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Antidiabetic effects of Morus alba fruit polysaccharides on high-fat diet- and streptozotocin-induced type 2 diabetes in rats
Journal of Ethnopharmacology 199 (2017) 119–127
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Antidiabetic effects of Morus alba fruit polysaccharides on high-fat dietand streptozotocin-induced type 2 diabetes in rats
MARK
Yukun Jiaoa,b,c, Xueqian Wanga, Xiang Jianga, Fansheng Konga, Shumei Wangb,c,⁎⁎, ⁎ Chunyan Yana,b,c, a b c
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China Key Laboratory of Digital Quality Evaluation of Chinese Materia Medica of State Administration of TCM, China Engineering & Technology Research Center for Chinese Materia Medica Quality of the Universities of Guangdong Province, China
A R T I C L E I N F O
A BS T RAC T
Chemical compounds: Ethanol (PubChem CID: 702) Glucose (PubChem CID: 107526) Streptozotocin (PubChem CID: 29327) Trisodium citrate dehydrate (PubChem CID: 71474) Citric acid (PubChem CID: 22230) Methanol (PubChem CID: 887) Hematoxylin (PubChem CID: 10603) Eosin Y (PubChem CID: 11048) Tween-20 (PubChem CID: 443314) Glycerol (PubChem CID: 753)
Ethnopharmacological relevance: Type 2 diabetes mellitus (T2DM) is becoming a serious threat to human health. The fruit of Morus alba L. is widely used as a traditional Chinese medicine for the treatment of DM, dizziness, tinnitus, insomnia, and premature graying, as well as to protect the liver and kidneys. Several studies have demonstrated that the aqueous extracts of the roots bark, leaves, and ramuli of mulberry, which are known to contain polyphenols and polysaccharides, have antihyperglycemic and antihyperlipidemic activities. The aim of the present study was to further investigate the active polysaccharides from M. alba fruit by evaluating the antidiabetic activities of different fractions on T2DM rats and elucidate the mechanism underlying these activities. Materials and methods: Diabetic rats were treated with two fractions of M. alba fruit polysaccharides (MFP50 and MFP90). The disease models were induced by a high-fat diet and low dose injection of streptozotocin and were compared to normal rats and metformin-treated diabetic rats. After seven weeks, the fasting blood glucose (FBG), oral glucose tolerance test (OGTT), fasting serum insulin (FINS) levels, homeostasis model of assessment-insulin resistance (HOMA-IR), glycated serum protein (GSP), and serum alanine transaminase (ALT) levels, as well as serum lipid profiles and histopathological changes in the pancreas were measured. Next, the expressions of the insulin signaling pathway were measured by western blot analysis to elucidate the potential mechanism underlying these antidiabetic activities. Results: After seven weeks of treatment, a significant reduction in the FBG levels, OGTT-area under the curve (OGTT-AUC), FINS, HOMA-IR, ALT, and triglyceride (TG) values of the MFP50 group was observed. On the other hand, in the MFP90 group, the FBG, OGTT-AUC, FINS, HOMA-IR, GSP, and TG levels were significantly reduced. The level of high-density lipoprotein cholesterol (HDL-c) and the proportion of HDL-c to total cholesterol (TC) significantly increased in the MFP50 group. Moreover, MFP50 and MFP90 induced repair of damaged pancreatic tissues of the diabetic rats. The hypoglycemic effect of MFP50 was more stable than MFP90, whereas the hypolipidemic effect of MFP90 was slightly better than MFP50. Moreover, the expression levels of InsR, IRS-2, Akt and GLUT4 in the MFP90 group significantly increased relative to that of the T2DM group. Conclusions: MFP50 and MFP90 have markedly antihyperglycemic and antihyperlipidemic effects and can clearly relieve diabetes symptoms in the T2DM rat model. The M. alba fruit polysaccharides may potentially be utilized as an effective treatment for T2DM. Further research into the structures of active M. alba fruit polysaccharides and their mechanisms in promoting antidiabetic effects are underway.
Keywords: Antidiabetic activity Morus alba L. Mulberry Polysaccharides Antihyperlipidemia Type 2 diabetic rats
1. Introduction Diabetes mellitus is a chronic, systemic metabolic disease that is related to a variety of genetic factors and environmental factors. Its
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incidence has recently increased due to changes in diet, recent urbanization, and unhealthy lifestyles (Pantalone et al., 2015). Diabetes is characterized by hyperglycemia that can lead to a series of complications. Non-insulin-dependent diabetes mellitus [NIDDM;
Corresponding author at: School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China. Corresponding author. E-mail address: [email protected] (C. Yan).
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http://dx.doi.org/10.1016/j.jep.2017.02.003 Received 18 March 2016; Received in revised form 20 January 2017; Accepted 1 February 2017 Available online 02 February 2017 0378-8741/ © 2017 Elsevier B.V. All rights reserved.
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2. Materials and methods
also called type-2 diabetes mellitus (T2DM)] is characterized by insulin resistance (American Diabetes Association, 2014) and is the foremost type of diabetes around the world. Insulin resistance pertains to lowefficiency glucose utilization in organs and tissues that renders insulin insensitivity in an organism. The hallmark features of insulin resistance include high levels of blood glucose and serum insulin. T2DM does not only cause hyperglycemia but also hyperlipidemia, diabetic nephropathy (DN), and liver impairment (Bello et al., 2014). A recent report has indicated that T2DM has evolved into an early-onset disorder (Constantino et al., 2013). These diabetic complications may cause fatal damage to human health and even threaten life (Constantino et al., 2013). Among these complications, cardiovascular disease (CVD) (Taskinen and Smith, 1998) is the most universal and serious symptom and is caused by hyperlipidemia that results in coronary heart disease, heart failure, and arteriosclerosis (Al Qahtani et al., 2015). Therefore, alleviating the complications of T2DM is equally important to lowering blood glucose levels in long-term therapies. According to a previous study, the mechanisms of T2DM were mainly include glycogenesis, glycolysis, fatty acid biosynthesis, glucose uptake, lipogenesis, lipolysis, starch and sucrose metabolism, and insulin signaling pathway (Lu et al., 2016). The insulin signaling pathway is considered as the major mechanism that involved in the pathogenesis of T2DM. In addition, insulin receptor substrates (IRSs) play key roles in the insulin signaling pathway. Prior studies have demonstrated that the incidence of diabetes was mainly because the disorder of IRS-2 (Withers et al., 1999, 1998). Previous studies describe three signaling pathways (the PI3K/Akt pathway, CAP/Cb1/ Tc10 pathway, and Ras MAPK pathway) occur after the IRS-2 activation (Rondinone et al., 1997; Datta et al., 1995; Taniguchi et al., 2006). In addition, the PI3K is primarily involved in insulin signaling and this is the reason why we selected this pathway in investigating the mechanism underlying diabetogenesis. The PI3K pathway mainly regulates the effects of the phosphatidyl inositol system, glycogenesis, and glucose uptake, and it is also correlated with the fatty acid biosynthesis and lipogenesis (Lu et al., 2016). Morus alba, commonly known as mulberry, is a member of the Moraceae family and is widely cultivated in Asia, America, Europe, Africa, and India (Khan et al., 2013). The dry fruit of the mulberry is used as a traditional Chinese medicine for the treatment of dizziness, tinnitus, insomnia, premature graying, and diabetes mellitus, as well as protects the liver and kidneys (Chinese Pharmacopoeia Commission, 2015). In recent decades, several research studies have shown that various kinds of extracts and compounds from M. alba have excellent biological activities such as antioxidant effects, anti-aging effects, and immunoregulation activities (Guo et al., 2013; Ren et al., 2015; Zhang et al., 2014). The polysaccharides extracted from mulberry ramuli imparts cytoprotective effects on the pancreas by inactivating apoptosis-related components such as Bcl-2, Bax, JNK, p38, and caspase-3 (Xu et al., 2015). Moreover, numerous studies have shown that the polyphenols extracted from M. alba exhibit antioxidative activities such as peroxyl radical scavenging capacity and α-glucosidase inhibitory activity (Isabelle et al., 2008). However, reports on the in vivo activity of mulberry fruit polysaccharides are limited. Based on the wide range of bioactivities of polysaccharides and the extensive applications of the mulberry fruit as an adjuvant therapy for diabetes, we were prompted to further explore the activities of mulberry fruit polysaccharides and elucidate the detailed compounds of the active polysaccharides. Here, we investigated and evaluated the antidiabetic activity of two fractions of polysaccharides extracted from M. alba fruit, including their antihyperglycemic, antihyperlipidemic, pancreatic and hepatoprotective effects to lay the foundation for antidiabetic mechanistic studies or for further development of T2DM medicines.
