3. Regular endothelial function mainly depends on the capacity to produce nitric oxide (NO). Endothelial dysfunction has been defined as the change in concentration of chemical messengers produced by the endothelium and/or blunting of NO dependant vasodilatory response to acetylcholine. Regular endothelial function mainly depends on the capacity to produce nitric oxide (NO). Endothelial dysfunction has been defined as the change in concentration of chemical messengers produced by the endothelium and/or blunting of NO dependant vasodilatory response to acetylcholine. Introduction

4. DM causes endothelial dysfunction mainly through glucose-induced oxidative stress leading to decreased nitric oxide bioavailability. Lipoic acid is a potent antioxidant. Tadalafil is a specific inhibitor of phosphodiesterase enzyme type 5 (PDE-5). It is used in treatment of erectile dysfunction. Effects of both drugs are related to NO bioavailability. DM causes endothelial dysfunction mainly through glucose-induced oxidative stress leading to decreased nitric oxide bioavailability. Lipoic acid is a potent antioxidant. Tadalafil is a specific inhibitor of phosphodiesterase enzyme type 5 (PDE-5). It is used in treatment of erectile dysfunction. Effects of both drugs are related to NO bioavailability. Introduction

5. The present work was designed to investigate the effects of α-lipoic acid and tadalafil for 4 weeks on fasting plasma glucose, insulin and nitric oxide levels and reactivity of the isolated aortic ring to phenylephrine and acetylcholine in streptozotocin (STZ) -induced diabetic rats. Aim of the Work

6. Methods  (I) In-vivo study: Effects of α-lipoic acid and tadalafil on the fasting plasma glucose, insulin and nitric oxide levels in streptozotocin-induced diabetic rats:  (A) Induction of type II diabetes mellitus:  By a single intravenous injection of small dose of streptozotocin (35mg/kg). Blood glucose was measured 72 hours following the injection. Rats having blood glucose levels below 200mg/dl were considered non-diabetic.  (I) In-vivo study: Effects of α-lipoic acid and tadalafil on the fasting plasma glucose, insulin and nitric oxide levels in streptozotocin-induced diabetic rats:  (A) Induction of type II diabetes mellitus:  By a single intravenous injection of small dose of streptozotocin (35mg/kg). Blood glucose was measured 72 hours following the injection. Rats having blood glucose levels below 200mg/dl were considered non-diabetic.

7. Methods (B) The experimental design: 3 groups Group I: Streptozotocin (STZ)-induced diabetic rats. Group II:(STZ+  -lipoic acid group) : each diabetic rat received intraperitoneal  - lipoic (50mg/kg b.w/day) for 4 weeks. Group III:(STZ +tadalafil group) : each diabetic rat received oral tadalafil (2mg/kg b.w/day) for 4 weeks. (B) The experimental design: 3 groups Group I: Streptozotocin (STZ)-induced diabetic rats. Group II:(STZ+  -lipoic acid group) : each diabetic rat received intraperitoneal  - lipoic (50mg/kg b.w/day) for 4 weeks. Group III:(STZ +tadalafil group) : each diabetic rat received oral tadalafil (2mg/kg b.w/day) for 4 weeks. After four weeks, blood samples were collected after 6 hours fasting.

8. The following measurements were done: I.Estimation of plasma glucose: by enzymatic method. II.Estimation of plasma insulin: by ELISA. III.Estimation of plasma nitric oxide: by enzymatic method.  The enzymatic method based on the oxidation of NADPH during the conversion of nitrate to nitrite by nitrate reductase and measuring the decrease in NADPH absorbance at 340nm. The following measurements were done: I.Estimation of plasma glucose: by enzymatic method. II.Estimation of plasma insulin: by ELISA. III.Estimation of plasma nitric oxide: by enzymatic method.  The enzymatic method based on the oxidation of NADPH during the conversion of nitrate to nitrite by nitrate reductase and measuring the decrease in NADPH absorbance at 340nm. Methods

9. Methods (II) Ex-vivo study: Effects of  -lipoic acid and tadalafil pretreatment for four weeks on the perfused rat's aortic ring isolated from STZ-induced diabetic rats: (II) Ex-vivo study: Effects of  -lipoic acid and tadalafil pretreatment for four weeks on the perfused rat's aortic ring isolated from STZ-induced diabetic rats:

15. (I) In-vivo :Effects of intraperitoneal injection of  - lipoic acid (50mg/kg/day) and oral tadalafil (2mg/kg/day) for 4 weeks on fasting plasma glucose, insulin and nitric oxide levels in streptozotocin (STZ) induced diabetic rats: Results (1) Effects of  -lipoic acid and tadalafil on fasting glucose level in diabetic rats :  -lipoic acid prodeced significant decrease in contrast to a non- significant change by tadalafil.

