|Year : 2013 | Volume
| Issue : 6 | Page : 556-562
The effect of chinese medicine pu-ren-dan on pancreatic angiogenesis in high fat diet/streptozotocin-induced diabetic rats
Binan Lu, Yongfei Bai, Ziliang Du, Shu Chen, D Deligema, Zongran Pang
Institute of Chinese Minority Traditional Medicine, Minzu University of China, Beijing 100081, China
|Date of Submission||14-Dec-2012|
|Date of Decision||08-Apr-2013|
|Date of Acceptance||12-Sep-2013|
|Date of Web Publication||14-Nov-2013|
Institute of Chinese Minority Traditional Medicine, Minzu University of China, Beijing 100081
Source of Support: This work was partly supported by Beijing Natural Science Foundation (NO. 7122091) and Independent Research Project of Minzu University of China (NO. 112KYXJ09), and also supported in part by Academic Scholarship for Doctoral Candidates of Ministry of Education., Conflict of Interest: None
Objectives: The islet vascular system is critical for β-cell function. This study investigated the antidiabetic effect of the Chinese Pu-Ren-Dan (PRD) recipe by regulating the pancreatic angiogenic factors in T2DM rats.
Materials Methods: High fat diet/streptozotocin-induced obese type-2 diabetes mellitus rats were developed and treated with PRD for 4 weeks. Then glucolipid metabolism, insulin secretion, pancreatic blood flow, ultrastructure of islet β-cell, histological changes of islet and protein expressions of pancreatic angiogenic factors were investigated.
Results: PRD-reduced T2DM rats' body weight and blood glucose level resisted the lipid metabolism disturbance, and ameliorated the insulin resistance and β-cell function. In addition, the histological and morphological studies proved that PRD could maintain the normal distribution of endocrine cell in islet and normal ultrastructure of β cell. An increased pancreatic blood flow was observed after the PRD treatment. In the investigation of pancreatic angiogenic factors, PRD inhibited the decreased expression of VEGF and Ang-1, and reversed the reduction of VEGFR2 and Tie2 phosphorylation in T2DM rats; the Ang-2 and TGFβ expression were up-regulated by PRD while PKC was activated; endostatin and angiostatin were down-regulated by PRD.
Conclusions: The results suggest that increasing VEGF expression, regulating VEGF/VEGFR2 signaling, stimulating Ang-1/Tie-2 signaling pathway, and inhibiting PKC-TGFβ signaling and antiangiogenic factors might be the underlying mechanism of PRD's antidiabetic effect.
Keywords: Pancreatic angiogenic factors, traditional Chinese medicine Pu-Ren-Dan, type-2 diabetes mellitus
|How to cite this article:|
Lu B, Bai Y, Du Z, Chen S, Deligema D, Pang Z. The effect of chinese medicine pu-ren-dan on pancreatic angiogenesis in high fat diet/streptozotocin-induced diabetic rats. Indian J Pharmacol 2013;45:556-62
|How to cite this URL:|
Lu B, Bai Y, Du Z, Chen S, Deligema D, Pang Z. The effect of chinese medicine pu-ren-dan on pancreatic angiogenesis in high fat diet/streptozotocin-induced diabetic rats. Indian J Pharmacol [serial online] 2013 [cited 2020 Jan 27];45:556-62. Available from: http://www.ijp-online.com/text.asp?2013/45/6/556/121364
| ╗ Introduction|| |
Type-2 diabetes mellitus (T2DM) is characterized by a state of chronic hyperglycemia, accompanied by macro- and micro-angiopathy in multiple organs, including eyes, kidneys, nerves, peripheral arteries, heart, and pancreas.  Mature pancreatic islets are highly vascularized by a dense capillary network, which allows the β cell to quickly sense and respond to changes in blood glucose by secreting insulin, a critical regulator of this metabolism.  Multiple abnormalities of angiogenesis have been found in animal models of diabetes mellitus and could contribute to the pathogenesis of this disease. Angiogenesis is controlled by 'angiogenic switch', which is the balance between proangiogenic and antiangiogenic factors,  including vascular endothelial growth factors (VEGF), angiopoietins, transforming growth factor-β (TGF-β), endostatin, and angiostatin. During diabetes, the angiogenic answer to ischemia differs according to tissue, which is either excessive in certain organs such as the retina, or defective in other organs such as the heart.  Angiogenesis is important to maintain the islet structure and functions through regulation of local blood flow.  But, so far, the impairment of angiogenesis in pancreas during diabetes has remained largely unidentified. We have therefore focused our research on the diabetes reduced angiogenesis of pancreas, which is associated with defective proangiogenic factor expression, over-expression of antiangiogenic factor, and the impairment of angiogenic signaling transduction.
