Original Papers
269
Protective Effects of Oxymatrine on Experimental Diabetic Nephropathy
Authors
Changrun Guo 1, Fengyu Han 1, Chunfeng Zhang 1, Wei Xiao 2, Zhonglin Yang 1
Affiliations
1
Key words " oxymatrine l " diabetic nephropathy l " oxidative stress l " TNF‑α l " TGF‑β1 l " Sophora flavescens l " Fabaceae l
received revised accepted
October 27, 2013 January 7, 2014 January 13, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1360369 Published online February 17, 2014 Planta Med 2014; 80: 269–276 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Wei Xiao State Key Laboratory of Natural Medicines China Pharmaceutical University No. 24 Tongjia Lane Nanjing City 210009 P. R. China Phone: + 86 25 83 27 14 26 Fax: + 86 2 58 3 27 14 26 [email protected] Correspondence Zhonglin Yang State Key Laboratory of Natural Medicines China Pharmaceutical University No. 24 Tongjia Lane Nanjing City 210009 P. R. China Phone: + 86 25 83 27 14 26 Fax: + 86 25 83 27 14 26 [email protected]
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, P. R. China Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, P. R. China
Abstract
experimental diabetic nephropathy by multiple mechanisms.
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Diabetic nephropathy, one of the most common and serious vascular complications of both type 1 and type 2 diabetes mellitus, has become a major contributor of end-stage renal failure. The aims of this study were to investigate the effects and possible underlying action mechanism(s) of oxymatrine on renal damage in diabetic rats. Diabetes was induced in male Sprague-Dawley rats by administering a high-fat diet and an intraperitoneal 30 mg/kg streptozotocin injection. The animals were treated orally with saline, metformin hydrochloride, and oxymatrine at 50, 100, and 150 mg/ kg/day for 11 weeks. At the end of the treatment, renal tissue, blood, and urine samples were collected for histological and biochemical examination. The results revealed that oxymatrine significantly decreased blood glucose, urinary protein and albumin excretion, serum creatinine, and blood urea nitrogen in diabetic rats, and ameliorated diabetes-induced glomerular and tubular pathological changes. Furthermore, oxymatrine significantly prevented oxidative stress and reduced the contents of renal advanced glycation end products, transforming growth factor-β1, connective tissue growth factor, and inflammatory cytokines in diabetic rats. All these results indicate that oxymatrine has protective effects on
Introduction !
Diabetic nephropathy, one of the most common and serious vascular complications of both type 1 and type 2 diabetes mellitus, has become a major contributor to end-stage renal failure [1]. The symptoms of DN include renal hypertrophy, glomerular hyperfiltration, ECM accumulation, and albuminuria. Albuminuria, induced by an increase in the albumin excretion rate, is widely acknowledged as the earliest index of DN and is a major
Abbreviations !
AGEs: BUN: CAT: CCr: CTGF: DC: DN: ECM: GSH‑Px: HE: ICMA-1: IOD: MDA: Met: NC: OMT: PAS: Scr: SOD: STZ: TNF-α: TGF-β1:
advanced glycation end products blood urea nitrogen catalase creatinine clearance ratio connective tissue growth factor diabetic control diabetic nephropathy extracellular matrix glutathione peroxidase hematoxylin-eosin intercellular adhesion molecule-1 integrated optical density malondialdehyde metformin hydrochloride normal control oxymatrine periodic acid–Schiff serum creatinine superoxide dismutase streptozotocin tumor necrosis factor-α transforming growth factor-β1
biochemical feature that aids in the clinical diagnosis of DN [2, 3]. Although the mechanisms of DN are yet to be explained, studies have demonstrated that many factors contribute to the progression of DN, including hyperglycemia, oxidative stress, and inflammatory reaction. Among these factors, hyperglycemia is the key initiating factor in the development of DN, and oxidative stress, as a result of hyperglycemia, is regarded as the major factor contributing to the progression of DN [4, 5]. Previous studies have provided evi-
Guo C et al. Protective Effects of …
Planta Med 2014; 80: 269–276
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Original Papers
Fig. 1 Structure of OMT.
Fig. 2 Effect of OMT on fasting blood glucose in diabetic rats. Values are presented as mean ± SD for 8 rats in each group. ##P < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group.
dence that reactive oxygen species mediate the activation of TGFβ1, which is widely expressed in all kidney cells. Subsequently, TGF-β1 stimulates the synthesis of matrix components, the accumulation of ECM, and the formation of albuminuria [6]. TGF-β1 also has a bearing on the increase in Scr and BUN in patients with DN [7]. Currently, only a few drugs that are available are effective against DN; therefore, there is a need to search for safe drugs which are " Fig. 1), a major quinolizidine alkaeffective against DN. OMT (l loid in Sophora flavescens (Fabaceae), has been shown to exhibit numerous pharmacological effects, such as anti-inflammatory, antioxidation, neuroprotection, antitumor, and antiarrhythmia in modern pharmacological research [8–10]. In view of the pathogenesis of DN and the pharmacological effects of OMT, we hypothesized that OMT may have a beneficial effect on the progression of DN. This study was carried out to examine the effects of OMT in rats with DN induced by a high-fat diet and a low dose of STZ, and elucidate the potential action mechanism(s).
Results !
Before administering the respective drugs, the fasting blood glucose levels of the five diabetic groups (diabetic control, metformin, and three OMT groups) were almost identical and signifi-
cantly higher than that of the normal control group (p < 0.01). However, after treatment for 11 weeks, the fasting blood glucose levels significantly decreased in the metformin and three OMT " Fig. 2). groups compared to the diabetic control group (l " l Table 1 shows the results of the kidney index, BUN, and Scr in the studied groups. The kidney index of the diabetic control rats was 76% higher than the index of the normal control rats (p < 0.01), while treatment with OMT at doses of 100 mg/kg and 150 mg/kg for 11 weeks significantly reduced the increase of the kidney index. Likewise, the levels of BUN and Scr were significantly increased in the diabetic control group compared to the normal control group (p < 0.01). Treatment with OMT (100 mg/ kg and 150 mg/kg) for 11 weeks produced a significant reduction in BUN and Scr levels. Excretion of urinary protein and albumin are markers of glomer" Table 1, ular dysfunction and tubular impairment. As shown in l the excretion of urinary protein and albumin were markedly elevated in the diabetic control group (p < 0.01). However, treatment with OMT at the doses of 50, 100, and 150 mg/kg for 11 weeks significantly decreased the elevation of urinary protein and albumin excretion (p < 0.01) compared to the diabetic control group. CCr is also an indicator of glomerular dysfunction. In this study, the CCr of the diabetic control rats was significantly decreased compared to the normal control group (p < 0.01), which was significantly inhibited by OMT treatment.
Table 1 Effect of OMT on body weight, kidney weight, kidney index, heart weight, BUN, Scr, CCr, urine protein, and albumin in diabetic rats. Index
NC
DC
Met
OMT-50
OMT-100
OMT-150
Body weight (g) Kidney weight (g) KI (g/kg) Heart weight (g) BUN (mmol/L) Scr (µmol/L) CCr (mL/min/kg) Urine protein (mg/kg/24 h) Urine albumin (mg/kg/24 h)
560.1 ± 48.6 1.36 ± 0.07 2.44 ± 0.27 1.00 ± 0.20 4.74 ± 0.55 104.1 ± 13.9 3.24 ± 0.39 26.4 ± 10.3 0.42 ± 0.17
339.9 ± 54.6## 1.43 ± 0.08 4.31 ± 0.75## 0.65 ± 0.15 5.79 ± 0.64## 146.1 ± 27.9## 2.73 ± 0.22## 212.4 ± 45.1## 4.08 ± 0.76##
386.9 ± 84.7 1.20 ± 0.17** 3.29 ± 1.08* 0.63 ± 0.11 5.14 ± 0.49* 118.9 ± 22.4* 2.81 ± 0.67 138.6 ± 55.1** 3.45 ± 1.47
361.9 ± 63.8 1.32 ± 0.14 3.74 ± 0.69 0.64 ± 0.10 5.21 ± 0.48 116.8 ± 17.2* 2.90 ± 0.47 132.5 ± 50.8** 2.43 ± 0.88**
390.7 ± 53.8 1.35 ± 0.07 3.53 ± 0.47 0.61 ± 0.14 5.13 ± 0.29* 114.5 ± 13.2* 3.24 ± 0.30** 125.8 ± 36.8** 2.15 ± 1.47**
419.6 ± 62.9* 1.38 ± 0.11 3.32 ± 0.43* 0.66 ± 0.11 5.04 ± 0.41* 110.8 ± 18.5** 3.32 ± 0.44** 116.0 ± 47.6** 1.96 ± 1.10**
Values are presented as mean ± SD for 8 rats in each group. ##P < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group
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Fig. 4 Representative micrographs of kidney tissue stained with PAS (arrows show the thickened basement membrane). A NC group, B DC group, C Met group, D OMT-50 group, E OMT-100 group, and F OMT-150 group. (Color figure available online only.)
