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ARTICLE Sequoyitol ameliorates diabetic nephropathy in diabetic rats induced with a high-fat diet and a low dose of streptozotocin Xian-Wei Li, Yan Liu, Wei Hao, and Jie-Ren Yang
Abstract: Sequoyitol decreases blood glucose, improves glucose intolerance, and enhances insulin signaling in ob/ob mice. The aim of this study was to investigate the effects of sequoyitol on diabetic nephropathy in rats with type 2 diabetes mellitus and the mechanism of action. Diabetic rats, induced with a high-fat diet and a low dose of streptozotocin, and were administered sequoyitol (12.5, 25.0, and 50.0 mg·(kg body mass)−1·d−1) for 6 weeks. The levels of fasting blood glucose (FBG), serum insulin, blood urea nitrogen (BUN), and serum creatinine (SCr) were measured. The expression levels of p22phox, p47phox, NF-B, and TGF-1 were measured using immunohistochemisty, real-time PCR, and (or) Western blot. The total antioxidative capacity (T-AOC), as well as the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) were also determined. The results showed that sequoyitol significantly decreased FBG, BUN, and SCr levels, and increased the insulin levels in diabetic rats. The level of T-AOC was significantly increased, while ROS and MDA levels and the expression of p22phox, p47phox, NF-B, and TGF-1 were decreased with sequoyitol treatment both in vivo and in vitro. These results suggested that sequoyitol ameliorates the progression of diabetic nephropathy in rats, as induced by a high-fat diet and a low dose of streptozotocin, through its glucose-lowering effects, antioxidant activity, and regulation of TGF-1 expression. Key words: sequoyitol, diabetic nephropathy, oxidative stress, TGF-1. Résumé : Le séquoyitol peut diminuer le glucose sanguin, améliorer l'intolérance au glucose et accroitre la signalisation de l'insuline chez les souris ob/ob. Le but de la présente étude était d'examiner les effets du séquoyitol dans la néphropathie diabétique, dans le diabète de type 2 chez le rat, ainsi que son mécanisme d'action. Des rats rendus diabétiques par une diète riche en gras et de faibles doses de streptozotocine ont reçu du séquoyitol (12,5, 25,0 et 50,0 mg·(kg de masse corporelle)−1·jour−1) pendant 6 semaines. Le glucose sanguin a` jeun, l'insuline sérique, l'azote uréique sanguine et la créatinine sérique ont été mesurés. L'expression de p22phox, p47phox, NF-B et TGF-1 a été mesurée par immunohistochimie, PCR en temps réel ou buvardage Western. La capacité anti-oxydante totale, le malondialdéhyde et les niveaux d'espèces réactives d'oxygène ont aussi été déterminés. Les résultats ont montré que le séquoyitol diminuait significativement le glucose sanguin a` jeun, l'azote uréique sanguine et la créatinine sérique, et augmentait le niveau d'insuline chez les rats diabétiques. La capacité anti-oxydante totale était significativement accrue, alors que les niveaux d'espèces réactives d'oxygène et de malondialdéhyde, de même que l'expression de p22phox, p47phox, NF-B et TGF-1 étaient diminués en fonction du traitement au séquoyitol in vivo ou in vitro. Ces résultats suggèrent que le séquoyitol ralentit la progression de la néphropathie diabétique induite par une diète riche en gras et de faibles doses de streptozotocine chez le rat, par son effet sur l'abaissement du glucose, son activité anti-oxydante et la régulation de l'expression de TGF-1. [Traduit par la Rédaction] Mots-clés : séquoyitol, néphropathie diabétique, stress oxydant, TGF-1.
