- Email: [email protected]
Smad signaling
Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 Contents lists available at ScienceDirect 2 3 4 5 6 journal homepage: www.elsevier.com/locate/jep 7 8 9 10 11 12 13 14 15 Nannan Shen a,1, Xiaoguang Li a,1, Tong Zhou a, Muhammad U. Bilal a, Ning Du a, 16 Yingying Hu a, Wei Qin a, Yingming Xie b, Hongtao Wang c, Jianwei Wu a, Jiaming Ju a, 17 a b,n , Yong Zhang a,d,nn 18 Q1 Zhiwei Fang , Lihong Wang a 19 Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, 20 Ministry of Education), Harbin Medical University, Harbin 150081, China b Department of Endocrinology, the Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China 21 c Hebei Yiling Pharmaceutical Research Institute, Shijiazhuang 050035, China 22 d Institute of Cardiovascular Research, Harbin Medical University, Harbin 150081, China 23 24 25 art ic l e i nf o a b s t r a c t 26 27 Article history: Ethnopharmacological relevance: Shensong Yangxin Capsule (SSYX), a traditional Chinese herbal medi28 Received 14 June 2014 cine, has long been used clinically to treat arrhythmias in China. However, the effect of SSYX on 29 Received in revised form interstitial fibrosis in diabetic cardiomyopathy is unknown. The objective of this study was to investigate 29 July 2014 30 the effects of SSYX on myocardial fibrosis in diabetic rats. Accepted 15 September 2014 31 Materials and methods: The antifibrotic effect of SSYX was investigated in streptozocin (STZ)-induced 32 diabetic rats with high fat-diet (HFD). Fasting blood glucose, heart weight/body weight ratio, total 33 Chemical compounds studied in this article: cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL) Linarin (Pubchem CID: 5317025) were measured. Echocardiography and histology examination were carried out to evaluate heart 34 Paeoniflorin (Pubchem CID: 442534) function. Expression of Smad7, TGF-β1, collagen I (col-1), collagen III (col-3), MMP-2 and MMP-9mRNA 35 Schisantherin A (Pubchem CID: 151529) in heart tissues were measured by real time polymerase chain reaction (PCR). TGF-β1, Smad2/3, 36 Berberine hydrochloride (Pubchem CID: p-Smad2/3 and Smad7 protein levels were measured by western blot analysis. Proliferation of cardiac 37 12456) fibroblast was detected via immunofluorescence. Salvianolic acid B (Pubchem CID: 71433917 ) 38 Results: SSYX markedly decreased heart weight/body weight ratio and improved the impaired cardiac Spinosin (Pubchem CID: 155692) 39 function of T2DM rats. Transmission electron microscopy (TEM), haematoxylin and eosin (HE) and Ginsenoside Rb1 (Pubchem CID: 9898279 ) 40 Masson staining results showed that SSYX attenuated cardiac fibrosis and collagen deposition in T2DM Sodium danshensu (Pubchem CID: 41 23711819) rats. Moreover, mRNA levels of TGF-β1, col-1, col-3, MMP-2 and MMP-9 were downregulated, whereas 42 Saponin (Pubchem CID: 11007422) Smad7 expression was upregulated after treatment with SSYX in rats with cardiac fibrosis. Furthermore, Salvianolic acid E (Pubchem CID: 49770697) 43 SSYX decreased protein levels of TGF-β1 and p-Smad2/3, and increased Smad7 expression. Tanshinone I (Pubchem CID: 114917) 44 Conclusion: TGF-β1/Smad signaling is involved in the cardiac fibrosis in diabetic cardiomyopathy and Nardosinone (Pubchem CID: 168136) SSYX inhibits fibrosis and improves cardiac function via suppressing this pathway. Therefore, SSYX might 45 Schizandrin B (Pubchem CID: 108130) be considered an alternative therapeutic remedy for diabetic cardiomyopathy. 46 Oleanic acid (Pubchem CID: 10494) & 2014 Published by Elsevier Ireland Ltd. Columbamine (Pubchem CID: 72310) 47 Demethyleneberberine (Pubchem CID: 48 363209) 49 Avicularin (Pubchem CID: 5490064) 50 Quercetin (Pubchem CID: 5280343) 51 52 53 54 Abbreviations: ANOVA, analysis of variance; α-SMA, α-smooth muscle actin; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; EF, ejection fraction; 55 FBS, fetal bovine serum; FS, fractional shortening; HDL, high density lipoprotein; HE, haematoxylin and eosin; HFD, high fat-diet; HW/BW, heart weight to body weight; LDL, 56 low density lipoprotein; LVD;d, left ventricular diastolic diameter; LVD;s, left ventricular systolic diameter; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl 57 sulfate polyacrylamide gel electrophoresis; SSYX, Shensong Yangxin Capsule; STZ, Streptozocin; TC, total cholesterol; TCM, traditional Chinese medicine; T2DM, type-2 diabetes mellitus; TEM, transmission electron microscopy; TG, triglycerides; TGF-β1, transforming growth factor-β1; UPLC–MS/MS, ultra-performance liquid chromato58 graphy–mass spectrometry. 59 n Corresponding author. Tel.: þ 86 451 86228255; fax: þ 86 451 86228983. 60 Q2 nn Corresponding author at: Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular 61 Research, Ministry of Education), Harbin Medical University, Harbin 150081, China. Tel./fax: þ 86 451 86671354. E-mail addresses: [email protected] (L. Wang), [email protected] (Y. Zhang). 62 1 These authors contributed equally to this work. 63 64 http://dx.doi.org/10.1016/j.jep.2014.09.035 65 0378-8741/& 2014 Published by Elsevier Ireland Ltd. 66
Journal of Ethnopharmacology
Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/Smad signaling
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Keywords: Shensong Yangxin Capsule Diabetic cardiomyopathy Cardiac fibrosis TGF-β1 Smad
1. Introduction The prevalence of type-2 diabetes mellitus (T2DM) is rising rapidly, and T2DM patients are at high risk of cardiovascular diseases. Hyperlipidaemia and hyperglycemia cause diverse cardiovascular complications, e.g. atherosclerosis in large arteries and/ or coronary arteries, which increases the risk for limb loss, stroke, and myocardial infarction. Despite coronary artery disease and changes in blood pressure, T2DM also can disturb cardiac energy metabolism, which causes cardiac structural and functional remodeling, a condition called diabetic cardiomyopathy (Boudina and Abel, 2007). Diabetic cardiomyopathy is one of the most serious complications of T2DM and is becoming a major cause of diabetesrelated morbidity and mortality (Go et al., 2014). Diabetic cardiomyopathy is characterized by diastolic dysfunction and increasing myocardial stiffness leading to heart failure (Zhao et al., 2013b; Mori et al., 2014). Interstitial fibrosis, the excessive deposition of extracellular matrix (ECM) within tissues, is one of the major contributing factors implicated in the pathogenesis of diabetic cardiomyopathy. Cardiac fibroblasts are enmeshed in the myocardium and play an important role in preserving the integrity of interstitial matrix in the adult mammalian heart. Normal cardiac ECM maintains a homeostasis between synthesis and degradation of matrix proteins. However, in diabetics, pro-fibrotic myofibroblasts are mainly differentiated from cardiac fibroblasts, resulting in more poorly organized fibrotic matrices (Talior-Volodarsky et al., 2012). Moreover, nonenzymatic advanced glycosylation end products alter the structure and function of matrix proteins, and expression of collagen, which can lead to myocardial fibrosis and stiffness (Joshi et al., 2014). Drugs that maintain heart function and prevent myocardial fibrosis might decrease mortality associated with T2DM. Traditional Chinese medicines (TCMs) have been widely used because of their prominent advantages, stable curative effects and low toxicity. As multi-component formulas, TCM treat diseases through different pathways and multi-target regulation at the same time. Several formulas of TCM have been previously reported to be effective against various stages and complications of T2DM (Park et al., 2008; Jing et al., 2009; Cheng et al., 2012; Singh et al., 2013; Gao et al., 2014; Zhang et al., 2014). Shensong Yangxin Capsule (SSYX) has long been used in China as a traditional Chinese remedy to treat cardiac diseases. SSYX was designed and carefully formulated in accordance with the rule of the traditional Chinese medicine theory comprising the ingredients specified in Table 1. The bioactive components of SSYX including sodium danshensu, chlorognenic acid, paeoniflorin, spinosin, salvianolic acid B, berberine hydrochloride, ginsenoside Rb1, and schisantherin A were determined by ultra-performance liquid chromatography (UPLC) (Jiang et al., 2013). The excretion kinetics of constituents in rat urine after oral administration of SSYX was quantified by UPLC–mass spectrometry (UPLC–MS/MS) (Liu et al., 2013). SSYX was traditionally used in China for treating cardiac arrhythmias. Previous studies have reported that SSYX could block multiple ion channels, which may change the action potential duration and contribute to its antiarrhythmic effects. In addition, downregulation of the hHCN4 current by an SSYX treatment may be an anti-arrhythmic mechanism in the heart (Li et al., 2007; Sun et al., 2010). Previous studies also suggested that SSYX effectively
prevented bradycardia (Liu et al., 2014b) and reduced premature ventricular contractions (Zou et al., 2011). However, the influence of SSYX on diabetic cardiomyopathy remains unclear. The present study aimed to examine this notion by investigating the potential beneficial effects of SSYX in diabetic cardiomyopathy, specifically the anti-fibrosis properties, and deciphering the underlying molecular mechanisms.
2. Materials and methods 2.1. Plant material and preparation SSYX (Lot no. 1205019) was provided by Yiling Pharmaceutical Corporation (Shijiazhuang, China). The contents and the voucher numbers of SSYX are shown in Table 1 and are carefully identified by the Yiling Pharmaceutical Corporation. The preparation of SSYX is described as follows. Air-dried plants (1 kg) were decocted by refluxing with water for a period of 2 h for 3 times. The resultant extracts were filtered and concentrated. The mixture was blended with eupolyphaga sinensis Walker (Tu Bie Chong) which has been ground into powder, and dried, crushed into fine powder and cased in capsule in a proper ratio eventually. The entire process was supervised according to the policy of the State Food and Drug Administration of P.R. China. The chemical profile of the formulated SSYX was established by UPLC analysis and characteristic UPLC peaks (profile) for quality control/quality assurance are also presented (Liu et al., 2014a). 2.2. Reagents and preparation of SSYX Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and tissue culture reagents were purchased from Life Technologies Inc. (Gaithersburg, MD, USA). All other chemicals of reagent grade were purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). SSYX ultrafine powder, provided by Shijiazhuang Yiling Pharmaceutical Co. (Shijiazhuang, China), was dissolved in distilled water. Fresh suspensions of SSYX ultrafine powder were prepared every day. The herbal drugs were authenticated and standardized on marker compounds according to the Chinese Pharmacopoeia (National Pharmacopoeia Committee, 2005). 2.3. Animals Male Wistar rats (180–220 g) were obtained from the Animal Center of the 2nd Affiliated Hospital of Harbin Medical University (Harbin, China). Use of animals was in accordance with the regulations of the ethic committees of Harbin Medical University. Animals were conditioned for one week, subjected to a 12-h dark/light cycle. The room in which animals were housed was 2371 1C with constant humidity (5575%). Thirty Wistar rats were randomly divided into five groups: Control, DM, and SSYX (200, 100 and 50 mg/kg/d) groups. 2.4. Establishment of diabetic model According to the previous studies (Ai et al., 2005, 2009; Frode and Medeiros, 2008), rats were intragastrically administered with
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
fat emulsion (10 ml/d) for 15-d. Then the animals were subjected to intraperitoneal (i.p.) injection of 40 mg/kg/d streptozocin (STZ) in a 0.1 M citrate buffer solution (pH 4.2) for 2 d. Blood samples were collected and fasting blood glucose (FBG) level was detected at 72 h after the last injection of STZ to ensure induction of T2DM (glycemia Z16.7 mm). Fat emulsion, kept in a refrigerator at 4 1C, which was made up with 20 g lard, 5 g cholesterol, 1 g thyreostat, 5 g sucrose, 1 g sodium glutamate, 5 g saccharu, 20 ml tween-80, and 30 ml propylene glycol, adjust the final volume to 100 ml with distilled water.
