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Effects of Astragalus injection on TGF-β/Smads
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pathway in kidney in type 2 diabetic mice
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Yanna Nie1, Shuyu Li1, Yuee Yi1, Weilian Su1, Xinlou Chai1, Dexian Jia1 and Qian
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Wang1
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Beijing, China.
School of Preclinical Medicine, Beijing University of Chinese Medicine, 100029,
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10
 Corresponding author
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Email addresses:
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YN: love_rapunzel@163.com
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SL: lishuyu0706@163.com
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YY：yijin_tao@163.com
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WS：susulin2005@163.com
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XC：mmxin3@126.com
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DJ：jiadexian2002@yahoo.com.cn
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QW: wangqianchai@163.com
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Abstract
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Background
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In traditional Chinese medicine, astragalus injection is used to treat diabetic
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nephropathy. This study was carried out to determine whether astragalus injection has
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any effects on diabetic nephropathy by modulation of TGF -β/Smads signaling
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pathway.
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Methods
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The 14-week-old diabetic male KKAy mice were randomly divided into model group
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and astragalus injection treatment group, while the same age male C57BL/6J mice
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were selected as the normal group. The treatment group received daily intraperitoneal
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injection of astragalus (0.03 ml/10g.d), while the model group only received an
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injection of the same amount of saline. Mice were killed at 24th week respectively.
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The serum of each group were obtained and the blood glucose, creatinine and urea
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nitrogen were detected. The kidney was used for morphometric studies. The
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expression of TGF-β1, TGFβ-RⅠand Smad3/7 were detected by reverse
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transcription-polymerase chain reaction (RT-PCR) and western blot.
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Results
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Mice of model group became obese, suffered health disorders such as
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hyperglycaemia, polyuria and proteinuria. Astragalus injection treatment significantly
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reduced albuminuria, improved renal function and ameliorated renal histopathologic
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changes. Moreover, Astragalus injection increased the expression of Smad7, inhibited
2
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the expression of TGF-β RⅠ,Smad3 and its phosphorylation, TGF-β RⅠand
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decreased the mRNA level of TGF-β1.
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Conclusions
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TGF-β/Smads signal pathway plays an important role in the development of diabetic
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nephropathy (DN). Astragalus injections can preclude or mitigate DN by rebalancing
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TGF-β/Smads signalling and possess a protective effect on renal damage of DN in
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KKay mice.
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Background
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Diabetic nephropathy (DN) is a major microvascular complication of diabetes
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mellitus and the leading cause of end-stage renal disease [1]. A pathological change in
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diabetic nephropathy is the accumulation of normal and abnormal extracellular matrix
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components in the glomeruli and the interstitium of kidney [2]. TGF-β is a secreted
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protein that plays a critical role in the renal fibrosis and the accumulation of ECM [3].
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Intraperitoneal injection of TGF-β alone is sufficient to initiate a prominent renal
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fibrotic response [4]. TGF-β isoforms and their receptors are upregulated in both
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experimental and human DN [5,6]. We focus on TGF-β1 because it is the most highly
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expressed isoform in kidney and has been most closely linked to the pathophysiology
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of DN [6]. It was reported that TGF-β1 is stimulated by high glucose and is mainly
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expressed in renal tubular epithelial cells of diabetic mice [7].
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TGF-β1 binds to the TGFβ-RⅡ which recruits the binding of TGF-β RⅠ to
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form a heterotetramer. Then the TGF-β RⅠ phosphorylates the Smad proteins [8,9],
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after which the activated Smad2/Smad3 associate with Smad4 and this complex
3
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translocates to the nucleus where it is involved in mediating transcriptional responses
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on target genes [10]. Ultimately, the predominant effect of TGF-β is to promote
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matrix accumulation.
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Astragalus (Astragalus membranaceus) has long been known as an
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immune-modulating herb in traditional Chinese medicine. In clinical practice, it has
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been widely used in treating diabetes and kidney abnormalities caused by diabetes in
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the form of astragalus injection[11,12]. There are mainly polysaccharoses,
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astragaloside, isoflavones, and saponin glycosides extracted from astragalus [13].