Introduction Diabetic nephropathy (DN) is one of the most important complications for diabetics and is characterized histologically by excessive deposition of extracellular matrix (ECM) in the kidney, leading to glomerular mesangial expansion and tubulointerstitial fibrosis (Sun et al. 2013). Numerous experimental and clinical studies have shown that oxidative stress plays an important role in the development and progression of vascular complications and diabetic nephropathy (Forbes et al. 2008). It has been reported that treatment with acarbose helps to prevent the increase in oxidative stress and vascular dysfunction induced by hyperglycemia (Rösen and Osmers 2006). In addition, pathological changes in glomeruli and an increase in urinary albumin excretion in diabetic rats were ameliorated by dietary antioxidant supplementation with vitamin E or lipoic acid, supporting the role of oxida-
tive stress in the pathogenesis of diabetic nephropathy (Craven et al. 1997; Melhem et al. 2001). The degree of oxidative stress is determined by the balance between the production of reactive oxygen species (ROS) and the antioxidant defence system (Droge 2002). In experimental and human diabetes, the generation of ROS is known to be increased via multiple pathways, including glucose auto-oxidation, increased mitochondrial superoxide production, PKC-dependent activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, formation of advanced glycation end-products (AGEs), and stimulation of cellular ROS production by extracellular AGEs through their receptors (Brownlee 2001; Forbes et al. 2008). Among these, the upregulation of the subunits of NADPH oxidase, such as p47phox and p22phox, mainly contribute to the increase in glomerular superoxide production in experimentally diabetic rats (Satoh et al. 2005). In a rodent model of diabetes (streptozotocin in-
Received 17 August 2013. Accepted 18 March 2014. X.-W. Li,* Y. Liu,* W. Hao, and J.-R. Yang. Department of Pharmacology, Third-Grade Pharmacology Laboratory of State Administration of Traditional Chinese Medicine, Wannan Medical College, Wen-Chang West Road 22#, Wuhu 241002, China. Corresponding author: Jie-Ren Yang (e-mail: [email protected]). *These authors contributed equally to this work. Can. J. Physiol. Pharmacol. 92: 405–417 (2014) dx.doi.org/10.1139/cjpp-2013-0307
Published at www.nrcresearchpress.com/cjpp on 28 March 2014.
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by EAST CAROLINA UNIVERSITY on 04/20/15 For personal use only.
406
duced), the renal expression of p22phox and p47phox was increased, and this was associated with ROS-induced renal damage (Lee et al. 2010; Feng et al. 2013). There is a growing body of evidence indicating that herbal constituents hold a great promise for the treatment of type-2 diabetes, and the extracts from numerous plants have been reported to reduce blood glucose (Chen et al. 2008; Huyen et al. 2010). Sequoyitol is a natural compound present in many plants (e.g., Amentotaxus yunnanensis, Aristolochia arcuata, and Crossostephium chinensis), as a novel small molecule (molecular mass: 194) (Li et al. 2003; Yang et al. 2008). Both oral and subcutaneous administrations of sequoyitol ameliorate hyperglycemia and glucose intolerance in ob/ob mice with insulin resistance (Shen et al. 2012). Additionally, sequoyitol directly improves insulin sensitivity in cultured hepatocytes, adipocytes, and -cells, and protects -cells against oxidative injury (Sivakumar et al. 2010; Shen et al. 2012). In a previous study (Chen et al. 2013), we found that sequoyitol acts as an antioxidant protectant against hepatic injury through inhibiting the expression of p22phox and p47phox. These results suggest that sequoyitol has a therapeutic potential for the treatment of diabetic complications. However, whether sequoyitol attenuates diabetic kidney disease is still unclear. This study was undertaken to investigate whether sequoyitol reduces oxidative stress and ameliorates the development of nephropathy in experimentally diabetic rat model.
