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. 2022 Dec 22;13(1):22. doi: 10.1007/s13205-022-03438-2

Comparative evaluation of natural neuroprotectives and their combinations on chronic immobilization stress-induced depression in experimental mice

Ibrahim M Ibrahim 1, Mohammed Alsieni 1, Sami G Almalki 2, Yaser E Alqurashi 3, Vinay Kumar 4,✉
PMCID: PMC9780413  PMID: 36568496

Abstract

The present study evaluates the potential of neuroprotective phytochemicals-rutin (R), resveratrol (Res), 17β-estradiol (17β-E2), and their different combinations against chronic immobilization stress (CIS)-induced depression-like behaviour in male albino mice. Here, the mice were exposed to stress via immobilization of their four limbs under a restrainer for 6 h daily until 7 days of the induction after 30 min of respective drug treatment in different mice groups. The result found the protective effect of these phytoconstituents and their combinations against CIS-induced depression due to their ability to suppress oxidative stress, restore mitochondria, HPA-axis modulation, neurotransmitter level, stress hormones, and inflammatory markers. Also, the combination drug regimens of these phytoconstituents showed synergistic results in managing the physiological and biochemical features of depression. Thus, these neuroprotective could be utilized well in combination to manage depression-like symptoms during episodic stress. Furthermore, such results could be well justified when administered in polyherbal formulation with these neuroprotective as major components. In addition, an advanced study can be designed at the molecular and epigenetics level using a formulation based on these neuroprotective.

Keywords: Chronic stress, Depression, Neuroprotectants, Rutin, Resveratrol, 17-β estradiol

Introduction

Depression is generally illustrated by the symptoms such as depressed mood, loss of interest, feelings of guilt or worthlessness, disordered sleep, appetite, fatigue, malaise, lack of focus, suicidal ideations, etc. (Fava and Kendler 2000). It is extremely troublesome to evade animal model, which perfectly mimics the clinical signs of depression (Yan et al. 2010). However, chronic or mild stress could induce depression-like behaviour in animals (Dhir et al. 2006; Kumari et al. 2007; Bhutani et al. 2009; Kumar and Garg 2009; Kumar et al. 2009; Kasala et al. 2014), which has been utilized to establish animal model via straining them using immobilization or exposure to cold/low temperature. In addition, the forced swim test (FST) is one of the paradigms to screen drugs with antidepressant potential against the various forms of stress exposure (Kumar and Goyal 2008). Also, the activation of the autonomic nervous system (ANS) and hypothalamic–pituitary–adrenal (HPA) axis is the hallmark of the stress response, causing the release of stress hormones (Gregus et al. 2005; Marais et al. 2008). In depression, corticosteroids get released and act on localized receptors in the brain's hippocampus, striatum, and cortex regions (Holsboer 2001). Thus, the corticosterone level in brain tissues and serum significantly increases post-CIS, recognized as the marker of episodic stressful events. Besides this, body weight also gets influenced, suggesting the significant interplay of the HPA axis in the pathophysiology of stress and depression (Bazhan and Zelena 2013). Also, the elevation in oxygen-free radicals has been observed in patients with depression (Michel et al. 2007; Szuster-Ciesielska et al. 2008).

The mitochondria are the critical pool of superoxide radicals accompanying oxidative stress. It is evident from the documents available that mitochondrial impairment is crucial in the pathophysiology of stress-related disorders, especially depression (Rezin et al. 2008, 2009; Seppet et al. 2009). Dysfunction of mitochondria dysfunction is correlated with simultaneous decreased production of ATP-elevated ROS production and is one of the features of apoptosis (Quiroz et al. 2008). It has been reported that various antidepressants also nullify the impact of stress-induced mitochondrial dysfunction. The degree of mitochondrial dysfunction can be very well correlated to the regulated activities of mitochondrial complexes (I–IV) (Scaini et al. 2011), enhanced CYT-C release, and caspase-3 activity (Levkovitz et al. 2005), uncoupling of OXPHOS, sensitizes Ca2+-mediated MPT, etc. (Li et al. 2012). In a nutshell, stress is often associated with elevated ROS and mitochondrial damage in stress-induced depression. Therefore, the role of ROS and mitochondrial restoration in chronic immobilization stress (CIS)-induced depression-like behaviour is still needed to be explored further.

The majority of depressed patients have been continuously noted with raised pro-inflammatory cytokines, enhanced expression of chemokines, and adhesion molecules, which are clear manifestations of the inflammatory response towards external stress/stimuli (Musselman et al. 2001; Alesci et al. 2005). Therefore, elevated serum levels of tumour necrosis factor (TNF-α) and prostaglandins have been reported in central nervous system disorders (Mikova et al. 2001; Tuglu et al. 2003; Raison et al. 2006). Besides all, depression is a complex disorder resulting from the altered nor-adrenergic, serotonergic, and dopaminergic or adrenomedullary innervation of the CNS. The decreased levels of dopamine (DA), noradrenaline (NA), and serotonin (5-HT) indicate a state of depression (Konstandi et al. 2000; Baik 2020).

Rutin (R) is a glycoside flavanol that is immensely found in many plants, and studies suggest antidepressant potential (Machado et al. 2008; Dimpfel 2009). Similarly, resveratrol (Res) is a polyphenol immensely found in red wine and grapes; it possesses antidepressant potential (Xu et al. 2010; Ge et al. 2013). Further, 17-β estradiol (17β-E2) has also been documented as an antidepressant in some ways (Molina-Hernández et al. 2009; Österlund 2010; Wang et al 2019). Therefore, we have employed the CIS model to induce depressive behaviour in albino mice to explore the efficacy of three natural neuroprotective agents, R, Res, 17-βE2, and their different combinations to attenuate the physiological and experimental changes induced as a consequence of CIS. The objective of the study involves the estimation of neurobehavioral parameters, oxidative stress, and mitochondrial enzymes complex in different brain regions of the respective study groups.

Materials and methods

Animals

Male albino mice weighing 25–35 g were procured from the animal house facility of KIET school of Pharmacy (KSOP), Ghaziabad, India, for carrying out the animal experimentation after approval was granted from the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA) with approval number IAEC/KSOP/2020-21/15. All animals were housed under standard laboratory conditions with open access to a standard laboratory pellet diet and water ad libitum.

Drugs and formulation

The phytochemicals—R, Res, 17β-E2, and imipramine (Imp)—were purchased from Sigma-Aldrich, St. Louis, MO, USA. Among them, only Imp was formulated as a solution in sterile 0.9% sodium chloride (NaCl) solution, while rest of the phytochemicals were formulated as a suspension in 0.5% w/v sodium carboxy-methyl-cellulose (CMC) solution in sterile water for injection.

