Cannabidiol (CBD) is a non- psychoactive component of the Canna- bis Sativa plant and has anti- inflammatory properties. This study was held to evaluate the ...
THE EGYPTIAN JOURNAL OF MEDICAL SCIENCES VOL. 36-No. 2 – DECEMBER 2015 (ISSN: 1110-0540)
9. Egypt. J. Med. Sci. 36 (2) December 2015: 659-684. THE PROTECTIVE EFFECTS OF CANNABIDIOL AGAINST THE HYPERGLYCEMIA OF THE STREPTOZOCIN-INDUCED DIABETES ARE THROUGH SUPPRESSION OF INFLAMMATION AND APOPTOSIS AND ENHANCEMENT OF -CELL REPLICATION IN THE ADULT ALBINO RAT By Noura M. S. Osman & Samah Shabana1&2 Department of Human Anatomy& Embryology, Faculty of Medicine, Minia University, El Minia, Egypt 1 Department of Pharmacognosy, Faculty of Pharmacy, Misr University for Science and Technology, 6th October City, Egypt 2 Department of Pharmacognosy, Pharmacy Program, Batterjee Medical College, Jeddah, Saudi Arabia ABSTRACT Cannabidiol (CBD) is a nonpsychoactive component of the Cannabis Sativa plant and has antiinflammatory properties. This study was held to evaluate the effects of CBD on mild diabetes induced by the administration of low doses of streptozocin (STZ) in rats. Fifty male adult albino rats were divided into: Group I: Normal control; Group II: Diabetic control& Group III: Diabetic +CBD (CBD was used in a dose of 20 mg/kg orally daily for 28 days). At the end of the experiment the results showed that the serum fasting glucose and insulin levels were normal in the Diabetic +CBD group. Histological examination revealed that CBD-treatment retains the normal islet architecture after STZ administration. Apoptotic cells were significantly lower in the islets of the Diabetic +CBD group compared to the Diabetic one as revealed by TUNEL reaction. Indicators of inflammation
were lower in the Diabetic +CBD group than that of the Diabetic control one as shown by the lower number of Mac-2 immune-positive macrophages infiltrating the islets and the significantly lower IFN-γ &TNF-α protein levels as observed by western blot analysis in the pancreas of Diabetic +CBD group. Restoration of the β- cell mass was observed and was proved to be a simple replication of the existing β-cells as indicated by double immunohistochemistry which showed the presence of larger number of cells that co-exist both Ki-67 (proliferation marker) and insulin (β-cell marker) in the islets of the Diabetic+ CBD group. This study found that CBD possesses an anti-inflammatory, anti-apoptotic and pro- β-cell replicative properties. INTRODUCTION More than 100 different cannabinoids can be extracted from Cannabis sativa plant, but most of them are known to have psychoactive activities (Elsohly
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and Slade, 2005). The FDA has not recognized or approved the Cannabis Sativa plant as a medicine. However, scientists are conducting preclinical and clinical trials using Cannabis Sativa and its extracts to treat a variety of diseases and conditions; specially treating the diseases of autoimmune basis such as HIV, demyelinating sclerosis and type-1 diabetes mellitus. Currently, there are two main cannabinoids of medical interest which are extracted and purified from the Cannabis Sativa plant. They are called the delta-9-tetrahydrocannabinol (THC) and the cannabidiol (Giacoppo et al. 2015). Cannabidiol (CBD) is the most abundant cannabinoid of Cannabis sativa and is used in the clinical trials in humans for the relief of pain and inflammation in multiple sclerosis (Barnes and Sativex, 2006). CBD has been found to ameliorate joint destruction in an animal model of rheumatoid arthritis (Weiss et al. 2006). Another study has demonstrated the ability of the CBD to protect the heart in the myocardial ischemia that induced in animals (Durst et al, 2007). In C57BL/6 mice, it was found that CBD suppressed multiple sclerosis-like disease and inhibited autoimmune inflammation associated with disease (Kozela et al. 2011). Cannabidiol also showed positive and protective effects in auto-immune encephalomyelitis- induced in a mouse model (Giacoppo et al. 2015). Regarding the pancreas, little investigations were held to find the effects of CBD on the pancreas and diabetes. An important fact about CBD is that: its administration does not result in the development of centrally mediated CB1- receptor activation in the brain (Pacher et al. 2006), therefore, it has no psychoactive effects in humans (Izzo et al. 2009).
