Efficacy of Probiotic Escherichia coli Nissle 1917 in Counteracting Reserpine-induced Depression-like Symptoms and Neuroinflammation in Zebrafish

Abstract

Background:

Depression is emerging as a significant global public health concern. Escherichia coli Nissle 1917 (EcN), a well-known probiotic, is recognized for its antioxidant and anti-inflammatory properties and has demonstrated efficacy in treating inflammatory bowel diseases and other gastrointestinal disorders.

Methods:

Reserpine, an obsolete antihypertensive medicine known to cause neurotoxicity, was used to induce depression-like symptoms in adult zebrafish. Our experimental approach encompassed various stages, including behavior assessment, histological examination, antioxidant assays, and enzyme-linked immunosorbent assay (ELISA).

Results:

Reserpine exposure resulted in significant behavioral deficits, increased oxidative stress, neuronal damage, and elevated pro-inflammatory cytokine levels in the zebrafish brain. EcN treatment significantly improved locomotor and exploratory behaviors and restored social preference compared with the reserpine group (p < .05). EcN markedly increased antioxidant enzyme activities (Catalase and Superoxide dismutase) while reducing lactate dehydrogenase, nitric oxide, and lipid peroxidation levels (p < .05). Furthermore, EcN significantly reduced tumor necrosis factor-α, interleukin-1β, and necrosis factor-κB levels in the brain relative to reserpine-treated fish (p < .001). These effects were comparable, though slightly less pronounced, than those observed with Venlafaxine.

Conclusions:

EcN significantly attenuated reserpine-induced depression-like behavior, oxidative stress, neuronal damage, and neuroinflammation in adult zebrafish, as evidenced by statistically significant improvements in behavioral, biochemical, and inflammatory endpoints. These findings support EcN as a promising psychobiotic with antidepressant-like properties, warranting further mechanistic and translational investigations.

Keywords

Reserpine depression neuroinflammation oxidative stress probiotics

Key Messages:

Few antihypertensive medications, like reserpine, have been recognized as potential contributors to depression.

A single dose of the probiotic EcN could significantly ameliorate depression-like behavior induced with reserpine in zebrafish.

EcN exerts its antidepressant activity by restoring the levels of oxidative and inflammatory markers.

Over the past decade, significant advances have been made in the diagnosis and treatment of psychiatric disorders, with a primary focus on changes in neurotransmitter levels, particularly in serotonin, norepinephrine, dopamine, and gamma-aminobutyric acid (GABA) secretion within the central nervous system.¹ Within the domain of Depressive Disorder (DD), distinct psychological alterations are observed, encompassing reduced activity, pervasive negative disposition, and impaired cognition, including delayed cognitive performance. At present, substantial therapeutic developments have been emerging to address DD. It includes diverse agents such as tricyclic antidepressants (TCAs), monoamine oxidase inhibitors (MAOIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), and selective serotonin reuptake inhibitors (SSRIs).² However, alongside their efficacy in alleviating depressive symptoms, these medications are associated with a range of adverse side effects, such as nausea, restlessness, somnolence, loss of appetite, and fatigue.^3,4 Consequently, the need to develop more efficacious antidepressant interventions has stimulated researchers to explore alternative avenues, notably probiotic-based therapies.

Gut-based probiotics have been shown to play a pivotal role in establishing a bidirectional communication link between the gut and the brain.^5,6 Increasingly, researchers acknowledge the profound influence of the vast array of bacteria residing in the gut on mental well-being.⁷ In this context, the gut bacterial repertoire, encompassing both benign and pathogenic microorganisms in the digestive tract, can synthesize and disseminate neurotrophic molecules such as serotonin and GABA. These molecules have the potential to modulate both the immune and central nervous systems, thereby exerting notable physiological effects.^8,9 Notably, two prior meta-analyses and a series of randomized controlled trials investigating the effects of probiotics on mood and anxiety disorders have underscored the therapeutic potential of probiotics in alleviating symptoms of DD, devoid of adverse effects.^10–12 Among the probiotics currently in use, strains such as Lactobacillus rhamnosus, Bifidobacterium bifidum, Bacillus subtilis, Lactobacillus plantarum, Streptococcus thermophilus, and EcN have received substantial attention.^13,14 In particular, the non-pathogenic, serum-negative fecal bacterium EcN is known for its potent probiotic properties, as demonstrated in animals and humans.^15–17 Clinical investigations employing EcN have yielded promising results, suggesting that probiotic-based immunotherapies could benefit individuals with persistent Crohn’s disease^18–20 and irritable bowel syndrome.²¹ Furthermore, EcN has attracted attention in recent studies for its anti-inflammatory and antioxidant properties, which may underlie neuroprotective mechanisms against stress-related psychiatric disorders.²² These properties are particularly relevant in the context of psychiatric disorders, where inflammation and oxidative stress are implicated in the pathophysiology. Notably, prior research has shown that EcN holds promise in alleviating neurotoxic effects in various experimental models.²³ In experimental models, reserpine-induced depression, attributed to diminished monoamine levels, serves as a valuable marker for inducing depression-like behavior.²³ Reserpine induces depression-like symptoms by depleting the levels of essential neurotransmitters, particularly serotonin, norepinephrine, and dopamine, which are known to be critical in the regulation of mood and behavior.²⁴ The depletion of these neurotransmitters results in a cascade of neurobiological changes, including altered receptor function and impaired cellular signaling, leading to the manifestation of depressive symptoms.²⁵ With this background knowledge, we hypothesize that the anti-inflammatory and antioxidative properties of EcN may counteract the behavioral or psychiatric abnormalities induced by reserpine treatment.