The sections from normal control rats showed a normal histology of the kidney. The glomerulus, glomerular capsules, and tubules " Figs. 3 A and 4 A). Representawere very clear and prominent (l tive glomerular and tubular changes were observed in the kid" Figs. 3 B and 4 B). Histopathoneys of the diabetic control rats (l logical examination of the kidneys from the diabetic control rats showed signs of tubular necrosis with vacuolar degeneration of the proximal tubules and a thickened basement membrane, and the glomerulus exhibited segmental sclerosis with basement membrane thickening and mesangial expansion. Treatment with OMT for 11 weeks significantly inhibited these changes in the " Figs. 3 and 4). kidneys (l Type IV collagen is a major component of basement membranes and is expressed in both the glomerular and tubular basement membranes. To investigate the effect of OMT on the expression of basement membrane components, type IV collagen was determined by an immunohistochemical technique. Immunohistochemical staining of type IV collagen showed that there was a significant increase in the expression of type IV collagen in the renal glomerulus and tubules of the diabetic control rats compared to the normal control rats, which was significantly inhibited by " Fig. 5). OMT (l In diabetic patients with nephropathy, the level of ICAM-1 was " Fig. 6, ICAM-1 positive staining in elevated [11]. As shown in l the diabetic rats was more prominent than that in the normal
control rats. The OMT treatment significantly reduced expression of ICAM-1 in the kidneys of diabetic rats. Oxidative stress is a key pathogenic factor in the development of " Table 2 shows the effects of OMT on SOD, GSH‑Px, CAT DN [12]. l activities, and MDA content in the kidneys. The activities of renal SOD, GSH‑Px, and CAT were markedly decreased, whereas renal MDA content in the diabetic control group was markedly increased compared to that in the normal control group (p < 0.01). Treatment with OMT for 11 weeks effectively elevated the activities of SOD, GSH‑Px, and CAT, and inhibited MDA content. These results indicate that treatment with OMT could significantly inhibit renal oxidative stress in diabetic rats. TNF-α plays an important role in the development of DN and is a therapeutic target for its treatment [13]. In this study, we investigated the effect of OMT on renal TNF-α in diabetic rats. Significantly elevated levels of renal TNF-α were found in the diabetic control rats; however, treatment with OMT for 11 weeks dosedependently reduced the content of TNF-α in the kidneys of the " Fig. 7). diabetic rats (l The TGF-β1/CTGF axis plays a critical role in progressive kidney " Table 3, when compared to the nordisease [14]. As shown in l mal control group, the levels of renal TGF-β1 and CTGF were markedly increased in the diabetic control group (p < 0.01). The increase in renal TGF-β1 and CTGF levels was effectively relieved by the administration of OMT.
Guo C et al. Protective Effects of …
Planta Med 2014; 80: 269–276
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Fig. 3 Representative micrographs of kidney tissue stained with HE (arrows show vacuolar degeneration of the proximal tubules). A NC group, B DC group, C Met group, D OMT-50 group, E OMT100 group, and F OMT-150 group. (Color figure available online only.)
Original Papers
Fig. 5 Effect of OMT on renal collagen IV in diabetic rats. The brown area is the positive expression area, enlarged 400× under a light microscope. A NC group, B DC group, C Met group, D OMT50 group, E OMT-100 group, F OMT-150 group, and G IOD of collagen IV. Values are presented as mean ± SD for 8 rats in each group. ##P < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group. (Color figure available online only.)
Fig. 6 Effect of OMT on renal ICAM-1 in diabetic rats. The brown area is the positive expression area, enlarged 400× under a light microscope. A NC group, B DC group, C Met group, D OMT-50 group, E OMT-100 group, F OMT-150 group, and G IOD of ICAM-1. Values are presented as mean ± SD for 8 rats in each group. ##P < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group. (Color figure available online only.)
Table 2 Effect of OMT on renal SOD, CAT, GSH‑Px, and MDA levels in diabetic rats. Index
NC
DC
Met
OMT-50
OMT-100
OMT-150
SOD (U/mg prot) CAT (U/mg prot) GSH‑Px (U/µg prot) MDA (nmol/mg prot)
206.6 ± 28.2 40.34 ± 7.10 1.85 ± 0.47 2.09 ± 0.27
147.4 ± 27.3## 31.28 ± 6.25# 1.33 ± 0.14# 3.18 ± 0.31##
142.1 ± 52.7 37.49 ± 9.43 1.52 ± 0.43 2.83 ± 0.53
183.2 ± 24.3 39.48 ± 5.48 1.75 ± 0.23 2.66 ± 0.50
186.9 ± 23.8* 39.90 ± 6.32* 1.85 ± 0.23* 2.42 ± 0.61*
187.2 ± 23.3* 40.88 ± 3.67* 1.86 ± 0.39* 2.40 ± 0.71*
Values are presented as mean ± SD for 8 rats in each group. #P < 0.05, ##p < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group
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Table 3 Effect of OMT on renal TGF-β1 and CTGF levels in diabetic rats. Groups
TGF-β1 (ng/mg prot)
CTGF (pg/mg prot)
NC DC Met OMT-50 OMT-100 OMT-150
3.05 ± 0.53 5.75 ± 0.48## 5.12 ± 0.47 4.86 ± 0.89* 4.66 ± 0.41** 4.55 ± 0.63**
84.8 ± 11.4 111.3 ± 14.5## 96.5 ± 14.4 108.8 ± 19.7 99.5 ± 8.9* 94.9 ± 6.0*
Values are presented as mean ± SD for 8 rats in each group. ##P < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group
AGEs are considered a marker of diabetic complications, includ" Fig. 8 shows the effect of OMT ing diabetes nephropathy [15]. l on renal content of AGEs in diabetic rats. The content of the renal AGEs in the diabetic control group was markedly increased compared to the normal control group (p < 0.01). However, OMT treatment markedly decreased the contents of the renal AGEs in the diabetic rats.
Discussion !
Although OMT is a toxic alkaloid, it has been used in humans as a safe oral drug in the treatment of hepatic fibrosis [16]. In our study, the rats did not show any signs of toxicity after the treatment period. Treatment with OMT for 11 weeks did not change " Table 1). the kidney weight, or the heart weight of diabetic rats (l Typical features of diabetic renal injuries include renal hypertrophy, glomerular hyperfiltration, albuminuria, basement membrane thickening, ECM accumulation, and tubulointerstitial fibrosis. In our study, the diabetic rats demonstrated these features, while OMT treatment provided renoprotection with suppression of albuminuria and alleviated glomerular and tubular injuries. A high blood glucose level is still considered to be the inducer of diabetic complications, including nephropathy. Historically, various hypotheses have been proposed to suggest potential mechanisms by which hyperglycemia causes diabetic complications. The four main hypotheses are increased polyol pathway flux, increased intracellular formation of AGEs, increased hexosamine pathway flux, and excessive activation of protein kinase C iso-
Fig. 8 Effect of OMT on the levels of the renal AGEs in diabetic rats. Values are presented as mean ± SD for 8 rats in each group. ##P < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group.