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Recent studies have shown that astragalus has antifibrotic effect in a rat model and
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can inhibit the expression of TGF-β1, reduce extracellular matrix (ECM) synthesis
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and block tubular epithelial-to-mesenchymal transition (EMT) process [14,15]. A
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meta-analysis results revealed that astragalus injection had more therapeutic effect in
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DN patients such as reducing urine protein and improving renal function [16].
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Astragaloside Ⅳ, one of the main actived ingredients of astragalus, was proved that
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could ameliorate podocyte apoptosis, prevent acute kidney injury and attenuate
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glycated albumin-induced EMT in renal proximal tubular cells [17,18].
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Therefore, understanding the mechanisms of the astragalus injection to treat DN
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is essential for clinical therapy. We employs diabetic nephropathy model to
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investigate the effects of astragalus injection on TGF-β/Smads pathway.
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Methods
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Chemicals and reagent
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Astragalus injections were purchased from the Chengdu di’aojiuhong pharmaceutical
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factory, Chengdu, China.
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Experimental animals and treatment
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All experiments were performed in accordance with the guidelines on Ethical
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Standards for the investigation in animals; the study was approved by the the animal
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research committee of the Beijing University of Chinese Medicine. Sixteen male
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KKAy mice (9-11 weeks of age) weighing 25-28 g were used. Eight male C57BL/6J
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mice (9-11 weeks of age) weighing 23-25 g were also used. All the mice were
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purchased from Animal Center of Chinese Academy of Medical Science (Beijing,
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China) and were raised in the Clinical Institute of China-Japan Friendship Hospital
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(Beijing, China). During the experimental protocol, the KKAy mice were allowed free
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access to high-fat diet (HFD) and pure water. As a control, the C57BL/6J mice were
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allocated a normal diet and pure water.
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At 14 weeks of age, the tail blood was taken to measure blood glucose. A mouse
whose glucose is higher than 13.9 mM can be seen as a diabetic mouse. Then the
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KKAy mice were randomly divided into the model group (MG, n=8) and the
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treatment group (TG, n=8) so that the averages of body weight and blood glucose
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levels were approximately equal. The C57BL/6J mice were used as the normal group
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(NG, n=8). The treatment group received daily intraperitoneal injection of astragalus
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(0.03 ml/10g.d), while the model group only received an injection of the same amount
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of saline. The mice were housed individually in plastic cages with free access to food
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and water throughout the experimental periods.
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Weekly measurements of body weight were conducted and no differences were
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detected among the groups. Samples for determination of the blood glucose were
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taken from the tip of the tail by using the BREEZE2 Blood Glucose Test Strips (Bayer
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HealthCare, USA) every four weeks. At 24 weeks of age, all the mice were deprived
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of food pellets for 10 h. Blood was then collected from the orbital plexus. Samples
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were kept on ice for 1 h, then the plasma was separated by centrifugation at 2000 rpm
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for 15 min at 4℃ and subsequently stored in tubes at -20℃ until analysis. Some
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kidney tissues were excised and instantly frozen in liquid nitrogen for the following
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polymerase chain reaction (PCR) and Western blotting assay. Others were fixed in 4%
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buffered paraformaldehyde for HE (hematoxylin and eosin) staining and Masson
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staining.
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Biochemical analysis
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Mice were executed after taking blood samples at 24 weeks of age, The levels of
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blood urea nitrogen (UREA) and plasma creatinine (CREA) were measured by
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Automated Biochemical Analyzer (Hitachi, Japan).
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Albumin urine analysis
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The metabolic cages method was used to collect the urine samples. The
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concentrations of albumin urine samples were assessed using an automatic
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biochemistry analyzer (DADE Xpand, USA). Albuminuria in mice was expressed as
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milligrams per 24 h.
