Dissection of the mechanism of traditional Chinese medical prescription-Yiqihuoxue formula as an effective anti-fibrotic treatment for systemic sclerosis
- Ting Wu†1,
- Haiyan Chu†1,
- Wenzhen Tu†2, 6,
- Mengmeng Song3,
- Dongdong Chen2,
- Jin Yuan3,
- Ling Yu2,
- Yanyun Ma1,
- Qingmei Liu1,
- Li Jin1,
- Xiaodong Zhou4, 6,
- Hejian Zou3, 5, 6,
- Wenyu Wu3, 5, 6, 7Email author and
- Jiucun Wang1, 5, 6Email author
© Wu et al.; licensee BioMed Central Ltd. 2014
Received: 12 October 2013
Accepted: 30 June 2014
Published: 7 July 2014
Systemic sclerosis (SSc) is a connective tissue fibrotic disease for which there is no effective treatment. Traditional Chinese Medicine (TCM), such as the Yiqihuoxue formula used in Shanghai TCM-integrated Hospital, has shown the efficacy of anti-fibrosis in clinical applications. This study was aiming to dissect the anti-fibrotic mechanism of Yiqihuoxue treatment for SSc.
Bleomycin-induced mice and SSc dermal fibroblasts were treated with Yiqihuoxue decoction; NIH-3T3 fibroblasts were exposed to exogenous TGF-β1, and then cultured with or without Yiqihuoxue decoction. Luciferase reporter gene assay was used to determine the activity of Smad binding element (SBE). Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the mRNA levels of extracellular matrix (ECM) genes. The protein levels of type I collagen, Smad3 and phosphorylated-Smad3 (p-Smad3) were detected by western blotting. Student’s t-tests were used to determine the significance of the results.
Bleomycin-induced mice, SSc dermal fibroblasts and TGF-β1-induced NIH/3T3 fibroblasts showed higher levels of ECM gene transcriptions and collagen production. In addition, the phosphorylation level of Smad3 and activity of SBE were significantly increased after exogenous TGF-β1 induction. Whereas, Yiqihuoxue treatment could obviously attenuate fibrosis in bleomycin-induced mice, down regulate ECM gene expressions and collagen production in SSc dermal fibroblasts and TGF-β1-induced NIH/3T3 fibroblasts. Furthermore, the aberrantly high phosphorylation level of Smad3 and activity of SBE in the TGF-β1-induced NIH/3T3 fibroblasts were also dramatically decreased by Yiqihuoxue treatment.
Yiqihuoxue treatment could effectively reduce collagen production via down-regulating the phosphorylation of Smad3 and then the activity of SBE, which are involved in the TGF-β pathway and constitutively activated in the progression of SSc.
KeywordsSystemic sclerosis Traditional Chinese Medicine Fibrosis Collagen TGF-β
Systemic sclerosis (SSc) is a connective tissue disease mainly characterized by extensive fibrosis in skin and internal organs, such as lung, heart, esophagus and kidney, among which skin fibrosis is the universal manifestation in SSc [1, 2]. Previous reports have shown that SSc confers a mortality risk and fibrosis of internal organs is the leading cause of death in SSc patients [3, 4]. Until now, the precise mechanisms of skin fibrosis in SSc remain unclear, and there are no generally accepted and effective medical treatments for fibrosis.
Fibrosis is usually caused by the excessive production, deposition and contraction of extracellular matrix (ECM) components, especially collagen . Fibroblast is the principal cell type responsible for turnover and composition of ECM. The “activated” fibroblast and its contractile and secretory counterpart, myofibroblast, are the primary cell types responsible for the persistent production and deposition of ECM. Tissue injury initiates the chronic inflammation generally involving the activation of inflammatory and immune cells which secrete cytokines, chemokines and growth factors. Then resident fibroblasts (quiescent fibroblasts), pericytes, fibrocytes, epithelial and endothelial cells are recruited, activated and finally differentiated into myofibroblasts. The “activated” fibroblast is an intermediate stage between resident fibroblast and myofibroblast. In addition, the “activated” fibroblast and myofibroblast also produce cytokines and growth factors such as TGF-β and CTGF to support further fibrogenesis [6–8].
