Journal of Ethnopharmacology 169 (2015) 229–238
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You-gui Pill ameliorates renal tubulointerstitial fibrosis via inhibition of TGF-β/Smad signaling pathway Li Wang a,1, Ai-li Cao a,1, Yang-Feng Chi b, Zheng-Cai Ju c, Pei-Hao Yin b, Xue-Mei Zhang d,n, Wen Peng a,b,nn a
Laboratory of Renal Disease, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China Department of Nephrology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China c Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China d Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 November 2014 Received in revised form 8 April 2015 Accepted 18 April 2015 Available online 25 April 2015
Ethnopharmacological relevance: You-gui Pill (YGP), a traditional Chinese medicinal prescription, was widely used to warm and recuperate “kidney-yang” clinically for hundreds of years in China. Recent studies found that YGP had a potential benefit for renoprotection. Aim of the study: The present study aimed to elucidate the in vivo and in vitro efficacy of YGP on renal tubulointerstitial fibrosis, and the molecular mechanism is also investigated. Materials and methods: Rat renal tubulointerstitial fibrosis model was elicited by unilateral ureteral obstruction (UUO). Sprague–Dawley rats underwent UUO and were studied after 14 days. Animals were randomly subjected to six groups: sham, UUO, UUO/YGP (0.14, 0.42, 1.26 g/kg/d), and UUO/enalapril (10 mg/kg/d). HE, Masson and ELISA were used for evaluate renal injury and function. Immunohistochemical analysis and western blot were used to detect the expressions of α-SMA, fibronectin, collagen matrix and Smads. In vitro studies were investigated in TGF-β1-stiumlated NRK-49F cell line. Results: Oral administration of YGP significantly decreased UUO-induced inflammatory cell infiltration, tubular atrophy and interstitial fibrosis, and there was no significant difference between YGP at 1.26 g/kg and enalapril at 10 mg/kg treatment (P4 0.05). Meanwhile, serum creatinine and blood urea nitrogen levels were reduced dramatically (Po 0.01). In coincide with the decreased of TGF-β1, α-SMA, fibronectin and collagen matrix expressions were also declined with YGP treatment in both UUO kidneys and TGFβ1-stimulated NRK-49F cell line. Additionally, nuclear translocation of p-Smad2/3 was markedly downregulated by YGP (P o0.001), with a relative mild up-regulated expression of Smad7 (P o0.05). Conclusions: Our findings demonstrate that YGP had a renoprotective effect in ameliorating renal tubulointerstitial fibrosis, and this activity possibly via suppression of the TGF-β and its downstream regulatory signaling pathway, including Smad2/3. & 2015 Published by Elsevier Ireland Ltd.
Chemical compounds studied in this article: Gallic acid (PubChem CID: 370) 5-HMF (PubChem CID: 237332) Morroniside (PubChem CID: 11228693) Sweroside (PubChem CID: 161036) Loganin (PubChem CID: 87691) Rutin (PubChem CID: 5280805) Cinnamic acid (PubChem CID: 444539) Quercerin (PubChem CID: 5280343) Kaempferide (PubChem CID: 5281666) Carboxymethyl cellulose (PubChem CID: 6328154) Keywords: You-gui Pill Renal tubulointerstitial fibrosis Unilateral ureteral obstruction TGF-β Smad2/3
1. Introduction Abbreviations: YGP, You-gui Pill; UUO, unilateral ureteral obstruction; TIF, tubulointerstitial fibrosis; ECM, extracellular matrix; α-SMA, α-smooth muscle actin; TCM, Traditional Chinese Medicine; 3D HPLC, three-dimensional high performance liquid chromatography; CMC, carboxymethyl cellulose; HRP, horseradish peroxidase; FBS, fetal bovine serum; LDH, lactate dehydrogenase; SDS-PAGE, SDS polyacrylamide gel electrophoresis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ANOVA, one-way analysis of variances; BUN, blood urea nitrogen; MMP-2, matrix metalloproteinase 2. n Corresponding author. Fax: þ 86 21 50980046. nn Corresponding author at: Department of Nephrology, Laboratory of Renal Disease, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, 164 LanXi Road, Shanghai 200062, China. Fax: þ 86 21 52665957. E-mail addresses: [email protected] (X.-M. Zhang), [email protected] (W. Peng). 1 Li Wang and Ai-li Cao contribute equally to this work. http://dx.doi.org/10.1016/j.jep.2015.04.037 0378-8741/& 2015 Published by Elsevier Ireland Ltd.
Renal tubulointerstitial fibrosis (TIF) is the final common pathway leading to end-stage renal failure irrespective of the initiating pathology (Liu, 2011). Predominating over glomerular and renal vascular disease, the degree of TIF is closely related with a loss of renal function (Bohle et al., 1987). Progression of TIF is a dynamic process, manifested as tubular loss and accumulation of extracellular matrix (ECM) proteins, including fibronectin and collagens (Wang et al., 2014b). Persistent of ECM production can be accelerated with severe renal injury, which results in large amounts of fibrinous tissue is generated and thus a vicious circle is formed progressively (Ishibe and Cantley, 2008). Therefore, it is essential to identify appropriate pharmacologic interventions to prevent TIF, especially to improve recovery of ECM following renal injury.
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Several cellular pathways, including TGF-β/Smad, Wnt/β-catenin and Integrin/ILK have been identified as major avenues for the generation of ECM, in which TGF-β is recognized as the most important profibrogenic cytokine in the kidney (Liu, 2010; Wang et al., 2014a, 2014b). A typical role of TGF-β is its biologic effects can exert through the Smad protein signaling pathways. Thus, inhibiting the TGF-β/Smad signaling pathway is helpful for preventing TIF and preserving renal function. In recent years, clinical and experimental studies have shown that Traditional Chinese Medicine (TCM) is beneficial for both the prevention and treatment of TIF. According to TCM theories, deficiency of “kidney-yang” is one of the key factors that is responsible for TIF. Therefore, TCM physicians take warm and recuperate “kidney-yang” as the main consideration in clinical practice. You-gui Pill (YGP) is a well-known prescription of TCM and originally recorded in “Jing-Yue Complete Works” at Ming dynasty. As a yang-tonic prescription clinically, previous studies have reported that YGP possesses a variety of pharmacological actions, such as in enhancing immune system, relieving states of asthma, ameliorating cerebral limbic system and improving body function in stress condition (Dai et al., 2006; Lin et al., 2012; Ou et al., 2003; Yao et al., 2005). Notably, evidence showed that YGP is a potential renal protective agent (Yang et al., 2009). However, the regulatory mechanism of YGP on renal disease remains largely unknown. Therefore, in the present study, we examined the efficacy of YGP on TIF in animal models of obstructive nephropathy and in TGFβ1-stiumlated NRK-49F cell. Furthermore, the underlying mechanism was also explored.