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Renal histological analysis
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Kidney sections were fixed in 4% buffered paraformaldehyde, embedded in paraffin
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and cut into 4-μm thick sections which were prepared for hematoxylin-eosin (HE) and
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Masson staining.
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Analysis of TGF-β1, TGFβ-RⅠ and Smad3 mRNA expressions by Reverse
transcriptase-PCR
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Total RNA was extracted from the kidney samples using Trizol reagent (Invitrogen,
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CA, USA). The total RNA concentration and purity were determined by measuring
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the OD260 and OD280 ratio. RNA was reverse-transcribed using GoScript Reverse
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Transcription System (Promega, USA) following the manufacturer instructions.
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Primers for PCR (Table 1)were designed and synthesized by Sangon Biotech Co. , Ltd
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(Shanghai, China). PCR reaction was performed using a thermal cycler (Bio-Rad
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Laboratory, USA) by the following contidions: 95℃ for 5 min; 95℃ for 30 s, 50℃
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for 30 s, 72℃ for 40 s, repeated for 36 cycles; and 72℃ for 8 min. Then a carefully
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prepared 1% agarose gel that would present the PCR products clearly was run. The
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quantity of specific mRNA was normalized as a ratio to the amount of β-actin mRNA.
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Western blot analysis for TGF-β1, TGFβ-RⅠ, Smad3/7 and p-Smad3
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The lysates were clarified by centrifugation and supernatants were collected. Protein
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concentrations were determined using the BCA Protein Assay (Applygen, Beijing,
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China). Equivalent amounts of tissue protein (80 μg) were resolved on SDS
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polyacrylamide gels and electroblotted to PVDF. The membranes were bloked in 5%
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(W/V) nonfat milk at room temperature for 1 h, and then incubated with the primary
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antibody against Smad3 (dilution 1: 200, Santa cruz, CA, USA), Smad7 (dilution
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1:200, Santa cruz, CA, USA), p-Smad3(dilution1: , epitomics, CA, USA) and β-actin
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(dilution 1: 1000, Santa cruz, CA, USA) overnight at 4℃ over night. After washing
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in 0.1% Tween TBS buffer, the membranes were incubated with horseradish
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peroxidase (HRP)-linked anti-mouse secondary antibody at 1:3000 dilution.
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Following washing in 0.1% Tween TBS buffer, the immunolabeled proteins were
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detected by enhanced chemiluminescense detection reagents (Applygen, Beijing,
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China). The density of bands was analyzed by Quantity One software.
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Statistical Analysis
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Numerical data are expressed as means ± standard deviation (SD) of at least three
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independent experiments. The significance of differences was examined using
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ANOVA. Values of P <0.05 were considered significant.
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Results
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Astragalus injection controls blood glucose levels and body weights
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The normal group didn’t display any obvious fluctuations in body status. However,
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the mice in the model group demonstrated retarded spirit, slow activities and
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lacklustre body hairs, which are typical manifestations of diabetes. Despite
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exhibitions of the similar symptoms to those of the model group, the treatment group
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showed much milder sufferings.
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The body weights of the mice in the model group were significantly higher than
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those in the normal group (P<0.01, Fig.1A), and the condition persisted during our
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experiment. Although the body weights of the mice in the treatment group also
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gradually increased, the mean weight of those was significantly lower than that of
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mice in the model group, as evidenced by measurement at 16,18,20 and 24 weeks
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respectively (Fig.1A). The blood glucose levels in the model group were also
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increased as compared with the normal group (P<0.01, Fig.1B), which were
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sigficantly reduced after the treatment with astragalus injections at 20 and 24 weeks
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(P<0.01, Fig.1B). However, the astragalus injection treatments were not able to bring
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glucose back to its normal level.