Materials and methods Reagents and materials Sequoyitol (MB1218, >98.0% purity) was provided by the Dalian Meilun Biotech Company (Dalian, China). Streptozotocin (STZ) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma (St. Louis, Missouri, USA). 2=7=-Dichlorofluorescein diacetate (DCFH-DA), and the kits for measuring total antioxidant capacity (T-AOC) and malondialdehyde (MDA) were purchased from the Beyotime Institute of Biotechnology (Haimen, China). Masson's trichrome stain kit was purchased from Nanjing KeyGEN Biotech (Nanjing, China). The kits for measuring blood urea nitrogen (BUN) and serum creatinine (SCr) were supplied by the Siemens Healthcare Diagnostics Company (Shanghai, China). The primers were purchased from the Shanghai Sangon Biological Engineering Company (Shanghai, China). The PrimeScript reverse transcription reagent kit and SYBR Premix Ex Taq were obtained from TaKaRa Biotechnology Company (Dalian, China). TGF-1 antibody, -actin, and the collagen IV antibody were purchased from Abcam (Hong Kong, China), and p22phox, p47phox, and nuclear factor B (NF-B) were obtained from Santa Cruz (California, USA). Animals Male Sprague–Dawley (SD) rats (aged 6–8 weeks, weighing 180– 220 g) were obtained from the Nanjing Qinglongshan Experimental Animal Company (certificate No: SCXK (jun) 2007-012; Nanjing, China). All experiments were conducted in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the experimental protocol was approved by the Medicine Animal Welfare Committee of Wannan Medical College. Experimental animals, grouping, and treatments The diabetic rat model was developed using a high-fat diet plus multiple low doses of streptozotocin, as described previously (Zhang et al. 2008). The high-fat diet consisted of 22% fat, 58% carbohydrate, and 20% protein with a total calorific value of 44.3 kJ·kg−1 (Nanjing Qinglongshan Experimental Animal Company, Nanjing, China). The control rats were given regular chow consisting of 5% fat, 53% carbohydrate, and 23% protein with a total calorific value of 25 kJ·kg−1. Following 4 weeks of dietary intervention, the diabetic group was injected intraperitoneally (i.p.) with low doses of strep-
Can. J. Physiol. Pharmacol. Vol. 92, 2014
tozotocin (30 mg·(kg body mass)−1, dissolved in 0.1 mol·L−1 sodium citrate buffer, pH 4.4; Sigma). One week later, blood samples for fasting blood glucose measurements (using the glucose oxidase peroxidase method) were collected by cutting the tail. Rats with a fasting blood glucose of
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by EAST CAROLINA UNIVERSITY on 04/20/15 For personal use only.
ARTICLE Sequoyitol ameliorates diabetic nephropathy in diabetic rats induced with a high-fat diet and a low dose of streptozotocin Xian-Wei Li, Yan Liu, Wei Hao, and Jie-Ren Yang
Abstract: Sequoyitol decreases blood glucose, improves glucose intolerance, and enhances insulin signaling in ob/ob mice. The aim of this study was to investigate the effects of sequoyitol on diabetic nephropathy in rats with type 2 diabetes mellitus and the mechanism of action. Diabetic rats, induced with a high-fat diet and a low dose of streptozotocin, and were administered sequoyitol (12.5, 25.0, and 50.0 mg·(kg body mass)−1·d−1) for 6 weeks. The levels of fasting blood glucose (FBG), serum insulin, blood urea nitrogen (BUN), and serum creatinine (SCr) were measured. The expression levels of p22phox, p47phox, NF-B, and TGF-1 were measured using immunohistochemisty, real-time PCR, and (or) Western blot. The total antioxidative capacity (T-AOC), as well as the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) were also determined. The results showed that sequoyitol significantly decreased FBG, BUN, and SCr levels, and increased the insulin levels in diabetic rats. The level of T-AOC was significantly increased, while ROS and MDA levels and the expression of p22phox, p47phox, NF-B, and TGF-1 were decreased with sequoyitol treatment both in vivo and in vitro. These results suggested that sequoyitol ameliorates the progression of diabetic nephropathy in rats, as induced by a high-fat diet and a low dose of streptozotocin, through its glucose-lowering effects, antioxidant activity, and regulation of TGF-1 expression. Key words: sequoyitol, diabetic nephropathy, oxidative