The effects of Nigella sativa hydro-alcoholic extract and thymoquinone on lipopolysaccharide - induced depression like behavior in rats.

BACKGROUND: Neuroimmune factors have been proposed as contributors to the pathogenesis of depression. Beside other therapeutic effects including neuroprotective, antioxidant, anticonvulsant and analgesic effects, Nigella sativa and its main ingredient, thymoquinone (TQ), have been shown to have anti-inflammatory effects. In the present study, the effects of Nigella sativa hydro-alcoholic extract and thymoquinone was investigated on lipopolysaccharide- induced depression like behavior in rats.
MATERIALS AND METHODS: 50 male Wistar rats were divided into 5 groups: Group 1 (control group) received saline instead of NS extract, thymoquinone or lipopolysaccharide. The animals in group 2 (lipopolysaccharide (LPS)) were treated by saline instead of NS extract and were injected LPS (100μg/kg, ip) 2 hours before conducting each forced swimming test. Groups 3 (LPS + NS 200) and 4 (LPS + NS 400) were treated by 200 and 400 mg/kg of NS (ip), respectively, from the day before starting the experiments and before each forced swimming test. These animals were also injected LPS 2hours before conducting each swimming test. The animals in group 5 received TQ instead of NS extract. Forced swimming test was performed 3 times for all groups (in alternative days), and immobility time was recorded. Finally, the animals were placed in an open- field apparatus, and the crossing number on peripheral and central areas was observed.
RESULTS: The immobility time in the LPS group was higher than that in the control group in all 3 times (P<0.001). The animals in LPS + NS 200, LPS + NS 400 and LPS + TQ had lower immobility times in comparison with LPS groups (P<0.01, and P<0.01). In the open- field test, the crossing number of peripheral in the LPS group was higher than that of the control one (P<0.01) while the animals of LPS + NS 200, LPS + NS 400 and LPS + TQ groups had lower crossing number of peripheral compared with the LPS group (P <0.05, and P<0.001). Furthermore, in the LPS group, the central crossing number was lower than that of the control group (P<0.01). In the animals treated by NS or TQ, the central crossing number was higher than that of the LPS group (P<0.05, and P<0.001).
CONCLUSIONS: The results of the present study showed that hydro-alcoholic extract of Nigella sativa can prevent LPS-induced depression like behavior in rats. These results support the traditional belief on the beneficial effects of Nigella sativa in the nervous system. Moreover, further investigations are required in order to better understand this protective effect.

Depression, the second most common chronic disease, is expanding in the world while about half of the patients with depression are unaware of their disease, or their disease are diagnosed else.[12] Depression occurs in children, adolescents, adults and in elderly as a result of the combination of states of sadness, loneliness, irritability, absurdity, despair, confusion and shame and reveals some physical symptoms.[1] Fortunately, proper diagnosis and treatment can significantly reduce the symptoms of depression.[3] From among the depressive patients who have been treated by drugs or psychotherapeutic methods, about 5% and 15% had complete and partial recovery and between 20% and 35% did not respond at all.[34]

Neuroimmune factors have been proposed as contributors to the pathogenesis of depression.[56] It has been shown that increased levels of the inflammatory markers such as C-reactive (CRP), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-a) are associated with depression.[78] It has also been shown that antidepressant medications affect cytokine levels, and that this mechanism appears to influence the treatment outcome in depression.[910] Neuroinflammation, the process, which is accompanied by brain immune response and glial cell activation, has been shown to play an important role in depression.[11] Moreover, antidepressant agents have anti-neuroinflammatory properties.[11] In vivo studies using animal models have demonstrated that different types of antidepressants exert on the expression of inflammatory mediator, such as cytokines, microgliosis and astrogliosis in the nervous system. In vitro studies on rodent glial cells have also demonstrated that some types of antidepressants decrease glial generation of inflammatory molecules.[11] Considering these findings, an inhibition for neuroinflammation seems to be a new mechanism of action of antidepressant treatment.

