Astragalus and Paeoniae Radix Rubra extract (APE) inhibits hepatic stellate cell activation by modulating transforming growth factor-β/smad pathway

Previous studies have shown that Astragalus and Paeoniae Radix Rubra extract (APE) is capable of protecting against liver fibrosis in rats. The hypothesis of the present study was that APE exerts its anti-fibrotic effect by mediating the transforming growth factor β (TGF-β)/Smad signaling pathway. In order to investigate this hypothesis, a series of assays were designed to detect the effects of APE on cell proliferation, cell invasion and the activation of hepatic stellate cells (HSCs). In addition, the effects of APE on the TGF-β/Smad signaling pathway were explored, with the aim of elucidating the underlying mechanisms. HSCs were initially isolated from normal rat liver. A number of assays were then employed in order to evaluate the effects of APE on the function of these cells. Cell proliferation was investigated using an MTT assay and cell invasion was observed with the use of transwell invasion chambers. Collagen synthesis was measured with a 3H-proline incorporation assay and expression of α-smooth muscle actin was used to determine the extent of HSC activation. Protein expression induced by TGF-β1 in HSCs was investigated by western blot and immunofluorescence analyses. Plasminogen activator inhibitor type1 (PAI-1) and urokinase-type plasminogen activator (uPA) transcriptional activity was measured using reverse transcription polymerase chain reaction. The results demonstrated that APE (5–80 μg/ml) significantly inhibited fetal bovine serum-induced cell proliferation in a dose-dependent manner. Cell invasion and activation of HSCs induced by TGF-β1 were disrupted by treatment with APE in a dose-dependent manner. TGF-β1 was observed to increase the phosphorylation of Smad2/3, while APE administered at higher doses produced inhibitory effects on Smad2/3 phosphorylation. In addition, administration of APE abrogated the TGF-β1-induced reduction in Smad-7 expression in a dose-dependent manner. The results further indicated that APE treatment not only reduced PAI-1 expression, but also increased uPA expression in a dose-dependent manner. In conclusion, APE exerted inhibitory effects on cell proliferation, invasion and activation of HSCs, and the mechanisms underlying these effects may involve the TGF-β1/Smad pathway.


Introduction
Hepatic fibrosis (HF) is recognized as one of the most common types of liver disease, as well as one that is resistant to the majority of current therapies, resulting in significant global morbidity (1). HF has been defined as a tissue-specific response to long-term injury or illnesses, including chronic viral hepatitis, alcoholic liver disease, cholestasis, circulatory disturbances, autoimmune liver disease or one of a number of nutritional disorders (2,3). Liver fibrosis is characterized by the excessive deposition of extracellular matrix (ECM) proteins, consisting predominantly of type I and type III collagen. These abnormal depositions disturb the structure of the hepatic lobule, misdirecting blood flow in the liver and thereby disturbing its healthy functioning. This leads to liver cirrhosis and, ultimately, to liver carcinoma (4). Although numerous therapeutic options are currently available for liver fibrosis, all have limited degrees of success and none were capable of producing a complete cure (5). Thus, there is an urgent need to develop better preventative options as well as treatment approaches, based on a more thorough understanding of the pathogenesis of hepatic fibrosis.
Although the exact pathophysiological mechanisms underlying the formation of hepatic fibrosis are elusive, there are a number of potential processes that may be worthy of investigation. Hepatic stellate cells (HSCs) are an important type of fibrogenic liver cell. They are found during liver injury and are known to be responsible for the progression of hepatic fibrosis (6). These cells may be activated, which induces their transdifferentiation into myofibroblasts (MFBs). MFBs are characterized by a number of fibrotic functions, including the induction of ECM deposition, α-smooth muscle actin (α-SMA) expression, as well as the synthesis and secretion of type I and type III collagen (7,8). A growing body of evidence has documented that inhibition of the transformation of HSCs may aid in the prevention and cure of liver fibrosis (9). However, HSCs are not the only mechanism through which fibrosis progresses. A number of studies have indicated that this process is complicated and involves numerous cytokines and signaling pathways (10,11).
