Rats of this group were injected with 0.1 ml of citrate buffer (pH; 4.5).

Rats of this group were injected intra-peritoneally with Streptozotocin (STZ).

Rats of this group were injected intra-peritoneally with STZ and after 3 days, they received powdered Amaryl^® tablet (Glimepiride) at 800µg/kg body weight orally once a day.

Rats of this group were injected intra-peritoneally with STZ and after 3 days they received the dried aqueous extract of Fenugreek at a dose rate of 500mg/kg.

Rats of this group were injected intra-peritoneal with STZ at single dose (40 mg/kg body weight) and after 3 days they received aqueous extract of Fenugreek at a dose rate of 500mg/kg and powdered Amaryl^® tablet (Glimepiride) at 800µg/kg body weight daily.

During the experiment, weights and random blood sugar levels of the rats were measured. At the end of the experiment, rats were sacrificed for the histological investigation of kidney, pancreas and testes. Hematological and biochemical assessments were studied.

The alteration induced by Streptozotocin led to some changes in the blood parameters. These changes are exhibited through a decrease in RBCs, Ht, Hb and platelets, on the other side, an increase in HbA1C. These changes could be due to oxidative stress, which lead to lipid peroxidation in RBCs membranes, auto oxidation of hemoglobin. The decrease in Mean Cell Hemoglobin Concentration (MCHC) values observed after administration of STZ is an indication of abnormal hemoglobin synthesis, failure of blood osmoregulation, and plasma osmolality. Oxidation of these proteins and hyper-glycaemia cause an increase in the production of lipid peroxides that lead to haemolysis of RBC 9. The study demonstrated that rats treated with Fenugreek or Glimepiride provided significant protection to the altered hematological variables (Table 1). Treatment with the combination of Fenugreek and Glimepiride were more effective than Fenugreek alone or Glimepiride alone. The anti-oxidant property of Fenugreek 7 inhibits lipid peroxidation of the erythrocytes. Fenugreek contains iron and it can improve anemia conditions. Glimepiride is able to improve the erythrocyte membrane integrity by the mitigation of oxidative damage to the erythrocytes membranes 10. Results showed that combination of Fenugreek and Glimepiride had better RBC, Hb, Ht, and MCHC levels of experimental groups showed no significant difference compared to normal control after eight weeks (Table 1).

In diabetes, there is an increased platelet activation and subsequent aggregation favoring thrombosis and also depression of bone marrow stem cells which could be contributory for a reduced platelet count observed. The decrease in circulating platelet number may also means an increase in aggregated platelet typical of atherosclerotic disorders of diabetes mellitus. In Fenugreek group, platelet count increased significantly (p < 0.01). The significant improvement in the levels of circulating platelets in blood could be attributed to inhibitory activity of certain constituents of T. foenum graecum on platelet aggregation 11. Glimepiride group showed an increase of the platelet count. Glimepiride exhibits inhibitory effects on human platelets aggregation through selective suppression of the cyclooxygenase pathway 12. Combination of Fenugreek and Glimepiride showed better platelet count with no significant difference compared with normal control group after eight weeks, (Table 1).

Regarding total WBCs, lymphocytes and monocytes; the results showed marked decrease with a distinct increase in the percentage of neutrophils and eosinophil's (Table 2). Fenugreek seeds play a role in protecting the spleen and increasing the lymphocytes. So, they might improve the immunity. These observations were in agreement with those obtained by Sindhu et al.13 and Kandhare et al.14. Fenugreek seeds influenced the hemoglobin and lymphocytes count, improving hematopoietic function and survival. The present study demonstrates that combination of Fenugreek and Glimepiride are more effective than Fenugreek alone or Glimepiride alone in protecting to the altered hematological variables, (Table 2).