stress, TGF-1. Résumé : Le séquoyitol peut diminuer le glucose sanguin, améliorer l'intolérance au glucose et accroitre la signalisation de l'insuline chez les souris ob/ob. Le but de la présente étude était d'examiner les effets du séquoyitol dans la néphropathie diabétique, dans le diabète de type 2 chez le rat, ainsi que son mécanisme d'action. Des rats rendus diabétiques par une diète riche en gras et de faibles doses de streptozotocine ont reçu du séquoyitol (12,5, 25,0 et 50,0 mg·(kg de masse corporelle)−1·jour−1) pendant 6 semaines. Le glucose sanguin a` jeun, l'insuline sérique, l'azote uréique sanguine et la créatinine sérique ont été mesurés. L'expression de p22phox, p47phox, NF-B et TGF-1 a été mesurée par immunohistochimie, PCR en temps réel ou buvardage Western. La capacité anti-oxydante totale, le malondialdéhyde et les niveaux d'espèces réactives d'oxygène ont aussi été déterminés. Les résultats ont montré que le séquoyitol diminuait significativement le glucose sanguin a` jeun, l'azote uréique sanguine et la créatinine sérique, et augmentait le niveau d'insuline chez les rats diabétiques. La capacité anti-oxydante totale était significativement accrue, alors que les niveaux d'espèces réactives d'oxygène et de malondialdéhyde, de même que l'expression de p22phox, p47phox, NF-B et TGF-1 étaient diminués en fonction du traitement au séquoyitol in vivo ou in vitro. Ces résultats suggèrent que le séquoyitol ralentit la progression de la néphropathie diabétique induite par une diète riche en gras et de faibles doses de streptozotocine chez le rat, par son effet sur l'abaissement du glucose, son activité anti-oxydante et la régulation de l'expression de TGF-1. [Traduit par la Rédaction] Mots-clés : séquoyitol, néphropathie diabétique, stress oxydant, TGF-1.
Introduction Diabetic nephropathy (DN) is one of the most important complications for diabetics and is characterized histologically by excessive deposition of extracellular matrix (ECM) in the kidney, leading to glomerular mesangial expansion and tubulointerstitial fibrosis (Sun et al. 2013). Numerous experimental and clinical studies have shown that oxidative stress plays an important role in the development and progression of vascular complications and diabetic nephropathy (Forbes et al. 2008). It has been reported that treatment with acarbose helps to prevent the increase in oxidative stress and vascular dysfunction induced by hyperglycemia (Rösen and Osmers 2006). In addition, pathological changes in glomeruli and an increase in urinary albumin excretion in diabetic rats were ameliorated by dietary antioxidant supplementation with vitamin E or lipoic acid, supporting the role of oxida-
tive stress in the pathogenesis of diabetic nephropathy (Craven et al. 1997; Melhem et al. 2001). The degree of oxidative stress is determined by the balance between the production of reactive oxygen species (ROS) and the antioxidant defence system (Droge 2002). In experimental and human diabetes, the generation of ROS is known to be increased via multiple pathways, including glucose auto-oxidation, increased mitochondrial superoxide production, PKC-dependent activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, formation of advanced glycation end-products (AGEs), and stimulation of cellular ROS production by extracellular AGEs through their receptors (Brownlee 2001; Forbes et al. 2008). Among these, the upregulation of the subunits of NADPH oxidase, such as p47phox and p22phox, mainly contribute to the increase in glomerular superoxide production in experimentally diabetic rats (Satoh et al. 2005). In a rodent model of diabetes (streptozotocin in-
Received 17 August 2013. Accepted 18 March 2014. X.-W. Li,* Y. Liu,* W. Hao, and J.-R. Yang. Department of Pharmacology, Third-Grade Pharmacology Laboratory of State Administration of Traditional Chinese Medicine, Wannan Medical College, Wen-Chang West Road 22#, Wuhu 241002, China. Corresponding author: Jie-Ren Yang (e-mail: [email protected]). *These authors contributed equally to this work. Can. J. Physiol. Pharmacol. 92: 405–417 (2014) dx.doi.org/10.1139/cjpp-2013-0307
Published at www.nrcresearchpress.com/cjpp on 28 March 2014.
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by EAST CAROLINA UNIVERSITY on 04/20/15 For personal use only.