2. Materials and methods 2.1. Preparation of extract of You-gui Pill (YGP) YGP comprises 10 kinds of herbs, namely Radix Rehmanniae Praeparata, Rhizoma Dioscoreae, Fructus Corni, Fructus Lycii, Semen Cuscutae, Cervi cornus colla, Eucommiae cortex, Cinnamomi cortex, Radix Angeliccae Sinensis and Radix Aconiti Lateralis Prreparata (See also in Table 1). All TCM herbs were supplied by Leiyunshang Pharmaceutical Co., Ltd. (Shanghai, China) and were authenticated by Dr. Bo Cui (college of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine). The dried plant samples with voucher number 2013005 were deposited in the herbarium of Laboratory of Renal Disease, Putuo Hospital, Shanghai University of Traditional Chinese Medicine. YGP extract was prepared as described previously (Wu et al., 2010). In brief, in total of 5.7 kg of dry TCM herb was powdered and suspended in 4 volumes of water. The suspension was boiled for 2 h and filtered. Then the residual herbs were subjected to the same decoction suspension and filtration procedure. The filtrate was mixed and extracted with 4 volumes of 95% ethanol (vol/wt) for 12 h for the third time. In the end, the extracts were concentrated at 65 1C with a vacuum rotary evaporator. A total amount of 1.0 g of extract powder
that was about 18.9% of the raw herb was obtained and the dried extract was stored at 20 1C. 2.2. HPLC analysis A three-dimensional high performance liquid chromatography (3D-HPLC) profile of YGP extract is shown in Fig. 1. The analysis procedure was conducted according to the previous reports (Liang et al., 2009; Lin et al., 2012). Briefly, YGP extract (2.0 g) was dissolved in 10 ml methanol under ultrasonication and centrifuged for 5 min at 9000 g, and then the supernatant was transferred into 10 ml volumetric flask and made up to volume with methanol. To acquire chromatograms and UV spectra, HPLC apparatus consisting of a CCPMII multisolvent delivery pump and a PD-8020 photodiode array detector was used (SD-8020 system, Tosoh Corp., Tokyo, Japan). The separation was performed on an YMC Carotenoid column (3.0 μm, 150 mm 4.6 mm I.D., Tokyo, Japan). The mobile phase consisted of acetonitrile (A) and 0.1% (v) formic acid (B). The gradient elution was programmed as follows: 0–30 min, 1–12% A; 30–32 min, 12–15% A; 32–55 min, 15–25% A; 55–60 min, 25–40% A; 60–70 min, 40–100% A at a flow rate of 1.0 ml/min. A pre-equilibration period of 15 min was set up between individual runs. The other analysis conditions were as follows: column temperature, 35 1C; injection volume, 10 μl; UV scan 254 nm. 2.3. Drugs and reagents Pulverized YGP extract was prepared as a suspension in 0.5% carboxymethyl cellulose (CMC) with needed concentration for animal oral administration or was dissolved in complete RPMI 1640 (Gibco, USA) at a concentration of 10,000 μg/ml for subsequent cellular usage. The latter was filtered through a 0.22 μm membrane pore (Millipore, Billerica, MA, USA), then was stored at 4 1C before use. Antibodies to TGF-β1, collagen I, collagen III, Smad4 and Smad7 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fibronectin, phosphorylated-Smad2/3 (p-Smad2/3), Smad2/3, GAPDH and horseradish peroxidase (HRP)-labeled goat anti-rabbit or -mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). α-SMA was purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant TGF-β1 was purchased from Peprotech (Hamburg, Germany). BCA protein assay kit was purchased from Pierce (Rockford, IL, USA). Rat renal fibroblasts (NRK-49F) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). 2.4. Animal and experimental protocols All experimental procedures were conducted in conformity with the ethics committee of Putuo Hospital, Shanghai University of Traditional Chinese Medicine and in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was performed according to previously described (Wang
Table 1 Consist of You-gui Pill (YGP). Chinese name
Botanical name
Common name
Weight (g)
Part used
Voucher numbers
Shu Di Huang Shan Yao Shan Zhu Yu Qou Qi Zi Tu Si Zi Lu Jiao Jiao Du Zhong Rou Gui Dang Gui Fu Zi
Rehmannia glutinosa (Gaertn).) DC. Dioscorea oppositifolia L. Cornus officinalis Sieb. Et Zucc. Lycium barbarum L. Cuscuta chinensis Lam. Cervus elaphus Linnaeus Eucommia ulmoides Oliv. Cinnamomum cassia (L.) J.PreslBark Angelica sinensis (Oliv.) Diels Aconitum carmichaeli Debx.
Radix Rehmanniae Praeparata Rhizoma Dioscoreae Fructus Corni Fructus Lycii Semen Cuscutae Cervi cornus colla Eucommiae cortex Cinnamomi cortex Radix Angeliccae Sinensis Radix Aconiti Lateralis Prreparata
8 4 3 4 4 4 4 2 3 2
Root Rootstock Fruit Fruit Fruit Horn Bark Bark Root Root
2012091711 2012081001 2012082006 2012091002 2012092101 20110902 2012042706 2012032302 2012091704 2011120426
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Fig. 1. 3D-HPLC profile of YGP. The analysis based on ultraviolet (UV) absorption clearly showed the presence of the following major constituents in YGP extract: Gallic acid, 5-HMF, Morroniside, Sweroside, Loganin, Rutin, Cinnamic acid, Quercerin and Kaempferide.
et al., 2014b). The selective doses of YGP for animal study were calculated based on the human optimal equivalent dose of 27 g raw herbs. The animals were divided randomly into six groups as follows: (1) sham group, (2) UUO group, (3) UUOþenalapril group (10 mg/kg/d, diluted in 0.5% CMC) as a positive control, and (4) UUOþYGP groups at doses of 0.14, 0.42, 1.26 g/kg/d (diluted in 0.5% CMC). All rats except for the sham-operated rats underwent UUO. At 14 days of post-surgery, the animals were sacrificed and the left kidneys were carefully removed. The kidneys were sagittally sliced into two parts, one part was snap frozen in liquid nitrogen and kept at 80 1C for protein extraction, and another part was immersed into 10% neutral-buffered formalin for histopathological and immunohistochemical examinations. 2.5. Renal function test The blood samples were taken from the abdominal aorta for determination of serum creatinine and blood urea nitrogen (BUN) at 14 days after UUO operation. Creatinine colorimetric assay kit (BioVision, USA) and BUN colorimetric detection kit (Arbor, USA) were used for serum creatinine and BUN test according to the manufacturer's protocol, respectively. 2.6. Cell culture and treatment The NRK-49F cells were cultured in complete RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco,
USA), 100 U/ml penicillin and 100 mg/ml streptomycin in an atmosphere of 5% carbon dioxide and 95% air at 37 1C. MTT and lactate dehydrogenase (LDH) assay were performed as previously described (Wang et al., 2014b). Briefly, cells were cultured at a density of 4 104 cells/ml in 100 mL 1640 containing 1.0% FBS in 96-well microplates. Then YGP at concentrations of 1, 3, 10, 30, 100, 300, 1000, 3000, 10,000 μg/ml was added individually to the medium. After 24 h of incubation, the supernatants were collected and centrifuged at 250 g for 10 min, and then the extents of cytotoxicity of YGP were measured by MTT assay kit (Sigma-Aldrich, St. Louis, MO, USA) and LDH release kit (BioVision, USA) using a microplate reader (Bio-Rad Laboratories, CA, USA). All measurements were performed in duplicate. To evaluate the inhibitory effect of YGP on TGF-β1-induced profibrogenic responses, the NRK-49F cells were pre-treated with YGP at concentrations of 100, 300 and 1000 μg/ml for 30 min, and then the TGF-β1 at 1.0 ng/ml was added. After treatment with YGP for 24 h, protein expression was measured. 2.7. Histology and immunohistochemistry For histological analysis, paraffin sections (3 μm thick) were performed with hematoxylin–eosin and Masson's modified trichrome-staining. Tubulointerstitial injury score, inflammatory cell count and collagen deposition area (%) were graded by the published criteria (Wang et al., 2014b). For immunohistochemistry analysis, antibodies to α-SMA (1:500 dilution) and p-Smad2/3 (1:250 dilution) were used. Color development procedure was conducted according to the instruction of a
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Vecta-Elite streptavidin-peroxidase kit (Vector Laboratories, Burlingame, CA, USA). The stained sections were developed with diaminobenzidine to produce brown products and counterstained with hematoxylin. Image Pro Plus 6.0 Multimedia Color Pathological image analysis software (Media Cybernetics, Inc. Silver Spring, MD, USA) was used for the semi-quantitative analysis. And at least five non-overlapping high-power fields at a magnification of 200 per section were analyzed for each animal. The mean optical density value of α-SMA was calculated and the percentage of positive cell units of p-Smad2/3 in the nucleus was calculated.
(1:1000 dilution), Smad2/3 (1:1000 dilution), Smad4 (1:500 dilution), Smad7 (1:500 dilution) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000 dilution). After three washes in TBS-Tween-20, the membranes were incubated with HRP-labeled goat anti-rabbit or -mouse IgG for 1 h at room temperature. Signals were scanned and visualized by enhanced ECL (Millipore, USA) and X-ray film. The ratio of the protein interested was subjected to GAPDH and was densitometrically analyzed by Multi-Analyst software program (Version 1.01; Bio-Rad). 2.9. Statistical analysis
2.8. Western blot analysis Proteins extracted from either renal tissues or cultured NRK-49F cells were analyzed by western blotting. The samples were lysed in a buffer (50 mM Tris at PH of 7.5, 1 mM EDTA, 150 mM NaCl, 20 mM NaF, 0.5% NP-40, 10% glycerol, 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail) by sonication. The supernatant was collected after centrifugation at 13,000 g at 4 1C for 20 min. Protein concentration was determined by BCA protein assay kit. Equal amounts of protein were separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore, USA). Following blocked with 5% BSA in TBS-Tween20 for 2 h, the membrane was incubated with primary antibodies at 4 1C overnight. Antibodies used in this study included: TGF-β1 (1:1000 dilution), α-SMA (1:800 dilution), fibronectin (1:10,000 dilution), collagen 1 (1:1000 dilution), collagen 3 (1:1000 dilution), p-Smad2/3
The data were presented as the mean7standard error. Statistical analysis was performed using GraphPad Prism software version 5.0 (GraphPad Prism software Inc., San Diego, Calif, USA). The differences among multiple groups were evaluated by one-way analysis of variances (ANOVA). The differences between two groups were determined by t test. Statistical significance was defined as Po0.05.