2.1. Preparation of M. alba fruit polysaccharides The M. alba fruit was purchased from a drugstore of Tong Ren Tang Chinese Medicine Co., Ltd. in Tianhe District, Guangzhou, Guangdong Province, China. Voucher specimens were kept at the Guangdong Pharmaceutical University's School of Pharmacy (Guangzhou, China). First, 10 kg of M. alba fruit was mixed with deionized water (1:10) and soaked for 8 h. Next, the sample was extracted with deionized water for 3 h at 80 °C. This was repeated 3 times. Then, the extracts were filtered and concentrated to 1/10 of the original volume at 60 °C. Moreover, the solution was precipitated with 95% ethanol to a final ethanol concentration of 50% (Zhang et al., 2014). The MFP70 and MFP90 were obtained by repeating the same process mentioned above with a final ethanol concentration of 70% and 90% respectively. 2.2. Animals Adult male Wistar (body weight: 225 ± 20 g) rats were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine (Certificate: SCXK20130034). The animals were housed in a specific pathogen-free (SPF) animal laboratory of the Experimental Animal Center of Guangdong Pharmaceutical University. The methods used in this experiment were approved by the Animal Ethics Committee of Guangdong Pharmaceutical University (No. GDPU 2013033). The animals were maintained at 12 h of light and dark cycles and at a room temperature maintained within the range of 24–26 °C, and relative humidity from 50% to 70%. Food and water was given ad libitum (Herling et al., 1997). 2.3. Acute toxicity testing An acute toxicity assay was conducted before the experiment to explore the potential toxicity of MFP. Here, the rats received a maximum dose of the M. alba polysaccharides intragastrically each day and respiratory distress, emaciation, posture, or mortality was recorded. The dose here was based on the solubility and the volume of extract that could be intragastrically administered to the rats. 2.4. Induction of T2DM Adult male Wistar rats were allowed to acclimatize to the laboratory environment for 2 weeks. Six rats were randomly separated as the normal control group (NC) and fed on a normal diet. The other rats were then fed on a high-fat diet (12% lard oil, 18% sugar, 70% normal diet) for 4 weeks with sufficient food and water. The animals were fasted for 12 h and then received an intra-peritoneal injection of streptozotocin (STZ) (Sigma-Aldrich, USA), dissolved in 0.1 M citric acid/sodium citrate buffer, pH 4.5 at a dose of 40 mg/kg (Srinivasan et al., 2005). After 72 h, the fasting blood glucose (FBG) levels of the animals was measured and recorded. The rats with an FBG of > 11.1 mmol/L were regarded as diabetic and then used in the subsequent analyses (Lu et al., 2016). 2.5. Experimental design Forty diabetic rats were randomly divided into four groups: diabetes mellitus group (DM), metformin group (Metformin), MFP50 group, and MFP90 group. Each group consisted of six rats. The metformin group received a metformin hydrochloride water solution (250 mg/kg) intragastrically, whereas the MFP50 group and MFP90 group received 400 mg/kg MFP50 and MFP90 respectively using the same route of administration. The DM group and NC group were treated with pure water. All animals were supplied with sufficient food, water, and clean cages every day until the end of the study. 120
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Proteins were transferred to a nitrocellulose membrane for 3 h at 300 mA by using a wet transfer equipment. The membrane was incubated in blocking solution containing 5% nonfat dried milk for 2 h at room temperature. Subsequently, the membrane was exposed to the desired primary antibodies in PBS including InsR antibody (1:1000) and IRS-2 antibody (1:1,500). These were incubated with the membrane at 4 °C overnight, and GAPDH (1:1000) was used as reference. After incubation with the secondary antibody for 2 h at room temperature, the membrane was exposed to the chemiluminescent reagent (ECL) for about 5–10 min. Protein expression was measured with fluorescence captured on X-ray photographic film in a dark room. The band densities were quantified.
2.6. Measurement of FBG levels All rats were fasted for 12 h every weekend and their FBG levels were measured by using a One-touch Ultra Glucometer (Johnson & Johnson). 2.7. Oral glucose tolerance test (OGTT) After 42 days of intragastric administration, the 12-h-fasted rats in all groups were intragastrically administered glucose (2.0 g/kg) (Salahuddin and Jalalpure, 2010). Blood samples were obtained at 0, 30, 60, 90, 120, 150, and 180 min after glucose administration. The FBG levels were immediately estimated using the blood glucometer.
2.11. Statistical analysis 2.8. Biochemical index All data were expressed as a mean ± standard deviation (SD). The statistical analyses were performed using the SPSS 19.0 for Windows. The significance between individual groups was analyzed by using the student's t-test.
The rats were sacrificed after 7 weeks of treatment. Blood samples were obtained and centrifuged (3000 rpm for 20 min) to isolate the serum, which were then stored at −80 °C until analysis. Fasting serum insulin (FINS) and glycated serum protein (GSP) levels were measured by using an enzyme-linked immunosorbent assay (ELISA) kit according to their manufacturer's instructions. The reagent kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The level of total cholesterol (TC), triglyceride (TG), highdensity lipoprotein cholesterol (HDL-c), and serum alanine transaminase (ALT) were analyzed using a biochemical analyzer (COBASc702, Roche). Insulin resistance was measured with the homeostasis model of assessment-insulin resistance (HOMA-IR) (Hanley et al., 2002). The HOMA-IR was estimated using the following equation: FBG (mmol/ L)×FINS (mIU/mL)/22.5.
3. Results 3.1. The yields of M. alba fruit polysaccharides In this study, we mainly presented two fractions of M. alba fruit polysaccharides (MFP50 and MFP90) with excellent activities in T2DM. The yields of MFP50 and MFP90 were 40.90% and 3.75%, respectively. 3.2. Acute toxicity tests
2.9. Histopathological studies
Acute toxicity studies revealed that the intragastric administration of polysaccharides of M. alba fruit (1000 mg/kg) did not cause significant changes to the behavior of the animals as observed by respiratory distress, emaciation, posture, or mortality. From 24 h to 1 week, all animals acted well without observable changes. No deaths occurred during the first week of the study, indicating that the intragastric administration of polysaccharides of M. alba fruit did not cause acute toxicity.
All animals were sacrificed, and the organs were appropriately stored. The pancreatic tissues were immediately stored in 10% buffered formalin. Paraffin sections of the pancreas were prepared and stained with hematoxylin and eosin (H & E) for the assessment of histopathological changes (Luna, 1968). 2.10. Western blot analysis
3.3. Antihyperglycemic effect of M. alba fruit polysaccharides The expression of various proteins, including InsR (156 kDa, Abcam, UK), ISR-2 (137 kDa, Abcam, UK), Akt (60 kDa, CST, USA), and GLUT4 (45 kDa, Abcam, UK) were analyzed by western blotting. Briefly, 100 mg of liver was homogenized in RIPA buffer for 10 min followed by centrifugation at 3000 rpm for 5 min at 4 °C. The supernatant was transferred, and total protein was extracted from the liver tissue samples. The concentrations were determined by using a BCA protein assay (Nanjing Jiancheng Bioengineering Institute). The tissue protein (40–70 μg) was subjected to 10% SDS-PAGE for 0.5 h at 80 V and for 2.5 h at 100 V to separate the target protein from the others.
