Introduction

∆9-tetrahydrocannabinol (THC) is the chief psychoactive constituent in the plant Cannabis sativa family Cannabaceae L which is linked to various health hazards (Volkow et al. 2014). According to the United Nations (2018), about 192.2 million people, which is about 3.9% of the world population, consume cannabis that makes it the most exceedingly abused illicit drug. THC manifests short- and long-term, physical and mental effects including impaired movement, memory, and attention as well as increased heart rate, depression, and anxiety (The National Academies of Sciences 2017). This is achieved by acting on specific cannabinoid receptors (CBRs) mainly CBR1 and CBR2. CBR1 plays a main part in the psychoactive and behavioral effects of cannabis (Tai and Fantegrossi 2014). It is mainly found centrally concentrated in the hippocampus which is involved in the memory and the cerebral cortex for cognition (Manzanares et al. 2006). CBR1 is also activated in the amygdala that is responsible for emotional responses. In addition, it is found in the limbic forebrain and cerebellum which are involved in motivation and motor coordination, respectively (Iversen 2012; Hu and Mackie 2015). On the other hand, CBR2 is primarily found peripherally in the immune system (Iversen 2012) and also in the gastrointestinal tract, heart, skin, and reproductive organs (Madras 2015).

Nicotine (Nic) is a naturally occurring alkaloid found primarily in the plant Nicotianatabacum, family Solanaceae L. It causes every year more than 7 million deaths globally; of which, 6 million are the result of the direct use of tobacco, while 890,000 are second-hand smokers (WHO 2018). Nicotine acts on the nicotinic acetylcholine receptor (nAChR) which resides both peripherally and centrally (Gotti and Clementi 2004). It has inverse dose-dependent protective antioxidant (Tunez et al. 2010) and deleterious pro-oxidant effects (Guan et al. 2003). Indeed, high Nic doses cause neurodegeneration via oxidative stress (Guan et al. 2003), while at a low-dose level, an anti-inflammatory effect through binding to neuronal α7 nAChRs is exerted (Viveros et al. 2006).

Cannabis and Nic are increasingly taken in combination, as Nic reduces the sedative effect of cannabis and increases and prolongs its rewarding effect (Tullis et al. 2003). Both induce complex dose-response effects, thus manifesting functional interactions in modulating the brain neurochemistry and behavioral aspects (Valjent et al. 2002). At the cellular level, CB1R and nAChRs are densely co-localized in the hippocampus to be involved in varied sets of modulatory processes (Filbey et al. 2015). Consequently, the current study investigated the potential modulatory effects of 30-days Nic co-administration with cannabis extract (10% THC) expressed as THC with emphases on the brain oxidative stress and inflammation status as well as behavioral outcomes in normal rats.

Material and methods

Animals

Adult male Wistar rats (150–200 g) were obtained from the animal house colony in the National Research Center (NRC; Giza, Egypt). Throughout the period of the investigation, the animals were housed in an ambient atmosphere, constant light cycle (12 h light/dark), and kept on a standard diet and tap water ad libitum.

Cannabis resin sampling and extraction

The dried cannabis resin sample (8,092,009/Azbakeyya Criminalistics) used in the current study was obtained from the Forensic Sciences of Ministry of Justice Laboratory (Cairo, Egypt). The chloroform extract was prepared with the modification previously described by Turner and Mahlberg (1984) at the laboratory of Toxicology and Narcotics Department (NRC, Cairo, Egypt). The dry extract was suspended in ethanol-saline (2%), and HPLC (Fischedick et al. 2009) quantification revealed that the mixture contained 10% THC.

Experimental design

Rats were classified into 6 groups, each included 9 rats that were daily injected intraperitoneally for 30 consecutive days with the tested compounds dissolved in saline. Rats that received saline served as the normal group, while those receiving 0.25 mg/kg Nic (File et al. 1998; Sigma-Aldrich, MO, USA) or THC (10 and 20 mg/kg; Pagotto et al. 2006) were designated as single-treatment regimen groups. Additionally, in the other 2 groups, the rats were injected by either Nic with THC10 or THC20.

