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Dendrobium officinale Kimura et Migo attenuates diabetic cardiomyopathy through inhibiting oxidative stress, inflammation and fibrosis in streptozotocin-induced mice
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Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
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Dendrobium officinale Kimura et Migo attenuates diabetic cardiomyopathy through inhibiting oxidative stress, inflammation and fibrosis in streptozotocin-induced mice Zhihao Zhanga , Duoduo Zhanga , Mengmeng Doua , Zhubo Lia , Jie Zhangb,1, Xiaoyan Zhaoa,* ,1 a b
College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China Department of Neurology, The Ninth People’s Hospital of Chongqing, Chongqing 400700, China
A R T I C L E I N F O
Article history: Received 20 September 2016 Received in revised form 21 October 2016 Accepted 24 October 2016 Keywords: Dendrobium officinale Diabetic cardiomyopathy Oxidative stress Lipid accumulation Inflammation Fibrosis
A B S T R A C T
Dendrobium officinale Kimura et Migo (Dendrobium catenatum Lindley), a prized traditional Chinese Medicine, has been used in China and Southeast Asian countries for centuries. The present study was aimed to investigate the effects and the possible mechanisms of the Dendrobium officinale extracts (DOE) on diabetic cardiomyopathy in mice. The diabetic model was induced by intraperitoneal injection of streptozotocin at the dose of 50 mg/kg body weight for 5 consecutive days. After 8 weeks treatment of DOE, mice were sacrificed, blood sample and heart tissues were collected. Our results showed that Streptozotocin-induced diabetic model was effectively achieved and serum CK and LDH levels were significantly increased in mice with diabetic cardiomyopathy. Pretreatment with DOE decreased the heart-to-body weight ratio (HW/BW) and showed an evident hypoglycemic effect. DOE pretreatment significantly decreased CK, LDH, TC and TG levels, limited the production of MDA and increased the activities of T-SOD. The histological analysis of Oil red O staining and Sirius red staining showed an obvious amelioration of cardiac injury, inhibition of cardiac lipid accumulation and deposition of collagen when pretreatment with DOE. In addition, Western blot detection and analysis showed that DOE downregulated the expression of TGF-b, collegan-1, fibronectin, NF-kB, TNF-a and IL-1b. In conclusion, our study suggested that DOE possesses the cardioprotective potential against diabetic cardiomyopathy, which may be due to the inhibition of oxidative stress, cardiac lipid accumulation, pro-inflammatory cytokines and cardiac fibrosis. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Diabetes mellitus is the most widespread metabolic disease worldwide. It is estimated that there are about 347 million people with diabetes worldwide [1]. It is reported that diabetes accompanies cardiovascular complications, including
Abbreviations: DCM, diabetic cardiomyopathy; DOE, Dendrobium officinale extracts; STZ, streptozotocin; TG, triglyceride; TC, total cholesterol; CK, creatine kinase; LDH, lactate dehydrogenase; SOD, superoxide dismutase; MDA, malondialdehyde; TNF-a, tumor necrosis factor a; IL-1b, interleukin 1b; NF-kB, nuclear factor kB; TGF-b, transforming growth factor b. * Corresponding author at: College of Pharmaceutical Sciences, Southwest University, No. 2, Tiansheng Road Beibei, Chongqing 400716, China. E-mail addresses: [email protected] (Z. Zhang), [email protected] (D. Zhang), [email protected] (M. Dou), [email protected] (Z. Li), [email protected] (J. Zhang), [email protected] (X. Zhao). 1 Authors made equal contribution to this study.
atherosclerosis, myocardial infarction, cardiomyopathy, and heart failure [2]. Cardiovascular complications are becoming the leading cause of morbidity and mortality in diabetic patients [3]. Diabetesinduced cardiomyopathy (DCM) mediated by hyperglycemia can induce the adverse architectural remodeling of heart, and ultimately lead to heart failure and death [4–6]. The main characteristics of diabetic cardiomyopathy are myocardial left ventricular dysfunction [7], cardiac injury, cardiomyocyte hypertrophy and cardiac cell apoptosis [8]. The probable mechanisms involved in the development of DCM include increased oxidative stress, activation of pro-inflammatory pathways and enhanced extracellular fibrosis [9]. Dendrobium officinale Kimura et Migo (also called Dendrobium catenatum Lindley) [10], one of the traditional Chinese medicinal herbs has been used as herbal medicine in many Asian countries for centuries [11] and due to its wide range of pharmacological and clinical effects, Dendrobium officinale is recognized as a high
http://dx.doi.org/10.1016/j.biopha.2016.10.074 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Z. Zhang, et al., Dendrobium officinale Kimura et Migo attenuates diabetic cardiomyopathy through inhibiting oxidative stress, inflammation and fibrosis in streptozotocin-induced mice, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.10.074
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quality health food in China and other south-east Asian countries [12]. Polysaccharide, the major constituent in Dendrobium species [13], was recently reported to possess various potent pharmacological effects, including anti-oxidative [14], anti-cancer [15,16] and immunomodulation effects [17]. Increasing evidence also showed that Dendrobium officinale polysaccharide possesses the hypoglycemic effect [14,18,19]. And according to Wu et al. [18], the hypoglycemic mechanisms may be by stimulating the secretion of insulin from b cells, inhibiting the secretion of glucagons from a cells, and decrease the decomposition of liver glycogen, increase the synthesis of muscle glycogen. In addition, Dendrobium officinale polysaccharides inhibits pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b and 6 [20]. However, no report was found about the cardio-protective effects of Dendrobium officinale and consequently, we hypothesized that Dendrobium officinale may be cardioprotective against diabetes-induced cardiomyopathy, thus, we explore the protective effect of Dendrobium officinale in DCM mice. In the present study, we have investigated the potential cardioprotective effects of water-soluble exacts of Dendrobium officinale (DOE) in streptozotocin (STZ) induced diabetic cardiomyopathy mouse models. Our data suggested that administration of DOE could significantly attenuate heart injury induced by diabetes, increase the levels of antioxidant enzyme activities and decrease the expression of pro-inflammatory and pro-fibrosis related proteins. Our findings underscore the potential of Dendrobium officinale for the prevention or treatment of DCM. 2. Materials and methods 2.1. Animals Kunming male mice, 8–10 weeks of age, weighting 20 2 g, were purchased from Chongqing Tengxin Biotechnology Co., Ltd [SCXK (Yu) 2011-0001]. The mice were housed at 22 C with a 12 h light/dark cycle with free access to food and tap water. All the animal protocols conformed to the National Institutes of Health (NIH) guidelines and were approved by the Ethical Committee for Animal of Southwest University. 2.2. Preparation of DOE and determination of total polysaccharides The dried material of Dendrobium officinale (batch No: XZ20140301) were purchased from Xi’an Xiaocao Botanical Development Co. Ltd., Xi’an, China, and authenticated in college of pharmaceutical science, Southwest University. The voucher specimens (No. 20140609) were submitted at the Herbarium of Materia Medica, Department of Traditional Chinese Medicine, College of Pharmaceutical Sciences, Southwest University, Chongqing, China. The dry stems were grinded into fine power through 350-mesh and then 100 g powder were pre-extracted by petroleum ether (Boiling range: 60–90 C) and 80% ethanol under 60 C condition. The residues were extracted 3 times with double distilled water. The crude extracts were filtered, concentrated and then dried by lyophilization. And 23.5 g power extracts were collected. The phenol-sulfuric acid method was used to quantify the polysaccharides [21], Glucose was used as the standard (Dglucose, Sigma, St. Louis, MO).