Methods

Zebrafish Maintenance and Experiments

Zebrafish (6–8 months old, ~50% male and female, total 65) were sourced from Beryl Aqua Fish Farm, Chennai, India, and housed under appropriate conditions. They were divided into five groups (13 fish each): Control, Probiotic (EcN alone), Reserpine (reserpine alone), Res-Pro (reserpine and EcN), and Res-Ven (reserpine and Venlafaxine). Ven was used as a positive control due to its well-established antidepressant efficacy in reversing reserpine-induced monoamine depletion and depression-like behavior. Additionally, its documented antioxidant and anti-inflammatory effects allow direct comparison with EcN across the mechanistic endpoints assessed in this study.²⁶ A group treated with Venlafaxine (Ven) alone was not included because the study’s primary focus was to evaluate the therapeutic potential of EcN against reserpine-induced neurotoxicity, in comparison to a standard antidepressant (Ven). Assessing the individual effect of Ven was not the main objective, as its efficacy as an antidepressant is already established in clinical practice.²⁷ The Reserpine group received reserpine (40 µg/mL) in the water for 20 minutes.²³ Subsequently, the Res-Pro group was treated with EcN, cultured at 37°C in Nutrient broth, and then measured for optical density (OD) at 600 nm. Fish were orally given 10 µL of EcN suspension (10⁷ cfu/mL). The Res-Ven group was treated with Venlafaxine (0.025 µg/mL) in the tank water for 7 days.²⁸ All groups were fed with fresh brine shrimp twice daily for 8 days. The experimental design is illustrated in Figure S1. All experimental procedures adhered to the guidelines stipulated by the Institutional Animal Ethics Committee (IAEC). Further, we confirm that all the procedures comply with the CCSEA guidelines and regulations. The fine chemicals were purchased from Sigma (St. Louis, MO, USA).

Behavioral Tests

Several assays for assessing behavioral changes have been reported in the literature.^23,29,30 The behavioral testing was conducted between 10:00 am and 4:00 pm (Indian Standard Time). All analyses were carried out using the ANY-maze→ software (Stoelting, CO, USA).

Novel Tank Test

After the respective treatments, each group of zebrafish was individually placed within a 1.5-l trapezoidal-shaped tank, and the experimental setup closely followed the methodology outlined in.³¹ Trapezoidal tanks, divided into two symmetrical sections (Figure 1), housed zebrafish whose swimming activity was recorded for 6 minutes using a video-tracking system. Behavior analysis focused on the time spent in the upper and lower sections of the tanks.

Figure 1.

Zone Preferences and Behavioral Changes Measured by (a) Novel Tank Test (b) Light and Dark Test, and (c) Open Field Test Between the Study Groups. Data Are Presented as Mean ± SD of Triplicates. Significance Levels (p < .05): * Denotes Significant Differences Between the Control and Probiotic Groups. The Symbol ω Represents Significant Differences With the Reserpine-exposed Group. φ Indicates Significant Differences Between the Res-Pro (Reserpine + Probiotic) and Res-Ven Groups (Reserpine + Venlafaxine). SD = Standard Deviation; s = seconds; m = meter; m/s = meter per second.

Open Field Test

Individually, the zebrafish were placed in an open field test (OFT) tank (30 × 30 × 10 cm), as specified.³² Zebrafish were carefully introduced and allowed to navigate freely for 6 minutes. A lateral-view video recording was used to capture their behavior, and key variables were analyzed, including distance traveled, average speed, freezing duration, and time spent immobile.

Social Preference Test

In a conventional social preference test (SPT), a 50 cm acrylic corridor was used, subdivided into five 10 × 10 cm units. The fish under study is placed within an interaction chamber referred to as the “conspecific box.” This chamber stands isolated from the rest of the experimental setup using a translucent divider, as illustrated in Figure 3.³³ Fish were introduced into a central zone of a drug-free tank, separated by movable doors. After a 30-second adaptation, the doors were raised, allowing 6 minutes of exploration, recorded from a lateral view. The analysis tracked entries and time spent in the conspecific arm, middle zone, and empty arm.

Color Preference Test

The experimental procedure was conducted in a 22.5 × 7.5 × 7 cm acrylic tank, commonly known as the Plus maze.²³ The tank, filled with 2.75 L of water and covered with blue, green, yellow, and red plates on each side, maintained routine zebrafish care temperatures. Individual zebrafish were placed centrally, and a 6-minute recording (dorsal view) assessed color preference by measuring the time spent near each color.