3
ejection fraction (EF), and LV fractional shortening (FS). M-mode recordings were performed at the level of the papillary muscles. 2.6. Blood biochemical estimation At the end of the study, all animals were euthanized, and blood samples were collected. Blood glucose, total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL) were analyzed using appropriate kits (Jiancheng Bioengineering Institute, Nanjing; China Beihuakangtai Clinical Reagent Ltd., Beijing; China and Dongou Diagnostic Products Co., Zhejiang, China).
2.5. Echocardiography 2.7. Histopathological and morphometric analysis Transthoracic echocardiography was performed with an ultrasound machine (Vivid 7, GE Medical, USA). The following parameters were measured and calculated: left ventricular systolic diameter (LVD;s), left ventricular diastolic diameter (LVD;d), LV
The hearts were excised and weighed to calculate the ratio of heart weight to body weight (HW/BW). Histopathological changes and collagen distribution were evaluated by haematoxylin and
Table 1 The Chinese medicines contained in SSYX. Latin name (Chinese name)
The part used
Collected place
Voucher numbers
Panax ginseng C.A.Mey. (Ren Shen) Salvia miltiorrhiza Bge. (Dan Shen) Nardostachys jatamansi Dc. (Gan Song) Cornus officinalis Sieb.et Zucc. (Shan Zhu Yu) Taxillus chinensis (DC.) Danser (Sang Ji Sheng) Paeonia lactiflora Pall. (Chi Shao) Schisandra sphenanthera Rehd.et (Wu Wei Zi) Coptis chinensis Franch. (HuangLian) Ophiopogon japonicas (Thunb.) Ker-Gawl. (Mai Dong) Polypodiodes chinensis (Long Gu) Eupolyphaga sinensis Walker (Tu Bie Chong) Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou (Suan Zao Ren)
Root Root Root Fruit Stem Root Fruit Root Tuber Skeleton Whole animal Fruit
Jilin Shandong Sichuan Shanxi Guangxi Hebei Henan Sichuan Sichuan Gansu Hebei Hebei
120103003 120124053 120124054 120124059 120124058 120124004 120124057 120124055 120124056 120124060 120101201 120124007
Fig. 1. Effect of SSYX on cardiac function evaluated by echocardiography. (A) Ultrasonic cardiogram in Control, DM, and SSYX (200 mg/kg/d) treatment group. (B) Ejection fraction (EF %) in Control, DM, and SSYX (200, 100 and 50 mg/kg/d) groups. (C) Fraction shortening (FS %) in each group. All values are expressed as mean 7 SEM. nnp o 0.01 vs Control, #p o 0.05 vs DM; t-test.
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Q6 66
eosin (H&E) and Masson's trichrome staining. The ventricles were fixed in 4% paraformaldehyde, then embedded with paraffin and cut cross-sectionally into 5 μm thick sections. Tissue sections were deparaffinized and stained with H&E or Masson's trichrome reagent. For Transmission electron microscopy (TEM) analysis, specimens were prepared by routine methods. 1 μm thick Epon embedded sections were stained with uranyl acetate, followed by lead citrate, and examined by JEOL 1200 electron microscope (JEOL Co., Japan).
overnight at 4 1C with the primary antibodies of Smad 2/3 (Cell Signaling Technology, Danvers, MA), p-Smad 2/3 (Cell Signaling Technology, Danvers, MA), Smad7 (Santa Cruz Biotechnology, Dallas, Texas, USA) and TGF-β1 (Cell Signaling Technology, Danvers, MA, USA). β-Actin (Santa Cruz Biotechnology, Dallas, Texas, USA) was used as internal control. The blotted proteins were detected and quantified using an Odyssey Infrared Imaging System (LI-COR, Lincoln, USA.).
2.8. Real-time polymerase chain reaction (PCR)
2.9. Western blot analysis
2.9.1. Fibroblast proliferation and differentiation assay Fibroblasts were isolated by collagenase as described elsewhere (Du et al., 2010). After appropriate treatment, fibroblasts were fixed with 4% paraformaldehyde followed by penetrated with 0.4% Triton 0 x-100. Fibroblast proliferation was determined using 5-Ethynyl-2 deoxyuridine (EdU) assay kit (Ribobio Co., Ltd. Guangzhou, China), and immunofluorescence of α-smooth muscle actin (α-SMA, Abcam, Cambridge, MA, USA) was used to detect fibroblast differentiation. Then the fibroblasts were probed with florescence labeled secondary antibody and observed by a confocal fluorescence microscope.
Total proteins extracted from heart tissues were prepared by standard procedures and quantified using the bicinchoninic acid (BCA) protein assay (Beyotime, Shanghai, China). Protein samples were fractionated by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12% polyacrylamide gels) and transferred to nitrocellulose filter membrane. The membranes were blocked with western blocking buffer for 2 h and incubated
2.9.2. Statistical analysis Data were expressed as mean 7SEM. Differences between groups were analyzed by Student's t test or by one-way analysis of variance (One-way ANOVA). p o0.05 was considered statistically significant. Data were analyzed using the GraphPad Prism 5.0.
Smad7, TGF-β1, col-1, col-3, MMP-2 and MMP-9mRNA expressions were determined by real-time PCR. Total RNA samples from heart tissues were dissolved in TRIzol reagent (Invitrogen, Carlsbad, USA). The SYBR Green PCR Master Mix Kit (Applied Biosystems, Cat. ♯4309155, Carlsbad, USA) was used in real-time PCR for relative quantification of RNA. GAPDH was used as an internal control. The sequences of primers were shown in Table S1.
Fig. 2. Effects of SSYX on histopathological changes. (A–C) Hematoxylin and eosin (H&E) staining of the cross-sectional tissue slices of heart tissues in the Control, DM, and SSYX (200 mg/kg/d) treatment group. Scale bar¼ 20 μm. (D–F) Masson staining for fibrosis of the cross-sectional tissue slices, fibrosis is indicated by blue area. Scale bar¼ 20 μm. (G–I) Transmission electron microscopy micrographs from the Control, DM and SSYX treatment group ( 10,000). Scale bar¼ 5 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
3. Results
5
animals, and SSYX normalized the diabetic morphological changes (Fig. 2A–C).
3.1. Effect of SSYX on cardiac function in STZ-induced diabetic rats 3.2. Effect of SSYX on cardiac interstitial fibrosis in diabetic rats In rats treated with STZ and fed with the high-fat diet, blood glucose levels increased significantly, accompanied by a decrease in body weight and an increase in water intake, indicating the successful establishment of diabetic models (Table S2). LDL, TG and TC were all significantly increased and HDL was decreased in DM group. SSYX did not affect these abnormal changes (Table S3). Echocardiography was performed 4 weeks post-SSYX administration. The ejection fraction (EF %) and fractional shortening (FS %) were dramatically decreased from 71.5870.99% and 41.9470.90% in the control group to 58.2871.14% and 29.0670.54% in the DM group, respectively, indicating impaired cardiac function. SSYX (200 mg/kg/d) treatment markedly improved cardiac function of the DM rats, with EF and FS restored to 67.1871.21% and 38.1270.77%, respectively (Fig. 1). Effects of SSYX on LVD;d and LVD;s are shown in Table S4. Moreover, HE staining of the hearts indicated clear structural abnormalities and cardiomyocyte hypertrophy in the DM rats compared with the control
Collagen production and deposition were evaluated by Masson trichrome staining of histological sections of the hearts. The interstitial fibrotic area in STZ hearts increased strikingly compared with the control group, and SSYX remarkably reduced the interstitial fibrosis (Fig. 2D–F). TEM images in the DM group displayed disordered sarcomere, swollen mitochondria, and lipid droplet, and increased interstitial collagen compared with the control group. The SSYX groups showed better integrity of sarcomere, slighter swelling of mitochondria, and reduced interstitial collagen production (Fig. 2G–I). In addition, the mRNA levels of col-1, col-3, MMP-2, MMP-9 and α-SMA were all significantly increased in the DM group compared with those in control hearts as shown in Fig. 3. Notably, SSYX treatment attenuated these changes in a dose-dependent manner.
Fig. 3. MRNA expression of col-1, col-3, MMP-2 MMP-9 and α-SMA. Relative mRNA levels of myocardial tissue were determined by real-time PCR in the Control, DM, and SSYX (200, 100 and 50 mg/kg/d) groups. (A) Col-1mRNA expression in each group. (B) Col-3mRNA expression in each group. (C) MMP-2mRNA expression. (D) MRNA expression of MMP-9 in each group. (E) α-SMA mRNA expression in each group. npo 0.05 nnp o 0.01 vs Control. #p o 0.05, ##p o0.01 vs DM; One-way ANOVA. All the values are presented as mean 7 SEM.
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 4. Immunofluorescence results of EdU assay and α-SMA staining. Nuclei were stained with DAPI (blue). EdU was used to detect proliferation of fibroblast (green). Confocal immunofluorescence labels with antibodies to α-SMA (red). Fibroblasts were stained with α-SMA specific primary antibody and rhodamine phalloidin-conjugated secondary antibody. (A–C) Immunostaining images were viewed at a magnification of 200 . (D–F) Immunostaining images were viewed at a magnification of 600 . Representative photomicrographs for Control, DM and SSYX (200 mg/kg/d). (G) Summarized data of Edu labeled cells ratio. (H) Summarized data of α-SMA þ cells ratio, n p o 0.05, nnp o 0.01 vs Control, ♯p o0.05 vs DM; t-test. All the values are represented as mean 7SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Effect of SSYX on TGF-β1 expression. (A) Effects of SSYX on TGF-β1 relative protein level measured by western blot analysis, normalized to β-Actin in the Control, DM, and SSYX (200, 100 and 50 mg/kg/d) groups. (B) Effects of SSYX on TGF-β1 mRNA expression measured by real-time PCR. Values are expressed as mean 7 SEM. npo 0.05, nn p o 0.01 vs Control, ♯p o 0.05, ♯♯po 0.01 vs DM. One-way ANOVA.