derivatives of mannose, glucose, galactose, galacturonic acid and arabinose (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) were prepared as described by Lin et al. [20]. Briefly, 0.12 g powder of stems were pre-extracted with 80% ethanol in a soxhlet extractor for 4 h. The dried residues and 1 mL glucosamine hydrochloride (12 mg/mL) (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) were added into 100 mL distilled water, and then decocted for 1 h. The mixture was diluted with distilled water to 100 mL at room temperature. 1 mL of mixture was react with 0.5 mL 3.0 M hydrochloric acid at 110 C for 1 h. The reactant mixture was neutralized by 3.0 M sodium hydroxide. Four hundreds microliter of the hydrolyzed DOE was mixed with 400 mL of 0.5 M PMP (dissolved in methanol) and 400 mL of 0.3 M sodium hydroxide, then kept at 70 C for 100 min. After cooled down to room temperature, the solution was neutralized with 500 mL of 0.3 M hydrochloric acid and washed with 2 mL chloroform for 3 times. The supernatant were obtained after centrifuging, and filtered through a 0.22 mm syringe filter. 2.4. HPLC analysis The PMP-derivatives of monosaccharide was separated by a RPHPLC (LC 20A, SHIMADZU, Japan) system equipped with Dikma Diamonsil C18 column (150 mm 4.6 mm; 5 mm; Dikma Technologies, China) and SPD-20A detector. The mobile phase consisted of 17% ammonium acetate (A, 0.02 M) and 83% acetonitrile (B), using a gradient elution (0–28 min, B 17%; 29–38 min, B 17%–19.4%; 39–65 min, B 19.4%) at a flow rate of 0.8 mL/min. The wavelength of the detector was 250 nm. The column temperature was 30 C. The quantitative analysis was performed with internal standard method [22]. The amount of mannose and glucose was expressed as percentage of the extracts of Dendrobium officinale. 2.5. Induction of diabetic cardiomyopathy models and DOE treatment Diabetic mice models was induced by intraperitoneal injection of streptozotocin (STZ, Sigma, St. Louis, MO) at the dose of 50 mg/ kg body weight (freshly dissolved in 0.1 M sodium citrate buffer pH 4.5) for 5 consecutive days while age-matched control mice were received multiple injections of the same volume of sodium citrate buffer. After 5 days, blood glucose levels were measured using glucometer (Sinocare Inc Co., Ltd., changsha, China) by tail vein puncture blood sampling, mice with blood sugar values >16.7 mmol/L were used for the study. It has been reported that DCM was start to develop from 4 weeks and reach the peak at 8 weeks [3]. The diabetic mice were randomly divided into four groups (n = 7–10/group): model group (normal saline), DOE low dose group (75 mg/kg), DOE medium dose group (150 mg/kg), DOE high dose group (300 mg/kg). The diabetic mice were received normal saline or DOE gavaged once a day for 8 weeks. And body weight of mice was record every day. The corresponding control groups were treated with either vehicle or DOE alone for the same duration. After 8 weeks of DOE or normal saline treatment, animals were euthanized, heart tissue and blood samples were collected. 2.6. Measurement of fasting blood glucose, triglyceride (TG) and total cholesterol (TC)
2.3. Preparation of PMP derivatives of monosaccharide The species of monosaccharide in DOE were analyzed. The amount of mannose and glucose were determined by 1-phenyl-3methyl-5-pyrazolone (PMP) pre-column derivatization method on the basis of the Pharmacopoeia of the People’s Republic of China (2015 Edition). Before HPLC analysis, PMP (Sigma, St. Louis, MO)
At the end of the experiment, the fasting blood glucose of mice were measured using glucometer (Sinocare Inc Co., Ltd., changsha, China) by tail vein puncture blood sampling. Blood samples were collected, and the serum was separated by centrifuging at 3000 rpm/min for 10 min at 4 C. The TC, TG levels in serum were determined by commercially available kit (Jiancheng Institute of
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Fig. 1. HPLC chromatograms of reference substances and PMP-labeled monosaccharide in DOE. (A) A mixture of reference substances: PMP (peak 1), mannose (peak 2), glucosamine hydrochloride (peak 3), galacturonic acid (peak 4), glucose (peak5), galactose (peak 6), arabinose (peak 7); (B) The species of monosaccharide in DOE: mannose (peak 2), glucose (peak 5).
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Biotechnology, Nanjing, China) in accordance with the manufacturer’s instructions. 2.7. Measurement of myocardial enzymes in serum The creatine kinase (CK) and lactate dehydrogenase (LDH) in serum were measured with an ultraviolet-visible spectrophotometer (Shanghai Jinghua Technology Instrument Co., Ltd., Shanghai, China) using commercial assay kits (Jiancheng Institute of Biotechnology, Nanjing, China) in accordance with the manufacturer’ s protocols. 2.8. Determination of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum In order to estimate the anti-oxidative activities of DOE, the levels of SOD and MDA were also determined by commercial assay kits (Jiancheng Institute of Biotechnology, Nanjing, China) with an ultraviolet-visible spectrophotometer (Shanghai Jinghua Technology Instrument Co., Ltd., Shanghai, China) according to the manufacturer’ s protocols.
2.12. Western blotting analysis Hearts tissues were collected and homogenized in RIPA lysis buffer and lysed on ice for 30 min, the lysates were centrifuged at 12,000 rpm at 4 C for 10 min. Total proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) (Millipore, Billerica, USA) membrane. After blocking with 5% nonfat milk for 1.5 h, the membranes were individually incubated with primary antibodies (Proteintech Group Inc, Wuhan, China. NF-kB, TNF-a, Fibronctin were from mouse sources; TGF-b, IL-1b, collegen1 were from rabbit sources) overnight at 4 C. Then the membranes were washed in PBST buffer for three times, and subsequently incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Proteintech Group Inc, Wuhan, China) for 2 h at room temperature. The specific proteins were detected by ECL chemiluminescence reagent (Advansta, CA, USA). GAPDH polyclonal antibody (1:5000), NF-kB Polyclonal antibody (1:1000), Fibronctin polyclonal antibody (1:2000), TGF-b polyclonal antibody (1:1000), IL-1b polyclonal antibody (1:1000), collegen1 polyclonal antibody (1:2000), TNF-a monoclonal antibody (1:2000) and HRP conjugated secondary antibodies (1:5000).
2.9. Histological analysis Heart tissues were collected from mice and washed with precooled saline, then fixed in 4% paraformaldehyde and embedded in paraffin. 5 mm slices of heart tissue were stained with haematoxylin and eosin (H&E). Morphological examination were performed using a light microscope (Olympus, Tokyo, Japan). 2.10. Oil red O staining Oil Red O staining was used to evaluate the cardiac lipid accumulation. Cryosections (10 mm) from heart tissue were embedded in optimal cutting temperature medium and fixed in 10% buffered formalin for 30 min and washed with water. Then the slices were immersed in 60% isopropanol and stained with Oil Red O solution at room temperature for 1 h. After washing with isopropanol twice, the slices were counterstained with hematoxylin for 30 s. An Olympus microscope (Olympus, Tokyo, Japan) was used to capture the Oil Red O-stained tissue sections. The areas of lipid accumulation was counted by Image Pro-plus (version 6.0).
2.13. Statistical analysis All data were presented as mean SD. Statistical significance of differences between groups was performed with Student’s t-test or one-way ANOVA followed by post hoc analysis. P < 0.05 was considered to be statistically significant. All statistical analyses were performed with SPSS Statistical Software (version 16.0 for Windows). 3. Results 3.1. Quality analysis of DOE Calibration curve for glucose was carried out with high sensitivity and consistency. The water extracts mainly consist of polysaccharide and the concentration of polysaccharide determined by phenol-sulfuric acid method was 72.1%.