Light and Dark Test

The dimensions of the experimental tank used in this analysis were 18 × 9 × 7 cm.³³ The tank, with a 3 cm water level and a sliding door dividing it into two equal parts, began testing by placing fish in the illuminated section. A 6-minute dorsal-view video was recorded during the test, measuring the time fish spent on the illuminated (light) and unilluminated (dark) sides of the tank.

Histopathological Analysis

Histopathological analysis was performed after 7 days (20 minutes of exposure to reserpine and 7 days of exposure to EcN/Ven) of treatment on the brain tissue of zebrafish. Initially, zebrafish, including control and treatment groups (n = 3/group), were anesthetized by immersion in ice water (4°C or less) for 20 minutes, followed by dissection to isolate the brain. Brain tissue was fixed in 10% formalin solution for 24 hours at room temperature. The fixed tissues were dehydrated and embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E).

Antioxidant Assays

Sample Preparation

For oxidative stress marker analysis, zebrafish brain tissue (n = 5/group, in triplicate) was homogenized in Tris-HCl buffer (pH 7.4) and centrifuged at 10,000 rpm for 10 minutes. The post-mitochondrial supernatant (PMS) was collected and stored at −20°C. A UV-1800 spectrophotometer (SHIMADZU, Kyoto, Japan) was used to measure absorbance at various wavelengths. The protein concentration was quantified based on Bradford’s method.³⁴

Catalase Assay

For the Catalase (CAT) assay, a reaction mixture comprising 50 mM PBS and 30 mM H₂O₂ was added to 10 µL of PMS, and the change in OD at 240 nm was monitored for 5 min. CAT activity is expressed in terms of U/mg of protein using a molar coefficient of 43.6 M⁻¹/cm⁻¹.²⁸

Lactate Dehydrogenase Assay

In lactate dehydrogenase (LDH), 50 µL of the reaction mixture, containing 10 mM of pyruvate and 250 µM of NADH, was added with 10 µL of the PMS, and the variation in OD was measured for 5 min at 340 nm.³⁵

Superoxide Dismutase

Superoxide dismutase (SOD) activity was determined by using 10 µL of PMS. It was mixed with 100 µL of reaction mixture containing 50 mM Tris HCl with 2.7 mM of pyrogallol, and the change in the OD was measured for 5 minutes at 420 nm.³⁶

Lipid Peroxidation Assay

One milliliter of 10% trichloroacetic acid (TCA) was mixed with 10 µL of PMS and centrifuged at 1000 rpm for 10 minutes. Subsequently, 1 ml of 0.67% thiobarbituric acid was added to 1 ml of the resultant supernatant, and was kept in a boiling water bath for 10 minutes. Then the tubes were immediately cooled, 1 ml of double-distilled water was added, and the OD at 532 nm was measured.³⁶

Nitric Oxide Assay

Nitric oxide (NO) levels were estimated by the Griess method.³⁷ 50 µL of PMS was added with Griess reagent containing sulphanilamide (1%) in phosphoric acid (5%), and N-(1-naphthyl) ethylenediamine dihydrochloride (0.1%), and the absorbance was read at 540 nm.³⁸

Glutathione Reduced Levels

Approximately 50 µL of PMS was precipitated with 25% TCA, followed by centrifugation at 8,000 g for 3 minutes at 4°C. Subsequently, a reaction mixture of 100 µL was prepared. This mixture contained 60 µM 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) and 50 mM potassium phosphate buffer saline, adjusted to pH 7.4. This reaction mixture was then combined with 10 µL of the enzyme supernatant. The absorbance of the resulting mixture was measured at a wavelength of 412 nm, referencing TCA as the blank solution. To compute the sulphydryl content, which was primarily constituted by 93% glutathione reduced (GSH), the molar extinction coefficient of DTNB (13,600 M⁻¹/cm⁻¹) was employed for estimation.³⁹

Glutathione Peroxidase Levels

Ten microliters of the acquired supernatant were added to a reaction mixture (110 µL) containing NADPH (150 µM), GSH (1 mM), and Sodium azide (100 mM), and the mixture was adjusted to pH 7.0. The ensuing absorbance kinetics were monitored at a wavelength of 340 nm over 2 minutes. The activity was then computed by the methodology outlined previously.⁴⁰

Glutathione S-transferase Assay

The Glutathione S-transferase (GST) activity within the head region was assessed using a well-established standard protocol as previously detailed.⁴¹ A 100 µL reaction mixture was prepared containing 10 mM GSH and 60 mM 1-chloro-2,4-dinitrobenzene. Subsequently, 20 µL of the supernatant was added to the reaction mixture. The alteration in absorbance was then tracked at a wavelength of 340 nm over 5 minutes.