406
duced), the renal expression of p22phox and p47phox was increased, and this was associated with ROS-induced renal damage (Lee et al. 2010; Feng et al. 2013). There is a growing body of evidence indicating that herbal constituents hold a great promise for the treatment of type-2 diabetes, and the extracts from numerous plants have been reported to reduce blood glucose (Chen et al. 2008; Huyen et al. 2010). Sequoyitol is a natural compound present in many plants (e.g., Amentotaxus yunnanensis, Aristolochia arcuata, and Crossostephium chinensis), as a novel small molecule (molecular mass: 194) (Li et al. 2003; Yang et al. 2008). Both oral and subcutaneous administrations of sequoyitol ameliorate hyperglycemia and glucose intolerance in ob/ob mice with insulin resistance (Shen et al. 2012). Additionally, sequoyitol directly improves insulin sensitivity in cultured hepatocytes, adipocytes, and -cells, and protects -cells against oxidative injury (Sivakumar et al. 2010; Shen et al. 2012). In a previous study (Chen et al. 2013), we found that sequoyitol acts as an antioxidant protectant against hepatic injury through inhibiting the expression of p22phox and p47phox. These results suggest that sequoyitol has a therapeutic potential for the treatment of diabetic complications. However, whether sequoyitol attenuates diabetic kidney disease is still unclear. This study was undertaken to investigate whether sequoyitol reduces oxidative stress and ameliorates the development of nephropathy in experimentally diabetic rat model.
Materials and methods Reagents and materials Sequoyitol (MB1218, >98.0% purity) was provided by the Dalian Meilun Biotech Company (Dalian, China). Streptozotocin (STZ) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma (St. Louis, Missouri, USA). 2=7=-Dichlorofluorescein diacetate (DCFH-DA), and the kits for measuring total antioxidant capacity (T-AOC) and malondialdehyde (MDA) were purchased from the Beyotime Institute of Biotechnology (Haimen, China). Masson's trichrome stain kit was purchased from Nanjing KeyGEN Biotech (Nanjing, China). The kits for measuring blood urea nitrogen (BUN) and serum creatinine (SCr) were supplied by the Siemens Healthcare Diagnostics Company (Shanghai, China). The primers were purchased from the Shanghai Sangon Biological Engineering Company (Shanghai, China). The PrimeScript reverse transcription reagent kit and SYBR Premix Ex Taq were obtained from TaKaRa Biotechnology Company (Dalian, China). TGF-1 antibody, -actin, and the collagen IV antibody were purchased from Abcam (Hong Kong, China), and p22phox, p47phox, and nuclear factor B (NF-B) were obtained from Santa Cruz (California, USA). Animals Male Sprague–Dawley (SD) rats (aged 6–8 weeks, weighing 180– 220 g) were obtained from the Nanjing Qinglongshan Experimental Animal Company (certificate No: SCXK (jun) 2007-012; Nanjing, China). All experiments were conducted in accordance with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the experimental protocol was approved by the Medicine Animal Welfare Committee of Wannan Medical College. Experimental animals, grouping, and treatments The diabetic rat model was developed using a high-fat diet plus multiple low doses of streptozotocin, as described previously (Zhang et al. 2008). The high-fat diet consisted of 22% fat, 58% carbohydrate, and 20% protein with a total calorific value of 44.3 kJ·kg−1 (Nanjing Qinglongshan Experimental Animal Company, Nanjing, China). The control rats were given regular chow consisting of 5% fat, 53% carbohydrate, and 23% protein with a total calorific value of 25 kJ·kg−1. Following 4 weeks of dietary intervention, the diabetic group was injected intraperitoneally (i.p.) with low doses of strep-
Can. J. Physiol. Pharmacol. Vol. 92, 2014
tozotocin (30 mg·(kg body mass)−1, dissolved in 0.1 mol·L−1 sodium citrate buffer, pH 4.4; Sigma). One week later, blood samples for fasting blood glucose measurements (using the glucose oxidase peroxidase method) were collected by cutting the tail. Rats with a fasting blood glucose of
