Introduction

Among anthracyclines, Adriamycin (ADR) is a powerful antineoplastic agent, widely used for the treatment of human’s solid tumors by blocking the process of replication in the malignant cells (Cutts et al. 2003). However, the clinical use of ADR is compromised by the development of life-threatening adverse effects, such as myelosuppression, fatigue, and cognitive impairments (Jansen et al. 2008). Recently, ADR-related neurobehavioral changes have been indicated using established rodent models (Liedke et al. 2009; Merzoug et al. 2011).

Several investigations have revealed that chemotherapeutic agents, including ADR, are responsible for brain tissue injury (Ahles and Saykin 2007). ADR modulates endogenous levels of cytokines, such as tumor necrosis factor alpha (TNF-α), leading to reactive oxygen and/or nitrogen species (ROS/RNS) production which results in brain oxidative stress (Joshi et al. 2005, 2007; Tangpong et al. 2006, 2007). It was also reported that ADR-mediated free radicals generation in the brain tissue increases lipid peroxidation and protein oxidation and alters the antioxidant defense system which can enhance the oxidative stress (Joshi et al. 2005, 2007, 2010) leading to neuropsychological changes (Dubovický 2010).

Chemotherapeutic drugs have been reported to induce toxic effects on immune cells in bone marrow and peripheral lymphoid tissues (Park et al. 2009). Interestingly, chemotherapy-associated myelotoxicity and immunosuppression might increase the release of inflammatory cytokines, which cross the blood–brain barrier and activate microglia, resulting in neuroinflammation and subsequent neuropsychiatric and cognitive symptoms (Ahles and Saykin 2007). Nowadays, it is well documented that the central nervous system (CNS) and the immune system (IS) are intimately linked through bidirectional chemical messengers (Banks and Erickson 2010). Importantly, the IS has been implicated in neurodegenerative diseases that accompany CNS injuries (Kipnis et al. 2008). Therein, preliminary experiments revealed that the hypothalamo-pituitary-adrenocortical (HPA) axis is involved in the protection of the rat against chemotherapeutic-induced toxic outcomes. In adrenalectomized or hypophysectomized rats, the lethal effects of anticancer drugs were considerably increased, probably due to the omitted corticosterone release (Navarra and Preziosi 1997). However, it is well known that stressful conditions, such as chemical stresses, result in enhanced HPA axis activity and, thus, lead to increased plasma glucocorticoids released from the adrenal cortex (Heim et al. 2000). Consequently, the behavioral outcomes of stress, such as anxiety and depression, are believed to be mediated by enhanced values of glucocorticoids (Berton and Nestler 2006).

Flavonoids belong to the family of natural polyphenols widely distributed in the plant kingdom, including apples, berries, onion, tea, seeds, and vegetables (Hollman and Kata 1999). Quercetin (Q, 3,5,7,3′,4′-pentahydroxyflavone) is the most abundant bioflavonoid in the human diet (Manach et al. 2004). Whatever the route of administration, a considerable amount of quercetin is metabolized during its first passage through the liver, resulting especially in the formation of methylated metabolites such as isorhamnetin and tamarixetin (van der Woude et al. 2004). Urinary and biliary excretions of either the unchanged form of quercetin or its conjugates were reported in previous pharmacokinetics studies (Gugler et al. 1975). Quercetin has a powerful antioxidant capacity, protecting the body against oxidative stress-related damages (Haleagrahara et al. 2009). With the ability of crossing the blood–brain barrier in vivo, quercetin and its metabolites appear in the brain after several hours of quercetin administration (Paulke et al. 2006), exerting beneficial actions on the CNS such as neuroprotection and anxiolytic and cognitive-enhancing effects (Cho et al. 2006). More recently, several experimental studies have demonstrated the efficacy of quercetin against depressive disorders in rodents. Indeed, it has been noted that quercetin-enriched foods, such as freeze-dried onion powder and Ginkgo biloba leaves, are regarded to possess an antidepressant-like activity in the forced swimming test (Rojas et al. 2011; Sakakibara et al. 2008), a behavioral paradigm for the assessment of antidepressant drugs efficacy. Moreover, it was reported that quercetin inhibits the corticotrophin releasing factor (CRF)-induced anxiety- and depression-like behaviors in mice and rats (Bhutada et al. 2010; Kawabata et al. 2010).