Traditional Chinese medicine (TCM) originated in ancient China and has evolved over thousands of years to promote health and treat disease. Pu-Ren-Dan (PRD), a traditional Chinese herbal medicine formula, has been used for the treatment of type-2 diabetes mellitus in China, which is composed of six medicinal materials [Table 1]. In our previous study, the PRD and its constitutive extracts showed a significant antidiabetic effect on diabetic patients.  We found that the PRD not only decreased blood glucose levels, but it also resisted the lipid metabolism disorders, improved insulin resistance and reduced diabetic complications.  Thus, it was necessary to investigate the effect and its mechanism using an animal model. In this study, a type-2 diabetic rat model was established by high-fat diet combined with single and quick injection in vena caudalis of low-dose Streptozotocin (STZ). The T2DM relevant indexes were observed in model rats after administration of the PRD preparation. Effects of PRD on pancreatic microcirculation and its mechanisms were also explored.
| ╗ Materials and Methods|| |
The PRD Extract Preparation
The PRD is composed of six crude drug materials as shown in [Table 1]. All crude drugs were purchase from Tong Ren Tang Traditional Chinese Medicine Co., Ltd. (Beijing, China). Momordicacharantia L. (Cucurbitaceae) was lyophilized to powder; Panax ginseng C. A. Meyer and Salvia miltiorrhiza Bge were grinded to small granular of approximately 5 mm and extracted with ten times the amount of distilled water three times for 2, 1.5, and 1 h, respectively; Puerariapeduncularis (Grah. ex Benth.) Benth. (Leguminosae), and Polygonummultiflorum Thunb were extracted with ten times the amount of 70% ethanol twice for 2 h and 1.5 h, respectively; Hirudomedicinalis was extracted with ten times the amount of 90% ethanol twice for 2 and 1.5 h, respectively. All the above extraction were combined and filtered, and then the filtrate was evaporated under reduced pressure and dried out by the spray-drying method. The PRD extract of proper concentration was prepared in distilled water.
Healthy male Wistar rats of specific pathogen-free (SPF) grade weighing 250 ± 10 g were purchased from Vital River Laboratories (Beijing, China). The rats were raised with sufficient food and water at an ambient temperature of 25°C ± 1°C, a relative humidity of 48% ± 2%, and under a 12 h light/dark cycle. All experimental measures were carried out in accordance with the approved guidelines (Guidelines for the Care and Use of Laboratory Animals) established by the Chinese Council on Animal Care.
Obese Rats with the Type-2 Diabetes Mellitus Model
The animal model of obesity and type-2 diabetes mellitus (T2DM) was developed by high-fat diet fed for 12 weeks combined with single and quick injection in vena caudalis of low-dose STZ (Sigma Chemicals, St. Louis, MO) dissolved in the citrate buffer (pH 4.4, Sigma Chemicals, St. Louis, MO) at a dose of 30 mg/kg B. W., while the control animals received citrate buffer alone. The animals were grown on high fat diet and water available ad libitum. After injection for 72 h, a blood sample was drawn from vena caudalis, and rats with a non-fasting glucose level >16.7 mmol/l over 2 days were chosen as T2DM animals.
Twenty normal rats were included in the control group, and 80 obese T2DM rats were randomly divided into four groups: the model group, PRD group, metformin (MF) group, and Jiang-tang Tong-mai tablet (JT) group. Each group contained 20 rats initially. PRD was administered intragastrically (i.g.) at a dose of 1770 mg/kg/d, which equals the everyday therapeutic dosage of an adult × animal-human dosage exchange ratio.  MF (140 mg/kg/d) and JT (420 mg/kg/d) was administered as the positive control. The control group and the model group were only administered an equal volume of distilled water. All operations continued for four weeks. During the 4 weeks, the model group and each treated group were continuously fed high fat diet, while the control group was fed normal diet. Body weight and the nonfasting blood glucose were measured every 2 weeks. After 4 weeks and 2 h after the final treatment, the blood of rats was drawn from inferior vena and then centrifuged at 3000 rpm for 10 min, and the obtained serum samples were stored at -80°C for the determination of blood biochemistry and serum insulin. After animals were sacrificed, the pancreases were promptly removed. A portion of the pancreas was fixed and the remaining tissues were stored at -80°C for western blotting assay.
