الفهرس 
Introduction
HypertensionOnly 14 pages are availabe for public view
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Abstract
This thesis is devoted to evaluate the potential protective effect of EGb761 against hypertension and hypertension associated with hypercholesterolemia-induced deleterious effects on the kidney function and structure. In addition, an attempt was undertaken to clarify the role of oxidative stress, NO, NOS isoforms and inflammation in renal damage induced by hypertension and by hypertension associated with hypercholesterolemia.
To induce hypertension, animals were treated daily with L-NAME at a dose level of 10 mg/kg ip for 12 weeks. Hypercholesterolemia was induced by feeding rats with a diet containing 1% cholesterol for 12 weeks.
In the first set of experiments, the animals were divided into 4 groups, 8 rats each. Animals of group-I were treated ip with 10 mg/kg/day L-NAME for 12 weeks. Group-II rats received orally 100 mg/kg/day EGb761 starting from the 9th week of continued daily treatment with 10 mg/kg L-NAME ip to the end of the 12 weeks treatment duration. Animals of group-III and IV received orally starting from the 9th to the 12th week of continued daily treatment with 10 mg/kg L-NAME ip, 1 mg/kg/day losartan and 1 mg/kg/day losartan in combination with 100 mg/kg/day EGb761, respectively. Control animals were treated likewise with the pure vehicles.
In the second set of experiments, the animals were divided into 4 groups, 8 rats each. Rats of group-I were treated daily with 10 mg/kg L-NAME ip and fed with a diet containing 1% cholesterol for 12 weeks. Group-II animals received orally 100 mg/kg/day EGb761 starting from the 9th week of daily treatment with 10 mg/kg L-NAME ip and feeding with a cholesterol-enriched diet to the end of the 12 weeks experimental duration. Animals of group-III were received orally 1 mg/kg/day losartan and 3 mg/kg/day simvastatin starting from the 9th week of daily treatment with 10 mg/kg L-NAME ip and feeding with a cholesterol-enriched diet to the end of 12 weeks experimental duration. Rats of group-IV received orally 1 mg/kg/day losartan, 3 mg/kg/day simvastatin and 100 mg/kg/day EGb761, starting from the 9th to the 12th week of daily treatment with 10 mg/kg L-NAME ip and feeding with a cholesterol-enriched diet. Control animals were treated likewise with the pure vehicles and fed with the normal diet.
The systolic, diastolic and mean arterial BP were measured by the tail-cuff method in all groups of rats. The systolic, diastolic and mean arterial BP of each rat was measured before and weekly after starting of the treatment. The average of 3 consecutive measurements was taken for presentation.
At the end of experimental duration and after recording the BP changes, animals were sacrificed by decapitation. Blood and kidney tissues were obtained from each animal for biochemical measurements. After centrifugation of blood samples for 10 minutes, the serum was collected for estimation the levels of urea, creatinine, TC, TGs, LDL-C, HDL-C and CRP. The samples could be used directly or stored at -20oC until assay.
One kidney from each animal was kept in 10% formalin for histopathological and immunohistochemical studies and the other kidney was rinsed in ice-cold saline, dissected and cleaned from fat and other tissues, then cut into small pieces, blotted carefully and weighed. A suitable weight of kidney tissue was homogenized in 10% w/v phosphate buffer (pH 7.4) or saline. The homogenate was divided into 2 portions. The first portion was centrifuged for 10 minutes at 10,000 rpm and the supernatant was used for estimation of MDA, nitrite, TNF-α, IL-6, IL-1β and ICAM-1 levels directly or stored at -20oC until assay. To the second part of homogenate an equal volume of perchloric acid (1 mol/l) was added and mixed by vortexing. The mixture was allowed to stand for 5 minutes at room temperature. After centrifugation for 10 minutes, the supernatant was collected carefully and used for estimation of intracellular GSH directly or stored at -20oC until assay.
Daily administration of 10 mg/kg L-NAME ip for 12 weeks produced a progressive increase in the systolic, diastolic and mean arterial BP (hypertensive rats). Also, daily treatment of rats with 10 mg/kg L-NAME ip and feeding with a diet containing 1% cholesterol resulted in a progressive increase in the systolic, diastolic and mean arterial BP with a compromised lipid profile (hypertensive hypercholesterolemic rats). Hypercholesterolemia enhanced the hypertensive effect of L-NAME.
After 8 weeks of treatment of animals with 10 mg/kg/day L-NAME, concurrent administration of 100 mg/kg/day EGb761 orally to the end of the treatment duration produced a progressive reduction in the systolic, diastolic and mean arterial BP. Similar results were obtained in hypertensive hypercholesterolemic rats.
Similar treatment of hypertensive rats with 1 mg/kg/day losartan produced a progressive reduction in the systolic, diastolic and mean arterial BP. EGb761 enhances the effects of losartan. Also, similar treatment of hypertensive hypercholesterolemic rats with 1 mg/kg/day losartan in combination with 3 mg/kg/day simvastatin produced a progressive reduction in the systolic, diastolic and mean arterial BP. Concurrent treatment with EGb761 enhances the effects of losartan with simvastatin. In addition, EGb761 improved the compromised serum lipid profile and enhanced the effects of losartan and simvastatin on the compromised lipid profile in hypertensive hypercholesterolemic rats.
