Daflon attenuates cisplatin-induced cerebellar neurotoxicity, anxiety-like behavior, and motor dysfunction by downregulating TLR4/NF-kB signaling

Abstract

Background

Although cisplatin is an effective chemotherapy, a major downside is its toxicity, including cerebellar neurotoxicity, which is mediated by the induction of inflammation and oxidative stress. On the other hand, daflon, a micronized purified flavonoid fraction, suppresses inflammation and oxidative stress. However, the effect of daflon on cisplatin-induced cerebellar neurotoxicity has not been documented.

Aim

The present study evaluated the effect of daflon in cisplatin-induced cerebellar toxicity. In addition, the role of TLR4/NF-kB signaling was explored.

Materials and methods

Twenty male Wistar rats were acclimatized for 2 weeks and then randomized into 4 equal groups: control, daflon-treated, cisplatin-treated, and cisplatin+daflon-treated.

Results

Daflon significantly improved cisplatin-induced distortions in cerebellar histology, evidenced by increased thickness in the molecular and intergranular layers, increased Purkinje cells, and reduced pyknotic neurons. Also, daflon attenuated cisplatin-induced rise in malondialdehyde and cisplatin-driven decline in glutathione, superoxide dismutase, and catalase activities. Furthermore, daflon ameliorated cisplatin-induced rise in myeloperoxidase activity and tumour-necrosis factor α, interleukin-1β 1β, and interleukin-6 levels. Additionally, daflon suppressed cisplatin-induced upregulation of toll-like receptor-4, nuclear factor-kappa B, cyclo-oxygenase-2, prostaglandin E2, and caspase-3 activity in the cerebellar tissue.

Conclusion

In conclusion, daflon confers neuroprotection against cisplatin-induced cerebellar neurotoxicity through the suppression of TLR4/NF-kB-mediated oxidative-inflammatory and apoptotic injury.

Introduction

Cisplatin is a widely used alkylating molecule for the treatment of malignancies [1]. As a chemotherapeutic agent, cisplatin is used either as a single antineoplastic or neoadjuvant therapy for solid tumors or hematologic malignancies [2]. However, cisplatin has been demonstrated to induce dose-related renal [3], gastrointestinal [4], ear-related [5], and brain [6] toxicities. Administration of cisplatin induces severe renal toxicity with associated renal failure evident by impairment of glomerular filtration rate and electrolyte imbalances [3]. Reproductive abnormalities such as ovarian failure, premature menopause, and impairment of spermatogenesis, as well as gastrointestinal side effects like nausea and vomiting [4], have also been linked to cisplatin exposure. In addition, increasing evidence shows that cisplatin use induces neurotoxicity [1, 7, 8].

Cisplatin exerts its toxicity in all brain parts, including the cerebellum. Chemotherapy-associated neurotoxicity is a prevalent side effect in cancer patients, and it is usually characterized by peripheral neuropathy and cognitive decline [8]. Neurotoxicity due to exposure to cisplatin with associated nerve damage has been demonstrated in nerve conduction studies [1]. Cisplatin-induced neurotoxicity, including hearing disorders and visual impairment, has been demonstrated to occur via the induction of oxidative stress [7]. Exposure to cisplatin increases sphingosine-1-phosphate levels in the central nervous system, which is responsible for post-chemotherapy cognitive impairment [9]. Besides, inflammation has also been implicated in the pathogenesis of cisplatin-induced neurotoxicity [10]. Cisplatin upregulates toll-like receptor 4 (TLR4), which in turn activates the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) [11]. According to the findings of Davies et al. [12], cisplatin enhances free radical generation and activates NF-kB. The generation of free radicals is characterized by the compromise of the antioxidant enzyme-mediated defense, deregulation of heme oxygenase-1 (HO-1), and nuclear factor erythroid 2-related factor (Nrf2) [13]. It has also been shown that cisplatin-induced rise in NF-kB activates myeloperoxidase activity and promotes inflammation by upregulating inflammatory chemokines and cytokines such as tumor necrosis factor-alpha (TNFα) and interleukin-1beta (IL-1β), which in turn activate cellular macrophages and neutrophil infiltration [14].