The FBG levels of each group are illustrated in Table 1. At the end of the experiment, MFP50 and MFP90 groups showed a 31.9% (P < 0. 01) and 47.5% (P < 0.01) decrease in FBG levels compared to the DM group. The OGTT levels and the oral glucose tolerance test-area under the curve (OGTT-AUC) are shown in Figs. 1 and 2A. The values of OGTT-AUC in the MFP50 and MFP90 groups were 158.71 and 157.53, respectively, which were significantly lower than that of the DM group (176.83). The GSP levels were reduced by 18.6% (P < 0.01), 31.7% (P < 0.01), and 29.0% (P < 0.05) in the MFP50, MFP90, and metformin
Table 1 FBG levels (mmol/L) of each group (n =6). DM 0 week 1st week 3rd week 5th week 6th week 7th week #
MFP50 ##
18.49 ± 3.49 22.23 ± 1.72## 21.45 ± 1.24## 19.87 ± 1.95## 22.97 ± 2.06## 21.58 ± 2.05##
MFP90 ##
Metformin ##
17.29 ± 3.10 12.88 ± 3.64** 17.23 ± 3.06* 12.35 ± 3.47** 11.85 ± 4.19** 14.70 ± 4.03**
17.72 ± 3.73 17.65 ± 1.98** 16.08 ± 3.10* 12.30 ± 3.96** 14.65 ± 6.12* 11.33 ± 5.44**
P < 0.05 vs. NC. ## P < 0.01 vs. NC. * P < 0.05 vs. DM. ** P < 0.01 vs. DM.
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NC ##
17.39 ± 1.83 17.08 ± 1.32* 17.05 ± 2.88* 9.68 ± 3.14** 11.15 ± 5.01** 9.63 ± 4.68**
4.76 ± 0.61 4.72 ± 0.51 4.91 ± 0.28 5.20 ± 0.72 5.07 ± 0.67 5.11 ± 0.56
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3.5. Antihyperlipidemic effects of M. alba fruit polysaccharides The lipid profiles of each group are shown in Fig. 4. At the end of the study, the TC (P < 0.05) and TG (P < 0.01) levels of the diabetic rats significantly increased, whereas that of the HDL-c levels decreased (P < 0.01) compared to the control rats. After seven weeks of treatment, the rats in the MFP50 group exhibited a significant (P < 0.01) reduction in TC/HDL-c and TG levels, whereas the HDL-c levels significantly (P < 0.01) increased. These values were more similar to those of the control rats than the diabetic rats. The rats in the MFP90 group exhibited a significant reduction in TC/HDL-c (P < 0.01), TC (P < 0.05), and TG (P < 0.01) levels. These findings indicated that the M. alba fruit polysaccharides affected lipid metabolic parameters and effectively ameliorated lipid metabolism in T2DM rats. 3.6. Assessment of the hepatic function of T2DM rats
Fig. 1. Oral glucose tolerance test curve of each group (n =6). 0.05 vs. DM. **P < 0.01 vs. DM.
##
Fig. 5 shows that the serum ALT levels of the DM group were significantly higher than those of the NC group (P < 0.01), whereas the MFP50 group exhibited significantly lower serum ALT levels (P < 0.05). The serum ALT levels in the metformin and MFP90 groups were also reduced. These findings indicated that the MFP50 could improve hepatic function in T2DM rats.
P < 0.01 vs. NC. *P <
groups relative to that of the DM group, respectively. The decrease in the GSP levels of the MFP90 group was more pronounced than that in the positive control and metformin group (29.0%, P < 0.05), thereby indicating that the treatment with M. alba fruit polysaccharides effectively reduces blood glucose and GSP levels in T2DM rats.
3.7. Histopathological changes in pancreas tissues The H & E stained sections of the pancreatic tissue samples are shown in Fig. 6. Significant differences in the pattern and number of pancreas islets were observed among the five groups. The DM group rarely showed islets with numerous vacuous areas (Fig. 6B). However, in the control, the pancreas islets were round and showed clear boundaries. The islets were bigger and more prevalent than in other groups (Fig. 6A). In the metformin group, there were a few islets but these were erratically shaped and contained numerous central particles (Fig. 6C). These findings indicated that metformin could repair the damage incurred by the pancreatic islets due to exposure to STZ, whereas the groups fed with MFP50 and MFP90 (Fig. 6D, E) showed better effects than the metformin group in terms of the recovery of STZ-induced impairment of pancreatic islets (40 mg/kg). These findings show that M. alba fruit polysaccharides can enhance the restoration of islets in the hyperplastic pancreas in T2DM rats.
3.4. Ameliorative effect on insulin resistance Fig. 3A shows that the FINS level of the DM group (12.89 ± 1.36 mIU/L) was significantly higher than that in the NC group (10.42 ± 1.07 mIU/L) after seven weeks of treatment (P < 0.01). However, relative to the DM group, the FINS of both the MFP50 (9.48 ± 2.28 mIU/L, P < 0.05) and MFP90 (8.70 ± 1.32 mIU/L, P < 0.01) groups were significantly reduced. At the end of the experiment, the FINS levels of the MFP50 and MFP90 groups further decreased compared to that in the NC group. The FINS levels decreased in the metformin group (11.65 ± 2.07 mIU/L), although this was not determined to be statistically significant. Fig. 3B shows that the HOMA-IR value of the DM group was significantly higher than that in the NC group (P < 0.01). After seven weeks of treatment, the HOMA-IR values of the MFP90 group (4.35 ± 1.62) were markedly lower than that in the metformin group (4.70 ± 2.43). These findings indicated that the M. alba fruit polysaccharides could effectively decrease the FINS and HOMA-IR values in T2DM rats.
3.8. M. alba fruit polysaccharides upregulated the expression of insulin signaling pathway The alternations of InsR, IRS-2, Akt, and GLUT4 in liver tissues in various treatment groups are shown in Fig. 7. A significant decrease in
Fig. 2. Area under the curve of OGTT in each group (A), glycated serum protein levels of each group (B), (n =6),
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P < 0.01 vs. NC. *P < 0.05 vs. DM. **P < 0.01 vs. DM.
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Fig. 3. Serum insulin levels of each group (A) and values of HOMA-IR in each group (B), (n =6).
##
P < 0.01 vs. NC. *P < 0.05 vs. DM. **P < 0.01 vs. DM.
Fig. 4. Serum HDL-c levels in each group (A), (n =6), serum TC levels in each group (B), (n =5), the ratio of TC/HDL-c in each group (C), (n =6), and serum TG levels in each group (D), (n =6). ##P < 0.01 vs. NC. *P < 0.05 vs. DM. **P < 0.01 vs. DM.
addition, the MFP50 group showed a significant upregulation in the expressions of Akt and GLUT4 (P < 0.05) by 131.3% and 262.1%, respectively.
InsR (P < 0.01) and IRS-2 (P < 0.01) expression of DM, Metformin, MFP50, and MFP90 groups were observed compared to that in the controls. The expressions levels of InsR and IRS-2 of the DM group decreased by 47.0% and 51.0%, respectively. After seven weeks, metformin upregulated the expressions of InsR, IRS-2, Akt, and GLUT4 by 115.3%, 86.9%, 113%, and 410.5%, respectively. The MFP90 group showed an upregulation of the expressions of InsR, IRS-2, Akt, and GLUT4 by 107.1%, 98.2%, 179%, and 420.7%, respectively. However, the MFP50 group showed a slight upregulated in the expression of IRS-2 (46.67%, P < 0.05), although this was not significant, as well as in the expression of InsR (28.96%, P > 0.05). In
4. Discussion Diabetes is one of the third healthy-killers and the diabetic was increased more and more sharply. Moreover, in the clinic, metformin, sulfonylurea, rosiglitazone, and α-glucosidase inhibitors all impart severe side-effects after prolonged treatment. M. alba, as a traditional Chinese medicine, is famous for its stable antidiabetic effect. In the 123
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Fig. 5. Serum alanine transaminase (ALT) levels in each group, (n =6), NC. *P < 0.05 vs. DM. **P < 0.01 vs. DM.