Study design

Mice were randomly divided into twelve experimental groups (I–XII) with twelve animals in each groups as follows: Group I: naive; Group II: vehicle control (0.5% CMC w/v + 0.9% w/v NaCl) + CIS; Group III: Imp-10 (Imipramine-10 mg/kg, i.p.) + CIS; Group IV: R-20 (Rutin-20 mg/kg, p.o.) + CIS; Group V: R-40 (Rutin-40 mg/kg, p.o.) + CIS; Group VI: Res-5 (Resveratrol-5 mg/kg, p.o.) + CIS; Group VII: Res-10 (Resveratrol-10 mg/kg, p.o.) + CIS; Group VIII: 17β-E2-40 (17β-estradiol-40 mg/kg, p.o.) + CIS; Group IX: 17β-E2-80 (80 mg/kg, p.o.) + CIS; Group X: R-40 + Res-10 + CIS; Group XI: R-40 + 17β-E2-80 + CIS; Group XII: Res-10 + 17β-est-80 + CIS. The mice groups II to XII were exposed to 6 h of chronic immobilization stress (CIS) daily for 7 days under ambient temperature (25 °C), in which all four limbs of mice were tied separately and then fixed using zinc oxide medical tape, followed by putting in restraint chamber with the tail being tied onto the side to immobilize the mice completely. The same routine was followed for 7 days after 30 min (min) of dosing the drug treatment as scheduled accordingly in each group. The parameters, including body weight and behavioural assessments (locomotor activity and forced swim test), were conducted on days 1, 3, and 7 of the study. After 7 days, blood was collected through the tail vein to isolate serum from each mouse and kept starved overnight. The next day (8th day), all mice were euthanized under ketamine (80 mg/kg, i.m.): xylazine (10 mg/kg, i.m.) anaesthesia, followed by dissecting the brain section under chilled ice (4 °C) environment (Navarro et al. 2021). The schematic representation of the experimental protocol followed is described in Fig. 1.

Fig. 1.

Fig. 1

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HYPERLINK "sps:id::fig1||locator::gr1||MediaObject::0" The schematic representation of experimental design along with treatment schedule. Where CIS (Chronic immobilization stress); Imp-10 (Imipramine-10 mg/kg, i.p.); R-20 (Rutin-20 mg/kg, p.o.); R-40 (Rutin-40 mg/kg, p.o.); Res-5 (Resveratrol-5 mg/kg, p.o.); Res-10 (Resveratrol-10 mg/kg, p.o.); 17β-E2-40 (17β-estradiol-40 mg/kg, p.o.); and 17β-E2-80 (80 mg/kg, p.o.)

Behavioural assessments

Locomotor activity

The effect of drug treatment on locomotor activity of CIS-depressive mice was evaluated by photoelectric activity cages, i.e. actophotometer, National Scientific Apparatus Works, India. Mice were placed in an actophotometer firstly for 3 min of run time as a habituation period and later next 5 min of run time for recording actual locomotor activity after 30 min of drug treatment (Mundugaru et al. 2018).

Forced-swim behaviour assessment

The test procedure was carried out using the description given by Kulkarni and his team (Kulkarni et al. 2008). In a nutshell, the mice were dropped to swim for 2–3 min in a rectangular glass jar of dimension (25 × 12 × 25 cm3) maintained at 15 cm water level and 23–25 °C. After that, animals displayed a phase of immobility with no or minimum movements. The mice were considered immobile when they remained floating passively with a slightly hunched and upright position, having a nose above the surface of the water. The duration of the immobility period was recorded for 6 min with the help of a stop watch (Goyal and Kumar 2007; Kumar et al. 2008, 2009; Aggarwal et al. 2010).

Total protein estimation in different brain parts

The total protein content of each brain part (hippocampus, striatum, and cortex) was measured using BSA as standard (Shang et al. 2018).

Measurement of oxidative stress parameters

Animals were killed with prior anesthetization followed by cervical dislocation, and the brains were extracted from the skull, rinsed in isotonic saline, and weighed containing hippocampus, striatum, and cortex collected, and then, 100 mg tissue was immediately homogenized. 10% (w/v) tissue homogenates of hippocampus, striatum, and cortex were prepared in phosphate buffer (0.1 M, pH = 7.4) with centrifugation at 3000 rpm for 10 min at 4–8 °C, and the supernatant was utilized for different biochemical estimations, including lipid peroxidation (LPO), nitrite (NO), catalase (CAT), and reduced glutathione (GSH) assays (Goyal and Kumar 2007). The total protein amount in each homogenate was measured according to the Lowry method as previously prescribed (Shang et al. 2018).

Estimation of various mitochondrial complexes in different brain regions

Protocol for isolation of mitochondria from individual brain parts

The isolated cryopreserved brain samples of the hippocampus, cortex, and striatum were separately put into the cold lysis buffer (5 ml/g) and vortexed for 5 min immediately. Then, tissues will be homogenized 8–10 times by an electro-homogenizer. Then, centrifugation of homogenate at 1500g was done for 10 min at 4 °C. Next, collect the supernatant and centrifuge at 10,000g for 10 min at 4 °C. Collect the pellet (crude mitochondria) and re-suspend with 3 ml store medium and centrifuge at 10,000g for 5 min at 4 °C. The finally obtained pellet will be the purified mitochondria. It will be re-suspended with a store buffer (1 ml). The activity and purity of mitochondrial will be assessed by Janus green B stain and electro-scope detection. The mitochondrial protein concentration will be determined using the Bradford method. Finally, the activities of Complex I (NADH dehydrogenase), Complex II (Succinate dehydrogenate), and Complex IV (Cytochrome oxidase) were detected according to the instructions based on the enzyme–substrate reaction (Wang et al. 2016).

Mitochondrial redox activity (MTT) assay

The in vitro approach used in this study is based on the assessment of live cells through their capability of mitochondria to produce NADPH, which can be assessed by the number of purple formazan crystals formed upon enzymatic reaction of MTT tetrazolium salt with mitochondrial dehydrogenase present in viable cells seeded in the culture medium. The amount of formazan crystals produced is dissolved by DMSO, which is measured by reading the absorbance at 580 nm in ELISA reader (Martínez‐Martos et al. 2000).

