International Journal of Biological Macromolecules 65 (2014) 436–440
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Protective effects of polysaccharides from Lilium lancifolium on streptozotocin-induced diabetic mice Ting Zhang a , Jie Gao a , Zheng-Yu Jin b , Xue-Ming Xu b , Han-Qing Chen a,∗ a
School of Biotechnology and Food Engineering, Hefei University of Technology, 193 Tunxi Road, Hefei, Anhui 230009, PR China State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China b
a r t i c l e
i n f o
Article history: Received 5 January 2014 Received in revised form 24 January 2014 Accepted 30 January 2014 Available online 6 February 2014 Keywords: Lilium lancifolium Polysaccharide Diabetes mellitus
a b s t r a c t In this study, the protective effect of Lilium lancifolium polysaccharides (LLP) on streptozotocin (STZ)induced diabetic mice and possible mechanism were investigated. The diabetic mice were administered with LLP for 28 days. The results showed that oral administration of LLP could significantly decrease blood glucose level and increase body weight loss in STZ-induced diabetic mice. LLP also significantly increased the activities of antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) and decreased the level of malondialdehyde (MDA) in serum, liver, and kidney in STZ-induced diabetic mice. Moreover, histopathological examination showed that LLP could markedly improve the structure integrity of pancreatic islet tissue in STZ-induced diabetic mice. However, LLP had no significant effect on organ weight of liver and pancreas of diabetic mice, but significantly decreased kidney weight compared with diabetic control mice. This study suggested that LLP had hypoglycemic and antioxidant properties and could provide protective effect on STZ-induced diabetic mice. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Diabetes mellitus (DM) is a chronic metabolic disorder characterized by hyperglycemia resulting from defective insulin secretion, resistance to insulin action or both. Clinically, DM can be classified to type 1 and type 2. Type 1 diabetes is typically caused by an autoimmune assault against the -cells, inducing progressive -cell death [1]; in type 2 diabetes insulin resistance and -cell dysfunction are the two major pathophysiological changes. At present, DM can be treated by exercise, diet and pharmaceutical therapy. However, because many current oral hypoglycemic agents are synthetic drugs with certain adverse side effects, therefore, interests in alternative medicines and natural therapies have become very popular. The plant kingdom has become the focus of the search for new drugs and biologically active compounds. In recent years, more and more studies suggested that some botanical polysaccharides isolated from Ophiopogon japonicus [2,3], Acanthopanax senticosus [4], Lycium barbarum [5,6], Astragalus membranaceus [7], and Opuntia dillenii [8] exhibited hypoglycemic activity.
∗ Corresponding author. Tel.: +86 551 62901516; fax: +86 551 62901516. E-mail addresses: [email protected], [email protected] (H.-Q. Chen). 0141-8130/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2014.01.063
Lilium lancifolium Thunb. is a well-known edible and medicinal plant, which belongs to the genus Lilium of the family Liliaceae. It is widely distributed in China and cultivated in Taihu Lake Basin. The bulb of L. lancifolium contains not only high content of starch but also a considerable amount of proteins. Moreover, the bulb contains a variety of bioactive ingredients, such as polysaccharide, saponin, and colchicine. Recent studies reported chemical characterization and antioxidant activities of polysaccharides from Lilium davidii [9,10]. However, few studies reported the hypoglycemic activity of polysaccharides from the bulb of L. lancifolium and the possible mechanisms. In the present study, we investigated the possible protective effect of polysaccharides from the bulb of L. lancifolium (LLP) on streptozotocin-induced diabetic mice.
2. Materials and methods 2.1. Material and chemicals Fresh bulbs of L. lancifolium Thunb. were obtained from Yixing City, Jiangsu Province, China. Accu-Chek® Active Blood Glucose Meter was purchased from Roche Diagnostics GmbH, Mannheim, Germany. Streptozotocin (STZ) was purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA), and glibenclamide tablet was purchased from Tianjin Pacific Pharmacy Co., Ltd. (Tianjin,
T. Zhang et al. / International Journal of Biological Macromolecules 65 (2014) 436–440
China). The assay kits for superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). All other chemicals used were of analytical grade. 2.2. Preparation of L. lancifolium polysaccharide (LLP) 10 kg fresh bulbs of L. lancifolium were dried and ground to powder, and extracted with 95% ethanol at 40 ◦ C for 2 h to remove pigments and lipids. The precipitate was extracted with distilled water at 70 ◦ C for three times and 3 h each time. After centrifugation at 3500 rpm for 20 min, the supernatant was collected and concentrated to one tenth of the original volume under reduced pressure with a rotary evaporator and then mixed with 4 volumes of 95% ethanol at 4 ◦ C overnight. The precipitate was dissolved in distilled water and macroporous resin of NKA-9 was added into the solution, shaking in bath shaker for 4 h to decolorize. The supernatant was collected and removed protein by the Sevag method [11], then dialyzed against tap water for 48 h and distilled water for 24 h. The resultant was concentrated and lyophilized. The powder obtained was the L. lancifolium polysaccharide (LLP). 2.3. Animals Male Kunming mice (5-week old, body weight 30 ± 2 g) were purchased from the Experimental Animal Center of Anhui Medical University, Hefei, China. During the experiment the mice were housed in polypropylene cages under controlled environmental conditions (temperature of 23 ± 2 ◦ C and relative humidity of 55 ± 5%) and a 12 h light/dark cycle. The animals were fed with a commercial pellet diet and water ad libitum. Before starting the experiment, the mice were acclimatized for one week. During the whole experiment, all animals were treated according to the guidelines of the National Institute of Health for the care and use of laboratory animals and their experimental use was approved by the animal Ethics Committee of Hefei University of Technology. 2.4. Establishment of the diabetic mice model Diabetic mice were induced by the intraperitoneal injection of STZ dissolved in freshly prepared 0.1 M sodium citrate buffer solution (pH 4.5) at a dose of 140 mg/kg body weight. After 72 h of STZ injection, development of diabetes was confirmed by measuring fasting glucose level of blood from the tail vein. The mice with blood glucose levels higher than 16.8 mmol/L were considered diabetic and were selected for further experiments. 2.5. Experimental design Diabetic mice were randomly divided into five groups (10 mice per group), and 10 normal mice were used as the normal control. Group 1: Normal control (NC), the normal mice were treated with distilled water for 28 days. Group 2: Diabetic control (DC), the diabetic mice were treated with distilled water for 28 days. Group 3: Positive control (PC), the diabetic mice were treated with glibenclamide (10 mg/kg/day) for 28 days. Group 4: LLP-L, the diabetic mice were treated with LLP (50 mg/kg/day) for 28 days. Group 5: LLP-M, the diabetic mice were treated with LLP (100 mg/kg/day) for 28 days.