forms. Overproduction of superoxide by the mitochondrial electron transport chain is considered to be the common element linking these four mechanisms. Studies have conclusively shown that oxidative stress is a key pathogenic factor in the development of DN [12]. In the present study, treatment with OMT inhibited the oxidative stress state in DN rats (as evidenced by decreased renal MDA contents), and improved antioxidant defenses (as evidenced by increased activities of renal SOD, GSH‑Px, and CAT). These results indicate that OMT imparts renoprotection against oxidative stress in DN. AGEs are also one of the key pathogenic factors responsible for diabetes nephropathy, which is indicated by the fact that the AGEs inhibitor aminoguanidine ameliorates diabetes-induced renal damage [5, 17]. The formation of AGEs can interfere with matrix-matrix interactions and matrix-cell interactions [15]. It is well known that hyperglycemia-induced oxidative stress facilitates the AGEs formation; on the other hand, the interactions between AGEs and receptors for AGEs induce the activation of oxidative stress [5]. This mutual interaction leads to ECM accumulation and mesangial cell hypertrophy. In our study, treatment with OMT reduced the formation of AGEs, and this could be beneficial for the prevention of DN. As a typical feature of DN, ECM accumulation can lead to glomerular fibrosis and loss of renal function. TGF-β1, both a fibrogenic and an inflammatory cytokine, is stimulated by high glucose, AGEs, and reactive oxygen species in diabetes. Several studies have demonstrated that TGF-β1 plays an important role in the accumulation of ECM and renal fibrosis [7]. For example, excessive deposition of type IV collagen caused by TGF-β1 is one of the reasons for diabetic glomerulopathy. In our experiments, treatment with OMT could inhibit the accumulation of TGF-β1, type IV collagen, and ECM, and ameliorate mesangial expansion. CTGF, a member of the CCN family, is increasingly being implicated in structural and functional changes in DN. Both in diabetic animal models and in human diabetic renal tissue, the levels of CTGF are elevated, which is related to hyperglycemia, AGEs formation, and hemodynamic change [14]. Several studies have identified the role of CTGF in fibroblast proliferation, migration, adhesion, and ECM formation [18]. As a crucial downstream mediator for TGF-β1 stimulation, CTGF not only mediates the induction of ECM protein expression, but also inhibits matrix degradation. Therefore, TGF-β1 and CTGF form a major axis in renal fibrosis [14]. In our study, the expression of CTGF in the diabetic control group was markedly increased, while for treatment with
Guo C et al. Protective Effects of …
Planta Med 2014; 80: 269–276
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Fig. 7 Effect of OMT on renal TNF-α levels in diabetic rats. Values are presented as mean ± SD for 8 rats in each group. ##P < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group.
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OMT, the increase of CTGF was ameliorated significantly. These results suggest that OMT may ameliorate renal fibrosis by mediating the TGF-β1/CTGF axis. Experimental and clinical studies demonstrate that inflammatory cytokines, such as TNF-α and ICAM-1, play a prominent role in the development and progression of DN [19]. In addition to infiltrating macrophages, TNF-α is synthesized by endothelial, mesangial, glomerular, and tubular epithelial cells, with the stimulation of AGEs and high glucose levels. Overexpression of TNF-α can result in hemodynamic alterations, damage to the glomerular permeability barrier, and apoptosis of renal cells [13]. ICAM-1 also promotes the recruitment of monocytes and macrophages in diabetic kidneys. These monocytes and macrophages synthesize a variety of growth factors, including TGF-β1, which contributes to the glomerular damage in diabetes [11]. Our study indicates that treatment with OMT significantly inhibited the expression of TNF-α and ICAM-1 in diabetic renal tissue, and thus improved the structural and functional abnormalities of the diabetic kidney. The interaction among hyperglycemia, oxidative stress, AGEs, TGF-β1, CTGF, and inflammatory cytokines has become a focus area in the research of DN [20]. Under hyperglycemic conditions, oxidative stress and AGEs mutually interact leading to upregulation, which can activate the TGF-β1/CTGF axis and lead to overexpression of inflammatory cytokines, subsequently inducing changes in renal structure and function [13, 18]. Our study demonstrated that OMT attenuated the formation of reactive oxygen species and AGEs, and reduced TGF-β1, CTGF, and inflammatory cytokine expression in diabetic kidneys. Therefore, by way of these mechanisms, OMT imparts renoprotection.
Materials and Methods
Pre-experiment To select the better doses of OMT, we performed a pre-experiment. In the pre-experiment, the rats were injected with STZ (65 mg/kg, dissolved in 0.01 M sodium citrate buffer, pH 4.4) intraperitoneally. After 72 hours, fasting blood glucose was measured. The rats with a fasting blood glucose level > 11.1 mmol/L were considered diabetic and selected for further pharmacological studies. According to the instructions for taking oxymatrine tablets (a drug already on the market), the adult dose of OMT is 600 or 900 mg/day. The ratʼs dose of OMT, calculated by body surface area, was 66 or 99 mg/kg/day. Therefore, we chose 66 mg/kg/ day ÷ 1.5 ≈ 50 mg/kg/day, 66 mg/kg/day × 1.5 ≈ 100 mg/kg/day, and 99 mg/kg/day × 1.5 ≈ 150 mg/kg/day as the low, middle, and high dose, respectively. Treatment with OMT at the three doses for 10 weeks significantly decreased blood glucose and the excretion of urinary albumin. Therefore, we chose the three doses at 50, 100, and 150 mg/kg/day for further studies.
Induction of experimental diabetes After one week of adaptive feeding, the animals were randomly divided into two groups: a control group and an experimental group. The control group rats were given a regular diet, while the experimental group rats were fed a high-fat diet (consisting of 70 % standard laboratory food, 15 % carbohydrate, 10 % lard, and 5 % yolk powder) [22]. After 4 weeks of high-fat diet feeding, the experimental group rats were injected with STZ (30 mg/kg, dissolved in 0.01 M sodium citrate buffer, pH 4.4) intraperitoneally, while the control group rats were injected with the vehicle citrate buffer [23]. Fasting blood glucose was measured one week after injection. The rats with a fasting blood glucose level > 11.1 mmol/L were considered diabetic and selected for further pharmacological studies. The animals were fed their respective diets until the end of the study [24, 25].
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Chemicals and reagents
Experimental design and treatment protocol
OMT (98.2 % purity) was supplied by Qingze Pharmaceutical, Inc. Met (99.1% purity), the positive control in these experiments, was obtained from Jingfeng Pharmaceutical, Inc. OMT and Met were dissolved in normal saline (0.9% NaCl saline solution) for the animal experiment. STZ was purchased from Sigma-Aldrich, Inc. ELISA kits for TNF-α and TGF-β1 were obtained from Abcam, Inc., while those for the AGEs, urinary albumin, and CTGF were obtained from Biocompare, Inc. Kits for blood glucose, SOD, MDA, GSH‑Px, CAT, creatinine, BUN, and urine protein were purchased from Nanjing Jiancheng Institute. The rabbit anti-rat ICAM-1 monoclonal antibody and the rabbit anti-rat type IV collagen monoclonal antibody were obtained from Santa Cruz Biotechnology, Inc.
The normal and diabetic rats were randomly divided into six groups of eight rats each: NC group, normal rats treated with saline in a matched volume; DC group, diabetic rats treated with saline in a matched volume; Met group, diabetic rats administered Met 200 mg/kg/day; OMT-50 group, diabetic rats administered OMT 50 mg/kg/day; OMT-100 group, diabetic rats administered OMT 100 mg/kg/day; and OMT-150 group, diabetic rats administered OMT 150 mg/kg/day. All drugs were administered orally via an orogastric cannula, continuously for 11 weeks. At the end of the treatment, urine samples were collected from the rats housed in individual metabolic cages after 24 h for analysis of urine volume and other biochemical parameters. The rats were fasted for 12 h and then were anesthetized to collect their blood. Post-fasting blood was collected via the abdominal aorta for blood biochemical parameters analysis, and then the kidneys were removed. After being weighed, the right kidneys were collected for histology examination and immunohistochemistry. The renal cortex of the left kidneys was separated and stored at − 70 °C for biochemical estimations.
Animals Adult male Sprague-Dawley rats (weight 180–220 g), with animal quality certificate number [(Su)SCKX2008–0010], were purchased from Nantong University laboratory animal center and maintained at a constant temperature of 23 ± 2 °C on a 12-h light/dark cycle with free access to normal laboratory food and water. All procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals [21] and were approved by the ethics committee of the China Pharmaceutical University (CEAE-093, Jan 10, 2013).
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Planta Med 2014; 80: 269–276
Analysis of blood and urine samples The blood samples were centrifuged at 4000 g for 10 min to separate the serum. The blood glucose, Scr, and BUN levels were determined using commercially available kits according to the manufacturerʼs instructions. Urine samples were centrifuged at 1600 g for 5 min to separate the supernatant. The urinary albumin concentration was determined using an ELISA kit. Urinary
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protein and creatinine concentrations were determined using commercially available kits. CCr was calculated usuing the following formula: CCr = urinary creatinine (mg/mL) × urine volume (mL/kg)/Scr (mg/mL)/1440 (min) [26, 27].
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Acknowledgements !
This work was supported by a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. The assistance of the staff is gratefully acknowledged.
