Anti-diabetic effect of Wen-pi-tang-Hab-Wu-ling-san extract in streptozotocin-induced diabetic rats.

OBJECTIVES: Wen-pi-tang-Hab-Wu-ling-san (WHW) is an oriental herbal prescription formulated using 14 herbs and has been used to cure chronic renal failure in Korean oriental medicine. In this study, we investigated the anti-diabetic effect of WHW in the streptozotocin-induced diabetic rats.
MATERIALS AND METHODS: Diabetes was induced by streptozotocin (STZ, 60 mg/kg, i.p.) in rats. WHW extract (100 mg/kg) was orally dosed once a day for four weeks. The results were compared with standard antidiabetic drug, glibenclamide (3 mg/kg, p.o).
RESULTS: Significant decrease in body weight and insulin levels and increase in blood glucose, triglycerides, urea nitrogen (BUN), and creatinine were detected in STZ-induced diabetic rats with disruption and disappearance of pancreatic and kidney cells and decrease in insulin producing beta cells. However, these diabetic changes were significantly inhibited by treatment with WHW extract. In the oral glucose tolerance test, the extract produced a significant decrease in glycemia 60 minutes after the glucose pulse.
CONCLUSIONS: Based on these results, we suggest that WHW extract has favorable effects in protecting the STZ-induced hyperglycemia, renal damage, and beta-cell damage in rats.

Introduction

Diabetes mellitus (DM) is a major health problem worldwide. Globally, the estimated incidence of diabetes and projection for year 2010, as given by International Diabetes Federation (IDF) is 239 million.[1] It is a metabolic disorder characterized by chronic hyperglycemia with disturbances of carbohydrate, fat, and protein metabolism resulting from defects in insulin secretion, insulin action or both.[2] Epidemiological studies and clinical trials strongly support the notion that hyperglycemia is the principal cause of complications. Therefore, effective blood glucose control is the key for preventing or reversing diabetic complications and improving quality of life in patients with diabetes. Thus, sustained reduction in hyperglycemia will decrease the risk of developing microvascular complications.[3]

In modern medicine, the beneficial effects on glycemic levels are well documented; the preventive activity of drugs against progressive nature of diabetes and its complications are modest and not always effective.[4] Treatment with sulfonylureas, biguanides, and insulin possess undesirable side effects.[5] So the management of diabetes without side effects is yet a challenge to the medical system. There is an increasing demand to use the natural products with antidiabetic activity. Plants are useful sources for the development of antidiabetic drugs. Particularly, the use of medicinal plants and oriental medicine prescriptions in modern medicine suffers from the fact that though hundreds of plants are used in the world to prevent or to cure diseases, scientific evidence in terms of modern medicine is lacking in most cases. However, today it is necessary to provide scientific proof as to whether it is justified to use plant active principles or traditional prescriptions. A search for natural products that have antidiabetic properties and low toxicity are under evaluation. Wen-pi-tang-Hab-Wu-ling-san (WHW), an oriental medicine prescription, has been used to cure several renal diseases including chronic renal failure and diabetic nephropathy in Korean oriental medicine clinics. Recently, we reported that WHW extract attenuates the epithelial to mesenchymal transition (EMT) induced by transforming growth factor-β (TGF-β) in Madin-Darby canine kidney (MDCK) cells;[6] protects kidneys from ischemia/ reperfusion (I/R) injury by a reduction of post-ischemic oxidative stress and up-regulation of heat-shock protein-27 (HSP-27) or HSP-72;[7] inhibits the production of inflammatory mediators from lipopolysaccharide (LPS)-stimulated mouse macrophages with involvement in the blocking of MAPK and NF-kappaB pathway;[8] and prevents the post-ischemic renal damage and ureteral obstructive renal fibrosis in mice by reduction of oxidative stress, inflammation and TGF-beta/Smad2/3 signaling.[910]

In the present study, we investigated the anti-diabetic effect of WHW extract in STZ-induced diabetic rats with hyperglycemia, cytoprotective, and pancreatic insulin localization. We hope to provide the therapeutic potential of WHW extract in diabetes including diabetic nephropathy.

