Effect of TBE, TMP, and metformin on blood glucose
High blood glucose level was a significant and dramatic character of diabetic mellitus and the control of blood glucose levels was a key step to cure diabetic mellitus and its complications. In the present study, an experimental model of diabetic rats was induced by intraperitoneal administration of alloxan. TBE was administered at the dose of 100 mg/Kg body weight. This dose of TBE was chosen since the results obtained by us in the earlier related studies suggested that it would be effective (Zhang and Zhang 2006). At the start of experiment, all groups of rats had nearly the same blood glucose levels (P > 0.05). In contrast with the blood glucose level of normal rats, administration of alloxan led to 5-fold elevation of blood glucose levels, which was maintained over a period of 4 wk. It was attested that the model rats were stable and usable. In comparison to the untreated group, the blood glucose levels of administrating TBE, TMP, and metformin were significantly decreased (P < 0.05). However, there was no significant difference between the blood glucose levels of TBE, TMP, and metformin groups (P > 0.05) (Table 1).
Table 1—. Effect of TBE on blood glucose in diabetic rats.
|Normal|| 5.89 ± 0.89||5.97 ± 1.03||5.93 ± 1.06||5.97 ± 1.08||6.01 ± 1.14|
|Untreated||22.34 ± 6.24||23.71 ± 6.11 ||24.63 ± 7.02 ||25.79 ± 5.12 ||27.05 ± 3.84 |
|TBE||22.58 ± 5.63||13.71 ± 6.22a||13.24 ± 6.75a||14.83 ± 6.76a||15.48 ± 6.52a|
|TMP||22.76 ± 5.60||15.80 ± 5.21a||16.89 ± 5.34a||18.21 ± 3.82a||20.29 ± 3.09a|
|Metformin||22.68 ± 5.29||15.49 ± 7.03a||15.87 ± 6.87a||16.38 ± 7.01a||17.88 ± 6.89a|
Effect of TBE, TMP, and metformin on body weight
The basal body weight was 200.8 ± 15.64 (Table 2) and there is no variation from one group to another. The body weight of the untreated and TMP groups was slightly changed at the end of the 4th week (P > 0.05) in comparison with initial body weight. But intragastrically administrating of TBE and metformin for 4 wk resulted in a significant increase in body weight (P < 0.05) as compared with the untreated group. No significant difference was observed between TBE, TMP, and metformin groups. While enhancing body weight, TBE also rectified the abnormal food intake efficiency of diabetic rats (Table 2). A significant difference in both body weight gain and food intake was observed between the control groups and normal animals. The food intake amount of the diabetic untreated rats significantly increased in contrast with the normal rats (P < 0.05). After administrating TBE, TMP, and metformin, the amount of food intake was markedly lower as compared with diabetic untreated group. Especially in TBE group, the weight gain was not lower than in TMP and metformin, but the food intake was lower than in TMP and metformin. This result demonstrated that TBE could significantly increase food efficiency ration and be in favor of animal growth.
Table 2—. Effect of TBE, TMP, and metformin on the body weight gain and food intake in diabetic rats for 28 d.
