الفهرس 
DoxorubicinOnly 14 pages are availabe for public view
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Abstract
DOX has a broad spectrum effect and high potency against wide range of human neoplasm. However, the clinical efficacy is limited by dose dependent cardiotoxicity. It was proved recently that Vit D plays an important role as anti-oxidant, anti apoptotic effect as well as its role in maintaining cardiovascular system functions. The present study was designed to investigate the potential protective effect of Vit D against DOX -induced cardiotoxcity in rats and to elucidate the possible underlying molecular mechanisms via studying its effects on different oxidative stress and apoptotic markers.
The present study aimed to screen the cardioprotective effect of Vit D against acute DOX cardiotoxicity.
Forty eight adult male rats weighing 120- 180 gm were used in this study. Rats were kept in the animal house of the physiology department, faculty of medicine, Ain Shams University.Rats were randomly assigned to four groups each group (n = 12):
group I: Normal control rats.
group II: Doxorobucin- treated rats.
group III: Vit D pretreated - DOX - treated rats.
group IV: Vit D treated rats.
The normal control rats group I were received saline for the whole length of the protocol and considered as the control group. DOX group II (EBEWE Pharma Ges 50 mg/25ml) was used in a single intraperitoneal injection in a dose of 15 mg/kg. The rats of this group were sacrificed 24 hours following the injection. Rats of the group III, received a daily intraperitoneal injection of Vit D (Memphis for Pharmaceuticals & chemical industries) in a dose of 10,000 IU/100 g BW for a consecutive four days,in addition, in the forth day, the rats were received a single intraperitoneal injection of DOX in a dose of 15 mg/kg BW, one hour after the last dose of Vit D injection, and sacrificed after 24 hours. Rats of the group IV, received a daily intraperitoneal injection of Vit D for a consecutive four days in the same regimen as group III. On the day of sacrifice (24 hrs after DOX injection), both systolic and diastolic arterial blood pressure were measured for all rats. ECG recording was done under anaesthesia (thiopental sodium, 40 mg/kg B.W.i.p.). Blood samples were collected from the abdominal aorta and the plasma was separated and stored at - 80°c until used. Then, rats were sacrificed and heart tissue were dissected and washed with normal saline 0.9% and dried by filter paper. It was cleaned of the fat and fibrous tissue, and preserved at -80°c in parafilm for subsequent testing. In addition, heart specimens from different groups were fixed in 10% buffered formalin for histopathological and immunohistochemical.
The following parameters were investigated:
1. Blood pressure measurement:
Systolic blood pressure
Diastolic blood pressure 2. Cardiotoxicity markers:
ECG
Heart rate
QRS duration
QTc interval
PR interval
3. Serum cardiotoxicity markers:
Creatinine Kinase MB (CK-MB)
Troponin I
4. Total plasma calcium
5. Histopathological examination:
Hematoxylin and Eosin (H & E)
Electron microscopic examination.6. Oxidative stress markers and antioxidant enzymes:
Reduced Glutathione (GSH)
Superoxide dismutase (SOD)
Lipid Peroxides (MDA)
7. Apoptotic marker
Bax
Caspase 3
Cytochrome C
8. Nuclear factor kappa B.
The finding of the present study can be summarized as follows:1- Rats treated with a single dose of DOX showed a significant increase in both the systolic and diastolic blood pressures, after 24 hours, compared to their control rats. On the other hand, the significant increase in both the systolic and diastolic blood pressures was abolished in the Vit D pretreatment- DOX treated rats compared to the DOX rats.
2- ECG tracing showed that DOX induced several ECG changes including bradycardia, widening of the QRS complex and prolongation of both QTc and PR intervals compared to control group. Meanwhile, both the control group and Vit D treated groups showed normal cardiac activity. These ECG abnormalities were normalized on the intoxicated animals pretreated with Vit D.
3- Cardiotoxicity markers showed significant increase of both CK-MB and Troponin I in rats treated with a single dose of DOX after 24 hours compared to the control rats.On the other hand, this significant increase was counteracted in the Vit D pretreatment- DOX treated rats compared to the control rats.
4- Interestingly, the total plasma calcium in rats treated with a single dose ofDOX, showed a significant decrease in the plasma calcium level compared to the control rats. On the other hand, the significant decrease in the plasma calcium level was changed into a significant increase in the Vit D pretreatment- DOX treated rats compared to the DOX rats.5- Histopathological examination using Hematoxylin and Eosin (H & E) stain revealed focal degeneration with inflammatory cells infiltration in the myocardium associated with congestion of the myocardial blood vessels. Interestingly, the pretreatment with Vit D pretty preserved the normal architecture of the myocardium.
6- Electron microscopic examination of cardiomyocytes of DOX intoxification caused severe ultrastructure changes in the cardiac cells in the form of myofibrillar disarray, loss of normal striation pattern, vacuolization, irregular folding of the nuclear surface and chromatin condensation and margination which are typical features of apoptosis. Besides the mitochondria in DOX - treated rats were condensed with inner wrinkled crista and showed heterogeneity both in size and shape. In addition, some extracellular matrix are observed between the cardiomyocytes. Vit D pretreated -DOX treated rats showed pretty normalization of the ultrastructure of cardiac myocytes both in myofibrils and their striation,nuclei and mitochondrial morphology in comparison with DOX-treated group.
7- DOX induced significant reduction in reduced GSH level and increased lipid peroxides level compared to control group. Moreover, DOX induced a significant decrease in the anti-oxidant enzyme activities (SOD) compared to control group. Meanwhile, pretreatment of intoxicated animals with Vit D restored the normal levels of GSH, MDA and SOD as compared to control and DOX groups. Animals treated with Vit D only did not show significant alteration in SOD and MDA but significantly increased the level of GSH to the double compared to control group.
8- DOX intoxification induced marked apoptosis in cardiomyocytes as it showed an increased expression in the cardiac tissues of Bax M, Cytochrome c and Caspase enzyme, which was evident from intense brown staining compared to the control group. However, pretreatment with Vit D significantly reduced the expression, when compared to Doxorubicin -intoxicated group. The immunohistochemical staining was quanitified as an optical density (OD) of the stained regions using the image analysis software.
9- NF-KB was assessed using immunohistochemical technique and revealed that Doxorubicin induced an increased expression in the cardiac tissues. However, pretreatment with Vit D significantly reduced the expression of NF-kB, when compared to Doxorubicin -intoxicated group.Finally, it can be concluded that vitD is an efficient cardio-protective agent against DOX induced cardiotoxicity in rats.