employed to show the data. Inference analysis was performed to compare the biochemical indicators of the control group with the experimental animal groups. Contrast of hypothesis tests such as Fisher analysis of variance (ANOVA) or Kruskal-Wallis test after variance homogeneity verification was used for this purpose. Tukey-Kramer or Steel-Dwass tests was employed as Post hoc contrasts test. Any associated probability value p < 0.05 was considered statistically significant. Analysis was performed using Sigma Plot Statistical v12 software [32].
Results
The result of Interleukin- 6, triglycerides, glucose and hemoglobin levels in the blood of rats treated with mix flavonoids + Salmonella T. in the presence of 3-nitropropionic acid are presented in Table 1. In animal groups treated with Mix flavonoids + Salmonella typhimurium + 3 -NPA, Interleukin – 6 levels decreased (p = 0.009) with significant differences when compared with the control group.
Dopamine levels in brain regions of rats treated with mixed flavonoids + Salmonella typhimurium in the presence of 3-nitropropionic acid are presented in Table 2. Dopamine diminished significantly (p = 0.012) in cortex region of animals that received Salmonella typhimurium combined with 3-nitropropionic acid when compared with the control group.
GABA levels in brain regions of animals treated with mixed flavonoids + Salmonella typhimurium in the presence of 3-nitropropionic acid are shown in Table 3. GABA increased significantly (p = 0.001) in Cerebellum regions of animals that received Salmonella typhimurium alone, or combined with Mix Flavonoids, or 3-NPA compounds when compared with the combination of mix Flavonoids+ Salmonella typhimurium + 3-NPA group.
Table 4 shows the levels of 5-HIAA in brain regions of rats treated with mix flavonoids + Salmonella typhimurium in the presence of 3-nitropropionic acid, where no significant difference (p > 0.05) was observed between them and the control group.
GSH levels in brain regions and duodenum of rats treated with mix flavonoids + Salmonella typhimurium in the presence of 3-nitropropionic acid are presented in Table 5. There were no significant differences (p > 0.05) between the levels in the experimental animals and in the control group. However, GSH levels were observed to decreased only in duodenum.
The total ATPase levels (Table 6) in brain regions, duodenum and stomach of rats treated with mix flavonoids + Salmonella typhimurium in the presence of 3-nitropropionic acid. ATPase increased significantly (p = 0.013) in cerebellum regions, in animal groups treated with Salmonella T. alone or combined with Mix flavonoids or 3-nitropropionic acid with respect to the control group.
Lipoperoxidation levels in brain regions of rats treated with mix flavonoids + Salmonella typhimurium in the presence of 3-nitropropionic acid are shown in Table 7. Lipoperoxidation diminished significantly (p = 0.043) in cerebellum regions of animals that received Salmonella typhimurium combined with mixed flavonoids and 3-nitropropionic acid when compared with the control group.
Table 8 depicts the levels of Catalase in brain regions of rats treated with mixed flavonoids + Salmonella typhimurium in the presence of 3-nitropropionic acid. Catalase activity diminished significantly (p = 0.043) in cortex region of animals treated with Salmonella typhimurium in combination with mixed flavonoids or 3-NPA groups and (p = 0.002) in cerebellum region of animals that received Salmonella typhimurium alone or combined with mixed flavonoids when compared with the same group with 3-nitropropionic acid. Besides, histological changes revealed marked lesions of neuronal cells in experimental animals treated with nitropropionic acid.
Discussion
The pathogenesis on neurological disorders involves the intricate interplay between neuroinflammation and energy metabolism, triggering metabolic dysregulation and activating neuroinflammatory responses, ultimately resulting in cellular damage and further metabolic disturbances [33], emphasizing the importance of in-depth research into their interaction to provide new treatment strategies to minimize neural damage and promote recovery.
Recent studies reveal that rutin suppresses the production of tumor necrosis factor-α (TNF-α) and inhibits the lipopolysaccharide (LPS)-induced activation of nuclear factor-κB (NF-κB) [34]. Neuroinflammatory responses involve the activation of the interleukins. In this study, interleukin is decreased in the animals that received diosmin, hesperidin, rutin and Salmonella typhimurium. Hence, we suggest that the successful colonization of Salmonella typhimurium may enable the rational design of effective therapeutic strategies [35]. These findings were in line with the results obtained by Wu et al, [36], who in their study reported that rutin significantly reduced the levels of reactive oxygen species and improved locomotion recovery. They suggest that in appropriate dosage conditions, the mechanism may be related to the alleviation of inflammation and oxidative stress.
