Vol. 27, no. 2, 195-201 (2014)
INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY
EFFECT OF A HIGH DOSE OF VITAMIN D ON A RABBIT MODEL OF ATHEROSCLEROSIS H.A. MALEK and A. SHATA
Clinical Pharmacology Department, Mansoura University, Egypt Received April 14, 2014 - Accepted May 20, 2014 Multifactorial factors have been involved in atherosclerosis. An association has been shown between osteoporosis and carotid atherosclerosis. This work evaluates the effect of vitamin D on regression of atherosclerosis. Forty-eight male rabbits were divided into: Group Ia: (Standard diet + saline for 4 weeks); Group I b: (Standard diet + a high dose of vitamin D3 daily for4 weeks); Group IIa: (Cholesterolenriched diet for 4 weeks]; Group lIb: (Cholesterol-enriched diet + a single high dose of vit D3, daily for 4 weeks. At the end of 4 weeks, the rabbits were sacrificed for assay in serum lipid profile, C reactive protein (CRP), vitamin D3 metabolite, calcium, soluble adhesion molecules (sVCAM and slCAM) and nitrite (NO) and malondialdehyde (MDA) released from isolated aortic rings. Results showed that vitamin D produced a significant reduction in the sera of lipid profile, CRP, and adhesion molecules, associated with a non-significant change in serum calcium and a significant increase in the body level of vitamin D3. Addition of vitamin D to the incubated aortic rings of the atherosclerotic rabbits resulted in a significant increase in NO and decrease in MDA release. It could be concluded that vitamin D has anti-atherosclerotic effects, and may exert these effects by inhibiting lipid peroxidation and stimulation of nitric oxide, resulting in attenuation of the inflammatory atherosclerotic process. and has a shorter duration of action than 0 3 (2). Vitamin 0 may be an ideal therapy to combat a number of diseases. For its natural resources, it is not surprising that its acceptance amongst patients is high, while side effects like hypercalcemia may be prevented on adjusting the dosage regimen. Osteoporosis and atherosclerosis are two multifactorial and degenerative entities. These diseases accompany the aging process and several common pathophysiological factors have been suggested to be associated with both of them. These include similar molecular pathways involving bone and vascular mineralization, estrogen deficiency, parathyroid hormone, lipid oxidative and inflammatory process, as well as vitamin therapy.
Vitamin 0 is a fat-soluble vitamin that plays a role in many important body functions. Vitamin 0 builds and maintains strong body bones, together with calcium. Vitamin 0 is also involved in regulating the immune system and cells, where it may help to prevent cancer. The body stores vitamin 0 and can make it when the skin is exposed to sunlight. There are two forms of vitamin 0: ergocalciferol (vitamin 02) and cholecalciferol (vitamin 03) (I). Ergocalciferol is made from ergosterol ultraviolet irradiation in yeast, while cholecalciferol is made from 7-dehydrocholesterol irradiation from lanolin and the chemical conversion of cholesterol. Vitamin O2 and 0 3 preparations are equivalent clinically. Studies mention that vitamin O2 is much less potent
Key words: vitamin D, atherosclerosis, rabbit, C reactive protein, adhesion molecules, aortic ring Mailing address: assoc. Prof Hala Abdel Malek, Clinical Pharmacology Department, Mansoura University, Gomhorea Street, 35516 Mansoura, Egypt Fax: +20(50)2223613-124 e-mail: [email protected]
0394-6320 (2014)
195
Copyright (0 by BIOLIFE. s.a.s. This publication andlor article is for individual use only and may not be further reproduced without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties DISCLOSURE: ALL AUTHORS REPORT NO CONFLICTS OF INTEREST RELEVANT TO THIS ARTICLE.
H.A. MALEK ET AL.
196
Moreover, vitamin D receptor gene mutations have been discovered and are associated with increased risk of arterial hypertension (1, 3). Atherosclerosis is an inflammatory disease that affects the major arteries of the vasculature. Reduction of the antioxidant defence system and many inflammatory markers and cytokines are involved, such as vascular cell adhesion molecule-l (VCAM-l) that is a major player in atherosclerosis, in starting and progression of the plaques, leading to adverse cardiovascular events (4, 5). Meanwhile, hyperlipidemia and the liability for lipoprotein oxidation and accumulation in bone is responsible for osteoporosis by inhibiting osteoblastic differentiation (6). On the other hand, it has been reported that vitamin D supplementation has shown deleterious pro-atherogenic effects in rodents, and its deficiency is correlated with the progression of atherogenesis process (1, 7). These contradictory findings on the progression of atherosclerosis were evaluated in this work. The present hypothesis is based on the investigation of the effect of vitamin D on regression of atherosclerosis. MATERIALS AND METHODS Experiments were performed according to the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, National Research Council, Washington, DC: National Academy Press, no. 85-23, revised 1996). Our local committee (Animal Care and Use Committee) approved all protocol. The drugs and chemicals used were: Vitamin D3 group (Devarol-S; Cholecalciferol (600000IU/ampule, Memphis company, Egypt); Cholesterol dry powder (E1 Gomhorea laboratory chemicals company, Egypt): and Phosphate butTered Solution (PBS), (Sigma chemical co. St. Louis, USA).
