Effect of Cannabis sativa on oxidative stress and ...

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Comp Clin Pathol DOI 10.1007/s00580-013-1745-1

ORIGINAL ARTICLE

Effect of Cannabis sativa on oxidative stress and organ damage after systemic endotoxin administration in mice Omar M. E. Abdel-Salam & Somaia A. Nada & Neveen A. Salem & Marawa El-Sayed El-Shamarka & Enayat Omara

Received: 24 July 2012 / Accepted: 3 April 2013 # Springer-Verlag London 2013

Abstract The effect of Cannabis sativa extract on oxidative stress and organ tissue damage during systemic inflammation was studied. For this purpose, Swiss mice were challenged with a single intraperitoneal dose of lipopolysaccharide (LPS; 200 μg/kg) to mimic aspects of mild systemic infection. Cannabis resin extract (5, 10, or 20 mg/kg) (expressed as Δ 9 -tetrahydrocannabinol) was given via subcutaneous route for 2 days prior to and at the time of endotoxin administration. Mice were euthanized 4 h after LPS injection. Malondialdehyde (MDA), reduced glutathione (GSH), and nitric oxide (nitrite/nitrate) in the brain, liver, kidney, lung, and heart as well as brain glucose were measured. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were measured in liver homogenates. Histopathological examination of different organs was performed, and immunohistochemical techniques were used to evaluate expression levels of inducible nitric oxide synthase (iNOS) and caspase-3 in the brain and liver. The administration of only cannabis (20 mg/kg) decreased MDA, increased GSH, and decreased glucose level in the brain. No significant effects were observed for cannabis alone on MDA, GSH, or nitric oxide in other organs or on liver enzymes. The administration of LPS increased MDA and nitric oxide, while GSH decreased in different organs. Brain glucose increased by endotoxin. AST, ALT, and ALP were markedly increased in the liver tissue. In LPS-treated mice, cannabis (20 mg/kg) O. M. E. Abdel-Salam (*) : N. A. Salem : M. E.-S. El-Shamarka Department of Toxicology and Narcotics, National Research Centre, Tahrir St., Dokki, Cairo, Egypt e-mail: [email protected] S. A. Nada Department of Pharmacology, National Research Centre, Cairo, Egypt E. Omara Department of Pathology, National Research Centre, Cairo, Egypt

decreased MDA. GSH increased in the brain, kidney, and lung, nitric oxide decreased in the brain and lung while brain glucose decreased after the highest dose of cannabis. Cannabis failed to alter the level of liver enzymes. Histological damage in the brain, kidney, heart, lung, and liver due to endotoxin is increased by cannabis. Increased immunoreactivity of caspase-3 in the cytoplasm of the hepatocytes was observed after LPS and cannabis cotreatment compared with the LPS only group. Caspase-3 immunoreactivity markedly increased in degenerating neurons of the cortex following cannabis and LPS cotreatment. iNOS inmmunoreactivity increased after LPS and more intense iNOS expression was detected in hepatocytes after cannabis and LPS cotreatment. iNOS expression increased after cannabis and LPS treatment especially in the cerebral cortex. Thus, the administration of cannabis decreased tissue oxidative stress but increased organ damage after endotoxin injection in mice. Keywords Cannabis . Lipopolysaccharide . Oxidative stress . Mice

Introduction Cannabis preparations are the most commonly used illicit drugs worldwide. They are derived from the flowering tops, leaves, and resin from the female plant of Cannabis sativa L. (family Cannabidaceae). Cannabis is abused for its mood altering properties. These psychotropic effects of cannabis are mediated by its main psychoactive constituent Δ9-tetrahydrocannabinol (Δ9-THC) (Mechoulam and Gaoni 1967; Ashton 2001; Huestis 2002). Cannabis also contains over 60 other cannabinoids, a C21 terpenophenolic compound that is uniquely produced by the cannabis plant, such as cannabinol, cannabidiol, cannabigerol, and cannabichromene. Some of these cannabinoids have been shown to possess important