Estimation for serum corticosterone, PGF-2α, TNF-α, and tissue levels of biogenic amines

Biogenic amines, namely DA, NA, and 5-HT, were estimated using the apparatus HPLC Waters System, which was operated at high pressure using the isocratic pump, C18 reverse phase column, sample (20 μl) injector valve, an electrochemical detector (+ 0.75 V and 5–50 nÅ of sensitivity with separation at 0.8 ml/min flow rate), and the mobile phase (4.5 pH sodium citrate buffer and acetonitrile in 87:13 v/v). On sample run day, the frozen brain samples were thawed, followed by homogenization for 5 min in 0.2 M perchloric acid mobile phase at 12,000 gauge. The supernatant obtained was filtered through 0.22-μm nylon filters before injecting into the HPLC injection pump. The recorded data were analysed using Empower software (Huang et al. 2019).

Statistical analysis of data

All the data values were analysed by applying one-way analysis of variance (ANOVA) along with Tukey’s test and were expressed as mean ± SEM, with p < 0.05 being considered statistically significant.

Results

Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on body weight of CIS-depressed mice

CIS (6 h daily for 7 days) on mice significantly (p < 0.05) reduced the body weight compared to naive animals. The subsequent oral administration of R-40, Res-10, and 17β-E2-80 for 7 days in CIS-depressed mice significantly (p < 0.05) recovered bodyweight towards naive mice compared to CIS-vehicle-treated mice shown in Table 1. The protective effects of R-40, Res-10, and 17β-E2-80 treatment were comparable with that of the Imp-10-treated mice group for 7 days. However, low-dose treatments such as R-20, Res-5, and 17β-E2-40 to CIS-mice groups did not significantly (p > 0.05) affect the body weight compared to vehicle-treated-CIS mice group (Table 1).

Table 1.

Interaction of neuroprotective interventions on body weight of CIS-depressed mice

S. no. Treatment groups (mg/kg) Body weight (gm)
1st Day 3rd Day 7th Day
1 Naive 28 ± 0.31 32 ± 0.62 36 ± 0.19
2 CIS 30 ± 0.22 28 ± 0.49 20 ± 0.82a
3 Imip-10 + CIS 30 ± 0.81 32 ± 0.74 36 ± 0.91b
4 R-20 + CIS 28 ± 0.52 26 ± 0.76 22 ± 0.19 ns
5 R-40 + CIS 28 ± 0.53 30 ± 0.91 34 ± 0.47b
6 Res-5 + CIS 28 ± 0.48 30 ± 0.92 24 ± 0.81 ns
7 Res-10 + CIS 28 ± 0.49 32 ± 0.62 34 ± 0.76b
8 17β-E2-40 + CIS 30 ± 0.73 28 ± 0.68 22 ± 0.71 ns
9 17β-E2-80 + CIS 28 ± 0.84 32 ± 0.73 34 ± 0.62b
10 R-40 + Res-10 + CIS 28 ± 0.36 34 ± 0.51 34 ± 0.17b
11 R-40 + 17β-E2-80 + CIS 29 ± 0.58 32 ± 0.21 36 ± 0.94b
12 Res-10 + 17β-E2-80 + CIS 27 ± 0.79 32 ± 0.67 34 ± 0.84b
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Values are expressed mean ± S.E.M. (One-way ANOVA followed by Tukey’s test)

Where CIS (Chronic immobilization stress); Imp-10 (Imipramine-10 mg/kg, i.p.); R-20 (Rutin-20 mg/kg, p.o.); R-40 (Rutin-40 mg/kg, p.o.); Res-5 (Resveratrol-5 mg/kg, p.o.); Res-10 (Resveratrol-10 mg/kg, p.o.); 17β-E2-40 (17β-estradiol-40 mg/kg, p.o.); 17β-E2-80 (80 mg/kg, p.o.)

aMeans p < 0.05 as compared to Naïve; bMeans p < 0.05 as compared to CIS; nsMeans not-significant, i.e. p > 0.05

Further, the combination treatments (R-40 + Res-10, R-40 + 17β-E2-80, and Res-10 + 17β-E2-80) significantly (p < 0.05) improved their body weight on the 7th day as compared to the vehicle-treated-CIS mice group (Table 1). Besides, per se treatment of R-40, Res-10, and 17β-E2-80 did not produce any significant (p > 0.05) effect on body weight compared to the naive group.

Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on locomotor activity of CIS-depressed mice

CIS (6 h daily for 7 days) on mice impaired locomotor activity on 1st, 3rd, and 7th day compared to naive mice group. The low-dose treatment groups, such as R-20, Res-5, and 17β-E2-40, for 7 days did not significantly (p > 0.05) affect the locomotor activity compared to the IS-induced vehicle-treated mice group (Fig. 2). However, their high-dose treatment, such as R-40, Res-10, and 17β-E2-80 for 7 days significantly (p < 0.05), improved the locomotor activity on the 3rd and 7th day compared to the vehicle-treated-CIS mice group (Fig. 2). Further, the protective effects of these drugs were comparable with that of Imp-10 treatment for 7 days.

Fig. 2.

Fig. 2

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Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on locomotor activity of CIS-depressed mice. Values are expressed as mean ± S.E.M. using one-way ANOVA along with Tukey’s test. aMeans p < 0.05 compared to Naive; bMeans p < 0.05 compared to CIS; cMeans p < 0.05 compared to R-20 + CIS; dMeans p < 0.05 compared to Res-5 + CIS; eMeans p < 0.05 compared to 17β-E2-40 + CIS; fMeans p < 0.05 compared to R-40 + CIS; gMeans p < 0.05 compared to Res-10 + CIS; hMeans p < 0.05 compared to 17β-E2-80 + CIS; nsMeans non-significant, i.e., p > 0.05. Where CIS (Chronic immobilization stress); Imp-10 (Imipramine-10 mg/kg, i.p.); R-20 (Rutin-20 mg/kg, p.o.); R-40 (Rutin-40 mg/kg, p.o.); Res-5 (Resveratrol-5 mg/kg, p.o.); Res-10 (Resveratrol-10 mg/kg, p.o.); 17β-E2-40 (17β-estradiol-40 mg/kg, p.o.); and 17β-E2-80 (80 mg/kg, p.o.)

Further, the combinations (R-40 + Res-10, 17β-E2-80 + R-40, and Res-10 + 17β-E2-80) significantly (p < 0.05) improved the locomotor activity on the 3rd and 7th day, which was significant (p < 0.05) compared to their effect per se (Fig. 2). However, the R-40, Res-10, and 17β-E2-80 per se treatment group did not significantly (p > 0.05) affect the locomotor activity compared to the naive group.

Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on immobility period CIS-depressed mice

CIS (6 h daily for 7 days) on mice significantly (p < 0.05) increased immobility compared to the naive group. Oral administration of R-40, Res-10, and 17β-E2-80 for 7 days significantly (p > 0.05) attenuated immobility compared to CIS-induced vehicle-treated mice (Fig. 3). However, low-dose treatments such as R-20, Res-5, and 17β-E2-40 did not show any significant (p > 0.05) effect on the immobility period compared to CIS-induced vehicle-treated mice (Fig. 3). Further, the protective effects of these drugs were comparable with that of Imp-10 treatment for 7 days.

Fig. 3.

Fig. 3

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Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on FST behaviour period of CIS-depressed mice. Values are expressed as mean ± S.E.M. using one-way ANOVA along with Tukey’s test. a means p < 0.05 compared to Naive; b means p < 0.05 compared to CIS; c means p < 0.05 compared to R-20 + CIS; d means p < 0.05 compared to Res-5 + CIS; e means p < 0.05 compared to 17β-E2-40 + CIS; f means p < 0.05 compared to R-40 + CIS; g means p < 0.05 compared to Res-10 + CIS; h means p < 0.05 compared to 17β-E2-80 + CIS; ns means non-significant, i.e. p > 0.05. Where CIS (Chronic immobilization stress); Imp-10 (Imipramine-10 mg/kg, i.p.); R-20 (Rutin-20 mg/kg, p.o.); R-40 (Rutin-40 mg/kg, p.o.); Res-5 (Resveratrol- 5 mg/kg, p.o.); Res-10 (Resveratrol-10 mg/kg, p.o.); 17β-E2-40 (17β-estradiol-40 mg/kg, p.o.) and 17β-E2-80 (80 mg/kg, p.o.)

Further, the combination (R-40 + Res-10, 17β-E2-80 + R-40, and Res-10 + 17β-E2-80) significantly (p < 0.05) potentiated their protective effect as evidenced with the significant attenuation of their immobility period compared to their effect per se (Fig. 3). However, the R-20, R-40, Res-5, Res-10, 17β-E2-40 and, 17β-E2-80 per se treatment group did not elicit any significant (p < 0.05) immobility compared to the naive group.

Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on anti-oxidant system of CIS-depressed brain regions of mice

CIS (6 h daily for 7 days) on mice significantly (p < 0.05) caused oxidative damage, as evident with the elevated LPO, NO accumulation, depletion of GSH and CAT activity in brain regions, i.e. hippocampus, cerebral cortex, and striatum, and as compared to the naive group. On the other hand, the low-dose treatment such as R-20, Res-5, and 17β-E2-40 did not significantly (p > 0.05) affect oxidative damage in the cerebral cortex, striatum, and hippocampus regions compared to the CIS-induced mice group (Table 2). However, high-dose treatment such as R-40, Res-10, and 17β-E2-80 significantly (p < 0.05) attenuated LPO, NO accumulation, and restored GSH, and CAT activity in the cerebral cortex, striatum, and hippocampus regions of the brain as compared to the CIS-induced mice group (Table 2). Further, the protective effects of these drugs were comparable with that of Imp (10 mg/kg) treatment for 7 days. However, R-40, Res-10, and 17β-E2-80 per se treatment group did not significantly (p > 0.05) affect the oxidative stress markers in the cerebral cortex, striatum, and hippocampus regions of the brain compared to the naive group.

Table 2.

Interaction of neuroprotective interventions on antioxidant levels of CIS-depressed brain regions of mice

Treatment group (mg/kg) Different brain area LPO
(n mole of MDA/mg protein)		 Nitrate
(μ mole of Nitrite/mg protein)		 Catalase
(μ mole of H2O2/min/mg protein)		 GSH
(μ mole of GSH/mg protein)

N

A

I

V

E

 Hippocampus 0.162 ± 0.023 315 ± 61 0.701 ± 0.034 0.062 ± 0.0017
Striatum 0.159 ± 0.041 310 ± 12 0.697 ± 0.051 0.058 ± 0.0021
Cortex 0.165 ± 0.022 319 ± 17 0.708 ± 0.041 0.068 ± 0.0034
CIS Hippocampus 0.622 ± 0.03a 638 ± 13a 0.123 ± 0.021a 0.014 ± 0.0012
Striatum 0.618 ± 0.045 633 ± 21a 0.119 ± 0.031a 0.010 ± 0.0037a
Cortex 0.627 ± 0.062a 642 ± 14a 0.127 ± 0.052 a 0.017 ± 0.0036a
Imip-10 + CIS Hippocampus 0.186 ± 0.061b 357 ± 19b 0.689 ± 0.046b 0.059 ± 0.0087b
Striatum 0.175 ± 0.021b 359 ± 12b 0.684 ± 0.016b 0.056 ± 0.008b
Cortex 0.191 ± 0.062b 362 ± 18b 0.697 ± 0.016b 0.064 ± 0.008b
R-20 + CIS Hippocampus 0.612 ± 0.019 ns 629 ± 12 ns 0.138 ± 0.025 ns 0.016 ± 0.002 ns
Striatum 0.607 ± 0.046 ns 631 ± 18 ns 0.124 ± 0.025 ns 0.013 ± 0.003 ns
Cortex 0.615 ± 0.092 ns 634 ± 16 ns 0.135 ± 0.015 ns 0.019 ± 0.001 ns
R-40 + CIS Hippocampus 0.326 ± 0.071b 456 ± 17b 0.289 ± 0.017b 0.029 ± 0.006b
Striatum 0.317 ± 0.017b 458 ± 28b 0.251 ± 0.027b 0.024 ± 0.006b
Cortex 0.324 ± 0.034b 461 ± 15b 0.271 ± 0.012b 0.037 ± 0.0011b
Res-5 + CIS Hippocampus 0.614 ± 0.021 ns 626 ± 52 ns 0.142 ± 0.033 ns 0.015 ± 0.0014 ns
Striatum 0.607 ± 0.033 ns 629 ± 32 ns 0.129 ± 0.019 ns 0.014 ± 0.0010 ns
Cortex 0.615 ± 0.041 ns 631 ± 14 ns 0.135 ± 0.021 ns 0.017 ± 0.0014 ns
Res-10 + CIS Hippocampus 0.329 ± 0.052b 458 ± 25b 0.291 ± 0.042b 0.028 ± 0.0028b
Striatum 0.321 ± 0.043b 461 ± 18b 0.257 ± 0.031b 0.026 ± 0.0037b
Cortex 0.328 ± 0.034b 463 ± 19b 0.271 ± 0.031b 0.038 ± 0.0028b
17β-E2-40 + CIS Hippocampus 0.616 ± 0.062 ns 631 ± 32 ns 0.135 ± 0.013 ns 0.017 ± 0.0014 ns
Striatum 0.611 ± 0.066 ns 633 ± 42 ns 0.121 ± 0.019 ns 0.012 ± 0.0010 ns
Cortex 0.619 ± 0.081 ns 635 ± 17 ns 0.135 ± 0.022 ns 0.02 ± 0.0014 ns
17β-E2-80 + CIS Hippocampus 0.330 ± 0.067b 460 ± 25b 0.284 ± 0.032b 0.03 ± 0.0028b
Striatum 0.320 ± 0.074b 462 ± 26b 0.248 ± 0.021b 0.025 ± 0.0037b
Cortex 0.327 ± 0.063b 464 ± 15b 0.271 ± 0.011b 0.039 ± 0.0028b
R-40 + Res-10 + CIS Hippocampus 0.185 ± 0.031b,f,g 369 ± 13b,f,g 0.658 ± 0.15b,f,g 0.057 ± 0.004b,f,g
Striatum 0.199 ± 0.041b,f,g 371 ± 18b,f,g 0.654 ± 0.011b,f,g 0.054 ± 0.0041b,f,g
Cortex 0.194 ± 0.034b,h,g 373 ± 11b,f,g 0.674 ± 0.013b,f,g 0.061 ± 0.005b,f,g
R-40 + 17β-E2-80 + CIS Hippocampus 0.192 ± 0.073b,h,g 374 ± 26b,h,g 0.664 ± 0.024b,h,g 0.056 ± 0.0074b,h,g
Striatum 0.204 ± 0.057b,h,g 376 ± 17b,h,g 0.651 ± 0.015b,h,g 0.051 ± 0.0093b,h,g
Cortex 0.197 ± 0.061b,f,g 378 ± 15b,h,g 0.681 ± 0.051b,h,g 0.064 ± 0.0074b,h,g
Res-10 + 17β-E2-80 + IS Hippocampus 0.196 ± 0.014b,f,g 364 ± 12b,f,g 0.641 ± 0.013b,f,g 0.057 ± 0.0094b,f,g
Striatum 0.206 ± 0.043b,f,g 366 ± 37b,f,g 0.661 ± 0.019b,f,g 0.053 ± 0.0073b,f,g
Cortex 0.16 ± 0.023b,f,g 368 ± 19b,f,g 0.678 ± 0.017b,f,g 0.062 ± 0.0014b,f,g
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Values are expressed as Mean ± S.E.M. using one-way ANOVA along with Tukey’s test