2.11. Sirius red staining
3.2. The monosaccharide species and the content of mannose and glucose in DOE
Sirius Red staining was used to evaluate the cardiac fibrosis. Heart tissues from mice were fixed in 10% neutral buffered formalin, and embedded in paraffin. 4 mm cross-sections were stained with 0.1% picrosirius red, and photographed by a light microscopy. The areas of collagen deposition was measured by Image Pro-plus (version 6.0).
According to the result of RP-HPLC (Fig. 1), it can be seen that the PMP-labeled monosaccharide in DOE were well separated by HPLC. The result indicated that DOE contains two main monosaccharides including mannose and glucose. And the content of mannose and glucose is 19.51% and 14.03% respectively.
Table 1 Effects of DOE treatment on body weight, heart weight, Heart/Body Weight Ratio, fasting blood glucose and lipids in DCM mice.
Body Weight (g) Heart Weight (mg) Heart/Body Weight Ratio(mg/g) Fasting Blood Glucose (mmol/L) TC (mmol/L) TG (mmol/L)
Control Group
STZ Group
STZ + DOM(mg/kg) Group 75
150
300
40.40 3.70 12.42 1.35 0.2923 0.01468 4.6 0.80 2.339 0.202 1.222 0.136
22.64 3.09* 8.82 1.70* 0.3929 0.02475* 23.8 2.39* 3.793 0.279* 4.680 0.716*
22.72 2.30* 8.02 1.21* 0.3592 0.01081*,# 24.0 1.98* 3.486 0.502* 3.094 0.918*,#
27.77 5.79*,# 9.13 2.16* 0.3377 0.02007*,# 19.2 4.71*,# 3.167 0.339*,# 2.638 0.708*,#
26.53 3.88* 8.90 1.34* 0.3266 0.01523*,# 19.7 3.40*,# 2.788 0.480# 2.207 0.529*,#
Date are expressed as mean SD (each n > 6). * P < 0.01 vs. control group. # P < 0.05 vs. STZ group.
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3.3. General features of diabetes and the effects of DOE on DCM mice During the experiments, body weight and fasting blood glucose were measured (Table 1) and it was found that the body and heart weight of STZ-induced diabetic mice were significantly decreased compared to the non-diabetic mice in control group (P < 0.05). The heart-to-body weight ratio (HW/BW) in the diabetic group was significantly higher than the control group (P < 0.05). Furthermore, the diabetic mice showed an obviously increase of fasting blood glucose, TC and TG compared to the non-diabetic mice (P < 0.05). After 8 weeks treatment with DOE, fasting blood glucose was significantly decreased in the middle dose group and high dose group compared with control group (P < 0.05). Meanwhile, the increased serum TC and TG levels were also decreased when pretreatment with DOE for 8 weeks. Moreover, the heart-to-body weight ratio (HW/BW) was significantly decreased in DOE treatment group compared with the STZ group. The data indicated that DOE may protect heart against cardiac hypertrophy. However, significant changes were not found on body and heart weight when pretreatment with DOE. 3.4. DOE protects against myocardial injury in DCM mice Myocardial injury was evaluated by H&E staining. Serum CK and LDH activities were also measured. In control group, the
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myocardial structural was clear and well organized, without infiltration of inflammatory cells (Fig. 2A). In contrast, perinuclear vacuolization, necrosis and inflammatory infiltration were obvious in STZ group. DOE treatment normalized alterations in cardiac tissues. The myocardial structural abnormalities such as necrotic were rare, vacuolization and myofibrillar loss were nearly not detected under the microscope in DOE treatment group. Myocardial enzymes CK and LDH, are regarded as biochemical indicators of myocardial injury. As compared to control group, the activities of CK and LDH were significantly elevated (Fig. 2B, C) in the STZ group (P < 0.05). After treatment with DOE, the activities of CK and LDH in middle and high dose group were almost decreased to the same levels as the control group, no significant changes in LDH (Fig. 2C) activities was found in the low dose of DOE group. 3.5. DOE attenuates cardiac oxidative stress in DCM mice Oxidative stress has been proved to be associated with the pathogenesis of diabetic cardiomyopathy [23]. Hence, MDA and T-SOD were measured to evaluate anti-oxidative effect of DOE. As showed in Fig. 3, diabetic mice in the STZ group showed significantly increased MDA activity (P < 0.05) (Fig. 3A) and significantly decreased T-SOD levels (P < 0.05) (Fig. 3B). Conversely, DOE treated mice exhibited markedly decrease of MDA content and an up-regulated T-SOD activity. However, the low dosage DOE
Fig. 2. Effect of DOE treatment on cardiac injury. (A) Paraffin-embedded heart tissue was stained with haematoxylin and eosin and examined under light microscope (200, bar = 100 mm). Arrows indicate cytoplasmic vascuolization, cardiomyocyte necrosis and inflammatory infiltration. (B) Effect of DOE treatment on LDH level in serum. (C) Effect of DOE treatment on CK level in serum. Date are expressed as mean SD (each n = 7–10). * P < 0.05 vs. control group; # P < 0.05 vs. STZ group.
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Fig. 3. DOE inhibits cardiac oxidative stress. (A) Effect of DOE treatment on MDA level in serum. (B) Effect of DOE treatment on T-SOD level in serum. Date are expressed as mean SD (each n = 7–10). * P < 0.05 vs. control group; # P < 0.05 vs. STZ group.
Fig. 4. DOE treatment attenuate cardiac lipid accumulation in heart tissue. Cardiac lipid accumulation was detected by Oil Red O staining (200, bar = 100 mm) in cryosections. Area of Oil Red O was quantified as fold of control. Date are expressed as mean SD (each n = 7–10). * P < 0.05 vs. control group; # P < 0.05 vs. STZ group.
group seems have no influence on T-SOD activity, this may contributes to the error in the STZ group was quite big and might be the reason why no significance was obtained. 3.6. DOE attenuates DCM-induced cardiac lipid accumulation One of the typical features of DCM is cardiac lipid accumulation. Oil Red O staining was performed to evaluate cardiac lipid accumulation. Image-Pro Plus (version 6.0) analysis showed a significant increase in lipid accumulation in cardiomyocyte in STZ group (P < 0.05) compared to the control group (Fig. 4). Treatment of DCM mice with DOE markedly lowered the lipid accumulation in cardiac tissues. 3.7. DOE inhibits DCM-induced cardiac inflammation Inflammation is considered to be an important factor in development of various cardiovascular disorders. Here, in this study, the inflammatory-associated proteins NF-kB, TNF-a and IL-1b were measured by western blot analysis (Fig. 5). The results indicated that NF-kB, TNF-a and IL-1b were significantly upregulated in diabetic mice in STZ group (P < 0.05), treatment of DOE could prevent cardiac inflammation.