Fruits and vegetables rich in quercetin are believed to protect the brain tissue from oxidative stress-related neurodegenerative diseases (Heo and Lee 2004; Youdim et al. 2004). Importantly, quercetin prevented stress-induced oxidative damage in neurons by inhibiting hydroperoxide formation, scavenging free radicals, and preserving antioxidant enzymes (Dok-Go et al. 2003; Haleagrahara et al. 2009). Quercetin also attenuated neuronal damage in rodents’ hippocampus following cerebral ischemia in vivo (Cho et al. 2006; Pu et al. 2007), and it is therefore suggested to be of therapeutic benefit in the treatment of Alzheimer’s and Parkinson’s diseases (Choi et al. 2012; Haleagrahara et al. 2011).

Previous investigations highlighted the immunostimulatory effects of quercetin by enhancing lymphocyte activation and/or secretion of the multipotent cytokine interferon-γ (Cherng et al. 2008), neutrophil chemotaxis and oxidative burst activity (Akbay et al. 2003), macrophage stimulation (Oršolić and Bašić 2005), and natural killer cell activity (Yu et al. 2010). Other studies have shown that quercetin inhibits the production of pro-inflammatory cytokines (Cho et al. 2003; Min et al. 2007) through inhibition of neuroendocrine signaling pathways (Spencer 2007), which may lead to normalization of stressor-mediated overactive HPA axis.

In this respect, the current study was aimed to investigate the possible beneficial effect of quercetin against ADR-related anxio-depressive-like behavior, plasma corticosterone hyperactivity, brain oxidative stress, and immune alterations in male Wistar rats.

Methods

Animals and housing

Sixty-eight adult male Wistar rats were obtained from Pasteur Institute (Algiers, Algeria) and individually housed in transparent cages in a temperature-controlled facility room (23 ± 1 °C). Rats were maintained on a 12-h/12-h light cycle (lights on at 07:30 a.m.) with access to standard rodents chow and tap water ad libitum. The experimental protocol was carried out according to the NIH revised Guidelines for the Care and Use of Laboratory Animals (No. 80-23, 1996).

Drugs

Adriamycin (doxorubicin hydrochloride) and quercetin (quercetin dihydrate) were purchased from Sigma-Aldrich Co. (Steinheim, Germany). Both ADR (7 mg/kg of body weight) and quercetin (60 mg/kg of body weight) were dissolved in saline solution (0.9 %) and injected intraperitoneally (i.p.) in a volume of 1 ml/kg of body weight according to the experimental procedure detailed below. Seven milligrams per kilogram of body weight of ADR was chosen based on our previous study, in which this dose has been indicated to induce anxiety-like behavior and locomotor anomalies with a concomitant brain oxidative injury and immune depletion (Merzoug et al. 2011). In many rodent studies, it was reported that the most effective physiological dose of quercetin is 60 mg/kg of body weight (Filho et al. 2008; Song et al. 2002).

Experimental procedure

The entire study includes three experiments in parallel. The 68 rats were randomly divided into four groups: vehicle-treated control (C) rats, ADR-treated rats (ADR), quercetin (Q)-treated rats, and quercetin-injected rats pretreated with ADR (ADR + Q). Animals received a single i.p. injection of saline (groups C and Q) or ADR (groups ADR and ADR + Q). A treatment with saline (groups C and ADR) or quercetin (groups Q and ADR + Q) was therefore conducted at specific time points in order to evaluate the ability of this flavonoid to reverse ADR-related neurobehavioral and immune disorders according to the procedure of each experiment (Fig. 1).

Fig. 1
figure 1

Scheme of experimental procedure

Full size image

Experiment 1

This experiment aimed at investigating the effect of quercetin on ADR-induced depression-like behavior in the forced swim test (FST). Seventy-two hours post-ADR injection, 24 rats (n = 6) were individually subjected to the first session of FST (i.e., the pretest session) as described below. Quercetin (groups Q and ADR + Q) or saline (groups C and ADR) injections were performed 24, 5, and 1 h before the second session of FST (i.e., the test session) during which the animals’ behavior was evaluated (Fig. 1). These time points of quercetin treatment are based on the method described by Porsolt et al. (1977) for the evaluation of antidepressant effects. It is interesting to note that the cumulative dose (i.e., 180 mg/kg of body weight) has no toxicological effects in rats (Russo et al. 2012).

Experiment 2

The second experiment aimed at evaluating the effect of quercetin on ADR-induced anxiety-like behavior. Seventy-two hours post-ADR injection, animals (n = 6) directly received quercetin (groups Q and ADR + Q) or saline (groups C and ADR) injections at the same time points as described in the experiment 1, then behaviorally tested in the open field (OF) and the elevated plus maze (EPM) tasks instead of the second FST session (Fig. 1).