The cerebellum is a vital part of the brain and it plays a central role primarily in motor function, and in memory, especially fear and anxiety [15]. It contains more neurons than other parts of the brain [15] and it is particularly vulnerable to intoxication, especially the cerebellar cortex and Purkinje neurons [21]. Cisplatin causes ROS generation that exceeds the antioxidative capacity of cerebellar cells, leading to cerebellar oxidative damage [16]. Primarily, this causes damage to the Purkinje and glial cells, and results in motor deficits [17]. Exposure to cisplatin also activates inflammatory signaling pathways, particularly NF-kB, leading to the activation of its downstream, TNF-α, IL-1β and IL-6 and suppression of cellular antioxidants [18]; thus exacerbating neuronal injury by promoting microglial and astrocyte activation, including reactive astrogliosis marked by hypertrophied Bergmann glial fibers [16]. This inflammatory response further activates ROS-driven oxidative injury and amplifies cerebellar damage. Additionally, cisplatin disrupts calcium ion homeostasis via modulation of glutamate receptors (NMDA and AMPA receptors), resulting in mitochondrial dysfunction that in turn promotes promotes the release of caspases, impairs energy metabolism and potentiates cerebellar oxidative damage [19]. Activation of caspase 3 by cisplatin drives apoptotic degeneration of the Purkinje cells, the hallmark of cisplatin neurotoxicity [16]. Cisplatin induces cytotoxicity of the cerebellar cortex via apoptosis of the proliferating granular cells and degeneration of the Purkinje cells [16, 20]. These events correlate with locomotor deficits observed in cisplatin-exposed experimental animals [16].

On the other hand, daflon (also called micronized flavonoid-rich fraction) contains 10% hesperidin and 90% diosmin and is used in the management of inflammatory disorders [21]. The flavonoids in daflon, hesperidin, and diosmin exert anti-inflammatory and antioxidant properties [22]. The findings of Ahmed et al. [23] showed that diosmin from daflon decreases NF-kB-mediated inflammation in Wistar rats. Oral administration of daflon has also been shown to suppress TLR4-mediated acute and chronic inflammatory responses and downregulate TNF-α and cytokines by microglia [24].

Despite the aforementioned cisplatin toxicity, its mechanism of action, and the anti-inflammatory and antioxidant effects of daflon, no study has reported the impact of daflon co-treatment in cisplatin-induced cerebellar neurotoxicity. Thus, this study aimed to investigate the potential of daflon as a therapeutic candidate for the management of cisplatin-associated cerebellar neurotoxicity. The possible involvement of the TLR4/NF-kB signaling was also explored.

Materials and methods

Drugs, reagents, and kits

Cisplatin (Naman Pharma Drugs, Princess Street, Mumbai-2, India) and micronized purified flavonoid fraction (Daflon®) (Servier Egypt Industries Limited, 6th October City, Giza, Egypt; under the license of Les Laboratories Servier, France) were procured from commercial sources. ELISA kits were also purchased from commercial sources (Elabscience Biotechnology Inc., USA) for the assays of TNF-α, IL-1β, IL-6, cyclo-oxygenase-2 (COX-2), prostaglandin 2 (PGE2), NF-kB, TLR4, and caspase-3. All other reagents were of high analytical grade and procured from standard commercial sources.

Animal handling and experimental protocol

Twenty albino Wistar rats of similar body weights were obtained from the institution’s Animal House and kept in the department’s Animal Holdings under a natural light/dark cycle. The rats were allowed to feed on rat feed and drink water ad’libitum. The Ethics Review Committee of the Faculty of Basic Medical Sciences, Ladoke Akintola University of Technology, Nigeria, approved the study protocol (ERCFBMSLAU TECH: 061/08/2024). The National Institutes of Health’s (NIH) Guide for the Care and Use of Laboratory Animals was followed. The rats were allotted into four equal groups after two weeks of acclimatization. The vehicle-treated animals received 0.5 mL of distilled water per os daily, the daflon (DAF)-treated animals received 100 mg/kg/day of daflon per os for 14 days, the cisplatin (CIS)-treated rats received 7 mg/kg of cisplatin i.p on day 8, and the CIS+DAF-treated rats received daflon and cisplatin as those in the daflon and cisplatin groups. Daflon was prepared by determining the body weight of each rat, and the appropriate dose of daflon (at 100 mg/kg) was determined and dissolved in 0.5 mL of the vehicle (distilled water). Daflon contains 90% of diosmin and 10% of hesperidin only. The doses and route of administration of daflon [25] and cisplatin [26] were as previously reported. Efforts were made to ensure that the minimum number of rats were used and that they had humane care.