Histological examination
Immunohistochemical analysis of ICAM-1 and type IV collagen Immunostaining was performed on 4-µm renal sections after deparaffinization and rehydration. The sections were microwaved for antigen retrieval in citrate buffer (pH 6.0) for 10 min and then were treated with 3% H2O2 buffer for 10 min. After that, the sections were thoroughly washed in tap water and blocked with 10 % (w/v) normal goat serum for 1 h. After blocking, the sections were incubated with rabbit monoclonal ICMA-1 and type IV collagen antibody at 4 °C overnight, followed by goat anti-rabbit IgG horseradish peroxidase for 30 min at 37 °C. To visualize ICMA-1 and type IV collagen, the sections were stained with chromogen diaminobenzidine for 10 min and counterstained with hematoxylin. At least 20 random fields of each section were examined under a light microscope and analyzed using Image-Pro Plus 6.0 software to calculate the value of the positive IOD.
Analysis of renal oxidative stress The renal cortex was sliced into pieces and homogenized with appropriate Tris–HCl (5 mmol/L containing 2 mmol/L EDTA, pH 7.4). Homogenates were centrifuged at 4000 g for 10 min at 4 °C. The supernatants were separated and used immediately for the assays for SOD, CAT, GSH‑Px, and MDA. They were determined using commercially available kits according to the manufacturerʼs instructions and were normalized as U/mg protein or nmol/mg protein. The protein content in the homogenate was measured by the Bradford method.
Analysis of cytokine levels and AGEs in kidney tissues The levels of TNF-α, TGF-β1, CTGF, and AGEs in renal homogenates (the same samples were used for renal oxidative stress analysis) were measured using commercial ELISA kits according to the manufacturerʼs protocols. The levels of these cytokines and AGEs in renal tissues were normalized to the protein content.
Statistical analysis Data are expressed as mean ± SD for eight animals in each group. Statistically significant differences between the two groups were determined by one-way analysis of variance, followed by a post hoc test (Dunnettʼs test). In all cases, probability values of p < 0.05 were considered statistically significant.
Conflict of Interest !
All authors have no conflicts of interest.
References 1 Lopes A. End-stage renal disease due to diabetes in racial/ethnic minorities and disadvantaged populations. Ethn Dis 2009; 19: S1–S47 2 Leese G, Savage M, Chattington P, Vora J. The diabetic patient with hypertension. Postgrad Med J 1996; 72: 263–268 3 Mishra A, Bhatti R, Singh A, Ishar M. Ameliorative effect of the cinnamon oil from Cinnamomum zeylanicum upon early stage diabetic nephropathy. Planta Med 2010; 76: 412–417 4 UKPDS. United Kingdom Prospective Diabetic Study. Lancet 1998; 352: 837–853 5 Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001; 414: 813–820 6 Tahara A, Tsukada J, Tomura Y, Yatsu T, Shibasaki M. Vasopressin increases type IV collagen production through the induction of transforming growth factor-beta secretion in rat mesangial cells. Pharmacol Res 2008; 57: 142–150 7 Hu Y, Ye S, Zhao L, Zheng M, Chen Y. Hydrochloride pioglitazone decreases urinary TGF-beta1 excretion in type 2 diabetics. Eur J Clin Invest 2010; 40: 571–574 8 Wang Y, Zhao W, Xue R, Zhou Z, Liu F, Han Y, Ren G, Peng Z, Cen S, Chen H. Oxymatrine inhibits hepatitis B infection with an advantage of overcoming drug-resistance. Antivir Res 2011; 89: 227–231 9 Hong-Li S, Lei L, Lei S, Dan Z, De-Li D, Guo-Fen Q, Yan L, Wen-Feng C, BaoFeng Y. Cardioprotective effects and underlying mechanisms of oxymatrine against ischemic myocardial injuries of rats. Phytother Res 2008; 22: 985–989 10 Gan R, Dong G, Yu J, Wang X, Yang S. Oxymatrine, the main alkaloid component of Sophora roots, protects heart against arrhythmias in rats. Planta Med 2011; 77: 226–230 11 Sugimoto H, Shikata K, Hirata K, Akiyama K, Matsuda M, Kushiro M, Shikata Y, Miyatake N, Miyasaka M, Makino H. Increased expression of intercellular adhesion molecule-1 in diabetic rat glomeruli: glomerular hyperfiltration is a potential mechanism of ICAM‑1 upregulation. Diabetes 1997; 46: 2075–2081 12 Onozato M, Tojo A, Goto A, Fujita T, Wilcox S. Oxidative stress and nitric oxide synthase in rat diabetic nephropathy: effects of ACEI and ARB. Kidney Int 2002; 61: 186–194 13 Navarro JF, Mora-Fernández C. The role of TNF-α in diabetic nephropathy: pathogenic and therapeutic implications. Cytokine Growth Factor Rev 2006; 17: 441–450 14 Qi W, Chen X, Poronnik P, Pollock CA. Transforming growth factor-β/connective tissue growth factor axis in the kidney. Int J Biochem Cell Biol 2008; 40: 9–13 15 Daroux M, Prévost G, Maillard L, Gaxatte C, Agati V, Schmidt A, Boulanger É. Advanced glycation end-products: implications for diabetic and nondiabetic nephropathies. Diabetes Metab J 2010; 36: 1–10 16 Mao Y, Zeng M, Lu L, Wan M, Li C, Chen C, Fu Q, Wang J, She W, Cai X. Capsule oxymatrine in treatment of hepatic fibrosis due to chronic viral hepatitis: a randomized, double blind, placebo-controlled, multicenter clinical study. World J Gastroenterol 2004; 10: 3269–3273 17 Wu D, Wen W, Qi C, Zhao R, Lü J, Zhong C, Chen Y. Ameliorative effect of berberine on renal damage in rats with diabetes induced by high-fat diet and streptozotocin. Phytomedicine 2012; 19: 712–718 18 Thomson S, McLennan S, Kirwan P, Heffernan S, Hennessy A, Yue D, Twigg S. Renal connective tissue growth factor correlates with glomerular basement membrane thickness and prospective albuminuria in a non-human primate model of diabetes: possible predictive marker for incipient diabetic nephropathy. J Diabetes Complications 2008; 22: 284–294
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HE and PAS staining can demonstrate different histopathological alterations of kidneys. PAS staining mainly shows basement membrane thickening and mesangial expansion, while HE staining mainly shows tubular necrosis with vacuolar degeneration of proximal tubules, fatty infiltration, and inflammatory cell infiltrate. We preformed both HE and PAS staining in our study. In brief, the renal samples were fixed in 10 % neutral formalin, dehydrated in gradual ethanol, embedded in paraffin, cut into 4-µm thick sections, and stained with HE and PAS. The sections were imaged using a light microscope, and at least 20 random glomeruli from each section were checked by an investigator blinded to the origin of the sections.
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19 Mensah B, Obineche E, Galadari S, Chandranath E, Shahin A, Ahmed I, Patel S, Adem A. Streptozotocin-induced diabetic nephropathy in rats: the role of inflammatory cytokines. Cytokine 2005; 31: 180–190 20 Hao H, Shao Z, Tang D, Lu Q, Chen X, Yin X, Wu J, Chen H. Preventive effects of rutin on the development of experimental diabetic nephropathy in rats. Life Sci 2012; 91: 959–967 21 Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the care and use of laboratory animals. Washington: The National Academies Press; 2011 22 Wu D, Wen W, Qi C, Zhao R, Lü J, Zhong C, Chen Y. Ameliorative effect of berberine on renal damage in rats with diabetes induced by high-fat diet and streptozotocin. Phytomedicine 2012; 19: 712–718 23 Tahara A, Matsuyama Y, Shibasaki M. Effects of antidiabetic drugs in high-fat diet and streptozotocin-nicotinamide-induced type 2 diabetic mice. Eur J Pharmacol 2011; 655: 108–116
24 Veerapur V, Prabhakar K, Thippeswamy B, Bansal P, Srinivasan K, Unnikrishnan M. Antidiabetic effect of Ficus racemosa Linn. stem bark in high-fat diet and low-dose streptozotocin-induced type 2 diabetic rats: A mechanistic study. Food Chem 2012; 132: 186–193 25 Wang Y, Campbell T, Perry B, Beaurepaire C, Qin L. Hypoglycemic and insulin sensitizing effects of berberine in high-fat diet- and streptozotocin-induced diabetic rats. Metabolism 2011; 60: 298–305 26 Zhang S, Yang J, Li H, Li Y, Liu Y, Zhang D, Zhang F, Zhou W, Chen X. Skimmin, a coumarin, suppresses the streptozotocin-induced diabetic nephropathy in Wistar rats. Eur J Pharmacol 2012; 692: 78–83 27 Honoré S, Cabrera W, Genta S, Sánchez S. Protective effect of yacon leaves decoction against early nephropathy in experimental diabetic rats. Food Chem Toxicol 2012; 50: 1704–1715
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269
Protective Effects of Oxymatrine on Experimental Diabetic Nephropathy
Authors
Changrun Guo 1, Fengyu Han 1, Chunfeng Zhang 1, Wei Xiao 2, Zhonglin Yang 1
Affiliations
1
Key words " oxymatrine l " diabetic nephropathy l " oxidative stress l " TNF‑α l " TGF‑β1 l " Sophora flavescens l " Fabaceae l
received revised accepted
October 27, 2013 January 7, 2014 January 13, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1360369 Published online February 17, 2014 Planta Med 2014; 80: 269–276 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Wei Xiao State Key Laboratory of Natural Medicines China Pharmaceutical University No. 24 Tongjia Lane Nanjing City 210009 P. R. China Phone: + 86 25 83 27 14 26 Fax: + 86 2 58 3 27 14 26 [email protected] Correspondence Zhonglin Yang State Key Laboratory of Natural Medicines China Pharmaceutical University No. 24 Tongjia Lane Nanjing City 210009 P. R. China Phone: + 86 25 83 27 14 26 Fax: + 86 25 83 27 14 26 [email protected]
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, P. R. China Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, P. R. China
Abstract
experimental diabetic nephropathy by multiple mechanisms.