Comp Clin Pathol

pharmacological effects (Ashton 2001; Pertwee 2005). Interest in the medicinal uses of cannabis is growing. Studies suggest that medical cannabis patients seek more information about various substances, including cannabis (Janichek and Reiman 2012). Medicinal use of cannabis was reported by patients with chronic pain, multiple sclerosis, depression, arthritis, and neuropathy (Ware et al. 2005). Lifetime or current use of cannabis has also been reported by patients with Parkinson’s disease (Venderová et al. 2004), psychotic disorders (Machielsen et al. 2010), fibromyalgia Fiz et al. (2011), sickle cell disease (Howard et al. 2005), ulcerative colitis, or Crohn’s disease for symptom relief (Lal et al. 2011). Sativex, an oromucosal spray of whole plant extract containing equal proportions of Δ9-tetrahydrocannabinol and cannabidiol, has been used in multiple sclerosis patients for bladder dysfunction (Brady et al. 2004), intractable neurogenic symptoms (Wade et al. 2003), and for treatment-resistant spasticity (Collin et al. 2010; Sastre-Garriga et al. 2011). Smoking cannabis has also been utilized to reduce neuropathic pain (Ellis et al. 2009; Ware et al. 2010). Nabilone, a synthetic THC for oral administration, is effective in alleviating nausea and vomiting associated with chemotherapy and as an analgesic in pain conditions (Ware et al. 2010). Cannabis preparations are thus used by different patient populations having conditions characterized by systemic or neuroinflammation. Cannabis and cannabinoids can influence the immune system both in the periphery as well as in the central nervous system (Klein et al. 2003; Chuchawankul et al. 2004; Klein and Cabral 2006; Lu et al. 2006; Ignatowska-Jankowska et al. 2009) by acting on cannabinoid CB2 receptors. The latter are mainly expressed in peripheral tissues on immune cells, one of their roles being to modulate cytokine release and inflammatory responses (Devane et al. 1988; Matsuda et al. 1992; Munro et al. 1993; Pertwee 2005; Downer 2011). Marijuana and its active constituent Δ9-THC suppress cell-mediated immune responses and cytokine mechanisms, leading to the hypothesis that these agents may be of value in the management of chronic inflammatory diseases (Klein and Cabral 2006). Cannabis, however, is likely to enhance infectious agents. In this context, Δ9-THC injection suppressed type 1 T-helper 1 (Th1) immunity to Legionella pneumophila (Klein et al. 2003), whereas a single dose of cannabis resin was as effective as Δ9-THC in enhancing the severity and duration of illness in vaccinia virus-infected mice (Huemer et al. 2011). Lipopolysaccharide (LPS), a product of the Gramnegative bacterial cell wall, potently stimulates the innate immune system, eliciting both pro- and anti-inflammatory responses (Ulevitch and Tobias 1995; Heine et al. 2001). Lipopolysaccharide can be recognized by immune cells such as monocytes, macrophages, neutrophils, and dendritic cells as a pathogen-associated molecule through Toll-like receptor

4 (TLR4). Mammalian TLR4 is the signal-transducing receptor that when activated by the bacterial LPS triggers the acute inflammatory cascade, alerting the host to infection by Gramnegative bacteria. The response from the host immune system depends on both the severity of infection and the particular structure of LPS of the invading bacteria (Wang and Quinn 2010). Systemic administration of LPS activates immune cells in the periphery to release pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1β, and IL-6 (Hua et al. 1996). Systemic injection of LPS causes neuroinflammation and thus represents a useful model for studying the effect of systemic inflammation on brain function (Qin et al. 2007; Noble et al. 2007; Jacewicz et al. 2009; Jeong et al. 2010). In view of the widespread use of cannabis, it looked pertinent to evaluate its effect on mild systemic inflammation. In the present study, the effect of cannabis resin was evaluated on organ damage and oxidative stress induced in mice by subseptic dose of LPS so as to produce a state of mild systemic inflammatory illness.

Materials and methods Animals Swiss male albino mice 22–25 g of body weight (age 5– 6 weeks) were used. Mice were obtained from the animal house colony of the National Research Centre (Cairo, Egypt). Standard laboratory food and water were provided ad libitum. Animal procedures were performed in accordance with the Ethics Committee of the National Research Centre and followed the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985). Drugs and chemicals A purified lyophilized Escherichia coli endotoxin (Serotype 055:B5, Sigma, St Louis, MO, USA) was used and dissolved in sterile saline, aliquoted, and frozen at −20 °C. Cannabis resin was supplied by the Ministry of Justice (A.R.E.). Study design Mice were randomly divided into five equal groups (six mice each). Mice were treated with vehicle (group 1) or C. sativa (5, 10, or 20 mg/kg) (expressed as Δ9-tetrahydrocannabinol) (groups 2, 3, and 4) via s.c. route for 2 days prior to and at the time of endotoxin administration (LPS, 200 μg/kg, i.p.). The fifth group (n=6) received only the vehicle (−ve control). Mice were euthanized 4 h after LPS or vehicle injection by

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decapitation under ether anesthesia, and the brain, lungs, heart, kidneys, and liver were then removed, washed with ice-cold phosphate buffered saline (PBS, pH 7.4), weighed, and stored at −80 ° until the biochemical analyses. The tissues were homogenized with 0.1 M phosphate buffer saline at pH 7.4, to give a final concentration of 10 %w/v for the biochemical assays.

groups of GSH to form 2-nitro-S-mercaptobenzoic acid, and the nitromercaptobenzoic acid anion has an intense yellow color which can be determined spectrophotometrically. GSH concentration was calculated by comparison with a standard curve.