Transforming growth factor (TGF)-β1 has been identified as the most significant factor involved in the activation and promotion of the transformation of HSCs (12). Previous studies have demonstrated that TGF-β1 is highly expressed in numerous tissues, which exhibit fibrosis. Furthermore, TGF-β1 has been shown to have a key role in the development of fibrosis by promoting the proliferation of tissue fibroblasts and the synthesis of collagen (13). The following stages of the cell-signaling pathway of TGF-β1 have been elucidated: The initial step is the transmission of a signal from the activated transmembrane receptor, TβR. This signal is then transmitted to the nucleus, predominantly through intracellular members of the Smad family. Further evidence for this mechanism has been provided by studies in which the TGF-β/Smad pathway was inhibited via RNA interference. This resulted in a significant decrease of type I and III collagen expression in fibrotic livers (14). Based on these findings, subsequent studies have confirmed the potential use of members of the Smad family as biomarkers with which to predict liver disease progression (15). Thus, the TGF-β/Smad pathway is hypothesized to be a target for the treatment and cure of hepatic fibrosis.
Astragalus and Paeoniae Radix Rubra extract (APE) is produced from a variety of herbs (Astragali Radix, Paeoniae Radix Rubra, Curcumae Rhizoma, Bupleuri Radix and Eupolyphaga) with a standard ratio of 30:30:15:12:10 by crude herb weight and contains the active components paeoniflorin, astragalosides and curzenone (16). Astragali Radix is thought to have restorative properties in Chinese Medicine, and studies have demonstrated that it is a hepatoprotective agent (17,18). Extracts from Paeoniae Radix Rubra, Curcumae Rhizoma, Bupleuri Radix and Eupolyphaga also have the potential to suppress liver fibrosis and have been considered potent herbs with which to treat liver disease (19). Previous in vivo experiments have been conducted, which demonstrated that APE administered at a ratio of 30:30:15:12:10 exhibited superior antifibrotic effects compared to administration of individual herbs in a model of CCl 4 -induced chronic liver injury (16). However, the molecular mechanisms underlying the hepatoprotective effects of APE have remained elusive. Therefore, the present study aimed to investigate the effects of APE on proliferation, invasion and activation of HSCs. In addition, the mechanisms underlying the effect of APE on the TGF-β/Smad pathway were examined.

Materials and methods
Preparation of APE. The following herbs were purchased at Xi'an Chinese Medicine Corporation (Xi'an, China): Astragali Radix, Paeoniae Radix Rubra, Curcumae Rhizoma, Bupleuri Radix and Eupolyphaga. Each herb was identified by Dr Genquan Qiu, an author of the study and a specialist in Traditional Chinese Herbal Medicine. Herb samples were preserved in the specimen room of the Institute of Clinical Pharmacology at the Xi'an Medical College (Xi'an, China). The process of extracting and preparing the APE components from the five herbs was as follows: A total amount 8.45 kg dried sliced crude herbs (Astragali Radix, Paeoniae Radix Rubra, Curcumae Rhizoma, Bupleuri Radix and Eupolyphaga) in a standard ratio of 30:30:15:12:10 were decocted in 80 l water three times at 95˚C for 35 min. The decocted solution was filtered though 150 µm gauze ( Sigma-Aldrich, St. Louis, MO, USA) and the combined filtrates were then concentrated to a mass of 4.22 kg in a vacuum desiccator at 70˚C. The sediment was dried into power using a spray drier at a temperature range of 80-160˚C. This process yielded 2.11 kg of dry powder. It should be noted that in all subsequent in vitro cell experiments, APE powder was dissolved in Hank's solution (Sigma-Aldrich).
Cell culture. Male Sprague-Dawley rats were obtained from the Experimental Animal Center of Anhui Medical University (Anhui, China) and had a weight range of 160-200g. HSCs were separated from normal rat liver and maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich), supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) (20). Cells were cultured in an environment of 95% air and 5% CO 2 at 37˚C. All experiments were performed during the exponential growth phase, once the cells had been plated for 24 h.