|Normal||215.6 ± 16.5 ||223.5 ± 17.6||239.8 ± 19.2||256.4 ± 21.6 ||273.2 ± 24.9 ||2.06 ± 0.3 ||7.63 ± 0.65 ||0.27 ± 0.04 |
|Untreated||193.2 ± 13.04||194.5 ± 18.9||201.5 ± 20.8||206.4 ± 26.5 ||208.1 ± 27.8 ||1.06 ± 0.51a ||11.24 ± 0.92a ||0.09 ± 0.05a |
|TBE||200.2 ± 11.82|| 216.3 ± 15.1b|| 232.5 ± 27.6b||246.5 ± 27.5b ||254.0 ± 31.1b||1.92 ± 0.37bc ||8.94 ± 0.74abc||0.21 ± 0.04abc |
|TMP||199.3 ± 11.26|| 207.5 ± 18.47||214.4 ± 20.5||224.7 ± 24.8 ||234.2 ± 26.5b||1.25 ± 0.32ab ||9.76 ± 0.83ab ||0.13 ± 0.03ab |
|Metformin||210.8 ± 19.89||220.0 ± 30.4|| 234.8 ± 27.7b||248.4 ± 25.64b||259.1 ± 29.6b||1.73 ± 0.28abc||8.85 ± 0.86a ||0.20 ± 0.03ac |
Effect of TBE, TMP, and metformin on lipid levels
Abnormal lipid metabolism is a common syndrome of diabetic mellitus. There is evidence that not only excessive consumption of fats may eventually lead to the development of insulin resistance (Uauy and Diaz 2005) but also endogenous lipid molecules induce cytosolic phosphoenolpyruvate carboxykinase gene transcription, which plays key roles in gluconeogenesis, glyceroneogenesis, and cataplerosis, and attenuate the insulin action (Chen 2007). Clinically, it has been observed that there is present altered fat metabolism in type 1 and type 2 diabetes leading to the variation of serum cholesterol and triglyceride levels (Eisenbarth and others 1994). Hypercholesterelomia and hypertriglyceredemia have been reported to occur in streptozotocin-induced diabetic rats (Sharma and others 1997). In insulin-deficient subjects, insulin deficiency fails to activate lipoprotein lipase and causes hypertriglyceredemia. Hence, it is necessary to estimate serum cholesterol and triglyceride in the rats suffering chronic type 1 diabetic mellitus. Comparisons with untreated diabetic rats and TMP rats, the levels of total cholesterol, phospholipid, and triglyceride in the TBE group were significantly lower (P < 0.01), almost reaching normal levels (P > 0.05) (Table 3). These results demonstrated that TBE could decrease the levels of total cholesterol and triglyceride in the blood of diabetic rats.
Table 3—. Effect of TBE, TMP, and metformin on levels of cholesterol, phospholipids, and triglyceride in the plasma of diabetic rats.
|Normal||90.1 ± 12.6||87.3 ± 8.5 ||85.3 ± 6.1 |
|Untreated||143.9 ± 10.8a ||135.6 ± 13a ||175.2 ± 27.4a |
|TBE||94.3 ± 9.4bc||91.2 ± 8.5bc|| 82.5 ± 13.6bc|
|TMP||108.6 ± 10.7ab||121.6 ± 9.8ab ||143.1 ± 18.9ab|
|Metformin||99.8 ± 8.5b ||104.2 ± 11abc||131.4 ± 17.6ab|
Attenuation of activities of TBE, TMP, and metformin and its effect on oxidative stress
Recently, much attention has been paid to the role of oxidative stress, which resulted from an imbalance in oxygen free radical production, and it has been suggested that oxidative stress may be the key and common factor constraining the pathogenesis of different diabetic complications (Ding and others 2007; Cvetkovic and others 2008). Under hyperglycemic conditions such as diabetes, free radical production may arise from glucose self-oxidation (Hunt and others 1988), oxidative degradation of Amadori products (Elgawish and others 1996), advanced glycation end-proteins–receptor of glycation end-proteins interaction (Tan and others 2007), and redox potential increase (Linnane and Eastwood 2006). If not completely scavenged, redundant free radicals can bring about great damages to DNA, protein, or lipids. Eucaryotic cells contain antioxidant defenses that can protect these cells from oxidative damage. Antioxidants may be either nonenzymatic (vitamins C and E and reduced glutathione) or enzymatic (glutathione reductase and superoxide dismutase). In the course of diabetes, both types of antioxidants have been reported to be reduced (Makar and others 1995; Mooradian 1995) or enhanced (Sechi and others 1997), depending on the tissues studied or diabetes duration. So, in our investigations, superoxide dismutase, glutathione reductase, catalase and glutathione peroxidise, total antioxidant capability, and malondialdehyde were indexed to compare the impact of TBE, TMP, and metformin on oxidative press in diabetic rats.
The total antioxidant capability of diabetic untreated group was significantly debased compared with normal group in plasma, liver, kidney, and pancreas (P < 0.05) (Table 4). When giving tested drugs, total antioxidant capabilities in different tissue showed uptrend, but the uptrend displayed markedly significant difference in liver and kidney, and the significant difference between TBE and TMP was not observed.