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So, there is a growing interest in the CBD chemical as a safe agent to treat various diseases which has an autoimmune basis. Because there are few studies which were done to investigate the effects of CBD on the diabetes and the pancreas. Because CBD relieves inflammation and its administration is safe and devoid of any abnormal psychomotor effects, so this study was done to find out its effects on the rat model of induced type-1 diabetes mellitus. Type-1 diabetes was induced in this study by streptozocin which was previously used as an antibiotic but if administered in repeated low doses it produces a picture similar to autoimmune inflammatory β-cell destruction in humans and rodents (Yin et al. 2010). MATERIALS AND METHODS Ethics statement This study was done in the Central Lab of Faculty of Medicine; Minia University, Egypt. Experiments with animals were approved by the institutional Committee for Ethics in Animal Experimentation and conform to the Guidelines for the Care and Use of Laboratory Animals. Animals Fifty male adult Westar albino (12 weeks old and 220-240 gm weight) were used in this study and kept at 24o C on a 12h light⁄ dark cycle. The rats had free access to food and water. Experimental Design : Group I (Normal control group): Ten adult male albino rats were used in this study. Intraperitoneal (I.P.) injection of sodium citrate buffer were administered for 28 days.
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Group II (Diabetic group): Twenty adult male albino rats were used in this study. Type-1 Diabetes was induced by administration of Streptozocin (STZ) and left untreated. The drug was administered as follows: 40 mg/kg. STZ dissolved in 10 mmol/l sodium citrate (pH 4.5). The injection was done I.P. within 15 minutes of the STZ solution preparation for five consecutive days (Herold et al. 1995). Fasting serum glucose was measured (OneTouch Ultra, Johnson and Johnson, Bucks, UK) from a tail venesection of the animals (200- 290 mg/dl considered as having mild hyperglycemia).
lanized glass. The first section from each series was stained with H&E staining. The second, third fourth, fifth and sixth. seventh sections were used for immunohistochemistry.
Group III (Diabetic +CBD group): Twenty adult albino rats were used in this study. Type-1 diabetes was induced by administration of STZ as explained previously in group II. At the same time the rats were treated with CBD (20 mg/kg) daily I. P. for 28 days. The CBD was isolated by Dr. Samah Shabana; Pharmacognosy Department; Pharmacy program, Batterjee medical college, Jeddah, Saudi Arabia; as described earlier by Gaoni and Mechoulam 1971 (it was completely pure CBD and free from any psychogenic components to avoid illegal handling of the substance)
Hematoxylin& Eosin (H&E) staining
All rats of the three groups were sacrificed after 28 days of onset of the experiment. The blood was collected after sacrificing the animals and immediately centrifuged. The pancreases from each group were excised, cleared of fat and lymph nodes, immersion-fixed for 12 h in Bouin’s fixative solution, dehydrated and embedded in paraffin. At 250 um intervals, seven serial sections (5 um thick) were cut on a rotary microtome and adhered to individual normal or si-
Biochemical Analysis Fasting serum glucose in mg/dl was estimated using a commercially available kit according to the method of Trinder (1969). Serum insulin level in µIU/ml were detected by radioimmunoassay (RIA), utilizing guinea-pig anti-rat insulin antibody and rat insulin as standard (Revees, 1983). According to Bancroft and Cook (1994), serial sections (of 5 um thickness) were done for the whole pancreases of all the studied groups and every first section was stained by H&E. The slides containing paraffin were placed in a slide holder (glass or metal) and deparaffinized then mounted in Xylene. Then the slides were rehydrated in different grades of 100% ethanol, 95% ethanol, 80% ethanol. Then the slides were mounted in deionized H2O. Hematoxylin staining was done by Hematoxylin dye (Poly Scientific, Bayshore, NY, #s212A; Harris hematoxylin with glacial acetic acid), then rinsed by deionized water, then tap water. Then dipped in 12x (fast) Acid ethanol (to destain) then rinsed in tap water. Eosin staining was done by mounting 30 sec in Eosin (Poly Scientific, Bayshore, NY; #s176; Eosin Phloxine stain,) and then dehydrated by 95% ethanol, 100% ethanol finally mounted in xylene. Then the slides were covered using Permount (Fisher Scientific #SP15 ‐100; Histological mounting medium).
