Targeting TLR4/NF-κB and Nrf2/HO-1 Crosstalk via Trimetazidine alleviates LPS-induced Depressive Like Behaviors in mice

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Mohamed, Nora O. Abdel Rasheed, Weam W. Ibrahim, Nesma A. Shiha This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4440337/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Sep, 2024 Read the published version in Journal of Neuroimmune Pharmacology → Version 1 posted 12 You are reading this latest preprint version Abstract Depression is a global psychiatric disorder that imposes a substantial economic burden. Trimetazidine (TMZ); a well-known anti-ischemic drug, could exert neuroprotection in cerebral ischemia. Aims: This study aimed to investigate the effect of TMZ in lipopolysaccharide (LPS) mouse model of depression with empathises on its ability to regulate toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) as well as nuclear factor erythroid 2 related factor 2 (Nrf2)/ heme oxygenase-1(HO-1) signaling pathways. Main methods: Male Swiss albino mice were injected with LPS (500 µg/kg, i.p) every other day alone or concurrently with oral doses of either TMZ (20 mg/kg/day) or escitalopram (Esc) (10 mg/kg/day) for 14 days. Key findings: Administration of TMZ attenuated LPS-induced animals' despair with decreased immobility time in forced swimming test. Additionally, TMZ diminished LPS–induced neuro-inflammation via inhibition of TLR4/NF-κB pathway contrary to Nrf2/HO-1 cascade activation with consequent increase in reduced glutathione (GSH) and HO-1 levels whereas the pro-inflammatory cytokines; tumor necrosis factor –α (TNF-α) and interleukin (IL)-1β were markedly reduced. Besides, TMZ replenished brain serotonin levels owing to serotonin transporter (SERT) inhibition. Thus, TMZ hindered LPS-induced neuro-inflammation, oxidative stress, serotonin deficiency in addition to its anti-apoptotic effect which was reflected by decreased caspase-3 level. The formerly mentioned neuroprotective effects of TMZ were verified by the histological photomicrographs which revealed prominent neuronal survival with minimal records of neuronal damage. Significance: Consequently, TMZ is proposed as an affluent nominee for depression management via targeting TLR4/NF-κB and Nrf2/HO-1 pathways. LPS Trimetazidine TLR4/NF-КB Nrf2/HO-1 Blood-brain barrier. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Depression is a common disabling mental disorder that is estimated to affect 5% of adults across the world [1] . It is characterized by a cluster of symptoms that includes low mood and energy, loss of interest and pleasure, sleeping and eating disturbances, cognitive impairment, and suicidal ideation [2,3] . Although, the pathophysiological causes of depression remain poorly understood, numerous studies have reported a significant association between neuro-inflammation and depression [2,4–6] . The core features of inflammatory responses such as increased pro-inflammatory cytokine levels and abnormal changes in oxidative stress parameters including malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione-S-transferase (GST) have been reported in blood and brain samples of patients with major depressive disorder (MDD) [7] . In accordance, several studies have demonstrated that administration of the bacterial endotoxin lipopolysaccharide (LPS) can cause depressive symptoms in rodents [8–10]. LPS triggers immune responses by acting on toll-like receptor 4 (TLR4), a vital pattern recognition receptor (PRR) [11,12]. Upon binding to TLR4, LPS can activate nuclear factor-κB (NF-κB) which induces the production of inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), eventually promoting oxidative stress and decreasing the expression of brain-derived neurotrophic factor (BDNF) [13,14]. Oxidative stress is a major contributor to the pathogenesis of various neurological diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and depression [15,16]. It results from the imbalance between the production of free radicals, particularly reactive oxygen species (ROS) and antioxidant defense systems [15].The increased ROS levels is known to damage biological macromolecules such as nucleic acids, proteins, and lipids resulting in cellular damage [17,18]. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a key transcription factor that regulates cellular antioxidant responses [17]. It activates antioxidant response element (ARE)-dependent gene expression of various antioxidant and cytoprotective proteins such as SOD-1, glutathione peroxidase (GPx), GST, and heme oxygenase-1 (HO-1) [19]. The latter is an enzyme that possesses anti-inflammatory and antioxidant activities [17]. Nrf2 inhibition is reportedly involved in the development of MDD. Indeed, decreased prefrontal cortex expression of Nrf2 has been documented in both MDD patients and experimental models of depression. On the other hand, compounds activating Nrf2 have shown considerable antidepressant effects in preclinical studies, suggesting that targeting Nrf2 could be a promising strategy to combat depression [20]. Blood–brain barrier (BBB) is essential for maintenance of the central nervous system (CNS) homeostasis [21]. It is formed by specialized endothelial cells that line the cerebral microvasculature and regulates the passage of molecules and cells between the blood and the brain [22,23]. Therefore, the BBB protects the brain from toxins, pathogens, inflammation, injury, and diseases [23]. The integrity of the BBB is maintained mainly by tight junction proteins such as claudin-5, whose loss has been implicated in loosening of the BBB and increased permeability [24,25]. BBB disruption has been involved in the pathogenesis of different CNS diseases such as depression, epilepsy and schizophrenia [26]. Moreover, peripheral inflammation including LPS-induced inflammation is reported to disrupt the BBB via various pathways [21]. Indeed, LPS reportedly reduced tight junction proteins such as claudin-5 and occludin in young and old mice thus affecting BBB integrity [24,27] . Trimetazidine (TMZ) is a cytoprotective anti-ischemic drug widely used in the treatment of coronary artery disease [28]. It is a selective inhibitor of long‐chain 3‐ketoacyl coenzyme A thiolase; an enzyme which catalyzes the final step in β-oxidation, thus shifting energy production from free fatty acids oxidation towards glucose oxidation leading to suppression of oxygen demands and enhancement of myocardial efficiency [29]. Cardioprotective effects of TMZ have also been attributed to its reported antioxidant and anti-inflammatory effects [30]. Besides its cardioprotective actions, the neuroprotective properties of TMZ have been getting more attention over the past years. TMZ has exhibited neuroprotective effects against focal cerebral ischaemia–reperfusion injury in rats [31] and anxiolytic effects in animal models of increased anxiety, owing to its antioxidant activity [32]. It has also been reported that treatment with TMZ ameliorated cognitive impairment in diabetic epileptic rats by hampering the inflammatory process [33]. Importantly, TMZ was recently identified as a potential drug that may be repurposed to treat bipolar depression using a combination of transcriptomics, drug screening and in vitro as well as in vivo mechanistic studies conducted by [34]. Accordingly, the current study sought to investigate the potential benefit of TMZ in attenuating LPS-induced depressive-like behaviors, neuro-inflammation, apoptosis, oxidative stress and BBB dysfunction in mice. Special emphasis was set on the possible involvement of the TLR4/NF-κB and Nrf2/HO-1 pathways in the protective effects of TMZ. 2. Materials and Methods 2.1. Animals Adult male Swiss albino mice weighing 22–25 g were used in the current study. They were obtained from the animal facility of the National Research Centre, Giza, Egypt and allowed to acclimate for 1 week before starting the experiment. Animals were maintained under temperature- and humidity-controlled conditions on a 12/12 light-dark cycle and provided access to food and water ad libitum. The experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 2011) and were approved by the Ethics Committee for Animal Experimentation of Faculty of Pharmacy, Cairo University (PT3522). Efforts were made to reduce animal suffering and decrease the number of animals used. 2.2. Materials Lipopolysaccharide (LPS) (Escherichia coli, serotype 0127: B8) was purchased from Sigma-Aldrich, USA. Trimetazidine dihydrochloride (TMZ) and escitalopram (Esc) were acquired from Servier and Multi-Apex Pharmaceutical Company, Egypt, respectively. They were all freshly prepared using physiological saline before use. 2.3. Experimental Design Mice were randomly divided into 5 groups (n = 15/group) as follows; control, TMZ, LPS, LPS+ Esc, and LPS+ TMZ groups ( Fig. 1) . Induction of depression was achieved by intraperitoneal injection of LPS at a dose of 500 µg/kg, every other day for 14 days, for a total of 7 injections [35,36] . Mice were treated orally with either Esc (10 mg/kg/day) [37,38] or TMZ (20 mg/kg/day) [32,39,40] for 14 days covering the LPS treatment period. Following treatments, mice were behaviorally tested using open field test (OFT) and forced swimming test (FST). After performing the behavioral tests, animals were euthanized by decapitation under anaesthesia then the whole brains were carefully collected and washed with ice-cold saline. Brains of three mice from each group were kept in 10% formalin for histopathological examination of the hippocampal region and for determination of mean intact neurons count. The hippocampi of the remaining mice were dissected out, flash frozen in liquid nitrogen and stored at −80°C for further biochemical investigations. Six of these hippocampi were then homogenized in cold phosphate-buffered saline to prepare a 10% homogenate to be used for enzyme-linked immunosorbent assay (ELISA) tests while the remaining six were used for real-time PCR analysis. 