Animals Forty-eight male New Zealand white rabbits, (3-5 months old, weighing 2 kg) were used. The rabbits were housed individually with free access to water. The animals received a standard diet without cholesterol during 2-weeks "pretreatment period" for adaptation to the environment. The rabbits were randomly divided to one of the two diet regimens: standard diet (n=24), or cholesterol-enriched diet (n=24). The cholesterol diet was prepared by adding
cholesterol to com oil and conjugating with the standard diet (2% cholesterol + 4% com oil + standard diet) - this mixture when given for a period of 4 weeks, results in a marked elevation in serum cholesterol and induction of diffuse aortic atherosclerosis (8-10). The rabbits were classified into two major groups: Group I (24). I-a (n=12); rabbits received a standard diet and 0.9% NaCL intramuscularly. They served as a negative control group throughout the 4 weeks of the study. I-b (n=12); rabbits received a standard diet and 50000 IU/kg of vitamin D3 intramuscularly, once per day during the 4 weeks of the experiment. Group II (cholesterol-enriched diet group, n = 24): II-a (n = 12); rabbits received an atherogenic diet "cholesterol enriched diet" for 4 weeks for the development of atherosclerosis. II-b (n = 12); rabbits received a "cholesterol enriched diet" and 50000 IU/kg of vitamin D3 intramuscularly, once per day during the 4 weeks of the experiment (11). At the end, all rabbits were sacrificed after 14 hours fasting. Trunk blood of each rabbit was collected and centrifuged. The sera of the samples were used for the assay of triglycerides, total-cholesterol, high density lipoprotein-cholesterol (HDL- cholesterol), low density Lipoprotein-cholesterol (LDL- cholesterol), soluble inter cellular adhesion molecule-l (sICAM-l), vascular cell adhesion molecule (sVCAM-l), 25 oH vitamin D3, calcium and CRP. The whole aorta was excised, from the ascending part ofthe arch up to the abdominal bifurcation, for nitrite and malondialhyde in vitro assay.
Biochemical studies Lipid profile levels were measured spectrophotometrically with the use ofBio-diagnostic Kits (Egypt). The determination of serum soluble intracellular adhesion molecules was performed using a commercially available solid phase sandwich ELISA kit (Immunotech, Marsille, and Cedex, France). Determination of serum calcium was carried out according to Wu et al. (12), and CRP was performed with a commercial kit (Biodiagnostic, Egypt). Determination ofserum 25 oH vitamin D was performed using a commercially available ELISA kit (Neobiolab company, Cambridge, MA, USA). Isolation and incubation ofthe aorta The aorta from each rabbit was removed and dissected carefully free from connective tissues and the thoracic aorta was cut into rings :::::20 mg in weight (l3, 14). Rings were placed in a small package of 80 urn nylon mesh, transferred into a tissue bath containing Phosphate butTered solution (PBS) [pH =7. 4 and to which 0.1% bovine serum albumin was added], and oxygenated with 95% 02/5% C02 for 30 min in C02 incubator (Revco Habitat). The
Int. J. ImmunopathoI. PharmacoI.
tissue rings (wet weight) were placed in plastic tubes, each 5 ml and containing 500 III of PBS as control or 450 III of PBS +50 ul of vitamin D (l umol/L) to demonstrate its effect on nitrite (NO) and malondialdehyde (MDA), then NO (14) and MDA (15) were assayed by Jenway 6405 UVNis spectrophotometer.
Statistical analysis Data were expressed as means ± SD and compared by one-way analysis of variance (ANOVA) followed by Tukey post hoc test. P :s 0.05 was considered as the level of significance between the non-treated and treated grousp. Statistical analysis was performed using the SPSS for Windows 9.05 package program.
