Article pubs.acs.org/JAFC
Polysaccharides of Trametes versicolor Improve Bone Properties in Diabetic Rats Chung-Hwan Chen,†,‡,§,∥ Lin Kang,⊥ Hui-Chen Lo,# Tai-Hao Hsu,∇ Fang-Yi Lin,∇ Yi-Shan Lin,∥ Zai-Jie Wang,∥ Shih-Tse Chen,○ and Chwan-Li Shen*,◆ †
Department of Orthopaedics, Kaohsiung Municipal Ta-Tung Hospital, ‡Department of Orthopedics, Kaohsiung Medical University Hospital, §Departments of Orthopedics, College of Medicine, and ∥Orthopaedic Research Center, Kaohsiung Medical University, Kaohsiung 807, Taiwan, Republic of China ⊥ Department of Obstetrics and Gynecology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan # Department of Nutritional Science, Fu Jen Catholic University, New Taipei City 510, Taiwan, Republic of China ∇ Department of Bioindustry Technology and Department of Medicinal Botanicals and Health Care, Da-Yeh University, Dacun 515, Taiwan, Republic of China ○ Department of Psychiatry, National Taiwan University Hospital Hsin-Chu Branch, Hsin Chu 300, Taiwan, Republic of China ◆ Department of Pathology, Texas Tech University Health Sciences Center, 1A096B, 3601 4th Street, Lubbock, Texas 79430-8115, United States ABSTRACT: This study investigates the effects of Trametes versicolor (L.:Fr.) Pilát (TVP, also known as Yunzhi) on bone properties in diabetic rats. Forty-five male Wistar rats (8 weeks old) were fed either a chow diet (control) or a high-fat diet throughout the study period of 28 days. Animals in the high-fat-diet group were injected with nicotinamide and streptozotocin to induce diabetes mellitus (DM). The DM rats were divided into a group receiving distilled water (vehicle) and another group receiving TVP at 0.1 g/kg weight by gavage. Relative to the vehicle group, TVP gavage lowered postprandial blood sugar (225 ± 18 mg/dL for TVP vs 292 ± 15 mg/dL for vehicle, p < 0.001) on day 26. Compared to the vehicle group, TVP mitigated DMinduced bone deterioration as determined by increasing bone volume of proximal tibia (22.8 ± 1.4% for TVP vs 16.8 ± 1.3% for vehicle, p = 0.003), trabecular number (p = 0.011), and femoral bone strength (11% in maximal load, 22% in stiffness, 14% in modulus, p < 0.001), and by reducing loss of femoral cortical porosity by 25% (p < 0.001). Our study demonstrates the protective effect of TVP on bone properties was mediated through, in part, the improvement of hyperglycemic control in DM animals. KEYWORDS: cortical porosity, diabetes, microarchitecture, polysaccharopeptides, Trametes versicolor
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INTRODUCTION Diabetes mellitus (DM) is a pandemic metabolic disease causing substantial morbidity and mortality. According to World Health Organization, approximately 150 million people have DM worldwide, and this number may well double by the year 2025.1 Patients with DM exhibit various skeletal disorders with osteopenia and osteoporosis being the most prevalent.2,3 Emerging evidence shows that DM is an independent risk factor for fracture.3 Women with type 1 or type 2 DM have shown higher risk for hip fracture compared to those without DM.4 Recently, the study by Schneider et al. supports the recommendation of American Diabetes Association for assessment of fracture risk and implementation of prevention strategies in patients with type 2 DM, particularly those with poor glucose control.5 DM is characterized by peripheral insulin resistance with variable degrees of hyperinsulinemia (high insulin) and hyperglycemia (high blood sugar) as well as impaired insulin secretion after metabolic challenge by glucose. These characteristics of DM may have a direct, adverse impact on bone and the bone marrow microenvironment including (i) advanced glycation end products of bone matrix proteins, (ii) chronic © 2015 American Chemical Society
inflammation and abnormal cytokine and adipokine production resulting in detrimental effects on bone cells, (iii) impaired neuromuscular/skeletal interactions,2,6,7 and (iv) excessive production of reactive oxygen species.8 Such negative effects would lead to significant bone loss, poor bone quality, and impaired biomechanical properties, thereby significantly increasing the risk of bone fracture in patients with DM.9 In recent years, the use of dietary supplements, nutraceuticals, and functional foods have become an alternative approach to prevent and mitigate the complication of hyperglycemia,10,11 to alleviate inflammation,12 to maintain the oxidant−antioxidant balance,13 and to mitigate bone loss.14 Among different functional foods, mushroom polysaccharides have demonstrated their antioxidant, anti-inflammatory, antihyperglycemic, and antilipidemic activities via in vitro and in vivo studies.15 However, the effects of mushroom polysaccharides on DMinduced bone loss are not clear. Received: Revised: Accepted: Published: 9232