Measurements of HbA1c and Serum Biochemical Indices
Blood plasma Hemoglobin A 1c (HbA1c) was determined with a Bio-Rad D-10 Hemoglobin Testing System (Bio-Rad, USA); glucose (GLU), triglyceride (TG), total cholesterol (CHO), high-density lipoprotein-cholesterol (HDL-c), and low-density lipoprotein-cholesterol (LDL-c) were determined with a Hitachi 7020 Automatic Biochemistry Analyzer (Hitachi, Japan).
Serum Insulin Assay
Serum insulin was measured using a SN-695B g-counter (China). Fasting insulin (FINS), fasting blood glucose (FBG), insulin sensitivity index (ISI), and homeostasis model assessment insulin resistance index (HOMA-IR) were calculated as described previously. ISI = ln1/(FINS × FBG), HOMA-IR = (FINS × FBG)/22.5. 
Measurement of Pancreatic Blood Flow
The animals were deeply anesthetized by intraperitoneal (i.p.) injection of 10% chloral hydrate (30 mg/kg), and then the pancreas of rats was exposed by surgery. A MoorFLPI laser-Doppler flowmeter (Moor Instruments, UK) was used to measure the blood flow. Values of control animals were used as baseline, and the same pancreas was measured three times and averaged to give the value.
Transmission Electron Microscopy
The LV anterior wall tissue from four rats in each group was cut rapidly into small 1 mm 3 pieces, and immersed in glutaraldehyde. The ultrastructure in the different groups of pancreas tissue was observed using a JEOL JEM-1230 transmission electron microscope (JEOL Ltd., Japan).
Double Staining Immunohistochemical Staining
Immunohistochemical staining was performed on 10% formalin-fixed and paraffin-embedded tissues. Deparaffinized sections were incubated in 3% H 2 O 2 dissolved in PBS for 30 min and normal goat serum to block nonspecific protein binding for 30 min. Then the sections were incubated at 4°C overnight with anti-insulin and antiglucagon antibodies, followed by incubation in the mixture of horseradish peroxidase (HRP) affiniPure goat antirabbit Ig G and alkaline phosphatase (AP) affiniPure goat antimouse Ig G. Then, DAB Kit and AP-Red kit, used as the chromogen, was counterstained by hematoxylin. Image was taken by OLYMPUS BX51 microscope (Olympus, German).
Western Blotting Assay
For Western blotting assay, proteins of pancreas tissues were collected and separated electrophoretically on SDS gel, then transferred to a PVDF membrane (Millipore, USA). After block nonspecific protein binding in 10 mM TBS with 1.0% Tween 20 and 1.0% bovine serum albumin (BSA), the PVDF membrane was incubated overnight at 4°C with primary antibodies including p-VEGFR2 (Abcam, 1:1000), VEGFR2 (Abcam, 1:1000), VEGF (Abcam, 1:2000), p-PKCα (Abcam, 1:1000), PKCα (CST, 1:1000), Ang-1 (Santa, 1:2000), Ang-2 (Santa, 1:200), p-Tie2 (Abcam, 1:1000), Tie2 (Abcam, 1:1000), TGFβ (CST, 1:10000), Endostatin (Santa, 1:200), Angiostatin (Abcam, 1:1000) and GAPDH (SiNoble, 1:5000). Blots were then incubated with horseradish peroxidase-conjugated antibodies for 2 h at room temperature. Detection was performed by an enhanced chemiluminescence (ECL) method and photographed by Bio-Spectrum Gel Imaging System (UVP, USA). To eliminate the variations due to protein quantity and quality, the data were adjusted to GAPDH expression (IOD of objective protein versus IOD of GAPDH protein).