2.4. Behavioral Assessment 2.4.1. Open Field Test Locomotor function of the mice was evaluated using the OFT. The test was performed in a wooden box (80 × 80 × 40 cm) with red walls and a black floor. The floor of the box was divided into 16 equal squares by white lines. Each mouse was placed in the middle of the open field and left to explore for 3 minutes. The arena was cleaned carefully using 20 % alcohol between each mouse to get rid of any remnant odors. The animal's locomotor function was assessed by recording the frequencies of ambulation and rearing [26,41] . 2.4.2. Forced Swimming Test Forced swimming test was performed to assess the depressive-like behaviors in rodents [8] . Mice were individually placed in a transparent cylinder (height: 40 cm, diameter: 30 cm) containing about 35 cm of water (25 ± 1°C) for 5 min. The total amount of time each animal remained immobile was recorded as the immobility time. The animal was considered immobile when floating motionless or making only small movements needed to keep its head above the water level [42,43] . 2.5. Histopathological Examination Brain samples were fixed in neutral buffered formalin (10%) for 72 hours. Samples were then processed in serial grades of ethanol and cleared in xylene. Afterwards, they were infiltrated and embedded into Paraplast tissue embedding media. A rotatory microtome was used to cut 5 μm-thick serial sagittal brain sections for demonstration of hippocampal regions in different samples and mounted on glass slides. Sections were then stained with hematoxylin and eosin (H&E) as a general staining method for tissue examination. Standard procedures for samples fixation and staining were carried out according to [44]. Additionally, sections were stained with toluidine blue and six randomly selected non-overlapping fields from the CA3 hippocampal region of each sample were assessed to determine mean intact neurons count [45] . All light microscopic examination and data were obtained using a full HD microscopic imaging system and Leica application module for tissue section analysis (Leica Microsystems GmbH, Germany). 2.6. Biochemical Assays 2.6.1. Enzyme-Linked Immunosorbent Assay Hippocampal NF-κB and matrix metalloproteinase-9 (MMP-9) contents were measured using mouse ELISA kits supplied by LifeSpan Biosciences (USA) and Elabscience (China), respectively. Moreover, mouse MyBioSource ELISA kits (USA) were used for the assessment of TNF-α, Nrf2, HO-1, GSH and claudin-5 contents in the hippocampus, while IL-1β and Caspase 3 contents were estimated using R&D systems mouse ELISA kits (USA). The procedures were performed following the protocols provided by the manufacturers. Protein content was determined using the method described by [46] . 2.6.2. Quantitative Real-Time PCR Analysis Total RNA was extracted from the mice hippocampi using SV total RNA isolation system (Promega, USA) and purity of the obtained RNA was detected spectrophotometrically at 260/280 nm. Using Reverse Transcription System (Promega, USA), the extracted RNA was reverse transcribed into complementary DNA. To evaluate gene expression of TLR4 and serotonin transporter (SERT), quantitative real-time PCR was performed using the SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich, USA) as per the manufacturer's instructions. The used primers' sequences are illustrated in Table 1. The relative expression of the target genes was estimated using the 2 −∆∆CT formula [47] with β-actin used as a housekeeping gene. Table 1: Sequences of the primers used for quantitative real time-PCR analysis. Gene Primer sequence TLR4 Forward: 5′-ATGCATGGATCAGAAACTCAGCAA-3′ Reverse: 5′- AAACTTCCTGGGGAAAAACTCTGG-3′ SERT Forward: 5′-AAGCCCCACCTTGACTCCTCC-3′ Reverse: 5′-CTCCTTCCTCTCCTCACATATCC-3′ β-actin Forward: 5′-CACTGTCGAGTCGCGTCC-3′ Reverse: 5′-CGCAGCGATATCGTCATCCA-3′ 2.7. Statistical Analysis Data sets that met the requirements for parametric criteria were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. Results were presented as mean ± SD. Statistical analysis was performed using GraphPad Prism software, version 9 (GraphPad Software Inc., USA) with the significance level fixed at P < 0.05. For each effect, the F-value (F), the degree of freedom, and the statistical significance (p) were reported, moreover, pairwise comparisons were provided in each corresponding figure. 3. Results The results displayed by TMZ group were presented herein concurrently with the other experimental groups. They were insignificantly different as compared to those of the control group upon histological, behavioral, and biochemical investigations. Thus, comparisons were done as related to the control group. 3.1. Trimetazidine alleviated LPS-induced depressive-like behavior in forced swimming test whereas it exerted null effect on locomotor activity in open field test LPS injection in mice instigated depressive-like symptoms which were investigated behaviorally using the FST. In comparison with the control group, LPS-injected mice showed a state of despair in FST where they displayed a marked rise in the time of immobility by 2.1-folds (F (4, 70) = 16.33, p< 0.0001). Treatment with either Esc or TMZ ameliorated LPS-induced depressive-like behavior restoring the values of immobility time to the normal range. As compared to LPS group, Esc- or TMZ-treated mice exhibited reduced immobility duration by 51 and 45%, respectively (Fig. 2A) . In the open field test, the ambulation and rearing frequencies were documented to evaluate the impact of LPS, TMZ, or Esc on spontaneous locomotor activity of rats. There was insignificant variation among the experimental groups in either the frequency of ambulation (F (4, 70) = 2.658, p = 0.0398) or rearing (F (4, 70) = 1.528, p = 0.2035). These findings rule out the possibility that the depressive-like anomalies detected herein can be attributed to changes in locomotor function (Figs. 2B and C) . 3.2. Trimetazidine alleviated LPS-induced hippocampal histopathological changes in mice Representative photomicrographs of the H&E-stained samples of different groups were microscopically examined, in addition to using toluidine-blue stain for determining the intact neurons count in CA3 hippocampal area. The control group showed normal histological appearance of the hippocampal layers along with many records of apparent intact pyramidal neurons having well recognized nuclear and cytoplasmic details (black arrows), in addition to an intact brain matrix with the absence of cellular infiltrates. Similarly, the hippocampal sections of TMZ group presented comparable histopathological appearance with that of the control group without recording any abnormal changes. In contrast, the histological examination of LPS group samples showed obvious dispersed neuronal degeneration as indicated by ample of damaged or necrotic neurons having pyknotic perikarya missing its subcellular details, along with mild perineuronal edema (red arrows) in contrast to reduced scattered records of intact cells (black arrows). Additionally, LPS group samples demonstrated a remarkable increase in reactive microglial cell infiltrates (arrowhead). Upon examining the toluidine blue-stained sections, LPS group presented a noticeable decline in the intact neurons mean count by 96% as compared to the control group (F (4, 25) = 382.8, p< 0.0001). Notably, treatment with either Esc or TMZ afforded equivalent neuroprotective efficacy against LPS-induced histopathological changes. The brain samples of Esc-or TMZ-treated mice exhibited few sporadic records of damaged neurons (red arrow) and higher records of apparent intact neurons (black arrow) with intact intercellular brain matrix and minimal infiltrates (arrowhead), as compared to the LPS group (Figs. 3 and 4) . 3.3. Trimetazidine mitigated LPS-induced activation of hippocampal TLR4/NF-κB pathway The LPS injection led to a profound activation of TLR4/NF-κB pathway as evidenced by producing considerable increase in the hippocampal gene expression of TLR4 by 8-folds, with subsequent increment in the hippocampal content of NF-κBp65 by 1.8-folds, as compared to the control group (for TLR4: F (4, 25) = 112.8, p< 0.0001; for NF-κBp65: F (4, 25) = 98.69, p< 0.0001). Upon treating the LPS-exposed mice with TMZ, a marked drop in TLR4 gene level as well as NF-κBp65 content by 66 and 34%, respectively, was detected producing an equivalent effect with that displayed by the LPS+Esc group (Fig. 5). 3.4. Trimetazidine reduced LPS-induced neuro-inflammation, apoptosis and oxidative stress The LPS depression model was accompanied by aggravated responses of neuro-inflammation, apoptosis, and oxidative stress. In comparison to the control group, LPS-injected mice showed profound rise in the hippocampal contents of inflammatory markers, TNF-α (4.2-folds) and IL-1β (3.9-folds), along with significant increment in the proapoptotic caspase-3 concentration by 6.1-folds (F (4, 25) = 137.0, 318.4, and 119.9, respectively, p< 0.0001). On the contrary, they showed a considerable decrease in the antioxidant variables; namely Nrf2, HO-1, and GSH levels by 68, 70, and 50%, respectively, in comparison to their control counterparts (F (4, 25) = 256.3, 94.84, and 204.8, respectively, p< 0.0001). Treatment of LPS-injected mice with TMZ exerted neuroprotective effects leading to about 54% depression in TNF-α and IL-1β concentration along with marked decline in caspase-3 content (66%). Additionally, TMZ mitigated LPS-induced oxidative stress producing significant rise in Nrf2 (2.5-folds), HO-1 (2.6-folds), and GSH levels (1.7-folds). Such effects of TMZ on the previously mentioned markers were almost comparable with those displayed by Esc treatment except for Nrf2 where Esc outweighed TMZ effect producing 10% increase in Nrf2 content as compared to TMZ-treated group (Fig. 6) . 