May 29, 2015 August 5, 2015 August 25, 2015 August 26, 2015 DOI: 10.1021/acs.jafc.5b02668 J. Agric. Food Chem. 2015, 63, 9232−9238
Article
Journal of Agricultural and Food Chemistry
a 12:12 h light−dark cycle. After acclimation for 5 days, 35 rats were fed a high-fat diet (40.6% of total calories from fat, TD96132, Harlan Teklad, Madison, WI, USA), and the remaining 10 rats were fed a normal chow diet (the control group) throughout the 28 day study period. After 1 week, all rats were fasted overnight, weighed, and anesthetized under light ether. Rats in the high-fat-diet group were intraperitoneally injected with nicotinamide (200 mg/kg of body weight) 15 min before the injection of streptozotocin (65 mg/kg of body weight) via tail vein. Nicotinamide and streptozotocin were dissolved in a saline buffer and 0.01 M citric saline buffer (pH 4.5), respectively, and were filter-sterilized.26 Rats in the control group were intraperitoneally injected with saline to control for the stress of injections. Five days later, the fasting blood glucose levels were determined using an Accu-chek Advantage II glucose meter and strips (Roche Diagnostics, Indianapolis, IN, USA). Subsequently, rats were intragastrically administered 30% glucose solution (2 g/kg of body weight), and blood samples were collected via tail vein at 120 min for the determination of the 2 h postprandial blood glucose levels. Those animals (n = 30) having 2 h postprandial blood glucose level higher than 200 mg/100 mL were categorized as diabetic animals. The diabetic animals were then divided into two groups and intragastrically administered 0.5 mL of distilled water (the vehicle group, n = 14) or TVP LH-1 ePSP (0.1 g/kg of body weight; the TVP group, n = 16) daily for 28 days (the study period). Animals in the control group were administered 0.5 mL of distilled water daily to control for the stress of gavage. The final sample size in the control, vehicle, and TVP groups was 10, 12, and 15, respectively, with the loss of 3 rats for the intragastric administration. Throughout the study, body weights and feed consumption of animals were recorded every week. The 2 h postprandial blood sugar samples were acquired on days 1, 20, and 26 after the vehicle/TVP treatment began. At the end of study (viz. 28 days after the vehicle/ TVP treatment began), animals were euthanized, and tibia and femora were harvested, cleaned of adhering soft tissues, and kept at 4 °C for later bone scans and bone strength tests. Assessment of Bone Microarchitecture by Microcomputed Tomogrphy. The bone microarchitecture of the proximal tibia was assessed by microcomputed tomography (Skyscan 1076: Skyscan, Antwerp, Belgium). Data were collected at every 0.5° rotation increment through 180°. The scanning width was 34 mm, and the height was 17 mm. The voxel size was isotropic and fixed at 8.7 μm. The trabecular bone parameters were calculated using the Skyscan software, CTan (Skyscan). Morphometric indices of the trabecular bone region were determined from the microtomographic data sets using direct 3D morphometry. Trabecular bone volume fraction (BV/ TV, %), trabecular thickness (Tb.Th, μm), trabecular number (Tb.N, n/mm), and separation (Tb.Sp, μm) were calculated. Coefficients of variation were 2.0% (BV/TV), 1.1% (Tb.N), 0.66% (Tb.Th), and 1.30% (Tb.Sp) for morphometric parameters. Cortical bone volume fraction (BV/TV, %) and cortical thickness were also calculated using the Skyscan software CTan (Skyscan).27 Cortical porosity was measured using previously described techniques based on the cortical pore volume and mineralized cortical bone volume.28 Assessment of Femoral Mechanical Property Using 3-Point Bending Test. Femoral quality (bone strength) was evaluated by 3point bending test using an Instron 4466 (Instron, Canton, Massachusetts). The femur bone was positioned onto two supports, and a single-pronged loading device was applied to the opposite surface precisely in the middle between the two supports. The distance between the two supports was 4 cm. The loading force was 1 N with a speed of 1 mm/min. The experimental procedure involved the measurement of the deflection of the bone at the point of load application and the concurrent measurement of the load, yielding a force−deflection graph. Parameters obtained from this graph include whole-bone stiffness (defined as the slope of the early, linear portion of the load−deflection curve), yield point, maximal load, and fracture load. The Young’s modulus of the material from the geometry of the loading device and the stiffness of the bone were measured. Data calculations were all based on beam theory and Hooke’s law.