3. Results 3.1. Effect of YGP on renal histology and renal function in rat UUO model As shown in Fig. 2A, significant tubulointerstitial injury, including tubular atrophy, inflammatory cells infiltration and interstitial fibrosis appeared in UUO kidneys, whereas those morphological changes were
Fig. 2. Treatment of rats with YGP attenuates renal injury in a dose-dependent manner in UUO kidneys. (A) Representative hematoxylin–eosin (HE) stained kidney sections from Sham (a), UUO (b), Enalapril (c), YGP (0.14, 0.42 and 1.26 g/kg; (d)–(f)) groups. (B) Histopathological scores and inflammatory cells count within the tubulointerstitium from sections similar to those shown in (A). (C) Representative Masson's modified trichrome stained kidney sections from sham (a), UUO (b), Enalapril (c), YGP (0.14, 0.42 and 1.26 g/kg; (d)–(f)) groups. (D) Quantitative analysis of the extent of renal interstitial expansion (area%) from sections similar to those shown in (C). Data represent mean 7SEM for at least 10 independent experiments. nnnP o0.001, compared with sham group; #Po 0.05, ##Po 0.01, ###P o0.001 compared with UUO group. Magnification: 400.
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not presented in sham-operative kidneys. Meanwhile, compared with vehicle-treated UUO kidneys, semiquantitative histomorphometry analysis demonstrated that YGP treatment decreased the tubulointerstitial injury scores and inflammatory response dose-dependently in UUO kidneys (Po0.01 or 0.001) (Fig. 2B). Increase of collagen synthesis is another typical characteristic in UUO kidneys (Wang et al., 2014b). Compared with sham-operative kidneys, collagen expression is prominent in the interstitial and tubular areas of UUO kidneys. In contrast, the appearance was significantly improved in the YGP-treated rat kidneys (Fig. 2C). This result was also confirmed by semiquantitative assessment of the fibrotic area on the Masson's trichrome-stained kidney sections (Fig. 2D). Renal function was assessed by serum creatinine and the BUN analysis (Fig. 3). In UUO kidneys, serum creatinine and BUN rose more than 2.2-fold at 14 days compared with sham-operative kidneys (Po0.001). However, the levels showed a significant, dosedependent reduction with treatment of YGP, even at 0.14 g/kg (Po0.01 and 0.05, respectively). It is worth noting that treatment with YGP at 0.14 g/kg caused a marked improvements on pathological and functional injuries compared to rat in UUO group (Po0.05 or 0.01), and there was no significant difference between YGP at 1.26 g/kg and enalapril at 10 mg/kg treatment (P40.05), indicating that YGP treatment had almost the same efficacy to that of enalapril.
Fig. 3. Treatment of rats with YGP improves renal function in a dose-dependent manner in UUO kidneys. Serum creatinine (A) and BUN (B) in the Sham, UUO, Enalapril and YGP (0.14, 0.42 and 1.26 g/kg) groups. Data represent mean 7SEM for at least 10 independent experiments. nnnP o 0.001, compared with the sham group; # P o 0.05, ##Po 0.01, ###Po 0.001 compared with the UUO group.
3.2. Effect of YGP on expression of α-SMA and ECM accumulation in rat UUO model and cultured NRK-49F cells Activation of myofibroblast cells, as manifested by α-smooth muscle actin (α-SMA) induction, is one of the representative events
Fig. 4. Treatment of rats with YGP decreases α-SMA expression by immunohistochemistry in UUO kidneys. (A) Representative photomicrographs of α-SMA immunohistochemistry on kidney sections from the Sham (a), UUO (b), Enalapril (c), YGP (0.14, 0.42 and 1.26 g/kg; (d)–(f)) groups. (B) Quantitative analysis of α-SMA expression from sections similar to those shown in (A). Data represent mean 7 SEM for at least 10 independent experiments. nnnPo 0.001, compared with the sham group; ## Po 0.01, ###P o0.001 compared with the UUO group. Magnification: 400.
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in ECM overexpression of fibrotic kidney (Li et al., 2002; Liu, 2011). As shown in Fig. 4, α-SMA is mainly expressed in interstitial cells of UUO kidneys. And immunohistochemistry and the western blot analysis showed that α-SMA was barely detectable in sham-operative kidneys, compared with the dramatic expression in UUO kidneys (Po0.001). However, this increase of α-SMA expression was significantly attenuated by YGP treatment, especially at the doses of 0.42 g/kg (Po0.01) and 1.26 g/kg (Po0.001). Moreover, as the pro-fibrotic protein expression and accumulation of ECM components are both important to TIF formation, here we further observed those changes. As shown in Fig. 5, the level of TGF-β1 increased dramatically in the UUO kidneys compared with those in sham-operative kidneys (Po0.01), but decreased with YGP treatment at 0.42 g/kg (Po0.05) and 1.26 g/kg (Po0.01). Same tendency was observed in fibronectin, collagen I and III expression. And our immunohistochemistry analysis revealed that the expression levels of fibronectin, collagen I and III were significantly increased in the UUO kidneys compared with sham-operative kidneys (Po0.001), and treatment with YGP at a dose of 1.26 g/kg decreased the integrated optical density of the three proteins by 47.570.3%, 60.970.2% and 55.270.1%, respectively (Po0.001, data not shown). Our previous study has revealed that TGF-β1 at 1.0 ng/ml could dramatically stimulate fibrogensis in NRK-49F (Wang et al., 2014b). Here, to clarify cytotoxicity of YGP on the NRK-49F cells for in vitro
experiment, MTT and LDH arrays were first performed (Fig. 6A and B). We found that YGP at and below 1000 μg/ml is safety to cell growth. Subsequently, the inhibitory effect of YGP on TGF-β1induced overexpression of α-SMA and ECM proteins in the NRK49F cells was determined. Similar to the in vivo study, YGP could also dose-dependently attenuate α-SMA, fibronectin, collagen I and III protein expressions in the TGF-β1-stimulated NRK-49F cells (Fig. 6C–G). 3.3. Blockade of p-Smad2/3 activation is a key mechanism by which YGP prevents renal tubulointerstitial fibrosis TGF-β/Smad signaling is the typical pathway related with renal fibrosis, thus we determined YGP may exert efficacy through this pathway. As expected, the immunohistochemistry analysis indicated that UUO markedly induced the nuclear translocation of p-Smad2/3 (Po0.001), and treatment of YGP reduced the expression to 24.8% at 0.14 g/kg, 29.6% at 0.42 g/kg and 51.0% at 1.26 g/kg, respectively (Fig. 7A and B). Western blot analysis confirmed that UUO-induced p-Smad2/3 was abolished upon administration of YGP. Meanwhile, the total expression of Smad2/3 was not affected by UUO injury and YGP treatment (Fig. 7C). Consistent with this observation, in the NRK-49F cells YGP treatment could inhibit TGF-β1-stimulated Smad2/3 phosphorylation level distinctly at 300 μg/ml and 1000 μg/ml (Po0.01)
Fig. 5. Treatment of rats with YGP decreases TGF-β1, fibronectin, α-SMA, collagen I and III protein expression in UUO kidneys. (A) Representative western blot photographs showing levels of TGF-β1, fibronectin, α-SMA, collagen I and III protein expression. (B)–(F) Quantitative analysis of TGF-β1, α-SMA, fibronectin, collagen I and III protein expression from sections similar to those shown in (A). Data represent mean 7SEM for at least 3 independent experiments. nnP o 0.01, nnnPo 0.001, compared with the sham group; #P o0.05, ##P o 0.01 compared with the UUO group.
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Fig. 6. YGP dose-dependently decreases TGF-β-induced α-SMA, fibronectin, collagen I and III expression in NRK-49F cells. (A) and (B) MTT and LDH release assays showed the dose-dependent effects of YGP on cytotoxicity of NRK-49F cells. (C) Representative western blot photographs showing levels of α-SMA, fibronectin, collagen I and III protein expressions in TGF-β1 (1 ng/ml)-treated cells. (D)–(G) Summary histograms showing the ratio of α-SMA, fibronectin, collagen I and III expressions relative to GAPDH from experiments similar to those shown in (c). Data represent mean 7 SEM for at least 3 independent experiments. nnPo 0.01, nnnPo 0.001, compared with non-treated control; # P o 0.05, ##Po 0.01, ###Po 0.001 compared with TGF-β1 (1 ng/ml)-treated cells.