Cell proliferation assay. HSCs were seeded in 96-well plates at a density of 1x10 4 cells/well and cultured in 200 µl DMEM supplemented with 10% FBS. Following overnight starvation in serum-free medium, HSCs were incubated with 10% FBS with various concentrations of APE (5,10,20,40 or 80 µg/ml) for 16 h. Control conditions consisted of cells which had an equal volume of serum-free medium added without the addition of APE. Following incubation, cell proliferation was assessed using an MTT assay (Abcam, Cambridge, UK), as previously described (21). Absorbance was measured in an ELISA microplate reader at 562 nm (FlexStation 3; Molecular Devices, Sunnyvale, CA, USA). Data are expressed as the mean of a minimum of three independent experiments.
Cell invasion assay. Transwell invasion chambers with 8-µm membrane pores coated with Matrigel™ (BD Biosciences, San Jose, CA, USA) were placed in 24-well plates. HSCs were starved overnight in serum-free DMEM. Cells were then seeded in the upper compartment of the chamber at a concentration of 1x10 5 cells/well and incubated with or without the administration of APE at various concentrations (10, 20, 40 or 80 µg/ml) for 24 h. Medium containing TGF-β1 (40 pmol/l) was placed into the lower compartment of the chamber. Serum-free medium was used for the control group. Following incubation, cells on the upper side of the membrane were completely removed using a cotton swab. Cells which had crossed the Matrigel barrier and migrated to the lower side of the chamber were fixed with 100% methanol for 1 min and stained with hematoxylin and eosin (H&E; Sigma-Aldrich). The number of invading cells was counted in five randomly selected fields at x100 magnification (CX31; Olympus Corp., Tokyo, Japan). The mean number of invading cells in these fields was calculated and the experiments were run in triplicate.
Collagen synthesis assay. Collagen synthesis was evaluated using a 3 H-proline incorporation assay (22). In brief, HSC cells were seeded in 96-well plates at a density of 1x10 4 cells per well for at least 24 h. Cells were synchronized by culturing in serum-free medium overnight prior to incubation with 40 pmol/l TGF-β1 in the presence of APE at various concentrations (10, 20, 40 or 80 µg/ml) for 24 h. The control group received an equal volume of serum-free medium without APE. The aforementioned cultures were subsequently exposed to 3 H-proline (37 kBq; Atom-Hitech, Beijing, China) for 12 h. Following proline exposure, the cells were washed twice with phosphate-buffered saline (PBS; Sigma-Aldrich), treated with ice-cold 5% trichloroacetic acid (Sigma-Aldrich) for 1 h and then washed twice more with distilled water. Finally, cells were lysed with 0.25% trypsogen and counted in a liquid scintillation counter (LS6000SE; Beckman Coulter, Fullerton, CA, USA).
Immunofluorescence. HSCs were seeded onto 24-well plates at 5x10 3 cells per well. Cells were then incubated in serum-free medium for 24 h with or without treatment with APE at various concentrations (10, 20 or 40 µg/ml) and stimulated with 60 pmol/l TGF-β1 for 1 h. Control cells were incubated in serum-free medium with neither TGF-β1 nor APE. Following incubation, cells were fixed with 4% paraformaldehyde for 30 min, permeabilized and then blocked with 0.1% saponin and 0.5% bovine serum albumin (Sigma-Aldrich) in PBS for 30 min at 4˚C. Cells were incubated overnight at 4˚C with the primary antibodies described above, and then incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (1:100) for 2 h at room temperature. Slides were then mounted with 80% phosphoglycerol, and visualized under a fluorescence microscope (CX31; Olympus Corp.). In each experiment, five randomly selected fields were analyzed from each sample.