Behavioral tests

Open field, forced swimming, and Morris water maze (MWM) tests were performed at the end of the experimental period to assess motor coordination and exploratory behaviors as well as depression and memory task.

Open field test

The open field test (Simonin et al. 1998) was carried out in a white squared wooden arena (80 cm wide, 80 cm long, and 40 cm high), with red walls divided by black lines into a total of 16 equal squares on the white floor. The test was performed under white light in a quiet room to avoid any external stimuli. The behaviors were recorded during an observation period of 5 min which included ambulation, rearing, and grooming frequencies.

Forced swimming test

In the forced swimming test, a 10-min pretest was done 24 h prior to the test day. The rats then were forced to swim individually in a plastic cylinder (46 cm in height with a 21-cm internal diameter), filled with water (25 °C) to a depth of 30 cm from which there was no escape. The passive (immobility) behavior was recorded for 5 min for each rat (Slattery and Cryan 2012).

Morris water maze test

Typically, the maze consists of a large pool with side walls of the 70 × 40 × 20 cm dimensions. The transparent platform’s top surface was hidden 1 cm below the water surface and was made rigid to make it easy to climb on. After 3 days of training, for evaluating memory performance, the time taken to reach the platform was recorded for each rat (Graziano et al. 2003).

Preparation of samples

After 24 h from the last dose administration, rats were deeply anesthetized for blood collection and euthanized by cervical dislocation to excise the brains for homogenization in ice-cold 0.1 M phosphate buffer saline at pH 7.4.

Determination of brain oxidative stress biomarkers

Brain oxidative stress biomarkers were assessed in brain homogenates by determining malondialdehyde (MDA), ascorbic acid, and superoxide dismutase (SOD). MDA was determined by measuring thiobarbituric reactive species using the method described by Ruiz-Larrea et al. (1994) in which the thiobarbituric acid reactive substances react with thiobarbituric acid to produce a red-colored complex having a peak absorbance at 532 nm. The main principle of ascorbic acid methodology (Harris and Ray 1935) is the redox reaction of ascorbate with 2,6-dichlorophenolindophenol, where ascorbate is oxidized to dehydroascorbate, while 2,6-dichlorophenol indophenol is reduced to a colorless leuco base. SOD is determined using a commercially available kit, colorimetric (Biodiagnostic, Cairo, Egypt), according to the method described by Nishikimi et al. (1972).

Determination of brain interleukin 1-beta

Brain interleukin (IL)-1β was determined using a commercially available ELISA kit (Sun Red Biological Technology, Shanghai, PRC), according to the manufacturer’s protocol.

Determination of butyrylcholinesterase and acetylcholinesterase activities

Serum butyrylcholinesterase (BChE; Knedel and Bottger 1967) activity was assessed depending on the hydrolysis of butyrylthiocholine by butyrylcholinesterase to give thiocholine and butyrate. A second reaction occurs between the resultant thiocholine and 5,5′-dithio-bis 2-nitrobenzoic acid (DTNB), yielding 2-nitro-5-mercaptobenzoate (a yellow compound) which can be measured at 405 nm. The determination of acetylcholinesterase activity in serum is a modification of the Ellman et al. (1961) method as described by Gorun et al. (1978). The principle depended on the measurement of thiocholine produced upon the hydrolysis of acetyl thiocholine where the color was read immediately at 412 nm.

Determination of brain glucose content

Brain glucose content (Trinder 1969) was assessed by its conversion to peroxide and gluconic acid in the presence of glucose oxidase. The produced hydrogen peroxide reacted with phenol and 4-aminoantipyrine in the presence of peroxidase to yield a colored quinonemine which is measured spectrophotometrically at 510 nm.

Statistical analysis

Data were presented as means ± standard error of the means (SEM) or median (minimum–maximum). Comparison between groups was carried out using the parametric one-way ANOVA test followed by Tukey multiple comparison test except for the open field test where nonparametric Kruskal-Wallis one-way ANOVA was carried out followed by Dunn multiple comparison test. Differences were considered significant when P ˂ 0.05. Graph-pad prism 6.00 for Windows software (CA, USA) was used to plot graphs and carry out these statistical tests.