Neurobehavioral assessment

A day before the termination of the study, anxiety-like behavior and motor function were assessed using elevated plus maze, open field, and hanging wire tests as reported by Kang et al. [27].

The elevated plus maze was used to assess the anxiety-like behavior of the rats. The elevated plus maze consists of two open arms and two closed arms (50 cm long and 10 cm wide) that are at right angles to one another, and 50 cm above the ground. Each rat was placed in the center of the maze with its head facing the open arms at the beginning of the trail. Animals were allowed to move freely from one arm to another. The events were captured with a camcorder for 5 minutes, and later viewed to determine the number of entries and time spent in the open arms.

The open field test was used to assess the locomotor activity and anxiety-like behavior of the rats. Each rat was positioned in the centre of the open field apparatus, which is a 40 cm cube. The rats were allowed to freely roam within the apparatus for 5 minutes. The events were captured with a camcorder and later viewed to determine the overall exploration distance, the number of rearing, and the number of centre field entries.

The hanging wire test was employed as an index of muscular strength and motor neuron integrity [28]. Rats used their forelimbs to suspend their body weight on a wire (0.5 mm in diameter, 60 cm in length) that was stretched between two posts and located 20 cm above a foam pillow. The rats were left for 60 seconds, and the events were captured with a camcorder. The time (in seconds) until the rat fell was determined and recorded as latency to fall.

Sacrifice and sample collection

Since Amiel and Barbe [29] reported that the effect of daflon may last about 24 hrs, animals were sacrificed 24 hrs after the last dose of treatment under euthanasia (40 mg/kg of ketamine and 4 mg/kg of xylazine i.p). The brain was harvested, and the cerebellum was dissected out immediately. The cerebellum was divided into two halves, and one half was homogenized in homogenization buffer (0.25 M sucrose, 0.5 mM EDTA, 5 mM histidine, and PI dissolved in PBS, pH 7.4), while the other half was fixed in 10% formo-saline for histological evaluation. The homogenates were spun at 10,000 g for 15 minutes at 4 °C in a cold centrifuge to obtain the supernatant for biochemical assays. The experts who carried out the biochemical assays and histopathological examinations were blinded to the study protocol.

Histopathological evaluation

The fixed tissues were cleaned with toluene, immersed in paraffin wax, and incubated overnight at 60 °C. About a 5 µm-thick section was obtained from each tissue, stained with hematoxylin and eosin (H&E), and dried. The tissues were viewed under a light microscope (Novel Microscope, China), and photomicrographs were obtained with a digital camera (Hayear, China) attached to the microscope. The photomicrographs were imported into ImageJ software for cell count by using similar principles as reported by Ricken et al. [30]. The number of Purkinje cells and pyknotic neurons was determined by evaluating five different fields and reported as normalized control (NC). The thickness of the molecular and intergranular layers was also determined.

Markers of oxidative stress, inflammation, and apoptosis

Cerebellar levels of malondialdehyde (MDA) and reduced glutathione (GSH) were determined by colorimetry as described by Akhigbe and Ajayi [31] and Beutler et al. [32], respectively. Superoxide dismutase (SOD) [33] and catalase [34] activities in the cerebellar tissues were assayed using established protocols.

Cerebellar TNF-α, IL-1β, IL-6, COX-2, PGE2, NF-kB, and TLR4 levels and caspase-3 activities were assayed with ELISA kits following the manufacturers’ guidelines.

Statistical analysis

The sample size was determined by power analysis using G*Power (version 3.1.9.4). The size determined was aligned with ethical recommendations. We used an effect size of 0.9 from our pilot study using Cohen’s d calculator, with 95% power and 5% type 1 error. Statistical analyses were performed using GraphPad Prism (version 8.0.2). One-way analysis of variance (ANOVA) and Tukey’s post hoc test were used to compare the means of the variables across and within the groups. p values of 0.05 or lower were considered to be statistically significant. Data are shown as means ± standard deviation (SD).