Materials and Methods

Preparation of Wen-pi-tang-Hab-Wu-ling-san Extract

Wen-pi-tang-Hab-Wu-ling-san extract was prepared using the following 14 herbs: 150 g of Codonopsis pilosulae radix (Codonopsis pilosula FR. NANNF), 150 g of Salviae miltiorrhizae radix (Salvia miltiorrhiza BGE), 100 g of Pinelliae rhizome (Pinellia ternata THUNB. BREIT.), 60 g of Coptis rhizome (Coptis chinensis FRANCH), 160 g of Epimedii herba (Epimedium koreanum NAKAI), 100 g of Rhei radix et rhizoma (Rheum palmatum L.), 100 g of Perillae folium (Perilla frutescens L. BRITT.), 50 g of Glycyrrhizae radix (Glycyrrhiza uralensis FISCH), 300 g of Artemisiae capillaris herba (Artemisia capillaris THUNB.), 200 g of Alismatis rhizome (Alisma plantago-aquatica var. orientale SAMUELS), 80 g of Poria (Poria cocos SCHW.), 80 g of Atractylodis macrocephalae rhizome (Atractylodes macrocephala KOIDZ.), 80 g of Polyporus (Polyporus umbellatus PERS. FRIES), and 40 g of Cinnamomi ramulus (Cinnamomum cassia PRESL). Plants were purchased from Medicinal Materials Company (Kwangmyungdang Medicinal Herbs, Ulsan, Republic of Korea) and were authenticated. A voucher specimen was deposited at the herbarium of Department of Herbology, College of Oriental Medicine, Dongguk University (DUCOM), with the registration number OB05-1. WHW was extracted from a crude herb mixture (1700 × g) by boiling in water for 5 h followed by filtering through a two-layer mesh and with concentration in a boiling water bath to obtain residues (yields of 18.5%). These extracts were stored at 4 °C before use. For supplementation of WHW, the extract was suspended in 0.9% NaCl.

Animals and Diets

Male Sprague-Dawley (SD) rats weighing 180-200 g (Orient Bio Inc., Gyeonggi-do, Republic of Korea) were used. The animals were housed at an ambient temperature of 22 ± 3 °C with humidity of 60 ± 5% under a daily 12 h light-dark cycle with free access to food and water. All animals were handled according to the animal welfare guidelines issued by the Korean National Institute of Health and the Korean Academy of Medical Sciences for the care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee of the Dongguk University.

Induction of Diabetes

Diabetes mellitus was induced in overnight fasted rats by a single intraperitoneal injection (i.p) of freshly prepared STZ (60 mg/kg b.w.). STZ was dissolved in citrate buffer (pH 4.5). Hyperglycemia was confirmed by the elevated glucose levels in plasma, determined at 72 h and then on day 7 after injection. The animals with blood glucose concentration more than 200 mg/dL were used for the study.

Experimental Design

A total of 24 rats (six rats in each group) were used. The rats were divided into four groups after the induction of diabetes with STZ. The experimental period was four weeks. Group I (normal control rats) received saline; group II were diabetic control rats; group III diabetic rats received WHW extract (100 mg/kg body weight) orally in saline for four weeks; and group IV diabetic rats were administered glibenclimide (3 mg/kg body weight) orally in saline for four weeks. At the end of four weeks, the animals were deprived of food overnight and sacrificed by decapitation. Blood was collected in tubes containing potassium oxalate and sodium fluoride mixture for the estimation of blood glucose, insulin, triglycerides, BUN, and creatinine. Pancreas and kidneys were immediately dissected out and washed in ice-cold saline to remove the blood.

Bodyweight Changes

Weight of individual animals was measured gravimetrically on 0, 2, and 4 weeks.

Determination of Oral Glucose Tolerance Test (OGTT)

The oral glucose tolerance test was performed on the second week of treatment and the same rats were further treated for four weeks. Prior to OGTT rats were fasted overnight (at least 12 h). Thirty min following the various treatments schedules, each rat was given an oral glucose load, 2 g/kg body weight according to Du Vigneaud and Karr[11] and Al-awadi et al .[12] Blood samples were collected from the tail vein at time 0 (prior to glucose load), 30, 60, 90, and 120 min after the glucose load. Blood glucose was determined by using commercial diagnostic kits (Asan Pharmaceutical, Republic of Korea).