Myocardial fibrosis is the major pathological characteristic of diabetic cardiomyopathy, involving proliferation and differentiation of fibroblasts. To investigate the effects of SSYX on fibroblast
proliferation during diabetic cardiac fibrogenesis, we performed EdU assay in cardiac fibroblasts. The proliferation of fibroblasts was significantly increased in the DM group, by calculating the
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
percentage of EdU positive cells stained in green, compared with the control group, and this increase was substantially reversed by SSYX (Fig. 4.). 3.3. SSYX inhibits cardiac fibroblast to myofibroblast differentiation
α-SMA is a myofibroblast marker for differentiation of fibroblast to myofibroblast. Immunostaining assay was used to assess the essential role of SSYX in differentiation of fibroblast to myofibroblast. Intriguingly, the number of α-SMA positive cells was significantly increased in the DM group compared with the control group, and SSYX treatment ameliorated this change (Fig. 4). Previous studies have indicated the involvement of TGF-β1 in the process of fibroblast differentiation (Hutcheson et al., 2012). Consistently, we found that both mRNA and protein levels of TGF-β1 in the SSYX group were significantly decreased compared with the DM group, suggesting that SSYX attenuated differentiation of fibroblast to myofibroblast by effectively inhibiting expression of TGF-β1 (Fig. 5). 3.4. Smad signaling pathway underlies the molecular mechanism of SSYX's anti-fibrosis properties TGF-β1/Smad signaling, specifically Smad2/3 and Smad7, plays an important role in the development of fibrosis. We therefore examined the effects of SSYX on the levels of phosphorylated Smad2/3 (p-Smad2/3) and Smad2/3. Fig. 6 shows that p-Smad2/3 was significantly increased in the DM group compared with the
7
control group, which was restored in the SSYX group, while the expression levels of total Smad2/3 remained unaltered. Moreover, it has been reported that Smad7 prevents Smad2/3 from activation. Fig. 7 shows that Smad7 protein and mRNA levels were significantly decreased in DM rats compared with the control group, which were rescued by SSYX.
4. Discussion In this study, we made an effort to understand whether SSYX prevents diabetic cardiomyopathy and the underlying anti-fibrotic mechanisms. The main findings of this study include: (1) SSYX markedly restored heart function and inhibited cardiac fibrosis in diabetic rats; (2) SSYX effectively attenuated interstitial fibrosis by inhibiting collagen production, cardiac fibroblast proliferation, and myofibroblast formation; and (3) the underlying mechanism may involve suppression of TGF-β1/Smad signaling. Our study thus unraveled SSYX as an anti-diabetic cardiomyopathy TCM capable of inhibiting the fibrotic TGF-β1/Smad signaling pathway (Fig. 8). T2DM is a group of metabolic diseases characterized by high blood glucose, which can cause multi-organ disorders. Major complications caused by T2DM include cardiovascular diseases, diabetic neuropathy, diabetic nephropathy (chronic renal failure), and diabetic retinopathy (retinal damage). Several TCMs/phytomedicines were reported to have beneficial effects on different stages of T2DM and related complications. Refined-JinQi-JiangTang
Fig. 6. Examine of p-Smad2/3 and Smad2/3 protein expression by western blot analysis. (A) Levels of phosphorylated or activated Smad2/3 protein (p-Smad2/3) measured by western blot analysis, normalized to β-Actin in the Control, DM, and SSYX (200, 100 and 50 mg/kg/d) groups. (B) Total protein levels of Smad2/3. *po 0.05 vs Control, ♯ p o 0.05 vs DM. Upper panel, examples of western blot bands; lower panel, the statistical figure expressed as mean 7 SEM. Data are presented as relative levels.
Fig. 7. Effect of SSYX on Smad7 expression. (A) Smad7 protein level was detected by western blot analysis, normalized to β-Actin in the Control, DM, and SSYX (200, 100 and 50 mg/kg/d) groups. (B) Relative mRNA expression levels of Smad7 measured by real-time PCR in each group. *p o 0.05, **p o 0.01 vs Control, ♯po 0.05, ♯♯po 0.01 vs DM. Data are presented as mean 7 SEM.
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 8. A schematic diagram depicting the effect of Shensong Yangxin Capsule on diabetic fibrosis. Shensong Yangxin Capsule effectively prevented diabetic cardiomyopathy by inhibiting myocardial fibrosis through TGF-β/Smad signaling pathway.
tablet ameliorates prediabetes by reducing insulin resistance and improving beta cell function (Gao et al., 2014). Pu-Ren-Dan promotes the sensitivity of insulin and decreases blood glucose level by ameliorating the pancreatic blood flow and maintaining the normal structure of islet and β cells (Lu et al., 2013). Jiao Tai Wan inhibits lipogenic gene expression and thus attenuates hepatic lipid accumulation in diabetic rats and patients (Huang et al., 2013). Liuwei Dihuang decoction was reported to have neuroprotective effect by attenuating the neural apoptosis in diabetic encephalopathy rats (Liu et al., 2013). Tangnaikang can inhibit TGF-β1 induced transdifferentiation of human renal tubular epithelial cells, with the effect of treating renal interstitial fibrosis (Yang et al., 2013). Gaoshan Hongjingtian inhibits retinal
capillary degeneration in the experimental diabetic retinopathy (Zhao et al., 2013a). Our previous study has suggested that Daming capsule could preserve impaired heart function in streptozocininduced diabetic rats with hyperlipidemia. SSYX has been shown to be effective in the treatment of arrhythmias, including bradycardia (Liu et al., 2014b) and premature ventricular contractions (Zou et al., 2011). Recent study demonstrated SSYX has clinical efficacy in treating diabetic arrhythmia in senile patients (Jiang et al., 2010). The anti-arrhythmic mechanism of SSYX may be to block pacemaker channels encoded by human HCN4 gene (Sun et al., 2010). However, it is unknown whether SSYX has a role in preventing myocardial fibrosis and subsequent cardiac dysfunctions in diabetic hearts. Therefore, the
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 Q3123 124 125 126 127 128 129 130 131 132
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 Q4 56 57 58 59 60 61 62 63 64 65 66
present study was carried out to test whether SSYX has beneficial effects on diabetic cardiomyopathy in a rat model of T2DM. Ginseng Radix et Rhizoma, one of the herb components in SSYX formula, improves cardiac energy metabolism in ischemic rat heart tissues (Wang et al., 2012). The active ingredient of Salviae Miltiorrhizae Radix et Rhizoma, such as Tanshinone IIA, protects cardiomyocytes from post-infarction injury (Zhang et al., 2010). The impaired heart function was restored by SSYX in the diabetic rats. Myocardial fibrosis is one of the main pathological changes in diabetic cardiomyopathy. TGF-β1 has a crucial role in cardiac fibrosis by activating fibroblasts and producing collagen (Du et al., 2010). Expression of TGF-β1 was found increased in long-term STZ-induced T2DM myocardium (Ares-Carrasco et al., 2009). Many Chinese herbal medicines can inhibit cardiac fibrosis, such as Gualou Xiebai Decoction by blocking TGF-β1/Smad signaling (Ding et al., 2013) and scutellarin by inhibiting TGF-β1 expression (Pan et al., 2011). In the present study, TGF-β1 and the downstream collagen I, collagen III, MMP-2 and MMP-9 genes were all inhibited by SSYX in the diabetic hearts. At the same time, cardiac fibroblast proliferation and α-SMA expression level were both inhibited in SSYX treated diabetic rats. These results were consistent with previous studies showing that type II diabetes promotes a myofibroblast phenotype in cardiac fibroblasts (Yue et al., 2011; Fowlkes et al., 2013). The involvement of Smad signaling activation in the pathogenesis of cardiac fibrosis has previously been studied (Van Linthout et al., 2008; Talior-Volodarsky et al., 2012). When TGF-β1 receptor is activated, Smad7 dissociates from the TGF-β1 receptor, and then Smad 2 and Smad 3 are activated/phosphorylated (Li et al., 2013). But direct evidence that Smad phosphorylation is causal for thebeneficial effects of SSYX on diabetic cardiomyopathy is still missing. The results presented in this study showed that inhibition of Smad 2/3 phosphorylation can prevent cardiac dysfunction. TGF-β1 has been shown to induce endothelial cells to undergo EndMT (Endothelial-to-mesenchymal transition), a mechanism for the progression of cardiac fibrosis (Zeisberg et al., 2007). Diastolic dysfunction is correlated with cardiac fibrosis, which is common in patients with cardiac failure (Vasan and Benjamin, 2001). It is recognized that anti-fibrotic therapies might be useful in improving cardiac function of the diseased heart. Our present study not only provided evidences for the therapeutic potential of SSYX on diabetic cardiomyopathy, but also elucidated an underlying mechanism. In conclusion, the present work suggests that the TGF-β1/Smad signaling pathway is involved in diabetic cardiomyopathy and that SSYX treatment improves impaired heart function and inhibits fibrosis in diabetes. Moreover, SSYX is able to reduce collagen accumulation and should afford beneficial effects in diabetic cardiomyopathy.
Acknowledgments This work was supported by the Funds for Creative Research Groups (Grant number 81121003) and the Major Program (Grant number 81130088) of National Natural Science Foundation of China, the National Natural Science Foundation of China (Grant numbers 30901208, 81270042, 81200593), and the National Basic Research Program of China (973 Program, Grant number 2012CB518606).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2014.09.035.