Results

Nic improves locomotion in cannabis-treated rats

Nic-treated animals tended to show an increase in ambulation (Fig. 1a), rearing (Fig. 1b), and grooming (Fig. 1c) in comparison to the normal group in the open field test. On the other hand, THC10 administered alone reduced rearing by 93% relative to the normal group, while the co-administration of THC10 with Nic decreased ambulation, rearing, and grooming frequencies by 84, 85, and 68%, respectively, relative to Nic. However, Nic/THC20 increased ambulation and grooming by 5.2- and 7.7-folds, respectively, relative to THC20.

Fig. 1
figure 1

Effects of 30-day nicotine and/or tetrahydrocannabinol administration on ambulation (a), rearing (b), and grooming (c) frequencies in open field test in rats. Rats were intraperitoneally injected daily with nicotine (N; 0.25 mg/kg) and/or tetrahydrocannabinol (T; 10 and 20 mg/kg) for 30 days. OFT was carried out on the 30th day of treatments. Results (n = 6 rats, per group) are expressed as boxplots presented with median (minimum–maximum) as well as 25th and 75th percentile values of ambulation (Fig. 1a), rearing (Fig. 1b), and grooming (Fig. 1c) frequencies among groups. Statistical analysis was carried out by nonparametric Kruskal-Wallis one-way ANOVA followed by Dunn multiple comparison test. *,@,^P ˂ 0.05, compared to normal, N0.25, and T20 groups respectively. OFT, open field test

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Nic reduces depressive-like behavior in cannabis-treated rats

Animals receiving THC20 showed an increase in floating time by 67% relative to the normal group while it was decreased by 44% (THC10) and 31% (THC20) in THC/Nic combinations relative to the corresponding cannabinoid dose. Notably, THC20/Nic increased the floating time by 130% relative to Nic0.25 group (Fig. 2).

Fig. 2
figure 2

Effects of 30-day nicotine and/or tetrahydrocannabinol administration on forced swimming test in rats. Rats were intraperitoneally injected daily with nicotine (N; 0.25 mg/kg) and/or tetrahydrocannabinol (T; 10 and 20 mg/kg) for 30 days. FST was carried out on the 30th day of treatments. Results are expressed as mean ± SEM (n = 6 rats). Statistical analysis was carried out by one-way ANOVA followed by Tukey multiple comparison test. *,@,$,^P ˂ 0.05, compared to normal, N0.25, and T10/20 groups respectively. FST, forced swimming test

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Nic improves memory in cannabis-treated rats

In the MWM test, THC10 experienced increased latency by 62% relative to the normal group, while the administration of Nic/THC10 decreased it by 45% relative to the corresponding dose of THC10 (Fig. 3).

Fig. 3
figure 3

Effects of 30-day nicotine and/or tetrahydrocannabinol administration on escape latency on Morris water maze test in rats. Rats were intraperitoneally injected daily with nicotine (N; 0.25 mg/kg) and/or tetrahydrocannabinol (T; 10 and 20 mg/kg) for 30 days. Morris water maze test was carried out on the 30th day of treatments. Results are expressed as mean ± SEM (n = 6 rats). Statistical analysis was carried out by one-way ANOVA followed by Tukey multiple comparison test. *,$P ˂ 0.05, compared to normal and T10 groups, respectively. MWM, Morris water maze test

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Nic decreases cannabis-induced oxidative stress

THC10 elevated MDA by 47% (Fig. 4a), where the addition of Nic decreased lipid peroxidation by 25% (Nic/THC10) relative to THC10 and 43% (Nic/THC20) compared to Nic. Tough THC10 only elevated ascorbic acid by 124% (Fig. 4b); in THC20-treated animals, both ascorbic acid (34%; Fig. 4b) and SOD (172%; Fig. 4c) contents were raised relative to that of the normal group. The co-administration of Nic with THC10 suppressed ascorbic acid (69%) relative to THC10, an effect that was lower than both THC10-treated and normal rats. Meanwhile, Nic co-administered to normal rats receiving THC reduced the brain SOD content by 58% (Nic/THC10) and 74% (Nic/THC20) compared to their counter partners as well as to normal rats and their respective single regimens. Notably, SOD in Nic/THC20-treated animals was higher (53%) than Nic alone.