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Group 6: LLP-H, the diabetic mice were treated with LLP (200 mg/kg/day) for 28 days. All groups were intragastrically administered once every day. The body weights were determined once every week during the experiment. The blood glucose levels were determined on the 14th day and the 28th day, respectively. On the last day of the experiment, the mice were fasted overnight. Blood was collected in sterilized polystyrene tubes without the anticoagulant from the eye, then the mice were sacrificed by cervical dislocation. The blood was centrifuged at 3000 rpm for 8 min to obtain serum. Samples were stored at −70 ◦ C until assayed. The pancreas, liver, kidney and spleen were excised quickly, blotted dry and weighed. The pancreas was used for the pathological histology by HE stain. The liver and kidney tissues were homogenized in nine volumes of ice-cold 0.9% saline solution by using a motor-driven Teflon glass homogenizer to yield a 10% (w/v) homogenate. The homogenate was then centrifuged at 3500 rpm and 4 ◦ C for 10 min. The supernatant obtained was used for assays of SOD, GPx, CAT, and MDA. 2.6. Analytical methods Blood samples were collected from the tail vein of the mice and the blood glucose level was determined by blood glucose meter (Accu-Chek® Active, Roche Diagnostics GmbH, Hannheim, Germany). The antioxidant enzyme activities were determined by commercially available kits purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). The determination of total SOD activity was based on the production of O2 •− anions by the xanthine/xanthine oxidase system [12]. One unit of SOD was defined as the amount of enzyme required to inhibit 50% the rate of reduction under the specific conditions. The activity of CAT was estimated by measuring the interaction product of H2 O2 and ammonium molybdate at 405 nm [13]. One unit of catalase was defined as the amount of enzyme required to breakdown 1 mol H2 O2 /second under specific conditions. The activity of GPx was measured according to the following principle. GPx catalyzed the reduced glutathione to oxidized glutathione by H2 O2 -induced oxidation [14]. One unit of GPx activity was defined as the amount of the enzyme which caused a decrease in reduced glutathione concentration of 1 mol/L/min under specific conditions. Lipid peroxidation was determined by measuring MDA concentration. The level of MDA was measured according to the principle described by Niehaus and Samuelsson [15]. Under the conditions of high temperature and acidic environment, MDA reacts with 2thiobarbituric acid (TBA) and the absorbance of colored product is tested at 532 nm. The determination of MDA was performed according to the instructions of MDA assay kit purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). 2.7. Histopathological examination of pancreas The pancreas tissues were cleaned and fixed in 10% neutral formalin solution, embedded in paraffin, serially sectioned, stained with hematoxylin and eosin (H.E.). Samples were observed at 400× magnification by optical microscope. 2.8. Statistical analysis Results were expressed as mean ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple-range test using the SPSS 17.0 statistical
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Table 1 Effect of LLP on body weight of normal and STZ-induced diabetic mice. Groups
−7
NC DC PC LLP-L LLP-M LLP-H
30.38 30.24 29.93 29.94 30.43 29.69
0 ± ± ± ± ± ±
0.96 1.22 1.38 1.03 1.19 1.23
35.43 26.56 26.24 26.39 27.91 25.75
7 ± ± ± ± ± ±
3.93 3.46## 3.43## 4.11## 2.70## 2.32##
14
39.38 30.45 29.80 31.79 32.87 33.35
± ± ± ± ± ±
3.53 2.79## 3.44## 3.38## 2.48## 2.04#
42.52 27.67 30.34 32.52 32.5 33.72
21 ± ± ± ± ± ±
3.75 3.06## 2.47## 4.61## 2.90## 2.51# , *
28
45.18 27.14 30.61 32.87 31.34 37.81
± ± ± ± ± ±
4.71 3.21## 3.00## 3.46## 3.92## 3.86**
46.93 28.74 28.88 33.28 30.61 42.14
± ± ± ± ± ±
3.05 3.80## 5.03## 3.93## 3.94## 5.56## , **
All values represent the mean ± SD (n = 10). # Significantly different vs. normal control at p < 0.05. ## Significantly different vs. normal control at p < 0.01. * Significantly different vs. diabetic control at p < 0.05. ** Significantly different vs. diabetic control at p < 0.01. Table 2 Effect of LLP on the organ weight of normal and STZ-induced diabetic mice. Group
Spleen (g/100 g BW)
NC DC PC LLP-L LLP-M LLP-H
0.33 0.16 0.31 0.25 0.22 0.26
± ± ± ± ± ±
0.07 0.04## 0.05** 0.09 0.05 0.08
Kidney (g/100 g BW) 0.71 0.86 0.68 0.77 0.76 0.69
± ± ± ± ± ±
Liver (g/100 g BW)
0.04 0.04## 0.12 0.15 0.08 0.07**
4.85 5.44 4.94 4.97 5.12 5.00
± ± ± ± ± ±
Pancreas (g/100 g BW)
0.46 0.73 0.77 0.66 0.61 0.91
0.57 0.38 0.47 0.41 0.39 0.46
± ± ± ± ± ±
0.10 0.09 0.05 0.05 0.08 0.10
All values represent the mean ± SD (n = 10). # Significantly different vs. normal control at p < 0.05. ## Significantly different vs. normal control at p < 0.01. * Significantly different vs. diabetic control at p < 0.05. ** Significantly different vs. diabetic control at p < 0.01.
software program. Differences were considered significant at p < 0.05. 3. Results 3.1. Effect of LLP on body weight and organ weight of STZ-induced diabetic mice As shown in Table 1, there is no significant difference in initial body weight of mice among groups. The body weight of mice in NC group increased regularly during the experiment. Compared to normal control mice, DC group mice exhibited a significant loss in body weight (p < 0.01). At the end of the experiment, the body weight of the mice in PC, LLP-L and LLP-M groups have no significant increase compared with DC group. However, LLP-H group mice that administered by LLP of 200 mg/kg/day showed a significant increase in body weight compared with DC group (p < 0.01). The organ weights of all groups mice were shown in Table 2. No significant differences were found in the weight of the liver and pancreas among all groups. Compared with the NC group, the spleen weight of DC group was significantly decreased, while the kidney weight was increased significantly. At the end of the experiment, the kidney weight of LLP-H group was decreased significantly as compared to the DC group (p < 0.01). No significant differences were observed in kidney weight among other groups. 3.2. Effect of LLP on blood glucose (BG) level of STZ-induced diabetic mice The effect of LLP on blood glucose level of STZ-induced diabetic mice was presented in Table 3. At the beginning, the BG level of STZ-induced diabetic mice was significantly increased compared with NC group (p < 0.01). By the end of the experiment, the BG level of DC group was significantly higher than that of NC group. However, after two weeks of LLP treatment, the BG levels of mice in PC and LLP groups were decreased significantly. Compared with DC group, the BG levels of mice in LLP-L, LLP-M and LLP-H groups were reduced by 32.64% (p < 0.01), 28.28% (p < 0.01) and 45.05% (p < 0.01),
Table 3 Effect of LLP on blood glucose level of normal and STZ-induced diabetic mice. Group
0
NC DC PC LLP-L LLP-M LLP-H
5.29 26.24 24.31 25.61 24.14 23.84
14 d ± ± ± ± ± ±
0.35 2.53## 3.78## 3.86## 4.32## 2.46##
5.40 28.68 22.55 19.32 20.57 15.76
28 d ± ± ± ± ± ±
0.52 2.57## 3.06## , * 1.84## , ** 2.36## , ** 2.76## , **
5.11 28.26 19.87 17.20 18.33 10.59
± ± ± ± ± ±
0.59 2.71## 1.88## , ** 3.82## , ** 2.75## , ** 2.59# , **
All values represent the mean ± SD (n = 10). # Significantly different vs. normal control at p < 0.05. ## Significantly different vs. normal control at p < 0.01. * Significantly different vs. diabetic control at p < 0.05. ** Significantly different vs. diabetic control at p < 0.01.
