Ergosterol is the active compound of cultured mycelium cordyceps sinensis on antiliver fibrosis.
Liver fibrosis is a wound-healing response to liver injury that involves a wide range of etiologic agents, including viruses, drugs, metabolic disorders, and immune attacks . Liver fibrosis involves an increased production of extracellular matrix (ECM) components, particularly the deposition of collagens in liver . Activated hepatic stellate cells (HSCs), portal fibroblasts, and myofibroblasts of bone marrow origin have been identified as the major collagen-producing cells in the injured liver . Above all, HSCs, especially the activated HSCs, were identified as the main collagen-producing cells in the liver . In carbon tetrachloride (C[Cl.sub.4]) model of liver fibrosis, quiescent HSCs are activated to become fibrogenic myofibroblasts , which express a-smooth muscle actin ([alpha]-SMA). Since liver fibrosis is closely linked with severe pathological changes including the development of hepatic carcinoma, it is essential to elucidate the mechanisms of restraining the activation and elimination of HSCs, which is considered as a target for the treatment of liver fibrosis .
Cordyceps sinensis (Berk.) Sacc., as a well-known tonic herb in TCM, is a highly valued fungus in China in regulating the immune function. In recent years, cultured mycelium Cordyceps sinensis (CMCS) was successfully developed and widely used as the alternative of natural Cordyceps sinensis (Berk.) Sacc. . Accumulated evidences from both animal and human studies indicated that Cordyceps sinensis was capable of antifibrosis  and anti-inflammation . Whether the active ingredients of CMCS could inhibit HSCs activation and liver fibrosis remained unknown.
In this study, we aimed to elucidate the effects of CMCS on C[Cl.sub.4]-induced liver fibrosis in vivo. Then, we evaluated the effects of the active compounds of CMCS on HSCs activation and hepatic fibrosis in vitro and in vivo. To clarify our aim, several questions should be formulated. Specifically, they were as follows. (1) Could CMCS inhibit hepatic fibrosis? (2) What were the ingredients of CMCS? (3) Which might be the active antifibrotic compound of CMCS that inhibited HSCs activation and hepatic fibrosis? The results demonstrated that both CMCS and ergosterol, an active ingredient of CMCS, reduced liver injury and hepatic fibrosis. These findings might suggest its potent effects on antifibrosis and exceed its role for designing better antifibrotic drugs.
2. Material and Methods
2.1. Reagents. C[Cl.sub.4] of analytical reagent grade was obtained from Sinopharm Group Co., Ltd. All the other chemicals and solvents used were of analytical grade.
2.2. Drug Preparation. The powder of cultured mycelium Cordyceps sinensis was purchased from Shanghai Sundise Chinese Medicine Technology Development Co., Ltd. The extraction process of CMCS and determination and quantitation of active compounds in CMCS analyzed by HPLC were all as previously described . Adenosine, ergosterol, vernine, and uridine were all purchased from Shanghai Winherb Medical Science Co., Ltd. The purity of these standards determined by HPLC was more than 98% as per the manufacturer's specification.
2.3. Mice. Male C57BL/6 mice (10 weeks of age) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China). Mice were housed in a room under controlled temperature (22-25[degrees] C), humidity (46-52%), and lighting (12-hour artificial light and dark cycle), with free access to tap water and mouse chow. The standard diet pellets contained not less than 20% protein, 5% fibers, 3.5% fats, and 6.5% ash and vitamins mixture. Our experiment was conducted according to the local ethical guidelines (Shanghai University of TCM, Shanghai, China).
2.4. Induction of Hepatic Fibrosis. For liver fibrosis, mice were intraperitoneally injected with 10% C[Cl.sub.4]/olive oil 2mL/kg body weight three times a week, for 4 weeks . Following pentobarbital sodium anesthesia, all mice were sacrificed 24 hours after the last injection. Serum and liver samples were harvested. For histology, tissue specimens were fixed in buffered formalin and embedded in paraffin wax. For fluorescence, liver specimens were embedded in OCT compound. Serum and liver samples were kept frozen at -70[degrees]C until assayed.