Results

Daflon co-administration improves anxiety-like behavior and motor function in cisplatin-treated rats

The vehicle-treated and daflon-treated rats showed a similar number of open arm entries and time spent in the open arms, but cisplatin exposure significantly reduced the entries and time spent in the open arms. Cisplatin-induced alteration in predictors of anxiety was attenuated by daflon in cisplatin-treated rats. Also, the number of rearing, centre square entry, and distance travelled were comparable between the control and daflon-treated rats. However, cisplatin treatment considerably reduced these indices, but co-treatment with daflon improved these variables in cisplatin-exposed rats. In addition, latency to fall was similar between the control and daflon-treated rats but reduced in the cisplatin-treated rats. Nonetheless, co-treatment with daflon attenuated cisplatin-induced decline in the latency to fall (Fig. 1).

Fig. 1.

Effect of cisplatin (CIS) with or without daflon (DAF) on open arm entry (A), and time spent in open arm (B) in the elevated maze test, rearing (C), centre square entry (D), and distance travelled (E) in the open field test, and latency to fall (F) in the wire hanging test. ^*p < 0.05 vs control, ^#p < 0.05 vs daf, ^~p < 0.05 vs cis. Data are shown as means ± SD of 5 replicates per group

Daflon co-administration improves cisplatin-induced histopathological injury of the cerebellar tissue

The control and daflon-treated rats showed preserved cerebellar tissues with normal molecular and granular layers. The Purkinje cells and neurons in the molecular layer appear normal. However, cisplatin-treated animals had distorted cerebellar tissues with several pyknotic neurons and few normal neurons in the molecular layer, and focal areas of inflammatory cell infiltration and hemorrhagic lesions. The granular layer also has a few Purkinje cells. Nonetheless, co-treatment with cisplatin improved cisplatin-induced alterations in cerebellar histology, evidenced by normal molecular layer neurons in the molecular layer and normal Purkinje cells (Fig. 2A).

Fig. 2.

Effect of cisplatin (CIS) with or without daflon (DAF) on cerebellar tissue; (A) photomicrographs (H&E, Mag.: x 400), (B) purkinje cells population (C) number of pyknotic neurons, and the thickness of the molecular layer, ML (D) and intergranular layer, IGL (E). ^*p < 0.05 vs control, ^#p < 0.05 vs DAF, ^~p < 0.05 vs CIS. Data are shown as means ± SD of 5 replicates per group. The control and daflon-treated (DAF) animals showed normal cerebellar tissues with normal molecular (M) and granular (G) layers. The purkinje cells (black arrow) and neurons (blue arrow) in the molecular layer appear normal. The cisplatin-treated (CIS) rats showed distorted cerebellar tissues. The molecular layer (M) shows several pyknotic neurons (blue circle) and few normal neurons (blue arrow) with focal areas of inflammatory cell infiltration (black circle) and hemorrhagic lesion (red circle). The granular layer (G) also appears distorted and there are few purkinje cells (black arrow). Co-treatment with cisplatin and daflon (CIS+DAF) improved CIS-induced alterations in cerebellar histology evidenced by normal molecular (M) with normal neurons (blue arrow) and normal purkinje cells (black arrow) granular (G) layers, although there are focal areas of inflammatory cell infiltration (black circle) in the molecular layer (M)

In addition, cisplatin led to a decline in the Purkinje cell population when compared with the control and daflon-treated rats. The decrease in Purkinje cells elicited by cisplatin treatment was attenuated by daflon co-therapy (Fig. 2B). Moreover, cisplatin significantly increased the number of pyknotic neurons in the molecular layer of the cerebellum when compared with the control and daflon-treated rats. Nonetheless, co-treatment with daflon prevented cisplatin-induced neuronal pyknosis (Fig. 2C). More so, cisplatin treatment significantly reduced the thickness of the molecular layer, but co-administration of daflon attenuated this effect (Fig. 2D). It was also observed that cisplatin treatment caused a reduction in the thickness of the intergranular layer, which was also improved by daflon co-administration (Fig. 2E).