respectively, at 14 days and by 39.14% (p < 0.01), 35.14% (p < 0.01) and 62.53% (p < 0.01), respectively, at 28 days. 3.3. Effects of LLP on the activities of antioxidant enzymes and the level of MDA in serum, liver and kidney of diabetic mice The effects of LLP on the activities of antioxidant enzymes and the level of MDA in serum, liver and kidney of diabetic mice were shown in Table 4. Compared with the NC mice, the activities of SOD, CAT, GPx in serum, liver and kidney of DC mice were significantly decreased (p < 0.01). However, oral administration of LLP (200 mg/kg/day) or glibenclamide (10 mg/kg/day) for 28 days significantly increased (p < 0.05 or p < 0.01) the activities of SOD, CAT, GPx in serum, liver and kidney of diabetic mice compared with the diabetic control mice. We also determined the level of MDA, the product of lipid peroxidation, in serum, liver, and kidney of the mice. As shown in Table 4, the level of MDA in serum, liver, and kidney of the diabetic control mice was significantly increased (p < 0.01) compared with the normal control mice. But after 28 days of LLP (200 mg/kg/day) or glibenclamide (10 mg/kg/day) treatment, compared with the diabetic control mice, the level of MDA in serum, liver and kidney of diabetic mice were reduced by 47.69% (p < 0.05), 63.44% (p < 0.01),
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Table 4 Effects of LLP on the activities of antioxidant enzymes and the level of MDA in serum, liver and kidney of normal and STZ-induced diabetic mice. Group
NC
Serum SOD (U/mL) CAT (U/mL) GPx (U/mL) MDA (nmol/mL)
DC
PC
LLP-L
LLP-M
LLP-H
175.29 16.38 425.18 4.28
± ± ± ±
7.10 1.13 21.89 1.41
133.07 5.36 132.81 19.52
± ± ± ±
10.77## 0.61## 10.96## 4.36##
167.40 9.77 214.06 9.14
± ± ± ±
6.67** 1.38## , ** 27.44## , ** 1.07## , **
150.70 9.19 196.88 14.20
± ± ± ±
7.11## 1.14## , ** 13.26## , ** 3.64##
153.64 11.27 215.62 13.42
± ± ± ±
8.96## , * 1.28## , ** 29.04## , ** 2.71##
155.24 11.66 273.44 10.21
± ± ± ±
8.57## , * 1.65## , ** 25.44## , ** 2.07## , *
Liver SOD (U/mg protein) CAT (U/mg protein) GPx (U/mg protein) MDA (nmol/mg protein)
411.25 162.60 552.45 4.26
± ± ± ±
31.93 22.22 24.6 1.38
218.36 71.72 406.63 24.51
± ± ± ±
29.42## 16.48## 31.75## 5.97##
385.03 125.38 509.34 8.11
± ± ± ±
29.42** 26.36* 30.92** 2.61**
246.49 116.78 460.52 16.13
± ± ± ±
40.64## 14.71# , ** 32.41## 4.37##
217.19 114.52 503.81 13.54
± ± ± ±
31.73## 8.61# , ** 13.17# , ** 1.58## , *
293.41 125.83 505.30 8.96
± ± ± ±
35.40## , * 18.67** 25.22** 2.45# , **
Kidney SOD (U/mg protein) CAT (U/mg protein) GPx (U/mg protein) MDA (nmol/mg protein)
166.05 123.52 432.41 2.72
± ± ± ±
8.17 11.39 17.61 0.19
121.55 76.03 254.93 7.10
± ± ± ±
6.75## 12.03## 25.44## 0.60##
152.45 99.04 406.49 2.95
± ± ± ±
10.12** 12.28#* 33.30** 0.12**
140.16 99.52 346.93 5.60
± ± ± ±
8.89## , * 7.56# , * 23.46## , ** 0.86##*
134.34 92.78 383.33 5.48
± ± ± ±
12.82## 14.64# 27.40# , ** 0.59##**
156.00 110.01 402.62 4.36
± ± ± ±
10.39** 9.58** 19.14** 0.64## , **
All values represent the mean ± SD (n = 10). # Significantly different vs. normal control at p < 0.05. ## Significantly different vs. normal control at p < 0.01. * Significantly different vs. diabetic control at p < 0.05. ** Significantly different vs. diabetic control at p < 0.01.
and 38.59% (p < 0.01), respectively, and by 53.18% (p < 0.01), 66.91% (p < 0.01), and 58.45% (p < 0.01), respectively. 3.4. Effect of LLP on the change in histopathology of pancreas of STZ-induced diabetic mice The effects of LLP and Glibenclamide on pancreatic tissues with HE staining were showed in Fig. 1. As revealed in Fig. 1, a clear decrease in the area occupied by the beta-cells was observed in the pancreatic section of STZ-induced diabetic mice. Moreover, the number and the size of pancreatic islets were significantly decreased in diabetic mice compared with the normal control mice. However, treatment with 200 mg/kg/day LLP or 10 mg/kg/day Glibenclamide for 28 days markedly repaired the islet damage, and improved the structure integrity of pancreatic islet beta-cells and tissues. 4. Discussion Diabetes mellitus (DM) is one of the most common human metabolic diseases. It is characterized by hyperglycemia due to
defects in insulin secretion and/or activation, resulting in abnormalities in carbohydrate, lipid and protein metabolism. The precise cellular and molecular mechanism underlying the etiology and progression of diabetes is still not fully understood. However, increasing evidence suggests that oxidative stress plays a crucial role in the pathogenesis and progression of diabetes and its complications [16–22]. Hyperglycemia-induced auto-oxidation of glucose and glycation of proteins result in the formation of reactive oxygen and nitrogen species. Uncontrolled reactive oxygen species (ROS) production often leads to damage in cellular macromolecules (DNA, lipids and proteins), contributing to the progress of diabetic complications and different organ damage [23]. STZ, an antibiotic produced by Streptomyces achromogenes, is frequently used to induce DM in experimental animals through its toxic effects on pancreatic -cells. The cytotoxic action of STZ is associated with the generation of ROS causing oxidative damage [24]. In this study, we observed a significant increase in the concentration of blood glucose and a decrease in body weight in STZ-induced diabetic mice. However, treatment with LLP for 28 days not only significantly decreased the level of blood glucose, but
Fig. 1. Effects of LLP on the changes in the histopathology of the pancreas tissues in STZ-induced diabetic mice with H & E staining (magnification 400×). (A) Normal control group; (B) diabetic control group; (C) diabetic mice were administered by glibenclamide (10 mg/kg/day); (D) diabetic mice were administered by LLP (50 mg/kg/day); (E) diabetic mice were administered by LLP (100 mg/kg/day); (F) diabetic mice were administered by LLP (200 mg/kg/day).