Fig. 4
figure 4

Effects of 30-day nicotine and/or tetrahydrocannabinol administration on brain malondialdehyde (a), ascorbic acid (b) contents, and superoxide dismutase activity (c) in rats. Rats were intraperitoneally injected daily with nicotine (N; 0.25 mg/kg) and/or tetrahydrocannabinol (T; 10 and 20 mg/kg) for 30 days. Results are expressed as mean ± SEM (n = 6 rats). Statistical analysis was carried out by one-way ANOVA followed by Tukey multiple comparison test. *,@,$,^ P ˂ 0.05, compared to normal, N0.25, and T10/20 groups, respectively. MDA, malondialdehyde; SOD, superoxide dismutase

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Nic decreases cannabis-induced inflammation

Notably, THC10/20 increased IL-1β to reach 2.5- and 1.7-folds in comparison to the control, respectively (Fig. 5). These values were leveled off to 39% (THC10/Nic) and 36% (THC20/Nic) when co-administered with Nic in comparison to THC10 and THC20, respectively.

Fig. 5
figure 5

Effects of 30-day nicotine and/or tetrahydrocannabinol administration on brain interleukin-1β content in rats. Rats were intraperitoneally injected daily with nicotine (N; 0.25 mg/kg) and/or tetrahydrocannabinol (T; 10 and 20 mg/kg) for 30 days. Results are expressed as mean ± SEM (n = 6 rats). Statistical analysis was carried out by one-way ANOVA followed by Tukey multiple comparison test. *,@,$,^P ˂ 0.05, compared to normal, N0.25, and T10/20 groups, respectively. IL-1β, interleukin-1 beta

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Nic prevents cannabis-mediated serum BChE reduction and elevates brain glucose

Though all treatments did not alter serum AChE activity, THC10/20 suppressed BChE activity by 49 and 47%, respectively, when compared to the normal group, while Nic was able to normalize its level when combined with both cannabinoid doses (Fig. 6). Finally, although serum glucose was not changed, Nic/THC combination elevated brain glucose level relative to normal, Nic, and/or THC (Fig. 7).

Fig. 6
figure 6

Effects of 30-day nicotine and/or tetrahydrocannabinol administration on serum acetylcholinesterase (a) and butyrylcholinesterase (b) activity in rats. Rats were intraperitoneally injected daily with nicotine (N; 0.25 mg/kg) and/or tetrahydrocannabinol (T; 10 and 20 mg/kg) for 30 days. Results are expressed as mean ± SEM (n = 6 rats). Statistical analysis was carried out by one-way ANOVA followed by Tukey multiple comparison test. *,$P ˂ 0.05, compared to normal and T10 groups, respectively. AChE, acetylcholinesterase; BChE, butyrylcholinesterase

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Fig. 7
figure 7

Effects of 30-day nicotine and/or tetrahydrocannabinol administration on brain glucose content in rats. Rats were intraperitoneally injected daily with nicotine (N; 0.25 mg/kg) and/or tetrahydrocannabinol (T; 10 and 20 mg/kg) for 30 days. Results are expressed as mean ± SEM (n = 6 rats). Statistical analysis was carried out by one-way ANOVA followed by Tukey multiple comparison test. *,@,$,^P ˂ 0.05, compared to normal, N0.25, and T10/20 groups, respectively

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Discussion

The present study clearly indicated that Nic co-administration with THC counteracted depression, memory deficits, and hypo-locomotion induced by THC in naïve rats. Nic co-treatment also decreased THC-induced brain oxidative stress and neuro-inflammation.