Data are shown as the mean ± standard deviation (SD). Multigroup comparisons were performed through One-way ANOVA test, while couple comparisons were performed through t-test. Differences were considered significant at P<0.05. Statistical analysis was performed using the Statistical Package for Social Sciences software, version 18.0 (SPSS, USA).
| ╗ Results|| |
Effect of PRD on Body Weight, Glucolipid Metabolism and Insulin Secretion
During the experiment, the body weight of rats in control group was steady, while rats in other four groups began to lose weight after the second week, but only the PRD group obtained the significant decrease in body weight at the fourth week [Figure 1]a. Blood glucose levels in the model group remained significantly high throughout the experiment. After each treatment for two weeks, blood glucose levels were reduced, but only PRD treated group has significant differences compared to the T2DM group. At the fourth week, blood glucose levels in each treated group were descending, and the effect of PRD were equivalent or even better than the effects produced by MF (140 mg/kg) or JT (420 mg/kg) [Figure 1]b. To further estimate the mean blood glucose concentration during the treatment, HbA1c level was measured and a significant increase of HbA1c level were observed in model group, but reduced by PRD treatment [Figure 1]c. In addition, lipid profiles were measured, as we found, CHO, TC, and LDL-c levels were significantly increased in model group compared with control group. After PRD treatment, CHO, TC, LDL-c levels were reduced, but HDL-c level had no significant differences between each group during the experiment [Figure 1]d.
|Figure 1: Effect of PRD on body weight, glucolipid metabolism, and insulin secretion. (a) Body weight, the red line represents the average body weight during the 4 weeks; (b) non-fasting blood glucose; (c) The level of HbA1c in blood; (d) The levels of CHO, TG, HDL-c and LDL-c in serum. Each value is expressed as mean ± S.D. (n = 20). *P<0.05, **P<0.01, ***P<0.001|
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Effect of PRD on Ultrastructure of Islet β Cell and Insulin Secretion
As observed using transmission electron microscopy (TEM), islet β cells in the control group possessed rounded or oval nuclei with a moderately distinct nucleolus. The chromatin was finely dispersed and the nuclear membranes and pores were easily discerned. The endoplasmic reticulum was mostly of rough type and either vesicular or lamellar. The mitochondria are medium-sized, rounded, oval, or elongated and possess a moderate number of mainly transverse cristae. Mature secretory granules in islet β cells were diffusely distributed in the cytoplasm, and the granules were composed of a central core and an external single-layered membrane with a rather large space between the core and the membrane [Figure 2]a-1. In the T2DM model group, islet β cell contained scanty organelles and secretory granules, and apoptosis of acinar cell was observed [Figure 2]a-2. After the treatment of PRD, islet β cell contained remarkably increased the number of immature secretory granules, mitochondria and the hypertrophied cytoplasmic organelles such as Golgi complex and endoplasmic reticulum, which were also observed in metformin group and JT group [Figure 2]a-3-5. The insulin secretion function of islet β cell was evaluated by ISI and HOMA-IR. Compared with the control group, the ISI of model group decreased, but HOMA-IR significantly increased. After 4 weeks treatment, there were no significant differences in FINS between the PRD and model group; however, the ISI of the PRD group was higher than the model group, while the HOMA-IR was lower with significant differences [Figure 2]b.
|Figure 2: Effect of PRD on islet β cell and pancreas islet. (a) Electron micrograph of islets. Magnification × 8,000. Bar = 1 μm; (b) FINS in serum and insulin secretion index: HOMA-IR and ISI; (c) immunohistochemical stainings of islet. Each pancreas was stained with antibodies against insulin (brown) and glucagon (red). Magnification ×400. Bar = 100 μm; (d) pancreatic blood flow. Each value is expressed as mean ± S.D. (n = 20). *P<0.05, **P<0.01, ***P<0.001|
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Effect of PRD on Morphous of Pancreatic Islet and Pancreatic Blood Flow
On double staining immunohistochemical staining, it was observed that the distribution of endocrine cells was like normal pattern in each islet of rats in control group: β cells located centrally and α cells located at the periphery [Figure 2]c-1. However, the distribution of both endocrine cells was changed to a mixed pattern and emerged destructed islets with fibrosis and poor margination in the T2DM model group [Figure 2]c-2. After the treatment with each drug, the sections of pancreas in PRD group and other two positive medicine groups showed islets with a relatively well-preserved typical distribution, and some islets were enlarged and showed newly developed blood vessels and ducts in the periphery of islet [Figure 2]c-3-5. The blood flow in pancreas tissue was markedly decreased in the model group compared with the control group (390.58 ± 108.96 vs. 1356.05 ± 135.13). This change was inhibited by PRD treatment (720.86 ± 176.51 vs. 390.58 ± 108.96) [Figure 2]d.