3.8. DOE prevents DCM-induced cardiac fibrosis The development of interstitial fibrosis is also a structural hallmark of diabetes cardiomyopathy. It played a key role in diabetes-induced cardiac pathogenesis [1]. The fibrotic effect of myocardium was measured by Sirius-red staining for collagen. As shown in Fig. 6A, significant collagen deposition in heart tissue were seen in STZ group mice, and this can be obviously inhibited by treatment of DOE (P < 0.05). Followed by Sirius-red staining, cardiac fibrosis markers TGF-b, collegan-1 and fibronectin were measured by western blot analysis (Fig. 6B). In STZ group, the expression of TGF-b, collegan-1 and fibronectin were significantly up-regulated (P < 0.05). The up-regulation of these proteins were remarkably inhibited in high dose and middle dose of DOE group. However, low dose of DOE has little effect on them. 4. Discussion Diabetic cardiomyopathy is a typical cardiovascular system complications mediated by hyperglycemia [4], and characterized by myocardial left ventricular dysfunction, cardiac injury, cardiomyocyte hypertrophy, cardiac cell apoptosis and microvascular abnormalities [7,8,23]. Increasing evidence indicated that
Please cite this article in press as: Z. Zhang, et al., Dendrobium officinale Kimura et Migo attenuates diabetic cardiomyopathy through inhibiting oxidative stress, inflammation and fibrosis in streptozotocin-induced mice, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.10.074
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Fig. 5. DOE prevents DCM-induced cardiac inflammation. The expression of nuclear factor kB p65 (NF-kB p65), tumor necrosis factor a (TNF-a) and interleukin-1b (IL-1b) were examined by Western blot assay and quantified by densitometric analysis. Date are expressed as mean SD (each n = 7–10). * P < 0.05 vs. control group; # P < 0.05 vs. STZ group.
oxidative stress, cardiac fibrosis, lipid accumulation and inflammation play vital role in the development of diabetic cardiomyopathy [3,9]. Though therapeutic agents for the specific metabolic and structural derangements of DCM are encouraging, there is still no specific treatment strategy to manage DCM [24]. Dendrobium officinale Kimura et Migo, a traditional Chinese herb widely used in Asian countries [11], has been shown to possess anti-oxidant and hypoglycemic effects [14]. Moreover, Dendrobium officinale polysaccharides was also reported to possess the anti-inflammatory effects by inhibiting the proinflammatory cytokines [20]. However, no report was found about the cardio-protective effect of Dendrobium officinale and previous work from our laboratory (unpublished) showed that Dendrobium officinale has a potential cardioprotective effect against myocardial ischemia. In the present study, we have investigated the effects of DOE on myocardial injury, oxidative stress, cardiac fibrosis, cardiac lipid accumulation and inflammation in STZ induced diabetic cardiomyopathy mice models. The results showed that DOE could significantly inhibit oxidative stress, lipid accumulation, inflammation and cardiac fibrosis. The data also suggested that DOE attenuates cardiac injury in mice with diabetic cardiomyopathy (Fig. 2). These data indicated that DOE has a cardio-protective potential against diabetic cardiomyopathy and may have clinical applications. Many studies have shown that oxidative stress may play an important role during development of DCM. Oxidative stress often exists during the imbalance of oxidative and antioxidant defenses systems [25]. Increased oxidative stress often correlates with lipid overload [26]. It is well known that SOD is an important enzyme that scavenges reactive oxygen radicals [19]. The present study convincingly showed that DOE treatment can attenuate oxidative stress in STZ induced diabetic mice as illustrated by the decrease of MDA levels (Fig. 3A) and increased T-SOD activity (Fig. 3B). As it mentioned previously [26], lipid overload or accumulation is often correlates with oxidative stress. Cardiac lipid accumulation also plays a causative role in the development of DCM. It has been
reported that diabetes-associated lipid accumulation can lead to cardiac oxidative stress by increasing reactive oxygen species, which then cause lipid peroxidation [27]. The lipid peroxidation can generate MDA and can cause the cross-linking polymerization of proteins, nucleic acids and other macromolecules. The results of Oil Red O staining showed that treatment with DOE can significantly attenuate lipid accumulation in myocardium compared to STZ group (Fig. 4). The data revealed that DOE may exert the myocardial protective effect though inhibiting lipid peroxidation. Myocardial inflammation has been reported in streptozotocin induced diabetes [28]. Cardiac inflammation can be characterized by the increase of pro-inflammatory cytokines [29]. The influx of infiltrating inflammatory cells may up-regulate the levels of ROS. Here, in this study, we investigated the expression of some proinflammatory cytokines by western blotting analysis. It has been reported that nuclear factor kB (NF-kB) is one of the most important regulators that controls the expression of pro-inflammatory genes [30]. As reported by Dirk Westermann et al. [31], activation of NF-kB was often correlated with an increase of IL-1b and TNF-a. IL1-b is known to be an important mediator in inflammatory diseases. TNF-a not only amplify the inflammatory response to induce other inflammatory cytokines release, but also contributes to myocardial hypertrophy and fibrosis [32]. Our current study suggested that DOE significantly attenuates cardiac inflammation though inhibited the expression of NF-kB, as well as the levels of IL-1b and TNF-a (Fig. 5). Cardiac fibrosis is an important contributor to the development of cardiac dysfunction in DCM. Extracellular matrix (ECM) is composed of collagen, elastin, laminin and fibronectin [33], the abnormally elevated ECM deposition results in myocardial fibrosis, in particular the deposition of collagen, which increases myocardial stiffness [23]. TGF-b1 is an important factor in regulating collagen generation in DCM [34], TGF-b1 induces the differentiation of fibroblasts into myofibroblasts, which have a higher activity for collagen production than fibroblasts [35,36]. Thus, in the present study, we measured the collagen deposition by Sirius-red
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Fig. 6. DOE prevents DCM-induced cardiac fibrosis. (A): Collagen was measured by Sirius-red staining (200, bar = 100 mm), followed by semi-quantitative analysis. (B): The expression of transforming growth factor b (TGF-b), collagen I and fibronectin were examined by Western blot assay and quantified by densitometric analysis. Date are expressed as mean SD (each n = 7–10). * P < 0.05 vs. control group; # P < 0.05 vs. STZ group.
staining (Fig. 6A). The expression of TGF-b1, collagen-1 and fibronectin were also evaluated by western blotting analysis. Our data showed an obviously deposition of collagen and up-regulation of TGF-b1, collagen-1, fibronectin in mice in STZ group (Fig. 6B). However, the deposition of collagen and up-regulation of fibrosis associated proteins were significantly attenuated by the administration of DOE, the result indicate that DOE has the potential protective effect on myocardium against fibrosis, and this protective effect may due to the inhibition of TGF-b1 mediated collagen production and deposition. In conclusion, the present study demonstrated that oral administration of DOE could significantly prevent the development of DCM induced by STZ. The probable mechanism may be due to the inhibition of oxidative stress, cardiac fibrosis and downregulation of pro-inflammatory cytokines. This is the first report indicating that DOE may possess the cardio-protective potential
against DCM and can be a candidate for therapeutic use. Currently, our research is extremely limited, further studies still need to be carried out to unveil the specific molecular mechanisms. Conflict of interest The authors declare no conflicts of interest. Acknowledgements The present study was supported by the Chongqing Science and Technology Commission (no. cstc2016jcyjA0296), the Fundamental Research Funds for the Central University (nos. XDJK2014B024 and XDJK2016E137), and the Innovative Project on Designing and Screening Drug Candidates of Chongqing (no. cstc2015zdcyztzx120003).