The administration of 10 mg/kg THC elevated lipid peroxidation and ascorbic acid in the brains of naïve rats. The redox imbalance could be explained by THC-induced oxidative stress that goes in line with a previous report (López-Malo et al. 2016). However, by doubling the THC dose, only ascorbic acid and SOD were increased. Herein, implying that the increased antioxidant defenses might have neutralized the free radicals that mediated the damage of the lipid cell membrane was consistent with the study of Wolff et al. 2015. Free radicals, including reactive oxygen species (ROS), had been previously reported to be implicated in several behavioral changes (Tanasawet et al. 2017). These include depression, memory decline, and locomotion deficits (Degenhardt et al. 2002; Schoeler and Bhattacharyya 2013; Freedland et al. 2002). Accordingly, it could be speculated that the oxidative stress associated by THC consumption could be the culprit for the behavioral changes seen in the present work following the cannabis extract administration.

At the molecular level, the depression, cognitive, and locomotion impairments were depicted to be the consequences of CB1R activation by THC (Iversen 2003). Of note, the activation of the CBR is known to stimulate the transcriptional activation of nuclear factor (NF) κB (Jean-Gilles et al. 2015), which initiates the production of ROS via the activation of NADPH oxidase and the inflammatory cytokine IL-1β (Sedeek et al. 2013; Chen et al. 2015); both of which increase the transcriptional activity of NFκB again to amplify their production (Kaushal and Bansal 2014). Indeed, in this investigation, THC was also able to increase IL-1β in the brains of rats indicating the development of neuro-inflammation that plays an essential role in depression, memory loss (Bevan-Jones et al. 2017), and hypo-locomotion (Bonsall et al. 2015). The effect of the cannabinoid extract on the cytokine was supported by previous data (Cabral 2001). Henceforth, the inhibition of oxidative and inflammatory processes was likely implicated in the treatment of memory deficits, depression (Kruk-Slomka et al. 2016), and hypo-locomotion (Patel et al. 2016), where co-treatment with Nic, in the present study, could provide therapeutic benefits against the reported behavioral deficits induced by THC. Indeed, this alkaloid was able to reduce both neuro-inflammation and oxidative damage as manifested by the decreased IL-1β, MDA, and the antioxidant defenses as well, when co-administered with THC in the current study. These beneficial effects might be linked to the ability of Nic to activate α7nAChR subunit that represses NFκB (Yoshikawa et al. 2006). Such an effect activates the cholinergic anti-inflammatory pathway to lend a plausible explanation to Nic antioxidant and anti-inflammatory activities to hamper THC devastating effects on memory, euthymic mood, and decreased movement, as shown in the present work. In fact, Nic has been accounted for its therapeutic use in some inflammatory and oxidative stress-mediated diseases (Yoshikawa et al. 2006). Besides the antioxidant effect of Nic, as depicted herein and earlier (Kacham 2013), the stimulant nature of the alkaloid small doses (Rose et al. 2003) can lend credit to the increased locomotion in both single and Nic/THC20 groups.

THC did not alter serum AChE activity but suppressed that of BChE; both of which catalyze the hydrolysis of acetylcholine (Ach) (Chen et al. 2011). On the other hand, only Nic co-administered with THC10 normalized BChE. Though serum levels of both esterases are reflections of their brain contents, Abdel-Salam et al. (2016) reported that the hydrolysis of ACh was mainly carried out faster by the specific enzyme with a negligible role for the pseudo-enzyme after THC administration in naïve rats, indicating a minor role for BChE activation by Nic co-administration in memory consolidation.

Of note, decreased brain glucose is linked to impaired cerebral energy metabolism and memory deficit, while on the contrary, its elevation enhanced memory (Abdel-Salam et al. 2013). Previous data showed that the administration of THC had variable dose- and region-dependent effects related to the CB1R (Miederer et al. 2017) on the brain glucose content ranging from low to high compared to untreated animals. However, our investigation showed that the cannabis extract failed to alter the whole brain glucose content, whereas only the combined administration of THC/Nic was able to elevate the brain glucose content. Accordingly, this effect might afford a further explanation to the enhanced memory consolidation seen in THC/Nic rats suggesting an interaction between these addictive and recreational drugs.

The present data provides evidence for the facilitatory effects of Nic on memory and locomotion impaired by THC, besides euthymia via antioxidant and anti-inflammatory potentials. This was reflected by functional as well as neurochemical and neuronal enhancements when Nic was co-adjunct with THC to provide important insights for understanding the consequences of habitual cannabis and/or Nic consumption.