Effect of PRD on Angiogenic Factors of Pancreas
In order to evaluate the effect of PRD on angiogenesis factors of pancreas, the expression of some relative proteins were investigated [Figure 3]. Up-regulations of endostatin, angiostatin, TGFβ, Ang-2, and p-PKC, as well as down-regulations of VEGF, Ang-1, p-VEGFR2, and p-TIE2 were existed in rats of the T2DM model group. But all these changes in protein expressions were significantly reversed by PRD, compared with the model group, expressions of endostatin (1.52 fold vs. 3.33 fold), angiostatin (1.01 fold vs. 1.64 fold), TGFβ (1.46 fold vs. 1.92 fold) and Ang-2(0.73 fold vs. 1.26 fold) were down-regulated and the phosphorylation of PKC (0.77 fold vs. 2.28 fold) was decreased, while VEGF (1.26 fold vs. 0.39 fold) and Ang-1 (0.88 fold vs. 0.43 fold) were up-regulated and the phosphorylation of TIE2 (0.63 fold vs. 0.25 fold) and VEGFR2 (0.57 fold vs. 0.28 fold) were increased after the treatment of PRD. The regulation effect of PRD on these proteins was similar to the effect of other two drugs.
|Figure 3: Effect of PRD on angiogenic factors of pancreas. The data were adjusted to GAPDH expression (IOD of objective protein versus IOD of ROTGAPDH protein). Each value is expressed as mean ± S.D. (n = 3). *P<0.05, **P<0.01, ***P<0.001|
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| ╗ Discussion|| |
T2DM is a metabolic disease with hyperglycemia and lipid disorders; the pathogenesis of T2DM is the mechanisms underlying the maintenance of insulin secretion in the presence of insulin resistance and the mechanisms that lead to the failure of this adaptive response.  In this study, we have confirmed the remarkable antidiabetic effect of PRD, which was in coincident with our previous reports. PRD reduced the body weight and blood glucose level, and resisted the lipid metabolism disturbance of high fat diet/streptozotocin-induced obese T2DM rats.
Failure of β cell function is a fundamental abnormality in the pathogenesis of T2DM. Normal ultrastructure of islet β cell is prerequisite for its insulin secretion function. In this study, damaged ultrastructure of islet β cell and failure of insulin secretion were both observed in diabetic rat, and after PRD repaired the ultrastructure damage of β cells, the insulin secretion was restored subsequently. Since islets are imbedded in pancreas, the adequate functional islet β cell would require a vasculature that can grow in response to specific signals arising from the islet parenchyma or external signals.  The vasculature is indispensable for maintaining the health of mature tissue by providing nutrients and oxygen, as well as additional growth signals. The islet vascular system is critical for islet β-cell function.  In this study, pancreatic blood flow of T2DM rats was decreased and the morphous of pancreatic islet were anomalous, which indicated that the pancreatic vascular system was damaged and receded during diabetes. After the treatment of PRD, the increased pancreatic blood flow and normal morphous of pancreatic islet revealed that PRD improved the pancreatic vascular system, providing a euangiotic environment for β-cells, which could be an underlying mechanism of the protective effect of PRD on islet β-cells.