Experiment 3

During the third experiment, the rats (n = 5) directly received quercetin injections at the same time points as described in the experiment 1, then sacrificed by decapitation without being tested in the FST, the OF, nor the EPM (Fig. 1). Blood samples were collected into ethylenediaminetetraacetic acid (EDTA)-coated tubes to quantify white blood cells (WBCs), lymphocytes (LYM), monocytes (MONO), and granulocytes (GRAN) using an automatic cell counter (ERMA Inc., Model PCE-210, Tokyo, Japan). Plasma samples were used to determine corticosterone concentrations in duplicate using a commercially available RIA kit according to the manufacturer’s instructions (Sigma, USA). Immediately after sacrifice, the brains were dissected out and rinsed with ice-cold isotonic saline. Brain samples were prepared with ice-cold 0.1 mol/l phosphate buffer (pH 7.4) to obtain 1:10 (w/v) homogenates, and then centrifuged at 10,000 g (4 °C) for 15 min. The supernatant aliquots were used for assessing the oxidative stress parameters spectrophotometrically (UV/Vis spectrophotometer, Optizen 2120 UV, Mecasys Co., Ltd, Korea).

Behavioral assessment

The rats’ behavior in all tests was videotaped and analyzed using ANYmaze™ computer software (Stoelting Co., USA).

Forced swim test

The FST is a well-established behavioral paradigm used to test the efficacy of antidepressant drugs. In our study, the FST was conducted according to the method of Porsolt et al. (1977). The behavioral apparatus was a clear glass aquarium (height, 54.0 cm; length, 34.0 cm; width 60.0 cm) filled with water to a depth of 40.0 cm maintained at 24.0 ± 1 °C (Molina-Hernández et al. 2004). Rats were forced to swim for 15 min (pretest). Twenty-four hours later, they were reinforced to swim for a 5-min period (test) during which the time spent in immobility, swimming, or struggling was measured. The number of defecations as a sign of emotional reactivity was also registered. The rats were judged to be immobile whenever they remained passively floating in a slightly hunched but upright position with their heads being just above the water surface. The struggling behavior was registered when the animals made active and exaggerated movements with their forepaws in an attempt to escape the stressful situation. They were considered to be in a swimming position when they made active swimming or circular movements (Cryan et al. 2002). After each session, the rats were removed from the water, dried with towels, and placed in a warmed enclosure for 20 min before being returned to their home cages. The aquarium was emptied and cleaned with an alcoholic solution between rats.

Open field test

Spontaneous locomotor activity of rats was tested in an OF, which consisted of a gray square Plexiglas arena (70 cm × 70 cm × 40 cm) divided into a 35 × 35 cm central zone and the surrounding peripheral one. The room was illuminated by a red bulb (25 W) located 130 cm above the center of the apparatus (Sáenz et al. 2006). The rats were individually placed in the center of the arena and allowed to freely explore for 5 min. The following behavioral parameters were measured: the locomotive distance, the number of rearings, the time spent in central zone, and the time spent in peripheral zone. After each test, the animals were returned to their home cages, and the apparatus was cleaned with an alcoholic solution followed by wet and dry paper towels.

Elevated plus maze test

As previously described (Patin et al. 2005), the elevated plus maze consisted of four arms (50 cm × 10 cm) extending from a central platform (10 cm × 10 cm). Two opposing arms were closed-in by 45-cm high Plexiglas walls, while the other two arms were open with 3–5 mm high railing around the perimeter to prevent the rat from falling. The entire apparatus was elevated to 50 cm above the ground level. The test room was lit by a 60-W electric bulb placed directly 175 cm above the central area of the apparatus (Estanislau and Morato 2005). Rats were individually placed in the center of the maze facing an open arm, and their activity over the entire 5-min test period was evaluated. The following behavioral parameters were measured: the number of entries into and the time spent in open arms, and the number of entries into and the time spent in closed arms. After each test, the animals were returned to their home cages, and the apparatus was cleaned with an alcoholic solution followed by wet and dry paper towels.

Oxidative stress analyses

Glutathione-S-transferase assay

The glutathione-S-transferase activity was estimated at 37 °C according to the method of Habig et al. (1974). The reaction mixture (1 ml) contained 0.334 ml of 100 mM phosphate buffer (pH 6.5), 0.033 ml of 30 mM 1-chloro-2-4-di-nitrobenzene (CDNB), and 0.033 ml of reduced glutathione. After preincubating the mixture for 2 min, 0.01 ml of supernatant was added, and the absorbance was measured for three consecutive minutes at 340 nm. The activity of GST is expressed as nanomole per min per milligram of protein, using an extinction coefficient of 9.6/Mm/cm.