Statistical analyses. Statistical analysis was performed using SPSS software (SPSS standard version 17.0; SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation. Comparisons between groups were made using Student's t-test and the Mann-Whitney rank sum test, the latter of which was used to compare the degree of staining intensity.  P<0.05 was considered to indicate a statistically significant difference.

APE suppresses HSC proliferation induced by FBS.
In order to detect the effect of APE on HSCs proliferation, cells were stimulated with 10% FBS and treated with varying concentrations of APE (5-80 µg/ml). As shown in Table I

APE inhibits cell invasion induced by TGF-β1.
The invasion capability of HSCs was investigated using a Transwell inva-sion assay. As shown in Fig. 1A, HSCs moved to the lower compartment of the chambers across the Matrigel-coated polycarbonate membrane when stimulated by treatment with TGF-β1. Treatment with APE (10, 20, 40, 80 µg/ml) significantly reduced the number of TGF-β1-stimulated cells that invaded across the polycarbonate membrane. In accordance with previous results, this effect occurred in a dose-dependent manner (23). Thus, TGF-β1-induced HSC invasion into the bottom chamber and across the collagen IV/collagen I-coated polycarbonate membrane was inhibited by APE ( Fig. 1A and B).

APE reduces TGF-β1-induced HSC activation.
Collagen synthesis was measured using a 3 H-proline incorporation assay. As shown in Fig. 1C, collagen synthesis in TGF-β1-stimulated HSCs was markedly increased compared with that of control cells (P<0.01). Furthermore, treatment with APE (10, 20, 40, 80 µg/ml) significantly reduced TGF-β1-induced collagen synthesis in a dose-dependent manner. Protein expression of α-SAM was then used to measure HSC activation. As shown in Fig. 1D, the expression of the α-SAM protein following treatment with TGF-β1 alone was 3.5-fold higher than that in the control group. Furthermore, administration of APE (10, 20, 40, 80 µg/ml) decreased α-SAM expression in a dose-dependent manner. These results indicate that APE inhibits liver fibrosis by suppressing HSC activation and collagen synthesis.

APE reduces TGF-β1-induced receptor regulated (R)-Smad phosphorylation in
HSCs. R-Smad phosphorylation was detected by western blot and immunofluorescence analyses. As shown in Fig. 2A and B, western blotting results demonstrated that TGF-β1 significantly increased the levels of Smad-2 phosphorylation and moderately enhanced Smad-3 phosphorylation. This increase in R-Smad phosphorylation was subsequently suppressed by the administration of APE (20, 40 µg/ml) in a dose-dependent manner. Of note, a 10 µg/ml dosage of APE significantly reduced Smad-3, but not Smad-2 phosphorylation. Immunofluorescence also demonstrated that the expression of phosphorylated R-Smad was elevated as a result of TGF-β1 treatment (Fig. 2C). Treatment with APE (20, 40 µg/ml) significantly reduced the fluorescence intensity of phosphorylated Smad2 and Smad3 in a dose-dependent manner. In accordance with the results from the western blotting experiments, a 10 µg/ml dosage of APE reduced the fluorescence intensity of phosphorylated Smad3, but had no detectable effect on that of phosphorylated Smad-2. These results indicated that APE decreased TGF-β1-induced R-Smad phosphorylation.
APE enhances Smad7 expression in HSCs, reversing downregulation of Smad7 by TGF-β1. As shown in Fig. 3A, western blot analysis demonstrated that the protein expression of Smad7 was significantly reduced following treatment with TGF-β1 compared with that in the controls. However, administration of high doses of APE (20 and 40 µg/ml) was able to inverse this effect, leading to an increase in the expression of Smad7. Immunofluorescence demonstrated that TGF-β1 reduced the fluorescence intensity of Smad7, whereas treatment with APE (10, 20 or 40 µg/ml) increased Smad7 fluorescence intensity ( Fig. 3B and C). These results indicated that APE at high doses increased the expression of Smad-7.