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et al., 1977). Our results indicate that MFP50 and MFP90 can significantly ameliorate hyperlipidemia. Fig. 4 shows that MFP50 reduced TG levels (P < 0.01) and lowered the ratio of TC/HDL-c (P < 0.05), but significantly increased HDL-c levels (P < 0.01). MFP90 reduced TC (P < 0.05) and TG (P < 0.01) levels and lowered the ratio of TC/HDL-c (P < 0.01). Metformin reduced TG levels (P < 0.05). In summ, both MFP50 and MFP90 demonstrated significant effects in reducing FBG, alleviating insulin resistance, and improving the ability of lipid metabolism, which were in agreement with a previous study using total M. alba fruit polysaccharides (Guo et al., 2013). Interestingly, MFP50 also improved glycometabolism and insulin resistance as indicated by changes in FBG, OGTT, FINS, HOMA-IR levels, whereas MFP90 enhanced lipid metabolism, including those of HDL-c, TG and TC levels. Dysregulation of lipid metabolism can result in systemic disruption of insulin and glucose metabolism. Therefore, the cure of hyperlipidemia was indispensable as hyperglycemia. The high level of ALT is a hallmark feature of liver dysfunction (Mangus et al., 2015). Herein, we selected ALT levels as a bioindicator of hepatic function in an animal model for T2DM. MFP50 significantly decreased ALT levels in T2DM rats, indicating that it imparts a potential ameliorative effect on hepatic function. In addition, histopathologic observations were performed to determine the effects of the polysaccharide extracts on pancreatic islet cells. We have thus elucidated that MFP50 and MFP90 have significant antihyperglycemic, antihyperlipidemic, pancreas protective, and potential hepatoprotective effects. Furthermore, M. alba fruit polysaccharides probably could prevent complications caused by hyperlipidemia such as cardiovascular disease (CVD) (Howard et al., 2000) or at least reduce the risk of CVD (Getz et al., 2010). However, more studies are needed to confirm these hypotheses. The underlying mechanism of these anti-diabetes effects required further exploration. Therefore, we measured the effects of MFP50 and MFP90 on the insulin signaling pathway firstly. By definition, insulin resistance is a defect of signal transduction. Insulin action is initiated through binding to and activation of cell-surface receptors and insulin receptors (InsR). When InsR was activated, a series of biochemical reactions were initiated to promote glucose utilization by reducing hepatic glucose output (via decreased gluconeogenesis and glycogenolysis) and increasing the rate of glucose uptake in muscle and adipose tissue (Pessin and Saltiel, 2000). Insulin receptor substances (IRS) are a series of proteins that play a key role in insulin signaling pathway (White, 1998). IRS-2 is mainly expressed in the liver and pancreatic β cells. Recent studies have suggested that the expression of IRS-2 plays an extremely important role in insulin resistance and T2DM (Withers et al., 1998). Knocking out the gene of IRS-1 and IRS-2 in mice not only results in insulin resistance, but also reduced the number of β cells and symptoms of T2DM. These studies indicated that if only IRS-2 function is abnormal, then the diabetic symptoms would present. According to previous studies, glucose transporter type 4 (GLUT4), which is primarily found in adipose and striated muscle, is an insulinregulated glucose transporter (Bogan et al., 2003). Moreover, it serves as the rate-limiting step of glucose metabolism; therefore, it was crucial to evaluate its expression levels in the present T2DM study. Lastly, GLUT4 is mainly regulated by the insulin signaling pathway (Perrini et al., 2004). A prior study has revealed that Akt plays a critical role in the insulin signaling pathway, and ultimately, insulin sensitivity (George et al., 2004). A decrease in Akt expression could lead to a downregulation of GLUT4 (Sano et al., 2003). Here, MFP90 significantly upregulated the expression of all four proteins, and MFP50 also upregulated the expression of IRS-2, Akt, and GLUT4 (Fig. 7). However, this integrated study on the insulin signaling pathway of MFP50 and MFP90 requires further validation.
P < 0.01 vs.
present study, we first extracted the M. alba fruit polysaccharides by using the classical method of water extraction and ethanol precipitation via graded ethanol precipitation. In addition, we evaluated the antihyperglycemic, antihyperlipidemic, pancreas protective, potential liver protective effects, and the protein expressions of insulin signaling pathway of the two fraction polysaccharides for the first time. Currently, metformin is considered as the gold standard for the treatment of T2DM. It also has antihyperlipidemic activity (American Diabetes Association, 2015; Stern and Murphy, 2015). Diabetic animal models play an important role in pharmacological activity evaluation. In this study, the diabetic rats exhibited significantly higher FBG levels (Table 1), worse OGTT (Figs. 1 and 2A), higher GSP levels (Fig. 2B), more serious insulin resistance (Fig. 3B), higher levels of hyperlipidemia (Fig. 4), and severe pancreatic impairment (Fig. 6) compared to the control rats. These results indicated that the model for type 2 diabetes was created successfully. The hypoglycemic activity experiments of the M. alba fruit polysaccharides in the diabetic mice produced an significant (P < 0.05) decrease in FBG levels compared to the T2DM group. The MFP50 and MFP90 groups showed a 31.9% (P < 0. 01) and 47.5% (P < 0.01) reduction in FBG levels, respectively. Metformin reduced it by 55.4% (P < 0.01) at the end of the experiment. Over these seven weeks of treatment, the hypoglycemic effects of the M. alba fruit polysaccharides were relatively stable as was metformin. Moreover, the MFP90 significantly reduced GSP levels 31.7% (P < 0.01) even more than 29.0% (P < 0.05) in metformin. The GSP levels can effectively reflect the blood glucose levels over a period of time (Lapolla et al., 1988). These findings suggest that the hypoglycemic activity of M. alba fruit polysaccharides were significantly reliable in treating T2DM. T2DM is characterized by insulin resistance. Thus, decreasing insulin resistance is a vital method to treat T2DM. Here, MFP50 and MFP90 lowered the serum insulin levels at a rate of 26.45% and 32.51% versus the DM group (Fig. 3A). The HOMA-IR values were greatly reduced (Fig. 3B), indicating that MFP50 and MFP90 can ameliorate insulin resistance in T2DM rats. Moreover, MFP50 and MFP90 repaired the damages that were incurred by the pancreatic islets (Fig. 6), even better than metformin. Based on these findings, we hence conclude that M. alba fruit polysaccharides could improve insulin sensibility and protect the pancreas, but not increase insulin secretion. Furthermore, these observations indicated that the M. alba fruit polysaccharides imparted antihyperglycemic effects by improving insulin sensibility. Dyslipidemia is one of the hallmark features of T2DM and the primary cause of cardiovascular diseases in patients with T2DM (Taskinen and Smith, 1998). Hyperlipidemia is characterized by high levels of TC, LDL-c, and TG, coupled with low levels of HDL-c (Castelli
5. Conclusions In summary, this study has proven that M. alba fruit polysacchar124
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Fig. 6. Histological analysis of pancreatic tissues sections in each group. Conventional H & E staining was performed (Magnification: 200×). NC group (A), DM group (B), Metformin group (C), MFP50 group (D), and MFP90 group (E). Black arrows are pancreatic islet cells.
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Fig. 7. InsR (A), IRS-2 (B), Akt (C) and GLUT4 (D) relative expression of each group in liver tissues (n=4). #P < 0.05 vs. NC. ##P < 0.01 vs. NC. ###P < 0.001 vs. NC. *P < 0.05 vs. DM. **P < 0.01 vs. DM. ***P < 0.01 vs. DM.
Acknowledgments
ides have good antihyperglycemic activity, antihyperlipidemic effects, and pancreatic protective effects on T2DM rats. This treatment can ameliorate insulin resistance. Although MFP50 and MFP90 revealed significant effects on T2DM, the major mechanisms might be different. We lay the foundation for mechanistic research into their antidiabetic activities. The findings of the present study indicated that MFP50 may ameliorate T2DM only through the PI3K/Akt pathway, and MFP90 has multiple pathways. Moreover, the M. alba fruit polysaccharides might possess hepatic protective activity. Our work provides a scientific basis for the further development of M. alba fruit polysaccharides as a new effective and low-toxicity agent to treat T2DM and also for the exploration of the detailed polysaccharides’ structure with active bioactivities. However, the integrated mechanism of its antihyperglycemic and antihyperlipidemic activity, improved insulin resistance, and pancreatic protective effects have not yet fully elucidated and is thus one of our future research directions. Furthermore, we plan to further purify the active constituents of the mulberry extract described in this study.
This work was financially supported by the National Natural Science Foundation of China (Nos. 81673557, 81102779 and 81274060), the Guangdong Natural Science Foundation (No. 9451022401003453), the Pearl River S & T Nova Program of Guangzhou (No. 2013J2200035), the Innovation Program of the University of Guangdong Province (No. 2014KTSCX118), the Science and Technology Program of Guangdong Province (No. 2014A050503067 and 2015A020211032) and the High-level Talents Program of the University of Guangdong Province (No. 2013). References Al Qahtani, M., Al Backer, T., Al Anazi, T., Al Johani, N., Binsalih, S., AlGobain, M., Alshammari, H., 2015. Impact of lipid disorders on mortality among Saudi patients with heart failure. J. Saudi. Heart Assoc. 27, 91–95. American Diabetes Association, 2014. Diagnosis and classification of diabetes mellitus. Diabetes Care 37, S81–S90.