The current study showed some changes in the biochemical measurements due to the injection of Streptozotocin (Figure 2). Group (5); Fenugreek and Glimepiride combination improved the random blood sugar to reach a value near the normal control values. The random blood sugar level of group (3) and group (4) showed no significant difference from each other. Injection with STZ induces activation of poly adenosine diphosphate ribosylation and nitric oxide release. As a result, pancreatic cells are destroyed by necrosis 15. Fenugreek seeds water extract acts as an insulin secretor. Hypoglycemic effect of Fenugreek may be mediated through stimulating insulin synthesis and increasing β pancreatic cells secretion. The primary mechanism of action of Glimepiride in lowering the blood glucose appears to be dependent on stimulating the release of insulin from functioning pancreatic beta-cells 16. Glimepiride binds to sulfonylurea receptors on β-cells leading to blocking of K⁺ ATP channels, opening of voltage-gated calcium channels and increase in Ca²⁺ influx leading to insulin release from pancreatic β-cells.

The present study demonstrated that STZ injection induced a significant decrease in the concentration of insulin in serum when compared with the control values. Groups (3 and 4) showed partial improvement in the concentration of insulin when compared with STZ group. However, the insulin concentration of group (5) reached the same value as the control group values (Figure 2).

Serum levels of total protein and albumin in the STZ group were significantly lowered while the administration of Fenugreek in group (3) or Glimepiride in group (4) revealed significant increase in the serum levels of albumin and total protein (Table 3). However, their levels in the combination treatment were near to the value of the control group. In Streptozotocin induced diabetes rats, body cells are unable to utilize glucose as a source of energy so proteins are spared as the energy source. The amino acids released from the proteins are used for gluconeogenesis. The decrease in the total protein might be due to micro-proteinuria which was an important clinical marker of diabetic nephropathy and /or might be due to increase in protein catabolism. This leads to a decrease in the protein storage which in turn reduces body weight. Albumin decrease due to its glycation during diabetes may be considered as an important factor responsible for oxidative stress related to diabetes 17.

Following the treatment with Fenugreek or Glimepiride, total protein and albumin level were increased significantly. The elevated serum TP level in Fenugreek groups may be due to high level of protein and other nutrients in T. foenum-graecum L seeds 18.

STZ administration associated with a high significant increase in serum lipid profile (Table 4); total cholesterol, triglyceride, low density lipoproteins LDL and Ratio (cholesterol/HDL) except high density lipoproteins (HDL). Fenugreek treatment improved these elevations. Also, Glimepiride significantly decrease these parameters as compared to the control group. However, group (5) significantly improve all these parameters specially triglycerides which reached a values near the control ones. On the other hand, the high-density lipoproteins (HDL) concentration of diabetic group showed an apparent decrease and groups (3, 4 and 5) improve this decrease.

The lipid changes associated with diabetes mellitus are attributed to an increase of free fatty acids into the liver and to insulin deficiency/resistance. This results in excess fatty acid accumulation in the liver. Krishnakumar et al. 19 reported that diabetes-induced hyperlipidemia is attributable to excess mobilization of fat from the adipose due to the underutilization of glucose. Fenugreek extract causes significant decrease in serum triglycerides, total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-c) 20. These effects may be due to sapogenins, which increase biliary cholesterol excretion in liver, leading to lowered serum cholesterol levels. On the other side, oral administration of Glimepiride causes significant decrease in the serum levels of triglycerides, total cholesterol and LDL-cholesterol in contrast to significant elevation in HDL-cholesterol. Glimepiride improved HDL-c level via improvement of plasma adiponectin level as adiponectin could increase HDL-c levels directly via increased lipoprotein lipase and decreased hepatic lipase activity 21. In addition, the anti-lipidemic action of Glimepiride may reside in their ability to stimulate insulin secretion and action.