Measurement of Biomarkers

Blood glucose was estimated colorimetrically using commercial diagnostic kits (Asan Pharmaceutical, Republic of Korea). Plasma insulin was assayed by ELISA using a Boehringer–Mannheim kit with an ES300 Boehringer analyzer (Mannheim, Germany). The levels of serum triglycerides, blood urea nitrogen (BUN), and creatinine in control and experimental groups were estimated spectrophotometrically using commercial diagnostic kits (Asan Pharmaceutical, Republic of Korea).

Histology and Immunohistochemistry

Samples from the splenic lobes of the pancreas and kidneys were perfused via the left ventricle with 30 mL phosphate buffered saline (PBS) for 2 min at 37 °C and then with PLP (2% paraformaldehyde, 75 mM L-lysine, 10 mM sodium periodate) fixative. The tissues were then excised, placed in PLP overnight at 4 °C, washed and stored in PBS containing 0.02% sodium azide at 4 °C. Half-hemisected fixed tissue samples were washed with PBS three times for five min each, placed in PBS overnight, embedded in paraffin and cut into 4 μm sections using a microtome. The sections were then stained with hematoxylin and eosin (H and E) staining following standard protocols. Ten fields (0.4 mm2/field) per tissue were used.

Pancreatic sections were used for immunstaining using the peroxidase anti-peroxidase (PAP) method. Blocking of nonspecific peroxidase reactions was performed with methanol containing 0.1% H2O2, and to avoid non-specific reactions with the background, the sections were incubated with normal goat serum prior to incubation with specific antibodies against insulin (dilution, 1:2,000, Diasorin, Chicago, IL). After rinsing in phosphate buffered saline (PBS; 0.01M, pH 7.4), they were incubated with secondary antibodies (goat anti-rabbit IgG or goat anti-guinea pig IgG, dilution, 1:200; Sigma, USA). Sections were then washed in PBS buffer and finally incubated with PAP complex (dilution 1:200; Sigma, St. Luis, MO). The paroxidase reaction was carried out using a solution of 3,3′-diaminobenzidine tetrahydrochloride containing 0.01% H2O2 in Tris-HCl buffer (0.05 M, pH 7.6). After staining, the sections were analyzed with a light microscope. After they were immunostained, the sections were lightly counterstained with Mayer's hematoxylin, and the immunoreactive cells were observed under a light microscope. The specificity of each immunohistochemical reaction was determined as recommended by Sternberger,[13] including the replacement of specific antiserum by the same antiserum, which had been preincubated with its corresponding antigen, insulin.

Statistical Analysis

All data analysis was completed using the Graphpad Instat software. Data are expressed as mean SEM. The significance level of treatment effects was determined using one way analysis of variance (ANOVA) followed by Tukey's post-hoc analysis and P value less than 0.05 was considered statistically significant. All experiments were performed a minimum of three times.

Results

Changes in Bodyweight

A significant decrease in bodyweight was detected in diabetic group as compared to the control group from day 0 to 2 and 4 weeks (P < 0.001) [Table 1]. The bodyweights of WHW extract supplemented at 100 mg/kg to diabetic group were increased significantly (P < 0.05) at end of four weeks compared to the diabetic group. In glibenclamide 3 mg/kg dosing group, no significant changes were detected as compared to the diabetic group.

Table 1

Effect of WHW extract on body weight changes in normal and diabetic rats

Changes in OGTT

Table 2 shows blood glucose levels of control, diabetic, and WHW treated groups after oral administration of glucose (1 g/kg body weight) at the end of seventh day. The blood glucose levels reached peak at 30 min after glucose administration in the diabetic group. WHW extract supplementation at 100 mg/kg showed a significant decrease in glucose levels at 60 min to 120 min after oral glucose administration when compared to the diabetic group. In glibenclamide (3 mg/kg) group, a decrease in glucose levels was also detected at 60 min to 120 min after glucose administration.

Table 2

Effect of WHW extract on glucose tolerance in normal and diabetic rats

Changes in Biomarkers

There was a significant elevation (P < 0.001) in blood glucose with significant decrease (P < 0.001) in plasma insulin levels in STZ-induced diabetic group, when compared to control group [Table 3]. Oral supplementation of WHW extract at 100 mg/kg to diabetic group significantly reversed the above biochemical changes. The glibenclimide (3 mg/kg) group also showed significant changes in blood glucose and plasma insulin levels when compared to diabetic group.