TGF-β is an important regulator in collagen production, and normal fibroblasts stimulated by TGF-β displayed features of SSc fibroblasts [9, 10]. Previous research suggested that the aberrant expression of TGF-β led to the activation of ECM synthesis and dysfunction of ECM degradation simultaneously , resulting in the unbalance between ECM production and degradation and thus fibrosis. Although the precise mechanism of fibrosis in SSc remains unknown, those results suggest a potential clue to uncover it. Latent TGF-β is cleaved to release active TGF-β, then TGF-β binds to the type II TGF-β receptor (TGFBRII) and activates the type I TGF-β receptor (TGFBRI). Activated TGFBRI transduces signals to the Smad proteins, that is, phosphorylates Smad3. The phosphorylation of Smad3 allows it to form a heterodimer with Smad4, which translocates into nucleus and interacts with Smad binding element (SBE) in the promoter origin of the target genes, resulting in the induction of gene expressions including ECM genes [12–14]. Smad3 seems to be a key signal transducer involved in the Smad-dependent TGF-β signaling pathway. Indeed, Smad3-deficient mice exhibited attenuated lung fibrosis induced by bleomycin , and overexpression of Smad3 significantly increased the activity of type I collagen promoter .
CTGF is a key effector in the downstream of TGF-β and plays a role in the regulation of fibroblast proliferation and migration as well as TGF-β-dependent ECM production . In addition, Ctgf siRNA also ameliorated fibrosis in skin and reduced inflammation in lungs of bleomycin-induced mice . SPARC, secreted protein, acidic and rich in cysteine, is one of the major components in ECM. Elevated expression of SPARC has been reported in numerous animal models of fibrotic diseases and human fibrotic tissues . Furthermore, our previous work and others’ also showed SPARC siRNA effectively reduced fibrosis in SSc dermal fibroblasts, TGF-β1-stimulated fibroblasts, skin and lung tissues from bleomycin-induced mice [18, 20, 21].
It is thorny and challenging to find an effective therapy for SSc, because of its complicated interaction of vascular, immunologic and fibrotic components. Traditional Chinese medicine (TCM) has a long history of dealing with diseases for several thousand years. More importantly, TCM with combinations of various components and multiple drug targets shows an overwhelming advantage of treating complicated diseases, such as acute promyelocytic leukemia (APL) . Currently, TCM has been applied to treat SSc patients in many hospitals in China. The prescription of Yiqihuoxue formula provided by the Shanghai TCM-integrated Hospital has been shown a satisfactory capability of attenuating SSc-associated fibrosis in clinical applications. The two main constituents of Yiqihuoxue formula are Astragalus membranaceus and Salvia miltiorrhiza, which have been proven effective in treating diseases. Compound Astragalus and Salvia miltiorrhiza extract could attenuate liver fibrosis induced by carbon tetrachloride via inhibiting Smad2 phosphorylation and α-SMA expression ; a standardized extract from Paeonia lactiflora and Astragalus membranaceus exerted an anti-fibrotic effect in rats induced by porcine serum via down-regulating PDGFR-β, inhibiting HSC proliferation and MAPK activation ; constituents of Radix Salviae Miltiorrhizae inhibited proliferation and procollagen synthesis in SSc dermal fibroblasts ; one of the main active components from Radix Salviae miltiorrihizae called Salvianolic Acid B could attenuate liver fibrosis by inhibiting Angiotensin II signaling . In order for the modernization and generalization of Yiqihuoxue formula in treating SSc, the precise anti-fibrotic mechanism remained to be addressed by using molecular approaches.
Aiming to explore the underlying mechanism of TCM in treatment of SSc-associated fibrosis, we used the treatment of Yiqihuoxue formula in vivo and in vitro in the present study.