STZ administration associated with a significant increase in the urea, creatinine and uric acid levels when compared with the control group (Table 5). Treatment in groups (3, 4 and 5) exhibited a high drop in this elevation especially when compared with the diabetic group. Fenugreek and Glimepiride supplement succeeded partially in improving urea, creatinine and uric acid levels when compared with the diabetic group. Group (5) showed a significant decrease in these levels when compared with groups (3 and 4). Raised serum creatinine, urea and uric acid levels in diabetic group may indicate a pre-renal problem such as volume depletion. The high creatinine levels observed in diabetic may be due to impaired function of the nephrons. The increase in urea nitrogen may be accounted for by enhanced catabolism of both liver and plasma proteins that accompany gluconeogenesis 22. Uric acid is an independent predictor of incident type 2 DM. The present data showed that uric acid levels were increased in diabetic rats. Fenugreek seed extract showed decrease in urea, creatinine and uric acid levels, demonstrating renal protective effects of Fenugreek. Nephron protective effect of Fenugreek is due to its antioxidant and hypoglycemic properties and its constructive effects on collagen which prevent ROS induced destruction of filtration and basement membrane of kidneys 2324. Fenugreek and Glimepiride supplements succeeded partially in improving the creatinine level in comparing with the diabetic group. The combination of Fenugreek and Glimepiride group showed a significant decrease in the creatinine level (Table 5).

The reported present data (Table 6) indicated that administration of Streptozotocin provoked a significant increase in serum ALT, AST and ALP. Treatment with Fenugreek and Glimepiride induced a partial recovery but treatment using combination of Fenugreek and Glimepiride showed significant improvement of the ALT, AST and ALP values. Diabetes is developed due to obstruction of glucose utilization by the tissues through glycolysis and its over-production through excessive hepatic gluconeogenesis. This may be attributed to the relative deficiency of insulin which is responsible for increasing the expression of the glycolytic enzymes in the liver 25. Serum transaminases and alkaline phosphatase activities were generally increased in Streptozotocin-induced diabetic rats. Fenugreek therapy restores the activity of these enzymes. These seeds may inhibit the liver damage induced by Streptozotocin and improved liver function with return of gluconeogenesis toward its normal level 26. Treatment of diabetic rats with sulfonylurea drugs was explained by increased insulin secretion, hepatic uptake of glucogenic amino acids and stimulation of amino acids incorporation into protein 27.

The elevation of biomarker enzymes such as SGOT, SGPT, and ALP was observed in diabetic rats and indicates the hepatocellular damage. Fenugreek and Glimepiride induced a partial recovery and their combination showed a significant improvement. Fenugreek may improve gluconeogenesis toward its normal level. Glimepiride increase insulin secretion, hepatic uptake of glucogenic amino acid and stimulation of amino acid incorporation.

Oxidative stress is enhanced through various sources such as hyperglycemia, dyslipidemia, hyper-insulinemia, insulin resistance, and impaired antioxidant defense. Oxidative stress in DM adversely affects the physiological and biochemical processes of cell, and among those islets cells of pancreas which are more prone to be damaged due to the lowest intrinsic antioxidant defense mechanism. Chronic hyperglycemia and oxidative stress might be resulted due to the multiple biochemical pathways. The concentration of Malondialdehyde (MDA) andMyeloperoxidase (MPO) significantly decreased in pancreas homogenates of group (5) compared with group (3), group (4) and diabetic group (Table 7). The increase induced by Strepotozotocin is attributed to its ability to generate ROS, which induce oxidative damage in several tissues by enhancing lipid peroxidation (LPO) and also decrease of the antioxidant enzymes. Fenugreek led to decrement in MDA level in treated rats, which could be due to the consumption of the extracellular antioxidant, ceruloplasmin, by the oxidants to combat oxidative stress 28. (Table 8) Fenugreek has been reported to have antioxidant and free radical scavenging activity. Fenugreek combats oxidative stress and the antioxidant activity of Glimepiride could be explained through normalization of elevated hyperglycemia as glucose auto oxidation is the major source of free radicals and peroxides. Rats which treated with Glimepiride showed significant increase in the antioxidant enzymes.