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Antidiabetic effects of Morus alba fruit polysaccharides on high-fat dietand streptozotocin-induced type 2 diabetes in rats
MARK
Yukun Jiaoa,b,c, Xueqian Wanga, Xiang Jianga, Fansheng Konga, Shumei Wangb,c,⁎⁎, ⁎ Chunyan Yana,b,c, a b c
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China Key Laboratory of Digital Quality Evaluation of Chinese Materia Medica of State Administration of TCM, China Engineering & Technology Research Center for Chinese Materia Medica Quality of the Universities of Guangdong Province, China
A R T I C L E I N F O
A BS T RAC T
Chemical compounds: Ethanol (PubChem CID: 702) Glucose (PubChem CID: 107526) Streptozotocin (PubChem CID: 29327) Trisodium citrate dehydrate (PubChem CID: 71474) Citric acid (PubChem CID: 22230) Methanol (PubChem CID: 887) Hematoxylin (PubChem CID: 10603) Eosin Y (PubChem CID: 11048) Tween-20 (PubChem CID: 443314) Glycerol (PubChem CID: 753)
Ethnopharmacological relevance: Type 2 diabetes mellitus (T2DM) is becoming a serious threat to human health. The fruit of Morus alba L. is widely used as a traditional Chinese medicine for the treatment of DM, dizziness, tinnitus, insomnia, and premature graying, as well as to protect the liver and kidneys. Several studies have demonstrated that the aqueous extracts of the roots bark, leaves, and ramuli of mulberry, which are known to contain polyphenols and polysaccharides, have antihyperglycemic and antihyperlipidemic activities. The aim of the present study was to further investigate the active polysaccharides from M. alba fruit by evaluating the antidiabetic activities of different fractions on T2DM rats and elucidate the mechanism underlying these activities. Materials and methods: Diabetic rats were treated with two fractions of M. alba fruit polysaccharides (MFP50 and MFP90). The disease models were induced by a high-fat diet and low dose injection of streptozotocin and were compared to normal rats and metformin-treated diabetic rats. After seven weeks, the fasting blood glucose (FBG), oral glucose tolerance test (OGTT), fasting serum insulin (FINS) levels, homeostasis model of assessment-insulin resistance (HOMA-IR), glycated serum protein (GSP), and serum alanine transaminase (ALT) levels, as well as serum lipid profiles and histopathological changes in the pancreas were measured. Next, the expressions of the insulin signaling pathway were measured by western blot analysis to elucidate the potential mechanism underlying these antidiabetic activities. Results: After seven weeks of treatment, a significant reduction in the FBG levels, OGTT-area under the curve (OGTT-AUC), FINS, HOMA-IR, ALT, and triglyceride (TG) values of the MFP50 group was observed. On the other hand, in the MFP90 group, the FBG, OGTT-AUC, FINS, HOMA-IR, GSP, and TG levels were significantly reduced. The level of high-density lipoprotein cholesterol (HDL-c) and the proportion of HDL-c to total cholesterol (TC) significantly increased in the MFP50 group. Moreover, MFP50 and MFP90 induced repair of damaged pancreatic tissues of the diabetic rats. The hypoglycemic effect of MFP50 was more stable than MFP90, whereas the hypolipidemic effect of MFP90 was slightly better than MFP50. Moreover, the expression levels of InsR, IRS-2, Akt and GLUT4 in the MFP90 group significantly increased relative to that of the T2DM group. Conclusions: MFP50 and MFP90 have markedly antihyperglycemic and antihyperlipidemic effects and can clearly relieve diabetes symptoms in the T2DM rat model. The M. alba fruit polysaccharides may potentially be utilized as an effective treatment for T2DM. Further research into the structures of active M. alba fruit polysaccharides and their mechanisms in promoting antidiabetic effects are underway.
Keywords: Antidiabetic activity Morus alba L. Mulberry Polysaccharides Antihyperlipidemia Type 2 diabetic rats
1. Introduction Diabetes mellitus is a chronic, systemic metabolic disease that is related to a variety of genetic factors and environmental factors. Its
⁎
incidence has recently increased due to changes in diet, recent urbanization, and unhealthy lifestyles (Pantalone et al., 2015). Diabetes is characterized by hyperglycemia that can lead to a series of complications. Non-insulin-dependent diabetes mellitus [NIDDM;
Corresponding author at: School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China. Corresponding author. E-mail address: [email protected] (C. Yan).
⁎⁎
http://dx.doi.org/10.1016/j.jep.2017.02.003 Received 18 March 2016; Received in revised form 20 January 2017; Accepted 1 February 2017 Available online 02 February 2017 0378-8741/ © 2017 Elsevier B.V. All rights reserved.
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2. Materials and methods
also called type-2 diabetes mellitus (T2DM)] is characterized by insulin resistance (American Diabetes Association, 2014) and is the foremost type of diabetes around the world. Insulin resistance pertains to lowefficiency glucose utilization in organs and tissues that renders insulin insensitivity in an organism. The hallmark features of insulin resistance include high levels of blood glucose and serum insulin. T2DM does not only cause hyperglycemia but also hyperlipidemia, diabetic nephropathy (DN), and liver impairment (Bello et al., 2014). A recent report has indicated that T2DM has evolved into an early-onset disorder (Constantino et al., 2013). These diabetic complications may cause fatal damage to human health and even threaten life (Constantino et al., 2013). Among these complications, cardiovascular disease (CVD) (Taskinen and Smith, 1998) is the most universal and serious symptom and is caused by hyperlipidemia that results in coronary heart disease, heart failure, and arteriosclerosis (Al Qahtani et al., 2015). Therefore, alleviating the complications of T2DM is equally important to lowering blood glucose levels in long-term therapies. According to a previous study, the mechanisms of T2DM were mainly include glycogenesis, glycolysis, fatty acid biosynthesis, glucose uptake, lipogenesis, lipolysis, starch and sucrose metabolism, and insulin signaling pathway (Lu et al., 2016). The insulin signaling pathway is considered as the major mechanism that involved in the pathogenesis of T2DM. In addition, insulin receptor substrates (IRSs) play key roles in the insulin signaling pathway. Prior studies have demonstrated that the incidence of diabetes was mainly because the disorder of IRS-2 (Withers et al., 1999, 1998). Previous studies describe three signaling pathways (the PI3K/Akt pathway, CAP/Cb1/ Tc10 pathway, and Ras MAPK pathway) occur after the IRS-2 activation (Rondinone et al., 1997; Datta et al., 1995; Taniguchi et al., 2006). In addition, the PI3K is primarily involved in insulin signaling and this is the reason why we selected this pathway in investigating the mechanism underlying diabetogenesis. The PI3K pathway mainly regulates the effects of the phosphatidyl inositol system, glycogenesis, and glucose uptake, and it is also correlated with the fatty acid biosynthesis and lipogenesis (Lu et al., 2016). Morus alba, commonly known as mulberry, is a member of the Moraceae family and is widely cultivated in Asia, America, Europe, Africa, and India (Khan et al., 2013). The dry fruit of the mulberry is used as a traditional Chinese medicine for the treatment of dizziness, tinnitus, insomnia, premature graying, and diabetes mellitus, as well as protects the liver and kidneys (Chinese Pharmacopoeia Commission, 2015). In recent decades, several research studies have shown that various kinds of extracts and compounds from M. alba have excellent biological activities such as antioxidant effects, anti-aging effects, and immunoregulation activities (Guo et al., 2013; Ren et al., 2015; Zhang et al., 2014). The polysaccharides extracted from mulberry ramuli imparts cytoprotective effects on the pancreas by inactivating apoptosis-related components such as Bcl-2, Bax, JNK, p38, and caspase-3 (Xu et al., 2015). Moreover, numerous studies have shown that the polyphenols extracted from M. alba exhibit antioxidative activities such as peroxyl radical scavenging capacity and α-glucosidase inhibitory activity (Isabelle et al., 2008). However, reports on the in vivo activity of mulberry fruit polysaccharides are limited. Based on the wide range of bioactivities of polysaccharides and the extensive applications of the mulberry fruit as an adjuvant therapy for diabetes, we were prompted to further explore the activities of mulberry fruit polysaccharides and elucidate the detailed compounds of the active polysaccharides. Here, we investigated and evaluated the antidiabetic activity of two fractions of polysaccharides extracted from M. alba fruit, including their antihyperglycemic, antihyperlipidemic, pancreatic and hepatoprotective effects to lay the foundation for antidiabetic mechanistic studies or for further development of T2DM medicines.