Coriolus versicolor is the one of the most popular medicinal mushrooms because of its various biologically active components.16−18 The intracellular polysaccharopeptides of Coriolus versicolor has been shown to have immune-regulatory function in normal19 and type 1 DM rats.20 Among Coriolus versicolor, Trametes versicolor (L.:Fr.) Pilát (TVP) (also named Yunzhi in China) has been successfully grown in submerged fermentation as mycelial biomass.21 Recently, a novel TVP strain, LH-1, and its fermented extracellular polysaccharopeptides (ePSP) have different compositions of simple sugar, protein, and beta-glucan compared to those of intracellular PSP.21,22 Antihyperglycemic activity of TVP LH-1 ePSP at different dosages (0.1, 0.5, and 1 g/kg/day orally) has been reported in diabetic rats.23 Up to now, no study has ever been conducted to investigate the effects of mushroom polysaccharides on bone properties, especially in DM animals or patients. Therefore, the purpose of the present study was to investigate the potential benefit of TVP LH-1 ePSP on bone microstructure and bone quality in diabetic rats. We hypothesized that supplementation of TVP LH-1 ePSP will mitigate DM-induced bone loss, microstructural deterioration, and compromised bone strength in diabetic rats. The novelty of this study was to demonstrate beneficial impacts of TVP supplementation on DM-induced unfavorable hyperglycemia and simultaneously to elucidate TVP’s osteo-protective role. Studying the effect of TVP LH-1 ePSP on bone properties in diabetic rats will advance the understanding of TVP LH-1 ePSP’s effects on skeletal biology and possibly lead to the establishment of their beneficial effects with regard to minimizing bone loss in human with DM.
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MATERIALS AND METHODS
Preparation of Polysaccharides TVP LH-1 ePSP. Preparation of TVP LH-1 ePSP has been described in our previous studies.21,24,25 Briefly, the culture of T. versicolor LH1 was maintained on potato dextrose agar plates at 25 °C. For seeding, the cultures were cultured in the culture medium (4.0% glucose, 0.3% peptone, 0.15% KH2PO4, and 0.15% MgSO4·7H2O) in Erlenmeyer flasks at 25 °C on a rotary shaker at 150 rpm; 5 days old cultures were used. Batch fermentation of T. versicolor LH1 was carried out in a 20 L fermenter (Biotop, Taiwan) in the same culture medium at 25 °C with initial medium of pH 4.5 at 100 rpm for 7 days. After fermentation, mycelium was removed by centrifugation at 6000 g for 30 min, and the supernatant from fermented culture broth was processed for TVP LH-1 ePSP preparations. To the supernatant were added 4 equivalent volumes of 95% ethanol, and this was allowed to settle at 4 °C for 24 h. The precipitates were collected by centrifugation at 9000 g for 30 min, then lyophilized and assigned as TVP LH-1 ePSP. Chemicals. The chemical composition analysis of TVP LH-1 ePSP has been described in our previous studies.21,24 In brief, TVP LH-1 ePSP mainly contains polysaccharide with 13.8% protein, and the monosaccharide composition of polysaccharide was composed of mainly glucose (82.27 mg/g), galactose (8.67 mg/g), mannose (8.18 mg/g), and xylose (0.87 mg/g).21,24 Nicotinamide and streptozotocin were purchased from SigmaAldrich (St. Louis, MO, USA). Accu-chek Advantage II glucose meter and strips were purchased from Roche Diagnostics (Indianapolis, IN, USA). Animals and Experimental Design. The animal facilities and protocols of the present study were approved by the Institutional Animal Care and Use Committee in Fu Jen Catholic University, New Taipei City, Taiwan. Forty-five Wistar male rats (8 weeks old, ∼280 g each; BioLASCO Taiwan Co., Ltd., Taipei, Taiwan) were housed in individual stainless-steel cages with free access to water and a normal chow diet (13.5% of total calories from fat, 5001 Laboratory Rodent diet, Labdiet, Richmond, IN, USA) in a room maintained at 22 °C on 9233
DOI: 10.1021/acs.jafc.5b02668 J. Agric. Food Chem. 2015, 63, 9232−9238
Article
Journal of Agricultural and Food Chemistry Statistical Analysis. Data are expressed as mean ± standard error of the mean (SEM). All data were analyzed using SigmaStat, version 2.03 (Systat Software, Inc., San Jose, CA, USA). Normality of distribution and homogeneity of variance were tested. Data of blood glucose and bone parameters were analyzed by one-way ANOVA followed by Fisher’s least significant difference (LSD) post hoc test to evaluate the effects of treatment. The level of significance was set at P < 0.05 for all statistical tests.