(Fig. 8A and B). In addition, we also found that TGF-β1 induced a down-regulation of Smad7 protein expression (Po0.05), and which was restored dose-dependently by treatment of YGP, especially at 1000 μg/ml (Po0.05). However, the change of Smad7 is milder than that of p-Smad2/3, so we considered that blockade of TGF-β/Smad signaling via suppressing the expression level of p-Smad2/3 is the most possible mechanism for YGP.
4. Discussion In the present study, we first investigated the effect of YGP on UUO-induced TIF. The result showed that YGP exerts marked
activities by improving tubulointerstitial injury and renal function. As the tubulointerstitium comprises about 90% of the volume of the kidney, it is reasonable accepted the correlation between TIF and the deterioration of renal function (Bohle et al., 1987). So treatment with YGW may be beneficial to suppress TIF. Inflammatory cell recruitment and accumulation of ECM proteins in the tubulointerstitium are one of the most typical characteristics in the progression of renal disease. Coincide with renal blood flow and glomerular filtration rate decline, these processes lead to renal fibrosis and ultimately contribute to kidney failure (Liu, 2011). From our result, histopathological scores, inflammatory cells count and renal interstitial expansion increased significantly in UUO kidneys, whereas YGW treatment decreased
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Fig. 7. Treatment of rats with YGP blocks activation of p-Smad2/3 in UUO kidneys. (A) Representative photomicrographs of p-Smad2/3 immunohistochemistry on kidney sections from the Sham (a), UUO (b), Enalapril (c), YGP (at 0.14, 0.42 and 1.26 g/kg; (d)–(f)) groups. (B) Nuclear p-Smad2/3 positive cells (%) analysis from sections similar to those shown in A. (C) Representative western blot photographs showing levels of p-Smad2/3 protein expressions in UUO kidneys. Data represent mean 7 SEM for at least 10 independent experiments. nnnPo 0.001, compared with the sham group; #P o0.05, ##P o 0.01, ###Po 0.001 compared with the UUO group. Magnification: 400.
the pathological changes, indicting the antifibrotic role of this TCM. The infiltrating inflammatory cell produces a variety of proinflammatory cytokines, including TGF-β. TGF-β is elevated in various experimental and human kidney diseases relating with glomerular and interstitial fibrosis, such as diabetic nephropathy (Masola et al., 2014; Zhou et al., 2014), rat models of obstructive kidney disease (Chevalier, 2006; Kumpers et al., 2007), renal ischemia-reperfusion injury and five–sixths nephrectomy (Tu et al., 2008; Wen et al., 2010). It has been widely accepted that TGF-β1, the major isoform of the TGFβ superfamily, is produced by fibroblasts under various pathophysiological conditions. And activation of TGF-β1 is responsible for cellular differentiation, proliferation and migration (Yang and Liu, 2001). Notably, the dynamics of ECM protein expression is tightly regulated by TGF-β1 (Liu, 2010; Zavadil and Bottinger, 2005). Consistent with these studies, our data showed that TGF-β1 protein is overexpressed in the UUO kidney. In contrast, the expression declined with the YGP treatment accompanied by the down-regulation of α-SMA, fibronectin and collagen matrix. Those findings suggest that YGP may act as an antifibrotic agent via interfering with TGF-β1 expression and activity. Although TGF-β action may involve multiple downstream signaling pathways and crosstalk, the intracellular Smad pathway is considered as the most important one which contributes to TGF-β-dependent structural manifestation of renal fibrotic responses (Fu et al., 2006; Kamato et al., 2013). Upon TGF-β stimulating, Smad 2 and Smad 3 undergo phosphorylation, heteroligomerizing with the common partner Smad 4 and those complexes are translocated into the nucleus to regulate target gene expression. The inhibitory Smads, such as Smad 7, can also be activated and then suppress Smad 2 and Smad 3 phosphorylation during the process (Kamato et al., 2013). TGF-β-
mediated renal fibrosis involved in Smads has been demonstrated previously, which is related to the TGF-β-induced ECM protein synthesis in mesangial cells, tubular epithelial cells and podocyte apoptosis during glomerulosclerosis (Li et al., 2002; Poncelet et al., 1999; Schiffer et al., 2001). A novel and significant finding in our study was the identification that TGF-β/Smad signaling is involved in YGP efficacy. The present study showed that the nuclear localization of p-Smad2/3 was evident. And p-Smad2/3 protein expression increased in both UUO rats and TGF-β1-stimualted NRK-49F cell line. These observations are consistent with the recent finding that activation of Smad protein contributes to accelerate TGF-β1-induced fibrosis (Jin Lim et al., 2014). Moreover, Smad2 and Smad3 are different functionally in profibrogenesis. Smad2 plays a crucial in TGF-β1-induced matrix metalloproteinase 2 (MMP-2) syntheses, but Smad3 is involved in stimulating Smad7 gene promoter (Piek et al., 2001; von Gersdorff et al., 2000). A recent report showed that Smad3 thr388 phosphorylation can regulate renal fibrosis dramatically, in which TGF-β1 induced phosphorylation of Smad3 T388 in a biphasic pattern. Furthermore, T388 phosphorylation was required for TGF-β-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts (Qu et al., 2014). Conversely, a previous study reported that Smad2 may exhibit function in suppressing Smad3-mediated ECM production and fibrosis by competitively inhibits Smad3 phosphorylation, nuclear translocation and transcriptional activity (Meng et al., 2010). However, our data not shown revealed that the phosphorylated Smad2 and Smad3 expressions are both down-regulated with YGP treatment, respectively, just as our another work about the two Smads expression (Wang et al., 2014b). Thus, Smad2 and Smad3, at least in part, are targets of YGP in the anti-fibrosis process.
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in vitro. And the mechanism is closely related to inhibit TGF-β/Smad signaling, which further underscoring the potential clinical benefits of YGP in the treatment of renal fibrosis. Author contributions XMZ and WP participated in the design and coordination, and evaluated the statistical analysis and critically revised the manuscript and guaranteed of integrity of the entire study. LW, ALC, YFC and ZCJ carried out the experiment. PHY performed statistical analysis and edited the manuscript. LW involved in designing the study and drafting the manuscript. All authors read and approved the final manuscript.
Conflicts of interest No.
Acknowledgments This work was supported by the National Natural Science Foundation of China (81403235); General Medicine of Key Discipline Construction Project, State Administration of Traditional Chinese Medicine of the People's Republic of China; Leading Academic Discipline Project of State Administration of Traditional Chinese Medicine of China, Talent Project of Integrative Medicine of Shanghai Municipal Health Bureau (ZYSNXD012-RC-ZXY); Key Medical Discipline Project of Shanghai Municipal Health Bureau (ZK2012A34) and Putuo Hospital Fund (2013SR123I, 2013GQ007I). Fig. 8. Treatment with YGP blocks expression of p-Smad2/3 in NRK-49F cells. (A) Representative western blot photographs showing levels of p-Smad2/3, Smad2/3, Smad4 and Smad7 protein expressions in TGF-β1-treated cells. (B) and (C) Quantitative analysis of p-Smad2/3 and Smad7 protein expression from sections similar to those shown in (A). Data represent mean7SEM for at least 3 independent experiments. *Po0.05, nnPo0.01, compared with non-treated control; #Po0.05, ##Po0.01 compared with TGF-β1 (1 ng/ml)-treated cells.
YGW has long been used for the treatment of “kidney-Yang” deficiency syndrome. Accumulating evidences from the ethnopharmacological aspect indicate that the herbs and their chemical constituents are beneficial for the recovery of renal function, including the pathological regression of renal fibrosis. Morroniside and loganin isolated from Fructus Corni have been used as renoprotective agents in type 2 diabetes (Xu et al., 2006; Yamabe et al., 2010; Yokozawa et al., 2010). And gallic acid also from Fructus Corni can exert activities in the long term treatment of chronic kidney disease (Peng et al., 2012). Cuscuta chinensis Lam. and its extracts, such as rutin, quercerin and cinnamic acid have been reported to attenuate renal damage induced by ischemia/reperfusion injury, experimental diabetic nephropathy and renal fibrosis in rats with UUO (Donnapee et al., 2014; Hao et al., 2012; Korkmaz and Kolankaya, 2013; Yan et al., 2014). Therefore, it is reasonable to assume that the improving effect of YGW on the TIF in our research may be caused via a synergistic effect derived from these herbs. Thus, the complicated composition of YGP confers the further work on identifying the active ingredient which exerts the real efficacy. Moreover, a more intensive human clinical trial may be also required to testify the renoprotective efficacy of YGP in vivo.