9
References Ai, J., Wang, N., Yang, M., Du, Z.M., Zhang, Y.C., Yang, B.F., 2005. Development of Wistar rat model of insulin resistance. World Journal of Gastroenterology 11, 3675–3679. Ai, J., Yan, X., Zhao, L., Lu, Y., Liang, F., Cai, B., Li, G., Yang, B., 2009. The protective effect of Daming capsule on heart function in streptozocin-induced diabetic rats with hyperlipidemia. Biological & Pharmaceutical Bulletin 32, 1354–1358. Ares-Carrasco, S., Picatoste, B., Benito-Martin, A., Zubiri, I., Sanz, A.B., Sanchez-Nino, M.D., Ortiz, A., Egido, J., Tunon, J., Lorenzo, O., 2009. Myocardial fibrosis and apoptosis, but not inflammation, are present in long-term experimental diabetes. American Journal of Physiology – Heart and Circulatory Physiology 297, H2109–H2119. Boudina, S., Abel, E.D., 2007. Diabetic cardiomyopathy revisited. Circulation 115, 3213–3223. Cheng, Y.Z., Chen, L.J., Lee, W.J., Chen, M.F., Jung Lin, H., Cheng, J.T., 2012. Increase of myocardial performance by Rhodiola–ethanol extract in diabetic rats. Journal of Ethnopharmacology 144, 234–239. Ding, Y.F., Peng, Y.R., Li, J., Shen, H., Shen, M.Q., Fang, T.H., 2013. Gualou Xiebai Decoction prevents myocardial fibrosis by blocking TGF-beta/Smad signalling. Journal of Pharmacy and Pharmacology 65, 1373–1381. Du, J., Xie, J., Zhang, Z., Tsujikawa, H., Fusco, D., Silverman, D., Liang, B., Yue, L., 2010. TRPM7-mediated Ca2 þ signals confer fibrogenesis in human atrial fibrillation. Circulation Research 106, 992–1003. Fowlkes, V., Clark, J., Fix, C., Law, B.A., Morales, M.O., Qiao, X., Ako-Asare, K., Goldsmith, J.G., Carver, W., Murray, D.B., Goldsmith, E.C., 2013. Type II diabetes promotes a myofibroblast phenotype in cardiac fibroblasts. Life Sciences 92, 669–676. Frode, T.S., Medeiros, Y.S., 2008. Animal models to test drugs with potential antidiabetic activity. Journal of Ethnopharmacology 115, 173–183. Gao, L.H., Liu, Q., Liu, S.N., Chen, Z.Y., Li, C.N., Lei, L., Sun, S.J., Li, L.Y., Liu, J.L., Shen, Z.F., 2014. A refined-JinQi-JiangTang tablet ameliorates prediabetes by reducing insulin resistance and improving beta cell function in mice. Journal of Ethnopharmacology 151, 675–685. Go, A.S., Mozaffarian, D., Roger, V.L., Benjamin, E.J., Berry, J.D., Blaha, M.J., Dai, S., Ford, E.S., Fox, C.S., Franco, S., Fullerton, H.J., Gillespie, C., Hailpern, S.M., Heit, J.A., Howard, V.J., Huffman, M.D., Judd, S.E., Kissela, B.M., Kittner, S.J., Lackland, D.T., Lichtman, J.H., Lisabeth, L.D., Mackey, R.H., Magid, D.J., Marcus, G.M., Marelli, A., Matchar, D.B., McGuire, D.K., Mohler 3rd, E.R., Moy, C.S., Mussolino, M.E., Neumar, R.W., Nichol, G., Pandey, D.K., Paynter, N.P., Reeves, M.J., Sorlie, P.D., Stein, J., Towfighi, A., Turan, T.N., Virani, S.S., Wong, N.D., Woo, D., Turner, M.B., 2014. Heart disease and stroke statistics – 2014 update: a report from the American Heart Association. Circulation 129, e28–e292. Huang, Z., Xu, X., Lu, F., Wang, N., Chen, G., Zhao, Y., Zou, X., Wang, K., Dong, H., Xu, L., 2013. Jiao tai wan attenuates hepatic lipid accumulation in type 2 diabetes mellitus. Evidence-Based Complementary and Alternative Medicine 2013, 567045. Hutcheson, J.D., Ryzhova, L.M., Setola, V., Merryman, W.D., 2012. 5-HT(2B) antagonism arrests non-canonical TGF-beta1-induced valvular myofibroblast differentiation. Journal of Molecular and Cellular Cardiology 53, 707–714. Jiang, X.G., Jia, J.M., YS., L., 2013. Simultaneous determination of eight bioactive constituents in shensong yangxin capsule by uplc. Chinese Herbal Medicines, 212–216. Jiang, X.L., Sun, X.H., Gu, Y.C., Zou, S.R., 2010. Clinical observation of Shensong Yangxin Capsule in treating senile diabetic arrhythmia. Hebei Journal of Traditional Chinese Medicine 32, 1046–1048. Jing, A., Li-Mei, Z., Yan-Jie, L., Ben-Zhi, C., Yong, Z., Bao-Feng, Y., 2009. A randomized, multicentre, open-label, parallel-group trial to compare the efficacy and safety profile of daming capsule in patients with hypercholesterolemia. Phytotherapy Research 23, 1039–1042. Joshi, M., Kotha, S.R., Malireddy, S., Selvaraju, V., Satoskar, A.R., Palesty, A., McFadden, D.W., Parinandi, N.L., Maulik, N., 2014. Conundrum of pathogenesis of diabetic cardiomyopathy: role of vascular endothelial dysfunction, reactive oxygen species, and mitochondria. Molecular and Cellular Biochemistry 386, 233–249. Li, N., Ma, K.J., Wu, X.F., Sun, Q., Zhang, Y.H., Pu, J.L., 2007. Effects of Chinese herbs on multiple ion channels in isolated ventricular myocytes. Chinese Medical Journal (English Edition) 120, 1068–1074. Li, Q., Zhang, D., Wang, Y., Sun, P., Hou, X., Larner, J., Xiong, W., Mi, J., 2013. MiR-21/ Smad 7 signaling determines TGF-beta1-induced CAF formation. Scientific Reports 3, 2038. Liu, J.P., Feng, L., Zhang, M.H., Ma, D.Y., Wang, S.Y., Gu, J., Fu, Q., Qu, R., Ma, S.P., 2013. Neuroprotective effect of Liuwei Dihuang decoction on cognition deficits of diabetic encephalopathy in streptozotocin-induced diabetic rat. Journal of Ethnopharmacology 150, 371–381. Liu, M., Li, S., Zhao, S., Wang, H., Tu, P., 2014a. Studies on excretion kinetics of ten constituents in rat urine after oral administration of Shensong Yangxin Capsule by UPLC–MS/MS. Biomedical Chromatography 28, 525–533. Liu, Y., Li, N., Jia, Z., Lu, F., Pu, J., 2014b. Chinese medicine shensongyangxin is effective for patients with bradycardia: results of a randomized, double-blind, placebo-controlled multicenter trial. Evidence-Based Complementary and Alternative Medicine 2014, 605714. Lu, B., Bai, Y., Du, Z., Chen, S., Deligema, D., Pang, Z., 2013. The effect of Chinese medicine Pu-Ren-Dan on pancreatic angiogenesis in high fat diet/streptozotocin-induced diabetic rats. Indian Journal of Pharmacology 45, 556–562. Mori, J., Patel, V.B., Abo Alrob, O., Basu, R., Altamimi, T., Desaulniers, J., Wagg, C.S., Kassiri, Z., Lopaschuk, G.D., Oudit, G.Y., 2014. Angiotensin 1–7 ameliorates
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 Q5 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
diabetic cardiomyopathy and diastolic dysfunction in db/db mice by reducing lipotoxicity and inflammation. Circulation: Heart Failure 7, 327–339. Pan, Z., Zhao, W., Zhang, X., Wang, B., Wang, J., Sun, X., Liu, X., Feng, S., Yang, B., Lu, Y., 2011. Scutellarin alleviates interstitial fibrosis and cardiac dysfunction of infarct rats by inhibiting TGFbeta1 expression and activation of p38-MAPK and ERK1/2. British Journal of Pharmacology 162, 688–700. Park, S.Y., Choi, Y.H., Lee, W., 2008. Dangnyohwan improves glucose utilization and reduces insulin resistance by increasing the adipocyte-specific GLUT4 expression in Otsuka Long-Evans Tokushima Fatty rats. Journal of Ethnopharmacology 115, 473–482. Singh, R., Kaur, N., Kishore, L., Gupta, G.K., 2013. Management of diabetic complications: a chemical constituents based approach. Journal of Ethnopharmacology 150, 51–70. Sun, L.P., Li, N., Wu, Y.L., Pu, J.L., 2010. Effects of Shensong Yangxin capsule on pacemaker channels encoded by human HCN4 gene. Chinese Medical Journal (English Edition) 123, 3148–3150. Talior-Volodarsky, I., Connelly, K.A., Arora, P.D., Gullberg, D., McCulloch, C.A., 2012. alpha11 integrin stimulates myofibroblast differentiation in diabetic cardiomyopathy. Cardiovascular Research 96, 265–275. Van Linthout, S., Seeland, U., Riad, A., Eckhardt, O., Hohl, M., Dhayat, N., Richter, U., Fischer, J.W., Bohm, M., Pauschinger, M., Schultheiss, H.P., Tschope, C., 2008. Reduced MMP-2 activity contributes to cardiac fibrosis in experimental diabetic cardiomyopathy. Basic Research in Cardiology 103, 319–327. Vasan, R.S., Benjamin, E.J., 2001. Diastolic heart failure–no time to relax. New England Journal of Medicine 344, 56–59. Wang, J.R., Zhou, H., Yi, X.Q., Jiang, Z.H., Liu, L., 2012. Total ginsenosides of Radix Ginseng modulates tricarboxylic acid cycle protein expression to enhance cardiac energy metabolism in ischemic rat heart tissues. Molecules 17, 12746–12757. Yang, L., Ma, X., Cheng, T., Liu, T., Wu, L., Sun, W., Joseph, M.P., 2013. Effect of tangnaikang on TGF-beta1-induced transdifferentiation of human renal tubular epithelial HK-2 cells. Journal of Traditional Chinese Medicine 33, 388–393.
Yue, L., Xie, J., Nattel, S., 2011. Molecular determinants of cardiac fibroblast electrical function and therapeutic implications for atrial fibrillation. Cardiovascular Research 89, 744–753. Zeisberg, E.M., Tarnavski, O., Zeisberg, M., Dorfman, A.L., McMullen, J.R., Gustafsson, E., Chandraker, A., Yuan, X., Pu, W.T., Roberts, A.B., Neilson, E.G., Sayegh, M.H., Izumo, S., Kalluri, R., 2007. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nature Medicine 13, 952–961. Zhang, H., Zhao, T., Gong, Y., Dong, X., Zhang, W., Sun, S., Wang, H., Gu, Y., Lu, X., Yan, M., Li, P., 2014. Attenuation of diabetic nephropathy by Chaihuang-Yishen granule through anti-inflammatory mechanism in streptozotocin-induced rat model of diabetics. Journal of Ethnopharmacology 151, 556–564. Zhang, Y., Zhang, L., Chu, W., Wang, B., Zhang, J., Zhao, M., Li, X., Li, B., Lu, Y., Yang, B., Shan, H., 2010. Tanshinone IIA inhibits miR-1 expression through p38 MAPK signal pathway in post-infarction rat cardiomyocytes. Cellular Physiology and Biochemistry 26, 991–998. Zhao, H.S., Shi, X.Y., Wei, W.B., Wang, N.L., 2013a. Effect of the regimen of Gaoshan Hongjingtian on the mechanism of poly (ADP-ribose) polymerase regulation of nuclear factor kappa B in the experimental diabetic retinopathy. Chinese Medical Journal (English Edition) 126, 1693–1699. Zhao, Y., Zhang, L., Qiao, Y., Zhou, X., Wu, G., Wang, L., Peng, Y., Dong, X., Huang, H., Si, L., Zhang, X., Li, J., Wang, W., Zhou, L., Gao, X., 2013b. Heme oxygenase-1 prevents cardiac dysfunction in streptozotocin-diabetic mice by reducing inflammation, oxidative stress, apoptosis and enhancing autophagy. PLoS One 8, e75927. Zou, J.G., Zhang, J., Jia, Z.H., Cao, K.J., 2011. Evaluation of the traditional Chinese Medicine Shensongyangxin capsule on treating premature ventricular contractions: a randomized, double-blind, controlled multicenter trial. Chinese Medical Journal (English Edition) 124, 76–83.