!
Diabetic nephropathy, one of the most common and serious vascular complications of both type 1 and type 2 diabetes mellitus, has become a major contributor of end-stage renal failure. The aims of this study were to investigate the effects and possible underlying action mechanism(s) of oxymatrine on renal damage in diabetic rats. Diabetes was induced in male Sprague-Dawley rats by administering a high-fat diet and an intraperitoneal 30 mg/kg streptozotocin injection. The animals were treated orally with saline, metformin hydrochloride, and oxymatrine at 50, 100, and 150 mg/ kg/day for 11 weeks. At the end of the treatment, renal tissue, blood, and urine samples were collected for histological and biochemical examination. The results revealed that oxymatrine significantly decreased blood glucose, urinary protein and albumin excretion, serum creatinine, and blood urea nitrogen in diabetic rats, and ameliorated diabetes-induced glomerular and tubular pathological changes. Furthermore, oxymatrine significantly prevented oxidative stress and reduced the contents of renal advanced glycation end products, transforming growth factor-β1, connective tissue growth factor, and inflammatory cytokines in diabetic rats. All these results indicate that oxymatrine has protective effects on
Introduction !
Diabetic nephropathy, one of the most common and serious vascular complications of both type 1 and type 2 diabetes mellitus, has become a major contributor to end-stage renal failure [1]. The symptoms of DN include renal hypertrophy, glomerular hyperfiltration, ECM accumulation, and albuminuria. Albuminuria, induced by an increase in the albumin excretion rate, is widely acknowledged as the earliest index of DN and is a major
Abbreviations !
AGEs: BUN: CAT: CCr: CTGF: DC: DN: ECM: GSH‑Px: HE: ICMA-1: IOD: MDA: Met: NC: OMT: PAS: Scr: SOD: STZ: TNF-α: TGF-β1:
advanced glycation end products blood urea nitrogen catalase creatinine clearance ratio connective tissue growth factor diabetic control diabetic nephropathy extracellular matrix glutathione peroxidase hematoxylin-eosin intercellular adhesion molecule-1 integrated optical density malondialdehyde metformin hydrochloride normal control oxymatrine periodic acid–Schiff serum creatinine superoxide dismutase streptozotocin tumor necrosis factor-α transforming growth factor-β1
biochemical feature that aids in the clinical diagnosis of DN [2, 3]. Although the mechanisms of DN are yet to be explained, studies have demonstrated that many factors contribute to the progression of DN, including hyperglycemia, oxidative stress, and inflammatory reaction. Among these factors, hyperglycemia is the key initiating factor in the development of DN, and oxidative stress, as a result of hyperglycemia, is regarded as the major factor contributing to the progression of DN [4, 5]. Previous studies have provided evi-
Guo C et al. Protective Effects of …
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Fig. 1 Structure of OMT.
Fig. 2 Effect of OMT on fasting blood glucose in diabetic rats. Values are presented as mean ± SD for 8 rats in each group. ##P < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group.
dence that reactive oxygen species mediate the activation of TGFβ1, which is widely expressed in all kidney cells. Subsequently, TGF-β1 stimulates the synthesis of matrix components, the accumulation of ECM, and the formation of albuminuria [6]. TGF-β1 also has a bearing on the increase in Scr and BUN in patients with DN [7]. Currently, only a few drugs that are available are effective against DN; therefore, there is a need to search for safe drugs which are " Fig. 1), a major quinolizidine alkaeffective against DN. OMT (l loid in Sophora flavescens (Fabaceae), has been shown to exhibit numerous pharmacological effects, such as anti-inflammatory, antioxidation, neuroprotection, antitumor, and antiarrhythmia in modern pharmacological research [8–10]. In view of the pathogenesis of DN and the pharmacological effects of OMT, we hypothesized that OMT may have a beneficial effect on the progression of DN. This study was carried out to examine the effects of OMT in rats with DN induced by a high-fat diet and a low dose of STZ, and elucidate the potential action mechanism(s).
Results !
Before administering the respective drugs, the fasting blood glucose levels of the five diabetic groups (diabetic control, metformin, and three OMT groups) were almost identical and signifi-
cantly higher than that of the normal control group (p < 0.01). However, after treatment for 11 weeks, the fasting blood glucose levels significantly decreased in the metformin and three OMT " Fig. 2). groups compared to the diabetic control group (l " l Table 1 shows the results of the kidney index, BUN, and Scr in the studied groups. The kidney index of the diabetic control rats was 76% higher than the index of the normal control rats (p < 0.01), while treatment with OMT at doses of 100 mg/kg and 150 mg/kg for 11 weeks significantly reduced the increase of the kidney index. Likewise, the levels of BUN and Scr were significantly increased in the diabetic control group compared to the normal control group (p < 0.01). Treatment with OMT (100 mg/ kg and 150 mg/kg) for 11 weeks produced a significant reduction in BUN and Scr levels. Excretion of urinary protein and albumin are markers of glomer" Table 1, ular dysfunction and tubular impairment. As shown in l the excretion of urinary protein and albumin were markedly elevated in the diabetic control group (p < 0.01). However, treatment with OMT at the doses of 50, 100, and 150 mg/kg for 11 weeks significantly decreased the elevation of urinary protein and albumin excretion (p < 0.01) compared to the diabetic control group. CCr is also an indicator of glomerular dysfunction. In this study, the CCr of the diabetic control rats was significantly decreased compared to the normal control group (p < 0.01), which was significantly inhibited by OMT treatment.
Table 1 Effect of OMT on body weight, kidney weight, kidney index, heart weight, BUN, Scr, CCr, urine protein, and albumin in diabetic rats. Index
NC
DC
Met
OMT-50
OMT-100
OMT-150
Body weight (g) Kidney weight (g) KI (g/kg) Heart weight (g) BUN (mmol/L) Scr (µmol/L) CCr (mL/min/kg) Urine protein (mg/kg/24 h) Urine albumin (mg/kg/24 h)
560.1 ± 48.6 1.36 ± 0.07 2.44 ± 0.27 1.00 ± 0.20 4.74 ± 0.55 104.1 ± 13.9 3.24 ± 0.39 26.4 ± 10.3 0.42 ± 0.17
339.9 ± 54.6## 1.43 ± 0.08 4.31 ± 0.75## 0.65 ± 0.15 5.79 ± 0.64## 146.1 ± 27.9## 2.73 ± 0.22## 212.4 ± 45.1## 4.08 ± 0.76##
386.9 ± 84.7 1.20 ± 0.17** 3.29 ± 1.08* 0.63 ± 0.11 5.14 ± 0.49* 118.9 ± 22.4* 2.81 ± 0.67 138.6 ± 55.1** 3.45 ± 1.47
361.9 ± 63.8 1.32 ± 0.14 3.74 ± 0.69 0.64 ± 0.10 5.21 ± 0.48 116.8 ± 17.2* 2.90 ± 0.47 132.5 ± 50.8** 2.43 ± 0.88**
390.7 ± 53.8 1.35 ± 0.07 3.53 ± 0.47 0.61 ± 0.14 5.13 ± 0.29* 114.5 ± 13.2* 3.24 ± 0.30** 125.8 ± 36.8** 2.15 ± 1.47**
419.6 ± 62.9* 1.38 ± 0.11 3.32 ± 0.43* 0.66 ± 0.11 5.04 ± 0.41* 110.8 ± 18.5** 3.32 ± 0.44** 116.0 ± 47.6** 1.96 ± 1.10**
Values are presented as mean ± SD for 8 rats in each group. ##P < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group
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Fig. 4 Representative micrographs of kidney tissue stained with PAS (arrows show the thickened basement membrane). A NC group, B DC group, C Met group, D OMT-50 group, E OMT-100 group, and F OMT-150 group. (Color figure available online only.)