Table 3

Effect of WHW extract on serum glucose, insulin, triglycerides, BUN, creatinine levels in normal and diabetic rats

A significant (P < 0.001) increase in level of serum triglycerides and renal functional markers such as BUN and creatinine was observed in STZ-induced diabetic group when compared with control group (Table 3). This increase was significantly (P < 0.01 and P < 0.001) decreased in group treated with WHW extract and glibenclimide as compared to the diabetic group.

Histopathological Changes of the Pancreas and Kidney

In the diabetic group, a decrease in pancreatic islet numbers and size, atrophy and vacuolation, and invasion of connective tissues in the parenchyma of pancreatic islets was detected, but these abnormal histological signs dramatically decreased in the group treated with WHW extract as compared to the diabetic group [Figure 1]. Significant but lesser effects than WHW extract supplemented group in histopathological changes were observed in the glibenclamide treated group.

Figure 1

Effect of WHW extract on histological changes of pancreatic islets in STZ-induced diabetic rats. H&E staining of pancreas was performed following 4 weeks of administration. Group I (control; A), group II (diabetic; B), group III (diabetic + WHW extract; C), and group IV (diabetic + glibenclamide; D) rats, original magnification ×200

In kidney, compared to control group, modest glomerular lesions were noted in diabetic group. Glomerular capillaries were irregular, widened, and attached to the Bowman's capsule [Figure 2]. Furthermore, mesangial cell number was slightly higher in diabetic group. The degree of tubulointerstitial damage was modest. There were only few widened tubuli with incipient atrophy of the epithelial cells. In addition, slight focal interstitial fibrosis was observed. Intrarenal arterial vessel showed modest thickening of the walls. The above changes were decreased significantly in WHW extract treated group and moderately in glibenclamide treated group.

Figure 2

Effect of WHW extract on histological changes of kidneys in STZ-induced diabetic rats. H&E staining of kidneys was performed following 4 weeks of administration. Group I (control; A), group II (diabetic; B), group III (diabetic + WHW extract; C), and group IV (diabetic + glibenclamide; D) rats, original magnification ×200.

Changes in Insulin Content of the Islets

Insulin content of the β cells was evaluated by immunocytochemical intensity. In the diabetic group, the number of immunoreactive insulin-producing β cells was reduced and they were distributed in restricted pancreatic islets, but these abnormal changes were dramatically inhibited by WHW extract supplementation group [Figure 3]. However, there were slightly more insulin-producing β cells in the glibenclamide treated group than in the diabetic group.

Figure 3

Effect of WHW extract on insulin-producing beta cells in pancreatic islets. The pancreas was stained with anti-insulin antibody. Group I (control; A), group II (diabetic; B), group III (diabetic + WHW extract; C), and group IV (diabetic + glibenclamide; D) rats, original magnification ×200

Discussion

STZ selectively destroys β cells, insulin-producing pancreatic endocrine cells, and thus induces experimental diabetes mellitus.[14] It has been proposed that streptozotocin (STZ) acts as a diabetogenic agent owing to its ability to destroy pancreatic beta-cells.[15] Type 1 diabetes mellitus (DM) is a chronic disease characterized by high glucose levels due to an absolute or relative deficiency of circulating insulin levels. Although various types of oral hypoglycemic agents are currently available along with insulin for treating diabetes mellitus, there is a growing interest in herbal remedies owing to the side effects associated with the existing therapeutic hypoglycemic agents.[5]

In the present study, the induction of diabetes by STZ was confirmed as reflected by the hyperglycemia (OGTT) and body weight loss compared to the control rats. Oral supplementation of WHW extract caused a rapid decrease in the hyperglycemic peak after glucose loading in rats. A dose of 100 mg/kg caused a significant decrease in the blood glucose 60 min after the glucose pulse. The above finding suggests that the WHW extract may have therapeutic potential in post-prandial hyperglycemia. The results led us to suppose that the effect of the WHW extract could take place through a sulfonylurea like mechanism, since it caused a decrease in blood glucose levels during the glucose tolerance test. The body weight usually decreases as diabetes progresses,[16] but WHW extract significantly inhibited the decrease in body weight four weeks from the start of supplementation. This effect is also an indication of the anti-diabetic activity of WHW extract.