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
1 Contents lists available at ScienceDirect 2 3 4 5 6 journal homepage: www.elsevier.com/locate/jep 7 8 9 10 11 12 13 14 15 Nannan Shen a,1, Xiaoguang Li a,1, Tong Zhou a, Muhammad U. Bilal a, Ning Du a, 16 Yingying Hu a, Wei Qin a, Yingming Xie b, Hongtao Wang c, Jianwei Wu a, Jiaming Ju a, 17 a b,n , Yong Zhang a,d,nn 18 Q1 Zhiwei Fang , Lihong Wang a 19 Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, 20 Ministry of Education), Harbin Medical University, Harbin 150081, China b Department of Endocrinology, the Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China 21 c Hebei Yiling Pharmaceutical Research Institute, Shijiazhuang 050035, China 22 d Institute of Cardiovascular Research, Harbin Medical University, Harbin 150081, China 23 24 25 art ic l e i nf o a b s t r a c t 26 27 Article history: Ethnopharmacological relevance: Shensong Yangxin Capsule (SSYX), a traditional Chinese herbal medi28 Received 14 June 2014 cine, has long been used clinically to treat arrhythmias in China. However, the effect of SSYX on 29 Received in revised form interstitial fibrosis in diabetic cardiomyopathy is unknown. The objective of this study was to investigate 29 July 2014 30 the effects of SSYX on myocardial fibrosis in diabetic rats. Accepted 15 September 2014 31 Materials and methods: The antifibrotic effect of SSYX was investigated in streptozocin (STZ)-induced 32 diabetic rats with high fat-diet (HFD). Fasting blood glucose, heart weight/body weight ratio, total 33 Chemical compounds studied in this article: cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL) Linarin (Pubchem CID: 5317025) were measured. Echocardiography and histology examination were carried out to evaluate heart 34 Paeoniflorin (Pubchem CID: 442534) function. Expression of Smad7, TGF-β1, collagen I (col-1), collagen III (col-3), MMP-2 and MMP-9mRNA 35 Schisantherin A (Pubchem CID: 151529) in heart tissues were measured by real time polymerase chain reaction (PCR). TGF-β1, Smad2/3, 36 Berberine hydrochloride (Pubchem CID: p-Smad2/3 and Smad7 protein levels were measured by western blot analysis. Proliferation of cardiac 37 12456) fibroblast was detected via immunofluorescence. Salvianolic acid B (Pubchem CID: 71433917 ) 38 Results: SSYX markedly decreased heart weight/body weight ratio and improved the impaired cardiac Spinosin (Pubchem CID: 155692) 39 function of T2DM rats. Transmission electron microscopy (TEM), haematoxylin and eosin (HE) and Ginsenoside Rb1 (Pubchem CID: 9898279 ) 40 Masson staining results showed that SSYX attenuated cardiac fibrosis and collagen deposition in T2DM Sodium danshensu (Pubchem CID: 41 23711819) rats. Moreover, mRNA levels of TGF-β1, col-1, col-3, MMP-2 and MMP-9 were downregulated, whereas 42 Saponin (Pubchem CID: 11007422) Smad7 expression was upregulated after treatment with SSYX in rats with cardiac fibrosis. Furthermore, Salvianolic acid E (Pubchem CID: 49770697) 43 SSYX decreased protein levels of TGF-β1 and p-Smad2/3, and increased Smad7 expression. Tanshinone I (Pubchem CID: 114917) 44 Conclusion: TGF-β1/Smad signaling is involved in the cardiac fibrosis in diabetic cardiomyopathy and Nardosinone (Pubchem CID: 168136) SSYX inhibits fibrosis and improves cardiac function via suppressing this pathway. Therefore, SSYX might 45 Schizandrin B (Pubchem CID: 108130) be considered an alternative therapeutic remedy for diabetic cardiomyopathy. 46 Oleanic acid (Pubchem CID: 10494) & 2014 Published by Elsevier Ireland Ltd. Columbamine (Pubchem CID: 72310) 47 Demethyleneberberine (Pubchem CID: 48 363209) 49 Avicularin (Pubchem CID: 5490064) 50 Quercetin (Pubchem CID: 5280343) 51 52 53 54 Abbreviations: ANOVA, analysis of variance; α-SMA, α-smooth muscle actin; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; EF, ejection fraction; 55 FBS, fetal bovine serum; FS, fractional shortening; HDL, high density lipoprotein; HE, haematoxylin and eosin; HFD, high fat-diet; HW/BW, heart weight to body weight; LDL, 56 low density lipoprotein; LVD;d, left ventricular diastolic diameter; LVD;s, left ventricular systolic diameter; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl 57 sulfate polyacrylamide gel electrophoresis; SSYX, Shensong Yangxin Capsule; STZ, Streptozocin; TC, total cholesterol; TCM, traditional Chinese medicine; T2DM, type-2 diabetes mellitus; TEM, transmission electron microscopy; TG, triglycerides; TGF-β1, transforming growth factor-β1; UPLC–MS/MS, ultra-performance liquid chromato58 graphy–mass spectrometry. 59 n Corresponding author. Tel.: þ 86 451 86228255; fax: þ 86 451 86228983. 60 Q2 nn Corresponding author at: Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular 61 Research, Ministry of Education), Harbin Medical University, Harbin 150081, China. Tel./fax: þ 86 451 86671354. E-mail addresses: [email protected] (L. Wang), [email protected] (Y. Zhang). 62 1 These authors contributed equally to this work. 63 64 http://dx.doi.org/10.1016/j.jep.2014.09.035 65 0378-8741/& 2014 Published by Elsevier Ireland Ltd. 66
Journal of Ethnopharmacology
Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/Smad signaling
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Keywords: Shensong Yangxin Capsule Diabetic cardiomyopathy Cardiac fibrosis TGF-β1 Smad
1. Introduction The prevalence of type-2 diabetes mellitus (T2DM) is rising rapidly, and T2DM patients are at high risk of cardiovascular diseases. Hyperlipidaemia and hyperglycemia cause diverse cardiovascular complications, e.g. atherosclerosis in large arteries and/ or coronary arteries, which increases the risk for limb loss, stroke, and myocardial infarction. Despite coronary artery disease and changes in blood pressure, T2DM also can disturb cardiac energy metabolism, which causes cardiac structural and functional remodeling, a condition called diabetic cardiomyopathy (Boudina and Abel, 2007). Diabetic cardiomyopathy is one of the most serious complications of T2DM and is becoming a major cause of diabetesrelated morbidity and mortality (Go et al., 2014). Diabetic cardiomyopathy is characterized by diastolic dysfunction and increasing myocardial stiffness leading to heart failure (Zhao et al., 2013b; Mori et al., 2014). Interstitial fibrosis, the excessive deposition of extracellular matrix (ECM) within tissues, is one of the major contributing factors implicated in the pathogenesis of diabetic cardiomyopathy. Cardiac fibroblasts are enmeshed in the myocardium and play an important role in preserving the integrity of interstitial matrix in the adult mammalian heart. Normal cardiac ECM maintains a homeostasis between synthesis and degradation of matrix proteins. However, in diabetics, pro-fibrotic myofibroblasts are mainly differentiated from cardiac fibroblasts, resulting in more poorly organized fibrotic matrices (Talior-Volodarsky et al., 2012). Moreover, nonenzymatic advanced glycosylation end products alter the structure and function of matrix proteins, and expression of collagen, which can lead to myocardial fibrosis and stiffness (Joshi et al., 2014). Drugs that maintain heart function and prevent myocardial fibrosis might decrease mortality associated with T2DM. Traditional Chinese medicines (TCMs) have been widely used because of their prominent advantages, stable curative effects and low toxicity. As multi-component formulas, TCM treat diseases through different pathways and multi-target regulation at the same time. Several formulas of TCM have been previously reported to be effective against various stages and complications of T2DM (Park et al., 2008; Jing et al., 2009; Cheng et al., 2012; Singh et al., 2013; Gao et al., 2014; Zhang et al., 2014). Shensong Yangxin Capsule (SSYX) has long been used in China as a traditional Chinese remedy to treat cardiac diseases. SSYX was designed and carefully formulated in accordance with the rule of the traditional Chinese medicine theory comprising the ingredients specified in Table 1. The bioactive components of SSYX including sodium danshensu, chlorognenic acid, paeoniflorin, spinosin, salvianolic acid B, berberine hydrochloride, ginsenoside Rb1, and schisantherin A were determined by ultra-performance liquid chromatography (UPLC) (Jiang et al., 2013). The excretion kinetics of constituents in rat urine after oral administration of SSYX was quantified by UPLC–mass spectrometry (UPLC–MS/MS) (Liu et al., 2013). SSYX was traditionally used in China for treating cardiac arrhythmias. Previous studies have reported that SSYX could block multiple ion channels, which may change the action potential duration and contribute to its antiarrhythmic effects. In addition, downregulation of the hHCN4 current by an SSYX treatment may be an anti-arrhythmic mechanism in the heart (Li et al., 2007; Sun et al., 2010). Previous studies also suggested that SSYX effectively
prevented bradycardia (Liu et al., 2014b) and reduced premature ventricular contractions (Zou et al., 2011). However, the influence of SSYX on diabetic cardiomyopathy remains unclear. The present study aimed to examine this notion by investigating the potential beneficial effects of SSYX in diabetic cardiomyopathy, specifically the anti-fibrosis properties, and deciphering the underlying molecular mechanisms.
2. Materials and methods 2.1. Plant material and preparation SSYX (Lot no. 1205019) was provided by Yiling Pharmaceutical Corporation (Shijiazhuang, China). The contents and the voucher numbers of SSYX are shown in Table 1 and are carefully identified by the Yiling Pharmaceutical Corporation. The preparation of SSYX is described as follows. Air-dried plants (1 kg) were decocted by refluxing with water for a period of 2 h for 3 times. The resultant extracts were filtered and concentrated. The mixture was blended with eupolyphaga sinensis Walker (Tu Bie Chong) which has been ground into powder, and dried, crushed into fine powder and cased in capsule in a proper ratio eventually. The entire process was supervised according to the policy of the State Food and Drug Administration of P.R. China. The chemical profile of the formulated SSYX was established by UPLC analysis and characteristic UPLC peaks (profile) for quality control/quality assurance are also presented (Liu et al., 2014a). 2.2. Reagents and preparation of SSYX Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and tissue culture reagents were purchased from Life Technologies Inc. (Gaithersburg, MD, USA). All other chemicals of reagent grade were purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). SSYX ultrafine powder, provided by Shijiazhuang Yiling Pharmaceutical Co. (Shijiazhuang, China), was dissolved in distilled water. Fresh suspensions of SSYX ultrafine powder were prepared every day. The herbal drugs were authenticated and standardized on marker compounds according to the Chinese Pharmacopoeia (National Pharmacopoeia Committee, 2005). 2.3. Animals Male Wistar rats (180–220 g) were obtained from the Animal Center of the 2nd Affiliated Hospital of Harbin Medical University (Harbin, China). Use of animals was in accordance with the regulations of the ethic committees of Harbin Medical University. Animals were conditioned for one week, subjected to a 12-h dark/light cycle. The room in which animals were housed was 2371 1C with constant humidity (5575%). Thirty Wistar rats were randomly divided into five groups: Control, DM, and SSYX (200, 100 and 50 mg/kg/d) groups. 2.4. Establishment of diabetic model According to the previous studies (Ai et al., 2005, 2009; Frode and Medeiros, 2008), rats were intragastrically administered with
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
fat emulsion (10 ml/d) for 15-d. Then the animals were subjected to intraperitoneal (i.p.) injection of 40 mg/kg/d streptozocin (STZ) in a 0.1 M citrate buffer solution (pH 4.2) for 2 d. Blood samples were collected and fasting blood glucose (FBG) level was detected at 72 h after the last injection of STZ to ensure induction of T2DM (glycemia Z16.7 mm). Fat emulsion, kept in a refrigerator at 4 1C, which was made up with 20 g lard, 5 g cholesterol, 1 g thyreostat, 5 g sucrose, 1 g sodium glutamate, 5 g saccharu, 20 ml tween-80, and 30 ml propylene glycol, adjust the final volume to 100 ml with distilled water.