Daflon co-administration attenuated cisplatin-induced oxidative stress

Daflon-treated rats showed similar MDA levels to the vehicle-treated control rats. However, cisplatin treatment led to a marked rise in MDA levels when compared with the control and daflon-treated rats. Co-administration of daflon ameliorated the cisplatin-induced rise in MDA levels in the cerebellar tissues (Fig. 3A).

Fig. 3.

Effect of cisplatin (CIS) with or without daflon (DAF) on cerebellar levels of malondialdehyde, MDA (A) and reduced glutathione, GSH (B), and superoxide dismutase, SOD (C) and catalase (D) activities. ^*p < 0.05 vs control, ^#p < 0.05 vs DAF, ^~p < 0.05 vs CIS. Data are shown as means ± SD of 5 replicates per group

In addition, cisplatin therapy significantly reduced GSH concentration, and SOD and catalase activities in the cerebellar tissues when compared with the control and daflon-treated rats. Notwithstanding, daflon co-treatment significantly attenuated cisplatin-induced reductions in these antioxidants (Fig. 3B–D).

Daflon co-administration ameliorated cisplatin-induced cerebellar inflammation and transcription factors

When compared with the control and daflon-treated rats, cisplatin significantly increased MPO activity, a marker of neutrophil accumulation, which was abrogated by daflon co-administration. More so, cisplatin therapy led to an increase in TNF-α, Il-1β, and IL-6 levels in the cerebellar tissue when compared to the control and daflon-treated rats. The rise in these pro-inflammatory cytokines caused by cisplatin treatment was observed to be abrogated by daflon co-treatment (Fig. 4).

Fig. 4.

Effect of cisplatin (CIS) with or without daflon (DAF) on cerebellar myeloperoxidase, MPO, activity (A), and tumor necrotic factor-alpha, TNF-α (B), interleukin-1beta, IL-1β (C) and interleukin-6, IL-6 (D) levels. ^*p < 0.05 vs control, ^#p < 0.05 vs DAF, ^~p < 0.05 vs CIS. Data are shown as means ± SD of 5 replicates per group

Daflon co-administration abrogated cisplatin-induced rise in NF-kB, TLR4, and caspase 3 activity in the cerebellum

In comparison with the control and daflon-treated rats, cisplatin led to a marked rise in NF-kB concentration. However, co-administration of daflon ameliorated the cisplatin-induced rise in NF-kB levels (Fig. 4A). Also, cisplatin significantly increased TLR4 levels when compared with the control and daflon-treated rats. Co-administration of daflon attenuated cisplatin-induced rise in TLR4 (Fig. 5).

Fig. 5.

Effect of cisplatin (CIS) with or without daflon (DAF) on cerebellar levels of nuclear factor-kappa B, NF-kB (A) and toll-like receptor 4, TLR4 (B). ^*p < 0.05 vs control, ^#p < 0.05 vs DAF, ^~p < 0.05 vs CIS. Data are shown as means ± SD of 5 replicates per group

Daflon co-administration blunted the cisplatin-induced rise in COX-2 and PGE2 in the cerebellar tissue

When compared with the control and daflon-treated rats, cisplatin treatment led to a significant rise in COX-2 in the cerebellar tissues. However, daflon co-treated attenuated the cisplatin-induced rise in COX-2 level. Also, cisplatin increased PGE2 in the cerebellar tissues compared with the control and daflon-treated rats, but daflon co-treatment attenuated cisplatin-induced PGE2 rise (Fig. 6).

Fig. 6.

Effect of cisplatin (CIS) with or without daflon (DAF) on cerebellar levels of cyclo-oxygenase-2 (COX-2) (A) and prostaglandin E2 (PGE2) (B). ^*p < 0.05 vs control, ^#p < 0.05 vs DAF, ^~p < 0.05 vs CIS. Data are shown as means ± SD of 5 replicates per group

Furthermore, cisplatin therapy markedly increased caspase 3 activity in the cerebellar tissue when compared with the control and daflon-treated rats. However, daflon co-treatment attenuated cisplatin-induced rise in caspase 3 activity (Fig. 7).

Fig. 7.