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Astragalus injection reduces albuminuria and renal function deterioration
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At 24 weeks of age, there were significant differences in the concentration of
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creatinine, blood urea nitrogen and albumin between the normal group and the model
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group (P<0.01, P<0.01, P<0.01, Fig.2). The treatment group showed significantly
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lower levels of plasma creatinine, blood urea nitrogen and urinary albumin after being
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administrated with astragalus injections, as compared to the model group. ( P<0.05,
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P<0.01, P<0.01, Fig.2).
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Astragalus injection prevents renal morphological changes in diabetic mice
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To identify the pathological damage in the kidney and to confirm the protective effect
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of astragalus injection on DN, HE and masson staining were performed. Compared
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with the normal group, a variety of damages of DN were detected in renal pathology
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of the model group, including thickening of basal membrane, vacuolar degeneration in
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the renal tubular epithelial cells (Fig.3 A-C). Masson staining revealed obvious
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glomerular sclerosis and interstitial fibrosis in KKay mouse. However, treatment with
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astragalus injection reversed these changes to a certain degree (Fig.3).
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Effects of astragalus injection on the expression of Smad3, Smad7, TGF-β1,
TGFβ-RⅠat mRNA level
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Using Reverse transcriptase-PCR , we found that astragalus injections significantly
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modulated the mRNA expression of Smad3, Smad7, TGF-β1, TGFβ-RⅠin kidneys of
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diabetic mice. The relative amounts of Smad3, TGF-β1, TGFβ-RⅠ mRNA were
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lower in diabetic mice treated with astragalus injections compared with the model
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group (P<0.01, P<0.01, P<0.05, Fig.4). Inversely, Smad7 expression was lower in
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KKay mice than in the normal mice (P<0.05, Fig.4), and astragalus injection
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treatment markedly induced Smad7 expression in diabetic mice (P<0.05, Fig.4).
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Effects of astragalus injection on the expression of Smad3, Smad7, TGF-β1,
TGFβ-RⅠ and p-Smad3 at protein level
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To examine the Smad3/7, TGF-β1 and TGFβ-RⅠexpressions, we employed western
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blot analysis. The model group showed higher levels of Smad3, TGF-β1, TGFβ-RⅠ
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(P<0.01, P<0.05, P<0.01, Fig.5 ) and lower level of Smad7 (P<0.01, Fig.5) compared
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with the normal group. The astragalus injection significantly reversed the increase in
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Smad3, TGFβ-RⅠ proteins (P<0.01, P<0.05) and the decrease in Smad7(P<0.01)
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protein in diabetic mice. But it had no effect on TGF-β1 expression (Fig.5).
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Smad3 is activated by phosphorylation. We then examined the expression of
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phosphorylated Smad3 by western blot. The results demonstrated that the
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phosphorylation of Smad3 as a fraction of total Smad3 was significantly increased in
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kidneys in the model group when compared with the normal group (P<0.01, Fig.6).
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Conversely, astragalus injection treatment significantly inhibited the Smad3 activation
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(P<0.01, Fig.6).
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Discussion
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Diabetic nephropathy, one of the most frequent chronic microvascular complications
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of diabetes mellitus, is the leading cause of end-stage kidney failure [19,20]. Its main
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pathological changes are ECM accumulation, degeneration of tubular epithelial cells,
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atrophy even disappearance of some tubules, thickening of basal membrane and
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infiltration with inflammatory cells in mesenchyme [21,22]. The KKAy mouse, which
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is known to serve as an excellent model of type-2 diabetes, was produced by
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transferring the yellow obese gene (Ay alele) into the KK/Ta mouse [23]. In the
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previous study, KKAy mice had developed obesity, hyperglycaemia and albuminuria
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by 14-week old. Furthermore, HE staining demonstrated the thickened glomerular
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basement membrance, vacuolar degeneration in the renal tubular epithelial cells. And
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Masson staining revealed obvious glomerular sclerosis and interstitial fibrosis in
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KKay mouse, a finding that is consistent with previous studies [22].