Composition and preparation of Yiqihuoxue formula
Yiqihuoxue formula was obtained from the Shanghai TCM-integrated Hospital and the full components are Astragalus membranaceus, Salvia miltiorrhizae, Angelica sinensis, Caulis spatholobi, Semen persicae, Tuyuan, Agkistrodon piscivorus, Centella asiatica, Ganoderma lucidum, Herba epimedii, Poria peel and Radix glycyrrhizae, of which Astragalus membranaceus and Salvia miltiorrhiza are two major components. The Yiqihuoxue decoction was boiled with ultrapure water as the doctor’s directions. In in vivo study, 250 μl Yiqihuoxue decoction was fed to mice twice a day at the concentration of 23 g/d/kg. In in vitro study, it was filtered through a filter membrane in 0.22 μm and diluted to the concentration of 10 mg/ml in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) with 10% fetal calf serum (FCS, Invitrogen).
Skin biopsy specimens were obtained from three diffuse SSc patients, who were fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) criteria for SSc . Skin biopsy specimens were collected from three normal controls who had no history of autoimmune and other dermal diseases. All patients provided written contents, and the study was approved by the School of Life Sciences, Fudan University, China.
Skin samples were transported in DMEM supplemented with 10% FCS for processing the same day. The skin samples were washed in 75% ethanol, phosphate buffered saline (PBS), and DMEM with 10% FCS. Cultured fibroblast strains were established by mincing tissues and placing them into 60-mm culture dishes secured by glass coverslips. Third- to fifth-passage human dermal fibroblasts were placed into 12-well culture plates at the density of 1 × 105 cells per well for gene and protein expression assays.
Bleomycin-induced dermal fibrosis mouse model establishment
Specific pathogen-free (SPF) mice on a C57BL/6 background were purchased from Sino-British Sippr/BK Lab Animal Ltd. (Shanghai, China). Skin fibrosis was induced in 7-week-old female mice by administering local injections of bleomycin for 3 weeks. Briefly, bleomycin dissolved in filter-sterilized saline at a concentration of 200 μg/ml, and 100 μl of bleomycin was administered daily by subcutaneous injection into defined areas of 1 cm2 on the upper back. Subcutaneous injections of 100 μl saline were used as controls. After 3 weeks, the mice were killed by chloral hydrate anesthesia and skin tissues were harvested in order to analyze gene and protein expression levels. All the animal protocols were approved by the School of Life Sciences, Fudan University, China.
Drug administration regimens
Preparation and treatment of recombinant TGF-β1 and TCM
Recombinant TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA) and diluted to the concentration of 10 ng/μl in DMEM medium with 1% FCS. SSc dermal fibroblasts were placed into a 12-well culture plate at the density of 1 × 105 cells per well and cultured for 24 hours followed by treatment with or without Yiqihuoxue decoction (10 mg/ml). These cells were then examined 24 hours later for gene and protein expressions. NIH/3T3 fibroblasts were also seeded at the same density as SSc fibroblasts and cultured with or without TGF-β1 (10 ng/μl) for 24 hours after starved overnight. Then the culture medium was replaced by the normal medium with or without Yiqihuoxue decoction (10 mg/ml) and examined 24 hours later for gene and protein expressions.
Luciferase reporter gene assay
pGL3-SBE4-Luc was a gift from Dr. Kiyoshi Higashi (Sumitomo Chemical Co., Ltd., Osaka, Japan), which consisted of four short tandem repeats of Smad binding element (SBE) (GTCTAGAC) with a minimal promoter (TATA box).The plasmid of pRL-SV40 was used as an internal control. NIH/3T3 fibroblasts were placed into a 24-well culture plate, and then 500 ng of pGL3-SBE4-Luc or pGL3-Basic plasmid were co-transfected with 10 ng of pRL-SV40 per well using Lipofectamin 2000 (Invitrogen) according to the manufacturer’s protocol. After 6 hours’ transfection, the culture medium was replaced by TGF-β1 medium with or without Yiqihuoxue decoction. Cell lysates were harvested 24 hours later and luciferase activity assay was performed according to the manufacturer’s protocol of Dual-luciferase reporter assay system (Promega, Madison, WI, USA) with a GloMax 20/20 Luminometer (Promega).