Chemistry

Nitric oxide estimated as nitrate/nitrite was determined in tissue homogenates according to Moshage et al. (1995). The first step converts nitrate to nitrite utilizing nitrate reductase. The second step uses Griess reagents to convert nitrite to a deep purple azo compound. The amount of the azochromophore accurately reflects nitric oxide amount in the samples. The samples were analyzed at 540 nm in a spectrophotometer.

Extraction Dry extract of C. sativa C. sativa extract was prepared from the dried resin. The method of extraction followed that described by Turner and Mahlberg (1984) with modification. In brief, 10 g of dried cannabis was divided into small pieces. Decarboxylation of the plant material was achieved by placing the sample in a glass test tube (30 ml) and covering it with aluminum foil. The test tubes were placed in boiling water bath (100 °C) for 2 h. Ten milliliters of analytical grade chloroform was added and allowed to react for 1 h. The dried cannabis was extracted three times with 20 ml chloroform, and the fractions were combined and filtered. The filtrate was evaporated under a gentle stream of nitrogen (on ice and protected from light), stored at 4 °C, and protected from light (in an aluminum-covered container). This provided the dry extract as residue.

Determination of nitric oxide

Determination of brain glucose Glucose was determined in serum according to the method of Trinder (1969). Glucose in the presence of glucose oxidase is converted to peroxide and gluconic acid. The produced hydrogen peroxide reacts with phenol and 4-amino-antipyrine in the presence of peroxidase to yield a colored quinonemine, which is measured spectrophotometrically. Determination of liver enzymes

Preparation of diluted extract for injection The residue (dry extract) was suspended in 2 ml of 96 % ethanol and the total volume in the volumetric flask was increased to 100 ml by adding distilled water. The extract was injected intraperitoneally at doses of 5, 10, and 20 mg/kg (expressed as delta-9THC). The injection volume was 0.2 ml/mice. THC content was quantified using GC mass. The THC content of the extract was 20 %.

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in liver were measured according to the Reitman–Frankel colorimetric transaminase procedure (Crowley 1967), whereas colorimetric determination of alkaline phosphatase (ALP) activity was done according to the method of Belfield and Goldberg (1971), using commercially available kits (BioMérieux, France).

Biochemical studies Histological assessment of liver injury Determination of lipid peroxidation Lipid peroxidation was assayed by measuring the thiobarbituric acid-reactive substances in tissue homogenates, as previously 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 peak absorbance at 532 nm. Determination of reduced glutathione Reduced glutathione (GSH) was determined in tissues by the method of Ellman (1959). The procedure is based on the reduction of Ellman’s reagent by –SH

The liver from each mouse was rapidly removed and fixed in freshly prepared 10 % neutral buffered formalin, processed routinely, and embedded in paraffin. Sections were cut 5 μm in thickness. The sections were stained with hematoxylin and eosin (H&E) for histopathological examination (Drury and Walligton 1980). All sections were investigated by a light microscope. Immunohistochemistry for caspase-3 and inducible nitric oxide synthase Immunohistochemical staining of anti-caspase-3 and anti iNOS antibodies was performed by streptoavidin–biotin. Four-

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micrometer–thick sections were deparaffinized and incubated with fresh 0.3 % hydrogen peroxide in methanol for 30 min at room temperature. The specimens were then incubated with anti-caspase-3 and inducible nitric oxide synthase (iNOS) antibody as the primer antibody at a 1:100 dilution. The specimens were counterstained with H&E. Negative controls were prepared by substituting normal mouse serum for each primary antibody.

USA). A probability value of less than 0.05 was considered statistically significant.

Results Biochemical results Brain

Statistical analysis Data are expressed as mean±SEM. The data were analyzed by one-way ANOVA followed by Duncan’s multiple range test, using SPSS software (SAS Institute Inc., Cary, NC,

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In saline-treated mice, the administration of only cannabis at the dose of 20 mg/kg decreased brain MDA by 20.4 % (21.9±1.4 vs. 27.5±1.5, p