Table 4—. Effect of TBE, TMP, and metformin on total antioxidant activities in tissues of diabetic rats (U/mL).
|Normal||21.89 ± 5.09||1.786 ± 0.15||1.029 ± 0.064||0.819 ± 0.176|
|Untreated|| 9.37 ± 6.74a||0.514 ± 0.33a||0.566 ± 0.143a||0.577 ± 0.165a|
|TBE||15.76 ± 5.97a||1.017 ± 0.43ab||0.976 ± 0.231b||0.704 ± 0.143|
|TMP||13.54 ± 6.27a||0.894 ± 0.32||0.872 ± 0.187b||0.639 ± 0.107|
|Metformin||12.86 ± 6.16a||0.943 ± 0.52b||0.738 ± 0.149a||0.677 ± 0.143|
Plasma, liver, kidney, and pancreas are endowed with innate antioxidant defense mechanisms, such as the presence of the enzymes catalase, superoxide dismutase, and glutathione peroxidase. A reduction in the activities of these enzymes is associated with the accumulation of highly reactive free radicals, leading to deleterious effects such as loss of integrity and function of cell membranes (Reedy and Lokesh 1992; Krishnakantha and Lokesh 1993; Sheela and Angusti 1995). Administration of alloxan leads to generation of reactive oxygen species such as H2O2, O •2−, and HO •, which are associated with inactivation of superoxide dismutase, catalase, and glutathione peroxidise. This probably explains the significantly reduced activities of superoxide dismutase, catalase, and glutathione peroxidise observed by us in rats challenged with alloxan (untreated group in Table 5 to 7). In rats receiving TBE, TMP, and metformin, Significant increase of superoxide dismutase was noticed in the TBE and metformin groups in different tissue (P < 0.05), simultaneous obvious differences also existed between the TBE and TMP groups (P < 0.05) in all examined tissue (Table 5); significant increase of catalase in the TBE, TMP, and metformin was only found in the liver and kidney (P < 0.05), and no markedly difference was found between TBE and TMP groups (P > 0.05) (Table 6); while significant increase of glutathione peroxidise activities was only found in all tissues of TBE group, as well as liver and plasma in the TMP group(Table 7).
Table 5—. Effect of TBE, TMP, and metformin on SOD activities in tissues of diabetic rats (U/mL).
|Normal||166.63 ± 24.11||17.75 ± 1.78||28.36 ± 3.48||11.23 ± 2.14 |
|Untreated|| 91.78 ± 12.87a|| 9.86 ± 2.04a||12.56 ± 3.53a||6.87 ± 2.04a|
|TBE||156.87 ± 11.83bc||16.34 ± 2.13bc||25.43 ± 4.72bc||9.65 ± 2.63b|
|TMP||121.56 ± 17.82ab||10.48 ± 2.41b||18.64 ± 3.72ab||8.97 ± 2.32b|
|Metformin||132.84 ± 13.76ab||14.18 ± 1.86ab||22.38 ± 4.27abc||8.45 ± 1.75b|
Table 6—. Effect of TBE, TMP, and metformin on catalase activities in tissues of diabetic rats (U/mL).
|Normal||20.894 ± 2.768||4.163 ± 0.276||1.784 ± 0.253||0.419 ± 0.176|
|Untreated||15.438 ± 2.515a||2.264 ± 0.282a||1.068 ± 0.199a||0.269 ± 0.147a|
|TBE||20.837 ± 2.587b||3.216 ± 0.276ab||1.497 ± 0.277ab||0.327 ± 0.174|
|TMP||18.364 ± 2.139||3.061 ± 0.307ab||1.412 ± 0.218ab||0.316 ± 0.156|
|Metformin||16.549 ± 2.132a||2.976 ± 0.284ab||1.423 ± 0.255ab||0.301 ± 0.163|
Table 7—. Effect of TBE, TMP, and metformin on glutathione peroxidase activities in tissues of diabetic rats (U/mL).