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Dendrobium officinale Kimura et Migo attenuates diabetic cardiomyopathy through inhibiting oxidative stress, inflammation and fibrosis in streptozotocin-induced mice Zhihao Zhanga , Duoduo Zhanga , Mengmeng Doua , Zhubo Lia , Jie Zhangb,1, Xiaoyan Zhaoa,* ,1 a b
College of Pharmaceutical Sciences, Southwest University, Chongqing 400716, China Department of Neurology, The Ninth People’s Hospital of Chongqing, Chongqing 400700, China
A R T I C L E I N F O
Article history: Received 20 September 2016 Received in revised form 21 October 2016 Accepted 24 October 2016 Keywords: Dendrobium officinale Diabetic cardiomyopathy Oxidative stress Lipid accumulation Inflammation Fibrosis
A B S T R A C T
Dendrobium officinale Kimura et Migo (Dendrobium catenatum Lindley), a prized traditional Chinese Medicine, has been used in China and Southeast Asian countries for centuries. The present study was aimed to investigate the effects and the possible mechanisms of the Dendrobium officinale extracts (DOE) on diabetic cardiomyopathy in mice. The diabetic model was induced by intraperitoneal injection of streptozotocin at the dose of 50 mg/kg body weight for 5 consecutive days. After 8 weeks treatment of DOE, mice were sacrificed, blood sample and heart tissues were collected. Our results showed that Streptozotocin-induced diabetic model was effectively achieved and serum CK and LDH levels were significantly increased in mice with diabetic cardiomyopathy. Pretreatment with DOE decreased the heart-to-body weight ratio (HW/BW) and showed an evident hypoglycemic effect. DOE pretreatment significantly decreased CK, LDH, TC and TG levels, limited the production of MDA and increased the activities of T-SOD. The histological analysis of Oil red O staining and Sirius red staining showed an obvious amelioration of cardiac injury, inhibition of cardiac lipid accumulation and deposition of collagen when pretreatment with DOE. In addition, Western blot detection and analysis showed that DOE downregulated the expression of TGF-b, collegan-1, fibronectin, NF-kB, TNF-a and IL-1b. In conclusion, our study suggested that DOE possesses the cardioprotective potential against diabetic cardiomyopathy, which may be due to the inhibition of oxidative stress, cardiac lipid accumulation, pro-inflammatory cytokines and cardiac fibrosis. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Diabetes mellitus is the most widespread metabolic disease worldwide. It is estimated that there are about 347 million people with diabetes worldwide [1]. It is reported that diabetes accompanies cardiovascular complications, including
Abbreviations: DCM, diabetic cardiomyopathy; DOE, Dendrobium officinale extracts; STZ, streptozotocin; TG, triglyceride; TC, total cholesterol; CK, creatine kinase; LDH, lactate dehydrogenase; SOD, superoxide dismutase; MDA, malondialdehyde; TNF-a, tumor necrosis factor a; IL-1b, interleukin 1b; NF-kB, nuclear factor kB; TGF-b, transforming growth factor b. * Corresponding author at: College of Pharmaceutical Sciences, Southwest University, No. 2, Tiansheng Road Beibei, Chongqing 400716, China. E-mail addresses: [email protected] (Z. Zhang), [email protected] (D. Zhang), [email protected] (M. Dou), [email protected] (Z. Li), [email protected] (J. Zhang), [email protected] (X. Zhao). 1 Authors made equal contribution to this study.
atherosclerosis, myocardial infarction, cardiomyopathy, and heart failure [2]. Cardiovascular complications are becoming the leading cause of morbidity and mortality in diabetic patients [3]. Diabetesinduced cardiomyopathy (DCM) mediated by hyperglycemia can induce the adverse architectural remodeling of heart, and ultimately lead to heart failure and death [4–6]. The main characteristics of diabetic cardiomyopathy are myocardial left ventricular dysfunction [7], cardiac injury, cardiomyocyte hypertrophy and cardiac cell apoptosis [8]. The probable mechanisms involved in the development of DCM include increased oxidative stress, activation of pro-inflammatory pathways and enhanced extracellular fibrosis [9]. Dendrobium officinale Kimura et Migo (also called Dendrobium catenatum Lindley) [10], one of the traditional Chinese medicinal herbs has been used as herbal medicine in many Asian countries for centuries [11] and due to its wide range of pharmacological and clinical effects, Dendrobium officinale is recognized as a high
http://dx.doi.org/10.1016/j.biopha.2016.10.074 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Z. Zhang, et al., Dendrobium officinale Kimura et Migo attenuates diabetic cardiomyopathy through inhibiting oxidative stress, inflammation and fibrosis in streptozotocin-induced mice, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.10.074
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quality health food in China and other south-east Asian countries [12]. Polysaccharide, the major constituent in Dendrobium species [13], was recently reported to possess various potent pharmacological effects, including anti-oxidative [14], anti-cancer [15,16] and immunomodulation effects [17]. Increasing evidence also showed that Dendrobium officinale polysaccharide possesses the hypoglycemic effect [14,18,19]. And according to Wu et al. [18], the hypoglycemic mechanisms may be by stimulating the secretion of insulin from b cells, inhibiting the secretion of glucagons from a cells, and decrease the decomposition of liver glycogen, increase the synthesis of muscle glycogen. In addition, Dendrobium officinale polysaccharides inhibits pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b and 6 [20]. However, no report was found about the cardio-protective effects of Dendrobium officinale and consequently, we hypothesized that Dendrobium officinale may be cardioprotective against diabetes-induced cardiomyopathy, thus, we explore the protective effect of Dendrobium officinale in DCM mice. In the present study, we have investigated the potential cardioprotective effects of water-soluble exacts of Dendrobium officinale (DOE) in streptozotocin (STZ) induced diabetic cardiomyopathy mouse models. Our data suggested that administration of DOE could significantly attenuate heart injury induced by diabetes, increase the levels of antioxidant enzyme activities and decrease the expression of pro-inflammatory and pro-fibrosis related proteins. Our findings underscore the potential of Dendrobium officinale for the prevention or treatment of DCM. 2. Materials and methods 2.1. Animals Kunming male mice, 8–10 weeks of age, weighting 20 2 g, were purchased from Chongqing Tengxin Biotechnology Co., Ltd [SCXK (Yu) 2011-0001]. The mice were housed at 22 C with a 12 h light/dark cycle with free access to food and tap water. All the animal protocols conformed to the National Institutes of Health (NIH) guidelines and were approved by the Ethical Committee for Animal of Southwest University. 2.2. Preparation of DOE and determination of total polysaccharides The dried material of Dendrobium officinale (batch No: XZ20140301) were purchased from Xi’an Xiaocao Botanical Development Co. Ltd., Xi’an, China, and authenticated in college of pharmaceutical science, Southwest University. The voucher specimens (No. 20140609) were submitted at the Herbarium of Materia Medica, Department of Traditional Chinese Medicine, College of Pharmaceutical Sciences, Southwest University, Chongqing, China. The dry stems were grinded into fine power through 350-mesh and then 100 g powder were pre-extracted by petroleum ether (Boiling range: 60–90 C) and 80% ethanol under 60 C condition. The residues were extracted 3 times with double distilled water. The crude extracts were filtered, concentrated and then dried by lyophilization. And 23.5 g power extracts were collected. The phenol-sulfuric acid method was used to quantify the polysaccharides [21], Glucose was used as the standard (Dglucose, Sigma, St. Louis, MO).