Effect of cisplatin (CIS) with or without daflon (DAF) on cerebellar caspase 3 activity. ^*p < 0.05 vs control, ^#p < 0.05 vs DAF, ^~p < 0.05 vs CIS. Data are shown as means ± SD of 5 replicates per group

Discussion

The results of this study are the first to show the ameliorative effects of daflon on cisplatin-induced cerebellar neurotoxicity. Our laboratory investigations showed that oral administration of cisplatin led to cerebellar neurotoxicity as depicted by a reduction in Purkinje cells and the molecular and granular layer thickness, and a rise in pyknotic neurons in the cerebellar tissue. This finding was associated with impaired motor function, anxiety-like behavior, and cerebellar oxidative injury (evidenced by a rise in MDA and reduction in antioxidant levels and activities), inflammation (as shown by an increase in MPO activity and TNF-α, IL-1β, and IL-6 levels), and apoptosis (demonstrated by upregulation of caspase 3 activity). These events were shown to be mediated by the activation of COX-2/PGE2 signaling and upregulation of TLR4 and NF-kB. However, co-administration of daflon attenuated cisplatin-induced cerebellar oxidative-inflammatory injury and apoptosis through the downregulation of COX-2/PGE2 pathway and TLR4/NF-kB signaling.

The present finding that cisplatin induces oxidative stress in the cerebellum agrees with previous findings [16, 20]. The cerebellum is a vital organ that is exposed to oxidative stress due to the high polyunsaturated lipid content in its membrane and its consumption of large amounts of oxygen, which makes it susceptible to reactive oxygen species (ROS) [35]. Cisplatin enhances ROS generation, leading to the accumulation of intracellular ROS and the destruction of essential biological molecules, including lipids, proteins, and DNA [36]. The ability of daflon to scavenge free radicals in the cerebellum, as demonstrated in this study, shows that it confers neuroprotection on the cerebellum. The increase in the concentrations of cerebellar GSH and the activities of SOD and catalase, with a concurrent decrease in lipid peroxidation, is suggestive of the effectiveness of daflon in buffering the levels of ROS in the cerebellum. This is in tandem with the findings of Shalkami et al. [37], who reported an increase in the activities of catalase and decreased levels of MDA following the use of diosmin, a constituent of daflon. In this study, the increase in the activities of SOD and catalase is likely due to the activation of the antioxidant buffer system in response to cisplatin-induced ROS generation, leading to the scavenging of ROS and preservation of the cerebellar tissue.

Inflammation of the brain tissue has been reported to be a key finding in neurodegenerative conditions [38]. Inflammation usually occurs with increased concentration of harmful free radicals, usually as a result of the presence of leukocytes at the site of inflammation. Acting as an antimicrobial oxidative enzyme, myeloperoxidase utilizes H₂O₂ in neutrophil phagolysosomes to synthesize hypochloric acid (Hawkins and Davies, 2021). The bactericidal MPO enzyme and the NADPH oxidase enzyme are generated when leukocytes are activated. After degranulation, MPO, an azurophilic granular constituent of leukocytes, is retained in the phagosome where it oxidizes hypochloric acid (HOCl) with H₂O₂ and chlorine, a potent bactericidal agent [12]. Hence, a rise in MPO activity is a pointer to an ongoing inflammatory process [39, 40].

The present findings that cisplatin enhanced myeloperoxidase activity, which was accompanied by a rise in TNF-α, IL-1β, and IL-6, demonstrate that cisplatin induces inflammation in the cerebellar tissue. The observation that cisplatin upregulated pro-inflammatory cytokines may be a consequence of cisplatin-induced oxidative stress. The overwhelming oxidative stress observed following cisplatin treatment may stimulate NF-kB-dependent inflammation, characterized by an increase in TNF-α, IL-1β, and IL-6 concentrations. TNF-α promotes inflammation by blocking GSK-3β-mediated phosphorylation via the NF-κB and Akt signaling pathways, which results in the stabilization of Snail and β-catenin proteins. TNF-α triggers NF-kB by attaching adaptor proteins to ligand-bound receptor complexes, which then recruit and activate IKK. IL-1β is a cytokine that induces inflammation by activating the transcription factor NF-κB. These events culminate in an inflammatory process.