2.5. Animal Groups and Experimental Design. To evaluate the effect of CMCS against liver fibrosis, mice were randomly divided into 4 groups (10 mice per group): normal control group (Normal control), CMCS-treated group (CMCS control), C[Cl.sub.4]-treated group (Model control), and C[Cl.sub.4] plus CMCS-treated group (CMCS treatment). Mice in CMCS control and CMCS treatment were treated orally with CMCS at a daily dose of 120 mg/kg of body weight, respectively, which was equivalent to the dosage of a 60 kg adult. Mice in Normal control and Model control were treated with dd[H.sub.2]O by gavage. To investigate the effect of ergosterol against liver fibrosis, mice were randomly divided into 3 groups (10 mice per group): normal control group (Normal control), C[Cl.sub.4]treated group (Model control), and C[Cl.sub.4] plus ergosterol-treated group (Ergosterol treatment). Mice in Ergosterol treatment were administered orally by gavage with ergosterol at a daily dose of 50 mg/kg of body weight.
2.6. Measurements of Serum Liver Function and Hydroxyproline Content. The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the levels of serum albumin (Alb) and total bilirubin (T.Bil) were quantitated by following the instructions provided by the manufacturer (Nanjing JianCheng Bioengineering Institute, Nanjing, China), including use of standardization. Levels of hydroxyproline (Hyp) in tissue were measured by Jamall's method .
2.7. Histopathological Assessment of Liver Injury. Liver specimens were fixed in 10% formaldehyde solution and dehydrated in a graded alcohol series, embedded in paraffin blocks, and cut into 4 [micro]m thick slices. For histopathological examination, slices were stained by using standard procedure of hematoxylin and eosin (H&E). For investigation of the hepatic collagen deposition, Sirius red-polarization staining was performed and a red color staining was considered where the collagen deposition. Images were analyzed with light microscope (Olympus BX40, Japan).
2.8. Cell Culture. The LX-2 cell line, a spontaneously immortalized human hepatic stellate cell, was a gift from Dr. Scott L. Friedman (Mount Sinai School of Medicine, New York, NY). LX-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 units/mL of streptomycin. The human hepatic cell line HL-7702, an immortal cell line derived from human adult hepatic tissue, was purchased from Institute of Biochemistry and Cell Biology, SIBS, CAS (Shanghai, China). HL-7702 cells were incubated in RPMI1640 medium containing 10% heat-inactivated FBS, 100 U/mL penicillin, 100 [micro]g/mL streptomycin, and 2 mM glutamine. All the cells were grown in a humidified incubator at 37[degrees]C in a 5% C[O.sub.2] atmosphere.
2.9. Drug Treatments. Cells were treated with 6 concentrations (2.5, 5, 10, 20, 40, and 80 [micro]M) of each compound, in duplicate, for a period of 24 h. The compounds were initially dissolved as concentrated stock solutions in dimethyl sulfoxide ([DMSO]; Sigma-Aldrich, USA).
2.10. Cell Viability and Proliferation Assay. For HL-7702 cells, cell viability and proliferation assays were performed by using the Cell Counting Kit-8 (CCK8, Beyotime Biotechnology, Jiangsu, China), according to the manufacturer's protocol. For LX-2 cells, cell viability and proliferation assays were detected by Thiazolyl Blue Tetrazolium Blue (MTT, SigmaAldrich, USA), according to the manufacturer's protocol. Besides, cytotoxicity of ergosterol on LX-2 was tested by Multiparameter Cytotoxicity II Kit (ThermoFisher Scientific, Germany), aiming to investigate the membrane physical state on lysosomal permeability, and cell proliferation was observed by Cell-Light EdU Apollo 643 DNA In Vitro Kit (RiboBio Co. LTD, Guangzhou, China), according to the manufacturer's protocol. Images of the permeability of the lysosomal membrane and EdU were taken by Cellomics ArrayScan VTI HCS Reader (ThermoFisher Scientific, USA) and data were analyzed by Cellomics Cell Health Profiling BioApplication Software.