Preparation and phosphorylation assay
Antibodies to Smad3 and p-Smad3 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). NIH/3T3 fibroblasts were placed into a 12-well culture plate at the density of 1 × 105 cells per well and grown until confluency. The culture medium was added by Yiqihuoxue decoction and cultured for 24 hours. After that, NIH/3T3 fibroblasts were treated with TGF-β1 for 1 hour, and cell lysates were harvested for western blotting.
Quantitative reverse transcription-polymerase chain reaction (RT-PCR)
RT-PCR was performed using an ABI Prism 7900 Detector Sysyem (Applied Biosystems, Foster City, CA, USA). The specific primers for each gene were designed using Primer 5 and synthesized by Generay Biotech Co., Ltd. (Shanghai, China). Total RNA samples were extracted from the cultured fibroblasts using TRIzol reagent (Invitrogen). Complementary DNA (cDNA) was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-PCR was performed using SYBR Premix ExTaq from TaKaRa Biotechnology Co., Ltd (Dalian, China). The data obtained from the assays were analyzed with SDS 2.3 software (Applied Biosystems). The amount of total RNA in each sample was normalized with GAPDH/Gapdh transcription levels.
Western blotting analysis
The protein concentration was determined using BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Equal amount of proteins from each sample was subject to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% polyacrylamide gel. Resolved proteins were transferred onto a polyvinylidenedifluoride (PVDF) membrane (Millipore, Billerica, MA, USA), and then blocked for 1 hour at room temperature using Tris Buffered Saline Tween (TBST) with 5% Bull Serum Albumin (BSA). After that, blotted proteins were incubated at 4°C overnight with a 1:1000 dilution of anti-mouse collagen antibody (Millipore), anti-Smad3 antibody (Cell Signaling Technology), and anti-p-Smad3 antibody (Cell Signaling Technology), as well as 1:500 dilution of anti-human collagen antibody (Millipore), respectively. Anti-GAPDH antibody (Cell Signaling Technology) was used as an internal control. After three washes with TBST for 30 minutes, the blotted proteins were incubated with the secondary antibody for 1 hour at room temperature, which was horse-radish peroxidase-conjugated anti-mouse, anti-rabbit or anti-goat IgG. Specific proteins were detected using an enhanced chemiluminescence system (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the intensity of bands was quantified using ImageQuantTL software (General Electric Company, Fairfield, CT, USA).
For the assessment of histopathological changes, skin tissues were embedded in paraffin after fixed with 4% paraformaldehyde. Then 4 um-thick skin sections were stained with hematoxylin/eosin or Masson’s trichrome staining for better visualization of the tissue structure. Dermal thickness was analyzed with a Nikon Eclipse 80i microscope (Nikon, Badhoevedorp, The Netherlands) by measuring the maximal distance between the epidermal–dermal junction and the dermal–subcutaneous fat junction at 4 different skin sections in each mouse, as previously described . Hypodermal thickness was determined by measuring the thickness of the subcutaneous connective tissue beneath the panniculus carnous at 4 different sites of the upper back in each mouse. The evaluation was performed by 2 independent examiners.