Where CIS (Chronic immobilization stress); Imp-10 (Imipramine-10 mg/kg, i.p.); R-20 (Rutin-20 mg/kg, p.o.); R-40 (Rutin-40 mg/kg, p.o.); Res-5 (Resveratrol- 5 mg/kg, p.o.); Res-10 (Resveratrol-10 mg/kg, p.o.); 17β-E2-40 (17β-estradiol-40 mg/kg, p.o.); 17β-E2-80 (80 mg/kg, p.o.)

aMeans p < 0.05 as compared to Naive; bMeans p < 0.05 as compared to CIS; cMeans p < 0.05 as compared to R-20; dMeans p < 0.05 as compared to Res-5; eMeans p < 0.05 as compared to 17β-E2-40; fMeans p < 0.05 as compared to R-40; gMeans p < 0.05 as compared to Res-10; hMeans p < 0.05 as compared to 17β-E2-80; nsMeans non-significant, i.e. p > 0.05

Further, the combinations (R-40 + Res-10, 17β-E2-80 + R-40, and Res-10 + 17β-E2-80) significantly (p < 0.05) potentiated their protective effect as evident with attenuation in elevated LPO levels and NO accumulation along with restoration of GSH and CAT activity in the cerebral cortex, striatum, and hippocampus regions of the brain as compared to their effect per se (Table 2).

Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on different mitochondrial enzyme complex activities of CIS-depressed brain regions of mice

CIS (6 h daily for 7 days) on mice significantly (p < 0.05) impaired mitochondrial enzyme complexes (I, II, and IV) and MTT (mitochondrial redox) expression in the cerebral cortex, striatum, and hippocampus as compared to the naive group. The treatment with R-40, Res-10, and 17β-E2-80 for 7 days significantly (p < 0.05) restored mitochondrial complex enzyme activity (I, II, and IV) and MTT activities in the hippocampus, striatum, and cerebral cortex regions of the brain as compared to the CIS-induced mice group (Fig. 5). However, low-dose treatments such as R-20, Res-5, and 17β-E2-40 for 7 days did not significantly (p > 0.05) affect the mitochondrial enzyme complexes (I, II, and IV) and MTT (mitochondrial redox) activities in different brain regions compared to the CIS-induced mice group. The protective effects of these drugs were comparable with that of Imp-10 treatment for 7 days. However, R-40, Res-10, and 17β-E2-80 per se treatment group did not produce any significant (p > 0.05) effect on the mitochondrial enzyme complexes (I, II, and IV) and MTT (mitochondrial redox) activities in the hippocampus, striatum, and cerebral cortex regions of the brain as compared to the naive group.

Fig. 5.

Fig. 5

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Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on different mitochondrial enzyme complex-II activities of CIS-depressed brain regions of mice. Values are expressed as mean ± S.E.M. using one-way ANOVA along with Tukey’s test. aMeans p < 0.05 compared to Naive; bMeans p < 0.05 compared to CIS; cMeans p < 0.05 compared to R-20 + CIS; dMeans p < 0.05 compared to Res-5 + CIS; eMeans p < 0.05 compared to 17β-E2-40 + CIS; fMeans p < 0.05 compared to R-40 + CIS; gMeans p < 0.05 compared to Res-10 + CIS; hMeans p < 0.05 compared to 17β-E2-80 + CIS; nsMeans non-significant, i.e. p > 0.05. Where CIS (Chronic immobilization stress); Imp-10 (Imipramine-10 mg/kg, i.p.); R-20 (Rutin-20 mg/kg, p.o.); R-40 (Rutin-40 mg/kg, p.o.); Res-5 (Resveratrol- 5 mg/kg, p.o.); Res-10 (Resveratrol-10 mg/kg, p.o.); 17β-E2-40 (17β-estradiol-40 mg/kg, p.o.); and 17β-E2-80 (80 mg/kg, p.o.)

Further, the combinations (R-40 + Res-10, 17β-E2-80 + R-40, and Res-10 + 17β-E2-80 mg/kg) significantly (p < 0.05) restored mitochondrial complex enzyme activity (I, II, and IV) and MTT activities as compared to their effect per se (CIS) (Figs. 4, 5, 6, and 7).

Fig. 4.