197 RESULTS
Vitamin D ameliorates lipid profile in experimental animals Vitamin D significantly suppressed total cholesterol, from 543.5±12.02 t0482.2±8.6 (P
INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY
EFFECT OF A HIGH DOSE OF VITAMIN D ON A RABBIT MODEL OF ATHEROSCLEROSIS H.A. MALEK and A. SHATA
Clinical Pharmacology Department, Mansoura University, Egypt Received April 14, 2014 - Accepted May 20, 2014 Multifactorial factors have been involved in atherosclerosis. An association has been shown between osteoporosis and carotid atherosclerosis. This work evaluates the effect of vitamin D on regression of atherosclerosis. Forty-eight male rabbits were divided into: Group Ia: (Standard diet + saline for 4 weeks); Group I b: (Standard diet + a high dose of vitamin D3 daily for4 weeks); Group IIa: (Cholesterolenriched diet for 4 weeks]; Group lIb: (Cholesterol-enriched diet + a single high dose of vit D3, daily for 4 weeks. At the end of 4 weeks, the rabbits were sacrificed for assay in serum lipid profile, C reactive protein (CRP), vitamin D3 metabolite, calcium, soluble adhesion molecules (sVCAM and slCAM) and nitrite (NO) and malondialdehyde (MDA) released from isolated aortic rings. Results showed that vitamin D produced a significant reduction in the sera of lipid profile, CRP, and adhesion molecules, associated with a non-significant change in serum calcium and a significant increase in the body level of vitamin D3. Addition of vitamin D to the incubated aortic rings of the atherosclerotic rabbits resulted in a significant increase in NO and decrease in MDA release. It could be concluded that vitamin D has anti-atherosclerotic effects, and may exert these effects by inhibiting lipid peroxidation and stimulation of nitric oxide, resulting in attenuation of the inflammatory atherosclerotic process. and has a shorter duration of action than 0 3 (2). Vitamin 0 may be an ideal therapy to combat a number of diseases. For its natural resources, it is not surprising that its acceptance amongst patients is high, while side effects like hypercalcemia may be prevented on adjusting the dosage regimen. Osteoporosis and atherosclerosis are two multifactorial and degenerative entities. These diseases accompany the aging process and several common pathophysiological factors have been suggested to be associated with both of them. These include similar molecular pathways involving bone and vascular mineralization, estrogen deficiency, parathyroid hormone, lipid oxidative and inflammatory process, as well as vitamin therapy.
Vitamin 0 is a fat-soluble vitamin that plays a role in many important body functions. Vitamin 0 builds and maintains strong body bones, together with calcium. Vitamin 0 is also involved in regulating the immune system and cells, where it may help to prevent cancer. The body stores vitamin 0 and can make it when the skin is exposed to sunlight. There are two forms of vitamin 0: ergocalciferol (vitamin 02) and cholecalciferol (vitamin 03) (I). Ergocalciferol is made from ergosterol ultraviolet irradiation in yeast, while cholecalciferol is made from 7-dehydrocholesterol irradiation from lanolin and the chemical conversion of cholesterol. Vitamin O2 and 0 3 preparations are equivalent clinically. Studies mention that vitamin O2 is much less potent
Key words: vitamin D, atherosclerosis, rabbit, C reactive protein, adhesion molecules, aortic ring Mailing address: assoc. Prof Hala Abdel Malek, Clinical Pharmacology Department, Mansoura University, Gomhorea Street, 35516 Mansoura, Egypt Fax: +20(50)2223613-124 e-mail: [email protected]
0394-6320 (2014)
195
Copyright (0 by BIOLIFE. s.a.s. This publication andlor article is for individual use only and may not be further reproduced without written permission from the copyright holder. Unauthorized reproduction may result in financial and other penalties DISCLOSURE: ALL AUTHORS REPORT NO CONFLICTS OF INTEREST RELEVANT TO THIS ARTICLE.
H.A. MALEK ET AL.
196
Moreover, vitamin D receptor gene mutations have been discovered and are associated with increased risk of arterial hypertension (1, 3). Atherosclerosis is an inflammatory disease that affects the major arteries of the vasculature. Reduction of the antioxidant defence system and many inflammatory markers and cytokines are involved, such as vascular cell adhesion molecule-l (VCAM-l) that is a major player in atherosclerosis, in starting and progression of the plaques, leading to adverse cardiovascular events (4, 5). Meanwhile, hyperlipidemia and the liability for lipoprotein oxidation and accumulation in bone is responsible for osteoporosis by inhibiting osteoblastic differentiation (6). On the other hand, it has been reported that vitamin D supplementation has shown deleterious pro-atherogenic effects in rodents, and its deficiency is correlated with the progression of atherogenesis process (1, 7). These contradictory findings on the progression of atherosclerosis were evaluated in this work. The present hypothesis is based on the investigation of the effect of vitamin D on regression of atherosclerosis. MATERIALS AND METHODS Experiments were performed according to the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, National Research Council, Washington, DC: National Academy Press, no. 85-23, revised 1996). Our local committee (Animal Care and Use Committee) approved all protocol. The drugs and chemicals used were: Vitamin D3 group (Devarol-S; Cholecalciferol (600000IU/ampule, Memphis company, Egypt); Cholesterol dry powder (E1 Gomhorea laboratory chemicals company, Egypt): and Phosphate butTered Solution (PBS), (Sigma chemical co. St. Louis, USA).