3.5. Trimetazidine diminished LPS-induced surge in hippocampal SERT expression The administration of LPS in mice notably augmented the hippocampal mRNA expression of SERT by 4.3-folds, in comparison to the control group (F (4, 25) = 124, p< 0.0001). This effect was inhibited by TMZ administration where LPS+TMZ group mice showed a noticeable reduction in SERT gene level by 53%, as compared to the LPS group. Likewise, Esc administration obviously decreased the SERT gene level by 60%, as compared to LPS group. Results afforded by the LPS+Esc and LPS+TMZ groups on SERT expression in the hippocampi were almost comparable where no substantial change was revealed between the two groups (Fig. 7) . 3.6. Trimetazidine attenuated LPS-induced blood-brain barrier dysregulation The LPS administration has a detrimental effect on BBB integrity and function. LPS-injected mice displayed 2.4-folds elevation in the hippocampal content of MMP-9, an important protein involved in maintaining BBB integrity and function (F (4, 25) = 122.4, p< 0.0001). This was associated with dysregulation in BBB tight junctional proteins as exemplified by showing a considerable depression in the hippocampal content of claudin-5 (67%) leading to BBB dysfunction (F (4, 25) = 146.9, p< 0.0001). Such effects were hindered by TMZ treatment which led to MMP-9 downregulation by 47%, while it enhanced the claudin-5 concentration in the hippocampi by 2.3-folds. These effects were almost analogous to that obtained by LPS-injected mice treated with Esc which reduced MMP-9 by 38%, while elevated claudin-5 level by 2.5-folds, as compared to LPS group (Fig. 8) . 4. Discussion Major depressive disorder (MDD) is a complex mental illness which imposes prominent disability and economic burden worldwide. It's associated with several clinical symptoms including feelings of worthlessness, fatigue as well as diminished ability to think or concentrate [48]. Despite the availability of various antidepressants, they are associated with multiple side effects and exhibit a delayed onset of action which hinders patients' compliance [3]. Accordingly, there is a consistent urge to investigate novel antidepressants which target various depressive pathological changes with fewer adverse effects hence enhancing patients' adherence [48]. The current study investigated the antidepressant activities of TMZ; an anti-ischemic drug used for coronary artery disease, in LPS-induced depressive behaviors in mice. Administration of LPS every other day for 2 weeks was linked to increased immobility time in FST reflecting animals' despair associated with LPS injection. Meanwhile, LPS injection didn't alter the animals' spontaneous locomotor activity and rearing frequency recorded during OFT [49]. Similarly, in the present study, animals injected with LPS exhibited significant elevation in the immobility time recorded in FST, yet they didn't reveal neither ambulation nor rearing frequencies derangements in OFT which empathizes that LPS-associated despair and lack of mobility in FST is contributed to its ability to induce depressive like behavior. On the other hand, TMZ administration alleviated LPS–induced animals despair witnessed in FST reflected by significant decrement in the immobility time of TMZ-treated mice, as compared to LPS-injected animals which didn't receive any treatment. TMZ-associated mood enhancement was comparable to the used reference antidepressant; Esc. Neuro-inflammation was reported as a promising target for MDD management [50]. LPS; a bacterial endotoxin, is implicated in depressive like behaviors via inducing multiple pro-inflammatory cytokines production [51]. It was reported to induce NF-κB nuclear translocation and activation with consequent elevation of TNF-α and IL-1β levels leading to augmented neuro-inflammation [52]. Moreover, TLR4; a critical receptor involved in neuro-inflammation, was correlated to depression pathogenesis [53]. Stimulation of TLR4 promotes the recruitment of inflammatory cells which is implicated in NF-κB activation with consequent release of the pro-inflammatory mediators namely; TNF-α, and IL-1β [54,55]. Accordingly, in the present study, LPS injection induced an obvious pro-inflammatory milieu owing to TLR4 upregulation which contributes to NF-κBp65 activation with consequent pro-inflammatory cytokines release as TNF-α, and IL-1β. Administration of TMZ attenuated LPS–induced neuro-inflammation with significant decrease in the formerly mentioned cytokines levels owing to TLR4 downregulation. TMZ-induced anti-neuro-inflammatory effects were equivalent to the reputable antidepressant drug; Esc. Activation of TLR4/NF-κB signaling is implicated in the marked disruption of BBB permeability associated with LPS injection [56]. The BBB constitutes a protective layer in the CNS which restricts molecular exchange between the circulating blood and the brain for maintaining an effective neural signaling. Thus, these proteins serve as a defensive layer that hinders the path of hazardous substances to the brain [57]. Claudin-5 is a chief tight junction protein of the BBB whose dysfunction has been implicated in neurodegenerative disorders such as Alzheimer’s disease and psychiatric diseases as depression [58,59]. MMP-9 has been correlated to BBB breakdown and inflammation in several CNS disorders [60]. Moreover, it was reported that LPS is implicated in MMP-9 induction via NF-κB leading to claudin-5 degeneration and BBB dysfunction [21,61]. Similarly, in the current investigation, LPS injection resulted in BBB disruption with elevated MMP-9 content contrary to claudin-5 decreased content owing to LPS induced activation of TLR4/NF-κB pathway. On the other hand, TMZ administration diminished LPS-induced neuro-inflammation and maintained BBB integrity as revealed by producing significant decrease in MMP-9 content contrary to claudin-5 enhanced content owing to TMZ-mediated inhibition of TLR4/NF-κB pathway which was comparable to Esc effects. LPS; probably via its well-known pro-inflammatory effects, contributes to upregulation of SERT which is responsible for serotonin reuptake leading to deficiency of this neurotransmitter in the brain with consequent depressive behaviors [62]. Moreover, TNF-α and IL-1β were reported to promote rapid activation of SERT [63]. Likewise, in the present study, LPS-injected animals revealed increased SERT expression which was noticeably reduced upon TMZ administration. Neuro-inflammation and oxidative stress relationship has been verified in depression pathogenesis [64]. An established cross link has been proposed between the pro-inflammatory transcriptional factor; NF-κB and the protective anti-oxidant; Nrf2 where LPS-induced neuro-inflammation is attributed principally to its inhibitory action on Nrf2 pathway contrary to NF-κB upregulation which is a key player in BBB dysfunction, neuro-inflammation, and apoptosis [65]. Nrf2 also regulates the expression of various antioxidant and anti-inflammatory mediators such as HO-1 and the antioxidant enzymes involved in GSH synthesis [66]. Besides, Nrf2 has the potential to inhibit LPS-induced up-regulation of pro-inflammatory cytokines as well as MMP-9 which induces BBB disruption and cell death [67]. Accordingly, in the current study, LPS-injected animals revealed extravagant oxidative stress and massive neuro-inflammatory milieu reflected by significant decrease in Nrf2, HO-1,and GSH contents owing to the culprit inflammatory cascade; TLR4/NF-κB and its downstream targets; namely MMP-9 and caspase-3 which were markedly elevated in LPS-injected mice. TMZ administration diminished LPS-induced triad of oxidative stress, neuro-inflammation, and apoptosis with augmented contents of Nrf2, HO-1, and GSH due to prominent inhibition of TLR4/NF-κB pathway along with MMP-9 and caspase-3 dismounted levels. TMZ neuroprotective effects were equivalent to Esc regarding the formerly mentioned parameters except for Nrf2 content whose elevation in LPS+Esc group out weighted that recorded in LPS+TMZ group. Finally, in the present investigation, photomicrographs of LPS-injected mice revealed diffuse neuronal damage with plentiful degenerated or necrotic neurons. Additionally, significant increase of reactive microglial cells infiltrates was recorded in animals receiving LPS. TMZ administration induced scarce sporadic neuronal damage with more apparent intact neurons besides minimal infiltration. Photomicrographs of LPS +TMZ group mimicked those witnessed in LPS +Esc group. Normal mice which received TMZ did not show morphological alterations as compared to normalcontrol animals. Consequently, the present study explored TMZ neuro-protective activity in LPS-induced depressive like behavior in mice. TMZ attenuated LPS-associated oxidative stress, neuro-inflammation, and apoptosis in addition to restoration of serotonin levels via down regulation of SERT. TMZ-induced neuroprotection can be attributed to its modulatory effects on TLR4/NF-κB and Nrf2/HO-1 pathways which were reflected by marked decrease in animals' immobility time recorded during FST. Conclusion TMZ; a well-known anti-ischemic drug, exerted mood boosting effects in LPS-injected mice. Moreover, it was able to overcome LPS-induced viscous cycle of oxidative stress, neuro-inflammation, and apoptosis probably via its ability to fine tune NF-κBp65 and Nrf2 correlation and replenish depression-associated neurotransmitter deficiency. Consequently, TMZ is a prosperous candidate that needs further investigations to authenticate its antidepressant activity. Declarations Funding No funding was received to assist with the preparation of this manuscript. Declaration of interest The authors have no conflicts of interest to declare that are relevant to the content of this article. Author contributions statement Sarah S. Mohamed, Nora O. Abdel Rasheed, Weam W. Ibrahim and Nesma A. Shiha: conceptualization. Sarah S. Mohamed: methodology. Nora O. Abdel Rasheed, Nesma A. Shiha, and Weam W. Ibrahim: data curation and writing-original draft preparation. Nora O. Abdel Rasheed, Nesma A. Shiha, Sarah S. Mohamed, and Weam W. Ibrahim: reviewing, and editing. All authors have read and agreed to the published version of the manuscript. Data Availability All data are available upon request. Limitations Extrapolation of the present study findings to humans is necessary to validate TMZ antidepressant effects in humans with emphasis on various depression aspects besides the neuroinflammatory milieu. Ethics approval The experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 2011) and were approved by the Ethics Committee for Animal Experimentation of Faculty of Pharmacy, Cairo University (PT3522). Efforts were made to reduce animal suffering and decrease the number of animals used. 