NS L. (NS) is an annual flowering plant, native to different regions of southern Europe and some parts of Asia. The flowers are delicate and are usually colored pale blue and white with small black seeds.[12] NS seeds are the source of active components such as 30-40% fixed oil, 0.5-1.5% essential oil, various sugars and proteins and pharmacologically active components containing thymoquinone (TQ), ditimoquinone (DTQ) and nigellin.[12-17] In traditional medicine, this herb was indentified to have healing power so that it has been used in the middle east and far east for treating diseases such as asthma, headache, dysentery, infections, obesity, back pain, hypertension and gastrointestinal problems. Finally, there is a common Islamic opinion that the NS is useful for all diseases except death.[18] The results of previous experimental studies have supported the pharmacological effects of the seeds of NS or thymoquinone. It has been reported that the extract of NS seeds and thymoquinone inhibit nitric oxide production and inducible nitric oxide synthase expression.[121920] The anti oxidant effects of NS and thymoquinone in CCl4-induced oxidative injury in rat liver,[21] isolated rat hepatocytes,[22] hypercholesterolemic rats[23] and gentamicin[24] and cyclosporine induced kidney injury have been reported as well.[2425] Hosseinzadeh et al.[26] found that NS oil and thymoquinone have antioxidant effects during cerebral ischemia-reperfusion injury in rat hippocampus.[26] Anticancer activity was reported against malignant cells in mice and humans.[2728] There is a line of evidence that NS and thymoquinone have anti-inflammatory activity and affect the immune system.[2930]

Based on [the properties of NS, which has been reported in traditional medicine and in experimental studies, the present study was designed in order to evaluate possible effects of hydro-alcoholic extract of NS and thymoquinone on lipopolysaccharide-induced depression-like behavior in rats.

Materials and Methods

Preparing the plant extract

Powdered seeds (100 g) of NS were extracted in a Soxhlet extractor with ethanol (70%). The resulting extract was concentrated under reduced pressure and kept at -20°C until being used (yielded 32%). Nigella sativa extract was dissolved in saline.[3132]

Animals and the experimental protocol

50 male Wistar rats (8 weeks old and weighted 230 ± 20 g) were kept at 22 ± 2°C and 12 h light/dark cycle at 7:00 am. They were randomly divided into 5 groups, and treated according to the experimental protocol for 1 week. All measurements were performed between 10 and 14 am.

Rats in group 1 (control group) received saline instead of both NS extract and thymoquinone from the day before starting the experiments. The animals of this group were injected saline instead of lipopolysaccharide as well. Group 2 (lipopolysaccharide; LPS) animals were injected by saline instead of NS extract and thymoquinone from the day before starting the experiments. The animals in this group were injected lipopolysaccharide (Sigma Chemical Co.) 2 hours before conducting the swimming test. Groups 3 (lipopolysaccharide + NS 200; LPS + NS 200) and 4 (lipopolysaccharide + NS 400; LPS + NS 400) animals were treated by 200 and 400 mg/kg of NS extract from the day before conducting the swimming test. The animals of this group were injected LPS (100 μg/kg) 2 hours before doing the swimming test. Group 5 (lipopolysaccharide + thymoquinone; LPS + TQ) animals were injected by thymoquinone (40 mg/kg) instead of NS extract. Forced swimming test (FST) was carried out 3 times for all the groups (in alternative days). Finally, the animals were placed in the open-field and were observed for 5 min, and the peripheral and central crossing number was recorded.

Behavioral procedures

Forced swimming test

All animals were compromised to the test room environment for 1 h before the beginning of the experiment. During the FST, the animals were placed in a glass cylindrical tank with 60 cm height and 38 cm width, which was filled with water (24 ± 1°C) to the depth of 40 cm. The water was changed between each animal. In the first day, the rats were placed inside the water cylinder for 15 min (pre-test), and then the animals were placed individually inside the water cylinder for 5 min for the following 3 alternative days (test days).[33] 2 hours before each test, the rats were injected with either LPS 100 μg/kg (LPS group, LPS + NS 200, LPS + NS 400 and LPS + TQ groups) or saline (control group).[34] The animals of LPS + NS 200, LPS + NS 400 and LPS + TQ groups were treated by 200 mg/kg NS, 400 mg/kg NS and 40 mg/kg thymoquinone from the day before doing the swimming test. The time of floating (immobility) during the FST was recorded for 5 min in each session.[3536] Rats were considered immobile when they floated in the water; they only performed movements that enabled them to keep their head above the water. After the FST, the rats were dried in a heated cage, and then they were returned to their home cages.[37]

Open-field

Open-field test was carried out for studying the depression-like behaviors of animals after the first FST test.[38] In the present study, the open-field measurement was done by a wooden apparatus with an area of 100 × 100 cm and height of 40 cm. Inside of the apparatus was divided into 16 equal squares using a black line. In addition, within the apparatus was divided into 2 zones called peripheral and central zones.[39-41] All the rats were familiarized with the test environment by being placing in the room for 1-h before beginning the experiment. During the experiment, a low-level light was used to reduce anxiety.[37] Each animal was placed in the central zone, and its movement was recorded by a digital camera for 5 min,[39-42] and the following criteria were calculated: (1) The crossing number in the central zone, (2) the crossing number in the peripheral zone, and (3) rearing counts: The number of vertical movement when the rat stood vertically on its hind paws on the floor and forepaws on the wall.[37] 2 hours before each test, the rats were injected either LPS 100 μg/kg (LPS group, LPS + NS 200, LPS + NS 400 and LPS + TQ groups) or saline (control group).[34] The animals in LPS + NS 200, LPS + NS 400 and LPS + TQ groups were treated by 200 mg/kg NS, 400 mg /kg NS and 40 mg/kg thymoquinone 2 hours before the open-field tests.