2.1. Preparation of M. alba fruit polysaccharides The M. alba fruit was purchased from a drugstore of Tong Ren Tang Chinese Medicine Co., Ltd. in Tianhe District, Guangzhou, Guangdong Province, China. Voucher specimens were kept at the Guangdong Pharmaceutical University's School of Pharmacy (Guangzhou, China). First, 10 kg of M. alba fruit was mixed with deionized water (1:10) and soaked for 8 h. Next, the sample was extracted with deionized water for 3 h at 80 °C. This was repeated 3 times. Then, the extracts were filtered and concentrated to 1/10 of the original volume at 60 °C. Moreover, the solution was precipitated with 95% ethanol to a final ethanol concentration of 50% (Zhang et al., 2014). The MFP70 and MFP90 were obtained by repeating the same process mentioned above with a final ethanol concentration of 70% and 90% respectively. 2.2. Animals Adult male Wistar (body weight: 225 ± 20 g) rats were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine (Certificate: SCXK20130034). The animals were housed in a specific pathogen-free (SPF) animal laboratory of the Experimental Animal Center of Guangdong Pharmaceutical University. The methods used in this experiment were approved by the Animal Ethics Committee of Guangdong Pharmaceutical University (No. GDPU 2013033). The animals were maintained at 12 h of light and dark cycles and at a room temperature maintained within the range of 24–26 °C, and relative humidity from 50% to 70%. Food and water was given ad libitum (Herling et al., 1997). 2.3. Acute toxicity testing An acute toxicity assay was conducted before the experiment to explore the potential toxicity of MFP. Here, the rats received a maximum dose of the M. alba polysaccharides intragastrically each day and respiratory distress, emaciation, posture, or mortality was recorded. The dose here was based on the solubility and the volume of extract that could be intragastrically administered to the rats. 2.4. Induction of T2DM Adult male Wistar rats were allowed to acclimatize to the laboratory environment for 2 weeks. Six rats were randomly separated as the normal control group (NC) and fed on a normal diet. The other rats were then fed on a high-fat diet (12% lard oil, 18% sugar, 70% normal diet) for 4 weeks with sufficient food and water. The animals were fasted for 12 h and then received an intra-peritoneal injection of streptozotocin (STZ) (Sigma-Aldrich, USA), dissolved in 0.1 M citric acid/sodium citrate buffer, pH 4.5 at a dose of 40 mg/kg (Srinivasan et al., 2005). After 72 h, the fasting blood glucose (FBG) levels of the animals was measured and recorded. The rats with an FBG of > 11.1 mmol/L were regarded as diabetic and then used in the subsequent analyses (Lu et al., 2016). 2.5. Experimental design Forty diabetic rats were randomly divided into four groups: diabetes mellitus group (DM), metformin group (Metformin), MFP50 group, and MFP90 group. Each group consisted of six rats. The metformin group received a metformin hydrochloride water solution (250 mg/kg) intragastrically, whereas the MFP50 group and MFP90 group received 400 mg/kg MFP50 and MFP90 respectively using the same route of administration. The DM group and NC group were treated with pure water. All animals were supplied with sufficient food, water, and clean cages every day until the end of the study. 120
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Proteins were transferred to a nitrocellulose membrane for 3 h at 300 mA by using a wet transfer equipment. The membrane was incubated in blocking solution containing 5% nonfat dried milk for 2 h at room temperature. Subsequently, the membrane was exposed to the desired primary antibodies in PBS including InsR antibody (1:1000) and IRS-2 antibody (1:1,500). These were incubated with the membrane at 4 °C overnight, and GAPDH (1:1000) was used as reference. After incubation with the secondary antibody for 2 h at room temperature, the membrane was exposed to the chemiluminescent reagent (ECL) for about 5–10 min. Protein expression was measured with fluorescence captured on X-ray photographic film in a dark room. The band densities were quantified.
2.6. Measurement of FBG levels All rats were fasted for 12 h every weekend and their FBG levels were measured by using a One-touch Ultra Glucometer (Johnson & Johnson). 2.7. Oral glucose tolerance test (OGTT) After 42 days of intragastric administration, the 12-h-fasted rats in all groups were intragastrically administered glucose (2.0 g/kg) (Salahuddin and Jalalpure, 2010). Blood samples were obtained at 0, 30, 60, 90, 120, 150, and 180 min after glucose administration. The FBG levels were immediately estimated using the blood glucometer.
2.11. Statistical analysis 2.8. Biochemical index All data were expressed as a mean ± standard deviation (SD). The statistical analyses were performed using the SPSS 19.0 for Windows. The significance between individual groups was analyzed by using the student's t-test.
The rats were sacrificed after 7 weeks of treatment. Blood samples were obtained and centrifuged (3000 rpm for 20 min) to isolate the serum, which were then stored at −80 °C until analysis. Fasting serum insulin (FINS) and glycated serum protein (GSP) levels were measured by using an enzyme-linked immunosorbent assay (ELISA) kit according to their manufacturer's instructions. The reagent kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The level of total cholesterol (TC), triglyceride (TG), highdensity lipoprotein cholesterol (HDL-c), and serum alanine transaminase (ALT) were analyzed using a biochemical analyzer (COBASc702, Roche). Insulin resistance was measured with the homeostasis model of assessment-insulin resistance (HOMA-IR) (Hanley et al., 2002). The HOMA-IR was estimated using the following equation: FBG (mmol/ L)×FINS (mIU/mL)/22.5.
3. Results 3.1. The yields of M. alba fruit polysaccharides In this study, we mainly presented two fractions of M. alba fruit polysaccharides (MFP50 and MFP90) with excellent activities in T2DM. The yields of MFP50 and MFP90 were 40.90% and 3.75%, respectively. 3.2. Acute toxicity tests
2.9. Histopathological studies
Acute toxicity studies revealed that the intragastric administration of polysaccharides of M. alba fruit (1000 mg/kg) did not cause significant changes to the behavior of the animals as observed by respiratory distress, emaciation, posture, or mortality. From 24 h to 1 week, all animals acted well without observable changes. No deaths occurred during the first week of the study, indicating that the intragastric administration of polysaccharides of M. alba fruit did not cause acute toxicity.
All animals were sacrificed, and the organs were appropriately stored. The pancreatic tissues were immediately stored in 10% buffered formalin. Paraffin sections of the pancreas were prepared and stained with hematoxylin and eosin (H & E) for the assessment of histopathological changes (Luna, 1968). 2.10. Western blot analysis
3.3. Antihyperglycemic effect of M. alba fruit polysaccharides The expression of various proteins, including InsR (156 kDa, Abcam, UK), ISR-2 (137 kDa, Abcam, UK), Akt (60 kDa, CST, USA), and GLUT4 (45 kDa, Abcam, UK) were analyzed by western blotting. Briefly, 100 mg of liver was homogenized in RIPA buffer for 10 min followed by centrifugation at 3000 rpm for 5 min at 4 °C. The supernatant was transferred, and total protein was extracted from the liver tissue samples. The concentrations were determined by using a BCA protein assay (Nanjing Jiancheng Bioengineering Institute). The tissue protein (40–70 μg) was subjected to 10% SDS-PAGE for 0.5 h at 80 V and for 2.5 h at 100 V to separate the target protein from the others.
The FBG levels of each group are illustrated in Table 1. At the end of the experiment, MFP50 and MFP90 groups showed a 31.9% (P < 0. 01) and 47.5% (P < 0.01) decrease in FBG levels compared to the DM group. The OGTT levels and the oral glucose tolerance test-area under the curve (OGTT-AUC) are shown in Figs. 1 and 2A. The values of OGTT-AUC in the MFP50 and MFP90 groups were 158.71 and 157.53, respectively, which were significantly lower than that of the DM group (176.83). The GSP levels were reduced by 18.6% (P < 0.01), 31.7% (P < 0.01), and 29.0% (P < 0.05) in the MFP50, MFP90, and metformin
Table 1 FBG levels (mmol/L) of each group (n =6). DM 0 week 1st week 3rd week 5th week 6th week 7th week #
MFP50 ##
18.49 ± 3.49 22.23 ± 1.72## 21.45 ± 1.24## 19.87 ± 1.95## 22.97 ± 2.06## 21.58 ± 2.05##
MFP90 ##
Metformin ##
17.29 ± 3.10 12.88 ± 3.64** 17.23 ± 3.06* 12.35 ± 3.47** 11.85 ± 4.19** 14.70 ± 4.03**
17.72 ± 3.73 17.65 ± 1.98** 16.08 ± 3.10* 12.30 ± 3.96** 14.65 ± 6.12* 11.33 ± 5.44**
P < 0.05 vs. NC. ## P < 0.01 vs. NC. * P < 0.05 vs. DM. ** P < 0.01 vs. DM.