Reduced glutathione assay

Reduced glutathione (GSH) concentration was determined as described earlier by Ellman (1959). A 1.0 ml supernatant was precipitated with 1.0 ml of 4 % sulfosalicylic acid and cold digested at 4 °C for 1 h. The samples were centrifuged at 1,200 g for 15 min at 4 °C. To 1 ml of this supernatant, 2.7 ml of 0.1 M phosphate buffer (pH 8.0) and 0.2 ml of 5-5′-dithio-bis-2-nitrobenzoic acid (DTNB) were added. The yellow color developed was immediately read at 412 nm. The results were expressed as nanomole of GSH per milligram of protein.

Lipid peroxidation assay

The malondialdehyde (MDA) levels were assayed as a product of lipid peroxidation (Draper and Hadley 1990). Briefly, 2.5 ml of 10 % trichloroacetic acid (TCA) was added to 0.5 ml of samples in each centrifuge tube and boiled in a water bath for 15 min. After cooling, the tubes were centrifuged at 1,000 g for 10 min, and 2.0 ml of the supernatant was added to 1.0 ml of 0.67 % thiobarbituric acid (TBA) solution and then boiled in a water bath for 15 min. After cooling, the absorbance of the TBA-MDA reaction was measured at 532 nm. Results were expressed as nanomole MDA per milligram of protein, using an extinction coefficient of 1.56 × 105/cm/M.

Brain protein assay

The brain protein content was measured according to the method of Bradford (1976) using bovine serum albumin (BSA) as standard. The Bradford reagent was diluted in deionized water (1:4). Twenty microliters of the protein extract was mixed with 1 ml of the diluted reagent, and the blue color formed was spectrophotometrically measured at 595 nm. A standard curve was determined using a serial dilution series (0.1–1.0 mg/ml) of BSA dissolved in phosphate buffer (0.1 mol/l, pH 7.4).

Statistical analysis

Results are presented as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used to compare key variable between groups, followed by the post-hoc Tukey–Kramer’s test when necessary. Additionally, an analysis of covariance (ANCOVA) was applied on FST, EPM, and OF measures to check for statistical relationships among the parameters of each behavioral test. Statistical significance was considered with a p < 0.05. All statistical analyses were performed using the Minitab software (version 15.1.1.0, Minitab Inc., USA).

Results

Body weight

One-way ANOVA registered significant differences among groups’ body weight change [F(3, 16) = 92.73, p < 0.001]. ADR-treated rats showed a significant decrease in body weight gain (p < 0.001) as compared to vehicle-treated ones. Moreover, treatment with quercetin in ADR-injected animals significantly alleviated body weight decrease (p < 0.001) when compared to that in ADR group (data not shown).

Depression-like behavior in the forced swim test

Animals’ behavior in the FST is presented in Fig. 2. One-way ANOVA indicated a significant difference among the groups in respect of immobility [F(3, 36) = 653.20; p < 0.001], swimming [F(3, 36) = 129.30, p < 0.001], struggling [F(3, 36) = 506.96, p < 0.001], and defecation [F(3, 36) = 21.23, p < 0.001]. When compared to C group, ADR injection significantly increased FST-related immobility (p < 0.001; Fig. 2a). Quercetin-injected rats exhibited a significant decrease in immobility time (p < 0.001) as compared to C rats and ADR-treated ones. Quercetin treatment in ADR-injected animals significantly reduced immobility time (p < 0.001) when compared to C and ADR groups. As shown in Fig. 2b, both ADR and quercetin treatments enhanced significantly the swimming time (p < 0.001) as compared to vehicle-treated rats, with further increase in quercetin-injected animals (p < 0.001) when compared to ADR-treated ones. Interestingly, the swimming time increased significantly in Q-injected rats pretreated with ADR (p < 0.001) when compared to that in vehicle- and ADR-injected ones, while decreased significantly (p < 0.01) as compared to Q group. ADR treatment significantly declined the struggling time (p < 0.001) when compared to C group (Fig. 2c). On the contrary, Q-injected rats spent longer time in struggling behavior as compared to vehicle- and ADR-injected ones. The struggling time in Q-injected rats pretreated with ADR decreased significantly (p < 0.001) as compared to that in C and Q groups, while enhanced significantly (p < 0.001) in comparison with ADR-injected animals. In addition, no significant difference was registered in respect to the number of fecal boli between ADR and C groups (Fig. 2d). Quercetin treatment alone suppressed FST-related defecation (p < 0.001) when compared to all other groups. An ANCOVA was applied to determine if the time spent immobile is influenced by the overall activity. When this activity (either the swimming time or the struggling time) was defined as a covariate, there was no change in significance (p ˂ 0.001) for the time of immobility, indicating that ADR-injected rats showed a depressive-like behavior.