16. Results (2) Effects of  -lipoic acid and tadalafil on fasting plasma insulin in diabetic rats:  -lipoic acid prodeced significant increase in contrast to a non- significant change by tadalafil ( 3) Effects of of  -lipoic acid and tadalafil on fasting plasma nitric oxide in diabetic rats: Both drugs prodeced significant increases

23. Results (II) Ex-vivo : 1-Effects of intraperitoneal injection of  -lipoic acid (50mg/kg/day) and oral tadalafil (2mg/kg /day) pretreatment for 4 weeks on phenylephrine induced contractile response (10nM-100  M) of the isolated perfused aortic ring preparation in STZ-induced diabetic rats : Both drugs produced significant incraeses in EC50 and significant decreases in E max. (II) Ex-vivo : 1-Effects of intraperitoneal injection of  -lipoic acid (50mg/kg/day) and oral tadalafil (2mg/kg /day) pretreatment for 4 weeks on phenylephrine induced contractile response (10nM-100  M) of the isolated perfused aortic ring preparation in STZ-induced diabetic rats : Both drugs produced significant incraeses in EC50 and significant decreases in E max.

24. The cumulative response curves for phenylephrine (10nM- 100  M) in rat aortic rings isolated from: (A)STZ-diabetic group.(B) STZ+  -lipoic group. (C)STZ +tadalafil group. The cumulative response curves for phenylephrine (10nM- 100  M) in rat aortic rings isolated from: (A)STZ-diabetic group.(B) STZ+  -lipoic group. (C)STZ +tadalafil group. Results A A B B C C

25. (1) Effects of intraperitoneal injection of  -lipoic acid (50mg/kg/day) and oral tadalafil (2mg/kg/day) for 4 weeks on the mean EC50 (  M) of phenylephrine induced contractions of isolated perfused aortic ring preparation in STZ-induced diabetic rats (n=6) Results (2) Effects of intraperitoneal injection of  -lipoic acid (50mg/kg/day) and oral tadalafil (2mg/kg/day) for 4 weeks on the Emax (g) of phenylephrine induced contractile response of the isolated perfused aortic ring preparation in STZ-induced diabetic rats (n=6).

26. 2-Effects of intraperitoneal injection of  - lipoic acid (50mg/kg/day) and oral tadalafil (2mg/kg /day) pretreatment for 4 weeks on % of acetylcholine induced relaxation (10nM- 100  M) of the isolated perfused aortic ring preparation in STZ-induced diabetic rats : Both drugs produced significant increases. 2-Effects of intraperitoneal injection of  - lipoic acid (50mg/kg/day) and oral tadalafil (2mg/kg /day) pretreatment for 4 weeks on % of acetylcholine induced relaxation (10nM- 100  M) of the isolated perfused aortic ring preparation in STZ-induced diabetic rats : Both drugs produced significant increases. Results

27. Endothelial-dependent relaxation induced by acetylcholine (10nM-100uM) in isolated perfused rat aortic rings precontracted by phenylephrine (10  M) in: (A)STZ-diabetic group.(B) STZ+  -lipoic group. (C)STZ +tadalafil group. Endothelial-dependent relaxation induced by acetylcholine (10nM-100uM) in isolated perfused rat aortic rings precontracted by phenylephrine (10  M) in: (A)STZ-diabetic group.(B) STZ+  -lipoic group. (C)STZ +tadalafil group. Results A A B B C C

28. Results Effects of intraperitoneal injection of  -lipoic acid (50mg/kg/day) and oral tadalafil (2mg/kg/day) for 4 weeks on % of acetylcholine induced relaxation of isolated perfused aortic ring preparation in STZ-induced diabetic rats (n=6).

30.  Lipoic acid improved the plasma glucose, insulin, and nitric oxide levels. It also improved the endothelial dysfunction in diabetic blood vessels.  In contrast tadalafil could not elicit any significant changes in the plasma glucose and insulin levels. Yet, it improved the plasma nitric oxide level as well as the endothelial dysfunction in diabetic vessels. Consequently, both drugs may be beneficial in diabetic patients. In addition α- lipoic acid may be a useful adjuvant in glycemic control in those patients.  Lipoic acid improved the plasma glucose, insulin, and nitric oxide levels. It also improved the endothelial dysfunction in diabetic blood vessels.  In contrast tadalafil could not elicit any significant changes in the plasma glucose and insulin levels. Yet, it improved the plasma nitric oxide level as well as the endothelial dysfunction in diabetic vessels. Consequently, both drugs may be beneficial in diabetic patients. In addition α- lipoic acid may be a useful adjuvant in glycemic control in those patients. Conclusion