Animals Forty-eight male New Zealand white rabbits, (3-5 months old, weighing 2 kg) were used. The rabbits were housed individually with free access to water. The animals received a standard diet without cholesterol during 2-weeks "pretreatment period" for adaptation to the environment. The rabbits were randomly divided to one of the two diet regimens: standard diet (n=24), or cholesterol-enriched diet (n=24). The cholesterol diet was prepared by adding
cholesterol to com oil and conjugating with the standard diet (2% cholesterol + 4% com oil + standard diet) - this mixture when given for a period of 4 weeks, results in a marked elevation in serum cholesterol and induction of diffuse aortic atherosclerosis (8-10). The rabbits were classified into two major groups: Group I (24). I-a (n=12); rabbits received a standard diet and 0.9% NaCL intramuscularly. They served as a negative control group throughout the 4 weeks of the study. I-b (n=12); rabbits received a standard diet and 50000 IU/kg of vitamin D3 intramuscularly, once per day during the 4 weeks of the experiment. Group II (cholesterol-enriched diet group, n = 24): II-a (n = 12); rabbits received an atherogenic diet "cholesterol enriched diet" for 4 weeks for the development of atherosclerosis. II-b (n = 12); rabbits received a "cholesterol enriched diet" and 50000 IU/kg of vitamin D3 intramuscularly, once per day during the 4 weeks of the experiment (11). At the end, all rabbits were sacrificed after 14 hours fasting. Trunk blood of each rabbit was collected and centrifuged. The sera of the samples were used for the assay of triglycerides, total-cholesterol, high density lipoprotein-cholesterol (HDL- cholesterol), low density Lipoprotein-cholesterol (LDL- cholesterol), soluble inter cellular adhesion molecule-l (sICAM-l), vascular cell adhesion molecule (sVCAM-l), 25 oH vitamin D3, calcium and CRP. The whole aorta was excised, from the ascending part ofthe arch up to the abdominal bifurcation, for nitrite and malondialhyde in vitro assay.
Biochemical studies Lipid profile levels were measured spectrophotometrically with the use ofBio-diagnostic Kits (Egypt). The determination of serum soluble intracellular adhesion molecules was performed using a commercially available solid phase sandwich ELISA kit (Immunotech, Marsille, and Cedex, France). Determination of serum calcium was carried out according to Wu et al. (12), and CRP was performed with a commercial kit (Biodiagnostic, Egypt). Determination ofserum 25 oH vitamin D was performed using a commercially available ELISA kit (Neobiolab company, Cambridge, MA, USA). Isolation and incubation ofthe aorta The aorta from each rabbit was removed and dissected carefully free from connective tissues and the thoracic aorta was cut into rings :::::20 mg in weight (l3, 14). Rings were placed in a small package of 80 urn nylon mesh, transferred into a tissue bath containing Phosphate butTered solution (PBS) [pH =7. 4 and to which 0.1% bovine serum albumin was added], and oxygenated with 95% 02/5% C02 for 30 min in C02 incubator (Revco Habitat). The
Int. J. ImmunopathoI. PharmacoI.
tissue rings (wet weight) were placed in plastic tubes, each 5 ml and containing 500 III of PBS as control or 450 III of PBS +50 ul of vitamin D (l umol/L) to demonstrate its effect on nitrite (NO) and malondialdehyde (MDA), then NO (14) and MDA (15) were assayed by Jenway 6405 UVNis spectrophotometer.
Statistical analysis Data were expressed as means ± SD and compared by one-way analysis of variance (ANOVA) followed by Tukey post hoc test. P :s 0.05 was considered as the level of significance between the non-treated and treated grousp. Statistical analysis was performed using the SPSS for Windows 9.05 package program.
197 RESULTS
Vitamin D ameliorates lipid profile in experimental animals Vitamin D significantly suppressed total cholesterol, from 543.5±12.02 t0482.2±8.6 (P