Yiqihuoxue treatment ameliorated skin fibrosis in bleomycin-induced mouse models
In this study, bleomycin-induced skin fibrosis mouse models were used to evaluate the anti-fibrotic effect of TCM treatment. Mice were randomly divided into two groups as described in Figure 1(1): Prevention (P) group and Prevention & Treatment (P & T) group. As measured by quantitative real-time RT-PCR, the transcripts of Col1a2, Col3a1, and Ctgf in both groups showed increased expressions in the skin tissues from bleomycin-induced mice, and Yiqihuoxue treatment recovered them nearly to the normal levels (Figure 1(2)). Moreover, the most significant effects of bleomycin and Yiqihuoxue treatment were detected in the P & T group, i.e., the fold changes of each gene in the skin tissues from bleomycin-induced mice were 5.3 ± 1.7-folds for Col1a2 (P = 0.016), 4.1 ± 0.4-folds for Col3a1 (P = 0.037), and 3.7 ± 1.5-folds for Ctgf (P = 0.007), respectively. After Yiqihuoxue treatment, significant reductions of Col1a2 (74.2% with P = 0.020), Col3a1 (67.1% with P = 0.031), and Ctgf (49.9% with P = 0.037) were found (Figure 1-2B). HE stain of skin tissues (Figure 1(3)) showed a significant dermal thickening after the induction of bleomycin in P & T group (P = 0.028), compared with the saline injection group. After Yiqihuoxue treatment, dermal thickening was notably improved (P = 0.024). Attenuation of dermal thickening also was found in P group after drug administration, though not very significant. Masson’s trichrome (Figure 1(3)) results were consistent with those of HE stain. Therefore, Yiqihuoxue treatment could significantly attenuate fibrosis and displayed an anti-fibrotic role in the treatment of SSc. More interestingly, Yiqihuoxue treatment not only exerted a powerful therapeutic action, but also presented a potential preventive effect in treating SSc.
Elevated expressions of ECM genes and excessive collagen production in cultured fibroblasts from the skin tissues of patients with SSc
Yiqihuoxue treatment reduced type I collagen production in the cultured fibroblasts from the skin tissues of patients with SSc
Yiqihuoxue treatment attenuated type I collagen production stimulated by exogenous TGF-β1 in the NIH/3T3 fibroblasts
Yiqihuoxue treatment attenuated type I collagen production via down-regulating the phosphorylation level of Smad3 and then the activity of SBE in the type I collagen promoter
SSc is a typical chronic fibrotic disease with multiple-system involvement, mainly including skin and various visceral organs such as lung, heart, esophagus and kidney. Its etiology remains to be unveiled so far. Currently, principal medical therapies for SSc are still restricted to certain immunomodulatory and anti-fibrotic agents, such as glucocorticoid, colchicine and cyclophosphamide, which are used to treat symptoms of SSc with limited therapeutic effects. What is worse, side effects of these medications undoubtedly add to the burden of patients’ lives in general. Applications of TCM to treat diseases have a long history in China. Based on compatible principles of Chinese Medicine and symptoms of patients, TCM formula is usually composed of several herbs or minerals, which can exert a comprehensive effect on the diseases. Although working slowly, it is mild and less harmful to the quality of patients’ lives. In China, TCM treatments have been applied to treat SSc patients by many hospitals, among which Yiqihuoxue formula is a good example and has been proven an anti-fibrotic efficacy in clinical applications. Therefore, it is imperative to gain a better understanding of the mechanism of TCM as an effective anti-fibrotic treatment for SSc.
In the progress of SSc, vascular dysfunction, autoimmunity and inflammation may jointly contribute to the fibrogenesis . Salvia miltiorrhiza is one of the main herbs in Yiqihuoxue formula, and its active component, salvianolic acid, has the function of removing blood stasis to improve vascular dysfunction [30, 31]. Several researches indicated that salvianolic acid B (SAB) could attenuate liver fibrosis via TGF-β-related signaling pathways [32, 33], and SAB addition inhibited Smad3 protein expression and its nuclear translocation in hepatic stellate cells . In this study, we also found that Yiqihuoxue treatment could significantly reduce the phosphorylation level of Smad3 and the activity of SBE (Figure 5), so that we assumed that Salvia miltiorrhiza in Yiqihuoxue formula exerts its anti-fibrotic effect via Smad-dependent TGF-β signaling pathway. In addition, Astragalus membranaceus is also one of the main components in Yiqihuoxue formula, and useful for immune enhancement and anti-inflammation [35, 36]. Astragalus membranaceus has been used in TCM for thousands of years. In modern Chinese Medicine, it has been used for cardiovascular and immunity system improvement [37, 38]. The related constituents from Astragalus membranaceus exhibited an anti-inflammatory effect by the inhibitory activity of NF-kB , whose inhibition could also down-regulate the expression of CTGF in SSc fibroblasts . Here, we proposed that Astragalus membranaceus attenuated fibrosis because of its immunomodulatory and anti-inflammatory properties. However, the precise mechanism of TCM treatment for vascular and immune system improvement remains unclear and deserves further efforts.