2.11. Immunocytochemistry and Immunofluorescence for [alpha]-SMA. For immunocytochemistry, LX-2 cells cultured on 96 wells were washed with cold PBS twice and fixed with cold methanol: acetone (1: 1) for 10 min on ice. After extensive washing with PBS three times, cells were permeated with 0.05% saponin for 15 min. The cells were then blocked with 5% bovine serum albumin in PBS buffer for 30 min at room temperature before being incubated with the alpha smooth muscle actin ([alpha]-SMA) (1: 100) primary antibodies (A2547, Sigma, USA). Cells were then stained with FITC-conjugated secondary antibodies. After washing, cells were double-stained with Hoechst 33258 (Beyotime Biotechnology, Jiangsu, China) to visualize the nuclei. Images were taken by Cellomics ArrayScan VTI HCS Reader. For immunofluorescence, liver tissue embedded in OCT compound was cut into 9 micrometers thick and fixed in 4% formaldehyde (Dingguo, Shanghai, China). After washing with PBS for three times, sections were permeabilized with 0.1% Triton X100 and blocked with 5% BSA in 0.1 M PBS for 20 minutes at room temperature to reduce nonspecific binding and then were incubated with primary antibodies against [alpha]-SMA (M0851, DAKO, Japan) followed by the biotinylated secondary antibody. To visualize the primary antibodies, cells were stained with FITC-conjugated secondary antibodies. After washing, cells were double stained with 4',6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. Images were obtained using a confocal microscope and photographs were taken with Olympus confocal software. The semiquantification data for [alpha]-SMA level in the liver tissue were determined with the computer-assisted image analysis system.
2.12. F-Actin Cytoskeleton Staining. LX-2 cells were grown in a 96-well chamber and treated with 5ng/mL transforming growth factor-beta1 (TGF-[beta]1) and drugs. After 24 hours, cells were fixed in 4% formaldehyde and permeabilized with 0.2% triton X-100. F-actin was stained with rhodamine phalloidin (1: 100) (Molecular Probes, Inc., Eugene, Oregon, USA) and the nucleus with DAPI, according to the manufacturer's protocol. Cells were visualized by Cellomics ArrayScan VTI HCS Reader and data were analyzed by Cellomics Cell Health Profiling BioApplication Software.
2.13. Statistical Analysis. All data were analyzed by using PASW Statistics 18 software. Differences between the groups were assessed by nonparametric one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) post hoc tests. Values in the text are means [+ or -] standard deviation (SD). Differences with P < 0.05 were considered to be statistically significant.
3.1. CMCS Attenuated C[Cl.sub.4]-Induced Liver Fibrosis in Mice. In order to evaluate the effect of CMCS on C[Cl.sub.4]-induced liver fibrosis, levels of serum liver functions were assayed by kits. Levels of serum ALT, AST, and T.Bil and higher levels of serum Alb were lower in Normal control compared with those in Model control (Figures 1(a), 1(b), 1(c), and 1(d)). The levels of ALT, AST, and T.Bil were significantly reduced in CMCS-treated mice. Besides, CMCS alleviated the ratios of liver/body weight (BW) (%) and spleen/BW (G) in C[Cl.sub.4]-induced hepatic fibrosis mice, respectively (Figures 1(e) and 1(f)). For investigating the inflammation and collagen accumulation in the liver tissue, H&E and Sirius Red staining were observed, respectively (Figures 1(g) and 1(h)). In Normal control and CMCS control groups, the hepatocytes were radially arranged around a central vein with no collagen fibers present, respectively. In Model control, hepatic plates were diffusely disrupted with pronounced ballooned/necrotic hepatocytes containing acidophilic hyaline inclusions, which were degenerated with widely mononuclear cell infiltration present in the portal area and fibrous septa (Figure 1(g)). Hepatic parenchyma was expanded by collagen fibers, and lobules were separated by fibrous septa that surrounded the hepatocytes (Figure 1(h)). Hepatic inflammation and fibrosis were significantly alleviated by CMCS treatment, as evidenced by reduced inflammatory cell infiltration and thinner fibrous septa (Figures 1(h) and 1(j)). Similarly, hepatic hydroxyproline content was dropped by CMCS treatment in model mice (Figure 1(l)).