Fig. 4

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Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on different mitochondrial enzyme complex-I activities of CIS-depressed brain regions of mice. Values are expressed as mean ± S.E.M. using one-way ANOVA along with Tukey’s test. aMeans p < 0.05 compared to Naive; bMeans p < 0.05 compared to CIS; cMeans p < 0.05 compared to R-20 + CIS; dMeans p < 0.05 compared to Res-5 + CIS; eMeans p < 0.05 compared to 17β-E2-40 + CIS; fMeans p < 0.05 compared to R-40 + CIS; g means p < 0.05 compared to Res-10 + CIS; h means p < 0.05 compared to 17β-E2-80 + CIS; nsMeans non-significant, i.e. p > 0.05. Where CIS (Chronic immobilization stress); Imp-10 (Imipramine-10 mg/kg, i.p.); R-20 (Rutin-20 mg/kg, p.o.); R-40 (Rutin-40 mg/kg, p.o.); Res-5 (Resveratrol-5 mg/kg, p.o.); Res-10 (Resveratrol-10 mg/kg, p.o.); 17β-E2-40 (17β-estradiol-40 mg/kg, p.o.); and 17β-E2-80 (80 mg/kg, p.o.)

Fig. 6.

Fig. 6

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Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on mitochondrial enzyme MTT activity of CIS-depressed brain regions of mice. Values are expressed as mean ± S.E.M. using one-way ANOVA along with Tukey’s test. aMeans p < 0.05 compared to Naive; bMeans p < 0.05 compared to CIS; cMeans p < 0.05 compared to R-20 + CIS; dMeans p < 0.05 compared to Res-5 + CIS; eMeans p < 0.05 compared to 17β-E2-40 + CIS; fMeans p < 0.05 compared to R-40 + CIS; gMeans p < 0.05 compared to Res-10 + CIS; hMeans p < 0.05 compared to 17β-E2-80 + CIS; ns means non-significant, i.e. p > 0.05. Where CIS (Chronic immobilization stress); Imp-10 (Imipramine-10 mg/kg, i.p.); R-20 (Rutin-20 mg/kg, p.o.); R-40 (Rutin-40 mg/kg, p.o.); Res-5 (Resveratrol- 5 mg/kg, p.o.); Res-10 (Resveratrol-10 mg/kg, p.o.); 17β-E2-40 (17β-estradiol-40 mg/kg, p.o.); and 17β-E2-80 (80 mg/kg, p.o.)

Fig. 7.

Fig. 7

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Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on different mitochondrial enzyme complex-IV activities of CIS-depressed brain regions of mice. Values are expressed as mean ± S.E.M. using one-way ANOVA along with Tukey’s test. aMeans p < 0.05 compared to Naive; bMeans p < 0.05 compared to CIS; cMeans p < 0.05 compared to R-20 + CIS; dMeans p < 0.05 compared to Res-5 + CIS; eMeans p < 0.05 compared to 17β-E2-40 + CIS; fMeans p < 0.05 compared to R-40 + CIS; gMeans p < 0.05 compared to Res-10 + CIS; hMeans p < 0.05 compared to 17β-E2-80 + CIS; nsMeans non-significant, i.e. p > 0.05. Where CIS (Chronic immobilization stress); Imp-10 (Imipramine-10 mg/kg, i.p.); R-20 (Rutin-20 mg/kg, p.o.); R-40 (Rutin-40 mg/kg, p.o.); Res-5 (Resveratrol- 5 mg/kg, p.o.); Res-10 (Resveratrol-10 mg/kg, p.o.); 17β-E2-40 (17β-estradiol-40 mg/kg, p.o.); and 17β-E2-80 (80 mg/kg, p.o.)

Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on serum corticosterone levels of CIS-depressed mice

CIS (6 h daily for 7 days) on mice for significantly (p < 0.05) increased serum corticosterone level compared to the naive group. Seven days of oral administration with R-40, Res-10, and 17β-E2-80 significantly (p < 0.05) attenuated serum corticosterone levels compared to the CIS-induced depressive mice group (Fig. 8). However, treatment with the lower dose of R-20, Res-5, and 17β-E2-40 did not significantly (p > 0.05) affect serum corticosterone levels compared to the CIS-induced depressive mice group. Further, their protective effects were comparable with that of Imp-10 treatment for 7 days. However, the R-40, Res-10, and 17β-E2-80 per se treatment group did not significantly (p > 0.05) effect serum corticosterone levels compared to the naive group. Further, combinations (R-40 + Res-10, 17β-E2-80 + R-40, and Res-10 + 17β-E2-80) significantly (p < 0.05) attenuated serum corticosterone level as compared to their effect per se (Fig. 8).

Fig. 8.

Fig. 8

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Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on serum corticosterone levels of CIS-depressed mice. Values are expressed as mean ± S.E.M. using one-way ANOVA along with Tukey’s test. aMeans p < 0.05 compared to Naive; bMeans p < 0.05 compared to CIS; cMeans p < 0.05 compared to R-20 + CIS; dMeans p < 0.05 compared to Res-5 + CIS; eMeans p < 0.05 compared to 17β-E2-40 + CIS; fMeans p < 0.05 compared to R-40 + CIS; gMeans p < 0.05 compared to Res-10 + CIS; hMeans p < 0.05 compared to 17β-E2-80 + CIS; nsMeans non-significant, i.e. p > 0.05. Where CIS (Chronic immobilization stress); Imp-10 (Imipramine-10 mg/kg, i.p.); R-20 (Rutin-20 mg/kg, p.o.); R-40 (Rutin-40 mg/kg, p.o.); Res-5 (Resveratrol- 5 mg/kg, p.o.); Res-10 (Resveratrol-10 mg/kg, p.o.); 17β-E2-40 (17β-estradiol-40 mg/kg, p.o.); and 17β-E2-80 (80 mg/kg, p.o.)

Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on serum pro-inflammatory markers (TNF-α and PGF-2α) of CIS-depressed mice

CIS (6 h daily for 7 days) on mice induces depression, which is marked with significant (p < 0.05) elevation in serum TNF-α and PGF2α levels as compared to the naive group. The 7 days oral administration of R-40, Res-10, and 17β-E2-80 in CIS-induced depressive mice significantly (p < 0.05) attenuated serum TNF-α and PGF2α levels compared to CIS-induced depressive mice group (Fig. 9). However, treatment with the low dose of R-20, Res-5, and 17β-E2-40 did not influence serum TNF-α and PGF2α remarkably compared to CIS-induced depressive mice. Further, their protective effects were comparable with that of Imp-10 treatment for 7 days. However, R (20 and 40 mg/kg), Res (5 and 10 mg/kg), and 17β-E2 (40 and 80 mg/kg) per se treatment group did not produce any significant (p < 0.05) effect on serum TNF-α and PGF2α levels as compared to naive group.