ELISA for TNF-α, IL-1β and NF-k-B

Concentrations of tumor necrosis factor-α (TNF-α) (MBS704369), interleukin-1β (IL-1β) (MBS700230), and necrosis factor-kappa-B (NF-k-B) (E-AB-40558) in the brain of reserpine zebrafish were determined in triplicate using ELISA kits from My BioSource (San Diego, CA, USA) and Elabscience (Texas, USA) following the manufacturer’s instructions. In brief, 50 µL of PMS was added to the designated sample wells, followed by 50 µL of streptavidin-HRP (Horseradish peroxidase) to both the sample and standard wells. The wells were covered and incubated at 37°C for 60 minutes, then washed five times with wash buffer. Subsequently, 50 µL of substrate solution A was added to each well, followed by 50 µL of substrate solution B. The plate was incubated in the dark at 37°C for 10 minutes. Afterwards, 50 µL of stop solution was added to each well, and OD values were measured using a microplate reader at a wavelength of 450 nm within 10 minutes of adding the stop solution.

Statistical Analysis

All experiments were performed in triplicate, and results are expressed as mean ± standard deviation; p < .05 were considered statistically significant. Behavioral, enzymatic, and biomarker datasets were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. GraphPad Prism software (Version 8.0.2) was used to perform the statistical analysis. The second author induced Reserpine and EcN, and the first author collected behavioral data under blinded conditions, ensuring that the observer was unaware of the treatment groups to minimize investigator bias.

Results

Primary Outcome (Behavioral, Oxidative Stress, and Inflammatory Responses)

EcN Improves the Behavioral Abnormalities Induced By Reserpine

The behavioral pattern of zebrafish before and after reserpine treatment, and the alleviation of this effect by EcN, was assessed using various behavioral tests as described in the methods. The results obtained from the NTT demonstrated distinct variations in the motility patterns of zebrafish exposed to reserpine in comparison to both the Res-Pro and Res-Ven groups (Figure 1a). Zebrafish exposed to reserpine exhibited a pronounced inclination to swim predominantly in the lower zone with a marked four-fold reduction in time spent in the upper zone of the NTT (p < .001). Remarkably, the administration of EcN increased the preference for the upper zone by approximately twofold compared with the reserpine-treated group, and the values were comparable to those observed in the Ven-treated group. Furthermore, in contrast to the reserpine-alone scenario, the probiotic and Ven treatments caused notable shifts in the preference to remain within the dark zone. Reserpine-induced changes were evident in terms of reduced time spent in the dark zone, coupled with an elevated number of crossings within intermediate concentrations in the light and dark test (L&D) (Figure 1b). There was about a 10-fold increase in freezing time in the reserpine-treated fish compared to the control, and that was reduced to 3-fold in the probiotic-treated fish. Comparison of data from the reserpine-only-treated group with that from the Res-Pro and Res-Ven-treated groups revealed a significant decrease in total distance traveled, average speed, and mobile time in the reserpine-exposed cohort. These parameters showed significant improvement after the administration of EcN (Figure 1c). In the case of the SPT, the EcN and Ven interventions exhibited a substantial rescue effect, driving reserpine-affected zebrafish to manifest a preference for the conspecific region within the SPT tank (Figure 2). The statistical differences between each group for all the evaluated behavioral tests have been indicated in Table S1. The representation of tanks and trajectory patterns is mentioned in Figure 3.

Figure 2.

Behavioral Changes Observed in the a. Social Preference Test Between the Reserpine and Probiotic-treated Groups. Data Are Presented as Mean ± SD of Triplicates. Significance Levels (p < .05): * Indicates Significant Differences Between the Control and Probiotic Groups. The Symbol ω Represents Significant Differences With the Reserpine-exposed Group. φ Indicates Significant Differences Between the Res-Pro (Reserpine + Probiotic) and Res-Ven (Reserpine + Venlafaxine) Groups. SD = Standard Deviation; s = seconds.

Figure 3.

Trajectory Pattern of the Different Behavioral Parameters Measured in This Study. (a) Novel Tank Test, (b) Open Field Test, (c) Social Preference Test, and (d) Color Preference Test. However, for the Light and Dark Test, the Software Did Not Detect a Trajectory Pattern. Res-Pro (Reserpine + Probiotic); Res-Ven (Reserpine + Venlafaxine).

The Antioxidant and Antiinflammatory Activity of EcN

Reserpine groups treated with both EcN and Ven exhibited heightened CAT and SOD activity, alongside a reduction in LDH, NO, and lipid peroxidation (LPO) activity, respectively. In terms of glutathione analysis, levels of GTPx, GSH, and GST were notably elevated following treatment with both EcN and Ven (Table S2).