Fig. 2
figure 2

Quercetin effect on forced swim test parameters in ADR-pretreated rats. Data are reported as mean ± SEM (n = 6). C (control), ADR (Adriamycin), Q (quercetin). cp < 0.001 versus C group; γp < 0.001 versus ADR group; ¶p < 0.01 and #p < 0.001 versus Q group

Full size image

Anxiety-like behavior in the open field test

Figure 3 shows the rats’ behavior in the OF. One-way ANOVA revealed significant differences among the groups in terms of the locomotive distance [F(3, 20) = 6.21, p < 0.01], the number of rearings [F(3, 20) = 6.96, p < 0.01], the central zone time [F(3, 20) = 36.46, p < 0.001], and the peripheral zone time [F(3, 20) = 36.54, p < 0.001]. Indeed, ADR-treated animals traveled lesser distance (p < 0.01; Fig. 3a) when compared to vehicle-treated ones. Quercetin administration in untreated and ADR-treated rats significantly enhanced the distance traveled in the apparatus (p < 0.001 and p < 0.05, respectively) as compared to that in ADR group. When compared to C animals, the number of rearings decreased significantly in ADR and ADR + Q groups (p < 0.001 and p < 0.001, respectively; Fig. 3b). Quercetin-injected rats showed greater number of rearings (p < 0.05) as compared to ADR-injected ones. ADR-treated rats spent shorter time in central area and longer time in peripheral one (p < 0.001; Fig. 3c, d) as compared to vehicle-treated animals. When compared to ADR group, Q-treated rats spent longer time in the central area and shorter time in the peripheral one (p < 0.001). Q-injected rats pretreated with ADR spent less time in the central zone and more time in the peripheral one as compared to C and Q groups (p < 0.01 and p < 0.05 respectively), while they spent longer time in the central area and shorter time in the peripheral one (p < 0.001) when compared to ADR-treated animals. An ANCOVA was performed to demonstrate that the behavioral responses of ADR-injected rats were due to the anxiety disorders rather than the fatigue. Taking the locomotive distance as a covariate, there was no change in significance (p < 0.001), indicating that the difference in the time spent in both central and peripheral zones among all the groups did not depend on the difference observed for the traveled distance.

Fig. 3
figure 3

Quercetin effect on open field parameters in ADR-pretreated rats. Data are reported as mean ± SEM (n = 6). C (control), ADR (Adriamycin), Q (quercetin). bp < 0.01 and cp < 0.001 versus C group; αp < 0.05 and γp < 0.001 versus ADR group; *p < 0.05 versus Q group

Full size image

Anxiety-like behavior in the elevated plus maze test

The rats’ behavior in the elevated plus maze is presented in Fig. 4. One-way ANOVA among all groups indicated a significant effect of treatment on the number of entries into [F(3, 20) = 12.02, p < 0.001] and the time spent in [F(3, 20) = 65.58, p < 0.001] the open arms, and the number of entries into [F(3, 20) = 18.54, p < 0.001] and the time spent in [F(3, 20) = 147.44, p < 0.001] the closed arms. Interestingly, ADR treatment decreased significantly the number of entries into and the time spent in the open arms (p < 0.001; Fig. 4a, b) as compared to C group. Q-treated rats exhibited lower number of entries into the open arms (p < 0.05) when compared to vehicle-treated ones, while they showed greater number of entries into (p < 0.05) and longer time spent in the open arms (p < 0.001) as compared to ADR-treated rats. When compared to C group, Q-injected rats pretreated with ADR showed lower number of entries into and less time spent in the open arms (p < 0.001). In addition, the time they spent in the open arms was longer than that registered in the ADR group (p < 0.001) and shorter than that noted in Q-treated animals (p < 0.001). On the other hand, ADR treatment significantly increased the number of entries into (p < 0.05) and the time spent in the closed arms (p < 0.001; Fig. 4c, d) when compared to controls. Q-injected rats showed lower number of entries into the closed arms as compared to C group. Moreover, they exhibited lower number of entries into and longer time spent in the closed arms (p < 0.001) when compared to ADR group. Q-injected animals pretreated with ADR indicated longer time spent in the closed arms (p < 0.001) as compared to controls. In addition, they showed lower number of entries into (p < 0.05) and longer time spent in the closed arms (p < 0.001) when compared to ADR-treated rats, with greater number of entries into and longer time spent in the closed arms (p < 0.001) when compared to Q-treated rats. An ANCOVA was applied to determine if the time spent in arms is influenced by the overall movement. When controlling for both open and closed arms entries, there was no change in significance (p ˂ 0.001) for the time spent in these arms.