As we know, impaired angiogenesis leading to microvascular insufficiency represents a major cause of end-stage organ failure among diabetics; meanwhile the alterations of islet angiogenesis could further lead to a progressive loss of β-cell function.  VEGF is important for the development and function of the endocrine pancreas, as well as islet endothelial cells.  In our study, reductions of VEGF expression and VEGFR2 phosphorylation were found in T2DM rats, which played a key role in the diabetes-associated impairment of angiogenesis. PRD could increase the expression of VEGF and regulate the VEGF/VEGFR2 signaling to produce its effect on pancreatic vascular system. The diabetes-associated impairment of angiogenesis is also associated with disruption of Angiopoietin-1 (Ang-1)/Tie-2 signaling pathway. The increased expression of Ang-2 in T2DM rats might be a cause of the reduction of Tie-2 phosphorylation. We speculated that reduction of Tie-2 expression and disruption of Ang-1/Tie-2 signaling under hyperglycemic conditions might be the underlying cause of impaired angiogenesis in diabetics. The up-regulated expression of Ang-1 or the down-regulated expression of Ang-2 by PRD might induce the increase of Tie-2 phosphorylation, and stimulated the Ang-1/Tie-2 signaling pathway and angiogenesis. Thickening of capillary basement membranes and matrix expansion are the most prominent structural abnormalities of the vasculatures in diabetes.  Hyperglycemia or diabetes could induce PKC activation, which might be responsible for the increased levels of matrix protein synthesis mediated either directly or through over-expression of transforming growth factor β (TGFβ).  In this study, PKC activation and over-expressions of TGF-β were confirmed in pancreas of T2DM rats, which were inhibited by PRD. It is suggested that PKC activation may be involved in the diabetes-induced increase of TGFβ expressions, and might be one target of PRD. In contrast with proangiogenic factors, over-expression of antiangiogenic factors was associated with the impaired angiogenesis in diabetics. Angiostatin potently blocks neovascularization, tumor growth, and metastasis.  Endostatin inhibits migration and promotes apoptosis specifically in vascular endothelial cells via multiple pathways.  In this study, PRD reversed the increased expression of angiostatin and endostatin, which might contribute to improve the vasculature in diabetic pancreas. Studies have revealed that the constituents of PRD exhibit a variety of biological effects. Momordicacharantia L. (Cucurbitaceae) possessing antidiabetic effect  are mainly due to its insulin-mimetic active compounds, including phytochemical momordica, charantia, galactose-binding lectin, and insulin-like protein, which can increase β cell mass and stimulate insulin release.  Ginseng polypeptides from the root of Panax ginseng C.A. Meyercan decrease blood glucose, increased liver glycogen level and stimulated insulin secretion.  Another main component of Panax ginseng, Ginsenoside, such as Rg1, has the ability to promote angiogenesis by regulating VEGF protein expression.  2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-d-glucoside (TSG), an active component extract from Polygonummultiflorum Thunb, ameliorates diabetic nephropathy on account of its antioxidative and anti-inflammatory effects, which involved in the SIRT1 and TGF-β1 pathway.  Danshensu (DSS, 4-methoxy-2-hydroxyacetophenone) isolated from Salvia miltiorrhiza Bgecan attenuate oxidative stress, protect endothelial cells and vascular functions.  Puerarin isolated from Puerariapeduncularis (Grah. ex Benth.) Benth. (Leguminosae) may exert inhibitory effects on high glucose-induced vascular smooth muscle cell proliferation via interfering with PKCβ2/Rac1-dependent ROS pathways, thus resulting in the attenuation of neointimal formation in the context of hyperglycemia in diabetes mellitus.  Hirudomedicinalis has been proved to have markedly improvement effect on blood viscosity,  which might also involved in the increase of pancreatic blood flow induced by PRD. Unlike target-oriented western medicine, the traditional Chinese medicine PRD exerts antidiabetic effect through the holistic and synergistic effects of all active ingredients, such as polysaccharides, saponin, puerarin, and stilbene glycoside. However, in this study, we cannot clarify which ingredient acts most effectively. This requires further studies.
In conclusion, the PRD extract can promote the sensibility of insulin, decrease insulin resistance and blood glucose level in rats with high-fat-diet/STZ-induced diabetes, and it can also ameliorate the pancreatic blood flow and maintain the normal structure of islet and β cells. Regulating the expressions of angiogenic factors to improve pancreatic vascular system might be the underlying mechanisms of those effects of the PRD. It is suggested that maintaining a euangiotic environment for β-cells might be a new insight into the prevention and treatment of diabetes. ('A euangiotic environment' means affluent blood vessels around the β-cells.)
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[Figure 1], [Figure 2], [Figure 3]
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