Oxidative stress in diabetes is coupled with a decrease in the antioxidant status, which can increase the deleterious effects of free radicals. The SOD and CAT are the two major scavenging enzymes that remove free radicals. The antioxidant property of Fenugreek is due to the presence of many active phytochemicals including vitamins, flavonoids, terpenoids, carotenoids, lignin plant sterol 29. Antioxidants exert their mode of action by suppressing the formation of reactive oxygen species either by the inhibition of enzymes or by chelating trace elements. Glimepiride possesses antioxidant activity against the STZ-induced oxidative stress 30. This antioxidant effect of Glimepiride may be attributed to its activation of the redox sensitive transcription factor NF (Kappa) B, activation of antioxidant enzymes such as SOD which is responsible for dismutation of superoxide ion into oxygen and hydrogen peroxide. Thus, it protects the cell from damage caused by superoxide activity.

Histological examination of control rats' testis showed normal histological structure of the seminiferous tubules with an increase in the spermatogonia and other spermatocytes compared with the diabetic rats which showed an obvious decrease in the count of germinal cells generally and sloughing of germ cell in the lumen of seminiferous tubules in addition to absence of the mature sperms (Figure 3 and Figure 4). Rats tested of Fenugreek group and those treated with Glimepiride (Figure 5 and Figure 6) presented an increase in the number of spermatogenic cells and mature sperms with few focal necrotic cells. Whereas the combination therapy group showed normal histological structure of the seminiferous tubules with normal spermatogonia, presence of ledying cells and more mature of sperms (Figure 7). STZ reduced testosterone production, suggesting a decrease in the function of both Leydig (testosterone producing cell) and Sertoli (spermatogenesis) cells, which might be caused by a reduction in insulin secretion. These changes are probably due to increased Reactive Oxygen Species (ROS) production by accelerated Advanced Glycation End Products (AGE) formation 31 hexosamine and Protein Kinase C pathway. The administration of Fenugreek seeds to diabetic rats significantly decrease of sperm shape abnormality and improve the sperm count 32. Glimepiride reduced sperm abnormality and increased testis weights and sperm count by its antioxidant action 30. The potential protective efficacy of Fenugreek seed extract when added to Glimepiride was observed on reproductive systems.

Histological examination of pancreas of the control rats showed normal sized islets of pancreas (Ip) compared to the diabetic rats which showed markedly atrophic islets of pancreas (I.p.) (Figure 8 and Figure 9). Pancreas of in groups (3) and (4) rats showed mild atrophic islets of pancreas (I.p.) with inflammatory cellular infiltrate (Figure 10 and Figure 11). The pancreas of rats in group (5) showed nearly normal islets of pancreas (I.p.) with few inflammatory cellular infiltrate (I.c.) (Figure 12).

Histopathological observation in diabetic control showed degenerative changes in both endocrine and exocrine pancreases. A probable explanation may be related to oxidative stress resulting from hyperglycemia which decreases the antioxidants levels and increases ROS 33 and the activities of antioxidant enzyme were altered in diabetic rats. These effects further exacerbate the development and progression of diabetes complications and these may represent the causes of degeneration revealed in histological sections. Fenugreek improved blood glucose levels and insulin, lowered pancreatic islet and β-cells damage which may be attributed to its immune modulatory activity and insulin stimulation action along with its antioxidant potential 32. Glimepiride caused restoration of morphology of beta cell of diabetic and it increased percentage of beta cells 34. This protective effect could be attributed to the antioxidant properties of Glimepiride. Combination therapy improves the protective effect of both therapies alone.

Kidney sections of diabetic rats showed an increase in mesangial cell and matrix of glomeruli with increase in glycogen deposition and hyalinization of arterioles with thickened basement membranes of proximal and distal convoluted tubules. These changes will lead to progressive reduction in the filtration surface of the glomeruli 35. Histological examination of the kidney of the control rats showed normal structure of renal glomerular. The proximal and distal tubules were lined with normal epithelium (Figure 13). The diabetic rats showed tubular casts, inflammatory cellular infiltration and glomerular atrophy (Figure 14). Kidney of rats in groups (3 and 4) showed some glomeruli return to be normal (G). Focal tubules casts (T) in Fenugreek group and some normal tubules in the Glimepiride group with no inflammatory cellular infiltrate in both groups (Figure 15 and Figure 16). However, kidney of rats in group (5) showed normal glomerular (G), normal tubules (T) with no tubular casts (Figure 17).