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NC ##
17.39 ± 1.83 17.08 ± 1.32* 17.05 ± 2.88* 9.68 ± 3.14** 11.15 ± 5.01** 9.63 ± 4.68**
4.76 ± 0.61 4.72 ± 0.51 4.91 ± 0.28 5.20 ± 0.72 5.07 ± 0.67 5.11 ± 0.56
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3.5. Antihyperlipidemic effects of M. alba fruit polysaccharides The lipid profiles of each group are shown in Fig. 4. At the end of the study, the TC (P < 0.05) and TG (P < 0.01) levels of the diabetic rats significantly increased, whereas that of the HDL-c levels decreased (P < 0.01) compared to the control rats. After seven weeks of treatment, the rats in the MFP50 group exhibited a significant (P < 0.01) reduction in TC/HDL-c and TG levels, whereas the HDL-c levels significantly (P < 0.01) increased. These values were more similar to those of the control rats than the diabetic rats. The rats in the MFP90 group exhibited a significant reduction in TC/HDL-c (P < 0.01), TC (P < 0.05), and TG (P < 0.01) levels. These findings indicated that the M. alba fruit polysaccharides affected lipid metabolic parameters and effectively ameliorated lipid metabolism in T2DM rats. 3.6. Assessment of the hepatic function of T2DM rats
Fig. 1. Oral glucose tolerance test curve of each group (n =6). 0.05 vs. DM. **P < 0.01 vs. DM.
##
Fig. 5 shows that the serum ALT levels of the DM group were significantly higher than those of the NC group (P < 0.01), whereas the MFP50 group exhibited significantly lower serum ALT levels (P < 0.05). The serum ALT levels in the metformin and MFP90 groups were also reduced. These findings indicated that the MFP50 could improve hepatic function in T2DM rats.
P < 0.01 vs. NC. *P <
groups relative to that of the DM group, respectively. The decrease in the GSP levels of the MFP90 group was more pronounced than that in the positive control and metformin group (29.0%, P < 0.05), thereby indicating that the treatment with M. alba fruit polysaccharides effectively reduces blood glucose and GSP levels in T2DM rats.
3.7. Histopathological changes in pancreas tissues The H & E stained sections of the pancreatic tissue samples are shown in Fig. 6. Significant differences in the pattern and number of pancreas islets were observed among the five groups. The DM group rarely showed islets with numerous vacuous areas (Fig. 6B). However, in the control, the pancreas islets were round and showed clear boundaries. The islets were bigger and more prevalent than in other groups (Fig. 6A). In the metformin group, there were a few islets but these were erratically shaped and contained numerous central particles (Fig. 6C). These findings indicated that metformin could repair the damage incurred by the pancreatic islets due to exposure to STZ, whereas the groups fed with MFP50 and MFP90 (Fig. 6D, E) showed better effects than the metformin group in terms of the recovery of STZ-induced impairment of pancreatic islets (40 mg/kg). These findings show that M. alba fruit polysaccharides can enhance the restoration of islets in the hyperplastic pancreas in T2DM rats.
3.4. Ameliorative effect on insulin resistance Fig. 3A shows that the FINS level of the DM group (12.89 ± 1.36 mIU/L) was significantly higher than that in the NC group (10.42 ± 1.07 mIU/L) after seven weeks of treatment (P < 0.01). However, relative to the DM group, the FINS of both the MFP50 (9.48 ± 2.28 mIU/L, P < 0.05) and MFP90 (8.70 ± 1.32 mIU/L, P < 0.01) groups were significantly reduced. At the end of the experiment, the FINS levels of the MFP50 and MFP90 groups further decreased compared to that in the NC group. The FINS levels decreased in the metformin group (11.65 ± 2.07 mIU/L), although this was not determined to be statistically significant. Fig. 3B shows that the HOMA-IR value of the DM group was significantly higher than that in the NC group (P < 0.01). After seven weeks of treatment, the HOMA-IR values of the MFP90 group (4.35 ± 1.62) were markedly lower than that in the metformin group (4.70 ± 2.43). These findings indicated that the M. alba fruit polysaccharides could effectively decrease the FINS and HOMA-IR values in T2DM rats.
3.8. M. alba fruit polysaccharides upregulated the expression of insulin signaling pathway The alternations of InsR, IRS-2, Akt, and GLUT4 in liver tissues in various treatment groups are shown in Fig. 7. A significant decrease in
Fig. 2. Area under the curve of OGTT in each group (A), glycated serum protein levels of each group (B), (n =6),
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P < 0.01 vs. NC. *P < 0.05 vs. DM. **P < 0.01 vs. DM.
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Fig. 3. Serum insulin levels of each group (A) and values of HOMA-IR in each group (B), (n =6).
##
P < 0.01 vs. NC. *P < 0.05 vs. DM. **P < 0.01 vs. DM.
Fig. 4. Serum HDL-c levels in each group (A), (n =6), serum TC levels in each group (B), (n =5), the ratio of TC/HDL-c in each group (C), (n =6), and serum TG levels in each group (D), (n =6). ##P < 0.01 vs. NC. *P < 0.05 vs. DM. **P < 0.01 vs. DM.
addition, the MFP50 group showed a significant upregulation in the expressions of Akt and GLUT4 (P < 0.05) by 131.3% and 262.1%, respectively.
InsR (P < 0.01) and IRS-2 (P < 0.01) expression of DM, Metformin, MFP50, and MFP90 groups were observed compared to that in the controls. The expressions levels of InsR and IRS-2 of the DM group decreased by 47.0% and 51.0%, respectively. After seven weeks, metformin upregulated the expressions of InsR, IRS-2, Akt, and GLUT4 by 115.3%, 86.9%, 113%, and 410.5%, respectively. The MFP90 group showed an upregulation of the expressions of InsR, IRS-2, Akt, and GLUT4 by 107.1%, 98.2%, 179%, and 420.7%, respectively. However, the MFP50 group showed a slight upregulated in the expression of IRS-2 (46.67%, P < 0.05), although this was not significant, as well as in the expression of InsR (28.96%, P > 0.05). In
4. Discussion Diabetes is one of the third healthy-killers and the diabetic was increased more and more sharply. Moreover, in the clinic, metformin, sulfonylurea, rosiglitazone, and α-glucosidase inhibitors all impart severe side-effects after prolonged treatment. M. alba, as a traditional Chinese medicine, is famous for its stable antidiabetic effect. In the 123
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Fig. 5. Serum alanine transaminase (ALT) levels in each group, (n =6), NC. *P < 0.05 vs. DM. **P < 0.01 vs. DM.