3
ejection fraction (EF), and LV fractional shortening (FS). M-mode recordings were performed at the level of the papillary muscles. 2.6. Blood biochemical estimation At the end of the study, all animals were euthanized, and blood samples were collected. Blood glucose, total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL) were analyzed using appropriate kits (Jiancheng Bioengineering Institute, Nanjing; China Beihuakangtai Clinical Reagent Ltd., Beijing; China and Dongou Diagnostic Products Co., Zhejiang, China).
2.5. Echocardiography 2.7. Histopathological and morphometric analysis Transthoracic echocardiography was performed with an ultrasound machine (Vivid 7, GE Medical, USA). The following parameters were measured and calculated: left ventricular systolic diameter (LVD;s), left ventricular diastolic diameter (LVD;d), LV
The hearts were excised and weighed to calculate the ratio of heart weight to body weight (HW/BW). Histopathological changes and collagen distribution were evaluated by haematoxylin and
Table 1 The Chinese medicines contained in SSYX. Latin name (Chinese name)
The part used
Collected place
Voucher numbers
Panax ginseng C.A.Mey. (Ren Shen) Salvia miltiorrhiza Bge. (Dan Shen) Nardostachys jatamansi Dc. (Gan Song) Cornus officinalis Sieb.et Zucc. (Shan Zhu Yu) Taxillus chinensis (DC.) Danser (Sang Ji Sheng) Paeonia lactiflora Pall. (Chi Shao) Schisandra sphenanthera Rehd.et (Wu Wei Zi) Coptis chinensis Franch. (HuangLian) Ophiopogon japonicas (Thunb.) Ker-Gawl. (Mai Dong) Polypodiodes chinensis (Long Gu) Eupolyphaga sinensis Walker (Tu Bie Chong) Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou (Suan Zao Ren)
Root Root Root Fruit Stem Root Fruit Root Tuber Skeleton Whole animal Fruit
Jilin Shandong Sichuan Shanxi Guangxi Hebei Henan Sichuan Sichuan Gansu Hebei Hebei
120103003 120124053 120124054 120124059 120124058 120124004 120124057 120124055 120124056 120124060 120101201 120124007
Fig. 1. Effect of SSYX on cardiac function evaluated by echocardiography. (A) Ultrasonic cardiogram in Control, DM, and SSYX (200 mg/kg/d) treatment group. (B) Ejection fraction (EF %) in Control, DM, and SSYX (200, 100 and 50 mg/kg/d) groups. (C) Fraction shortening (FS %) in each group. All values are expressed as mean 7 SEM. nnp o 0.01 vs Control, #p o 0.05 vs DM; t-test.
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Q6 66
eosin (H&E) and Masson's trichrome staining. The ventricles were fixed in 4% paraformaldehyde, then embedded with paraffin and cut cross-sectionally into 5 μm thick sections. Tissue sections were deparaffinized and stained with H&E or Masson's trichrome reagent. For Transmission electron microscopy (TEM) analysis, specimens were prepared by routine methods. 1 μm thick Epon embedded sections were stained with uranyl acetate, followed by lead citrate, and examined by JEOL 1200 electron microscope (JEOL Co., Japan).
overnight at 4 1C with the primary antibodies of Smad 2/3 (Cell Signaling Technology, Danvers, MA), p-Smad 2/3 (Cell Signaling Technology, Danvers, MA), Smad7 (Santa Cruz Biotechnology, Dallas, Texas, USA) and TGF-β1 (Cell Signaling Technology, Danvers, MA, USA). β-Actin (Santa Cruz Biotechnology, Dallas, Texas, USA) was used as internal control. The blotted proteins were detected and quantified using an Odyssey Infrared Imaging System (LI-COR, Lincoln, USA.).
2.8. Real-time polymerase chain reaction (PCR)
2.9. Western blot analysis
2.9.1. Fibroblast proliferation and differentiation assay Fibroblasts were isolated by collagenase as described elsewhere (Du et al., 2010). After appropriate treatment, fibroblasts were fixed with 4% paraformaldehyde followed by penetrated with 0.4% Triton 0 x-100. Fibroblast proliferation was determined using 5-Ethynyl-2 deoxyuridine (EdU) assay kit (Ribobio Co., Ltd. Guangzhou, China), and immunofluorescence of α-smooth muscle actin (α-SMA, Abcam, Cambridge, MA, USA) was used to detect fibroblast differentiation. Then the fibroblasts were probed with florescence labeled secondary antibody and observed by a confocal fluorescence microscope.
Total proteins extracted from heart tissues were prepared by standard procedures and quantified using the bicinchoninic acid (BCA) protein assay (Beyotime, Shanghai, China). Protein samples were fractionated by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12% polyacrylamide gels) and transferred to nitrocellulose filter membrane. The membranes were blocked with western blocking buffer for 2 h and incubated
2.9.2. Statistical analysis Data were expressed as mean 7SEM. Differences between groups were analyzed by Student's t test or by one-way analysis of variance (One-way ANOVA). p o0.05 was considered statistically significant. Data were analyzed using the GraphPad Prism 5.0.
Smad7, TGF-β1, col-1, col-3, MMP-2 and MMP-9mRNA expressions were determined by real-time PCR. Total RNA samples from heart tissues were dissolved in TRIzol reagent (Invitrogen, Carlsbad, USA). The SYBR Green PCR Master Mix Kit (Applied Biosystems, Cat. ♯4309155, Carlsbad, USA) was used in real-time PCR for relative quantification of RNA. GAPDH was used as an internal control. The sequences of primers were shown in Table S1.
Fig. 2. Effects of SSYX on histopathological changes. (A–C) Hematoxylin and eosin (H&E) staining of the cross-sectional tissue slices of heart tissues in the Control, DM, and SSYX (200 mg/kg/d) treatment group. Scale bar¼ 20 μm. (D–F) Masson staining for fibrosis of the cross-sectional tissue slices, fibrosis is indicated by blue area. Scale bar¼ 20 μm. (G–I) Transmission electron microscopy micrographs from the Control, DM and SSYX treatment group ( 10,000). Scale bar¼ 5 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
3. Results
5
animals, and SSYX normalized the diabetic morphological changes (Fig. 2A–C).
3.1. Effect of SSYX on cardiac function in STZ-induced diabetic rats 3.2. Effect of SSYX on cardiac interstitial fibrosis in diabetic rats In rats treated with STZ and fed with the high-fat diet, blood glucose levels increased significantly, accompanied by a decrease in body weight and an increase in water intake, indicating the successful establishment of diabetic models (Table S2). LDL, TG and TC were all significantly increased and HDL was decreased in DM group. SSYX did not affect these abnormal changes (Table S3). Echocardiography was performed 4 weeks post-SSYX administration. The ejection fraction (EF %) and fractional shortening (FS %) were dramatically decreased from 71.5870.99% and 41.9470.90% in the control group to 58.2871.14% and 29.0670.54% in the DM group, respectively, indicating impaired cardiac function. SSYX (200 mg/kg/d) treatment markedly improved cardiac function of the DM rats, with EF and FS restored to 67.1871.21% and 38.1270.77%, respectively (Fig. 1). Effects of SSYX on LVD;d and LVD;s are shown in Table S4. Moreover, HE staining of the hearts indicated clear structural abnormalities and cardiomyocyte hypertrophy in the DM rats compared with the control
Collagen production and deposition were evaluated by Masson trichrome staining of histological sections of the hearts. The interstitial fibrotic area in STZ hearts increased strikingly compared with the control group, and SSYX remarkably reduced the interstitial fibrosis (Fig. 2D–F). TEM images in the DM group displayed disordered sarcomere, swollen mitochondria, and lipid droplet, and increased interstitial collagen compared with the control group. The SSYX groups showed better integrity of sarcomere, slighter swelling of mitochondria, and reduced interstitial collagen production (Fig. 2G–I). In addition, the mRNA levels of col-1, col-3, MMP-2, MMP-9 and α-SMA were all significantly increased in the DM group compared with those in control hearts as shown in Fig. 3. Notably, SSYX treatment attenuated these changes in a dose-dependent manner.
Fig. 3. MRNA expression of col-1, col-3, MMP-2 MMP-9 and α-SMA. Relative mRNA levels of myocardial tissue were determined by real-time PCR in the Control, DM, and SSYX (200, 100 and 50 mg/kg/d) groups. (A) Col-1mRNA expression in each group. (B) Col-3mRNA expression in each group. (C) MMP-2mRNA expression. (D) MRNA expression of MMP-9 in each group. (E) α-SMA mRNA expression in each group. npo 0.05 nnp o 0.01 vs Control. #p o 0.05, ##p o0.01 vs DM; One-way ANOVA. All the values are presented as mean 7 SEM.
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 4. Immunofluorescence results of EdU assay and α-SMA staining. Nuclei were stained with DAPI (blue). EdU was used to detect proliferation of fibroblast (green). Confocal immunofluorescence labels with antibodies to α-SMA (red). Fibroblasts were stained with α-SMA specific primary antibody and rhodamine phalloidin-conjugated secondary antibody. (A–C) Immunostaining images were viewed at a magnification of 200 . (D–F) Immunostaining images were viewed at a magnification of 600 . Representative photomicrographs for Control, DM and SSYX (200 mg/kg/d). (G) Summarized data of Edu labeled cells ratio. (H) Summarized data of α-SMA þ cells ratio, n p o 0.05, nnp o 0.01 vs Control, ♯p o0.05 vs DM; t-test. All the values are represented as mean 7SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Effect of SSYX on TGF-β1 expression. (A) Effects of SSYX on TGF-β1 relative protein level measured by western blot analysis, normalized to β-Actin in the Control, DM, and SSYX (200, 100 and 50 mg/kg/d) groups. (B) Effects of SSYX on TGF-β1 mRNA expression measured by real-time PCR. Values are expressed as mean 7 SEM. npo 0.05, nn p o 0.01 vs Control, ♯p o 0.05, ♯♯po 0.01 vs DM. One-way ANOVA.