Gamma-aminobutyric acid is the major inhibitory neurotransmitter in the central nervous system and various mechanisms have reported that GABA ameliorate inhibition of neuroinflammatory responses and repair of oxidative damage [37]. In this study GABA levels increased in animals treated with Salmonella typhimurium alone or combined with flavonoids plus Rutin® in cerebellum regions. This result agrees with the findings of other authors who suggest that GABA could be a good strategy to modulate immunological response in various inflammatory diseases, produced by microbial strain [38].
Transmitter dopamine metabolism by monoamine oxidase enzyme has been attributed to striatal damage in Huntington´s disease (HD) model associated with mitochondrial toxin [39]. HD is a destructive neurodegenerative disorder associated with progressive loss of neuronal functions, which eventually bring about the death of specific brain parts with the striatum and cerebral cortex being the principal targets [40]. These results can be in line with the reports of the present study, for the fact that dopamine levels diminished in cortex regions of animals that received 3-nitropropionic acid treatment in combination with Salmonella typhimurium ATCC14028. Although the metabolism of dopamine is recognized as one of the sources of reactive oxygen species through the Fenton mechanism [41].
Regarding the animals treated with diosmin, hesperidin, rutin and Salmonella typhimurium, ATPase activity decreased in cerebellum, probably as consequence of changes in the affinity of the enzyme [42]. In the animals that received the same treatment plus 3-NPA, there was a decrease in lipoperoxidation in cerebellum, and this may be since reactive oxygen species is the primary event in 3-NPA toxicity [43].
GSH serves as a critical regulator of ferroptosis-an iron-dependent form of cell death marked by excessive lipid peroxidation [44]. However, Ferroptosis refers to a novel way of cell death. It shows a close association with iron metabolism and oxidative damage, as marked by the significant increase of reactive oxygen species as NO [45]. confirming that ferroptosis is closely associated with high levels of reactive oxygen species and lipid peroxides. GSH levels reported in present study decreased in the duodenum of the animals treated with 3-NPA alone or combined. These results may have relation with the reports of Kumar et al. [46], who suggest that 3-NPA depleted the GSH in cortex.
ATPase enzyme is related to oxidative stress in neurological impairments [47], and Ca² ⁺ /Mg² ⁺ -dependent ATPase increased in the cerebellum of animals that received 3-NPA alone or combined. This result is in line with the reports of Naziroğlu et al. [48], who suggest that increased Ca 2+-ATPase activities is due to substances that induced brain injury by exhibiting free radical production, regulating calcium-dependent processes and supporting the antioxidant redox system.
Among endogenous antioxidants, enzymes such as catalase are central to disabling free radicals, thereby preventing oxidative damage to cellular lipids, proteins, and nucleic acids [49]. The Catalase activity decreased in the animals treated with diosmine, hesperidine and rutin in cortex and cerebellum regions, but increased in the presence of 3-NPA. These results coincide with the reports of Mascaraque et al. [50], who suggest that rutin has a significant protective effect in this neurological disease.
Huntington’s disease is an inherited neurodegenerative disease. It is characterized by excessive motor movements couple with cognitive and emotional deficits [51]. In addition, there is a marked neuronal loss among the medium-sized projection neurons of the dorsal striatum. In this study, however; diosmin, hesperidin, rutin and Salmonella typhimurium supplemented in vivo, protected the striatum. This suggests a mechanism that involves antioxidant activity by controlling the expression of antioxidant enzymes and other chaperones regulating proteostasis, with potential neuroprotective role [52]. Besides, histological changes revealed marked lesions of neuronal cells in experimental animals treated with nitropropionic acid.
Limitations: The authors do not analyze the flavonoids separately as groups to compare the effects of individual flavonoids and their combinations, then more research work must be carried out to thoroughly examine its neuroprotective mechanisms.
Conclusion
The protective role of antioxidant compounds by inhibiting inflammatory response and correcting the fundamental oxidant/antioxidant imbalance in patients suffering from neurodegenerative diseases are important vistas for further research.
We recommend further studies to investigate the possible relationship between flavonoids, rutin, inflammation by LPS and 3-NPA in different animal models. As a possible protective barrier against pro-inflammatory responses, it may be a new dietary strategy to combat Huntington’s disease.
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