Fig. 4
figure 4

Quercetin effect on elevated plus maze parameters in ADR-pretreated rats. Data are reported as mean ± SEM (n = 6). C (control), ADR (Adriamycin), Q (quercetin). ap < 0.05, bp < 0.01, and cp < 0.001 versus C group; αp < 0.05 and γp < 0.001 versus ADR group; #p < 0.001 versus Q group

Full size image

Corticosterone changes

One-way ANOVA revealed a significant effect of treatment on corticosterone level between groups [F(3, 16) = 18.98, p < 0.001]. Plasma corticosterone concentration significantly increased post-ADR injection (p < 0.05; Fig. 5) as compared to C and Q groups. Quercetin treatment in ADR-pretreated rats resulted in a significant reduction of corticosterone level when compared to that in C, ADR (p < 0.001), and Q (p < 0.01) groups.

Fig. 5
figure 5

Plasma corticosterone level in male rats. Data are reported as mean ± SEM (n = 5). C (control), ADR (Adriamycin), Q (quercetin). ap < 0.05 and cp < 0.001 versus C group; αp < 0.05 and γp < 0.001 versus ADR group; ¶p < 0.01 versus Q group

Full size image

Brain oxidative biomarkers

One-way ANOVA revealed significant differences for the GST activity [F(3,16) = 109.50, p < 0.001], GSH level [F(3,16) = 14.90, p < 0.001], and lipid peroxidation [F(3,16) = 40.34, p < 0.001]. When compared to controls, ADR and ADR + Q groups exhibited a significant increase in the GST activity (p < 0.001; Fig. 6a). Quercetin administration in saline- or ADR-pretreated rats significantly reduced the GST activity (p < 0.001) as compared to ADR-treated ones. In addition, ADR + Q group showed a significant increase in GST activity (p < 0.001) when compared to Q-injected animals. On the other hand, GSH level was significantly depleted in ADR and ADR + Q (p < 0.001 and p < 0.05 respectively; Fig. 6b), as compared to that in vehicle-injected ones. Quercetin treatment significantly enhanced GSH content in Q and ADR + Q groups (p < 0.001 and p < 0.01, respectively) when compared to that in ADR-treated animals. Furthermore, ADR + Q showed a significant decrease in GSH level (p < 0.05) in comparison to Q-treated ones. A significant increase in lipid peroxidation was registered in ADR and ADR + Q groups (p < 0.001 and p < 0.01, respectively; Fig. 6c) as compared to saline-injected rats. Quercetin injection in Q and ADR + Q significantly reduced lipid peroxidation (p < 0.001 and p < 0.01, respectively) in comparison to that in ADR-treated ones. Lipid peroxidation was significantly enhanced in ADR + Q (p < 0.01) when compared to that in Q-injected ones.

Fig. 6
figure 6

Brain oxidative parameters in male rats. Data are reported as mean ± SEM (n = 5). C (control), ADR (Adriamycin), Q (quercetin). ap < 0.05, bp < 0.01, and cp < 0.001 versus C group; βp < 0.01 and γp < 0.001 versus ADR group; *p < 0.05, ¶p < 0.01, and #p < 0.001 versus Q group

Full size image

Hematological changes

Total leukocyte counts and WBC relative counts (%) are presented in Table 1. One-way ANOVA revealed significant differences between groups in respect of total leukocytes [F(3, 16) = 6.02, p < 0.01], number and percentage of lymphocytes [F(3, 16) = 43.07; F(3, 16) = 53.52, p < 0.001] and monocytes [F(3, 16) = 9.92; F(3, 16) = 33.03, p < 0.001], and only the percentage of granulocytes [F(3, 16) = 11.22, p < 0.001]. ADR injection significantly decreased the WBC count (p < 0.01) as compared to vehicle-treated animals. Q and ADR + Q groups showed an increase in the total number of leukocytes (p < 0.05) when compared to ADR-treated rats. Furthermore, a significant lymphopenia was registered in the ADR and ADR + Q groups (p < 0.001) as compared to that in the controls. When compared to the ADR-treated rats, a significant increase in lymphocyte number (p < 0.001) and percentage (p < 0.001 and p < 0.05, respectively) was noticed in the Q and ADR + Q groups. On the other hand, the mean monocyte levels (p < 0.01) and the percentage of monocytes in leukocytes increased significantly in the ADR and ADR + Q groups (p < 0.001) as compared to those in the controls. When compared to ADR group, Q-injected animals showed lower monocyte values (p < 0.01) and relative count of monocytes in leukocytes (p < 0.001). The ADR + Q exhibited a significant decline in the percentage of monocytes (p < 0.05) as compared to the ADR-treated rats, while they had higher monocyte levels (p < 0.01) and relative monocyte counts (p < 0.001) when compared to the Q group. Moreover, the granulocyte relative counts were significantly enhanced in the ADR and ADR + Q (p < 0.001 and p < 0.01, respectively) as compared to those in the vehicle-injected group. The Q rats exhibited lower percentage of granulocytes (p < 0.01) as compared to the ADR-treated group. Furthermore, the Q-injected rats pretreated with ADR had higher granulocyte values and granulocyte relative counts (p < 0.05) when compared to the Q-treated rats.