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et al., 1977). Our results indicate that MFP50 and MFP90 can significantly ameliorate hyperlipidemia. Fig. 4 shows that MFP50 reduced TG levels (P < 0.01) and lowered the ratio of TC/HDL-c (P < 0.05), but significantly increased HDL-c levels (P < 0.01). MFP90 reduced TC (P < 0.05) and TG (P < 0.01) levels and lowered the ratio of TC/HDL-c (P < 0.01). Metformin reduced TG levels (P < 0.05). In summ, both MFP50 and MFP90 demonstrated significant effects in reducing FBG, alleviating insulin resistance, and improving the ability of lipid metabolism, which were in agreement with a previous study using total M. alba fruit polysaccharides (Guo et al., 2013). Interestingly, MFP50 also improved glycometabolism and insulin resistance as indicated by changes in FBG, OGTT, FINS, HOMA-IR levels, whereas MFP90 enhanced lipid metabolism, including those of HDL-c, TG and TC levels. Dysregulation of lipid metabolism can result in systemic disruption of insulin and glucose metabolism. Therefore, the cure of hyperlipidemia was indispensable as hyperglycemia. The high level of ALT is a hallmark feature of liver dysfunction (Mangus et al., 2015). Herein, we selected ALT levels as a bioindicator of hepatic function in an animal model for T2DM. MFP50 significantly decreased ALT levels in T2DM rats, indicating that it imparts a potential ameliorative effect on hepatic function. In addition, histopathologic observations were performed to determine the effects of the polysaccharide extracts on pancreatic islet cells. We have thus elucidated that MFP50 and MFP90 have significant antihyperglycemic, antihyperlipidemic, pancreas protective, and potential hepatoprotective effects. Furthermore, M. alba fruit polysaccharides probably could prevent complications caused by hyperlipidemia such as cardiovascular disease (CVD) (Howard et al., 2000) or at least reduce the risk of CVD (Getz et al., 2010). However, more studies are needed to confirm these hypotheses. The underlying mechanism of these anti-diabetes effects required further exploration. Therefore, we measured the effects of MFP50 and MFP90 on the insulin signaling pathway firstly. By definition, insulin resistance is a defect of signal transduction. Insulin action is initiated through binding to and activation of cell-surface receptors and insulin receptors (InsR). When InsR was activated, a series of biochemical reactions were initiated to promote glucose utilization by reducing hepatic glucose output (via decreased gluconeogenesis and glycogenolysis) and increasing the rate of glucose uptake in muscle and adipose tissue (Pessin and Saltiel, 2000). Insulin receptor substances (IRS) are a series of proteins that play a key role in insulin signaling pathway (White, 1998). IRS-2 is mainly expressed in the liver and pancreatic β cells. Recent studies have suggested that the expression of IRS-2 plays an extremely important role in insulin resistance and T2DM (Withers et al., 1998). Knocking out the gene of IRS-1 and IRS-2 in mice not only results in insulin resistance, but also reduced the number of β cells and symptoms of T2DM. These studies indicated that if only IRS-2 function is abnormal, then the diabetic symptoms would present. According to previous studies, glucose transporter type 4 (GLUT4), which is primarily found in adipose and striated muscle, is an insulinregulated glucose transporter (Bogan et al., 2003). Moreover, it serves as the rate-limiting step of glucose metabolism; therefore, it was crucial to evaluate its expression levels in the present T2DM study. Lastly, GLUT4 is mainly regulated by the insulin signaling pathway (Perrini et al., 2004). A prior study has revealed that Akt plays a critical role in the insulin signaling pathway, and ultimately, insulin sensitivity (George et al., 2004). A decrease in Akt expression could lead to a downregulation of GLUT4 (Sano et al., 2003). Here, MFP90 significantly upregulated the expression of all four proteins, and MFP50 also upregulated the expression of IRS-2, Akt, and GLUT4 (Fig. 7). However, this integrated study on the insulin signaling pathway of MFP50 and MFP90 requires further validation.
P < 0.01 vs.
present study, we first extracted the M. alba fruit polysaccharides by using the classical method of water extraction and ethanol precipitation via graded ethanol precipitation. In addition, we evaluated the antihyperglycemic, antihyperlipidemic, pancreas protective, potential liver protective effects, and the protein expressions of insulin signaling pathway of the two fraction polysaccharides for the first time. Currently, metformin is considered as the gold standard for the treatment of T2DM. It also has antihyperlipidemic activity (American Diabetes Association, 2015; Stern and Murphy, 2015). Diabetic animal models play an important role in pharmacological activity evaluation. In this study, the diabetic rats exhibited significantly higher FBG levels (Table 1), worse OGTT (Figs. 1 and 2A), higher GSP levels (Fig. 2B), more serious insulin resistance (Fig. 3B), higher levels of hyperlipidemia (Fig. 4), and severe pancreatic impairment (Fig. 6) compared to the control rats. These results indicated that the model for type 2 diabetes was created successfully. The hypoglycemic activity experiments of the M. alba fruit polysaccharides in the diabetic mice produced an significant (P < 0.05) decrease in FBG levels compared to the T2DM group. The MFP50 and MFP90 groups showed a 31.9% (P < 0. 01) and 47.5% (P < 0.01) reduction in FBG levels, respectively. Metformin reduced it by 55.4% (P < 0.01) at the end of the experiment. Over these seven weeks of treatment, the hypoglycemic effects of the M. alba fruit polysaccharides were relatively stable as was metformin. Moreover, the MFP90 significantly reduced GSP levels 31.7% (P < 0.01) even more than 29.0% (P < 0.05) in metformin. The GSP levels can effectively reflect the blood glucose levels over a period of time (Lapolla et al., 1988). These findings suggest that the hypoglycemic activity of M. alba fruit polysaccharides were significantly reliable in treating T2DM. T2DM is characterized by insulin resistance. Thus, decreasing insulin resistance is a vital method to treat T2DM. Here, MFP50 and MFP90 lowered the serum insulin levels at a rate of 26.45% and 32.51% versus the DM group (Fig. 3A). The HOMA-IR values were greatly reduced (Fig. 3B), indicating that MFP50 and MFP90 can ameliorate insulin resistance in T2DM rats. Moreover, MFP50 and MFP90 repaired the damages that were incurred by the pancreatic islets (Fig. 6), even better than metformin. Based on these findings, we hence conclude that M. alba fruit polysaccharides could improve insulin sensibility and protect the pancreas, but not increase insulin secretion. Furthermore, these observations indicated that the M. alba fruit polysaccharides imparted antihyperglycemic effects by improving insulin sensibility. Dyslipidemia is one of the hallmark features of T2DM and the primary cause of cardiovascular diseases in patients with T2DM (Taskinen and Smith, 1998). Hyperlipidemia is characterized by high levels of TC, LDL-c, and TG, coupled with low levels of HDL-c (Castelli
5. Conclusions In summary, this study has proven that M. alba fruit polysacchar124
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Fig. 6. Histological analysis of pancreatic tissues sections in each group. Conventional H & E staining was performed (Magnification: 200×). NC group (A), DM group (B), Metformin group (C), MFP50 group (D), and MFP90 group (E). Black arrows are pancreatic islet cells.
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Fig. 7. InsR (A), IRS-2 (B), Akt (C) and GLUT4 (D) relative expression of each group in liver tissues (n=4). #P < 0.05 vs. NC. ##P < 0.01 vs. NC. ###P < 0.001 vs. NC. *P < 0.05 vs. DM. **P < 0.01 vs. DM. ***P < 0.01 vs. DM.
Acknowledgments
ides have good antihyperglycemic activity, antihyperlipidemic effects, and pancreatic protective effects on T2DM rats. This treatment can ameliorate insulin resistance. Although MFP50 and MFP90 revealed significant effects on T2DM, the major mechanisms might be different. We lay the foundation for mechanistic research into their antidiabetic activities. The findings of the present study indicated that MFP50 may ameliorate T2DM only through the PI3K/Akt pathway, and MFP90 has multiple pathways. Moreover, the M. alba fruit polysaccharides might possess hepatic protective activity. Our work provides a scientific basis for the further development of M. alba fruit polysaccharides as a new effective and low-toxicity agent to treat T2DM and also for the exploration of the detailed polysaccharides’ structure with active bioactivities. However, the integrated mechanism of its antihyperglycemic and antihyperlipidemic activity, improved insulin resistance, and pancreatic protective effects have not yet fully elucidated and is thus one of our future research directions. Furthermore, we plan to further purify the active constituents of the mulberry extract described in this study.
This work was financially supported by the National Natural Science Foundation of China (Nos. 81673557, 81102779 and 81274060), the Guangdong Natural Science Foundation (No. 9451022401003453), the Pearl River S & T Nova Program of Guangzhou (No. 2013J2200035), the Innovation Program of the University of Guangdong Province (No. 2014KTSCX118), the Science and Technology Program of Guangdong Province (No. 2014A050503067 and 2015A020211032) and the High-level Talents Program of the University of Guangdong Province (No. 2013). References Al Qahtani, M., Al Backer, T., Al Anazi, T., Al Johani, N., Binsalih, S., AlGobain, M., Alshammari, H., 2015. Impact of lipid disorders on mortality among Saudi patients with heart failure. J. Saudi. Heart Assoc. 27, 91–95. American Diabetes Association, 2014. Diagnosis and classification of diabetes mellitus. Diabetes Care 37, S81–S90.
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