derivatives of mannose, glucose, galactose, galacturonic acid and arabinose (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) were prepared as described by Lin et al. [20]. Briefly, 0.12 g powder of stems were pre-extracted with 80% ethanol in a soxhlet extractor for 4 h. The dried residues and 1 mL glucosamine hydrochloride (12 mg/mL) (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) were added into 100 mL distilled water, and then decocted for 1 h. The mixture was diluted with distilled water to 100 mL at room temperature. 1 mL of mixture was react with 0.5 mL 3.0 M hydrochloric acid at 110 C for 1 h. The reactant mixture was neutralized by 3.0 M sodium hydroxide. Four hundreds microliter of the hydrolyzed DOE was mixed with 400 mL of 0.5 M PMP (dissolved in methanol) and 400 mL of 0.3 M sodium hydroxide, then kept at 70 C for 100 min. After cooled down to room temperature, the solution was neutralized with 500 mL of 0.3 M hydrochloric acid and washed with 2 mL chloroform for 3 times. The supernatant were obtained after centrifuging, and filtered through a 0.22 mm syringe filter. 2.4. HPLC analysis The PMP-derivatives of monosaccharide was separated by a RPHPLC (LC 20A, SHIMADZU, Japan) system equipped with Dikma Diamonsil C18 column (150 mm 4.6 mm; 5 mm; Dikma Technologies, China) and SPD-20A detector. The mobile phase consisted of 17% ammonium acetate (A, 0.02 M) and 83% acetonitrile (B), using a gradient elution (0–28 min, B 17%; 29–38 min, B 17%–19.4%; 39–65 min, B 19.4%) at a flow rate of 0.8 mL/min. The wavelength of the detector was 250 nm. The column temperature was 30 C. The quantitative analysis was performed with internal standard method [22]. The amount of mannose and glucose was expressed as percentage of the extracts of Dendrobium officinale. 2.5. Induction of diabetic cardiomyopathy models and DOE treatment Diabetic mice models was induced by intraperitoneal injection of streptozotocin (STZ, Sigma, St. Louis, MO) at the dose of 50 mg/ kg body weight (freshly dissolved in 0.1 M sodium citrate buffer pH 4.5) for 5 consecutive days while age-matched control mice were received multiple injections of the same volume of sodium citrate buffer. After 5 days, blood glucose levels were measured using glucometer (Sinocare Inc Co., Ltd., changsha, China) by tail vein puncture blood sampling, mice with blood sugar values >16.7 mmol/L were used for the study. It has been reported that DCM was start to develop from 4 weeks and reach the peak at 8 weeks [3]. The diabetic mice were randomly divided into four groups (n = 7–10/group): model group (normal saline), DOE low dose group (75 mg/kg), DOE medium dose group (150 mg/kg), DOE high dose group (300 mg/kg). The diabetic mice were received normal saline or DOE gavaged once a day for 8 weeks. And body weight of mice was record every day. The corresponding control groups were treated with either vehicle or DOE alone for the same duration. After 8 weeks of DOE or normal saline treatment, animals were euthanized, heart tissue and blood samples were collected. 2.6. Measurement of fasting blood glucose, triglyceride (TG) and total cholesterol (TC)
2.3. Preparation of PMP derivatives of monosaccharide The species of monosaccharide in DOE were analyzed. The amount of mannose and glucose were determined by 1-phenyl-3methyl-5-pyrazolone (PMP) pre-column derivatization method on the basis of the Pharmacopoeia of the People’s Republic of China (2015 Edition). Before HPLC analysis, PMP (Sigma, St. Louis, MO)
At the end of the experiment, the fasting blood glucose of mice were measured using glucometer (Sinocare Inc Co., Ltd., changsha, China) by tail vein puncture blood sampling. Blood samples were collected, and the serum was separated by centrifuging at 3000 rpm/min for 10 min at 4 C. The TC, TG levels in serum were determined by commercially available kit (Jiancheng Institute of
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Fig. 1. HPLC chromatograms of reference substances and PMP-labeled monosaccharide in DOE. (A) A mixture of reference substances: PMP (peak 1), mannose (peak 2), glucosamine hydrochloride (peak 3), galacturonic acid (peak 4), glucose (peak5), galactose (peak 6), arabinose (peak 7); (B) The species of monosaccharide in DOE: mannose (peak 2), glucose (peak 5).
Please cite this article in press as: Z. Zhang, et al., Dendrobium officinale Kimura et Migo attenuates diabetic cardiomyopathy through inhibiting oxidative stress, inflammation and fibrosis in streptozotocin-induced mice, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.10.074
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Biotechnology, Nanjing, China) in accordance with the manufacturer’s instructions. 2.7. Measurement of myocardial enzymes in serum The creatine kinase (CK) and lactate dehydrogenase (LDH) in serum were measured with an ultraviolet-visible spectrophotometer (Shanghai Jinghua Technology Instrument Co., Ltd., Shanghai, China) using commercial assay kits (Jiancheng Institute of Biotechnology, Nanjing, China) in accordance with the manufacturer’ s protocols. 2.8. Determination of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum In order to estimate the anti-oxidative activities of DOE, the levels of SOD and MDA were also determined by commercial assay kits (Jiancheng Institute of Biotechnology, Nanjing, China) with an ultraviolet-visible spectrophotometer (Shanghai Jinghua Technology Instrument Co., Ltd., Shanghai, China) according to the manufacturer’ s protocols.
2.12. Western blotting analysis Hearts tissues were collected and homogenized in RIPA lysis buffer and lysed on ice for 30 min, the lysates were centrifuged at 12,000 rpm at 4 C for 10 min. Total proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) (Millipore, Billerica, USA) membrane. After blocking with 5% nonfat milk for 1.5 h, the membranes were individually incubated with primary antibodies (Proteintech Group Inc, Wuhan, China. NF-kB, TNF-a, Fibronctin were from mouse sources; TGF-b, IL-1b, collegen1 were from rabbit sources) overnight at 4 C. Then the membranes were washed in PBST buffer for three times, and subsequently incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Proteintech Group Inc, Wuhan, China) for 2 h at room temperature. The specific proteins were detected by ECL chemiluminescence reagent (Advansta, CA, USA). GAPDH polyclonal antibody (1:5000), NF-kB Polyclonal antibody (1:1000), Fibronctin polyclonal antibody (1:2000), TGF-b polyclonal antibody (1:1000), IL-1b polyclonal antibody (1:1000), collegen1 polyclonal antibody (1:2000), TNF-a monoclonal antibody (1:2000) and HRP conjugated secondary antibodies (1:5000).
2.9. Histological analysis Heart tissues were collected from mice and washed with precooled saline, then fixed in 4% paraformaldehyde and embedded in paraffin. 5 mm slices of heart tissue were stained with haematoxylin and eosin (H&E). Morphological examination were performed using a light microscope (Olympus, Tokyo, Japan). 2.10. Oil red O staining Oil Red O staining was used to evaluate the cardiac lipid accumulation. Cryosections (10 mm) from heart tissue were embedded in optimal cutting temperature medium and fixed in 10% buffered formalin for 30 min and washed with water. Then the slices were immersed in 60% isopropanol and stained with Oil Red O solution at room temperature for 1 h. After washing with isopropanol twice, the slices were counterstained with hematoxylin for 30 s. An Olympus microscope (Olympus, Tokyo, Japan) was used to capture the Oil Red O-stained tissue sections. The areas of lipid accumulation was counted by Image Pro-plus (version 6.0).
2.13. Statistical analysis All data were presented as mean SD. Statistical significance of differences between groups was performed with Student’s t-test or one-way ANOVA followed by post hoc analysis. P < 0.05 was considered to be statistically significant. All statistical analyses were performed with SPSS Statistical Software (version 16.0 for Windows). 3. Results 3.1. Quality analysis of DOE Calibration curve for glucose was carried out with high sensitivity and consistency. The water extracts mainly consist of polysaccharide and the concentration of polysaccharide determined by phenol-sulfuric acid method was 72.1%.