Myocardial fibrosis is the major pathological characteristic of diabetic cardiomyopathy, involving proliferation and differentiation of fibroblasts. To investigate the effects of SSYX on fibroblast
proliferation during diabetic cardiac fibrogenesis, we performed EdU assay in cardiac fibroblasts. The proliferation of fibroblasts was significantly increased in the DM group, by calculating the
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
percentage of EdU positive cells stained in green, compared with the control group, and this increase was substantially reversed by SSYX (Fig. 4.). 3.3. SSYX inhibits cardiac fibroblast to myofibroblast differentiation
α-SMA is a myofibroblast marker for differentiation of fibroblast to myofibroblast. Immunostaining assay was used to assess the essential role of SSYX in differentiation of fibroblast to myofibroblast. Intriguingly, the number of α-SMA positive cells was significantly increased in the DM group compared with the control group, and SSYX treatment ameliorated this change (Fig. 4). Previous studies have indicated the involvement of TGF-β1 in the process of fibroblast differentiation (Hutcheson et al., 2012). Consistently, we found that both mRNA and protein levels of TGF-β1 in the SSYX group were significantly decreased compared with the DM group, suggesting that SSYX attenuated differentiation of fibroblast to myofibroblast by effectively inhibiting expression of TGF-β1 (Fig. 5). 3.4. Smad signaling pathway underlies the molecular mechanism of SSYX's anti-fibrosis properties TGF-β1/Smad signaling, specifically Smad2/3 and Smad7, plays an important role in the development of fibrosis. We therefore examined the effects of SSYX on the levels of phosphorylated Smad2/3 (p-Smad2/3) and Smad2/3. Fig. 6 shows that p-Smad2/3 was significantly increased in the DM group compared with the
7
control group, which was restored in the SSYX group, while the expression levels of total Smad2/3 remained unaltered. Moreover, it has been reported that Smad7 prevents Smad2/3 from activation. Fig. 7 shows that Smad7 protein and mRNA levels were significantly decreased in DM rats compared with the control group, which were rescued by SSYX.
4. Discussion In this study, we made an effort to understand whether SSYX prevents diabetic cardiomyopathy and the underlying anti-fibrotic mechanisms. The main findings of this study include: (1) SSYX markedly restored heart function and inhibited cardiac fibrosis in diabetic rats; (2) SSYX effectively attenuated interstitial fibrosis by inhibiting collagen production, cardiac fibroblast proliferation, and myofibroblast formation; and (3) the underlying mechanism may involve suppression of TGF-β1/Smad signaling. Our study thus unraveled SSYX as an anti-diabetic cardiomyopathy TCM capable of inhibiting the fibrotic TGF-β1/Smad signaling pathway (Fig. 8). T2DM is a group of metabolic diseases characterized by high blood glucose, which can cause multi-organ disorders. Major complications caused by T2DM include cardiovascular diseases, diabetic neuropathy, diabetic nephropathy (chronic renal failure), and diabetic retinopathy (retinal damage). Several TCMs/phytomedicines were reported to have beneficial effects on different stages of T2DM and related complications. Refined-JinQi-JiangTang
Fig. 6. Examine of p-Smad2/3 and Smad2/3 protein expression by western blot analysis. (A) Levels of phosphorylated or activated Smad2/3 protein (p-Smad2/3) measured by western blot analysis, normalized to β-Actin in the Control, DM, and SSYX (200, 100 and 50 mg/kg/d) groups. (B) Total protein levels of Smad2/3. *po 0.05 vs Control, ♯ p o 0.05 vs DM. Upper panel, examples of western blot bands; lower panel, the statistical figure expressed as mean 7 SEM. Data are presented as relative levels.
Fig. 7. Effect of SSYX on Smad7 expression. (A) Smad7 protein level was detected by western blot analysis, normalized to β-Actin in the Control, DM, and SSYX (200, 100 and 50 mg/kg/d) groups. (B) Relative mRNA expression levels of Smad7 measured by real-time PCR in each group. *p o 0.05, **p o 0.01 vs Control, ♯po 0.05, ♯♯po 0.01 vs DM. Data are presented as mean 7 SEM.
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 8. A schematic diagram depicting the effect of Shensong Yangxin Capsule on diabetic fibrosis. Shensong Yangxin Capsule effectively prevented diabetic cardiomyopathy by inhibiting myocardial fibrosis through TGF-β/Smad signaling pathway.
tablet ameliorates prediabetes by reducing insulin resistance and improving beta cell function (Gao et al., 2014). Pu-Ren-Dan promotes the sensitivity of insulin and decreases blood glucose level by ameliorating the pancreatic blood flow and maintaining the normal structure of islet and β cells (Lu et al., 2013). Jiao Tai Wan inhibits lipogenic gene expression and thus attenuates hepatic lipid accumulation in diabetic rats and patients (Huang et al., 2013). Liuwei Dihuang decoction was reported to have neuroprotective effect by attenuating the neural apoptosis in diabetic encephalopathy rats (Liu et al., 2013). Tangnaikang can inhibit TGF-β1 induced transdifferentiation of human renal tubular epithelial cells, with the effect of treating renal interstitial fibrosis (Yang et al., 2013). Gaoshan Hongjingtian inhibits retinal
capillary degeneration in the experimental diabetic retinopathy (Zhao et al., 2013a). Our previous study has suggested that Daming capsule could preserve impaired heart function in streptozocininduced diabetic rats with hyperlipidemia. SSYX has been shown to be effective in the treatment of arrhythmias, including bradycardia (Liu et al., 2014b) and premature ventricular contractions (Zou et al., 2011). Recent study demonstrated SSYX has clinical efficacy in treating diabetic arrhythmia in senile patients (Jiang et al., 2010). The anti-arrhythmic mechanism of SSYX may be to block pacemaker channels encoded by human HCN4 gene (Sun et al., 2010). However, it is unknown whether SSYX has a role in preventing myocardial fibrosis and subsequent cardiac dysfunctions in diabetic hearts. Therefore, the
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 Q3123 124 125 126 127 128 129 130 131 132
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 Q4 56 57 58 59 60 61 62 63 64 65 66
present study was carried out to test whether SSYX has beneficial effects on diabetic cardiomyopathy in a rat model of T2DM. Ginseng Radix et Rhizoma, one of the herb components in SSYX formula, improves cardiac energy metabolism in ischemic rat heart tissues (Wang et al., 2012). The active ingredient of Salviae Miltiorrhizae Radix et Rhizoma, such as Tanshinone IIA, protects cardiomyocytes from post-infarction injury (Zhang et al., 2010). The impaired heart function was restored by SSYX in the diabetic rats. Myocardial fibrosis is one of the main pathological changes in diabetic cardiomyopathy. TGF-β1 has a crucial role in cardiac fibrosis by activating fibroblasts and producing collagen (Du et al., 2010). Expression of TGF-β1 was found increased in long-term STZ-induced T2DM myocardium (Ares-Carrasco et al., 2009). Many Chinese herbal medicines can inhibit cardiac fibrosis, such as Gualou Xiebai Decoction by blocking TGF-β1/Smad signaling (Ding et al., 2013) and scutellarin by inhibiting TGF-β1 expression (Pan et al., 2011). In the present study, TGF-β1 and the downstream collagen I, collagen III, MMP-2 and MMP-9 genes were all inhibited by SSYX in the diabetic hearts. At the same time, cardiac fibroblast proliferation and α-SMA expression level were both inhibited in SSYX treated diabetic rats. These results were consistent with previous studies showing that type II diabetes promotes a myofibroblast phenotype in cardiac fibroblasts (Yue et al., 2011; Fowlkes et al., 2013). The involvement of Smad signaling activation in the pathogenesis of cardiac fibrosis has previously been studied (Van Linthout et al., 2008; Talior-Volodarsky et al., 2012). When TGF-β1 receptor is activated, Smad7 dissociates from the TGF-β1 receptor, and then Smad 2 and Smad 3 are activated/phosphorylated (Li et al., 2013). But direct evidence that Smad phosphorylation is causal for thebeneficial effects of SSYX on diabetic cardiomyopathy is still missing. The results presented in this study showed that inhibition of Smad 2/3 phosphorylation can prevent cardiac dysfunction. TGF-β1 has been shown to induce endothelial cells to undergo EndMT (Endothelial-to-mesenchymal transition), a mechanism for the progression of cardiac fibrosis (Zeisberg et al., 2007). Diastolic dysfunction is correlated with cardiac fibrosis, which is common in patients with cardiac failure (Vasan and Benjamin, 2001). It is recognized that anti-fibrotic therapies might be useful in improving cardiac function of the diseased heart. Our present study not only provided evidences for the therapeutic potential of SSYX on diabetic cardiomyopathy, but also elucidated an underlying mechanism. In conclusion, the present work suggests that the TGF-β1/Smad signaling pathway is involved in diabetic cardiomyopathy and that SSYX treatment improves impaired heart function and inhibits fibrosis in diabetes. Moreover, SSYX is able to reduce collagen accumulation and should afford beneficial effects in diabetic cardiomyopathy.
Acknowledgments This work was supported by the Funds for Creative Research Groups (Grant number 81121003) and the Major Program (Grant number 81130088) of National Natural Science Foundation of China, the National Natural Science Foundation of China (Grant numbers 30901208, 81270042, 81200593), and the National Basic Research Program of China (973 Program, Grant number 2012CB518606).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2014.09.035.