Similar treatment of hypertensive rats with 100 mg/kg/day EGb761 resulted in a significant decrease in serum urea and creatinine levels. Losartan treatment produced similar results. EGb761 enhanced the effect of losartan.
Treatment of hypertensive hypercholesterolemic rats similarly with 100 mg/kg EGb761 or with 1 mg/kg/day losartan and 3 mg/kg/day simvastatin combination resulted in a significant decrease in serum urea and creatinine levels. EGb761 enhanced the effect of losartan with simvastatin.
The histopathological examination of the kidney tissue obtained from hypertensive rats showed glomerulosclerosis, cloudy swelling of tubules, interstitial inflammation and fibrosis. Treatment of rats with 100 mg/kg/day EGb761 orally starting from the 9th week of continued treatment with 10 mg/kg/day L-NAME ip to the end of 12 weeks experimental duration ameliorated these pathological changes. A similar protective effect was observed after treatment of rats with losartan. EGb761 enhanced the effect of losartan on the renal histopathological changes.
The histopathological examination of the kidney tissue obtained from hypertensive hypercholesterolemic rats showed glomerulosclerosis, cloudy swelling of tubules, interstitial inflammation and fibrosis. Treatment of rats with 100 mg/kg/day EGb761orally starting on the 9th week of continued daily treatment with 10 mg/kg L-NAME and a cholesterol-enriched diet to the end of the experimental duration reduced these pathological changes. Similarly, combined treatment with 1 mg/kg/day losartan and 3 mg/kg/day simvastatin produced the same ameliorative effects. Addition of EGb761 enhanced the combined effect of losartan and simvastatin treatment on these histopathological changes.
The renal tissue MDA and nitrite levels were increased in hypertensive and in hypertensive hypercholesterolemic rats, while the levels of renal tissue intracellular GSH were decreased.
Concomitant administration of 100 mg/kg/day EGb761 orally starting from the 9th to the 12th week of continued treatment of rats with 10 mg/kg/day L-NAME decreased renal tissue MDA and nitrite levels and increased intracellular GSH level. Similar results were obtained after treatment of hypertensive rats with 1 mg/kg/day losartan. EGb761 enhanced the effects of losartan.
In hypertensive hypercholesterolemic rats, similar treatment with 100 mg/kg/day EGb761 or combined treatment with 1 mg/kg/day losartan and 3 mg/kg/day simvastatin decreased the renal levels of MDA and nitrite and increased the renal level of intracellular GSH levels. EGb761 enhanced the effects of losartan with simvastatin.
The levels of renal tissue TNF-α, IL-6, IL-1β and ICAM-1 and serum level of CRP were increased in hypertensive and in hypertensive hypercholesterolemic rats.
Oral administration of 100 mg/kg/day EGb761 starting on the 9th week of continued daily treatment of rats with 10 mg kg/ L-NAME until the end of the 12 weeks treatment duration decreased the renal tissue TNF-α, IL-6, IL-1β and ICAM-1 and serum CRP levels. Similar results were obtained after similar treatment with 1 mg/kg/day losartan. EGb761 enhanced the effects of losartan.
In hypertensive hypercholesterolemic rats, similar treatment with 100 mg/kg/day EGb761 decreased the renal tissue TNF-α, IL-6, IL-1β and ICAM-1 levels and serum CRP levels. Also, similar results were obtained after similar combined treatment with 1 mg/kg/day losartan and 3 mg/kg/day simvastatin. EGb761 enhanced the effects of losartan with simvastatin.
In this study, the Immunohistochemical analysis of kidney tissues obtained from hypertensive rats showed a decrease in eNOS protein expression and an increase in the protein expression of iNOS and inflammatory cytokines, TNF-α, IL-6 and IL-1β. Similar results were obtained in hypertensive hypercholesterolemic rats.
Oral administration of 100 mg/kg/day EGb761 starting from the 9th week of continued treatment of rats with10 mg/kg L-NAME i.p. to the end of the experimental duration produced an increase in the protein expression of eNOS and a decrease in the protein expression of iNOS and inflammatory cytokines. Similar results were obtained by similar treatment with losartan. EGb761 enhanced the effects of losartan.
Treatment of hypertensive hypercholesterolemic rats similarly with 100 mg/kg/day EGb761 produced an increase in the protein expression of eNOS and a decrease in the protein expression of iNOS and inflammatory cytokines in the kidney tissue. Similar results were obtained after similar combined treatment with 1 mg/kg/day losartan and 3 mg/kg/day simvastatin. EGb761 enhanced the effect of losartan with simvastatin.
Results of this work lead to the following conclusions:
1- Ginkgo biloba extract (EGb761) has the ability to protect against hypertension and hypertension with hypercholesterolemia-induced kidney injury in rats.
2- The ability of EGb761 to provide this renoprotective effect may positively correlate to its ability to suppress renal oxidative stress, nitrosative stress and inflammation in addition to its antihypertensive and antihypercholesterolemic effects.
3- EGb761 has the ability to potentiate the antihypertensive effect of losartan and the antihypertensive antihypercholesterolemic effects of losartan with simvastatin and to potentiate their renoprotective effects. EGb761 may potentiate these effects through enhancement of their abilities to attenuate oxidative and nitrosative stresses and to suppress inflammatory responses in the kidney.
4- Thus, EGb761 appears to have a therapeutic potential as an adjuvant agent for protection against kidney injury in hypertensive and in hypertensive hypercholesterolemic cases.