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The Chinese herbal astragalus is an effective medical prescription which is
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clinically used to treat DN [16,24]. All of the major constituents of astragalus have
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been shown to differentially lower high blood glucose levels and improve impaired
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glucose tolerance in type 2 diabetic models [25,26]. In this study, astragalus injection,
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the aqueous extract of astragalus, administration of which for 10 weeks did have a
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mild hypoglycemic effect as suggested in our data above. However, good glycemic
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control with insulin has been demonstrated to ameliorate DN in STZ-DM rats [27],
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suggesting that astragalus injection possesses other renoprotective mechanism beyond
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the sugar lowering effect. Diabetic albuminuria is an early hallmark of DN and is
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always associated with the development of characteristic histopathologic features. The
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results that there were aggravated kidney injuries such as albuminuria, glomerular
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sclerosis and interstitial fibrosis in KKay mice supported this notion. However,
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astragalus injection treatment attenuated theses changes in diabetic kidneys.
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Supporting this result may be the idea that astragalus injection can improve renal
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function through inhibiting EMT process and collagen production [28]. Moreover, it
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was found that levels of serum creatinine and blood urea nitrogen were elevated
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significantly in KKay mouse and the treatment group showed milder symptoms than
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the model group after administration of astragalus injection, indicating that astragalus
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injection could ameliorate renal function deterioration. Taken together, astragalus
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injection may be appropriate to control blood glucose levels and body weight.
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Specifically, it can reverse renal histopathologic changes, attenuate albuminuria,
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through which renal function may be improved. So there is a need for investigating
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the molecular mechanism of astragalus injection in treating DN.
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Then we aimed to investigate the mechanism of the astragalus injection to treat
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DN by focusing on the TGFβ/Smad pathway. TGF-β, a multifunctional cytokine,
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which leads to renal fibrosis, plays a crucial role in the pathogenesis of DN.
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Interestingly, the administration of TGF-β neutralizing antibodies significantly
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reduced renal fibrosis [9]. In our previous studies, we concluded that TGF-β1 proteins
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were high in diabetic kidneys of the KKay mice by immunohistochemistry. Moreover,
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We also found that TGF-β1 was mainly expressed in the cytoplasm of the renal
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tubular epithelial cells and is rarely expressed in glomeruli [29]. In this study, the
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amount of TGF-β1 expression was higher in the model group than that in the normal
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group, all of which are consistent with others’ findings [30,31]. Furthermore,
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astragalus injection treatment significantly reduced mRNA expression of TGF-β1.
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This finding suggested that it may play a role in downregulation of TGF-β1 at
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transcriptional level. Our western blot studies confirmed that the model group showed
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upregulated TGF-β RⅠproteins, however increased TGF-β RⅠwas not significant at
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mRNA levels. This phenomenon may be ascribed to the possibility that the mRNA of
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TGF-β RⅠis apt to degrade due to its unstable nature.
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One of our aims in this experiment was to investigate the effect of astragalus
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injection on Smad3/7. Smad7, as one of the inhibitory Smads, blocks TGF-β signaling
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pathway by inhibiting Smad2/3 phosphorylation and thereby exerting its anti-fibrotic
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effect [32]. Increasing evidence has shown that disruption of Smad7 may accelerate
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renal fibrosis [33,34], Our results that KKay mice with down-regulated Smad7
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developed more severe renal dysfunction further confirmed this viewpoint. Besides
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the inhibitory Smads, there are receptor-regulated Smads that can transduce TGFβ
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signal, such as Smad2 and Smad3. Smad3 is a critical downstream mediator
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responsible for renal fibrosis and is proved to function in the diabetes-induced
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up-regulation of fibronectin and α3 (Ⅳ) collagen, and therefore may play a critical
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role in the early phase of DN [35,36]. It is well documented that smad3-deficient mice
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are protected from renal fibrosis by reduced EMT, collagen deposition, and the
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expression of profibrotic TGF-β target genes [37,38]. As shown in our results, the
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expression of Smad3 and p-Smad3 were increased in diabetic kidney. Furthermore,
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astragalus injection suppressed Smad3 expression and its phosphorylation and
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promoted Smad7 expression resulting in improved conditions provides further
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evidence for its effectiveness in treatment of DN.