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Cite Share Download PDF Status: Published Journal Publication published 23 Sep, 2024 Read the published version in Journal of Neuroimmune Pharmacology → Version 1 posted Editorial decision: Revision requested 27 Jun, 2024 Reviewers agreed at journal 24 Jun, 2024 Reviews received at journal 23 Jun, 2024 Reviews received at journal 21 Jun, 2024 Reviewers agreed at journal 20 Jun, 2024 Reviewers agreed at journal 20 Jun, 2024 Reviewers agreed at journal 20 Jun, 2024 Reviewers agreed at journal 19 Jun, 2024 Reviewers invited by journal 19 Jun, 2024 Editor assigned by journal 08 Jun, 2024 Submission checks completed at journal 28 May, 2024 First submitted to journal 18 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4440337","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":312124095,"identity":"00d92ba1-6ddf-47ec-b928-751f38ec7ce9","order_by":0,"name":"Sarah S. 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Shiha","email":"","orcid":"","institution":"Cairo University","correspondingAuthor":false,"prefix":"","firstName":"Nesma","middleName":"A.","lastName":"Shiha","suffix":""}],"badges":[],"createdAt":"2024-05-18 09:11:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4440337/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4440337/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11481-024-10149-3","type":"published","date":"2024-09-23T15:57:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58052344,"identity":"b743ac42-5d25-4206-85f8-85d74235d215","added_by":"auto","created_at":"2024-06-10 13:16:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":69816,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentation of the experimental design. \u003cem\u003eLPS\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003elipopolysaccharide, \u003cem\u003eTMZ\u003c/em\u003e Trimetazidine, \u003cem\u003eEsc \u003c/em\u003eescitalopram, \u003cem\u003eOFT\u003c/em\u003e open field test, \u003cem\u003eFST\u003c/em\u003eforced swimming test.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4440337/v1/73f8edc14de521674f55f49c.png"},{"id":58052345,"identity":"eb9d308a-f4db-46a4-aa09-7c02a68e4461","added_by":"auto","created_at":"2024-06-10 13:16:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":160274,"visible":true,"origin":"","legend":"\u003cp\u003eTrimetazidine alleviated LPS-induced behavioral changes\u003cstrong\u003e \u003c/strong\u003ein forced swimming and open field tests.\u003cstrong\u003e (A)\u003c/strong\u003eImmobility time, \u003cstrong\u003e(B)\u003c/strong\u003e Ambulation frequency, and \u003cstrong\u003e(C)\u003c/strong\u003e Rearing frequency. Data were expressed as mean ± SD (n = 15) using one-way ANOVA followed by Tukey's post-hoc test, P \u0026lt; 0.05.\u003cem\u003e LPS\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003elipopolysaccharide, \u003cem\u003eTMZ\u003c/em\u003e Trimetazidine, \u003cem\u003eEsc \u003c/em\u003eescitalopram.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4440337/v1/de22e5589ec40db820aa57d8.png"},{"id":58053068,"identity":"3b0a0508-50d6-4d16-82b1-95e6be1d3909","added_by":"auto","created_at":"2024-06-10 13:24:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12024869,"visible":true,"origin":"","legend":"\u003cp\u003eTrimetazidine alleviated LPS-induced hippocampal histopathological changes in mice. Representative H\u0026amp;E photomicrographs of all experimental groups (n = 3); Control, TMZ, LPS, LPS+Esc, and LPS+TMZ groups. Magnifications: ×400. \u003cem\u003eBlack arrows\u003c/em\u003e indicate intact neurons while \u003cem\u003ered arrows\u003c/em\u003e represent degenerated ones. \u003cem\u003eArrowheads\u003c/em\u003eshow reactive glial cells infiltrate.\u003cem\u003e LPS\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003elipopolysaccharide, \u003cem\u003eTMZ\u003c/em\u003e Trimetazidine, \u003cem\u003eEsc \u003c/em\u003eescitalopram.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4440337/v1/05fdec34f21c10356378c630.png"},{"id":58052347,"identity":"454677f7-7926-47cd-9307-7399bde3e61c","added_by":"auto","created_at":"2024-06-10 13:16:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":11717331,"visible":true,"origin":"","legend":"\u003cp\u003eTrimetazidine enhanced hippocampal neuronal survival in LPS-injected mice. Illustrative toluidine blue-stained photomicrographs of all experimental groups; Control, TMZ, LPS, LPS+Esc, and LPS+TMZ groups. Magnifications: ×400. \u003cem\u003eBlack arrows\u003c/em\u003e represent intact neurons while \u003cem\u003ered arrows\u003c/em\u003e show degenerated ones. A bar chart showing the mean count of intact neurons in each group where each bar with vertical line illustrating the mean ± SD (n = 6), using one-way ANOVA followed by Tukey's post-hoc test, P \u0026lt; 0.05.\u003cem\u003e LPS\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003elipopolysaccharide, \u003cem\u003eTMZ\u003c/em\u003e Trimetazidine, \u003cem\u003eEsc \u003c/em\u003eescitalopram.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4440337/v1/2ff586508f63db1110e003b1.png"},{"id":58052352,"identity":"edec7960-0f83-46e0-8de8-cdb39663aeb0","added_by":"auto","created_at":"2024-06-10 13:16:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":151255,"visible":true,"origin":"","legend":"\u003cp\u003eTrimetazidine mitigated LPS-induced alterations in the hippocampal \u003cstrong\u003e(A)\u003c/strong\u003e TLR4 gene expression and \u003cstrong\u003e(B)\u003c/strong\u003e NF-κBp65 content. Data were expressed as mean ± SD (n = 6), using one-way ANOVA followed by Tukey's post-hoc test, P \u0026lt; 0.05.\u003cem\u003e LPS\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003elipopolysaccharide, \u003cem\u003eTMZ\u003c/em\u003e Trimetazidine, \u003cem\u003eEsc \u003c/em\u003eescitalopram.\u003cem\u003eTLR4 \u003c/em\u003eToll-like receptor-4,\u003cem\u003e NF-κB \u003c/em\u003enuclear factor-kappa B.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4440337/v1/40f90980e7e2255a0a2ad35b.png"},{"id":58053067,"identity":"2a2ed6f5-77ea-49b8-bae4-1eea78cb155a","added_by":"auto","created_at":"2024-06-10 13:24:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":228098,"visible":true,"origin":"","legend":"\u003cp\u003eTrimetazidine reduced LPS-induced alterations in the hippocampal contents of \u003cstrong\u003e(A)\u003c/strong\u003e TNF-α, \u003cstrong\u003e(B)\u003c/strong\u003e IL-1β, \u003cstrong\u003e(C)\u003c/strong\u003e Caspase-3, \u003cstrong\u003e(D)\u003c/strong\u003e Nrf2, \u003cstrong\u003e(E)\u003c/strong\u003e HO-1, and \u003cstrong\u003e(F)\u003c/strong\u003e GSH.\u003csup\u003e \u003c/sup\u003eData were expressed as mean ± SD (n = 6), using one-way ANOVA followed by Tukey's post-hoc test, P \u0026lt; 0.05.\u003cem\u003e LPS\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003elipopolysaccharide, \u003cem\u003eTMZ\u003c/em\u003e Trimetazidine, \u003cem\u003eEsc \u003c/em\u003eescitalopram, \u003cem\u003eTNF-α\u003c/em\u003e tumor necrosis factor-α,\u003cem\u003e IL-1β\u003c/em\u003e interleukin-1β, \u003cem\u003eNrf2\u003c/em\u003e nuclear factor erythroid 2–related factor 2, \u003cem\u003eHO-1\u003c/em\u003e heme oxygenase-1, \u003cem\u003eGSH\u003c/em\u003e reduced glutathione.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4440337/v1/22a6c6232c1983a3f19ac7e6.png"},{"id":58052350,"identity":"b316edba-51b8-4a53-872d-8e6fbf8fd8ae","added_by":"auto","created_at":"2024-06-10 13:16:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":74819,"visible":true,"origin":"","legend":"\u003cp\u003eTrimetazidine diminished LPS-induced changes in the hippocampal SERT gene expression. Data were expressed as mean ± SD (n = 6) using one-way ANOVA followed by Tukey's post-hoc test, P \u0026lt; 0.05.\u003cem\u003e LPS\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003elipopolysaccharide, \u003cem\u003eTMZ\u003c/em\u003e Trimetazidine, \u003cem\u003eEsc \u003c/em\u003eescitalopram, SERT serotonin transporter.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-4440337/v1/9b44463f17786bc40e557319.png"},{"id":58052346,"identity":"17d944e2-7351-4119-a94a-484452016062","added_by":"auto","created_at":"2024-06-10 13:16:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":129854,"visible":true,"origin":"","legend":"\u003cp\u003eTrimetazidine attenuated LPS-induced changes in the hippocampal contents of (A) MMP-9 and (B) Claudin-5. Data were expressed as mean ± SD (n = 6) using one-way ANOVA followed by Tukey's post-hoc test, P \u0026lt; 0.05. LPS lipopolysaccharide, TMZ Trimetazidine, Esc escitalopram, MMP-9 matrix metallopeptidase 9.