The sections from normal control rats showed a normal histology of the kidney. The glomerulus, glomerular capsules, and tubules " Figs. 3 A and 4 A). Representawere very clear and prominent (l tive glomerular and tubular changes were observed in the kid" Figs. 3 B and 4 B). Histopathoneys of the diabetic control rats (l logical examination of the kidneys from the diabetic control rats showed signs of tubular necrosis with vacuolar degeneration of the proximal tubules and a thickened basement membrane, and the glomerulus exhibited segmental sclerosis with basement membrane thickening and mesangial expansion. Treatment with OMT for 11 weeks significantly inhibited these changes in the " Figs. 3 and 4). kidneys (l Type IV collagen is a major component of basement membranes and is expressed in both the glomerular and tubular basement membranes. To investigate the effect of OMT on the expression of basement membrane components, type IV collagen was determined by an immunohistochemical technique. Immunohistochemical staining of type IV collagen showed that there was a significant increase in the expression of type IV collagen in the renal glomerulus and tubules of the diabetic control rats compared to the normal control rats, which was significantly inhibited by " Fig. 5). OMT (l In diabetic patients with nephropathy, the level of ICAM-1 was " Fig. 6, ICAM-1 positive staining in elevated [11]. As shown in l the diabetic rats was more prominent than that in the normal
control rats. The OMT treatment significantly reduced expression of ICAM-1 in the kidneys of diabetic rats. Oxidative stress is a key pathogenic factor in the development of " Table 2 shows the effects of OMT on SOD, GSH‑Px, CAT DN [12]. l activities, and MDA content in the kidneys. The activities of renal SOD, GSH‑Px, and CAT were markedly decreased, whereas renal MDA content in the diabetic control group was markedly increased compared to that in the normal control group (p < 0.01). Treatment with OMT for 11 weeks effectively elevated the activities of SOD, GSH‑Px, and CAT, and inhibited MDA content. These results indicate that treatment with OMT could significantly inhibit renal oxidative stress in diabetic rats. TNF-α plays an important role in the development of DN and is a therapeutic target for its treatment [13]. In this study, we investigated the effect of OMT on renal TNF-α in diabetic rats. Significantly elevated levels of renal TNF-α were found in the diabetic control rats; however, treatment with OMT for 11 weeks dosedependently reduced the content of TNF-α in the kidneys of the " Fig. 7). diabetic rats (l The TGF-β1/CTGF axis plays a critical role in progressive kidney " Table 3, when compared to the nordisease [14]. As shown in l mal control group, the levels of renal TGF-β1 and CTGF were markedly increased in the diabetic control group (p < 0.01). The increase in renal TGF-β1 and CTGF levels was effectively relieved by the administration of OMT.
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Fig. 3 Representative micrographs of kidney tissue stained with HE (arrows show vacuolar degeneration of the proximal tubules). A NC group, B DC group, C Met group, D OMT-50 group, E OMT100 group, and F OMT-150 group. (Color figure available online only.)
Original Papers
Fig. 5 Effect of OMT on renal collagen IV in diabetic rats. The brown area is the positive expression area, enlarged 400× under a light microscope. A NC group, B DC group, C Met group, D OMT50 group, E OMT-100 group, F OMT-150 group, and G IOD of collagen IV. Values are presented as mean ± SD for 8 rats in each group. ##P < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group. (Color figure available online only.)
Fig. 6 Effect of OMT on renal ICAM-1 in diabetic rats. The brown area is the positive expression area, enlarged 400× under a light microscope. A NC group, B DC group, C Met group, D OMT-50 group, E OMT-100 group, F OMT-150 group, and G IOD of ICAM-1. Values are presented as mean ± SD for 8 rats in each group. ##P < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group. (Color figure available online only.)
Table 2 Effect of OMT on renal SOD, CAT, GSH‑Px, and MDA levels in diabetic rats. Index
NC
DC
Met
OMT-50
OMT-100
OMT-150
SOD (U/mg prot) CAT (U/mg prot) GSH‑Px (U/µg prot) MDA (nmol/mg prot)
206.6 ± 28.2 40.34 ± 7.10 1.85 ± 0.47 2.09 ± 0.27
147.4 ± 27.3## 31.28 ± 6.25# 1.33 ± 0.14# 3.18 ± 0.31##
142.1 ± 52.7 37.49 ± 9.43 1.52 ± 0.43 2.83 ± 0.53
183.2 ± 24.3 39.48 ± 5.48 1.75 ± 0.23 2.66 ± 0.50
186.9 ± 23.8* 39.90 ± 6.32* 1.85 ± 0.23* 2.42 ± 0.61*
187.2 ± 23.3* 40.88 ± 3.67* 1.86 ± 0.39* 2.40 ± 0.71*
Values are presented as mean ± SD for 8 rats in each group. #P < 0.05, ##p < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group
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Table 3 Effect of OMT on renal TGF-β1 and CTGF levels in diabetic rats. Groups
TGF-β1 (ng/mg prot)
CTGF (pg/mg prot)
NC DC Met OMT-50 OMT-100 OMT-150
3.05 ± 0.53 5.75 ± 0.48## 5.12 ± 0.47 4.86 ± 0.89* 4.66 ± 0.41** 4.55 ± 0.63**
84.8 ± 11.4 111.3 ± 14.5## 96.5 ± 14.4 108.8 ± 19.7 99.5 ± 8.9* 94.9 ± 6.0*
Values are presented as mean ± SD for 8 rats in each group. ##P < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group
AGEs are considered a marker of diabetic complications, includ" Fig. 8 shows the effect of OMT ing diabetes nephropathy [15]. l on renal content of AGEs in diabetic rats. The content of the renal AGEs in the diabetic control group was markedly increased compared to the normal control group (p < 0.01). However, OMT treatment markedly decreased the contents of the renal AGEs in the diabetic rats.
Discussion !
Although OMT is a toxic alkaloid, it has been used in humans as a safe oral drug in the treatment of hepatic fibrosis [16]. In our study, the rats did not show any signs of toxicity after the treatment period. Treatment with OMT for 11 weeks did not change " Table 1). the kidney weight, or the heart weight of diabetic rats (l Typical features of diabetic renal injuries include renal hypertrophy, glomerular hyperfiltration, albuminuria, basement membrane thickening, ECM accumulation, and tubulointerstitial fibrosis. In our study, the diabetic rats demonstrated these features, while OMT treatment provided renoprotection with suppression of albuminuria and alleviated glomerular and tubular injuries. A high blood glucose level is still considered to be the inducer of diabetic complications, including nephropathy. Historically, various hypotheses have been proposed to suggest potential mechanisms by which hyperglycemia causes diabetic complications. The four main hypotheses are increased polyol pathway flux, increased intracellular formation of AGEs, increased hexosamine pathway flux, and excessive activation of protein kinase C iso-
Fig. 8 Effect of OMT on the levels of the renal AGEs in diabetic rats. Values are presented as mean ± SD for 8 rats in each group. ##P < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group.