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also improved the body weight loss of diabetic mice, which were consistent with previous studies on polysaccharides from other plants [2,5,8,25–28]. As the most predominant characteristic of diabetes mellitus, hyperglycemia increases oxidative stress through the overproduction of ROS, which results in an imbalance between free radicals and the antioxidant defense systems of the cells. The administration of LLP decreases blood glucose concentration in diabetic mice suggesting the blood glucose-controlling ability of LLP and its role as an essential trigger for the liver, kidney and pancreas to revert to their normal antioxidative abilities. Lipid peroxidation is considered to be a hallmark of oxidative stress, in which ROS interact with polyunsaturated fatty acids, and leads to the formation of lipid products such as MDA, which then causing damages to the membrane components of the cell, cell necrosis and inflammation [29]. Extensive evidence has demonstrated that the increased lipid peroxidation plays an important role in the progression of diabetes by altering the transbilayer fluidity gradient, which could hamper the activities of membrane-bound enzymes and receptors [16,30]. In this study, there was a significant increase in lipid peroxidation in serum, liver and kidney of diabetic mice, as measured by MDA formation. However, after administration of LLP for 28 days, we observed that the level of MDA in serum, liver and kidney of diabetic mice were significantly decreased. These results are in agreement with previous studies on polysaccharides from other sources [2,5,8,27,31,32]. Taken together these findings suggest that LLP can provide protective effect on the tissues of diabetic mice against oxidative damage by decreasing lipid peroxidation. Antioxidative enzymes, including SOD, GPx and CAT, are regarded as the first line of the antioxidant defense system against ROS generated in vivo during oxidative stress. Their ability to decompose superoxide and peroxide while blocking lipid peroxidation as well as their involvement in cellular defense mechanisms helps to protect tissues against oxidative damage. SOD scavenges the superoxide radical by converting it to hydrogen peroxide and molecular oxygen. CAT is a hemeprotein, which catalyzes the reduction of hydrogen peroxide and protects the tissues from highly reactive hydroxyl radicals. GPx, an enzyme with selenium, works together with glutathione (GSH) in the decomposition of hydrogen peroxide (or) other organic hydroperoxides to non-toxic products at the expense of reduced glutathione. In this study, significant decrease in the activities of SOD, CAT and GPx were observed in serum, liver and kidney of STZ-induced diabetic mice. The reduced activities of these antioxidant enzymes may result in a number of deleterious effects due to the accumulation of superoxide radicals and hydrogen peroxides. In this context, other researchers also reported a decrease in the activities of these antioxidant enzymes (SOD, CAT and GPx) in the serum, liver and kidney of diabetic mice or rats [2,5,8,27,28,31,32]. However, in the present study, treatment with LLP for 28 days significantly increased the activities of SOD, CAT and GPx in the serum, liver and kidney of STZ-induced diabetic mice. The result of increased activities of SOD, CAT and GPx suggest that LLP has a free radical scavenging activity, which could exert a beneficial effect against pathological alterations caused by ROS. Previous studies have also demonstrated that the polysaccharides from other sources can ameliorate antioxidant enzymes activities in blood and tissues of diabetic mice or rats [2,5,8,27,28,31–33]. Moreover, recent study reported that the hypoglycemic mechanisms of polysaccharides were closely associated with their antioxidant activities [32]. Based on these findings, our study revealed that one mechanism of the anti-hyperglycemic actions of LLP may be closely related with its antioxidant activity and free radical scavenging ability, hence provide protective effect against oxidative damage.
In this study, histopathological examination showed that the pancreatic structure was destroyed by STZ, the distortion of the pancreatic islets of Langerhans were observed in the diabetic mice together with atrophy (reduction of cell size and number). However, after administration of LLP, the pancreatic damage was markedly repaired and the integrity of pancreatic islet cells and tissues were improved. This further confirmed that LLP could exert protective effect on the pancreatic tissue of diabetic mice against oxidative damage. In summary, the present study suggests that LLP exhibits hypoglycemic and protective effects in STZ-induced diabetic mice by reducing oxidative stress and preserving the structural integrity of pancreatic islet. The protective effect of LLP may be attributed to its antioxidant activity. However, further investigation is necessary to delineate the exact molecular mechanism underlying the protective effect of LLP on STZ-induced diabetic mice. Acknowledgements This study was supported by the Key Program of National Natural Science Foundation of China (No. 31230057), and the National Key Technology Research and Development Program for the 12th Five-Year Plan (Nos. 2012BAD37B02 and 2012BAD37B01). References [1] M. Cnop, N. Welsh, J.-C. Jonas, A. Jörns, S. Lenzen, D.L. Eizirik, Diabetes 54 (Suppl. 2) (2005) S97–S107. [2] X.M. Chen, J. Tang, W.Y. Xie, J.J. Wang, J. Jin, J. Ren, L.Q. Jin, J.X. Lu, Carbohydr. Polym. 94 (2013) 378–385. [3] J. Xu, Y. Wang, D.-S. Xu, K.-F. Ruan, Y. Feng, S. Wang, Int. J. Biol. Macromol. 49 (2011) 657–662. [4] J.F. Fu, J.F. Fu, Y. Liu, R.Y. Li, B. Gao, N.Y. Zhang, B.L. Wang, Y.Z. Cao, K.S. Guo, Y.Y. Tu, Carbohydr. Polym. 87 (2012) 2327–2331. [5] X.M. Li, Int. J. Biol. Macromol. 40 (2007) 461–465. [6] S. Zou, X. Zhang, W.B. Yao, Y.G. Niu, X.D. Gao, Carbohydr. Polym. 80 (2010) 1161–1167. [7] X.Q. Mao, F. Yu, N. Wang, Y. Wu, F. Zou, K. Wu, M. Liu, J.P. Ouyang, Phytomedicine 16 (2009) 416–425. [8] L.Y. Zhao, Q.J. Lan, Z.C. Huang, L.J. Ouyang, F.H. Zeng, Phytomedicine 18 (2011) 661–668. [9] J. Zhang, Y.X. Gao, X.J. Zhou, L.P. Hu, T.Z. Xie, Nat. Prod. Res. 24 (2010) 357–369. [10] B.T. Zhao, J. Zhang, X. Guo, J.L. Wang, Food Hydrocoll. 31 (2013) 346–356. [11] A.M. Staub, Methods Carbohydr. Chem. 5 (1965) 5–6. [12] Y. Kono, Arch. Biochem. Biophys. 186 (1978) 189–195. [13] R.F. Beers, I.W. Sizer, J. Biol. Chem. 195 (1952) 133–140. [14] D.E. Paglia, W.N. Valentine, J. Lab. Clin. Med. 70 (1967) 158–169. [15] W.G. Niehaus Jr., B. Samuelsson, Eur. J. Biochem. 6 (1968) 126–130. [16] F. Giacco, M. Brownlee, Circ. Res. 107 (2010) 1058–1070. [17] H. Kaneto, N. Katakami, D. Kawamori, T. Miyatsuka, K. Sakamoto, T. Matsuoka, M. Matsuhisa, Y. Yamasaki, Antioxid. Redox Signal. 9 (2007) 355–366. [18] A.C. Maritim, R.A. Sanders, J.B. Watkins, J. Biochem. Mol. Toxicol. 17 (2003) 24–38. [19] C.K. Roberts, K.K. Sindhu, Life Sci. 84 (2009) 705–712. [20] R.P. Robertson, Curr. Opin. Pharmacol. 6 (2006) 615–619. [21] A.P. Rolo, C.M. Palmeira, Toxicol. Appl. Pharmacol. 212 (2006) 167–178. [22] R.A. Simmons, Free Radic. Biol. Med. 40 (2006) 917–922. [23] J.W. Baynes, Diabetes 40 (1991) 405–412. [24] T. Szkudelski, Physiol. Res. 50 (2001) 537–546. [25] N. Yang, M. Zhao, B. Zhu, B. Yang, C. Chen, C. Cui, Y. Jiang, Innov. Food Sci. Emerg. 9 (2008) 570–574. [26] X.M. Chen, J. Jin, J. Tang, Z.F. Wang, J.J. Wang, L.Q. Jin, J.X. Lu, Carbohydr. Polym. 83 (2011) 749–754. [27] S.X. Xue, X.M. Chen, J.X. Lu, L.Q. Jin, Carbohydr. Polym. 75 (2009) 415–419. [28] D. Zhang, H. Meng, H.S. Yang, Int. J. Biol. Macromol. 50 (2012) 720–724. [29] G. Stark, J. Membr. Biol. 205 (2005) 1–16. [30] L.F. Dmitriev, V.N. Titov, Ageing Res. Rev. 9 (2010) 200–210. [31] W. Zhang, L.J. Zheng, Z.M. Zhang, C.-X. Hai, Carbohydr. Polym. 89 (2012) 890–898. [32] Y.T. Liu, J. Sun, S.Q. Rao, Y.J. Su, Y.J. Yang, Food Chem. Toxicol. 57 (2013) 39–45. [33] J. Jia, X. Zhang, Y.-S. Hu, Y. Wu, Q.-Z. Wang, N.-N. Li, Q.-C. Guo, X.-C. Dong, Food Chem. 115 (2009) 32–36.