\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-4440337/v1/5fa0c823d08bd78831d97607.png"},{"id":65628449,"identity":"0c833b4b-07b3-450a-96ed-93cb446ab6eb","added_by":"auto","created_at":"2024-09-30 16:19:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":37694147,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4440337/v1/59253aee-f3a9-46f1-b2e4-9122497a0c6e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting TLR4/NF-κB and Nrf2/HO-1 Crosstalk via Trimetazidine alleviates LPS-induced Depressive Like Behaviors in mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDepression is a common disabling mental disorder that is estimated to affect 5% of adults across the world\u0026nbsp;\u003cspan lang=\"EN-GB\"\u003e[1]\u003c/span\u003e.\u0026nbsp;It is characterized by a cluster of symptoms that includes\u0026nbsp;low mood and energy, loss of interest and pleasure, sleeping and eating disturbances, cognitive impairment, and suicidal ideation\u0026nbsp;\u003cspan lang=\"EN-GB\"\u003e[2,3]\u003c/span\u003e. Although, the pathophysiological causes of depression remain poorly understood, numerous studies have reported a significant association between neuro-inflammation and depression\u0026nbsp;\u003cspan lang=\"EN-GB\"\u003e[2,4\u0026ndash;6]\u003c/span\u003e. The core features of inflammatory responses such as increased pro-inflammatory cytokine levels and abnormal changes in oxidative stress parameters including malondialdehyde (MDA), superoxide dismutase (SOD), and\u0026nbsp;glutathione-S-transferase (GST)\u0026nbsp;have been reported in blood and brain samples of patients with major depressive disorder (MDD)\u003cspan lang=\"EN-GB\"\u003e[7]\u003c/span\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn accordance, several studies have demonstrated that administration of the bacterial endotoxin lipopolysaccharide (LPS) can cause depressive symptoms in rodents\u0026nbsp;[8\u0026ndash;10].\u0026nbsp;LPS triggers immune responses by acting on toll-like receptor 4 (TLR4), a vital pattern recognition receptor (PRR)\u0026nbsp;[11,12]. Upon binding to TLR4, LPS can activate nuclear factor-\u0026kappa;B (NF-\u0026kappa;B) which induces the production of inflammatory cytokines such as\u0026nbsp;interleukin (IL)-1\u0026beta;, IL-6, and tumor necrosis factor-\u0026alpha; (TNF-\u0026alpha;), eventually promoting oxidative stress and decreasing the expression of brain-derived neurotrophic factor (BDNF)\u0026nbsp;[13,14].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOxidative stress is a major contributor to the pathogenesis of various neurological diseases such as Alzheimer\u0026rsquo;s disease, Parkinson\u0026rsquo;s disease, multiple sclerosis, and depression\u0026nbsp;[15,16]. It results from the imbalance between the production of free radicals, particularly reactive oxygen species (ROS) and antioxidant defense systems\u0026nbsp;[15].The increased ROS levels is known to damage biological macromolecules such as nucleic acids, proteins, and lipids resulting in cellular damage\u0026nbsp;[17,18]. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a key transcription factor that regulates cellular antioxidant responses\u0026nbsp;[17]. It activates antioxidant response element (ARE)-dependent gene expression of various antioxidant and cytoprotective proteins such as SOD-1, glutathione peroxidase (GPx), GST, and heme oxygenase-1 (HO-1)\u0026nbsp;[19]. The latter is an enzyme that possesses anti-inflammatory and antioxidant activities\u0026nbsp;[17]. Nrf2 inhibition is reportedly involved in the development of MDD. Indeed, decreased prefrontal cortex expression of Nrf2 has been documented in both MDD patients and experimental models of depression. On the other hand, compounds activating Nrf2 have shown considerable antidepressant effects in preclinical studies, suggesting that targeting Nrf2 could be a promising strategy to combat depression\u0026nbsp;[20].\u003c/p\u003e\n\u003cp\u003eBlood\u0026ndash;brain barrier (BBB) is essential for maintenance of the central nervous system (CNS) homeostasis\u0026nbsp;[21]. It is formed by specialized endothelial cells that line the cerebral microvasculature and regulates the passage of molecules and cells between the blood and the brain\u0026nbsp;[22,23]. Therefore, the BBB protects the brain from toxins, pathogens, inflammation, injury, and diseases\u0026nbsp;[23]. The integrity of the BBB is maintained mainly by tight junction proteins such as claudin-5, whose loss has been implicated in loosening of the BBB and increased permeability\u0026nbsp;[24,25]. BBB disruption has been involved in the pathogenesis of different CNS diseases such as depression, epilepsy and schizophrenia\u0026nbsp;[26]. Moreover, peripheral inflammation including LPS-induced inflammation is reported to disrupt the BBB via various pathways\u0026nbsp;[21]. Indeed, LPS reportedly reduced tight junction proteins such as claudin-5 and occludin in young and old mice thus affecting BBB integrity\u0026nbsp;[24,27]\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTrimetazidine (TMZ) is a cytoprotective anti-ischemic drug widely used in the treatment of coronary artery disease\u0026nbsp;[28]. It is a selective inhibitor of\u0026nbsp;long‐chain 3‐ketoacyl coenzyme A\u0026nbsp;thiolase; an enzyme which catalyzes the final step in \u0026beta;-oxidation, thus shifting energy production from free fatty acids oxidation towards glucose oxidation leading to suppression of oxygen demands and enhancement of myocardial efficiency\u0026nbsp;[29]. Cardioprotective effects of TMZ have also been attributed to its reported antioxidant and anti-inflammatory effects\u0026nbsp;[30]. Besides its cardioprotective actions, the neuroprotective properties of TMZ have been getting more attention over the past years. TMZ has exhibited neuroprotective effects against focal cerebral ischaemia\u0026ndash;reperfusion injury in rats\u0026nbsp;[31]\u0026nbsp;and anxiolytic effects in animal models of increased anxiety, owing to its antioxidant activity\u0026nbsp;[32]. It has also been reported that treatment with TMZ ameliorated cognitive impairment in diabetic epileptic rats by hampering the inflammatory process\u0026nbsp;[33]. Importantly, TMZ was recently identified as a potential drug that may be repurposed to treat bipolar depression using a combination of transcriptomics, drug screening and in vitro as well as in vivo mechanistic studies conducted by\u0026nbsp;[34].\u003c/p\u003e\n\u003cp\u003eAccordingly, the current study sought to investigate the potential benefit of TMZ in attenuating LPS-induced depressive-like behaviors, neuro-inflammation, apoptosis, oxidative stress and BBB dysfunction in mice. Special emphasis was set on the possible involvement of the TLR4/NF-\u0026kappa;B and Nrf2/HO-1 pathways in the protective effects of TMZ.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdult male Swiss albino mice weighing 22\u0026ndash;25 g were used in the current study. They were obtained from the animal facility of the National Research Centre, Giza, Egypt and allowed to acclimate for 1 week before starting the experiment. Animals were maintained under temperature- and humidity-controlled conditions on a\u0026nbsp;12/12 light-dark cycle\u0026nbsp;and provided access to food and water ad libitum.\u0026nbsp;The experimental procedures were\u0026nbsp;performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 2011) and were approved by the Ethics Committee for Animal Experimentation of Faculty of Pharmacy, Cairo University (PT3522). Efforts were made to reduce animal suffering and decrease the number of animals used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLipopolysaccharide (LPS) (Escherichia coli, serotype 0127: B8) was purchased from Sigma-Aldrich, USA. Trimetazidine dihydrochloride (TMZ) and escitalopram\u0026nbsp;(Esc)\u0026nbsp;were acquired from\u0026nbsp;Servier\u0026nbsp;and Multi-Apex Pharmaceutical Company, Egypt, respectively.\u0026nbsp;They were all freshly prepared using physiological saline before use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Experimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Mice were randomly divided into 5 groups (n = 15/group) as follows; control, TMZ, LPS, LPS+ Esc, and LPS+ TMZ groups (\u003cstrong\u003eFig. 1)\u003c/strong\u003e. Induction of depression was achieved by intraperitoneal injection of LPS at a dose of 500 \u0026micro;g/kg, every other day for 14 days, for a total of 7 injections \u003cspan lang=\"EN-GB\"\u003e[35,36]\u003c/span\u003e. Mice were treated orally with either Esc (10 mg/kg/day) \u003cspan lang=\"EN-GB\"\u003e[37,38]\u003c/span\u003e or TMZ (20 mg/kg/day) [32,39,40] for 14 days covering the LPS treatment period. Following treatments, mice were behaviorally tested using open field test (OFT) and forced swimming test (FST). After performing the behavioral tests, animals were euthanized by decapitation under anaesthesia then the whole brains were carefully collected and washed with ice-cold saline. Brains of three mice from each group were kept in 10% formalin for histopathological examination of the hippocampal region and for determination of mean intact neurons count. The hippocampi of the remaining mice were dissected out, flash frozen in liquid nitrogen and stored at \u0026minus;80\u0026deg;C for further biochemical investigations. Six of these hippocampi were then homogenized in cold phosphate-buffered saline to prepare a 10% homogenate to be used for enzyme-linked immunosorbent assay (ELISA) tests while the remaining six were used for real-time PCR analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Behavioral Assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.1. Open Field Test\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLocomotor function of the mice was evaluated using the OFT. The test was performed in a wooden box (80 \u0026times; 80 \u0026times; 40 