Additionally, a key factor in neurotoxicity is the excessive secretion of pro-inflammatory cytotoxins, leading to neuroinflammation associated with chronic TLR4 activation [41]. TLR4 is activated by various endogenous damage-associated molecular patterns (DAMPs), which bind to TLR4 and activate downstream signaling pathways in glia, inducing secretion of ROS and pro-inflammatory cytokines like TNF-α and IL-1β that may lead to damage and death of neurons [42]. DAMPs are released into the extracellular space during neuronal death, and this can further stimulate TLR4/NF-kB signaling, thus aggravating neuroinflammation [43]. In this study, the administration of daflon attenuated myeloperoxidase activity and TNF-α, IL-1β, and IL-6 levels, possibly by inactivating TLR4/NF-kB signaling either directly or through the suppression of oxidative stress that may trigger this pathway.

It is likely that cisplatin-driven oxidative stress-mediated cytokine accumulation activated TLR4/NF-kB signaling, which promoted the transcription of several pro-inflammatory genes like COX-2 and PGE2 [44]. The upregulation of COX-2 promotes the conversion of arachidonic acid to PGE2, which possibly amplifies cisplatin-induced inflammatory response. TLR4 plays a critical and often detrimental role in neuroinflammation. It acts as a pattern recognition receptor (PRR), sensing both external threats (pathogen-associated molecular patterns, PAMPs) and internal danger signals (damage-associated molecular patterns, DAMPs). It may be activated by bacterial lipopolysaccharide, amyloid-beta (Aβ) fibrils, High-mobility group box 1 (HMGB1) released during ischemia, trauma, or neurodegeneration, heat shock proteins (HSPs), fibrinogen, and oxidized phospholipids [45, 46]. Upon activation, TLR4 activates the MyD88-dependent pathway or the TRIF-dependent pathway. The former pathways promote the recruitment of adaptor protein myeloid differentiation primary response gene 88 (MyD88) and subsequent activation of IL-1 receptor-associated kinase (IRAK) family proteins and TNF receptor-associated factor 6 (TRAF6) [47]. This results in the activation of the NF-κB and activator protein-1 (AP-1), leading to NF-κB translocation to the nucleus and promotion of the transcription of an array of pro-inflammatory genes, including chemokines, adhesion molecules, pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), and COX-2, leading to PGE2 production [48, 49]. The activation of TRIF-dependent pathway, an MyD88-independent pathway, results in the production of type I interferons (IFN-α/β) and some inflammatory cytokines, which culminate in the activation of interferon regulatory factor 3 (IRF3) and NF-κB [50] that in turn promotes the transcription of pro-inflammatory cytokines and COX-2, leading to PGE2 production.

Oxidative stress causes the depolarization of the mitochondrial membrane, resulting in cytochrome C release and caspase-3-dependent apoptosis [51–53]. Caspases play an important role as apoptotic mediators. They are synthesized as pro-activated by internal and external stimuli. Caspase 3 is the most significant effector of caspases, and the induction of its pathway is a hallmark of apoptosis and is employed as a marker of the death cascade [54]. Findings from this study revealed that cisplatin up-regulated the activity of cerebellar caspase 3. The protease may degrade the blocker of caspase-activated DNAse 1 (1CAD), which subsequently promotes caspase-activated DNAse I (CAD) (Larsen et al., 2010 [55]. CAD has been shown to degrade chromosomal DNA within the enzymes and generate chromatin condensation, which leads to DNA breakage and cell death. However, the result of our laboratory investigations showed that daflon significantly downregulated caspase 3 in the cerebellar tissues, possibly by inhibiting oxidative stress and blocking COX-2/PGE2 pathway and TLR4/NF-kB signaling, thus inhibiting cytochrome C release, which is required for activating caspase 3.