In the present study, it was found that the oral supplementations of WHW extract decrease the increased blood glucose concentration to normal glycemic concentration. In addition, WHW extract supplementation significantly increased the plasma insulin levels as compared to the diabetic control. The impaired insulin secretion of beta cells results in abnormal glucose homeostasis, leading to type I diabetes due to selective and progressive destruction of pancreatic beta cells.[17] The beta cells are highly susceptible to cytotoxic agents, such as STZ.[18] The elevated circulating insulin levels in WHW extract supplementation may have been due to increased insulin synthesis and secretion from beta cells in the treatment than in the diabetic group. The possible mechanism of the WHW extract may be an induction of insulin secretion through interaction with sulfonylurea receptor in the plasma membrane of the pancreatic beta cells.[19] WHW extract may promote insulin secretion by closure of K+ ATP channels, membrane depolarization, and stimulation of Ca2+ influx, an initial key step in insulin secretion.[20]

The hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels. In the present study, the STZ-induced diabetic rats exhibited significantly higher serum triglycerides compared to the control rats. However, the WHW extract supplementation lowered the serum values to a normal range. Increased triglycerides may be due to increase in biosynthesis and/or diminished clearance from the blood. Hypertriglyceridemia is often seen in uncontrolled diabetes, as well as in isolated lipid disorders. Marked elevation of serum triglyderides are also associated with an increased risk of pancreatitis. The derangements in lipid metabolism in diabetes mellitus are often important determinants of course and status of the disease.[17] According to numerous experimental and clinical observations, lipids, particularly oxidized lipids, cause glomerular injury[1621] and serum triglycerides play a role in the development and progression of renal disease in type I diabetes mellitus.[222]

In the present study, the diabetic rats had increased levels of blood urea nitrogen and serum creatinine, which are considered as significant markers of renal insufficiency. Urea is the major nitrogen containing metabolic product of protein metabolism; creatinine is endogenously produced and released into body fluids and its clearance measured as an indicator of renal function.[2324] Treatment with WHW extract significantly decreased these parameters which could be due to decreased disturbances in protein and nucleic acid metabolism owing to better glycemic control. However, it is still unclear which component is involved in this protection.

STZ-induced diabetes is a useful experimental model to study the anti-diabetic activity of several agents. In the present study, the pancreatic islet was apparently observed to be irregular and reduced in its shape. Atrophy and vacuolation and invasion of connective tissues in the parenchyma of pancreatic islets were detected when compared to the control rats. In contrast, these changes are dramatically inhibited by WHW extract supplementation. Therefore, these findings imply that WHW extract exhibited a protective effect against the diabetogenic activity of STZ that was seemingly related to an enhanced insulin secretion. However, further quantitation is needed to confirm the changes in increased beta cell staining.

In kidney of diabetic rats, modest glomerular lesions were noted with irregular glomerular capillaries, widened and attached to the Bowman's capsule.[25] Furthermore, mesangial cell number was slightly higher in STZ. The degree of tubulointerstitial damage was modest. There were only few widened tubuli with incipient atrophy of the epithelial cells. In addition, slight focal interstitial fibrosis was observed. Intrarenal arterial vessel showed modest thickening of the walls. The above changes were decreased significantly in WHW extract supplementation group.

The anti-diabetic activity of WHW extract has been demonstrated from the immunohistochemical changes in insulin-producing beta cells. Insulin–producing beta cells are generally located in the central regions of the pancreatic islets. However, the insulin-producing cells are destroyed in STZ-induced diabetes. Inhibition of histomorphometrical changes in insulin-producing cells by WHW extract are considered to be direct evidence that WHW extract protects from the destruction of these cells by STZ. This also corresponds to the changes in plasma insulin levels.

In conclusion, our results show that supplementation with WHW extract has favorable effects in preventing changes in blood glucose levels, body weight, lipids, and renal markers. In addition, WHW extract supplementation increased the number of insulin-producing beta cells. We speculate that such an anti-diabetogenic effect is achieved by the combined actions of components of WHW extract. Moreover, these results suggest that WHW extract might have a therapeutic potential in post-prandial hyperglycemia and could ameliorate the impaired renal function associated with diabetes and diabetic nephropathy.