Table 1 Immune cell counts in male rats
Full size table

Discussion

In the present study, we investigated the ability of quercetin to reverse the effects of a single i.p. injection of ADR (7 mg/kg) on neurobehavioral and immune aspects in male rats. The behavioral part of our experiments indicated that ADR treatment enhanced immobility and swimming with the struggling time being decreased, revealing that ADR administration could be associated with a mild depressive-like behavior. Quercetin treatment following ADR injection reduced the immobility time in the FST, suggesting that it produces a powerful antidepressant-like effect in rats. Interestingly, quercetin per se decreased the emotional reactivity in response to stress conditions, as indicated by the suppression of defecation during the test. Quercetin administered per os (10, 20, or 40 mg/kg) dose dependently reduced the immobility time in mice which were submitted to FST 60 min later (Bhutada et al. 2010). Moreover, quercetin (50 and 100 mg/kg, i.p.) dose dependently reduced the immobility during the FST in diabetic mice (Anjaneyulu et al. 2003). Rojas et al. (2011) have also reported that Ginkgo biloba extract (EGb761; 10 mg/kg, i.p.), a quercetin-enriched medicinal plant, manifests an antidepressant-like effect in the FST when administered for 17 consecutive days in mice.

In the OF test, ADR-treated animals traveled lesser distance and showed a reduced number of rearings in the apparatus, suggesting that ADR affects the locomotor and exploratory activities and thereby promotes the sickness behavior (Konat et al. 2008; Liedke et al. 2009). This hypolocomotion was accompanied with a shorter time in the central area and a longer time in the peripheral one, meaning that these animals tend to remain close to the apparatus walls. This finding is considered as an index of thigmotaxis (Simon et al. 1994). Meanwhile, the EPM test indicated that ADR treatment reduced the number of entries into and the time spent in the open arms, while enhanced the number of entries into and the time spent in the closed arms. These later results suggest that ADR-treated rats had an anxiety-like behavior. Indeed, we have recently reported that a single i.p. injection of ADR at 7 mg/kg reduced the locomotive activity and enhanced the anxiety in the OF and EPM tests 72 h post-treatment (Merzoug et al. 2011). It was also reported that prenatal exposure to ADR dose dependently increased anxiety and affected the emotional behavior in 3-month-old C57BL/6 J-mice (Van Calsteren et al. 2009). Our findings demonstrate that quercetin attenuates the ADR-related anxiety-like behavior and locomotor impairment in both OF and EPM tasks, except for the vertical exploration (i.e., the number of rearings). This anxiolytic effect has been found in rats at a dose of 300 mg/kg after oral or intranasal administration (Priprem et al. 2008). Quercetin has also been shown to ameliorate the time spent in and the number of entries into the open arms of EPM after a sub-chronic treatment in male rats (Wattanathorn et al. 2007). Using an animal model of aging, Sun et al. (2007) indicated that the long-term intake of quercetin attenuates the ambulatory impairment. Specifically, our findings suggest that the behavioral impairment following ADR treatment could be conferred to the significant increase in corticosterone levels, suggesting further that ADR is a chemical stress that activates the HPA axis. Then, the alleviating effect of quercetin may be related to its ability to restore the corticosterone level in ADR-exposed animals. It has been noted that quercetin supplementation attenuated the stress-induced behavioral depression by inhibiting the corticotrophin-releasing factor (CRF) mRNA expression in the hypothalamus (Kawabata et al. 2010). More recently, a sub-acute oral treatment with quercetin (50 mg/kg for 6 days) was found to be anxiolytic in predator-stressed pregnant rats when tested in the EPM and OF paradigms, an effect that was partially attributed to the blockade of corticosterone hypersecretion (Toumi et al. 2013).