cm) with red walls and a black floor. The floor of the box was divided into 16 equal squares by white lines. Each mouse was placed in the middle of the open field and left to explore for 3 minutes. The arena was cleaned carefully using 20 % alcohol between each mouse to get rid of any remnant odors. The animal\u0026apos;s locomotor function was assessed by recording the frequencies of ambulation and rearing \u003cspan lang=\"EN-GB\"\u003e[26,41]\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.2. Forced Swimming Test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForced swimming test was performed to assess the depressive-like behaviors in rodents \u003cspan lang=\"EN-GB\"\u003e[8]\u003c/span\u003e. Mice were individually placed in a transparent cylinder (height: 40 cm, diameter: 30 cm) containing about 35 cm of water (25 \u0026plusmn; 1\u0026deg;C) for 5 min. The total amount of time each animal remained immobile was recorded as the immobility time. The animal was considered immobile when floating motionless or making only small movements needed to keep its head above the water level \u003cspan lang=\"EN-GB\"\u003e[42,43]\u003c/span\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Histopathological Examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrain samples were fixed in neutral buffered formalin (10%) for 72 hours. Samples were then processed in serial grades of ethanol and cleared in xylene. Afterwards, they were infiltrated and embedded into Paraplast tissue embedding media. A rotatory microtome was used to cut 5 \u0026mu;m-thick serial sagittal brain sections for demonstration of hippocampal regions in different samples and mounted on glass slides. Sections were then stained with hematoxylin and eosin (H\u0026amp;E) as a general staining method for tissue examination. Standard procedures for samples fixation and staining were carried out according to [44]. Additionally, sections were stained with toluidine blue and six randomly selected non-overlapping fields from the CA3 hippocampal region of each sample were assessed to determine mean intact neurons count \u003cspan lang=\"EN-GB\"\u003e[45]\u003c/span\u003e. All light microscopic examination and data were obtained using a full HD microscopic imaging system and Leica application module for tissue section analysis (Leica Microsystems GmbH, Germany). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Biochemical Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.1. Enzyme-Linked Immunosorbent Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHippocampal NF-\u0026kappa;B and matrix metalloproteinase-9 (MMP-9) contents were measured using mouse ELISA kits supplied by LifeSpan Biosciences (USA) and Elabscience (China), respectively. Moreover, mouse MyBioSource ELISA kits (USA) were used for the assessment of TNF-\u0026alpha;, Nrf2, HO-1, GSH and claudin-5 contents in the hippocampus, while IL-1\u0026beta; and Caspase 3 contents were estimated using R\u0026amp;D systems mouse ELISA kits (USA). The procedures were performed following the protocols provided by the manufacturers. Protein content was determined using the method described by \u003cspan lang=\"EN-GB\"\u003e[46]\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6.2. Quantitative Real-Time PCR Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the mice hippocampi using SV total RNA isolation system (Promega, USA) and purity of the obtained RNA was detected spectrophotometrically at 260/280 nm. Using Reverse Transcription System (Promega, USA), the extracted RNA was reverse transcribed into complementary DNA. To evaluate gene expression of TLR4 and serotonin transporter (SERT), quantitative real-time PCR was performed using the SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich, USA) as per the manufacturer\u0026apos;s instructions. The used primers\u0026apos; sequences are illustrated in Table 1. The relative expression of the target genes was estimated using the 2\u003csup\u003e\u0026minus;∆∆CT\u003c/sup\u003e formula \u003cspan lang=\"EN-GB\"\u003e[47]\u003c/span\u003e with \u0026beta;-actin used as a housekeeping gene.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u0026nbsp;\u003c/strong\u003eSequences of the primers used for quantitative real time-PCR analysis.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.625678119349004%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.374321880651%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimer sequence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.625678119349004%\" valign=\"top\"\u003e\n \u003cp\u003eTLR4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.374321880651%\" valign=\"top\"\u003e\n \u003cp\u003eForward: 5\u0026prime;-ATGCATGGATCAGAAACTCAGCAA-3\u0026prime;\u003c/p\u003e\n \u003cp\u003eReverse: 5\u0026prime;- AAACTTCCTGGGGAAAAACTCTGG-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.625678119349004%\" valign=\"top\"\u003e\n \u003cp\u003eSERT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.374321880651%\" valign=\"top\"\u003e\n \u003cp\u003eForward: 5\u0026prime;-AAGCCCCACCTTGACTCCTCC-3\u0026prime;\u003c/p\u003e\n \u003cp\u003eReverse: 5\u0026prime;-CTCCTTCCTCTCCTCACATATCC-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.625678119349004%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026beta;-actin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"81.374321880651%\" valign=\"top\"\u003e\n \u003cp\u003eForward: 5\u0026prime;-CACTGTCGAGTCGCGTCC-3\u0026prime;\u003c/p\u003e\n \u003cp\u003eReverse: 5\u0026prime;-CGCAGCGATATCGTCATCCA-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7. Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData sets that met the requirements for parametric criteria were analyzed using one-way ANOVA followed by Tukey\u0026rsquo;s multiple comparisons test. Results were presented as mean \u0026plusmn; SD. Statistical analysis was performed using GraphPad Prism software, version 9 (GraphPad Software Inc., USA) with the significance level fixed at P \u0026lt; 0.05. For each effect, the F-value (F), the degree of freedom, and the statistical significance (p) were reported, moreover, pairwise comparisons were provided in each corresponding figure.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe results displayed by TMZ group were presented herein concurrently with the other experimental groups. They were insignificantly different as compared to those of the control group upon histological, behavioral, and biochemical investigations. Thus, comparisons were done as related to the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1. Trimetazidine \u003c/strong\u003e\u003cstrong\u003ealleviated LPS-induced depressive-like behavior in forced swimming test whereas it exerted null effect on locomotor activity in open field test \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLPS injection in mice instigated depressive-like symptoms which were investigated behaviorally using the FST. In comparison with the control group, LPS-injected mice showed a state of despair in FST where they displayed a marked rise in the time of immobility by 2.1-folds (F\u003csub\u003e(4, 70)\u003c/sub\u003e = 16.33, p\u0026lt; 0.0001). Treatment with either Esc or TMZ ameliorated LPS-induced depressive-like behavior restoring the values of immobility time to the normal range. As compared to LPS group, Esc- or TMZ-treated mice exhibited reduced immobility duration by 51 and 45%, respectively \u003cstrong\u003e(Fig. 2A)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eIn the open field test, the ambulation and rearing frequencies were documented to evaluate the impact of LPS, TMZ, or Esc on spontaneous locomotor activity of rats. There was insignificant variation among the experimental groups in either the frequency of ambulation (F\u003csub\u003e(4, 70)\u003c/sub\u003e = 2.658, p = 0.0398) or rearing (F\u003csub\u003e(4, 70)\u003c/sub\u003e = 1.528, p = 0.2035). These findings rule out the possibility that the depressive-like anomalies detected herein can be attributed to changes in locomotor function \u003cstrong\u003e(Figs. 2B and C)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Trimetazidine alleviated LPS-induced hippocampal histopathological changes in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative photomicrographs of the H\u0026amp;E-stained samples of different groups were microscopically examined, in addition to using toluidine-blue stain for determining the intact neurons count in CA3 hippocampal area. The control group showed normal histological appearance of the hippocampal layers along with many records of apparent intact pyramidal neurons having well recognized nuclear and cytoplasmic details (black arrows), in addition to an intact brain matrix with the absence of cellular infiltrates. Similarly, the hippocampal sections of TMZ group presented comparable histopathological appearance with that of the control group without recording any abnormal changes. In contrast, the histological examination of LPS group samples showed obvious dispersed neuronal degeneration as indicated by ample of damaged or necrotic neurons having pyknotic perikarya missing its subcellular details, along with mild perineuronal edema (red arrows) in contrast to reduced scattered records of intact cells (black arrows). Additionally, LPS group samples demonstrated a remarkable increase in reactive microglial cell infiltrates (arrowhead). Upon examining the toluidine blue-stained sections, LPS group presented a noticeable decline in the intact neurons mean count by 96% as compared to the control group (F\u003csub\u003e(4, 25)\u003c/sub\u003e = 382.8, p\u0026lt; 0.0001). Notably, treatment with either Esc or TMZ afforded equivalent neuroprotective efficacy against LPS-induced histopathological changes. The brain samples of Esc-or TMZ-treated mice exhibited few sporadic records of damaged neurons (red arrow) and higher records of apparent intact neurons (black arrow) with intact intercellular brain matrix and minimal infiltrates (arrowhead), as compared to the LPS group \u003cstrong\u003e(Figs. 3 and 4)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Trimetazidine mitigated LPS-induced activation of hippocampal TLR4/NF-\u0026kappa;B pathway \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe LPS injection led to a profound activation of TLR4/NF-\u0026kappa;B pathway as evidenced by producing considerable increase in the hippocampal gene expression of TLR4 by 8-folds, with subsequent increment in the hippocampal content of NF-\u0026kappa;Bp65 by 1.8-folds, as compared to the control group (for TLR4: F\u003csub\u003e(4, 25)\u003c/sub\u003e = 112.8, p\u0026lt; 0.0001; for NF-\u0026kappa;Bp65: F\u003csub\u003e(4, 25)\u003c/sub\u003e = 98.69, p\u0026lt; 0.0001). Upon treating the LPS-exposed mice with TMZ, a marked drop in TLR4 gene level as well as NF-\u0026kappa;Bp65 content by 66 and 34%, respectively, was detected producing an equivalent effect with that displayed by the LPS+Esc group \u003cstrong\u003e(Fig. 5).