5. Conclusion In summary, we demonstrate for the first time that YGP has a renoprotective effect in suppressing the fibrogenic process in vivo and
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
You-gui Pill ameliorates renal tubulointerstitial fibrosis via inhibition of TGF-β/Smad signaling pathway Li Wang a,1, Ai-li Cao a,1, Yang-Feng Chi b, Zheng-Cai Ju c, Pei-Hao Yin b, Xue-Mei Zhang d,n, Wen Peng a,b,nn a
Laboratory of Renal Disease, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China Department of Nephrology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China c Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China d Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 November 2014 Received in revised form 8 April 2015 Accepted 18 April 2015 Available online 25 April 2015
Ethnopharmacological relevance: You-gui Pill (YGP), a traditional Chinese medicinal prescription, was widely used to warm and recuperate “kidney-yang” clinically for hundreds of years in China. Recent studies found that YGP had a potential benefit for renoprotection. Aim of the study: The present study aimed to elucidate the in vivo and in vitro efficacy of YGP on renal tubulointerstitial fibrosis, and the molecular mechanism is also investigated. Materials and methods: Rat renal tubulointerstitial fibrosis model was elicited by unilateral ureteral obstruction (UUO). Sprague–Dawley rats underwent UUO and were studied after 14 days. Animals were randomly subjected to six groups: sham, UUO, UUO/YGP (0.14, 0.42, 1.26 g/kg/d), and UUO/enalapril (10 mg/kg/d). HE, Masson and ELISA were used for evaluate renal injury and function. Immunohistochemical analysis and western blot were used to detect the expressions of α-SMA, fibronectin, collagen matrix and Smads. In vitro studies were investigated in TGF-β1-stiumlated NRK-49F cell line. Results: Oral administration of YGP significantly decreased UUO-induced inflammatory cell infiltration, tubular atrophy and interstitial fibrosis, and there was no significant difference between YGP at 1.26 g/kg and enalapril at 10 mg/kg treatment (P4 0.05). Meanwhile, serum creatinine and blood urea nitrogen levels were reduced dramatically (Po 0.01). In coincide with the decreased of TGF-β1, α-SMA, fibronectin and collagen matrix expressions were also declined with YGP treatment in both UUO kidneys and TGFβ1-stimulated NRK-49F cell line. Additionally, nuclear translocation of p-Smad2/3 was markedly downregulated by YGP (P o0.001), with a relative mild up-regulated expression of Smad7 (P o0.05). Conclusions: Our findings demonstrate that YGP had a renoprotective effect in ameliorating renal tubulointerstitial fibrosis, and this activity possibly via suppression of the TGF-β and its downstream regulatory signaling pathway, including Smad2/3. & 2015 Published by Elsevier Ireland Ltd.
Chemical compounds studied in this article: Gallic acid (PubChem CID: 370) 5-HMF (PubChem CID: 237332) Morroniside (PubChem CID: 11228693) Sweroside (PubChem CID: 161036) Loganin (PubChem CID: 87691) Rutin (PubChem CID: 5280805) Cinnamic acid (PubChem CID: 444539) Quercerin (PubChem CID: 5280343) Kaempferide (PubChem CID: 5281666) Carboxymethyl cellulose (PubChem CID: 6328154) Keywords: You-gui Pill Renal tubulointerstitial fibrosis Unilateral ureteral obstruction TGF-β Smad2/3
1. Introduction Abbreviations: YGP, You-gui Pill; UUO, unilateral ureteral obstruction; TIF, tubulointerstitial fibrosis; ECM, extracellular matrix; α-SMA, α-smooth muscle actin; TCM, Traditional Chinese Medicine; 3D HPLC, three-dimensional high performance liquid chromatography; CMC, carboxymethyl cellulose; HRP, horseradish peroxidase; FBS, fetal bovine serum; LDH, lactate dehydrogenase; SDS-PAGE, SDS polyacrylamide gel electrophoresis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ANOVA, one-way analysis of variances; BUN, blood urea nitrogen; MMP-2, matrix metalloproteinase 2. n Corresponding author. Fax: þ 86 21 50980046. nn Corresponding author at: Department of Nephrology, Laboratory of Renal Disease, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, 164 LanXi Road, Shanghai 200062, China. Fax: þ 86 21 52665957. E-mail addresses: [email protected] (X.-M. Zhang), [email protected] (W. Peng). 1 Li Wang and Ai-li Cao contribute equally to this work. http://dx.doi.org/10.1016/j.jep.2015.04.037 0378-8741/& 2015 Published by Elsevier Ireland Ltd.
Renal tubulointerstitial fibrosis (TIF) is the final common pathway leading to end-stage renal failure irrespective of the initiating pathology (Liu, 2011). Predominating over glomerular and renal vascular disease, the degree of TIF is closely related with a loss of renal function (Bohle et al., 1987). Progression of TIF is a dynamic process, manifested as tubular loss and accumulation of extracellular matrix (ECM) proteins, including fibronectin and collagens (Wang et al., 2014b). Persistent of ECM production can be accelerated with severe renal injury, which results in large amounts of fibrinous tissue is generated and thus a vicious circle is formed progressively (Ishibe and Cantley, 2008). Therefore, it is essential to identify appropriate pharmacologic interventions to prevent TIF, especially to improve recovery of ECM following renal injury.
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Several cellular pathways, including TGF-β/Smad, Wnt/β-catenin and Integrin/ILK have been identified as major avenues for the generation of ECM, in which TGF-β is recognized as the most important profibrogenic cytokine in the kidney (Liu, 2010; Wang et al., 2014a, 2014b). A typical role of TGF-β is its biologic effects can exert through the Smad protein signaling pathways. Thus, inhibiting the TGF-β/Smad signaling pathway is helpful for preventing TIF and preserving renal function. In recent years, clinical and experimental studies have shown that Traditional Chinese Medicine (TCM) is beneficial for both the prevention and treatment of TIF. According to TCM theories, deficiency of “kidney-yang” is one of the key factors that is responsible for TIF. Therefore, TCM physicians take warm and recuperate “kidney-yang” as the main consideration in clinical practice. You-gui Pill (YGP) is a well-known prescription of TCM and originally recorded in “Jing-Yue Complete Works” at Ming dynasty. As a yang-tonic prescription clinically, previous studies have reported that YGP possesses a variety of pharmacological actions, such as in enhancing immune system, relieving states of asthma, ameliorating cerebral limbic system and improving body function in stress condition (Dai et al., 2006; Lin et al., 2012; Ou et al., 2003; Yao et al., 2005). Notably, evidence showed that YGP is a potential renal protective agent (Yang et al., 2009). However, the regulatory mechanism of YGP on renal disease remains largely unknown. Therefore, in the present study, we examined the efficacy of YGP on TIF in animal models of obstructive nephropathy and in TGFβ1-stiumlated NRK-49F cell. Furthermore, the underlying mechanism was also explored.