2.11. Sirius red staining
3.2. The monosaccharide species and the content of mannose and glucose in DOE
Sirius Red staining was used to evaluate the cardiac fibrosis. Heart tissues from mice were fixed in 10% neutral buffered formalin, and embedded in paraffin. 4 mm cross-sections were stained with 0.1% picrosirius red, and photographed by a light microscopy. The areas of collagen deposition was measured by Image Pro-plus (version 6.0).
According to the result of RP-HPLC (Fig. 1), it can be seen that the PMP-labeled monosaccharide in DOE were well separated by HPLC. The result indicated that DOE contains two main monosaccharides including mannose and glucose. And the content of mannose and glucose is 19.51% and 14.03% respectively.
Table 1 Effects of DOE treatment on body weight, heart weight, Heart/Body Weight Ratio, fasting blood glucose and lipids in DCM mice.
Body Weight (g) Heart Weight (mg) Heart/Body Weight Ratio(mg/g) Fasting Blood Glucose (mmol/L) TC (mmol/L) TG (mmol/L)
Control Group
STZ Group
STZ + DOM(mg/kg) Group 75
150
300
40.40 3.70 12.42 1.35 0.2923 0.01468 4.6 0.80 2.339 0.202 1.222 0.136
22.64 3.09* 8.82 1.70* 0.3929 0.02475* 23.8 2.39* 3.793 0.279* 4.680 0.716*
22.72 2.30* 8.02 1.21* 0.3592 0.01081*,# 24.0 1.98* 3.486 0.502* 3.094 0.918*,#
27.77 5.79*,# 9.13 2.16* 0.3377 0.02007*,# 19.2 4.71*,# 3.167 0.339*,# 2.638 0.708*,#
26.53 3.88* 8.90 1.34* 0.3266 0.01523*,# 19.7 3.40*,# 2.788 0.480# 2.207 0.529*,#
Date are expressed as mean SD (each n > 6). * P < 0.01 vs. control group. # P < 0.05 vs. STZ group.
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3.3. General features of diabetes and the effects of DOE on DCM mice During the experiments, body weight and fasting blood glucose were measured (Table 1) and it was found that the body and heart weight of STZ-induced diabetic mice were significantly decreased compared to the non-diabetic mice in control group (P < 0.05). The heart-to-body weight ratio (HW/BW) in the diabetic group was significantly higher than the control group (P < 0.05). Furthermore, the diabetic mice showed an obviously increase of fasting blood glucose, TC and TG compared to the non-diabetic mice (P < 0.05). After 8 weeks treatment with DOE, fasting blood glucose was significantly decreased in the middle dose group and high dose group compared with control group (P < 0.05). Meanwhile, the increased serum TC and TG levels were also decreased when pretreatment with DOE for 8 weeks. Moreover, the heart-to-body weight ratio (HW/BW) was significantly decreased in DOE treatment group compared with the STZ group. The data indicated that DOE may protect heart against cardiac hypertrophy. However, significant changes were not found on body and heart weight when pretreatment with DOE. 3.4. DOE protects against myocardial injury in DCM mice Myocardial injury was evaluated by H&E staining. Serum CK and LDH activities were also measured. In control group, the
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myocardial structural was clear and well organized, without infiltration of inflammatory cells (Fig. 2A). In contrast, perinuclear vacuolization, necrosis and inflammatory infiltration were obvious in STZ group. DOE treatment normalized alterations in cardiac tissues. The myocardial structural abnormalities such as necrotic were rare, vacuolization and myofibrillar loss were nearly not detected under the microscope in DOE treatment group. Myocardial enzymes CK and LDH, are regarded as biochemical indicators of myocardial injury. As compared to control group, the activities of CK and LDH were significantly elevated (Fig. 2B, C) in the STZ group (P < 0.05). After treatment with DOE, the activities of CK and LDH in middle and high dose group were almost decreased to the same levels as the control group, no significant changes in LDH (Fig. 2C) activities was found in the low dose of DOE group. 3.5. DOE attenuates cardiac oxidative stress in DCM mice Oxidative stress has been proved to be associated with the pathogenesis of diabetic cardiomyopathy [23]. Hence, MDA and T-SOD were measured to evaluate anti-oxidative effect of DOE. As showed in Fig. 3, diabetic mice in the STZ group showed significantly increased MDA activity (P < 0.05) (Fig. 3A) and significantly decreased T-SOD levels (P < 0.05) (Fig. 3B). Conversely, DOE treated mice exhibited markedly decrease of MDA content and an up-regulated T-SOD activity. However, the low dosage DOE
Fig. 2. Effect of DOE treatment on cardiac injury. (A) Paraffin-embedded heart tissue was stained with haematoxylin and eosin and examined under light microscope (200, bar = 100 mm). Arrows indicate cytoplasmic vascuolization, cardiomyocyte necrosis and inflammatory infiltration. (B) Effect of DOE treatment on LDH level in serum. (C) Effect of DOE treatment on CK level in serum. Date are expressed as mean SD (each n = 7–10). * P < 0.05 vs. control group; # P < 0.05 vs. STZ group.
Please cite this article in press as: Z. Zhang, et al., Dendrobium officinale Kimura et Migo attenuates diabetic cardiomyopathy through inhibiting oxidative stress, inflammation and fibrosis in streptozotocin-induced mice, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.10.074
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Fig. 3. DOE inhibits cardiac oxidative stress. (A) Effect of DOE treatment on MDA level in serum. (B) Effect of DOE treatment on T-SOD level in serum. Date are expressed as mean SD (each n = 7–10). * P < 0.05 vs. control group; # P < 0.05 vs. STZ group.
Fig. 4. DOE treatment attenuate cardiac lipid accumulation in heart tissue. Cardiac lipid accumulation was detected by Oil Red O staining (200, bar = 100 mm) in cryosections. Area of Oil Red O was quantified as fold of control. Date are expressed as mean SD (each n = 7–10). * P < 0.05 vs. control group; # P < 0.05 vs. STZ group.
group seems have no influence on T-SOD activity, this may contributes to the error in the STZ group was quite big and might be the reason why no significance was obtained. 3.6. DOE attenuates DCM-induced cardiac lipid accumulation One of the typical features of DCM is cardiac lipid accumulation. Oil Red O staining was performed to evaluate cardiac lipid accumulation. Image-Pro Plus (version 6.0) analysis showed a significant increase in lipid accumulation in cardiomyocyte in STZ group (P < 0.05) compared to the control group (Fig. 4). Treatment of DCM mice with DOE markedly lowered the lipid accumulation in cardiac tissues. 3.7. DOE inhibits DCM-induced cardiac inflammation Inflammation is considered to be an important factor in development of various cardiovascular disorders. Here, in this study, the inflammatory-associated proteins NF-kB, TNF-a and IL-1b were measured by western blot analysis (Fig. 5). The results indicated that NF-kB, TNF-a and IL-1b were significantly upregulated in diabetic mice in STZ group (P < 0.05), treatment of DOE could prevent cardiac inflammation.