|Normal||159.771 ± 13.168||67.131 ± 9.171||40.518 ± 5.946||10.342 ± 2.673 |
|Untreated||100.494 ± 12.611a||41.953 ± 10.622a||30.673 ± 6.783a||6.414 ± 2.917a|
|TBE||139.715 ± 12.839ab||61.739 ± 9.735b||38.361 ± 6.539ab||9.313 ± 2.37b |
|TMP||117.623 ± 11.723ab||58.552 ± 8.371b||35.412 ± 5.811|| 7.02 ± 2.356a|
|Metformin|| 197.28 ± 12.35.8a||58.269 ± 8.661b||34.347 ± 6.114||7.177 ± 2.537a|
Glutathione reductase, the enzyme responsible for recycling glutathione disulfide to glutathione, significantly decreased in plasma, liver, kidney, and pancreas of diabetic rats as compared with the normal rats (P < 0.01) (Table 8). However, after given drugs, TBE significantly changed almost to the normal levels as compared with the normal rats (P > 0.05), and the activities were stronger than the effects of TMP (P < 0.05).
Table 8—. Effect of TBE, TMP, and metformin on GR activities in tissues of diabetic rats (U/mL).
|Normal||221.99 ± 11.15||83.65 ± 5.35|| 37.5 ± 6.4||8.72 ± 1.13 |
|Untreated||177.59 ± 12.56a||49.54 ± 6.23a||18.54 ± 5.98a||5.12 ± 1.29a |
|TBE||218.93 ± 12.13bc||78.65 ± 6.87bc||32.23 ± 6.12bc||7.67 ± 1.08bc |
|TMP||188.34 ± 10.13a||57.13 ± 6.94ab||25.41 ± 5.83ab||6.02 ± 1.14ab |
|Metformin||197.28 ± 11.86ab||70.54 ± 6.07abc||30.65 ± 6.82ab||7.1 ± 0.97abc|
These results described previously possibly confer that these protective activities of TBE, by dampening the generation of free radicals induced by alloxan, were stronger than those of TMP and metformin.
Peroxidation of free radical mediated cell membrane lipids has been implicated under pathological conditions such as increased cell membrane rigidity, decreased cellular deformability, and lipid fluidity. Overproduction of reactive oxygen species due to glucose oxidation in the presence of the transition metals can cause membrane damage through the peroxidation of membrane lipid and protein glycation (Kolanjiappan and others 2002). Elevated lipid peroxidation in liver and kidney has been well demonstrated in alloxan-induced diabetic rats (Ananthan and others 2004). The extent of tissue damage has been assessed in terms of the measurements of lipid peroxidation products such as malondialdehyde and antioxidants (Gutteridge 1995). Thus, the observed increase of malondialdehyde in different tissues could be used as an index of lipid peoxidation.
Alloxan administration led to a significant increase in the malondialdehyde level of plasma, liver, kidney, and pancreas as compared with the normal rats (P < 0.01) (Table 9). The administration of TBE ameliorated the alloxan-induced elevation of lipid peroxidation in plasma, liver, and pancreas as compared to the untreated rats. However, no significant decrease was observed in MDA in TMP and metformin rats. The previously mentioned results presented in Table 9 show that TBE could decrease the oxidative stress induced by alloxan and protect tissues from damages mediated by free radicals.
Table 9—. Effect of TBE, TMP, and metformin on malondialdehyde levels in tissues of diabetic rats (nmol/mL).
|Normal||3.85 ± 1.67||0.64 ± 0.42||0.98 ± 0.31||0.23 ± 0.17|
|Untreated||6.26 ± 1.56a||1.25 ± 0.47a||1.56 ± 0.35a||0.48 ± 0.18a|
|TBE||4.18 ± 1.32bc||0.77 ± 0.48bc||1.32 ± 0.34||0.25 ± 0.14b|
|TMP||5.59 ± 1.09a||1.19 ± 0.33a||1.61 ± 0.38a||0.28 ± 0.19|
|Metformin||5.13 ± 1.14||1.06 ± 0.26||1.47 ± 0.34a||0.31 ± 0.17|