2. Materials and methods 2.1. Preparation of extract of You-gui Pill (YGP) YGP comprises 10 kinds of herbs, namely Radix Rehmanniae Praeparata, Rhizoma Dioscoreae, Fructus Corni, Fructus Lycii, Semen Cuscutae, Cervi cornus colla, Eucommiae cortex, Cinnamomi cortex, Radix Angeliccae Sinensis and Radix Aconiti Lateralis Prreparata (See also in Table 1). All TCM herbs were supplied by Leiyunshang Pharmaceutical Co., Ltd. (Shanghai, China) and were authenticated by Dr. Bo Cui (college of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine). The dried plant samples with voucher number 2013005 were deposited in the herbarium of Laboratory of Renal Disease, Putuo Hospital, Shanghai University of Traditional Chinese Medicine. YGP extract was prepared as described previously (Wu et al., 2010). In brief, in total of 5.7 kg of dry TCM herb was powdered and suspended in 4 volumes of water. The suspension was boiled for 2 h and filtered. Then the residual herbs were subjected to the same decoction suspension and filtration procedure. The filtrate was mixed and extracted with 4 volumes of 95% ethanol (vol/wt) for 12 h for the third time. In the end, the extracts were concentrated at 65 1C with a vacuum rotary evaporator. A total amount of 1.0 g of extract powder
that was about 18.9% of the raw herb was obtained and the dried extract was stored at 20 1C. 2.2. HPLC analysis A three-dimensional high performance liquid chromatography (3D-HPLC) profile of YGP extract is shown in Fig. 1. The analysis procedure was conducted according to the previous reports (Liang et al., 2009; Lin et al., 2012). Briefly, YGP extract (2.0 g) was dissolved in 10 ml methanol under ultrasonication and centrifuged for 5 min at 9000 g, and then the supernatant was transferred into 10 ml volumetric flask and made up to volume with methanol. To acquire chromatograms and UV spectra, HPLC apparatus consisting of a CCPMII multisolvent delivery pump and a PD-8020 photodiode array detector was used (SD-8020 system, Tosoh Corp., Tokyo, Japan). The separation was performed on an YMC Carotenoid column (3.0 μm, 150 mm 4.6 mm I.D., Tokyo, Japan). The mobile phase consisted of acetonitrile (A) and 0.1% (v) formic acid (B). The gradient elution was programmed as follows: 0–30 min, 1–12% A; 30–32 min, 12–15% A; 32–55 min, 15–25% A; 55–60 min, 25–40% A; 60–70 min, 40–100% A at a flow rate of 1.0 ml/min. A pre-equilibration period of 15 min was set up between individual runs. The other analysis conditions were as follows: column temperature, 35 1C; injection volume, 10 μl; UV scan 254 nm. 2.3. Drugs and reagents Pulverized YGP extract was prepared as a suspension in 0.5% carboxymethyl cellulose (CMC) with needed concentration for animal oral administration or was dissolved in complete RPMI 1640 (Gibco, USA) at a concentration of 10,000 μg/ml for subsequent cellular usage. The latter was filtered through a 0.22 μm membrane pore (Millipore, Billerica, MA, USA), then was stored at 4 1C before use. Antibodies to TGF-β1, collagen I, collagen III, Smad4 and Smad7 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fibronectin, phosphorylated-Smad2/3 (p-Smad2/3), Smad2/3, GAPDH and horseradish peroxidase (HRP)-labeled goat anti-rabbit or -mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). α-SMA was purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant TGF-β1 was purchased from Peprotech (Hamburg, Germany). BCA protein assay kit was purchased from Pierce (Rockford, IL, USA). Rat renal fibroblasts (NRK-49F) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). 2.4. Animal and experimental protocols All experimental procedures were conducted in conformity with the ethics committee of Putuo Hospital, Shanghai University of Traditional Chinese Medicine and in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was performed according to previously described (Wang
Table 1 Consist of You-gui Pill (YGP). Chinese name
Botanical name
Common name
Weight (g)
Part used
Voucher numbers
Shu Di Huang Shan Yao Shan Zhu Yu Qou Qi Zi Tu Si Zi Lu Jiao Jiao Du Zhong Rou Gui Dang Gui Fu Zi
Rehmannia glutinosa (Gaertn).) DC. Dioscorea oppositifolia L. Cornus officinalis Sieb. Et Zucc. Lycium barbarum L. Cuscuta chinensis Lam. Cervus elaphus Linnaeus Eucommia ulmoides Oliv. Cinnamomum cassia (L.) J.PreslBark Angelica sinensis (Oliv.) Diels Aconitum carmichaeli Debx.
Radix Rehmanniae Praeparata Rhizoma Dioscoreae Fructus Corni Fructus Lycii Semen Cuscutae Cervi cornus colla Eucommiae cortex Cinnamomi cortex Radix Angeliccae Sinensis Radix Aconiti Lateralis Prreparata
8 4 3 4 4 4 4 2 3 2
Root Rootstock Fruit Fruit Fruit Horn Bark Bark Root Root
2012091711 2012081001 2012082006 2012091002 2012092101 20110902 2012042706 2012032302 2012091704 2011120426
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Fig. 1. 3D-HPLC profile of YGP. The analysis based on ultraviolet (UV) absorption clearly showed the presence of the following major constituents in YGP extract: Gallic acid, 5-HMF, Morroniside, Sweroside, Loganin, Rutin, Cinnamic acid, Quercerin and Kaempferide.
et al., 2014b). The selective doses of YGP for animal study were calculated based on the human optimal equivalent dose of 27 g raw herbs. The animals were divided randomly into six groups as follows: (1) sham group, (2) UUO group, (3) UUOþenalapril group (10 mg/kg/d, diluted in 0.5% CMC) as a positive control, and (4) UUOþYGP groups at doses of 0.14, 0.42, 1.26 g/kg/d (diluted in 0.5% CMC). All rats except for the sham-operated rats underwent UUO. At 14 days of post-surgery, the animals were sacrificed and the left kidneys were carefully removed. The kidneys were sagittally sliced into two parts, one part was snap frozen in liquid nitrogen and kept at 80 1C for protein extraction, and another part was immersed into 10% neutral-buffered formalin for histopathological and immunohistochemical examinations. 2.5. Renal function test The blood samples were taken from the abdominal aorta for determination of serum creatinine and blood urea nitrogen (BUN) at 14 days after UUO operation. Creatinine colorimetric assay kit (BioVision, USA) and BUN colorimetric detection kit (Arbor, USA) were used for serum creatinine and BUN test according to the manufacturer's protocol, respectively. 2.6. Cell culture and treatment The NRK-49F cells were cultured in complete RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco,
USA), 100 U/ml penicillin and 100 mg/ml streptomycin in an atmosphere of 5% carbon dioxide and 95% air at 37 1C. MTT and lactate dehydrogenase (LDH) assay were performed as previously described (Wang et al., 2014b). Briefly, cells were cultured at a density of 4 104 cells/ml in 100 mL 1640 containing 1.0% FBS in 96-well microplates. Then YGP at concentrations of 1, 3, 10, 30, 100, 300, 1000, 3000, 10,000 μg/ml was added individually to the medium. After 24 h of incubation, the supernatants were collected and centrifuged at 250 g for 10 min, and then the extents of cytotoxicity of YGP were measured by MTT assay kit (Sigma-Aldrich, St. Louis, MO, USA) and LDH release kit (BioVision, USA) using a microplate reader (Bio-Rad Laboratories, CA, USA). All measurements were performed in duplicate. To evaluate the inhibitory effect of YGP on TGF-β1-induced profibrogenic responses, the NRK-49F cells were pre-treated with YGP at concentrations of 100, 300 and 1000 μg/ml for 30 min, and then the TGF-β1 at 1.0 ng/ml was added. After treatment with YGP for 24 h, protein expression was measured. 2.7. Histology and immunohistochemistry For histological analysis, paraffin sections (3 μm thick) were performed with hematoxylin–eosin and Masson's modified trichrome-staining. Tubulointerstitial injury score, inflammatory cell count and collagen deposition area (%) were graded by the published criteria (Wang et al., 2014b). For immunohistochemistry analysis, antibodies to α-SMA (1:500 dilution) and p-Smad2/3 (1:250 dilution) were used. Color development procedure was conducted according to the instruction of a
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Vecta-Elite streptavidin-peroxidase kit (Vector Laboratories, Burlingame, CA, USA). The stained sections were developed with diaminobenzidine to produce brown products and counterstained with hematoxylin. Image Pro Plus 6.0 Multimedia Color Pathological image analysis software (Media Cybernetics, Inc. Silver Spring, MD, USA) was used for the semi-quantitative analysis. And at least five non-overlapping high-power fields at a magnification of 200 per section were analyzed for each animal. The mean optical density value of α-SMA was calculated and the percentage of positive cell units of p-Smad2/3 in the nucleus was calculated.
(1:1000 dilution), Smad2/3 (1:1000 dilution), Smad4 (1:500 dilution), Smad7 (1:500 dilution) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000 dilution). After three washes in TBS-Tween-20, the membranes were incubated with HRP-labeled goat anti-rabbit or -mouse IgG for 1 h at room temperature. Signals were scanned and visualized by enhanced ECL (Millipore, USA) and X-ray film. The ratio of the protein interested was subjected to GAPDH and was densitometrically analyzed by Multi-Analyst software program (Version 1.01; Bio-Rad). 2.9. Statistical analysis
2.8. Western blot analysis Proteins extracted from either renal tissues or cultured NRK-49F cells were analyzed by western blotting. The samples were lysed in a buffer (50 mM Tris at PH of 7.5, 1 mM EDTA, 150 mM NaCl, 20 mM NaF, 0.5% NP-40, 10% glycerol, 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail) by sonication. The supernatant was collected after centrifugation at 13,000 g at 4 1C for 20 min. Protein concentration was determined by BCA protein assay kit. Equal amounts of protein were separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore, USA). Following blocked with 5% BSA in TBS-Tween20 for 2 h, the membrane was incubated with primary antibodies at 4 1C overnight. Antibodies used in this study included: TGF-β1 (1:1000 dilution), α-SMA (1:800 dilution), fibronectin (1:10,000 dilution), collagen 1 (1:1000 dilution), collagen 3 (1:1000 dilution), p-Smad2/3
The data were presented as the mean7standard error. Statistical analysis was performed using GraphPad Prism software version 5.0 (GraphPad Prism software Inc., San Diego, Calif, USA). The differences among multiple groups were evaluated by one-way analysis of variances (ANOVA). The differences between two groups were determined by t test. Statistical significance was defined as Po0.05.