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Conclusions
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In conclusion, we believe that the diabetic nephropathy is caused by the imbalanced
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TGF-β/Smad pathway, and as a result, FN increases and assembles, leading to
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glomerular sclerosis and interstitial fibrosis. In consistent with our expectation,
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astragalus injection alleviates DN by suppressing Smad3, p-Smad3 and TGF-β RⅠ
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expression. Concurrently, it also exerts its effect by reducing the mRNA level of
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TGF-β1 and promoting Smad7 expression, These results demonstrated that astragalus
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injection could be a potential agent for amelioration of DN via blockade of
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TGF-β/Smad pathway.
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Abbreviations
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TGF-β: transforming growth factor-beta; DN: Diabetic nephropathy; TGFβ-R:
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transforming growth factor-beta receptor; UREA: blood urea nitrogen; CREA: plasma
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creatinine.
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Competing interests
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The authors have declared that there is no conflict of interest.
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Authors' contributions
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YN carried out the animal experiments, performed the statistical analysis and drafted
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the manuscript. YY participated in the western blot assay. WS involved in the
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extraction of RNA and RT-PCR, XC and DJ participated in the HE staining and
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discussion of the experiment. SL and QW formulated the original ideas and working
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hypothesis. QW is the owner of the research grant and revised the draft of manuscript.
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All authors read and approved the final manuscript.
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Acknowledgements
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This study was supported by the National Natural Science Foundation of China (no.
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30672756; no. 81072926) and innovation team project funded by Beijing university of
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Chinese medicine (no. 2011-CXD-04).
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factor-β1/Smad3 in diabetic nephropathy. Exp Biol Med 2010, 235:
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31. SW Hong, M Isono, S Chen, FN Ziyadeh: Increased glomerular and
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tubular expression of TGF--β1, its type II receptor, and activation of the
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Smad signaling pathway in the db/db mouse. Am J Pathol 2001, 158:
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32. Schiffer M, Schiffer LE, Gupta A, et al: Inhibitory smads and TGF-beta
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33. Arthur C. K. Chung, Xiao R. Huang, Li Zhou, et al: Disruption of the
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35. Wang A, Ziyadeh FN, Lee EY, et al: Interference with TGF-beta signaling
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affecting albuminuria. Am J Physiol Renal Physiol 2007, 293: F1657–1665
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36. Fujimoto M, Maezawa Y, Yokote K, et al: Mice lacking Smad3 are
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protected against streptozotocin-induced diabetic glomerulopathy.
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37. Arany P. R, Flanders KC, DeGraff W, et al: Absence of Smad3 confers
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radioprotection through modulation of ERK-MAPK in primary dermal
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38. Inazaki K, Kanamaru Y, Kojima Y, et al: Smad3 deficiency attenuates renal
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Kidney Int 2004, 66: 597–604
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Figures
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Figure 1- Body weights and the blood glucose levels in different weeks (means ±
424
SD, n=6-8). NG: the normal group. MG: the model group. TG: the astragalus
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injection treatment group. The bargraphs summarize average values for body weight
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and blood glucose. A: body weights gradually increased in mice in the model group.
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Astragalus injection treatment could significantly inhibit body weight gains. B:
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diabetic mice (the model group) remained hyperglycemic and C57BL/6J mice (the
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normal group) remained normoglycemic throughout the period of study. The tendency
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for astragalus injection treatment to reduce the blood glucose concentration in diabetic
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mice was obvious. but the astragalus injection treatments can not return the blood
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glucose levels to normal. Compared with NG, *P<0.05, **P<0.01.Compared with
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MG, P<0.05, P<0.01.