The reserpine-treated adult zebrafish exhibited a significant level of inhibition of CAT activity in the brain (0.69 ± 0.05). However, the administration of EcN and Ven to the reserpine-exposed zebrafish demonstrated a reduction in the inhibition of CAT enzyme activity, resulting in measured values of 1.05 ± 0.05 (EcN) and 1.2 ± 0.05 (Ven). Differences in LDH activity were evident between the reserpine-induced zebrafish brain (2.08 ± 0.07) and the reserpine-exposed zebrafish treated with EcN (1.54 ± 0.04) and Ven (1.44 ± 0.02). Furthermore, there was a significant increase in SOD activity observed in the EcN (3.01 ± 0.11) and Ven (4.39 ± 0.04) treated groups of reserpine-exposed zebrafish (Figure 4). In terms of NO levels, the control fish brain exhibited a relatively stable concentration of around 0.14 ± 0.01. However, reserpine treatment increased NO levels (0.39 ± 0.01), which were subsequently reduced by EcN (0.27 ± 0.01) and Ven (0.18 ± 0.01). The reserpine-treated group exhibited a noteworthy increase in LPO production (15.43 ± 1.09) in comparison to the control group (9.48 ± 0.58). The administration of EcN (12.48 ± 0.46) and Ven (10.03 ± 0.25) significantly mitigated LPO production in reserpine-treated fish, thereby demonstrating a reduction in oxidative stress.

Figure 4.

Changes in Antioxidant Molecules Were Evaluated in the Zebrafish Brain After the Exposure Period. The Experimental Groups Compared Include Control (Untreated), Probiotic-Treated, Reserpine-exposed, Res-Pro (Reserpine + Probiotic), and Res-Ven (Reserpine + Venlafaxine) Groups. Statistical Analysis Employed a One-way ANOVA Followed by Tukey’s Multiple Comparison Tests. The Outcomes Are Presented as the Mean ± SD of Triplicate Samples. Significance Levels (p < .05): * Denotes Significant Differences Between the Control and Probiotic Groups. The Symbol ω Indicates Significant Differences Observed With the Reserpine-exposed Group, Whereas ε Highlights Significant Differences Between the Res-Pro and Res-Ven Groups, Respectively. CAT = Catalase; SOD = Superoxide Dismutase; LDH = Lactate Dehydrogenase, LPO = Lipid Peroxidation; NO = Nitrous Oxide, GSH = Glutathione Reduced; GTPx = Glutathione Peroxidase; GST = Glutathione Transferase; SD = Standard Deviation.

The glutathione activities within the zebrafish brain exhibited a significant reduction in the reserpine-induced group. The GSH level in the healthy group was 0.61 ± 0.02, whereas it decreased substantially in the reserpine-exposed group (0.35 ± 0.02). However, treatment with EcN and Ven demonstrated an appreciable recovery of GSH levels (0.50 ± 0.01 and 0.55 ± 0.01, respectively). A similar trend was observed in GTPx activity. Reserpine exposure decreased GTPx activity (3.69 ± 0.13), which was subsequently countered by the elevation of GTPx activity through treatment with EcN (6.15 ± 0.18) and Ven (8.40 ± 0.19). These patterns were paralleled in GST-specific enzyme activity. GST activity within the healthy zebrafish brain was quantified to be 6.44 ± 0.08, and the value decreased in response to reserpine exposure (2.77 ± 0.12). However, the application of EcN and Ven increased GST activity (4.51 ± 0.12 and 5.24 ± 0.23, respectively). The observed rise in glutathione levels was pivotal in facilitating the elimination of the reserpine effect in the zebrafish brain (Figure 4).

Remarkable Reduction in Reserpine-induced Inflammatory Response by EcN

In the zebrafish brain (as detailed in Table S3), a twofold increase in TNF-α levels was observed among zebrafish subjected to reserpine administration. However, the administration of probiotic and Ven treatments resulted in a marked reduction in TNF-α levels within the brain [F (4,10) = 331.8 (p < .001)] of zebrafish exposed to reserpine (Figure 5). Similarly, a noteworthy increase in IL-1β levels within the zebrafish brain was evident following reserpine treatment, in contrast to the co-treatment group involving probiotic and Ven. Through the application of probiotics and Ven, a significant decrease in IL-1β levels was achieved in the brain [F (4,10) = 116.7 (p < .001)] of zebrafish subjected to reserpine (Figure 5). The NF-kB factor exhibited a significant elevation in the [2.07 fold] zebrafish exposed to reserpine, as compared to the control group. However, the administration of probiotic and Ven treatments effectively limited the increase in NF-kB levels [F (4,10) = 143.5 (p < .001)] among zebrafish subjected to reserpine treatment (Figure 5).

Figure 5.

The Assessment of Pro-inflammatory Cytokines Alteration Encompassing (a) TNF-α, (b) IL-1β, and (c) NF-kβ in the Zebrafish Brain Under Reserpine-induced Conditions Was Conducted, Followed by Treatment With EcN and Ven. The Experimental Groups for Comparison Encompass the Control (Untreated), Probiotic-treated, Reserpine-exposed, Reserpine-probiotic (Reserpine + Probiotic), and Reserpine-Ven (Reserpine + Venlafaxine) Groups. For Statistical Analysis, a One-Way ANOVA Was Initially Utilized, Followed by Tukey’s Multiple Comparison Tests. The Results Are Presented as the Mean ± SD of Triplicate Samples. Significance Levels (p < .05): * Signify Noteworthy Differences Between the Control and Probiotic Groups. The Symbol ω Indicates Significant Differences Observed Within the Reserpine-exposed Group. Conversely, ε Highlights Notable Distinctions Between the Res-Pro and Res-Ven Groups, Respectively. TNFα = Tumor Necrosis Factor alpha; IL1β = Interleukin 1 beta; NF-kB = Nuclear Factor kappa-light-chain-enhancer of activated B cell; SD = Standard Deviation.