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Protective effects of polysaccharides from Lilium lancifolium on streptozotocin-induced diabetic mice Ting Zhang a , Jie Gao a , Zheng-Yu Jin b , Xue-Ming Xu b , Han-Qing Chen a,∗ a
School of Biotechnology and Food Engineering, Hefei University of Technology, 193 Tunxi Road, Hefei, Anhui 230009, PR China State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China b
a r t i c l e
i n f o
Article history: Received 5 January 2014 Received in revised form 24 January 2014 Accepted 30 January 2014 Available online 6 February 2014 Keywords: Lilium lancifolium Polysaccharide Diabetes mellitus
a b s t r a c t In this study, the protective effect of Lilium lancifolium polysaccharides (LLP) on streptozotocin (STZ)induced diabetic mice and possible mechanism were investigated. The diabetic mice were administered with LLP for 28 days. The results showed that oral administration of LLP could significantly decrease blood glucose level and increase body weight loss in STZ-induced diabetic mice. LLP also significantly increased the activities of antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) and decreased the level of malondialdehyde (MDA) in serum, liver, and kidney in STZ-induced diabetic mice. Moreover, histopathological examination showed that LLP could markedly improve the structure integrity of pancreatic islet tissue in STZ-induced diabetic mice. However, LLP had no significant effect on organ weight of liver and pancreas of diabetic mice, but significantly decreased kidney weight compared with diabetic control mice. This study suggested that LLP had hypoglycemic and antioxidant properties and could provide protective effect on STZ-induced diabetic mice. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Diabetes mellitus (DM) is a chronic metabolic disorder characterized by hyperglycemia resulting from defective insulin secretion, resistance to insulin action or both. Clinically, DM can be classified to type 1 and type 2. Type 1 diabetes is typically caused by an autoimmune assault against the -cells, inducing progressive -cell death [1]; in type 2 diabetes insulin resistance and -cell dysfunction are the two major pathophysiological changes. At present, DM can be treated by exercise, diet and pharmaceutical therapy. However, because many current oral hypoglycemic agents are synthetic drugs with certain adverse side effects, therefore, interests in alternative medicines and natural therapies have become very popular. The plant kingdom has become the focus of the search for new drugs and biologically active compounds. In recent years, more and more studies suggested that some botanical polysaccharides isolated from Ophiopogon japonicus [2,3], Acanthopanax senticosus [4], Lycium barbarum [5,6], Astragalus membranaceus [7], and Opuntia dillenii [8] exhibited hypoglycemic activity.
∗ Corresponding author. Tel.: +86 551 62901516; fax: +86 551 62901516. E-mail addresses: [email protected], [email protected] (H.-Q. Chen). 0141-8130/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2014.01.063
Lilium lancifolium Thunb. is a well-known edible and medicinal plant, which belongs to the genus Lilium of the family Liliaceae. It is widely distributed in China and cultivated in Taihu Lake Basin. The bulb of L. lancifolium contains not only high content of starch but also a considerable amount of proteins. Moreover, the bulb contains a variety of bioactive ingredients, such as polysaccharide, saponin, and colchicine. Recent studies reported chemical characterization and antioxidant activities of polysaccharides from Lilium davidii [9,10]. However, few studies reported the hypoglycemic activity of polysaccharides from the bulb of L. lancifolium and the possible mechanisms. In the present study, we investigated the possible protective effect of polysaccharides from the bulb of L. lancifolium (LLP) on streptozotocin-induced diabetic mice.
2. Materials and methods 2.1. Material and chemicals Fresh bulbs of L. lancifolium Thunb. were obtained from Yixing City, Jiangsu Province, China. Accu-Chek® Active Blood Glucose Meter was purchased from Roche Diagnostics GmbH, Mannheim, Germany. Streptozotocin (STZ) was purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA), and glibenclamide tablet was purchased from Tianjin Pacific Pharmacy Co., Ltd. (Tianjin,
T. Zhang et al. / International Journal of Biological Macromolecules 65 (2014) 436–440
China). The assay kits for superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). All other chemicals used were of analytical grade. 2.2. Preparation of L. lancifolium polysaccharide (LLP) 10 kg fresh bulbs of L. lancifolium were dried and ground to powder, and extracted with 95% ethanol at 40 ◦ C for 2 h to remove pigments and lipids. The precipitate was extracted with distilled water at 70 ◦ C for three times and 3 h each time. After centrifugation at 3500 rpm for 20 min, the supernatant was collected and concentrated to one tenth of the original volume under reduced pressure with a rotary evaporator and then mixed with 4 volumes of 95% ethanol at 4 ◦ C overnight. The precipitate was dissolved in distilled water and macroporous resin of NKA-9 was added into the solution, shaking in bath shaker for 4 h to decolorize. The supernatant was collected and removed protein by the Sevag method [11], then dialyzed against tap water for 48 h and distilled water for 24 h. The resultant was concentrated and lyophilized. The powder obtained was the L. lancifolium polysaccharide (LLP). 2.3. Animals Male Kunming mice (5-week old, body weight 30 ± 2 g) were purchased from the Experimental Animal Center of Anhui Medical University, Hefei, China. During the experiment the mice were housed in polypropylene cages under controlled environmental conditions (temperature of 23 ± 2 ◦ C and relative humidity of 55 ± 5%) and a 12 h light/dark cycle. The animals were fed with a commercial pellet diet and water ad libitum. Before starting the experiment, the mice were acclimatized for one week. During the whole experiment, all animals were treated according to the guidelines of the National Institute of Health for the care and use of laboratory animals and their experimental use was approved by the animal Ethics Committee of Hefei University of Technology. 2.4. Establishment of the diabetic mice model Diabetic mice were induced by the intraperitoneal injection of STZ dissolved in freshly prepared 0.1 M sodium citrate buffer solution (pH 4.5) at a dose of 140 mg/kg body weight. After 72 h of STZ injection, development of diabetes was confirmed by measuring fasting glucose level of blood from the tail vein. The mice with blood glucose levels higher than 16.8 mmol/L were considered diabetic and were selected for further experiments. 2.5. Experimental design Diabetic mice were randomly divided into five groups (10 mice per group), and 10 normal mice were used as the normal control. Group 1: Normal control (NC), the normal mice were treated with distilled water for 28 days. Group 2: Diabetic control (DC), the diabetic mice were treated with distilled water for 28 days. Group 3: Positive control (PC), the diabetic mice were treated with glibenclamide (10 mg/kg/day) for 28 days. Group 4: LLP-L, the diabetic mice were treated with LLP (50 mg/kg/day) for 28 days. Group 5: LLP-M, the diabetic mice were treated with LLP (100 mg/kg/day) for 28 days.