The observed cisplatin-induced biochemical perturbation in the cerebellar tissue was associated with distorted cerebellar histology characterized by the reduced thickness of the molecular and intergranular layers, reduced Purkinje cells, increased pyknotic neurons, and focal areas of hemorrhagic lesions and inflammatory cell infiltration. The induction of neuroapoptosis, evident by the reduced thickness of the molecular and intergranular layers and loss of neuronal and pyramidal cells in the cerebellum, establishes the loss of these structures in complex chemical insults. The pyramidal cells in the cerebellum are a type of multipolar neuron, and they are significant in the corticospinal tracts. These neurons are concerned with motor activities and are prominent in the premotor and motor functions of the cerebellum. Also, the granular layer of the cerebellum receives excitatory impulses from the mossy fiber, thus performing a complex spatiotemporal reconfiguration of incoming mossy fiber signals [56], while the molecular layer plays a major role in cerebellar information processing [57]. Also, the thickness of the molecular and intergranular layers of the cerebellum is the most widely used cortical morphological measurement for quantitative analysis of neurodegenerative diseases in clinical [58, 59] and preclinical settings [59, 60]. Hence, the present histopathological lesions observed following cisplatin exposure demonstrate that cisplatin induces neurodegeneration. This may be via the observed cisplatin-induced oxidative stress, inflammation, and apoptosis. In our results, the noted neurodegenerative changes caused by cisplatin were attenuated by daflon co-treatment, indicating that daflon preserves the structures in the cerebellar tissue by preventing atrophy of the cells via oxidative, inflammatory, and apoptotic damage.

The deranged biochemical variables and histological distortions may explain the observed motor dysfunction and anxiety-like symptoms. The observed neurobehavioral deficits, including reduced open-arm entry, center exploration, and locomotor activity, may be due to cisplatin-induced neurodegeneration. Cisplatin-induced cerebellar injury disrupts the neural circuits necessary for normal exploratory behavior and motor drive. However, attenuation of these deficits by Daflon suggests it provides a neuroprotective effect. It likely mitigates the underlying histological damage and inflammation, thereby preserving neuronal integrity and function, and restoring normal behavior in the exposed rats.

Overall, the neuroprotection conferred on the cerebellar tissue by daflon may be attributed to its constituent compounds, diosmin and hesperidin. These flavonoids exert antioxidant and anti-inflammatory activities by suppressing NF-kB activation, thus curtailing pro-inflammatory cytokine release [23, 61]. Therefore, it could be inferred from the present findings that daflon attenuates inflammation by downregulating TLR4/NF-kB/COX-2/PGE2 signaling. This is the first report documenting the protective effect of daflon in cisplatin-induced cerebellar neurodegeneration.

Conclusively, cisplatin induces cerebellar neurotoxicity through the induction of oxidative stress, inflammation, and apoptosis. Nonetheless, daflon exerts neuroprotective effects by mitigating cisplatin-induced cerebellar injury by ameliorating oxido-inflammation and caspase 3-mediated apoptosis by suppressing TLR4/NF-kB signaling. Despite the robustness of these present findings, there are still some gaps. Further experimental studies on the associated mechanisms of the ameliorative action of daflon on cisplatin-induced neurotoxicity, especially with protein assay (protein visual assays, such as Western blot), are recommended. There is a need for further evidence to establish the protective effects of daflon on cisplatin-induced neurotoxicity by probing its impact on protein denaturation, ferroptosis, and pyroptotic mechanisms. This would increase our understanding of the effectiveness of daflon on cisplatin-induced cerebellar toxicity and its possible use in the management of cisplatin-induced neurotoxicity.

Author contributions

Conceptualization and design: REA. Funding acquisition: FBF, TMA, AAO, PAO, and REA. Investigation: FBF, TMA, AAO, PAO, and REA. Methodology: TMA, OPA, and REA. Project administration: FBF, TMA, AAO, PAO, ASL, OPA, OA, OOA, OOO, and REA. Supervision: REA. Validation: OPA and REA. Writing-original draft: TMA, PAO, ASL, and REA. Writing-review and editing and final approval: FBF, TMA, AAO, PAO, ASL, OPA, OA, OOA, OOO, and REA.

Funding

This study was self-funded.

Data availability

The data used to support the findings of the present study are available from the corresponding author upon request.

Declarations

Ethical approval

The Ethics Review Committee of the Faculty of Basic Medical Sciences, Ladoke Akintola University of Technology, Nigeria, approved the study protocol (ERCFBMSLAU TECH: 061/08/2024). The National Institutes of Health’s (NIH) Guide for the Care and Use of Laboratory Animals was followed.

Competing interests

The authors declare no competing interests.