lower than that of the control group, and BV/TV of the TVP group is significantly higher than that of the vehicle group. Exhibited in Figure 2 are the 3D cortical structures (Figure 2A) and cortical porosity (Figure 2B) of femoral diaphysis. Figure 2A shows that the cortical bone of the TVP group is generally thicker than that of the vehicle group and comparable with that of the control group, whereas Figure 2B shows that the cortical porosity at femoral diaphysis of the vehicle group is higher than that of both the control and the TVP groups. The cortical porosity decreased from 24% in the vehicle group to 18% in the TVP group (P < 0.001). There was no difference in cortical porosity between the control and the TVP groups. Mechanical Strength of Femur. According to the results of 3-point bending test in Table 3, maximal load, stiffness, and Young’s modulus were lower in the vehicle group compared to the control group. TVP administration to the diabetic rats significantly increased maximal load, stiffness, and Young’s modulus relative to the vehicle group.
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RESULTS Body Weight. The body weights of rats increased as the rats aged. The vehicle and TVP groups had significantly greater body weights than the control group, whereas there was no difference in body weight between the TVP and the vehicle groups (control: 386 ± 11 g < vehicle: 432 ± 7 g = TVP: 437 ± 7 g). Postprandial Blood Glucose. Compared to the control group, both the vehicle and TVP groups had significantly higher values for 2 h postprandial blood glucose on days 1, 20, and 26 (Table 1). There was no difference in blood glucose
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DISCUSSION In the present study, diabetic rats were utilized to demonstrate beneficial impacts of TVP supplementation on DM-induced unfavorable hyperglycemia and to elucidate TVP’s osteoprotective role simultaneously. We found that mushroom polysaccharides, TVP LH-1 ePSP, can reduce 2 h postprandial blood sugar and femoral cortical porosity and improve proximal tibial microarchitecture and femoral bone strength in diabetic rats. This is the first report demonstrating the effects of TVP LH-1 ePSP on suppressing blood sugar and improving bone properties in diabetic animals simultaneously. The findings of the antihyperglycemic effect of TV LH-1 agrees with the results of Hsu et al.19 study’s that yellow brain culinary−medicinal mushroom, Tremella mesenterica Ritz.:Fr. (higher Basidiomycetes) is antihyperglycemic. In Hsu’s study,19 authors also reported that Tremella mesenterica Ritz.:Fr has immunomodulatory effects on type 1 DM rats via partially reversing the proliferation of T splenocytes and suppressing the production of pro-inflammatory cytokines in rats with impaired glucose tolerance. As expected, bone microarchitecture and bone strength of animals deteriorated after DM induction. In the present study, the findings that DM rats demonstrated lower bone volume (BV/TV) and higher cortical porosity, resulting in compromised bone strength is consistent with the results of Campbell et al.29 using Zucker Diabetic Fatty rats as well as Fowlkes et al.30 using STZ-induced mouse model of type 1 DM. The current work also corroborated with recent human studies.31−33 In their finite element analysis, Patsch et al.31 reported that cortical porosity impaired more significantly the mechanical strength of distal radius and distal tibia in DM patients with
Table 1. Postprandial Blood Glucose (mg/dL) on Days 1, 20, and 26 after Treatment Begana time
control
vehicle
TVP
P value
day 1 day 20 day 26
94.4 ± 4.8** 97.4 ± 3.8*** 116 ± 10***
252 ± 16* 201 ± 19* 292 ± 15*
252 ± 18* 157 ± 11** 225 ± 18**