Secondary/Exploratory Outcome (Histopathology and Color Preference Test)

Reserpine-mediated Neuronal Damage Is Partially Rescued by EcN

Histological sections of the brains from reserpine-treated fish indicated severe cellular damage, characterized by elongated nuclei, an elevated presence of pyknotic and necrotic cells, as well as clumping of basal glial cells in the striatum. Additionally, these sections exhibited moderate edema, marked tissue degeneration, and immune cell infiltration. Brain sections from the control fish showed clear cytoplasm containing intact oligodendrocytes, with unimpaired cellular morphology, as expected. Conversely, brain sections from zebrafish subjected to EcN and Ven treatments showed reduced pyknosis and necrosis, with mild edema and partial degeneration. These observations strongly support the notion that EcN can alleviate reserpine-induced pyknosis and edema, resulting in partial rescue of tissue degeneration in the zebrafish brain with the efficiency equivalent to Ven (Figure 6). The results of the Color preference test revealed a significant alteration in color preference induced by reserpine in zebrafish, wherein a preference for the red zone was observed. Notably, the administration of EcN (3-fold reduction in time spent in the red zone) and Ven ameliorated this condition, resulting in a more equitable distribution of time across the yellow, green, and blue zones (Figure S2).

Figure 6.

A Microscopic Analysis of Adult Zebrafish Brain Sections Exposed to Reserpine, Both With and Without EcN/Venlafaxine Treatment (At 400× Magnification), Was Conducted. The Untreated Control Fish Displayed an Intact Cellular Morphology. However, Exposure to Reserpine Led to Notable Alterations, Including Elongation of the Nuclei (NE) and the Presence of Necrotic Cells (N). Additionally, Edema (E) and Tissue Degeneration (D) Were Observed. In the Res-Pro (Reserpine + Probiotic) and Res-Ven (Reserpine + Venlafaxine) Groups, the Presence of Necrotic Cells Was Evident, Along With a Partial Amelioration of Degeneration. EcN Treatment Exhibited Cells With a Diffusely Arranged Pattern, Correlating With Improvements in Necrosis and Edema. Noteworthy Observations Encompassed Necrosis (N), Nucleus Elongation (NE), Degeneration (D), and Edema (E).

Discussion

The present study aimed to assess the psychological impact of probiotic EcN on the central nervous system of zebrafish following reserpine therapy. Since the 1940s, reserpine has been employed as a first-line medication for hypertension, and more recently, as a second-line treatment.⁴² Reserpine is no longer a first-line antihypertensive agent primarily due to its well-documented adverse neuropsychiatric effects, including depression, which led to its replacement by newer antihypertensive drugs with improved safety and tolerability profiles.²⁴ Initial studies, however, employed high dosages of reserpine (ranging from 0.75 to 10 mg daily), which seemed to elicit depressive symptoms and gastrointestinal side effects.⁴³ The research assessed reserpine’s antimicrobial properties by determining the minimum inhibitory concentration (MIC) and the zone of inhibition (ZOI) against Gram-positive and Gram-negative bacteria. S. aureus showed a 13 mm ZOI and 625 µg as MIC, while Escherichia coli had 10 mg as MIC.⁴⁴ Reserpine-induced alterations in the gut microbiota (GM) may have led to behavioral changes in zebrafish, which were effectively alleviated by EcN treatment. This highlights EcN’s significant role in combating anxiety arising from dysbiosis.⁴⁵ Dysbiosis, characterized by an imbalance within the gut microbial ecosystem, has been closely associated with an array of mental disorders, encompassing anxiety and depressive states.⁴⁶ Behavioral analyses in this study confirm that reserpine induces behavioral changes consistent with prior findings. Reserpine, while no longer a first-line treatment for hypertension in clinical settings due to the availability of newer drugs with improved side-effect profiles, remains a valuable pharmacological tool in research. Its unique mechanism of action, involving the depletion of monoamines such as serotonin, norepinephrine, and dopamine, is critical for modeling specific aspects of neurotransmitter dysfunction relevant to neuropsychiatric disorders.^47,48 In this study, we used reserpine to induce a depression-like state in zebrafish, thereby enabling us to investigate the potential therapeutic effects of the probiotic EcN, representing a novel approach to counteract reserpine-induced neurotoxicity. Zebrafish displayed enduring depressive-like symptoms post-reserpine administration, characterized by increased whole-body cortisol, social withdrawal, psychomotor impairment, and modified color preference.^23,49,50 A study indicates that although reserpine did not manifest acute behavioral effects, it significantly suppressed movement after 7 days. After 7 days, zebrafish exhibited notably reduced physical movement, along with evident signs of a deteriorated and depressed demeanor. Moreover, their exploratory activities showed reduced levels of vigor, collectively confirming the successful establishment of the reserpine-induced zebrafish depression model by the methodology introduced by Kyzar et al., 2013.⁵¹ EcN and Ven treatments significantly reduced the severity of reserpine-induced depression, increasing locomotor activity and enhancing exploratory behavior. The color preference test, utilized in cognitive assessment, has been valuable across various evaluations. Zebrafish typically show a preference for shorter-wavelength colors, particularly blue over red, yellow, and green, a pattern observed in numerous studies.⁵² In our study, control zebrafish displayed a strong preference for blue over yellow, with blue significantly favored over red and green. Zebrafish with depression -like behavior exhibited altered color preferences, favoring red followed by green. EcN treatment restored the conventional preference pattern to a marked extent, whereas Ven’s effect was less pronounced, highlighting EcN’s antidepressant activity. In the SPT, reserpine-exposed zebrafish tend to avoid social interactions, distancing themselves from their shoal. Conversely, both EcN- and Ven-treated groups showed increased social interactions, spending more time in the conspecific zone. Reserpine-treated zebrafish displayed reluctance to swim in the dark side tank, whereas EcN and Ven-treated fish showed willingness, indicating significant differences. These findings further underscore EcN’s potential in mitigating depression related cognitive impairment in zebrafish. Additionally, histological analysis revealed reserpine-induced alterations in brain cellular morphology.⁵³ Conversely, sections from the EcN group revealed cells distributed diffusely, with diminished pyknotic and necrotic features.⁵⁴ Notably, mild edema and partial degeneration were observed. The absence of nucleus elongation and inflammatory cell migration in both the EcN and Ven cohorts suggests a notable reduction in inflammation.⁵⁵ These observations collectively underscore that EcN mitigates brain damage resulting from reserpine exposure, thereby enhancing functional outcomes in behavioral manifestations, owing to its intrinsic antioxidant properties.