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Trimetazidine reduced LPS-induced neuro-inflammation, apoptosis and oxidative stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe LPS depression model was accompanied by aggravated responses of neuro-inflammation, apoptosis, and oxidative stress. In comparison to the control group, LPS-injected mice showed profound rise in the hippocampal contents of inflammatory markers, TNF-\u0026alpha; (4.2-folds) and IL-1\u0026beta; (3.9-folds), along with significant increment in the proapoptotic caspase-3 concentration by 6.1-folds (F\u003csub\u003e(4, 25)\u003c/sub\u003e = 137.0, 318.4, and 119.9, respectively, p\u0026lt; 0.0001). On the contrary, they showed a considerable decrease in the antioxidant variables; namely Nrf2, HO-1, and GSH levels by 68, 70, and 50%, respectively, in comparison to their control counterparts (F\u003csub\u003e(4, 25)\u003c/sub\u003e = 256.3, 94.84, and 204.8, respectively, p\u0026lt; 0.0001). Treatment of LPS-injected mice with TMZ exerted neuroprotective effects leading to about 54% depression in TNF-\u0026alpha; and IL-1\u0026beta; concentration along with marked decline in caspase-3 content (66%). Additionally, TMZ mitigated LPS-induced oxidative stress producing significant rise in Nrf2 (2.5-folds), HO-1 (2.6-folds), and GSH levels (1.7-folds). Such effects of TMZ on the previously mentioned markers were almost comparable with those displayed by Esc treatment except for Nrf2 where Esc outweighed TMZ effect producing 10% increase in Nrf2 content as compared to TMZ-treated group\u003cstrong\u003e (Fig. 6)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Trimetazidine diminished LPS-induced surge in hippocampal SERT expression \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe administration of LPS in mice notably augmented the hippocampal mRNA expression of SERT by 4.3-folds, in comparison to the control group (F\u003csub\u003e(4, 25)\u003c/sub\u003e = 124, p\u0026lt; 0.0001). This effect was inhibited by TMZ administration where LPS+TMZ group mice showed a noticeable reduction in SERT gene level by 53%, as compared to the LPS group. Likewise, Esc administration obviously decreased the SERT gene level by 60%, as compared to LPS group. Results afforded by the LPS+Esc and LPS+TMZ groups on SERT expression in the hippocampi were almost comparable where no substantial change was revealed between the two groups \u003cstrong\u003e(Fig. 7)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6. Trimetazidine attenuated LPS-induced blood-brain barrier dysregulation \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe LPS administration has a detrimental effect on BBB integrity and function. LPS-injected mice displayed 2.4-folds elevation in the hippocampal content of MMP-9, an important protein involved in maintaining BBB integrity and function (F\u003csub\u003e(4, 25)\u003c/sub\u003e = 122.4, p\u0026lt; 0.0001). This was associated with dysregulation in BBB tight junctional proteins as exemplified by showing a considerable depression in the hippocampal content of claudin-5 (67%) leading to BBB dysfunction (F\u003csub\u003e(4, 25)\u003c/sub\u003e = 146.9, p\u0026lt; 0.0001). Such effects were hindered by TMZ treatment which led to MMP-9 downregulation by 47%, while it enhanced the claudin-5 concentration in the hippocampi by 2.3-folds. These effects were almost analogous to that obtained by LPS-injected mice treated with Esc which reduced MMP-9 by 38%, while elevated claudin-5 level by 2.5-folds, as compared to LPS group \u003cstrong\u003e(Fig. 8)\u003c/strong\u003e.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMajor depressive disorder (MDD) is a complex mental illness which imposes prominent disability and economic burden worldwide. It's associated with several clinical symptoms including feelings of worthlessness, fatigue as well as diminished ability to think or concentrate [48]. Despite the availability of various antidepressants, they are associated with multiple side effects and exhibit a delayed onset of action which hinders patients' compliance [3]. Accordingly, there is a consistent urge to investigate novel antidepressants which target various depressive pathological changes with fewer adverse effects hence enhancing patients' adherence [48].\u003c/p\u003e\n\u003cp\u003eThe current study investigated the antidepressant activities of TMZ; an anti-ischemic drug used for coronary artery disease, in LPS-induced depressive behaviors in mice. Administration of LPS every other day for 2 weeks was linked to increased immobility time in FST reflecting animals' despair associated with LPS injection. Meanwhile, LPS injection didn't alter the animals' spontaneous locomotor activity and rearing frequency recorded during OFT [49]. Similarly, in the present study, animals injected with LPS exhibited significant elevation in the immobility time recorded in FST, yet they didn't reveal neither ambulation nor rearing frequencies derangements in OFT which empathizes that LPS-associated despair and lack of mobility in FST is contributed to its ability to induce depressive like behavior. On the other hand, TMZ administration alleviated LPS\u0026ndash;induced animals despair witnessed in FST reflected by significant decrement in the immobility time of TMZ-treated mice, as compared to LPS-injected animals which didn't receive any treatment. TMZ-associated mood enhancement was comparable to the used reference antidepressant; Esc.\u003c/p\u003e\n\u003cp\u003eNeuro-inflammation was reported as a promising target for MDD management [50]. LPS; a bacterial endotoxin, is implicated in depressive like behaviors via inducing multiple pro-inflammatory cytokines production [51]. It was reported to induce NF-\u0026kappa;B nuclear translocation and activation with consequent elevation of TNF-\u0026alpha; and IL-1\u0026beta; levels leading to augmented neuro-inflammation [52]. \u0026nbsp;Moreover, TLR4; a critical receptor involved in neuro-inflammation, was correlated to depression pathogenesis [53]. \u0026nbsp;Stimulation of TLR4 promotes the recruitment of inflammatory cells\u0026nbsp;which is implicated in NF-\u0026kappa;B activation with consequent release of the pro-inflammatory mediators namely; TNF-\u0026alpha;, and IL-1\u0026beta; [54,55]. Accordingly, in the present study, LPS injection induced an obvious pro-inflammatory milieu owing to TLR4 upregulation which contributes to NF-\u0026kappa;Bp65 activation with consequent pro-inflammatory cytokines release as TNF-\u0026alpha;, and IL-1\u0026beta;. Administration of TMZ attenuated LPS\u0026ndash;induced neuro-inflammation with significant decrease in the formerly mentioned cytokines levels owing to TLR4 downregulation. TMZ-induced anti-neuro-inflammatory effects were equivalent to the reputable antidepressant drug; Esc.\u003c/p\u003e\n\u003cp\u003eActivation of TLR4/NF-\u0026kappa;B signaling is implicated in the marked disruption of BBB permeability associated with LPS injection [56]. The BBB constitutes a protective layer in the CNS which restricts molecular exchange between the circulating blood and the brain for maintaining an effective neural signaling. Thus, these proteins serve as a defensive layer that hinders the path of hazardous substances to the brain [57]. Claudin-5 is a chief tight junction protein of the BBB whose dysfunction has been implicated in neurodegenerative disorders such as Alzheimer\u0026rsquo;s disease and psychiatric diseases as depression [58,59]. MMP-9 has been correlated to BBB breakdown and inflammation in several CNS disorders [60]. Moreover, it was reported that LPS is implicated in MMP-9 induction via NF-\u0026kappa;B leading to claudin-5 degeneration and BBB dysfunction [21,61]. Similarly, in the current investigation, LPS injection resulted in BBB disruption with elevated MMP-9 content contrary to claudin-5 decreased content owing to LPS induced activation of TLR4/NF-\u0026kappa;B pathway. On the other hand, TMZ administration diminished LPS-induced neuro-inflammation and maintained BBB integrity as revealed by producing significant decrease in MMP-9 content contrary to claudin-5 enhanced content owing to TMZ-mediated inhibition of TLR4/NF-\u0026kappa;B pathway which was comparable to Esc effects.\u003c/p\u003e\n\u003cp\u003eLPS; probably via its well-known pro-inflammatory effects, contributes to upregulation of SERT which is responsible for serotonin reuptake leading to deficiency of this neurotransmitter in the brain with consequent depressive behaviors [62]. Moreover, TNF-\u0026alpha; and IL-1\u0026beta; were reported to promote rapid activation of SERT [63]. Likewise, in the present study, LPS-injected animals revealed increased SERT expression which was noticeably reduced upon TMZ administration.