9
References Ai, J., Wang, N., Yang, M., Du, Z.M., Zhang, Y.C., Yang, B.F., 2005. Development of Wistar rat model of insulin resistance. World Journal of Gastroenterology 11, 3675–3679. Ai, J., Yan, X., Zhao, L., Lu, Y., Liang, F., Cai, B., Li, G., Yang, B., 2009. The protective effect of Daming capsule on heart function in streptozocin-induced diabetic rats with hyperlipidemia. Biological & Pharmaceutical Bulletin 32, 1354–1358. Ares-Carrasco, S., Picatoste, B., Benito-Martin, A., Zubiri, I., Sanz, A.B., Sanchez-Nino, M.D., Ortiz, A., Egido, J., Tunon, J., Lorenzo, O., 2009. Myocardial fibrosis and apoptosis, but not inflammation, are present in long-term experimental diabetes. American Journal of Physiology – Heart and Circulatory Physiology 297, H2109–H2119. Boudina, S., Abel, E.D., 2007. Diabetic cardiomyopathy revisited. Circulation 115, 3213–3223. Cheng, Y.Z., Chen, L.J., Lee, W.J., Chen, M.F., Jung Lin, H., Cheng, J.T., 2012. Increase of myocardial performance by Rhodiola–ethanol extract in diabetic rats. Journal of Ethnopharmacology 144, 234–239. Ding, Y.F., Peng, Y.R., Li, J., Shen, H., Shen, M.Q., Fang, T.H., 2013. Gualou Xiebai Decoction prevents myocardial fibrosis by blocking TGF-beta/Smad signalling. Journal of Pharmacy and Pharmacology 65, 1373–1381. Du, J., Xie, J., Zhang, Z., Tsujikawa, H., Fusco, D., Silverman, D., Liang, B., Yue, L., 2010. TRPM7-mediated Ca2 þ signals confer fibrogenesis in human atrial fibrillation. Circulation Research 106, 992–1003. Fowlkes, V., Clark, J., Fix, C., Law, B.A., Morales, M.O., Qiao, X., Ako-Asare, K., Goldsmith, J.G., Carver, W., Murray, D.B., Goldsmith, E.C., 2013. Type II diabetes promotes a myofibroblast phenotype in cardiac fibroblasts. Life Sciences 92, 669–676. Frode, T.S., Medeiros, Y.S., 2008. Animal models to test drugs with potential antidiabetic activity. Journal of Ethnopharmacology 115, 173–183. Gao, L.H., Liu, Q., Liu, S.N., Chen, Z.Y., Li, C.N., Lei, L., Sun, S.J., Li, L.Y., Liu, J.L., Shen, Z.F., 2014. A refined-JinQi-JiangTang tablet ameliorates prediabetes by reducing insulin resistance and improving beta cell function in mice. Journal of Ethnopharmacology 151, 675–685. Go, A.S., Mozaffarian, D., Roger, V.L., Benjamin, E.J., Berry, J.D., Blaha, M.J., Dai, S., Ford, E.S., Fox, C.S., Franco, S., Fullerton, H.J., Gillespie, C., Hailpern, S.M., Heit, J.A., Howard, V.J., Huffman, M.D., Judd, S.E., Kissela, B.M., Kittner, S.J., Lackland, D.T., Lichtman, J.H., Lisabeth, L.D., Mackey, R.H., Magid, D.J., Marcus, G.M., Marelli, A., Matchar, D.B., McGuire, D.K., Mohler 3rd, E.R., Moy, C.S., Mussolino, M.E., Neumar, R.W., Nichol, G., Pandey, D.K., Paynter, N.P., Reeves, M.J., Sorlie, P.D., Stein, J., Towfighi, A., Turan, T.N., Virani, S.S., Wong, N.D., Woo, D., Turner, M.B., 2014. Heart disease and stroke statistics – 2014 update: a report from the American Heart Association. Circulation 129, e28–e292. Huang, Z., Xu, X., Lu, F., Wang, N., Chen, G., Zhao, Y., Zou, X., Wang, K., Dong, H., Xu, L., 2013. Jiao tai wan attenuates hepatic lipid accumulation in type 2 diabetes mellitus. Evidence-Based Complementary and Alternative Medicine 2013, 567045. Hutcheson, J.D., Ryzhova, L.M., Setola, V., Merryman, W.D., 2012. 5-HT(2B) antagonism arrests non-canonical TGF-beta1-induced valvular myofibroblast differentiation. Journal of Molecular and Cellular Cardiology 53, 707–714. Jiang, X.G., Jia, J.M., YS., L., 2013. Simultaneous determination of eight bioactive constituents in shensong yangxin capsule by uplc. Chinese Herbal Medicines, 212–216. Jiang, X.L., Sun, X.H., Gu, Y.C., Zou, S.R., 2010. Clinical observation of Shensong Yangxin Capsule in treating senile diabetic arrhythmia. Hebei Journal of Traditional Chinese Medicine 32, 1046–1048. Jing, A., Li-Mei, Z., Yan-Jie, L., Ben-Zhi, C., Yong, Z., Bao-Feng, Y., 2009. A randomized, multicentre, open-label, parallel-group trial to compare the efficacy and safety profile of daming capsule in patients with hypercholesterolemia. Phytotherapy Research 23, 1039–1042. Joshi, M., Kotha, S.R., Malireddy, S., Selvaraju, V., Satoskar, A.R., Palesty, A., McFadden, D.W., Parinandi, N.L., Maulik, N., 2014. Conundrum of pathogenesis of diabetic cardiomyopathy: role of vascular endothelial dysfunction, reactive oxygen species, and mitochondria. Molecular and Cellular Biochemistry 386, 233–249. Li, N., Ma, K.J., Wu, X.F., Sun, Q., Zhang, Y.H., Pu, J.L., 2007. Effects of Chinese herbs on multiple ion channels in isolated ventricular myocytes. Chinese Medical Journal (English Edition) 120, 1068–1074. Li, Q., Zhang, D., Wang, Y., Sun, P., Hou, X., Larner, J., Xiong, W., Mi, J., 2013. MiR-21/ Smad 7 signaling determines TGF-beta1-induced CAF formation. Scientific Reports 3, 2038. Liu, J.P., Feng, L., Zhang, M.H., Ma, D.Y., Wang, S.Y., Gu, J., Fu, Q., Qu, R., Ma, S.P., 2013. Neuroprotective effect of Liuwei Dihuang decoction on cognition deficits of diabetic encephalopathy in streptozotocin-induced diabetic rat. Journal of Ethnopharmacology 150, 371–381. Liu, M., Li, S., Zhao, S., Wang, H., Tu, P., 2014a. Studies on excretion kinetics of ten constituents in rat urine after oral administration of Shensong Yangxin Capsule by UPLC–MS/MS. Biomedical Chromatography 28, 525–533. Liu, Y., Li, N., Jia, Z., Lu, F., Pu, J., 2014b. Chinese medicine shensongyangxin is effective for patients with bradycardia: results of a randomized, double-blind, placebo-controlled multicenter trial. Evidence-Based Complementary and Alternative Medicine 2014, 605714. Lu, B., Bai, Y., Du, Z., Chen, S., Deligema, D., Pang, Z., 2013. The effect of Chinese medicine Pu-Ren-Dan on pancreatic angiogenesis in high fat diet/streptozotocin-induced diabetic rats. Indian Journal of Pharmacology 45, 556–562. Mori, J., Patel, V.B., Abo Alrob, O., Basu, R., Altamimi, T., Desaulniers, J., Wagg, C.S., Kassiri, Z., Lopaschuk, G.D., Oudit, G.Y., 2014. Angiotensin 1–7 ameliorates
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 Q5 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
N. Shen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
diabetic cardiomyopathy and diastolic dysfunction in db/db mice by reducing lipotoxicity and inflammation. Circulation: Heart Failure 7, 327–339. Pan, Z., Zhao, W., Zhang, X., Wang, B., Wang, J., Sun, X., Liu, X., Feng, S., Yang, B., Lu, Y., 2011. Scutellarin alleviates interstitial fibrosis and cardiac dysfunction of infarct rats by inhibiting TGFbeta1 expression and activation of p38-MAPK and ERK1/2. British Journal of Pharmacology 162, 688–700. Park, S.Y., Choi, Y.H., Lee, W., 2008. Dangnyohwan improves glucose utilization and reduces insulin resistance by increasing the adipocyte-specific GLUT4 expression in Otsuka Long-Evans Tokushima Fatty rats. Journal of Ethnopharmacology 115, 473–482. Singh, R., Kaur, N., Kishore, L., Gupta, G.K., 2013. Management of diabetic complications: a chemical constituents based approach. Journal of Ethnopharmacology 150, 51–70. Sun, L.P., Li, N., Wu, Y.L., Pu, J.L., 2010. Effects of Shensong Yangxin capsule on pacemaker channels encoded by human HCN4 gene. Chinese Medical Journal (English Edition) 123, 3148–3150. Talior-Volodarsky, I., Connelly, K.A., Arora, P.D., Gullberg, D., McCulloch, C.A., 2012. alpha11 integrin stimulates myofibroblast differentiation in diabetic cardiomyopathy. Cardiovascular Research 96, 265–275. Van Linthout, S., Seeland, U., Riad, A., Eckhardt, O., Hohl, M., Dhayat, N., Richter, U., Fischer, J.W., Bohm, M., Pauschinger, M., Schultheiss, H.P., Tschope, C., 2008. Reduced MMP-2 activity contributes to cardiac fibrosis in experimental diabetic cardiomyopathy. Basic Research in Cardiology 103, 319–327. Vasan, R.S., Benjamin, E.J., 2001. Diastolic heart failure–no time to relax. New England Journal of Medicine 344, 56–59. Wang, J.R., Zhou, H., Yi, X.Q., Jiang, Z.H., Liu, L., 2012. Total ginsenosides of Radix Ginseng modulates tricarboxylic acid cycle protein expression to enhance cardiac energy metabolism in ischemic rat heart tissues. Molecules 17, 12746–12757. Yang, L., Ma, X., Cheng, T., Liu, T., Wu, L., Sun, W., Joseph, M.P., 2013. Effect of tangnaikang on TGF-beta1-induced transdifferentiation of human renal tubular epithelial HK-2 cells. Journal of Traditional Chinese Medicine 33, 388–393.
Yue, L., Xie, J., Nattel, S., 2011. Molecular determinants of cardiac fibroblast electrical function and therapeutic implications for atrial fibrillation. Cardiovascular Research 89, 744–753. Zeisberg, E.M., Tarnavski, O., Zeisberg, M., Dorfman, A.L., McMullen, J.R., Gustafsson, E., Chandraker, A., Yuan, X., Pu, W.T., Roberts, A.B., Neilson, E.G., Sayegh, M.H., Izumo, S., Kalluri, R., 2007. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nature Medicine 13, 952–961. Zhang, H., Zhao, T., Gong, Y., Dong, X., Zhang, W., Sun, S., Wang, H., Gu, Y., Lu, X., Yan, M., Li, P., 2014. Attenuation of diabetic nephropathy by Chaihuang-Yishen granule through anti-inflammatory mechanism in streptozotocin-induced rat model of diabetics. Journal of Ethnopharmacology 151, 556–564. Zhang, Y., Zhang, L., Chu, W., Wang, B., Zhang, J., Zhao, M., Li, X., Li, B., Lu, Y., Yang, B., Shan, H., 2010. Tanshinone IIA inhibits miR-1 expression through p38 MAPK signal pathway in post-infarction rat cardiomyocytes. Cellular Physiology and Biochemistry 26, 991–998. Zhao, H.S., Shi, X.Y., Wei, W.B., Wang, N.L., 2013a. Effect of the regimen of Gaoshan Hongjingtian on the mechanism of poly (ADP-ribose) polymerase regulation of nuclear factor kappa B in the experimental diabetic retinopathy. Chinese Medical Journal (English Edition) 126, 1693–1699. Zhao, Y., Zhang, L., Qiao, Y., Zhou, X., Wu, G., Wang, L., Peng, Y., Dong, X., Huang, H., Si, L., Zhang, X., Li, J., Wang, W., Zhou, L., Gao, X., 2013b. Heme oxygenase-1 prevents cardiac dysfunction in streptozotocin-diabetic mice by reducing inflammation, oxidative stress, apoptosis and enhancing autophagy. PLoS One 8, e75927. Zou, J.G., Zhang, J., Jia, Z.H., Cao, K.J., 2011. Evaluation of the traditional Chinese Medicine Shensongyangxin capsule on treating premature ventricular contractions: a randomized, double-blind, controlled multicenter trial. Chinese Medical Journal (English Edition) 124, 76–83.
Please cite this article as: Shen, N., et al., Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-β1/ Smad signaling. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.035i
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49