Like other vertebrate species, zebrafish possess CAT and SOD systems to counteract the effects of pathogenic reactive oxygen species (ROS). These mechanisms involve the conversion of O₂ to H₂O₂, which is subsequently transformed into water and oxygen.⁵⁶ Within the zebrafish brain, reserpine elicited a decrease in SOD and CAT activity,⁵⁷ however, when exposed to EcN or Ven, CAT and SOD activities increased and approached the levels observed in the control group.

The LDH catalyzes the interconversion of pyruvate and lactate, thereby generating electrical signals in living cells.⁵⁸ Heightened energy demand to counteract oxidative damage increases LDH production, which serves as an indicator of oxidative stress. Reserpine-treated zebrafish exhibited elevated LDH activity, suggesting oxidative damage in the brain. Co-treatment with EcN or Ven reduced LDH activity, thereby restoring ROS homeostasis. Excessive NO production can activate brain cells, leading to pro-inflammatory responses.⁵⁹ NO, produced by inducible nitric oxide synthase (iNOS), is an oxidative byproduct that functions as a pro-inflammatory mediator. Notably, iNOS can be stimulated by inflammatory cytokines, including IFN-γ, IL-1, and TNF-α. The controlled synthesis of NO in cells by iNOS contributes to the establishment of an effective defense mechanism against various bacterial intruders.⁶⁰ In this study, zebrafish exposure to reserpine induced increased NO production, thereby promoting inflammation. However, concurrent administration of EcN and Ven exposure may reduce NO production, thereby mitigating inflammation.^61,62 The observed increase in intracellular ROS and NO generation underscores that reserpine exposure induces inflammation in the zebrafish brain. An important parameter to assess the extent of damage stemming from oxidative stress is LPO. Due to the increased ROS levels, LPO was also noted to be elevated within the cranial region of reserpine-exposed zebrafish brains. Notably, both EcN and Ven treatments reduced ROS levels, thereby decreasing LPO production in the zebrafish brain.⁶³ Excessive ROS production and elevated LPO levels can trigger the activation of programmed cell death pathways. The family of glutathione-related enzymes, including GTPx and GST, plays a pivotal role in bolstering the intracellular antioxidant defense system. Perturbations within the homeostasis of the glutathione redox cycle have been linked to neurodegenerative processes.⁶⁴ GTPx, present in various cellular compartments, particularly neurons,⁶⁵ regulates the GSH equilibrium by facilitating the conversion of reduced glutathione to its oxidized form, glutathione disulfide (GSSG). Furthermore, GST and GSH collaborate to ameliorate oxidative damage through detoxification mechanisms.³⁵

Prior research has consistently demonstrated that Ven can restore brain glutathione levels, thereby countering oxidative stress conditions.⁶⁶ These findings are strikingly consistent with our observations, indicating that reserpine exposure impairs glutathione enzyme function, whereas subsequent exposure to Ven triggers activation of GTPx and GST. Similarly, treatment with EcN also stimulates the glutathione-related enzyme family, yielding results analogous to those observed with Ven.^67–69 In this study, we investigated the expression levels of glutathione-related antioxidant enzymes and found decreased expression of GST, GTPx, and GSR in the brains of reserpine-treated zebrafish. However, co-exposure with EcN or Ven restored their expression patterns to an appreciable level, suggesting EcN’s potential to trigger antioxidant defense mechanisms, thereby aiding in combating oxidative stress-induced neuronal damage.