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Group 6: LLP-H, the diabetic mice were treated with LLP (200 mg/kg/day) for 28 days. All groups were intragastrically administered once every day. The body weights were determined once every week during the experiment. The blood glucose levels were determined on the 14th day and the 28th day, respectively. On the last day of the experiment, the mice were fasted overnight. Blood was collected in sterilized polystyrene tubes without the anticoagulant from the eye, then the mice were sacrificed by cervical dislocation. The blood was centrifuged at 3000 rpm for 8 min to obtain serum. Samples were stored at −70 ◦ C until assayed. The pancreas, liver, kidney and spleen were excised quickly, blotted dry and weighed. The pancreas was used for the pathological histology by HE stain. The liver and kidney tissues were homogenized in nine volumes of ice-cold 0.9% saline solution by using a motor-driven Teflon glass homogenizer to yield a 10% (w/v) homogenate. The homogenate was then centrifuged at 3500 rpm and 4 ◦ C for 10 min. The supernatant obtained was used for assays of SOD, GPx, CAT, and MDA. 2.6. Analytical methods Blood samples were collected from the tail vein of the mice and the blood glucose level was determined by blood glucose meter (Accu-Chek® Active, Roche Diagnostics GmbH, Hannheim, Germany). The antioxidant enzyme activities were determined by commercially available kits purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). The determination of total SOD activity was based on the production of O2 •− anions by the xanthine/xanthine oxidase system [12]. One unit of SOD was defined as the amount of enzyme required to inhibit 50% the rate of reduction under the specific conditions. The activity of CAT was estimated by measuring the interaction product of H2 O2 and ammonium molybdate at 405 nm [13]. One unit of catalase was defined as the amount of enzyme required to breakdown 1 mol H2 O2 /second under specific conditions. The activity of GPx was measured according to the following principle. GPx catalyzed the reduced glutathione to oxidized glutathione by H2 O2 -induced oxidation [14]. One unit of GPx activity was defined as the amount of the enzyme which caused a decrease in reduced glutathione concentration of 1 mol/L/min under specific conditions. Lipid peroxidation was determined by measuring MDA concentration. The level of MDA was measured according to the principle described by Niehaus and Samuelsson [15]. Under the conditions of high temperature and acidic environment, MDA reacts with 2thiobarbituric acid (TBA) and the absorbance of colored product is tested at 532 nm. The determination of MDA was performed according to the instructions of MDA assay kit purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). 2.7. Histopathological examination of pancreas The pancreas tissues were cleaned and fixed in 10% neutral formalin solution, embedded in paraffin, serially sectioned, stained with hematoxylin and eosin (H.E.). Samples were observed at 400× magnification by optical microscope. 2.8. Statistical analysis Results were expressed as mean ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple-range test using the SPSS 17.0 statistical
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Table 1 Effect of LLP on body weight of normal and STZ-induced diabetic mice. Groups
−7
NC DC PC LLP-L LLP-M LLP-H
30.38 30.24 29.93 29.94 30.43 29.69
0 ± ± ± ± ± ±
0.96 1.22 1.38 1.03 1.19 1.23
35.43 26.56 26.24 26.39 27.91 25.75
7 ± ± ± ± ± ±
3.93 3.46## 3.43## 4.11## 2.70## 2.32##
14
39.38 30.45 29.80 31.79 32.87 33.35
± ± ± ± ± ±
3.53 2.79## 3.44## 3.38## 2.48## 2.04#
42.52 27.67 30.34 32.52 32.5 33.72
21 ± ± ± ± ± ±
3.75 3.06## 2.47## 4.61## 2.90## 2.51# , *
28
45.18 27.14 30.61 32.87 31.34 37.81
± ± ± ± ± ±
4.71 3.21## 3.00## 3.46## 3.92## 3.86**
46.93 28.74 28.88 33.28 30.61 42.14
± ± ± ± ± ±
3.05 3.80## 5.03## 3.93## 3.94## 5.56## , **
All values represent the mean ± SD (n = 10). # Significantly different vs. normal control at p < 0.05. ## Significantly different vs. normal control at p < 0.01. * Significantly different vs. diabetic control at p < 0.05. ** Significantly different vs. diabetic control at p < 0.01. Table 2 Effect of LLP on the organ weight of normal and STZ-induced diabetic mice. Group
Spleen (g/100 g BW)
NC DC PC LLP-L LLP-M LLP-H
0.33 0.16 0.31 0.25 0.22 0.26
± ± ± ± ± ±
0.07 0.04## 0.05** 0.09 0.05 0.08
Kidney (g/100 g BW) 0.71 0.86 0.68 0.77 0.76 0.69
± ± ± ± ± ±
Liver (g/100 g BW)
0.04 0.04## 0.12 0.15 0.08 0.07**
4.85 5.44 4.94 4.97 5.12 5.00
± ± ± ± ± ±
Pancreas (g/100 g BW)
0.46 0.73 0.77 0.66 0.61 0.91
0.57 0.38 0.47 0.41 0.39 0.46
± ± ± ± ± ±
0.10 0.09 0.05 0.05 0.08 0.10
All values represent the mean ± SD (n = 10). # Significantly different vs. normal control at p < 0.05. ## Significantly different vs. normal control at p < 0.01. * Significantly different vs. diabetic control at p < 0.05. ** Significantly different vs. diabetic control at p < 0.01.
software program. Differences were considered significant at p < 0.05. 3. Results 3.1. Effect of LLP on body weight and organ weight of STZ-induced diabetic mice As shown in Table 1, there is no significant difference in initial body weight of mice among groups. The body weight of mice in NC group increased regularly during the experiment. Compared to normal control mice, DC group mice exhibited a significant loss in body weight (p < 0.01). At the end of the experiment, the body weight of the mice in PC, LLP-L and LLP-M groups have no significant increase compared with DC group. However, LLP-H group mice that administered by LLP of 200 mg/kg/day showed a significant increase in body weight compared with DC group (p < 0.01). The organ weights of all groups mice were shown in Table 2. No significant differences were found in the weight of the liver and pancreas among all groups. Compared with the NC group, the spleen weight of DC group was significantly decreased, while the kidney weight was increased significantly. At the end of the experiment, the kidney weight of LLP-H group was decreased significantly as compared to the DC group (p < 0.01). No significant differences were observed in kidney weight among other groups. 3.2. Effect of LLP on blood glucose (BG) level of STZ-induced diabetic mice The effect of LLP on blood glucose level of STZ-induced diabetic mice was presented in Table 3. At the beginning, the BG level of STZ-induced diabetic mice was significantly increased compared with NC group (p < 0.01). By the end of the experiment, the BG level of DC group was significantly higher than that of NC group. However, after two weeks of LLP treatment, the BG levels of mice in PC and LLP groups were decreased significantly. Compared with DC group, the BG levels of mice in LLP-L, LLP-M and LLP-H groups were reduced by 32.64% (p < 0.01), 28.28% (p < 0.01) and 45.05% (p < 0.01),
Table 3 Effect of LLP on blood glucose level of normal and STZ-induced diabetic mice. Group
0
NC DC PC LLP-L LLP-M LLP-H
5.29 26.24 24.31 25.61 24.14 23.84
14 d ± ± ± ± ± ±
0.35 2.53## 3.78## 3.86## 4.32## 2.46##
5.40 28.68 22.55 19.32 20.57 15.76
28 d ± ± ± ± ± ±
0.52 2.57## 3.06## , * 1.84## , ** 2.36## , ** 2.76## , **
5.11 28.26 19.87 17.20 18.33 10.59
± ± ± ± ± ±
0.59 2.71## 1.88## , ** 3.82## , ** 2.75## , ** 2.59# , **
All values represent the mean ± SD (n = 10). # Significantly different vs. normal control at p < 0.05. ## Significantly different vs. normal control at p < 0.01. * Significantly different vs. diabetic control at p < 0.05. ** Significantly different vs. diabetic control at p < 0.01.