3. Results 3.1. Effect of YGP on renal histology and renal function in rat UUO model As shown in Fig. 2A, significant tubulointerstitial injury, including tubular atrophy, inflammatory cells infiltration and interstitial fibrosis appeared in UUO kidneys, whereas those morphological changes were
Fig. 2. Treatment of rats with YGP attenuates renal injury in a dose-dependent manner in UUO kidneys. (A) Representative hematoxylin–eosin (HE) stained kidney sections from Sham (a), UUO (b), Enalapril (c), YGP (0.14, 0.42 and 1.26 g/kg; (d)–(f)) groups. (B) Histopathological scores and inflammatory cells count within the tubulointerstitium from sections similar to those shown in (A). (C) Representative Masson's modified trichrome stained kidney sections from sham (a), UUO (b), Enalapril (c), YGP (0.14, 0.42 and 1.26 g/kg; (d)–(f)) groups. (D) Quantitative analysis of the extent of renal interstitial expansion (area%) from sections similar to those shown in (C). Data represent mean 7SEM for at least 10 independent experiments. nnnP o0.001, compared with sham group; #Po 0.05, ##Po 0.01, ###P o0.001 compared with UUO group. Magnification: 400.
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not presented in sham-operative kidneys. Meanwhile, compared with vehicle-treated UUO kidneys, semiquantitative histomorphometry analysis demonstrated that YGP treatment decreased the tubulointerstitial injury scores and inflammatory response dose-dependently in UUO kidneys (Po0.01 or 0.001) (Fig. 2B). Increase of collagen synthesis is another typical characteristic in UUO kidneys (Wang et al., 2014b). Compared with sham-operative kidneys, collagen expression is prominent in the interstitial and tubular areas of UUO kidneys. In contrast, the appearance was significantly improved in the YGP-treated rat kidneys (Fig. 2C). This result was also confirmed by semiquantitative assessment of the fibrotic area on the Masson's trichrome-stained kidney sections (Fig. 2D). Renal function was assessed by serum creatinine and the BUN analysis (Fig. 3). In UUO kidneys, serum creatinine and BUN rose more than 2.2-fold at 14 days compared with sham-operative kidneys (Po0.001). However, the levels showed a significant, dosedependent reduction with treatment of YGP, even at 0.14 g/kg (Po0.01 and 0.05, respectively). It is worth noting that treatment with YGP at 0.14 g/kg caused a marked improvements on pathological and functional injuries compared to rat in UUO group (Po0.05 or 0.01), and there was no significant difference between YGP at 1.26 g/kg and enalapril at 10 mg/kg treatment (P40.05), indicating that YGP treatment had almost the same efficacy to that of enalapril.
Fig. 3. Treatment of rats with YGP improves renal function in a dose-dependent manner in UUO kidneys. Serum creatinine (A) and BUN (B) in the Sham, UUO, Enalapril and YGP (0.14, 0.42 and 1.26 g/kg) groups. Data represent mean 7SEM for at least 10 independent experiments. nnnP o 0.001, compared with the sham group; # P o 0.05, ##Po 0.01, ###Po 0.001 compared with the UUO group.
3.2. Effect of YGP on expression of α-SMA and ECM accumulation in rat UUO model and cultured NRK-49F cells Activation of myofibroblast cells, as manifested by α-smooth muscle actin (α-SMA) induction, is one of the representative events
Fig. 4. Treatment of rats with YGP decreases α-SMA expression by immunohistochemistry in UUO kidneys. (A) Representative photomicrographs of α-SMA immunohistochemistry on kidney sections from the Sham (a), UUO (b), Enalapril (c), YGP (0.14, 0.42 and 1.26 g/kg; (d)–(f)) groups. (B) Quantitative analysis of α-SMA expression from sections similar to those shown in (A). Data represent mean 7 SEM for at least 10 independent experiments. nnnPo 0.001, compared with the sham group; ## Po 0.01, ###P o0.001 compared with the UUO group. Magnification: 400.
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in ECM overexpression of fibrotic kidney (Li et al., 2002; Liu, 2011). As shown in Fig. 4, α-SMA is mainly expressed in interstitial cells of UUO kidneys. And immunohistochemistry and the western blot analysis showed that α-SMA was barely detectable in sham-operative kidneys, compared with the dramatic expression in UUO kidneys (Po0.001). However, this increase of α-SMA expression was significantly attenuated by YGP treatment, especially at the doses of 0.42 g/kg (Po0.01) and 1.26 g/kg (Po0.001). Moreover, as the pro-fibrotic protein expression and accumulation of ECM components are both important to TIF formation, here we further observed those changes. As shown in Fig. 5, the level of TGF-β1 increased dramatically in the UUO kidneys compared with those in sham-operative kidneys (Po0.01), but decreased with YGP treatment at 0.42 g/kg (Po0.05) and 1.26 g/kg (Po0.01). Same tendency was observed in fibronectin, collagen I and III expression. And our immunohistochemistry analysis revealed that the expression levels of fibronectin, collagen I and III were significantly increased in the UUO kidneys compared with sham-operative kidneys (Po0.001), and treatment with YGP at a dose of 1.26 g/kg decreased the integrated optical density of the three proteins by 47.570.3%, 60.970.2% and 55.270.1%, respectively (Po0.001, data not shown). Our previous study has revealed that TGF-β1 at 1.0 ng/ml could dramatically stimulate fibrogensis in NRK-49F (Wang et al., 2014b). Here, to clarify cytotoxicity of YGP on the NRK-49F cells for in vitro
experiment, MTT and LDH arrays were first performed (Fig. 6A and B). We found that YGP at and below 1000 μg/ml is safety to cell growth. Subsequently, the inhibitory effect of YGP on TGF-β1induced overexpression of α-SMA and ECM proteins in the NRK49F cells was determined. Similar to the in vivo study, YGP could also dose-dependently attenuate α-SMA, fibronectin, collagen I and III protein expressions in the TGF-β1-stimulated NRK-49F cells (Fig. 6C–G). 3.3. Blockade of p-Smad2/3 activation is a key mechanism by which YGP prevents renal tubulointerstitial fibrosis TGF-β/Smad signaling is the typical pathway related with renal fibrosis, thus we determined YGP may exert efficacy through this pathway. As expected, the immunohistochemistry analysis indicated that UUO markedly induced the nuclear translocation of p-Smad2/3 (Po0.001), and treatment of YGP reduced the expression to 24.8% at 0.14 g/kg, 29.6% at 0.42 g/kg and 51.0% at 1.26 g/kg, respectively (Fig. 7A and B). Western blot analysis confirmed that UUO-induced p-Smad2/3 was abolished upon administration of YGP. Meanwhile, the total expression of Smad2/3 was not affected by UUO injury and YGP treatment (Fig. 7C). Consistent with this observation, in the NRK-49F cells YGP treatment could inhibit TGF-β1-stimulated Smad2/3 phosphorylation level distinctly at 300 μg/ml and 1000 μg/ml (Po0.01)
Fig. 5. Treatment of rats with YGP decreases TGF-β1, fibronectin, α-SMA, collagen I and III protein expression in UUO kidneys. (A) Representative western blot photographs showing levels of TGF-β1, fibronectin, α-SMA, collagen I and III protein expression. (B)–(F) Quantitative analysis of TGF-β1, α-SMA, fibronectin, collagen I and III protein expression from sections similar to those shown in (A). Data represent mean 7SEM for at least 3 independent experiments. nnP o 0.01, nnnPo 0.001, compared with the sham group; #P o0.05, ##P o 0.01 compared with the UUO group.
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Fig. 6. YGP dose-dependently decreases TGF-β-induced α-SMA, fibronectin, collagen I and III expression in NRK-49F cells. (A) and (B) MTT and LDH release assays showed the dose-dependent effects of YGP on cytotoxicity of NRK-49F cells. (C) Representative western blot photographs showing levels of α-SMA, fibronectin, collagen I and III protein expressions in TGF-β1 (1 ng/ml)-treated cells. (D)–(G) Summary histograms showing the ratio of α-SMA, fibronectin, collagen I and III expressions relative to GAPDH from experiments similar to those shown in (c). Data represent mean 7 SEM for at least 3 independent experiments. nnPo 0.01, nnnPo 0.001, compared with non-treated control; # P o 0.05, ##Po 0.01, ###Po 0.001 compared with TGF-β1 (1 ng/ml)-treated cells.