Statistical analysis

The data were expressed as mean ± SEM. For the FST, repeated measured ANOVA, and for the data of open-field test, the one-way ANOVA were run, followed by tukey post hoc comparisons test. The criterion for the statistical significance was P < 0.05.

Results

The results of the FST showed that the immobility times in the control group were 13.13 ± 1.92, 10.74 ± 1.3 and 11.58 ± 2.21 sec in the 3 times, respectively. There was no significant difference between these 3 times. The animals in the LPS group had 39.68 ± 7.97, 51.73 ± 6.23 and 56.78 ± 8.11 sec immobility times. As shown in the figure, the immobility times increased in 3 days. The repeated measures ANOVA also showed that immobility times in the LPS group were higher than that of the control group (P < 0.001). In the LPS + NS 200 group, the immobility time was 17.3 ± 3.22, 15.34 ± 4.36 and 18.95 ± 3.66 sec, respectively. There was no significant difference between these 3 times. The comparison using the repeated measure ANOVA showed that the immobility times in the LPS + NS 200 group were lower than those that of the LPS group (P < 0.01); however, there was no significant difference between the LPS + NS 200 and control groups. In the LPS + NS 400 group, the 3 measured immobility times were 16.33 ± 2.50, 19.83 ± 3.56 and 14.64 ± 4.45 sec, respectively, and there was no significant difference between the 3 times. Immobility times were lower than those of the LPS group (P < 0.01) when being compared using the repeated measures ANOVA. In TQ + NS 400 group, the immobility times were 11.81 ± 1.94, 12.93 ± 1.59 and 13.65 ± 2.39 sec, respectively, and they were significantly lower than those of the LPS group (P < 0.001) [Figure 1].

Figure 1

Comparison of immobility times in the forced swimming test between 5 groups. Data are expressed as mean ± SEM (N = 10 in each group). ***P < 0.001 comparison of LPS group with Saline group, ++P < 0.01 comparison of LPS + NS 200 and LPS + NS 400 groups with LPS group, &&&P < 0.001 comparison of LPS + TQ group with LPS group.

The results of the open-field test showed that the number of crossing in the peripheral zone in the LPS group was higher than that of the control group (P < 0.01). As shown in Figure 2, the crossing number of peripheral zone in LPS + NS 200 , LPS + NS 400 and LPS + TQ groups was significantly lower than that in the LPS group (P < 0.001, P < 0.05 and P < 0.001, respectively) [Figure 2].

Figure 2

Comparison of the number of crossing in the peripheral zone in the open-field test. Data are expressed as mean ± SEM (N = 10 in each group). **P < 0.01 compared to Saline group, +P < 0.05 and +++P < 0.001 compared to LPS group.

The results also showed that the number of crossing in the central zone in the LPS group was lower than that in the control group (4.38 ± 0.47 vs. 9.5 ± 1.92), (P < 0.01). The crossing number in the central zone by the animals of LPS + NS 200, LPS + NS 400 and LPS + TQ groups was 7.37 ± 0.97, 8.33 ± 0.85 and 11.03 ± 1.31, respectively; they were significantly higher than those of the LPS group (P < 0.05, P < 0.01 and P < 0.001) [Figure 3].

Figure 3

Comparison of the number of crossing in the central zone in the open-field test. Data are expressed as mean ± SEM (N = 10 in each group). **P < 0.01 compared to Saline group, +P < 0.05, ++P < 0.01 and +++P < 0.001 compared to LPS group

As shown in Figure 4, there was no significant difference between the groups in the rearing number.

Figure 4

Comparison of the number of rearing in the open-field test. Data are expressed as mean ± SEM (N = 10 in each group)