\u003c/p\u003e\n\u003cp\u003eNeuro-inflammation and oxidative stress relationship has been verified in depression pathogenesis [64]. An established cross link has been proposed between the pro-inflammatory transcriptional factor; NF-\u0026kappa;B and the protective anti-oxidant; Nrf2 where LPS-induced neuro-inflammation is attributed principally to its inhibitory action on Nrf2 pathway contrary to NF-\u0026kappa;B upregulation which is a key player in BBB dysfunction, neuro-inflammation, and apoptosis [65]. Nrf2 also regulates the expression of various antioxidant and anti-inflammatory mediators such as HO-1 and the antioxidant enzymes involved in GSH synthesis [66]. Besides, Nrf2 has the potential to inhibit LPS-induced up-regulation of pro-inflammatory cytokines as well as MMP-9 which induces BBB disruption and cell death [67]. Accordingly, in the current study, LPS-injected animals revealed extravagant oxidative stress and massive neuro-inflammatory milieu reflected by significant decrease in Nrf2, HO-1,and GSH contents owing to the culprit inflammatory cascade; TLR4/NF-\u0026kappa;B and its downstream targets; namely MMP-9 and caspase-3 which were markedly elevated in LPS-injected mice. TMZ administration diminished LPS-induced triad of oxidative stress, neuro-inflammation, and apoptosis with augmented contents of Nrf2, HO-1, and GSH due to prominent inhibition of TLR4/NF-\u0026kappa;B pathway along with MMP-9 and caspase-3 dismounted levels. TMZ neuroprotective effects were equivalent to Esc regarding the formerly mentioned parameters except for Nrf2 content whose elevation in LPS+Esc group out weighted that recorded in LPS+TMZ group.\u003c/p\u003e\n\u003cp\u003eFinally, in the present investigation, photomicrographs of LPS-injected mice revealed diffuse neuronal damage with plentiful degenerated or necrotic neurons.\u0026nbsp; \u0026nbsp;Additionally, significant increase of reactive microglial cells infiltrates was recorded in animals receiving LPS. TMZ administration induced scarce sporadic neuronal damage with more apparent intact neurons besides minimal infiltration. Photomicrographs of LPS +TMZ group mimicked those witnessed in LPS +Esc group. Normal mice which received TMZ did not show morphological alterations as compared to normalcontrol animals.\u003c/p\u003e\n\u003cp\u003eConsequently, the present study explored TMZ neuro-protective activity in LPS-induced depressive like behavior in mice. TMZ attenuated LPS-associated oxidative stress, neuro-inflammation, and apoptosis in addition to restoration of serotonin levels via down regulation of SERT. TMZ-induced neuroprotection can be attributed to its modulatory effects on TLR4/NF-\u0026kappa;B and Nrf2/HO-1 pathways which were reflected by marked decrease in animals' immobility time recorded during FST.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTMZ; a well-known anti-ischemic drug, exerted mood boosting effects in LPS-injected mice. Moreover, it was able to overcome LPS-induced viscous cycle of oxidative stress, neuro-inflammation, and apoptosis probably via its ability to fine tune NF-\u0026kappa;Bp65 and Nrf2 correlation and replenish depression-associated neurotransmitter deficiency. Consequently, TMZ is a prosperous candidate that needs further investigations to authenticate its antidepressant activity.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received to assist with the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare that are relevant to the content of this article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSarah S. Mohamed, Nora O. Abdel Rasheed, Weam W. Ibrahim and Nesma A. Shiha: conceptualization. Sarah S. Mohamed: methodology. Nora O. Abdel Rasheed, Nesma A. Shiha, and Weam W. Ibrahim: data curation and writing-original draft preparation. Nora O. Abdel Rasheed, Nesma A. Shiha, Sarah S. Mohamed, and Weam W. Ibrahim: reviewing, and editing. \u0026nbsp;All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtrapolation of the present study findings \u0026nbsp;to humans is necessary \u0026nbsp;to validate TMZ antidepressant \u0026nbsp;effects in humans with emphasis \u0026nbsp;on various depression aspects besides the neuroinflammatory milieu.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental procedures were\u0026nbsp;performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 2011) and were approved by the Ethics Committee for Animal Experimentation of Faculty of Pharmacy, Cairo University (PT3522). Efforts were made to reduce animal suffering and decrease the number of animals used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Generative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work, the authors didn\u0026rsquo;t use any AI tool or AI-assisted technologies.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWorld Health Organization, Depression, (2023). https://www.who.int/news-room/fact-sheets/detail/depression (accessed October 7, 2023).\u003c/li\u003e\n\u003cli\u003eE. Maffioletti, A. Minelli, D. Tardito, M. Gennarelli, Blues in the Brain and Beyond: Molecular Bases of Major Depressive Disorder and Relative Pharmacological and Non-Pharmacological Treatments., Genes (Basel) 11 (2020) 1089. https://doi.org/10.3390/genes11091089.\u003c/li\u003e\n\u003cli\u003eZ. Li, M. Ruan, J. Chen, Y. 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Trimetazidine (TMZ); a well-known anti-ischemic drug, could exert neuroprotection in cerebral ischemia.\u003c/p\u003e\n\u003cp\u003eAims: This study aimed to investigate the effect of TMZ in lipopolysaccharide (LPS) mouse model of depression with empathises on its ability to regulate toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) as well as nuclear factor erythroid 2 related factor 2 (Nrf2)/ heme oxygenase-1(HO-1) signaling pathways.\u003c/p\u003e\n\u003cp\u003eMain methods: Male Swiss albino mice were injected with LPS (500 µg/kg, i.p) every other day alone or concurrently with oral doses of either TMZ (20 mg/kg/day) or escitalopram (Esc) (10 mg/kg/day) for 14 days.\u003c/p\u003e\n\u003cp\u003eKey findings: Administration of TMZ attenuated LPS-induced animals' despair with decreased immobility time in forced swimming test. Additionally, TMZ diminished LPS–induced neuro-inflammation via inhibition of TLR4/NF-κB pathway contrary to Nrf2/HO-1 cascade activation with consequent increase in reduced glutathione (GSH) and HO-1 levels whereas the pro-inflammatory cytokines; tumor necrosis factor –α (TNF-α) and interleukin (IL)-1β were markedly reduced. Besides, TMZ replenished brain serotonin levels owing to serotonin transporter (SERT) inhibition. Thus, TMZ hindered LPS-induced neuro-inflammation, oxidative stress, serotonin deficiency in addition to its anti-apoptotic effect which was reflected by decreased caspase-3 level. The formerly mentioned neuroprotective effects of TMZ were verified by the histological photomicrographs which revealed prominent neuronal survival with minimal records of neuronal damage.\u003c/p\u003e\n\u003cp\u003eSignificance: Consequently, TMZ is proposed as an affluent nominee for depression management via targeting TLR4/NF-κB and Nrf2/HO-1 pathways.\u003c/p\u003e","manuscriptTitle":"Targeting TLR4/NF-κB and Nrf2/HO-1 Crosstalk via Trimetazidine alleviates LPS-induced Depressive Like Behaviors in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-10 13:16:53","doi":"10.21203/rs.3.rs-4440337/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-27T22:23:06+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"301398837286680512017785761012541835182","date":"2024-06-24T21:55:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-23T12:37:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-21T10:50:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338852321173333477381168321981992621337","date":"2024-06-20T06:24:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"279584228835691456102181251870161457517","date":"2024-06-20T05:25:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172357324407769685306935780744529126674","date":"2024-06-20T05:00:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"244274620937296401999503103893790024646","date":"2024-06-19T23:13:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-19T21:50:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-08T16:01:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-28T10:38:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuroimmune Pharmacology","date":"2024-05-18T09:09:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuroimmune-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnip","sideBox":"Learn more about [Journal of Neuroimmune Pharmacology](http://link.springer.com/journal/11481)","snPcode":"11481","submissionUrl":"https://submission.nature.com/new-submission/11481/3","title":"Journal of Neuroimmune Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2e035aca-9e76-41fa-a2ca-7a0c12859af6","owner":[],"postedDate":"June 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:13:40+00:00","versionOfRecord":{"articleIdentity":"rs-4440337","link":"https://doi.org/10.1007/s11481-024-10149-3","journal":{"identity":"journal-of-neuroimmune-pharmacology","isVorOnly":false,"title":"Journal of Neuroimmune Pharmacology"},"publishedOn":"2024-09-23 15:57:36","publishedOnDateReadable":"September 23rd, 2024"},"versionCreatedAt":"2024-06-10 13:16:53","video":"","vorDoi":"10.1007/s11481-024-10149-3","vorDoiUrl":"https://doi.org/10.1007/s11481-024-10149-3","workflowStages":[]},"version":"v1","identity":"rs-4440337","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4440337","identity":"rs-4440337","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}