Fig. 9.

Fig. 9

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Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on serum pro-inflammatory markers (TNF-α and PGF-2α) of CIS-depressed mice. Values are expressed as mean ± S.E.M. using one-way ANOVA along with Tukey’s test. aMeans p < 0.05 compared to Naive; bMeans p < 0.05 compared to CIS; cMeans p < 0.05 compared to R-20 + CIS; dMeans p < 0.05 compared to Res-5 + CIS; eMeans p < 0.05 compared to 17β-E2-40 + CIS; fMeans p < 0.05 compared to R-40 + CIS; g means p < 0.05 compared to Res-10 + CIS; h means p < 0.05 compared to 17β-E2-80 + CIS; ns means non-significant, i.e. p > 0.05. Where CIS (Chronic immobilization stress); Imp-10 (Imipramine-10 mg/kg, i.p.); R-20 (Rutin-20 mg/kg, p.o.); R-40 (Rutin-40 mg/kg, p.o.); Res-5 (Resveratrol-5 mg/kg, p.o.); Res-10 (Resveratrol-10 mg/kg, p.o.); 17β-E2-40 (17β-estradiol-40 mg/kg, p.o.); and 17β-E2-80 (80 mg/kg, p.o.)

Further, the combination of R-40 with Res-10, or 17β-E2-80 with Res-10, and or Res-10 with 17β-E2-80 significantly (p < 0.05) attenuated serum TNF-α and PGF2α levels as compared to their effect per se (Fig. 9).

Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on biogenic amines of CIS-depressed brain regions of mice

CIS (6 h daily for 7 days) on mice induces depression as evident with the significant (p < 0.05) declination in brain level of biogenic amines (serotonin, norepinephrine, and dopamine) in comparison with the naive group. The treatment having lower doses of R-20, Res-5, and 17β-E2-40 did not show any significant (p > 0.05) effects in biogenic amines, especially serotonin, norepinephrine and dopamine, compared to CIS-induced depression mice group. However, treatment with higher doses of R-40, Res-10, and 17β-E2-80 significantly (p < 0.05) elevated all biogenic amine levels in the brain compared to the CIS-induced depression mice group (Fig. 10). Further, the protective effects of these neuroprotective combinations were comparable to that of Imp-10 treatment. However, R-40, Res-10, and 17β-E2-80 per se treatment group did not produce any significant (p > 0.05) effects on the biogenic amines as compared to the naive group.

Fig. 10.

Fig. 10

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Interaction of different neuroprotectives (R, Res, 17β-E2) and their combinations on biogenic amines of CIS-depressed brain regions of mice. Values are expressed as mean ± S.E.M. using one-way ANOVA along with Tukey’s test. aMeans p < 0.05 compared to Naive; bMeans p < 0.05 compared to CIS; cMeans p < 0.05 compared to R-20 + CIS; dMeans p < 0.05 compared to Res-5 + CIS; eMeans p < 0.05 compared to 17β-E2-40 + CIS; fMeans p < 0.05 compared to R-40 + CIS; g means p < 0.05 compared to Res-10 + CIS; hMeans p < 0.05 compared to 17β-E2-80 + CIS; nsMeans non-significant, i.e. p > 0.05. Where CIS (Chronic immobilization stress); Imp-10 (Imipramine-10 mg/kg, i.p.); R-20 (Rutin-20 mg/kg, p.o.); R-40 (Rutin-40 mg/kg, p.o.); Res-5 (Resveratrol- 5 mg/kg, p.o.); Res-10 (Resveratrol-10 mg/kg, p.o.); 17β-E2-40 (17β-estradiol-40 mg/kg, p.o.); and 17β-E2-80 (80 mg/kg, p.o.)

Further, combination of R-40 with Res-10, or 17β-E2-80 with Res-10, and or Res-10 with 17β-E2-80 significantly (p < 0.05) elevated biogenic amine levels in brain compared to their individual effects (Fig. 10).

Discussion

CIS induces remarkable depression-like behaviour as evident through various experimental stress stimuli leading to behavioural and biochemical changes (Mineur et al. 2006). Nevertheless, the specific pathophysiological mechanism is still ineffectively comprehended by CIS-induced depression. The relationship between stress and depression has been explored by utilizing the animal model of CIS-induced depression (Kulkarni and Dhir 2008; Kumar and Goyal 2008; Kumar and Garg 2009; Kumar et al. 2010). In addition, different laboratories have very well documented the successful induction of depression through CIS by varying the time and days of exposure to immobilization (Chiba et al. 2012).

Here, the CIS-depressed mice exhibit remarkably reduced food intake and body weight compared to naïve mice as reported in many previous reports (Hu et al. 2000; Rather et al. 2013; Huang et al. 2017). The remarkable reduction of body weight in CIS-induced depressed mice is mediated through glucocorticoids, mobilization of energy stores, and increasing hepatic gluconeogenesis (Dallman et al. 2005; Torres and Nowson 2007). However, the neuroprotectives, namely R, Res, and 17β-E2, have been explored for their therapeutic efficacy in different neurological murine models (Garcia-Segura et al. 2001; Quincozes-Santos et al. 2009; Nakayama et al. 2011). The current study demonstrates that the 7-day oral administration of R, Res, and 17β-E2 significantly (p < 0.05) restored the loss in body weight compared to the stressed mice group, reflecting their potential in managing the body weight in stress conditions. However, the exact mechanism needs to be elucidated.

In the present study, it was observed that CIS for 7 days caused a significant (p < 0.05) alteration in locomotor activity (Kumari et al. 2007; Kulkarni and Dhir 2008; Kumar and Garg 2009). Such a paradigm can be reversed by 7-days treatment with R, Res, and 17β-E2. Further, their combinations synergistically potentiated the locomotor activity. The previous study by Capra and his group revealed that rutin (R) treatment improved locomotor activity (Capra et al. 2010; Pachauri et al. 2012). Similarly, Res and 17β-E2 showed improvement in locomotor activity in the spinal cord injury (SCI) rat model (Yune et al. 2004; Ates et al. 2006).