The anxio-depressive-like behavior in ADR-injected rats was associated with a pronounced brain oxidative stress. Indeed, ADR exposure enhanced the GST activity and lipid peroxidation (i.e., increased MDA level) while depleted the GSH level. MDA is the most commonly studied product of lipid peroxidation for quantifying the level of oxidative stress in vivo and in vitro (Del Rio et al. 2005). It has been reported that lipid peroxidation exerts cytotoxic effects on a wide range of organs including the brain, contributing therefore to cognitive disorders and neurodegenerative diseases (Sultana et al. 2006). The reduction in GSH level may be involved in neuronal death (Bains and Shaw 1997) and neurobehavioral and cognitive deficits in rats (Cruz-Aguado et al. 2001). In addition, the elevation of GST activity following ADR injection could be related to its protective role against oxidative injury in the brain. Interestingly, it was noted that GST can detoxify toxins and transport them away from axons and myelin sheaths (Sagara and Sugita 2001). Our findings corroborate the prior investigations indicating that ADR exposure generates lipid peroxides and alters the activity of antioxidant enzymes in cerebral tissues (Joshi et al. 2005, 2007, 2010). Quercetin is a well-known antioxidant flavonoid that protects brain cells against ROS-induced damage. Our data show that quercetin improved the brain oxidative status in ADR-treated rats. This effect of quercetin could be associated with its ability to stabilize high levels of GSH while scavenging lipid peroxides within the brain tissue (Sun et al. 2007). A neuroprotective activity of quercetin through ROS elimination has also been reported in isolated rat cortical cells that were exposed to H2O2 (Dok-Go et al. 2003). In addition, quercetin alleviated d-galactose-induced neuron damage in mice by scavenging oxygen radicals and increasing the activity of antioxidant enzymes such as superoxide dismutase (Lu et al. 2006). Interestingly, the antioxidant effect of quercetin in our study might in part be responsible for the behavioral improvement in OF and EPM tests. In fact, Singh et al. (2003) reported that a chronic treatment of mice with this flavonoid reverses their cognitive deficits due to ethanol intoxication and aging by ameliorating the forebrain level of GSH and activity of superoxide dismutase and catalase.

ADR treatment has been previously considered as immunosuppressive and myelosuppressive (Grant et al. 1991). In the current study, ADR injection reduced total WBC and the number and the percentage of lymphocytes, while it enhanced the number and the percentage of monocytes and only the percentage of granulocytes. We have previously demonstrated that the acute ADR treatment at the same dose is associated with immune alterations in rats (Merzoug et al. 2011). It has been noticed that ADR dose dependently depleted leukocytes and lymphocyte numbers in the spleen, thymus, lymph nodes, and peripheral blood of healthy mice (Pourtier-Manzanedo et al. 1995). Interestingly, ADR-associated lymphopenia might be due to the elimination of lymphocyte precursors and the destruction of mature lymphocyte populations leading to immunodeficiency (Steele 2002). Moreover, Asmis et al. (2006) have revealed that intravenous injections of ADR in female mice increased blood monocytes. ADR-induced monocytosis may contribute to T cell immunosuppression by the production of suppressive factors that inhibit T cell function (Mackall 2000). On the other hand, ADR has been indicated to cause pronounced marrow depression with peripheral blood granulocytopenia (Falkson et al. 1985), which may result in increased susceptibility to infections (Tsang et al. 2007). The IS has been reported to be involved in neurodegenerative conditions that accompany CNS injuries (Weiner and Cohen 2002). Recent investigations have revealed that an injection of T cells from wild-type donors into immune-depleted mice restored their cognitive functions (Kipnis et al. 2008), supporting the hypothesis that neurobehavioral diseases could be related to chemotherapy-mediated immunodeficiency (Ahles and Saykin 2007; Kipnis et al. 2008). The high level of corticosterone is likely to play a role in the obtained immune disturbance in ADR-injected rats, as corticosterone is thought to inhibit the cell-mediated immunity (CMI) and modulate monocyte and granulocyte counts (Song et al. 1994; Woiciechowsky et al. 1999). Quercetin treatment reversed leucopenia and slightly attenuated lymphopenia and monocytosis in ADR-exposed rats, while no effect was noted in respect of granulocytosis. The hematologic benefits of quercetin could be related to its potent ability to stabilize cell membranes by reducing lipid peroxidation (Chaudhuri et al. 2007) and DNA oxidative damage in human lymphocytes (Sato et al. 1997). Furthermore, quercetin has been indicated to enhance WBCs in X ray-exposed mice and human lymphocytes after radiotherapy (Fahlman and Krol 2009). The immune-stimulating effect of quercetin might also be due to its blockade of ADR-mediated HPA axis hyperactivation, a possible mechanism that was recently highlighted (Toumi et al. 2013).

In conclusion, we have demonstrated that quercetin administration attenuates ADR-associated behavioral disorders, corticosterone increase, brain oxidative injury, and immune cell count alteration in male Wistar rats. Further experimental studies are needed to clarify the potential effect of quercetin on chemotherapy-mediated cognitive and physiological impairments.