respectively, at 14 days and by 39.14% (p < 0.01), 35.14% (p < 0.01) and 62.53% (p < 0.01), respectively, at 28 days. 3.3. Effects of LLP on the activities of antioxidant enzymes and the level of MDA in serum, liver and kidney of diabetic mice The effects of LLP on the activities of antioxidant enzymes and the level of MDA in serum, liver and kidney of diabetic mice were shown in Table 4. Compared with the NC mice, the activities of SOD, CAT, GPx in serum, liver and kidney of DC mice were significantly decreased (p < 0.01). However, oral administration of LLP (200 mg/kg/day) or glibenclamide (10 mg/kg/day) for 28 days significantly increased (p < 0.05 or p < 0.01) the activities of SOD, CAT, GPx in serum, liver and kidney of diabetic mice compared with the diabetic control mice. We also determined the level of MDA, the product of lipid peroxidation, in serum, liver, and kidney of the mice. As shown in Table 4, the level of MDA in serum, liver, and kidney of the diabetic control mice was significantly increased (p < 0.01) compared with the normal control mice. But after 28 days of LLP (200 mg/kg/day) or glibenclamide (10 mg/kg/day) treatment, compared with the diabetic control mice, the level of MDA in serum, liver and kidney of diabetic mice were reduced by 47.69% (p < 0.05), 63.44% (p < 0.01),
T. Zhang et al. / International Journal of Biological Macromolecules 65 (2014) 436–440
439
Table 4 Effects of LLP on the activities of antioxidant enzymes and the level of MDA in serum, liver and kidney of normal and STZ-induced diabetic mice. Group
NC
Serum SOD (U/mL) CAT (U/mL) GPx (U/mL) MDA (nmol/mL)
DC
PC
LLP-L
LLP-M
LLP-H
175.29 16.38 425.18 4.28
± ± ± ±
7.10 1.13 21.89 1.41
133.07 5.36 132.81 19.52
± ± ± ±
10.77## 0.61## 10.96## 4.36##
167.40 9.77 214.06 9.14
± ± ± ±
6.67** 1.38## , ** 27.44## , ** 1.07## , **
150.70 9.19 196.88 14.20
± ± ± ±
7.11## 1.14## , ** 13.26## , ** 3.64##
153.64 11.27 215.62 13.42
± ± ± ±
8.96## , * 1.28## , ** 29.04## , ** 2.71##
155.24 11.66 273.44 10.21
± ± ± ±
8.57## , * 1.65## , ** 25.44## , ** 2.07## , *
Liver SOD (U/mg protein) CAT (U/mg protein) GPx (U/mg protein) MDA (nmol/mg protein)
411.25 162.60 552.45 4.26
± ± ± ±
31.93 22.22 24.6 1.38
218.36 71.72 406.63 24.51
± ± ± ±
29.42## 16.48## 31.75## 5.97##
385.03 125.38 509.34 8.11
± ± ± ±
29.42** 26.36* 30.92** 2.61**
246.49 116.78 460.52 16.13
± ± ± ±
40.64## 14.71# , ** 32.41## 4.37##
217.19 114.52 503.81 13.54
± ± ± ±
31.73## 8.61# , ** 13.17# , ** 1.58## , *
293.41 125.83 505.30 8.96
± ± ± ±
35.40## , * 18.67** 25.22** 2.45# , **
Kidney SOD (U/mg protein) CAT (U/mg protein) GPx (U/mg protein) MDA (nmol/mg protein)
166.05 123.52 432.41 2.72
± ± ± ±
8.17 11.39 17.61 0.19
121.55 76.03 254.93 7.10
± ± ± ±
6.75## 12.03## 25.44## 0.60##
152.45 99.04 406.49 2.95
± ± ± ±
10.12** 12.28#* 33.30** 0.12**
140.16 99.52 346.93 5.60
± ± ± ±
8.89## , * 7.56# , * 23.46## , ** 0.86##*
134.34 92.78 383.33 5.48
± ± ± ±
12.82## 14.64# 27.40# , ** 0.59##**
156.00 110.01 402.62 4.36
± ± ± ±
10.39** 9.58** 19.14** 0.64## , **
All values represent the mean ± SD (n = 10). # Significantly different vs. normal control at p < 0.05. ## Significantly different vs. normal control at p < 0.01. * Significantly different vs. diabetic control at p < 0.05. ** Significantly different vs. diabetic control at p < 0.01.
and 38.59% (p < 0.01), respectively, and by 53.18% (p < 0.01), 66.91% (p < 0.01), and 58.45% (p < 0.01), respectively. 3.4. Effect of LLP on the change in histopathology of pancreas of STZ-induced diabetic mice The effects of LLP and Glibenclamide on pancreatic tissues with HE staining were showed in Fig. 1. As revealed in Fig. 1, a clear decrease in the area occupied by the beta-cells was observed in the pancreatic section of STZ-induced diabetic mice. Moreover, the number and the size of pancreatic islets were significantly decreased in diabetic mice compared with the normal control mice. However, treatment with 200 mg/kg/day LLP or 10 mg/kg/day Glibenclamide for 28 days markedly repaired the islet damage, and improved the structure integrity of pancreatic islet beta-cells and tissues. 4. Discussion Diabetes mellitus (DM) is one of the most common human metabolic diseases. It is characterized by hyperglycemia due to
defects in insulin secretion and/or activation, resulting in abnormalities in carbohydrate, lipid and protein metabolism. The precise cellular and molecular mechanism underlying the etiology and progression of diabetes is still not fully understood. However, increasing evidence suggests that oxidative stress plays a crucial role in the pathogenesis and progression of diabetes and its complications [16–22]. Hyperglycemia-induced auto-oxidation of glucose and glycation of proteins result in the formation of reactive oxygen and nitrogen species. Uncontrolled reactive oxygen species (ROS) production often leads to damage in cellular macromolecules (DNA, lipids and proteins), contributing to the progress of diabetic complications and different organ damage [23]. STZ, an antibiotic produced by Streptomyces achromogenes, is frequently used to induce DM in experimental animals through its toxic effects on pancreatic -cells. The cytotoxic action of STZ is associated with the generation of ROS causing oxidative damage [24]. In this study, we observed a significant increase in the concentration of blood glucose and a decrease in body weight in STZ-induced diabetic mice. However, treatment with LLP for 28 days not only significantly decreased the level of blood glucose, but
Fig. 1. Effects of LLP on the changes in the histopathology of the pancreas tissues in STZ-induced diabetic mice with H & E staining (magnification 400×). (A) Normal control group; (B) diabetic control group; (C) diabetic mice were administered by glibenclamide (10 mg/kg/day); (D) diabetic mice were administered by LLP (50 mg/kg/day); (E) diabetic mice were administered by LLP (100 mg/kg/day); (F) diabetic mice were administered by LLP (200 mg/kg/day).