Reserpine’s role as a monoamine depletion disrupts the vesicular monoamine transporter, leading to dopamine autoxidation and oxidative catabolism. This process generates dopamine quinones and hydrogen peroxide, contributing to oxidative stress. Bagis et al. found elevated pentosidine and malondialdehyde levels and reduced serum SOD in patients with chronic pain compared to controls, suggesting a link between nitrosative stress and pain in depression.⁷⁰ This surge in advanced glycation end-product generation, stemming from heightened nitrosative stress, initiates the activation of the transcription factor NF-kB, subsequently triggering the expression of pro-inflammatory genes.⁷¹ This cascade encompasses the upregulation of cytokines and growth factors by macrophages and mesangial cells, including IL-1β, IGF-1 (Insulin-like growth factor-1), and TNF-α. We also detected elevated levels of IL-1β and TNF-α in reserpinized zebrafish brains, aligning with the findings of Huang et al., who identified increased IL-1β levels in the brains of reserpinized zebrafish.⁷² Notably, in our investigation, administration of EcN caused a significant reduction in TNF-α and IL-1β levels within the brains of reserpine-treated zebrafish, an effect attributed to the robust anti-inflammatory properties of both EcN and Ven.⁷³

Additionally, a marked increase in NF-κB levels was observed in the brain tissue of zebrafish administered reserpine, suggesting a potential role for the apoptotic pathway. Our findings align with those of Ruster et al., who reported activated NF-κB and elevated Nε-(carboxymethyl)lysine levels in the serum of individuals with chronic pain.⁷⁴ In this study, treatment with EcN and Ven substantially attenuated NF-κB levels in the brains of reserpinized zebrafish.⁷⁵ These outcomes are consistent with investigations by Bialek et al., who proposed Ven as a potent inhibitor of NF-κB in various cell types.⁷⁶ This study posits that excessive ROS production induced by reserpine leads to the generation of inflammatory and pro-inflammatory cytokines, contributing to neuronal damage and implicating neurotoxicity and neurological inflammation.⁷⁷ This study is limited by its focus on zebrafish, which may not fully replicate the complexities of mammalian depression. Future research investigating the specific mechanisms of EcN’s action, including its impact on neurotransmitter systems and neural pathways, and exploring its efficacy in mammalian models and clinical trials, is warranted. Further studies could also examine the long-term effects of EcN treatment and its potential interactions with other therapeutic agents.

Conclusions

This study highlights the multifaceted benefits of EcN, driven by its anti-inflammatory and antioxidant properties, particularly in counteracting reserpine-induced neuroinflammation. The modulation and restoration of the balance between pro-inflammatory and anti-inflammatory cytokines may mediate these effects. While Ven demonstrated superior improvements, EcN was equally effective in reversing reserpine-induced depression-like symptoms to an appreciable extent, with no observable side effects. Reserpine-induced neurochemical alterations and nitroductive inflammatory cascade might contribute to depression and pain symptoms in zebrafish. EcN’s multitargeted action intervenes in this cascade, potentially mitigating depression-like symptoms. Detailed research on the precise mechanism underlying the anti-inflammatory and antioxidant activities of EcN would further expand its role in the treatment of depression-like phenotypes associated with other neurological complications.

Supplemental Material

Supplemental material for this article available online.

Supplemental Material

Supplemental material for this article available online.

Footnotes

Acknowledgements

The authors thank Prof. Murugesan R for his guidance and support. The authors acknowledge Ardeypharm, Germany, for providing the EcN1917 strain. The authors sincerely thank Dr. Jesu Arockia Raj A, Professor, Department of Biotechnology, SRM University, Chennai, Tamil Nadu, for assistance with enzyme assays. The authors also thank Dr. Sujatha Sunil, Group Leader, Vector Borne Diseases, ICGEB, New Delhi, for her generous support in performing ELISA. The authors acknowledge the management of Chettinad Academy of Research and Education and the Department of Biotechnology, SRM University, for providing infrastructure facilities. Mohammed V acknowledges CARE for the fellowship support.

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Declaration Regarding the Use of Generative AI

No part of this article was written or generated by a generative AI tool. The authors take full responsibility for the accuracy, integrity, and originality of the published article.

Ethical Approval

Ethical approval for the study was obtained from the Institutional Animal Ethics Committee (IAEC) of Chettinad Hospital and Research Institute (Approval Ref. No.: IAEC 3/Proposal:86/A.Lr:62; Date: 22 August 2022).

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Informed Consent

Not applicable.

Prior Presentation

The authors confirm that this work has not been previously presented.

PROSPERO/CTRI details

Not Applicable.

Simultaneous Submission

The authors confirm that this manuscript is not under consideration elsewhere.

Status of your study (for Study Protocol)

Not Applicable.

Trial Registration

Not applicable.

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