More interestingly, we also found that the absence of the two animal products in Yiqihuoxue formula, Tuyuan and Agkistrodon piscivorus, had no effects on decreasing SBE activity and Smad3 phosphorylation in the TGF-β1-treated NIH/3T3 fibroblasts (Additional file 1: Figure S1), indicating that these two animal components were not the necessary parts of the treatment formulation for systemic sclerosis, which meant that Yiqihuoxue formula had some redundancies. Thus, studies on the isolation of active ingredients and optimization of treatment formulation will greatly benefit the broader prospect for Yiqihuoxue formula and other traditional Chinese medical treatment.
Fibrosis is the main and most universal manifestation of SSc, which is caused by the unbalanced state between ECM production and degradation. TGF-β is considered as a potent profibrotic cytokine involved in the pathogenesis of SSc, because of its fibrotic functionality of stimulation of ECM synthesis and inhibition of metalloproteinase production . Type I and type III collagens are two major proteins in the ECM components. CTGF is an important downstream cytokine which is regulated by TGF-β and has a synergistic effect with TGF-β [41, 42]. In this study, overexpressions of both type I and type III collagen, and other ECM genes such as CTGF/Ctgf, SPARC/Sparc, TGF-β1/Tgf-β1 were observed in bleomycin-induced mice, SSc dermal fibroblasts as well as TGF-β1-induced cell model (Figures 1, 2, 4), however, Yiqihuoxue treatment could recover them to the normal levels (Figures 1, 3, 4). Notably, the effects of Yiqihuoxue formula on the bleomycin-treated mice were more significant in the Prevention & Treatment group compared with those in the Prevention group (Figure 1), both in the reduction of ECM genes (Col1a2/Col3a1/Ctgf) by TCM and histopathological changes. It suggested that the effect of Yiqihuoxue formula was much better when administrated for long-term treatment, and it might exert its anti-inflammatory function in the early stage and anti-fibrotic function in the later stage.
Our studies preliminarily demonstrated that Yiqihuoxue treatment for SSc significantly reduced collagen production in both in vivo bleomycin-induced mouse models and in vitro SSc dermal firbroblasts and TGF-β1-induced NIH/3T3 fibroblasts, via down-regulating the phosphorylation of Smad3 and the activity of SBE in the type I collagen promoter. Although our studies were limited to mouse model and cell culture conditions, it was the first attempt to explore the mechanism of TCM treatment for SSc, which provided a valuable clue to find novel drug targets and develop new medications for SSc.
Acute promyelocytic leukemia
Connective tissue growth factor
Hematoxylin and eosin
- P & T:
Prevention & treatment
Small interfering RNA
Secreted protein, acidic and rich in cysteine
Traditional Chinese medicine
Transforming growth factor beta
We sincerely thank Dr. Hiyoshi Higashi from Sumitomo Chemical Co., Ltd., Osaka, Japan for providing pGL3-SBE4-Luc plasmid. This study was partially supported by the grants from the National Science Foundation of China (81270120), International S & T Cooperation Program of China (2013DFA30870), the Science and Technology Committee of Shanghai Municipality (10JC1402100), US NIH NIAID U01 (1U01AI090909), and 111 Project (B13016).
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