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Figure 2 - The concentration of plasma creatinine, blood urea nitrogen, urine
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albumin at 24 weeks of age (means ± SD, n=6-8). NG: the normal group. MG: the model
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group. TG: the astragalus injection treatment group. A: the mean of plasma creatinine was
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elevated in the model group. The increase in plasma creatinine with diabetes was prevented in
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the treatment group. B: blood urea nitrogen tracked with the plasma creatinine, increasing in
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model group but decreasing in the treatment group. C: urine albumin also increased in the
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model group, which was markedly reduced with astragalus injection treatment. Compared
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with NG, *P<0.05, **P<0.01. Compared with MG, P<0.05 , P<0.01.
20
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Figure 3. Renal pathology of different groups (HE , masson ×400). Figures show
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pathologic damages in KKAy mice at 24 weeks of age. Glomerulosclerosis and interstitial
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fibrosis are the main pathology in diabetic nephropathy. A-C: HE staining. A: the normal
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group. B: the model group. HE staining showed thickened basal membrane and vacuolar
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degeneration in the renal tubular epithelial cells. C: the treatment group. D-F: Masson
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staining. D: the normal group. E: the model group. Masson staining revealed collagen
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deposition (blue color) in the interstitium and glomeruli. F: the treatment group. It showed
449
more improved kidney pathological.
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Figure 4. Astragalus injection has effects on the mRNA expressions of
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Smad3/7, TGF-β1, TGFβ-RⅠ(means ± SD, n=3). Mice were sacrificed at 24 week. NG:
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the normal group. MG: the model group. The model group showed decreased
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expression of Smad7 and increased expression of Smad3, TGFβ-RⅠ, TGF-β1. TG: the
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astragalus injection treatment group. Compared with NG, *P<0.05, **P<0.01.
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Compared with MG, P<0.05 , P<0.01.
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Figure 5. Astragalus injection has effects on the protein expressions of
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Smad3/7, TGF-β1, TGFβ-RⅠ(means ± SD, n=3). Protein extracts from the renal cortex
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were analyzed to evaluate the expression of TGF-β/Smad proteins. NG: the normal
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group. MG: the model group. TG: the astragalus injection treatment group. The
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reduction in Smad3 and TGFβ-RⅠprotein levels was similarly observed in diabetic
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mice after astragalus injection treatment, but no significant change was observed in
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TGF-β1 level. Compared with NG, *P<0.05, **P<0.01. Compared with MG,
463
P<0.05
, P<0.01.
21
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Figure 6. Smad3 phosphorylation in diabetic mice was suppressed by
465
astragalus injection (means ± SD, n=3). NG: the normal group. MG: the model group.
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TG: the astragalus injection treatment group. The Smad3 was actived in diabetes. The
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ratio of phosphorylated Smad3 to total Smad3 was increased in KKay mice. In addition,
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Smad3 activation were significantly inhibited in astragalus injection treated diabetic mice.
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Representative western blots of p-Smad3 and total Smad3 are shown in figure6. Compared
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with NG, *P<0.05, **P<0.01. Compared with MG, P<0.05 , P<0.01.
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Tables
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Table 1 - PCR sequences and PCR products
name
TGF-β1
TGFβ-RⅠ
Smad7
Smad3
β-actin
length
493bp
172bp
309bp
232bp
243bp
Upstream primer（5’-3’）
TCCCTCAACCTCAAATTATTCA
GGCGAAGGCATTACAGTGTT
ACAGAAAGTGCGGAGCAAGAT
GGGCCAACAAGTCAACAAGT
GAAATCGTGCGTGACATTAAGG
Downstream primer（5’-3’）
GCGGTCCACCATTAGCAC
TGCACATACAAATGGCCTGT
CTGATGAACTGGCGGGTGTAG
CTGGCTGGCTAAGGAGTGAC
CACGTCACACTTCATGATGGAG
473
474
Additional files
475
476
477
478
Additional file 1 – revised manuscript with corrections red-marked
A revised version with corrections in red mark is for convenient scrutiny. It is in the
formats of MS word.
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