(Fig. 8A and B). In addition, we also found that TGF-β1 induced a down-regulation of Smad7 protein expression (Po0.05), and which was restored dose-dependently by treatment of YGP, especially at 1000 μg/ml (Po0.05). However, the change of Smad7 is milder than that of p-Smad2/3, so we considered that blockade of TGF-β/Smad signaling via suppressing the expression level of p-Smad2/3 is the most possible mechanism for YGP.
4. Discussion In the present study, we first investigated the effect of YGP on UUO-induced TIF. The result showed that YGP exerts marked
activities by improving tubulointerstitial injury and renal function. As the tubulointerstitium comprises about 90% of the volume of the kidney, it is reasonable accepted the correlation between TIF and the deterioration of renal function (Bohle et al., 1987). So treatment with YGW may be beneficial to suppress TIF. Inflammatory cell recruitment and accumulation of ECM proteins in the tubulointerstitium are one of the most typical characteristics in the progression of renal disease. Coincide with renal blood flow and glomerular filtration rate decline, these processes lead to renal fibrosis and ultimately contribute to kidney failure (Liu, 2011). From our result, histopathological scores, inflammatory cells count and renal interstitial expansion increased significantly in UUO kidneys, whereas YGW treatment decreased
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Fig. 7. Treatment of rats with YGP blocks activation of p-Smad2/3 in UUO kidneys. (A) Representative photomicrographs of p-Smad2/3 immunohistochemistry on kidney sections from the Sham (a), UUO (b), Enalapril (c), YGP (at 0.14, 0.42 and 1.26 g/kg; (d)–(f)) groups. (B) Nuclear p-Smad2/3 positive cells (%) analysis from sections similar to those shown in A. (C) Representative western blot photographs showing levels of p-Smad2/3 protein expressions in UUO kidneys. Data represent mean 7 SEM for at least 10 independent experiments. nnnPo 0.001, compared with the sham group; #P o0.05, ##P o 0.01, ###Po 0.001 compared with the UUO group. Magnification: 400.
the pathological changes, indicting the antifibrotic role of this TCM. The infiltrating inflammatory cell produces a variety of proinflammatory cytokines, including TGF-β. TGF-β is elevated in various experimental and human kidney diseases relating with glomerular and interstitial fibrosis, such as diabetic nephropathy (Masola et al., 2014; Zhou et al., 2014), rat models of obstructive kidney disease (Chevalier, 2006; Kumpers et al., 2007), renal ischemia-reperfusion injury and five–sixths nephrectomy (Tu et al., 2008; Wen et al., 2010). It has been widely accepted that TGF-β1, the major isoform of the TGFβ superfamily, is produced by fibroblasts under various pathophysiological conditions. And activation of TGF-β1 is responsible for cellular differentiation, proliferation and migration (Yang and Liu, 2001). Notably, the dynamics of ECM protein expression is tightly regulated by TGF-β1 (Liu, 2010; Zavadil and Bottinger, 2005). Consistent with these studies, our data showed that TGF-β1 protein is overexpressed in the UUO kidney. In contrast, the expression declined with the YGP treatment accompanied by the down-regulation of α-SMA, fibronectin and collagen matrix. Those findings suggest that YGP may act as an antifibrotic agent via interfering with TGF-β1 expression and activity. Although TGF-β action may involve multiple downstream signaling pathways and crosstalk, the intracellular Smad pathway is considered as the most important one which contributes to TGF-β-dependent structural manifestation of renal fibrotic responses (Fu et al., 2006; Kamato et al., 2013). Upon TGF-β stimulating, Smad 2 and Smad 3 undergo phosphorylation, heteroligomerizing with the common partner Smad 4 and those complexes are translocated into the nucleus to regulate target gene expression. The inhibitory Smads, such as Smad 7, can also be activated and then suppress Smad 2 and Smad 3 phosphorylation during the process (Kamato et al., 2013). TGF-β-
mediated renal fibrosis involved in Smads has been demonstrated previously, which is related to the TGF-β-induced ECM protein synthesis in mesangial cells, tubular epithelial cells and podocyte apoptosis during glomerulosclerosis (Li et al., 2002; Poncelet et al., 1999; Schiffer et al., 2001). A novel and significant finding in our study was the identification that TGF-β/Smad signaling is involved in YGP efficacy. The present study showed that the nuclear localization of p-Smad2/3 was evident. And p-Smad2/3 protein expression increased in both UUO rats and TGF-β1-stimualted NRK-49F cell line. These observations are consistent with the recent finding that activation of Smad protein contributes to accelerate TGF-β1-induced fibrosis (Jin Lim et al., 2014). Moreover, Smad2 and Smad3 are different functionally in profibrogenesis. Smad2 plays a crucial in TGF-β1-induced matrix metalloproteinase 2 (MMP-2) syntheses, but Smad3 is involved in stimulating Smad7 gene promoter (Piek et al., 2001; von Gersdorff et al., 2000). A recent report showed that Smad3 thr388 phosphorylation can regulate renal fibrosis dramatically, in which TGF-β1 induced phosphorylation of Smad3 T388 in a biphasic pattern. Furthermore, T388 phosphorylation was required for TGF-β-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts (Qu et al., 2014). Conversely, a previous study reported that Smad2 may exhibit function in suppressing Smad3-mediated ECM production and fibrosis by competitively inhibits Smad3 phosphorylation, nuclear translocation and transcriptional activity (Meng et al., 2010). However, our data not shown revealed that the phosphorylated Smad2 and Smad3 expressions are both down-regulated with YGP treatment, respectively, just as our another work about the two Smads expression (Wang et al., 2014b). Thus, Smad2 and Smad3, at least in part, are targets of YGP in the anti-fibrosis process.
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in vitro. And the mechanism is closely related to inhibit TGF-β/Smad signaling, which further underscoring the potential clinical benefits of YGP in the treatment of renal fibrosis. Author contributions XMZ and WP participated in the design and coordination, and evaluated the statistical analysis and critically revised the manuscript and guaranteed of integrity of the entire study. LW, ALC, YFC and ZCJ carried out the experiment. PHY performed statistical analysis and edited the manuscript. LW involved in designing the study and drafting the manuscript. All authors read and approved the final manuscript.
Conflicts of interest No.
Acknowledgments This work was supported by the National Natural Science Foundation of China (81403235); General Medicine of Key Discipline Construction Project, State Administration of Traditional Chinese Medicine of the People's Republic of China; Leading Academic Discipline Project of State Administration of Traditional Chinese Medicine of China, Talent Project of Integrative Medicine of Shanghai Municipal Health Bureau (ZYSNXD012-RC-ZXY); Key Medical Discipline Project of Shanghai Municipal Health Bureau (ZK2012A34) and Putuo Hospital Fund (2013SR123I, 2013GQ007I). Fig. 8. Treatment with YGP blocks expression of p-Smad2/3 in NRK-49F cells. (A) Representative western blot photographs showing levels of p-Smad2/3, Smad2/3, Smad4 and Smad7 protein expressions in TGF-β1-treated cells. (B) and (C) Quantitative analysis of p-Smad2/3 and Smad7 protein expression from sections similar to those shown in (A). Data represent mean7SEM for at least 3 independent experiments. *Po0.05, nnPo0.01, compared with non-treated control; #Po0.05, ##Po0.01 compared with TGF-β1 (1 ng/ml)-treated cells.
YGW has long been used for the treatment of “kidney-Yang” deficiency syndrome. Accumulating evidences from the ethnopharmacological aspect indicate that the herbs and their chemical constituents are beneficial for the recovery of renal function, including the pathological regression of renal fibrosis. Morroniside and loganin isolated from Fructus Corni have been used as renoprotective agents in type 2 diabetes (Xu et al., 2006; Yamabe et al., 2010; Yokozawa et al., 2010). And gallic acid also from Fructus Corni can exert activities in the long term treatment of chronic kidney disease (Peng et al., 2012). Cuscuta chinensis Lam. and its extracts, such as rutin, quercerin and cinnamic acid have been reported to attenuate renal damage induced by ischemia/reperfusion injury, experimental diabetic nephropathy and renal fibrosis in rats with UUO (Donnapee et al., 2014; Hao et al., 2012; Korkmaz and Kolankaya, 2013; Yan et al., 2014). Therefore, it is reasonable to assume that the improving effect of YGW on the TIF in our research may be caused via a synergistic effect derived from these herbs. Thus, the complicated composition of YGP confers the further work on identifying the active ingredient which exerts the real efficacy. Moreover, a more intensive human clinical trial may be also required to testify the renoprotective efficacy of YGP in vivo.
5. Conclusion In summary, we demonstrate for the first time that YGP has a renoprotective effect in suppressing the fibrogenic process in vivo and
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