APE suppresses PAI-1 and increases uPA expression, reversing the effects of TGF-β1 on HSCs. As shown in Fig. 4A, protein expression of PAI-1 was markedly increased in HSCs treated with TGF-β1 compared with that in the control group. However, treatment with APE (20 or 40 µg/ml) suppressed protein expression in a dose-dependent manner. TGF-β1 induced an increase of ~3.5-fold in PAI-1 mRNA expression compared with controls, while APE suppressed the transcriptional activity of PAI-1 is a dose-dependent manner. This inhibitory effect of APE was statistically significant at higher concentrations (20 and 40 µg/ml; Fig. 4B). In accordance with these results, TGF-β1 significantly reduced uPA protein levels in HSCs, while APE, at concentrations of 20 or 40 µg/ml, completely restored uPA protein expression levels in TGF-β1-treated cells (Fig. 4C). The transcriptional activity of uPA was also reduced following TGF-β1 treatment, while administration of APE at doses of 20 or 40 µg/ml resulted in an increase in uPA mRNA levels (Fig. 4D). These results demonstrated that APE treatment decreases PAI-1 expression and restores that of uPA.

Discussion
Liver fibrosis is a chronic disease that is a culmination of the insidious effects of liver degeneration. In recent years, the clinical focus has concentrated predominantly on the prophylaxis and treatment of primary liver disease, including chronic hepatitis, or in trying to avoid contact with hepatotoxic substances (24)(25)(26)(27). It has been shown that certain active components found in traditional herbal plants are effective in suppressing liver fibrosis and collagen synthesis, which indicated a potential therapeutic option for liver fibrosis (28)(29)(30).
One of these studies demonstrated that APE significantly reduced levels of serum glutamic pyruvic transaminase, glutamic oxaloacetic transaminase and hydroxyproline in liver homogenates (31). Of note, APE also attenuated the pathological changes characteristic of liver fibrosis, which had been induced by CCl 4 (16). In the present study, the results showed that APE markedly suppressed hepatic stellate cell proliferation and invasion, as well as their activation. Furthermore, these inhibitory effects may be due to the effects of APE on the TGF-β/Smad pathway. HF results from the excessive secretion of matrix proteins by HSCs, a process which is primarily triggered by TGF-β1. A recent study suggested that garlic extract exhibits therapeutic effects in liver fibrosis through inhibition of TGF-β1 in HSCs (32). Other studies have demonstrated that proanthocyanidin from grape seed extract exerts protective hepatocellular effects, which resulted in the amelioration of murine liver fibrosis, induced by TGF-β1 (33). In addition, using TGF-β1 small interfering RNA to inhibit the expression of TGF-β1 attenuated rat hepatic fibrosis induced by a high-fat diet and CCl 4 (34). The results of the present study confirmed that administration of TGF-β1 significantly increased collagen synthesis in HSCs, which was hypothesized to be involved in the formation of liver fibrosis. APE suppressed the proliferation induced by FBS and the cell invasion induced by TGF-β1 in HSCs in a dose-dependent manner. In addition, APE reduced Application of APE at dosages of 20 and 40 µg/ml were found to increase mRNA levels of uPA. ## P<0.05 compared with control and ** P<0.05 compared with group treated with TGF-β1 alone. APE, Astragalus and Paeoniae Radix Rubra extract; PAI-1, plasminogen activator inhibitor type 1; uPA, urokinase-type plasminogen activator; TGF-β1, transforming growth factor-β1; HSC, hepatic stellate cells. the collagen synthesis and the expression of α-SAM in HSCs, also in a dose-dependent manner. These results indicated that TGF-β1 exerts pro-fibrotic activity in HSCs, primarily through a dual action of collagen synthesis promotion and the inhibition of collagen degradation. These combined effects ultimately lead to excessive collagen deposition.