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also improved the body weight loss of diabetic mice, which were consistent with previous studies on polysaccharides from other plants [2,5,8,25–28]. As the most predominant characteristic of diabetes mellitus, hyperglycemia increases oxidative stress through the overproduction of ROS, which results in an imbalance between free radicals and the antioxidant defense systems of the cells. The administration of LLP decreases blood glucose concentration in diabetic mice suggesting the blood glucose-controlling ability of LLP and its role as an essential trigger for the liver, kidney and pancreas to revert to their normal antioxidative abilities. Lipid peroxidation is considered to be a hallmark of oxidative stress, in which ROS interact with polyunsaturated fatty acids, and leads to the formation of lipid products such as MDA, which then causing damages to the membrane components of the cell, cell necrosis and inflammation [29]. Extensive evidence has demonstrated that the increased lipid peroxidation plays an important role in the progression of diabetes by altering the transbilayer fluidity gradient, which could hamper the activities of membrane-bound enzymes and receptors [16,30]. In this study, there was a significant increase in lipid peroxidation in serum, liver and kidney of diabetic mice, as measured by MDA formation. However, after administration of LLP for 28 days, we observed that the level of MDA in serum, liver and kidney of diabetic mice were significantly decreased. These results are in agreement with previous studies on polysaccharides from other sources [2,5,8,27,31,32]. Taken together these findings suggest that LLP can provide protective effect on the tissues of diabetic mice against oxidative damage by decreasing lipid peroxidation. Antioxidative enzymes, including SOD, GPx and CAT, are regarded as the first line of the antioxidant defense system against ROS generated in vivo during oxidative stress. Their ability to decompose superoxide and peroxide while blocking lipid peroxidation as well as their involvement in cellular defense mechanisms helps to protect tissues against oxidative damage. SOD scavenges the superoxide radical by converting it to hydrogen peroxide and molecular oxygen. CAT is a hemeprotein, which catalyzes the reduction of hydrogen peroxide and protects the tissues from highly reactive hydroxyl radicals. GPx, an enzyme with selenium, works together with glutathione (GSH) in the decomposition of hydrogen peroxide (or) other organic hydroperoxides to non-toxic products at the expense of reduced glutathione. In this study, significant decrease in the activities of SOD, CAT and GPx were observed in serum, liver and kidney of STZ-induced diabetic mice. The reduced activities of these antioxidant enzymes may result in a number of deleterious effects due to the accumulation of superoxide radicals and hydrogen peroxides. In this context, other researchers also reported a decrease in the activities of these antioxidant enzymes (SOD, CAT and GPx) in the serum, liver and kidney of diabetic mice or rats [2,5,8,27,28,31,32]. However, in the present study, treatment with LLP for 28 days significantly increased the activities of SOD, CAT and GPx in the serum, liver and kidney of STZ-induced diabetic mice. The result of increased activities of SOD, CAT and GPx suggest that LLP has a free radical scavenging activity, which could exert a beneficial effect against pathological alterations caused by ROS. Previous studies have also demonstrated that the polysaccharides from other sources can ameliorate antioxidant enzymes activities in blood and tissues of diabetic mice or rats [2,5,8,27,28,31–33]. Moreover, recent study reported that the hypoglycemic mechanisms of polysaccharides were closely associated with their antioxidant activities [32]. Based on these findings, our study revealed that one mechanism of the anti-hyperglycemic actions of LLP may be closely related with its antioxidant activity and free radical scavenging ability, hence provide protective effect against oxidative damage.
In this study, histopathological examination showed that the pancreatic structure was destroyed by STZ, the distortion of the pancreatic islets of Langerhans were observed in the diabetic mice together with atrophy (reduction of cell size and number). However, after administration of LLP, the pancreatic damage was markedly repaired and the integrity of pancreatic islet cells and tissues were improved. This further confirmed that LLP could exert protective effect on the pancreatic tissue of diabetic mice against oxidative damage. In summary, the present study suggests that LLP exhibits hypoglycemic and protective effects in STZ-induced diabetic mice by reducing oxidative stress and preserving the structural integrity of pancreatic islet. The protective effect of LLP may be attributed to its antioxidant activity. However, further investigation is necessary to delineate the exact molecular mechanism underlying the protective effect of LLP on STZ-induced diabetic mice. Acknowledgements This study was supported by the Key Program of National Natural Science Foundation of China (No. 31230057), and the National Key Technology Research and Development Program for the 12th Five-Year Plan (Nos. 2012BAD37B02 and 2012BAD37B01). References [1] M. Cnop, N. Welsh, J.-C. Jonas, A. Jörns, S. Lenzen, D.L. Eizirik, Diabetes 54 (Suppl. 2) (2005) S97–S107. [2] X.M. Chen, J. Tang, W.Y. Xie, J.J. Wang, J. Jin, J. Ren, L.Q. Jin, J.X. Lu, Carbohydr. Polym. 94 (2013) 378–385. [3] J. Xu, Y. Wang, D.-S. Xu, K.-F. Ruan, Y. Feng, S. Wang, Int. J. Biol. Macromol. 49 (2011) 657–662. [4] J.F. Fu, J.F. Fu, Y. Liu, R.Y. Li, B. Gao, N.Y. Zhang, B.L. Wang, Y.Z. Cao, K.S. Guo, Y.Y. Tu, Carbohydr. Polym. 87 (2012) 2327–2331. [5] X.M. Li, Int. J. Biol. Macromol. 40 (2007) 461–465. [6] S. Zou, X. Zhang, W.B. Yao, Y.G. Niu, X.D. Gao, Carbohydr. Polym. 80 (2010) 1161–1167. [7] X.Q. Mao, F. Yu, N. Wang, Y. Wu, F. Zou, K. Wu, M. Liu, J.P. Ouyang, Phytomedicine 16 (2009) 416–425. [8] L.Y. Zhao, Q.J. Lan, Z.C. Huang, L.J. Ouyang, F.H. Zeng, Phytomedicine 18 (2011) 661–668. [9] J. Zhang, Y.X. Gao, X.J. Zhou, L.P. Hu, T.Z. Xie, Nat. Prod. Res. 24 (2010) 357–369. [10] B.T. Zhao, J. Zhang, X. Guo, J.L. Wang, Food Hydrocoll. 31 (2013) 346–356. [11] A.M. Staub, Methods Carbohydr. Chem. 5 (1965) 5–6. [12] Y. Kono, Arch. Biochem. Biophys. 186 (1978) 189–195. [13] R.F. Beers, I.W. Sizer, J. Biol. Chem. 195 (1952) 133–140. [14] D.E. Paglia, W.N. Valentine, J. Lab. Clin. Med. 70 (1967) 158–169. [15] W.G. Niehaus Jr., B. Samuelsson, Eur. J. Biochem. 6 (1968) 126–130. [16] F. Giacco, M. Brownlee, Circ. Res. 107 (2010) 1058–1070. [17] H. Kaneto, N. Katakami, D. Kawamori, T. Miyatsuka, K. Sakamoto, T. Matsuoka, M. Matsuhisa, Y. Yamasaki, Antioxid. Redox Signal. 9 (2007) 355–366. [18] A.C. Maritim, R.A. Sanders, J.B. Watkins, J. Biochem. Mol. Toxicol. 17 (2003) 24–38. [19] C.K. Roberts, K.K. Sindhu, Life Sci. 84 (2009) 705–712. [20] R.P. Robertson, Curr. Opin. Pharmacol. 6 (2006) 615–619. [21] A.P. Rolo, C.M. Palmeira, Toxicol. Appl. Pharmacol. 212 (2006) 167–178. [22] R.A. Simmons, Free Radic. Biol. Med. 40 (2006) 917–922. [23] J.W. Baynes, Diabetes 40 (1991) 405–412. [24] T. Szkudelski, Physiol. Res. 50 (2001) 537–546. [25] N. Yang, M. Zhao, B. Zhu, B. Yang, C. Chen, C. Cui, Y. Jiang, Innov. Food Sci. Emerg. 9 (2008) 570–574. [26] X.M. Chen, J. Jin, J. Tang, Z.F. Wang, J.J. Wang, L.Q. Jin, J.X. Lu, Carbohydr. Polym. 83 (2011) 749–754. [27] S.X. Xue, X.M. Chen, J.X. Lu, L.Q. Jin, Carbohydr. Polym. 75 (2009) 415–419. [28] D. Zhang, H. Meng, H.S. Yang, Int. J. Biol. Macromol. 50 (2012) 720–724. [29] G. Stark, J. Membr. Biol. 205 (2005) 1–16. [30] L.F. Dmitriev, V.N. Titov, Ageing Res. Rev. 9 (2010) 200–210. [31] W. Zhang, L.J. Zheng, Z.M. Zhang, C.-X. Hai, Carbohydr. Polym. 89 (2012) 890–898. [32] Y.T. Liu, J. Sun, S.Q. Rao, Y.J. Su, Y.J. Yang, Food Chem. Toxicol. 57 (2013) 39–45. [33] J. Jia, X. Zhang, Y.-S. Hu, Y. Wu, Q.-Z. Wang, N.-N. Li, Q.-C. Guo, X.-C. Dong, Food Chem. 115 (2009) 32–36.