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TUNEL staining For Assessment of apoptosis in the rat islets of the studied groups: The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit was used to evaluate the islet apoptosis. Briefly, 5 μm sections were incubated with TUNEL reagent for 1 h in dark. After rinsing with PBS, the 3,3’diaminobenzidine reagent was added. In addition, the positive control includes permeabilization of sections with deoxyribonuclease 1 to induce DNA strand breaks. The slides were mounted and photographed under light microscope. The TUNEL-positive cells and total islet cells were counted manually in ten randomly selected islets of each mouse under a 20× objective microscope by an investigator who was blinded to the experiments. The results were adjusted for β-cell area in serial sections stained with insulin and used to calculate the TUNEL -positive β-cells per islet (Kong et al. 2015). Immunohistochemistry Cellular distribution of insulin, SOX9, neurogenin-3 (Ngn-3) and Mac-2 was analyzed according to Bancroft and Cook (1994) using a standard indirect immuno-peroxidase method. After paraffin removal, the sections were rehydrated and blocked against endogenous peroxidase activity with 1% H2O2. After washing with 0.01 m phosphate buffered solution (PBS, pH 7.4) the sections were treated with 0.01 m sodium citrate buffer (0.05% Tween 20, pH 6.0) at 98 C for antigen retrieval. Sections were then incubated for 30 min with PBS (0.05% Tween 20 and 5% of dry skimmed milk) followed by primary antibody incubation for 2 h at room temperature (RT). The
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antibodies used were rabbit anti-insulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-Caspase-3 (Santa Cruz biotechnology), rabbit anti-SOX9 (1:150), anti- Ngn-3 (1:75) and anti-Mac -2 (1:150) (Dako Cytomation, Carpinteria, CA, USA) diluted in PBS with 2% of dry skimmed milk respectively. After washes in PBS, sections were incubated at RT for 30 min with LSAB (Dako Cytomation) with specific biotinylated secondary antibody. Sections were then treated with horseradish peroxidase (HRP)-streptavidin solution AB system (Dako Cytomation) for 30 min at RT. The streptavidin–biotin complexes were detected with diaminobenzidine (DAB) solution (0.1% DAB and 0.02% H2O2 in PBS). Finally, the sections were rapidly counterstained with Harris’ haematoxylin and mounted for microscopic observation. A negative immunohistochemical control of all samples was carried out by primary antibody omission. Calculation of insulin positive area percentage: Five pancreata were used from each group. On average, 15–20 sections per pancreas were analyzed for insulin. Immunohistochemically stained images of sections were scanned at the same magnification and exposure conditions. Quantification was done with ImageJ software (National Institutes of Health, Bethesda, MD). Results are expressed as the percentage of positive cells per total counted cells. The percentage of insulinspecific cells was then determined (Prasadan et al. 2002) Double label immunohistochemistry By using two peroxidase substrates (After Nakane 1968), this technique al-
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lowed the simultaneous visualization of nuclear and cytoplasmic antigens. Sections were incubated first with antisera to ki-67 and the bound antibody was visualized by a chromogenic dye DAB (brown precipitate staining the nucleus), followed by incubation with antisera to insulin, which was visualized with the red reaction product of the AP/Fast red in the cytoplasm (Dako, USA). The percentage of double labelled cells for Ki67 and insulin- positive was calculated for the pancreas with the ImageJ software. Western Blot Analysis According to Galuppo et al. (2014) all the extraction procedures were performed on the ice using ice-cold reagents. In brief, 5 pancreatic tissues from each group were suspended in extraction buffer containing 0.32 M sucrose, 10 mM Tris–HCl, pH 7.4, 1 mM EGTA, 2 mM EDTA, 5 mM NaN3, 10 mM 2mercaptoethanol, 50 mM NaF, protease inhibitor tablets (Roche Applied Science) and they were homogenized at the highest setting for 2 min. The homogenates were chilled on ice for 15 min and then centrifuged at 1000 g for 10 min at 4 °C, and the supernatant (cytosol + membrane extract from pancreas tissue) was collected to evaluate content of cytoplasmic proteins. The pellets were suspended in the supplied complete lysis buffer containing 1 % Triton X-100, 150 mM NaCl, 10 mM Tris–HCl, pH 7.4, 1 mM EGTA, 1 mM EDTA protease inhibitors (Roche), and then were centrifuged for 30 min at 15000 g at 4 °C. Then, supernatant containing nuclear extract was collected to evaluate the content of nuclear proteins. Supernatants were stored at −80 °C until use. Protein
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concentration in homogenate was estimated by BioRad Protein Assay (BioRad ) using BSA as standard, and 20 μg of cytosol and nuclear extract from each sample were analyzed. Proteins were separated on sodium dodecyl sulfatepolyacrylamide minigels and transferred onto PVDF membranes (Immobilon-P Transfer membrane, Millipore), blocked with PBS containing 5 % nonfat dried milk (PM) for 45 min at room temperature, and subsequently probed at 4 °C overnight with specific antibodies for TNF-α (1:500; Cell Signaling Technology) and IFN-γ (1:250; Santa Cruz Biotechnology Inc) in 1x PBS, 5 % (w/v) non-fat dried milk, 0.1 % Tween-20 (PMT). HRP-conjugated goat antimouse IgG, HRP-conjugated goat antirabbit IgG or HRP-conjugated chicken anti-rat were incubated as secondary antibody (1:2000; Santa Cruz Biotechnology Inc) for 1 h at room temperature. To ascertain that blots were loaded with equal amounts of protein lysates, they were also incubated with antibody for GAPDH HRP Conjugated (1:1000; Cell Signaling Technology) and beta-actin (1:1000; Santa Cruz Biotechnology, Inc). The relative expressions of protein bands, were visualized using an enhanced chemiluminescence system (Luminata Western HRP Substrates, Millipore) and protein bands were acquired and quantified with ChemiDoc™ MP System (Bio-Rad) and a computer program (ImageJ software) respectively. Blots are representative of three separate and reproducible experiments. The statistical analysis was carried out on three repeated blots performed on separate experiments. The percentage of bands density was expressed against GAPDH bands density.
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Statistical Analysis: Data are expressed as mean± SEM. Statistical differences between groups were analyzed by ANOVA test. A P value