TGF-β1 controls a diverse set of cellular process and its canonical signaling is mediated via the TGF-β-induced phosphorylation of receptor-activated Smad2 and Smad3 (35). It was recently shown that the expression of the phosphorylated Smad2 and Smad3 proteins was higher in samples of CCl 4 -induced liver fibrosis, compared with that in the controls (36). Furthermore, overexpression of Smad ubiquitin regulatory factor 2 suppressed TGF-β-mediated liver fibrosis (37). Finally, co-treatment with Boswellia serrata and Salvia miltiorrhiza extracts reduced dimethylnotrosamine-induced hepatic fibrosis in mice via downregulation of phosphorylated Smad3 (38). The present study showed that APE inhibited the phosphorylation of Smad2 and Smad3 which was induced by TGF-β1 in HSCs, in a dose-dependent manner. It was therefore hypothesized that the reduction of phosphorylated Smad2 and -3 by APE may underlie its inhibitory effects on HSCs.
Smad7 serves as the negative feedback regulator for TGF-β signaling, acting to antagonize the activity of the receptor-regulated Smads, which leads to a termination of the TGF-β signal (39). Suppression of Smad7 by DNA methyltransferase 1 promoted the phosphorylation of Smad2 and Smad3 that had been induced by HSC activation, or liver fibrosis in general (40). Recent studies have shown that hepatocytes are more sensitive to the effects of TGF-β. This resulted in enhanced cell death in S7DeltaE1 and wild-type mice that had a deletion of exon I from the endogenous Smad7 gene. A study demonstrated that hepatocytes are more sensitive to the effects of TGF-β. In these mice, an increase in oxidative stress and an increase in cell damage in response to CCl 4 was observed (41). Smad7-overexpression in common bile duct ligation rats reduced the expression of collagen and α-SMA, as well as the hydroxyproline content in the liver (42). The present study also showed that TGF-β1-induced reduction in Smad7 expression was ameliorated by treatment with APE in a dose-dependent manner. Smad7 has recently been used successfully to abrogate TGF-β signaling in a number of fibrotic diseases, resulting in decreased collagen or α-SMA expression as a result of inhibition of the phosphorylation of Smads (43). Therefore, it is possible that the inhibition of Smad activation caused by the application of APE may be partly due to an upregulation in Smad7 expression, with a resultant reduction in collagen production.
uPA and plasmin are involved in the cellular proteolytic degradation of ECM proteins and in the maintenance of tissue homeostasis. The activities of uPA/plasmin predominantly rely on the functioning of a potent inhibitor of PA, PAI-1 (44). Under normal physiological conditions, PAI-1 controls the activities of the uPA/plasmin system and thus maintains tissue homeostasis. Recently, a number of studies have demonstrated that co-expression of Smad7 and uPA attenuates CCl 4 -induced liver fibrosis in rats (45,46). Furthermore, the administration of Genistein was shown to modify liver fibrosis and improve liver function by inducing uPA expression in CCl 4 -treated rats (47). PAI-1 levels are known to be significantly elevated in fibrotic tissues and a lack of PAI-1 protects certain organs from fibrosis in response to injury-associated, pro-fibrotic signals (48). Liver fibrosis produces intrinsically high levels of PAI-1 and low levels of urokinase-type plasminogen activator. This altered ratio of activator and inhibitor activities is an important factor contributing to altered fibrin degradation and the subsequent ECM metabolism. The imbalance ultimately aids in the formation of liver fibrosis (49). In the present study, TGF-β1 was shown to increase mRNA and protein levels of PAI-1 and decrease those of uPA, while APE significantly suppressed TGF-β1-induced PAI-1 expression and increased uPA expression in the presence of TGF-β1 in a dose-dependent manner, therefore reversing the effects of TGF-β1. In view of growing evidence suggesting that elevated PAI-1 expression and reduced uPA expression are essential in collagen accumulation in HSCs, the APE-induced decrease in PAI-1 mRNA and protein expression may interfere with collagen deposition, thus preventing keloid formation.
In conclusion, the present study demonstrated that APE inhibited cell proliferation, invasion and collagen synthesis in HSCs, and that the mechanisms underlying these effects may involve the TGF-β/Smad signaling pathway. These results provided evidence for the promising potential therapeutic use of APE in liver fibrosis.