3.8. DOE prevents DCM-induced cardiac fibrosis The development of interstitial fibrosis is also a structural hallmark of diabetes cardiomyopathy. It played a key role in diabetes-induced cardiac pathogenesis [1]. The fibrotic effect of myocardium was measured by Sirius-red staining for collagen. As shown in Fig. 6A, significant collagen deposition in heart tissue were seen in STZ group mice, and this can be obviously inhibited by treatment of DOE (P < 0.05). Followed by Sirius-red staining, cardiac fibrosis markers TGF-b, collegan-1 and fibronectin were measured by western blot analysis (Fig. 6B). In STZ group, the expression of TGF-b, collegan-1 and fibronectin were significantly up-regulated (P < 0.05). The up-regulation of these proteins were remarkably inhibited in high dose and middle dose of DOE group. However, low dose of DOE has little effect on them. 4. Discussion Diabetic cardiomyopathy is a typical cardiovascular system complications mediated by hyperglycemia [4], and characterized by myocardial left ventricular dysfunction, cardiac injury, cardiomyocyte hypertrophy, cardiac cell apoptosis and microvascular abnormalities [7,8,23]. Increasing evidence indicated that
Please cite this article in press as: Z. Zhang, et al., Dendrobium officinale Kimura et Migo attenuates diabetic cardiomyopathy through inhibiting oxidative stress, inflammation and fibrosis in streptozotocin-induced mice, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.10.074
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Fig. 5. DOE prevents DCM-induced cardiac inflammation. The expression of nuclear factor kB p65 (NF-kB p65), tumor necrosis factor a (TNF-a) and interleukin-1b (IL-1b) were examined by Western blot assay and quantified by densitometric analysis. Date are expressed as mean SD (each n = 7–10). * P < 0.05 vs. control group; # P < 0.05 vs. STZ group.
oxidative stress, cardiac fibrosis, lipid accumulation and inflammation play vital role in the development of diabetic cardiomyopathy [3,9]. Though therapeutic agents for the specific metabolic and structural derangements of DCM are encouraging, there is still no specific treatment strategy to manage DCM [24]. Dendrobium officinale Kimura et Migo, a traditional Chinese herb widely used in Asian countries [11], has been shown to possess anti-oxidant and hypoglycemic effects [14]. Moreover, Dendrobium officinale polysaccharides was also reported to possess the anti-inflammatory effects by inhibiting the proinflammatory cytokines [20]. However, no report was found about the cardio-protective effect of Dendrobium officinale and previous work from our laboratory (unpublished) showed that Dendrobium officinale has a potential cardioprotective effect against myocardial ischemia. In the present study, we have investigated the effects of DOE on myocardial injury, oxidative stress, cardiac fibrosis, cardiac lipid accumulation and inflammation in STZ induced diabetic cardiomyopathy mice models. The results showed that DOE could significantly inhibit oxidative stress, lipid accumulation, inflammation and cardiac fibrosis. The data also suggested that DOE attenuates cardiac injury in mice with diabetic cardiomyopathy (Fig. 2). These data indicated that DOE has a cardio-protective potential against diabetic cardiomyopathy and may have clinical applications. Many studies have shown that oxidative stress may play an important role during development of DCM. Oxidative stress often exists during the imbalance of oxidative and antioxidant defenses systems [25]. Increased oxidative stress often correlates with lipid overload [26]. It is well known that SOD is an important enzyme that scavenges reactive oxygen radicals [19]. The present study convincingly showed that DOE treatment can attenuate oxidative stress in STZ induced diabetic mice as illustrated by the decrease of MDA levels (Fig. 3A) and increased T-SOD activity (Fig. 3B). As it mentioned previously [26], lipid overload or accumulation is often correlates with oxidative stress. Cardiac lipid accumulation also plays a causative role in the development of DCM. It has been
reported that diabetes-associated lipid accumulation can lead to cardiac oxidative stress by increasing reactive oxygen species, which then cause lipid peroxidation [27]. The lipid peroxidation can generate MDA and can cause the cross-linking polymerization of proteins, nucleic acids and other macromolecules. The results of Oil Red O staining showed that treatment with DOE can significantly attenuate lipid accumulation in myocardium compared to STZ group (Fig. 4). The data revealed that DOE may exert the myocardial protective effect though inhibiting lipid peroxidation. Myocardial inflammation has been reported in streptozotocin induced diabetes [28]. Cardiac inflammation can be characterized by the increase of pro-inflammatory cytokines [29]. The influx of infiltrating inflammatory cells may up-regulate the levels of ROS. Here, in this study, we investigated the expression of some proinflammatory cytokines by western blotting analysis. It has been reported that nuclear factor kB (NF-kB) is one of the most important regulators that controls the expression of pro-inflammatory genes [30]. As reported by Dirk Westermann et al. [31], activation of NF-kB was often correlated with an increase of IL-1b and TNF-a. IL1-b is known to be an important mediator in inflammatory diseases. TNF-a not only amplify the inflammatory response to induce other inflammatory cytokines release, but also contributes to myocardial hypertrophy and fibrosis [32]. Our current study suggested that DOE significantly attenuates cardiac inflammation though inhibited the expression of NF-kB, as well as the levels of IL-1b and TNF-a (Fig. 5). Cardiac fibrosis is an important contributor to the development of cardiac dysfunction in DCM. Extracellular matrix (ECM) is composed of collagen, elastin, laminin and fibronectin [33], the abnormally elevated ECM deposition results in myocardial fibrosis, in particular the deposition of collagen, which increases myocardial stiffness [23]. TGF-b1 is an important factor in regulating collagen generation in DCM [34], TGF-b1 induces the differentiation of fibroblasts into myofibroblasts, which have a higher activity for collagen production than fibroblasts [35,36]. Thus, in the present study, we measured the collagen deposition by Sirius-red
Please cite this article in press as: Z. Zhang, et al., Dendrobium officinale Kimura et Migo attenuates diabetic cardiomyopathy through inhibiting oxidative stress, inflammation and fibrosis in streptozotocin-induced mice, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.10.074
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Fig. 6. DOE prevents DCM-induced cardiac fibrosis. (A): Collagen was measured by Sirius-red staining (200, bar = 100 mm), followed by semi-quantitative analysis. (B): The expression of transforming growth factor b (TGF-b), collagen I and fibronectin were examined by Western blot assay and quantified by densitometric analysis. Date are expressed as mean SD (each n = 7–10). * P < 0.05 vs. control group; # P < 0.05 vs. STZ group.
staining (Fig. 6A). The expression of TGF-b1, collagen-1 and fibronectin were also evaluated by western blotting analysis. Our data showed an obviously deposition of collagen and up-regulation of TGF-b1, collagen-1, fibronectin in mice in STZ group (Fig. 6B). However, the deposition of collagen and up-regulation of fibrosis associated proteins were significantly attenuated by the administration of DOE, the result indicate that DOE has the potential protective effect on myocardium against fibrosis, and this protective effect may due to the inhibition of TGF-b1 mediated collagen production and deposition. In conclusion, the present study demonstrated that oral administration of DOE could significantly prevent the development of DCM induced by STZ. The probable mechanism may be due to the inhibition of oxidative stress, cardiac fibrosis and downregulation of pro-inflammatory cytokines. This is the first report indicating that DOE may possess the cardio-protective potential
against DCM and can be a candidate for therapeutic use. Currently, our research is extremely limited, further studies still need to be carried out to unveil the specific molecular mechanisms. Conflict of interest The authors declare no conflicts of interest. Acknowledgements The present study was supported by the Chongqing Science and Technology Commission (no. cstc2016jcyjA0296), the Fundamental Research Funds for the Central University (nos. XDJK2014B024 and XDJK2016E137), and the Innovative Project on Designing and Screening Drug Candidates of Chongqing (no. cstc2015zdcyztzx120003).
Please cite this article in press as: Z. Zhang, et al., Dendrobium officinale Kimura et Migo attenuates diabetic cardiomyopathy through inhibiting oxidative stress, inflammation and fibrosis in streptozotocin-induced mice, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.10.074
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Please cite this article in press as: Z. Zhang, et al., Dendrobium officinale Kimura et Migo attenuates diabetic cardiomyopathy through inhibiting oxidative stress, inflammation and fibrosis in streptozotocin-induced mice, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j. biopha.2016.10.074