The results from the ongoing study demonstrate that CIS induces oxidative damage to the brain due to high oxygen consumption and modest antioxidant defence of brain tissue (Lee et al. 2005). It was also evident from the present study that the CIS leads to oxidative damage via elevated oxidation of lipids, nitrite concentration, and reduction of glutathione and catalase activity in distinct parts of the brain. The compromised antioxidant mechanism plays a key role suggested in the pathophysiology of depression (Luca et al. 2013). Moreover, R, Res, and 17β-E2 are well-established antioxidants (Frémont 2000; Green and Simpkins 2000; Fukui et al. 2001; Andreazza et al. 2008; Tasset et al. 2008; Mikstacka et al. 2010). The neuroprotective effect of Quercetin on the oxidation of lipids in the cortex has been well reported (Franco et al. 2007). In relevance to the current study, 7-days oral administration of R, Res, 17β-E2 and their synergistic combination significantly (p < 0.05) reinstate reduced glutathione and catalase activity. It also diminished the enhanced lipid peroxidation and nitrite concentration in the hippocampus, striatum, and cortex, suggestive of their antioxidant property against CIS.

Increased activity in the hypothalamic–pituitary–adrenal (HPA) in patients with chronic depression is an important and consistent observation in biological psychiatry (Joseph and Whirledge 2017). Specifically, there is an increased cortisol concentration in plasma, urine, and CSF in patients with major depression because of an aggressive cortisol behaviour to adrenocorticotropic hormone (ACTH) and augmentation of both glands (adrenal and pituitary) (Barden 2004; Pariante and Lightman 2008). Furthermore, it is well established that an increased level of corticosterone hormone is the main indicator of stress (Heiderstadt et al. 2000). CIS (6 h for 7 days) significantly aggravated corticosterone magnitude, drained by 7 days of management with rutin and its combination with resveratrol and 17β-E2, suggesting their protective role in depression-like behaviour that was induced by chronic stress. Moreover, it is also suggested that the HPA axis might be involved in their neuroprotective effect in chronic stress-induced depression. Still, the mechanism of action R, Res, and 17β-E2 is expected to illustrate.

The novel approach of developing therapeutics to treat stress-related disorders by targeting mitochondrial function is possible (Einat et al. 2005). There are five multi-subunit enzyme complexes: NADH-ubiquinone reductase (complex I), succinate-ubiquinone reductase (complex II), ubiquinone-cytochrome-c reductase (complex III), and cytochrome-c oxidase (complex IV) in the respiratory chain (Mayevsky 2009). At present, 6 h of immobilization stress for 7 days significantly (p < 0.05) disoriented the mitochondrial enzyme complexes (I to IV) in the hippocampus, striatum, and cortex, suggesting their potential role in stress-related disorders, including depression. Chronic exposure to glucocorticoid leads to an increase in (Horchar and Wohleb 2019) lactate production after aerobic exercise and decrease in complex-I activity in skeletal muscle (Manoli et al. 2007).

Several studies reported a continuous depletion in the mitochondrial complexes I–V in neurodegenerative disorders (Picard and McEwen 2018). In addition, the deposition of oxidized products with reactive carbonyl groups could lead to inter- and intramolecular cross-links with protein amino groups, leading to loss of biochemical and physiological function in mitochondria. Therefore, the accumulation of age-related protein oxidation products in mitochondria also leads to loss of energy production and increased production of oxidants (Chen et al. 2017).

However, 7 days treatment with R, Res, and 17β-E2 significantly (p < 0.05) reinstate the mitochondrial enzyme complexes, suggestive of their potential role in the swapping of mitochondrial enzyme complexes. Moreover, Res, a strong antioxidant polyphenol, significantly reduced the immobility time in murine models of depression (Xu et al. 2010) and has potential neuroprotective effects in mitochondria-induced, calcium-induced nitrogen, and oxygen sensing (Ungvari et al. 2011). Also, flavonoids quercetin and rutin protect against DNA strand breaks induced by tert-BOOH in mitochondria that leads to mitochondrial damage. Moreover, quercetin, except rutin, also protected against menadione-induced DNA single-strand breaks by acting as a metal chelator and a radical scavenger (Aherne and O’Brien 2000).

It has been reported that excessive secretion of pro-inflammatory cytokines has also been responsible for depression (Felger and Lotrich 2013). Neuroinflammation is a critical player in the pathophysiology of stress and stress-related disorders (Munhoz et al. 2008). According to some studies, C-reactive protein, acute phase proteins, and pro-inflammatory cytokines were increased in depressed patients (Maes et al. 2011). In the current study, there is a significant (p > 0.05) increase in pro-inflammatory cytokines (TNF-α and PGF-2α) compared to naive animals in chronic stress-induced depression. Here, oral neuroprotective (R, Res, and 17β-E2) treatment for 7 days significantly (p < 0.05) attenuated serum pro-inflammatory cytokines suggesting their neuroprotective effect.

Stress also induces disturbances in various neurotransmitters like dopamine level, gamma-aminobutyric acid; GABA, noradrenaline, and adrenaline levels (Cecchi et al. 2002; Ma and Morilak 2005; Nestler and Carlezon 2006), and aggravated serotonin synthesis (Ramkumar et al. 2008) during stress and stress-related conditions. There is a correlation between depression and neurotransmitters; stressors may aggravate NE, DA, and 5-HT release in distinct areas of the brain (Mineur et al. 2006). In the current study, immobilization stress for 6 h for 7 days significantly (p < 0.05) decreased the DA, NE, and 5-HT levels in specific brain areas. Also, 7 days of treatment with R, Res, and 17β-E2 and their combination significantly attenuated DA, NE, and 5-HT levels in the brain, suggesting their potential role in stress-induced depression.

Conclusion

In conclusion, mice exposed to CIS-induced depression (6 h daily for 7 days) were found to be highly immobile, elevated oxidative stress, impaired locomotor activity, elevated corticosterone, proinflammatory cytokines, prostaglandins, and neurotransmitters levels in the cerebral cortex, striatum, and hippocampus regions of the brain which were reversed upon simultaneous daily oral administration of R, Res, and 17β-E2 and their combinations for 7 days. The protective effect of these phytochemicals against CIS-induced depression was evident with the suppression of oxidative stress, restoration of mitochondrial enzymes, modulation of HPA-axis, biogenic amines, and inflammatory pathways, suggesting that the drugs (R, Res, 17β-E2, and their combinations) can be used in the management of depression-like symptoms and novel formulation can be designed using these neuroprotective combinations.

Author contributions

IAI carried out the bench work; MA contributed equally with IAI in carrying out the wet laboratory work; SGA analysed the results and applied statistics; VK supervised the study.

Funding

This study was not supported by any funding agencies.

Data Availability Statement

Data will be provided by the corresponding author upon request.

Declarations

Conflict of interest

Authors have no conflict of interest.

Ethical approval

This study was undertaken through prior approval from the Institutional Animal Ethics Committee (IAEC) of KIET school of Pharmacy (KSOP), Ghaziabad, India, as per standard guidelines of the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA) with approval number- IAEC/KSOP/2020-21/15.

Informed consent

This article does not contain any studies with human participants.

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