forms. Overproduction of superoxide by the mitochondrial electron transport chain is considered to be the common element linking these four mechanisms. Studies have conclusively shown that oxidative stress is a key pathogenic factor in the development of DN [12]. In the present study, treatment with OMT inhibited the oxidative stress state in DN rats (as evidenced by decreased renal MDA contents), and improved antioxidant defenses (as evidenced by increased activities of renal SOD, GSH‑Px, and CAT). These results indicate that OMT imparts renoprotection against oxidative stress in DN. AGEs are also one of the key pathogenic factors responsible for diabetes nephropathy, which is indicated by the fact that the AGEs inhibitor aminoguanidine ameliorates diabetes-induced renal damage [5, 17]. The formation of AGEs can interfere with matrix-matrix interactions and matrix-cell interactions [15]. It is well known that hyperglycemia-induced oxidative stress facilitates the AGEs formation; on the other hand, the interactions between AGEs and receptors for AGEs induce the activation of oxidative stress [5]. This mutual interaction leads to ECM accumulation and mesangial cell hypertrophy. In our study, treatment with OMT reduced the formation of AGEs, and this could be beneficial for the prevention of DN. As a typical feature of DN, ECM accumulation can lead to glomerular fibrosis and loss of renal function. TGF-β1, both a fibrogenic and an inflammatory cytokine, is stimulated by high glucose, AGEs, and reactive oxygen species in diabetes. Several studies have demonstrated that TGF-β1 plays an important role in the accumulation of ECM and renal fibrosis [7]. For example, excessive deposition of type IV collagen caused by TGF-β1 is one of the reasons for diabetic glomerulopathy. In our experiments, treatment with OMT could inhibit the accumulation of TGF-β1, type IV collagen, and ECM, and ameliorate mesangial expansion. CTGF, a member of the CCN family, is increasingly being implicated in structural and functional changes in DN. Both in diabetic animal models and in human diabetic renal tissue, the levels of CTGF are elevated, which is related to hyperglycemia, AGEs formation, and hemodynamic change [14]. Several studies have identified the role of CTGF in fibroblast proliferation, migration, adhesion, and ECM formation [18]. As a crucial downstream mediator for TGF-β1 stimulation, CTGF not only mediates the induction of ECM protein expression, but also inhibits matrix degradation. Therefore, TGF-β1 and CTGF form a major axis in renal fibrosis [14]. In our study, the expression of CTGF in the diabetic control group was markedly increased, while for treatment with
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Fig. 7 Effect of OMT on renal TNF-α levels in diabetic rats. Values are presented as mean ± SD for 8 rats in each group. ##P < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. diabetic control group.
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OMT, the increase of CTGF was ameliorated significantly. These results suggest that OMT may ameliorate renal fibrosis by mediating the TGF-β1/CTGF axis. Experimental and clinical studies demonstrate that inflammatory cytokines, such as TNF-α and ICAM-1, play a prominent role in the development and progression of DN [19]. In addition to infiltrating macrophages, TNF-α is synthesized by endothelial, mesangial, glomerular, and tubular epithelial cells, with the stimulation of AGEs and high glucose levels. Overexpression of TNF-α can result in hemodynamic alterations, damage to the glomerular permeability barrier, and apoptosis of renal cells [13]. ICAM-1 also promotes the recruitment of monocytes and macrophages in diabetic kidneys. These monocytes and macrophages synthesize a variety of growth factors, including TGF-β1, which contributes to the glomerular damage in diabetes [11]. Our study indicates that treatment with OMT significantly inhibited the expression of TNF-α and ICAM-1 in diabetic renal tissue, and thus improved the structural and functional abnormalities of the diabetic kidney. The interaction among hyperglycemia, oxidative stress, AGEs, TGF-β1, CTGF, and inflammatory cytokines has become a focus area in the research of DN [20]. Under hyperglycemic conditions, oxidative stress and AGEs mutually interact leading to upregulation, which can activate the TGF-β1/CTGF axis and lead to overexpression of inflammatory cytokines, subsequently inducing changes in renal structure and function [13, 18]. Our study demonstrated that OMT attenuated the formation of reactive oxygen species and AGEs, and reduced TGF-β1, CTGF, and inflammatory cytokine expression in diabetic kidneys. Therefore, by way of these mechanisms, OMT imparts renoprotection.
Materials and Methods
Pre-experiment To select the better doses of OMT, we performed a pre-experiment. In the pre-experiment, the rats were injected with STZ (65 mg/kg, dissolved in 0.01 M sodium citrate buffer, pH 4.4) intraperitoneally. After 72 hours, fasting blood glucose was measured. The rats with a fasting blood glucose level > 11.1 mmol/L were considered diabetic and selected for further pharmacological studies. According to the instructions for taking oxymatrine tablets (a drug already on the market), the adult dose of OMT is 600 or 900 mg/day. The ratʼs dose of OMT, calculated by body surface area, was 66 or 99 mg/kg/day. Therefore, we chose 66 mg/kg/ day ÷ 1.5 ≈ 50 mg/kg/day, 66 mg/kg/day × 1.5 ≈ 100 mg/kg/day, and 99 mg/kg/day × 1.5 ≈ 150 mg/kg/day as the low, middle, and high dose, respectively. Treatment with OMT at the three doses for 10 weeks significantly decreased blood glucose and the excretion of urinary albumin. Therefore, we chose the three doses at 50, 100, and 150 mg/kg/day for further studies.
Induction of experimental diabetes After one week of adaptive feeding, the animals were randomly divided into two groups: a control group and an experimental group. The control group rats were given a regular diet, while the experimental group rats were fed a high-fat diet (consisting of 70 % standard laboratory food, 15 % carbohydrate, 10 % lard, and 5 % yolk powder) [22]. After 4 weeks of high-fat diet feeding, the experimental group rats were injected with STZ (30 mg/kg, dissolved in 0.01 M sodium citrate buffer, pH 4.4) intraperitoneally, while the control group rats were injected with the vehicle citrate buffer [23]. Fasting blood glucose was measured one week after injection. The rats with a fasting blood glucose level > 11.1 mmol/L were considered diabetic and selected for further pharmacological studies. The animals were fed their respective diets until the end of the study [24, 25].
!
Chemicals and reagents
Experimental design and treatment protocol
OMT (98.2 % purity) was supplied by Qingze Pharmaceutical, Inc. Met (99.1% purity), the positive control in these experiments, was obtained from Jingfeng Pharmaceutical, Inc. OMT and Met were dissolved in normal saline (0.9% NaCl saline solution) for the animal experiment. STZ was purchased from Sigma-Aldrich, Inc. ELISA kits for TNF-α and TGF-β1 were obtained from Abcam, Inc., while those for the AGEs, urinary albumin, and CTGF were obtained from Biocompare, Inc. Kits for blood glucose, SOD, MDA, GSH‑Px, CAT, creatinine, BUN, and urine protein were purchased from Nanjing Jiancheng Institute. The rabbit anti-rat ICAM-1 monoclonal antibody and the rabbit anti-rat type IV collagen monoclonal antibody were obtained from Santa Cruz Biotechnology, Inc.
The normal and diabetic rats were randomly divided into six groups of eight rats each: NC group, normal rats treated with saline in a matched volume; DC group, diabetic rats treated with saline in a matched volume; Met group, diabetic rats administered Met 200 mg/kg/day; OMT-50 group, diabetic rats administered OMT 50 mg/kg/day; OMT-100 group, diabetic rats administered OMT 100 mg/kg/day; and OMT-150 group, diabetic rats administered OMT 150 mg/kg/day. All drugs were administered orally via an orogastric cannula, continuously for 11 weeks. At the end of the treatment, urine samples were collected from the rats housed in individual metabolic cages after 24 h for analysis of urine volume and other biochemical parameters. The rats were fasted for 12 h and then were anesthetized to collect their blood. Post-fasting blood was collected via the abdominal aorta for blood biochemical parameters analysis, and then the kidneys were removed. After being weighed, the right kidneys were collected for histology examination and immunohistochemistry. The renal cortex of the left kidneys was separated and stored at − 70 °C for biochemical estimations.
Animals Adult male Sprague-Dawley rats (weight 180–220 g), with animal quality certificate number [(Su)SCKX2008–0010], were purchased from Nantong University laboratory animal center and maintained at a constant temperature of 23 ± 2 °C on a 12-h light/dark cycle with free access to normal laboratory food and water. All procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals [21] and were approved by the ethics committee of the China Pharmaceutical University (CEAE-093, Jan 10, 2013).
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Analysis of blood and urine samples The blood samples were centrifuged at 4000 g for 10 min to separate the serum. The blood glucose, Scr, and BUN levels were determined using commercially available kits according to the manufacturerʼs instructions. Urine samples were centrifuged at 1600 g for 5 min to separate the supernatant. The urinary albumin concentration was determined using an ELISA kit. Urinary
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protein and creatinine concentrations were determined using commercially available kits. CCr was calculated usuing the following formula: CCr = urinary creatinine (mg/mL) × urine volume (mL/kg)/Scr (mg/mL)/1440 (min) [26, 27].
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Acknowledgements !
This work was supported by a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. The assistance of the staff is gratefully acknowledged.