Discussion

The results of the present study showed that treating of the animals by lipopolysaccharide induces a depression-like behavior in rats. Lipopolysaccharide is a fragment from the cell wall of gram-negative bacteria,[43] which triggers the production of IL-1β, interleukin-6 (IL-6), and TNF-α.[4445] Both systemic and central administrations of LPS increase the levels of IL-1β and the related pro-inflammatory cytokines in several areas of the brain including the hippocampus, hypothalamus and diencephalic structures.[4647] The result is a complex of behavioral changes, comparable with the cytokine-induced depression-like behavior in rats.[48] A large number of evidence has confirmed the role of inflammation in depression. Treatment with interleukin (IL)-2 or interferon (IFN)-γ in patients with cancer leads to high depression rates.[4950] In the present study, the corresponding depressive symptoms were observed in lipopolysaccharide treated rats.[5152] It has been previously reported that administration of LPS in doses, lower than the doses used in the present study, was resulted in avoidance of the central area of the open-field by arts.[5354] It has also been shown that peripherally injection of LPS in doses, higher than doses which were used in the present study, induced sickness behavior including reduction in appetite and body weight, suppresses exploratory and social activity, fatigue and malaise, impairment in cognitive abilities, reduced libido and sexual behavior and anhedonia as well as depression like behaviors.[55-58] The results of the present study showed that immobility times in the LPS group were higher than those of the control group in the forced swimming test. It seems that peripherally administration of a medium dose of LPS also induce depressive like behavior. However, the effects of LPS on behavior has been repeatedly evaluated in a period lower than 24 h, it is likely that the injection of LPS in 3 times, which was carried out in the present study, resulted in a cumulative response because it has been previously reported that the effect of LPS may be seen even 28 days after injection.[53-78] The results of present study also showed that the crossing number in the central area of the open-field by the animals of the LPS group was lower than that of the control group, which may be another evidence for depression like behavior due to administration of LPS.

On the other hand, patients with depression were also found to exhibit all the cardinal features of inflammation.[49] Evidence suggests that various anti-inflammatory manipulations have antidepressant effects in experimental animals and humans. For example, genetic knockout of IL-6 in mice reduces depressive-like behavior in the forced swim, tail suspension, learned helplessness, and sucrose preference tests.[59] Knockout of the IL-1 receptor and administration of IL-1 receptor antagonist also blocks stress-induced depressive-like behavior in the sucrose preference, social exploration tests, escape deficits, anhedonia and reduces social behavior.[60-63]

Consistent with the pathophysiology of depression, these cytokine-induced behavioral changes are associated with alterations in the metabolism of serotonin, norepinephrine and dopamine in brain regions, which are essential to the regulation of emotion, including the limbic system (amygdala, hippocampus and nucleus accumbens), as well as the regulation of psychomotor function and reward, including the basal ganglia.[6465]

The seed extract of NS has been used for many years as a natural remedy for treating of a number of illnesses and conditions due to its pharmacological properties and being an immunostimulant, anti-tumor, anti-inflammatory and respiratory stimulant agent.[182866-68] The volatile oil is extracted from the seeds, and its main active ingredient, thymoquinone has been shown to exert anti-inflammatory effects in a number of diseases including bronchial asthma.[6669-71] It has been shown that IL-4, IL-5, and IL-13 protein levels in the fluid of bronchial lavages were significantly reduced in allergic mice after TQ administration.[72]

TQ supplementation also could alleviate hepatic toxicity, induced by LPS in the form of normalization of GSH hepatic level and reduction of liver function parameters. Also, LPS-induced hepatic lipid peroxides formation (in the form of MDA) and apoptosis (indicated by hepatic caspase-3 activity) were markedly reduced in rats, which received TQ along with LPS. In addition, serum TNF-α and inflammatory changes in the liver section were markedly reduced in rats, which were co-administered TQ and LPS in comparison with the LPS-treated animals.[73] Tekeoglu et al. (2006) also reported that TQ had anti-inflammatory effects on experimentally-induced arthritis in rats and decreased the levels of TNF-a and IL-1β in circulation.[74] Similarly, Mohamed et al. (2005) showed that treatment of rats with thymoquinone prevented the experimental autoimmune encephalomyelitis,[75] which was probably due to its antioxidant and anti-inflammatory properties.[76] The results of the present study also showed that pretreatment by both NS extract and thymoquinone prevented depression-like behavior induced by LPS in rats. Based on the results of present study, the mechanism(s) for the effect of NS extract or thymoquinone is unknown, but it seems that they can affect depression like behavior induced by LPS with common or different mechanisms.

Besides thymoquinone, the main ingredient, which has an important role in pharmacological effects of NS, other component(s) may also be involved in the results of the present study. More recently, melanin has been shown to occur abundantly in the seed coats of NS.[77] On the other hand, according to a number of botanical sources, melanin has been found to act as an immunologically active modulator of cytokines.[7879] Therefore, an involvement of this ingredient of NS, which modulates cytokine production,[77] may also be suggested as a possible compound, which has a role in the result of the present study.

Conclusion

The results of the present study showed that the hydro-alcoholic extract of NS prevented lipopolysaccharide-induced depression like behavior in rats. These results support the traditional belief about the beneficial effect of NS on the nervous system. Further studies are required for determining (confirming) the protective effect of NS.