Histological examination
Immunohistochemical analysis of ICAM-1 and type IV collagen Immunostaining was performed on 4-µm renal sections after deparaffinization and rehydration. The sections were microwaved for antigen retrieval in citrate buffer (pH 6.0) for 10 min and then were treated with 3% H2O2 buffer for 10 min. After that, the sections were thoroughly washed in tap water and blocked with 10 % (w/v) normal goat serum for 1 h. After blocking, the sections were incubated with rabbit monoclonal ICMA-1 and type IV collagen antibody at 4 °C overnight, followed by goat anti-rabbit IgG horseradish peroxidase for 30 min at 37 °C. To visualize ICMA-1 and type IV collagen, the sections were stained with chromogen diaminobenzidine for 10 min and counterstained with hematoxylin. At least 20 random fields of each section were examined under a light microscope and analyzed using Image-Pro Plus 6.0 software to calculate the value of the positive IOD.
Analysis of renal oxidative stress The renal cortex was sliced into pieces and homogenized with appropriate Tris–HCl (5 mmol/L containing 2 mmol/L EDTA, pH 7.4). Homogenates were centrifuged at 4000 g for 10 min at 4 °C. The supernatants were separated and used immediately for the assays for SOD, CAT, GSH‑Px, and MDA. They were determined using commercially available kits according to the manufacturerʼs instructions and were normalized as U/mg protein or nmol/mg protein. The protein content in the homogenate was measured by the Bradford method.
Analysis of cytokine levels and AGEs in kidney tissues The levels of TNF-α, TGF-β1, CTGF, and AGEs in renal homogenates (the same samples were used for renal oxidative stress analysis) were measured using commercial ELISA kits according to the manufacturerʼs protocols. The levels of these cytokines and AGEs in renal tissues were normalized to the protein content.
Statistical analysis Data are expressed as mean ± SD for eight animals in each group. Statistically significant differences between the two groups were determined by one-way analysis of variance, followed by a post hoc test (Dunnettʼs test). In all cases, probability values of p < 0.05 were considered statistically significant.
Conflict of Interest !
All authors have no conflicts of interest.
References 1 Lopes A. End-stage renal disease due to diabetes in racial/ethnic minorities and disadvantaged populations. Ethn Dis 2009; 19: S1–S47 2 Leese G, Savage M, Chattington P, Vora J. The diabetic patient with hypertension. Postgrad Med J 1996; 72: 263–268 3 Mishra A, Bhatti R, Singh A, Ishar M. Ameliorative effect of the cinnamon oil from Cinnamomum zeylanicum upon early stage diabetic nephropathy. Planta Med 2010; 76: 412–417 4 UKPDS. United Kingdom Prospective Diabetic Study. Lancet 1998; 352: 837–853 5 Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001; 414: 813–820 6 Tahara A, Tsukada J, Tomura Y, Yatsu T, Shibasaki M. Vasopressin increases type IV collagen production through the induction of transforming growth factor-beta secretion in rat mesangial cells. Pharmacol Res 2008; 57: 142–150 7 Hu Y, Ye S, Zhao L, Zheng M, Chen Y. Hydrochloride pioglitazone decreases urinary TGF-beta1 excretion in type 2 diabetics. Eur J Clin Invest 2010; 40: 571–574 8 Wang Y, Zhao W, Xue R, Zhou Z, Liu F, Han Y, Ren G, Peng Z, Cen S, Chen H. Oxymatrine inhibits hepatitis B infection with an advantage of overcoming drug-resistance. Antivir Res 2011; 89: 227–231 9 Hong-Li S, Lei L, Lei S, Dan Z, De-Li D, Guo-Fen Q, Yan L, Wen-Feng C, BaoFeng Y. Cardioprotective effects and underlying mechanisms of oxymatrine against ischemic myocardial injuries of rats. Phytother Res 2008; 22: 985–989 10 Gan R, Dong G, Yu J, Wang X, Yang S. Oxymatrine, the main alkaloid component of Sophora roots, protects heart against arrhythmias in rats. Planta Med 2011; 77: 226–230 11 Sugimoto H, Shikata K, Hirata K, Akiyama K, Matsuda M, Kushiro M, Shikata Y, Miyatake N, Miyasaka M, Makino H. Increased expression of intercellular adhesion molecule-1 in diabetic rat glomeruli: glomerular hyperfiltration is a potential mechanism of ICAM‑1 upregulation. Diabetes 1997; 46: 2075–2081 12 Onozato M, Tojo A, Goto A, Fujita T, Wilcox S. Oxidative stress and nitric oxide synthase in rat diabetic nephropathy: effects of ACEI and ARB. Kidney Int 2002; 61: 186–194 13 Navarro JF, Mora-Fernández C. The role of TNF-α in diabetic nephropathy: pathogenic and therapeutic implications. Cytokine Growth Factor Rev 2006; 17: 441–450 14 Qi W, Chen X, Poronnik P, Pollock CA. Transforming growth factor-β/connective tissue growth factor axis in the kidney. Int J Biochem Cell Biol 2008; 40: 9–13 15 Daroux M, Prévost G, Maillard L, Gaxatte C, Agati V, Schmidt A, Boulanger É. Advanced glycation end-products: implications for diabetic and nondiabetic nephropathies. Diabetes Metab J 2010; 36: 1–10 16 Mao Y, Zeng M, Lu L, Wan M, Li C, Chen C, Fu Q, Wang J, She W, Cai X. Capsule oxymatrine in treatment of hepatic fibrosis due to chronic viral hepatitis: a randomized, double blind, placebo-controlled, multicenter clinical study. World J Gastroenterol 2004; 10: 3269–3273 17 Wu D, Wen W, Qi C, Zhao R, Lü J, Zhong C, Chen Y. Ameliorative effect of berberine on renal damage in rats with diabetes induced by high-fat diet and streptozotocin. Phytomedicine 2012; 19: 712–718 18 Thomson S, McLennan S, Kirwan P, Heffernan S, Hennessy A, Yue D, Twigg S. Renal connective tissue growth factor correlates with glomerular basement membrane thickness and prospective albuminuria in a non-human primate model of diabetes: possible predictive marker for incipient diabetic nephropathy. J Diabetes Complications 2008; 22: 284–294
Guo C et al. Protective Effects of …
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HE and PAS staining can demonstrate different histopathological alterations of kidneys. PAS staining mainly shows basement membrane thickening and mesangial expansion, while HE staining mainly shows tubular necrosis with vacuolar degeneration of proximal tubules, fatty infiltration, and inflammatory cell infiltrate. We preformed both HE and PAS staining in our study. In brief, the renal samples were fixed in 10 % neutral formalin, dehydrated in gradual ethanol, embedded in paraffin, cut into 4-µm thick sections, and stained with HE and PAS. The sections were imaged using a light microscope, and at least 20 random glomeruli from each section were checked by an investigator blinded to the origin of the sections.
Original Papers
19 Mensah B, Obineche E, Galadari S, Chandranath E, Shahin A, Ahmed I, Patel S, Adem A. Streptozotocin-induced diabetic nephropathy in rats: the role of inflammatory cytokines. Cytokine 2005; 31: 180–190 20 Hao H, Shao Z, Tang D, Lu Q, Chen X, Yin X, Wu J, Chen H. Preventive effects of rutin on the development of experimental diabetic nephropathy in rats. Life Sci 2012; 91: 959–967 21 Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the care and use of laboratory animals. Washington: The National Academies Press; 2011 22 Wu D, Wen W, Qi C, Zhao R, Lü J, Zhong C, Chen Y. Ameliorative effect of berberine on renal damage in rats with diabetes induced by high-fat diet and streptozotocin. Phytomedicine 2012; 19: 712–718 23 Tahara A, Matsuyama Y, Shibasaki M. Effects of antidiabetic drugs in high-fat diet and streptozotocin-nicotinamide-induced type 2 diabetic mice. Eur J Pharmacol 2011; 655: 108–116
24 Veerapur V, Prabhakar K, Thippeswamy B, Bansal P, Srinivasan K, Unnikrishnan M. Antidiabetic effect of Ficus racemosa Linn. stem bark in high-fat diet and low-dose streptozotocin-induced type 2 diabetic rats: A mechanistic study. Food Chem 2012; 132: 186–193 25 Wang Y, Campbell T, Perry B, Beaurepaire C, Qin L. Hypoglycemic and insulin sensitizing effects of berberine in high-fat diet- and streptozotocin-induced diabetic rats. Metabolism 2011; 60: 298–305 26 Zhang S, Yang J, Li H, Li Y, Liu Y, Zhang D, Zhang F, Zhou W, Chen X. Skimmin, a coumarin, suppresses the streptozotocin-induced diabetic nephropathy in Wistar rats. Eur J Pharmacol 2012; 692: 78–83 27 Honoré S, Cabrera W, Genta S, Sánchez S. Protective effect of yacon leaves decoction against early nephropathy in experimental diabetic rats. Food Chem Toxicol 2012; 50: 1704–1715
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