3.2. CMCS Reduced Expression of [alpha]-SMA In Vivo. Since CMCS could attenuate C[Cl.sub.4]-induced liver fibrosis, we next examined the effect of CMCS on the expression of hepatic [alpha]-SMA, the gold marker of liver fibrosis and HSCs activation. In Normal control and CMCS control group, positive staining for [alpha]-SMA was mainly observed in vascular walls of central veins and portal regions, while the stronger [alpha]-SMA staining was seen in Model control, especially in the vicinity of the bile ductules and the fibrous septa (Figures 1(i) and 1(l)). The expression of [alpha]-SMA was significantly decreased in CMCS treatment group, compared with that in Model control (Figures 1(i) and 1(l)). These results revealed that CMCS could significantly inhibit the activation of HSCs.
3.3. Ergosterol Inhibited Activities of HSCs In Vitro. We previously detected the active compounds of CMCS by HPLC, finding that CMCS mainly contained four ingredients: adenosine, ergosterol, vernine, and uridine. In an effort to define the potential active substances of CMCS that might play the critical roles in resisting liver fibrosis, we observed the effects of these four monomers on human hepatocytes and HSC cell lines in vitro, respectively. Firstly, HL-7702 and LX-2 cells were preincubated with 2.5-80 [micro]M of adenosine, ergosterol, vernine, and uridine for 24 h, respectively.
After 24 h, the survival ratio of HL-7702 and LX-2 cells in incubations with 2.5-20 [micro]M of adenosine, ergosterol, vernine, and uridine for 24 h exceeded 80%, respectively (Figures 2(a) and 2(b)), which showed no obvious toxic to the cells (Table 1). Secondly, HL-7702 cells were exposed to 0.5 mM [H.sub.2][O.sub.2] for 30 min to induce hepatocellular injury in vitro. Subsequently, cells were incubated with 2.5-20 [micro]M adenosine, ergosterol, vernine, and uridine for 24 h. 24 hours later, the four active ingredients of CMCS were not able to protect against hepatocellular injury induced by [H.sub.2][O.sub.2] in vitro (Figure 2(c)). Finally, LX-2 cells were incubated with 5 ng/mL TGF-[beta]1 for 24 h to induce HSC activation. Then, cells were cultured with 2.5-20 [micro]M adenosine, ergosterol, vernine, and uridine for 24 h, respectively. 24 hours later, HSCs activation could not be restrained by the other monomers of CMCS except ergosterol (Figure 2(d)). Surprisingly, ergosterol had a dose-dependent inhibition on HSCs activation in vitro, which revealed that ergosterol might be able to act a vital role in resisting liver fibrosis in vitro. To confirm the antifibrotic effect of ergosterol in vitro, we proceeded to explore the effect of ergosterol on permeability of the lysosomal membrane in LX-2 cells. As expected, the permeability of the lysosomal membrane in LX-2 cells was altered by treatment with ergosterol for 24 h (Figures 2(e) and 2(f)). Incubating with 10 [micro]M ergosterol decreased the level of permeability of the lysosomal membrane but increased in the concentration of 20 [micro]M (Figures 2(e) and 2(f)). Besides, proliferation of activated LX2 cells stimulated by 5ng/mL TGF-[beta]1 for 24 h was inhibited after treatment with 10 [micro]M ergosterol for another 24 h unsurprisingly (Figure 3). In addition, cytoskeleton protein expression of LX-2 cells was significantly downregulated and levels of [alpha]-SMA showed a decreasing trend by cultured with ergosterol in vitro, respectively, which further proved that ergosterol, as a highly efficacious substance in CMCS, could inhibit HSCs activation in vitro (Figure 3).
3.4. Ergosterol Protected Liver against C[Cl.sub.4]-Induced Hepatic Fibrosis In Vivo. Treatment of intragastric administration with 50 mg/kg ergosterol was given to investigate the prevention of liver fibrosis induced by 10% C[Cl.sub.4] in vivo. Model control group had much higher serum levels of ALT, AST, T.Bil, liver/BW, and spleen/BW and lower Alb than those of the Normal control group (Figures 4(a), 4(b), 4(c), 4(d), 4(e), and 4(f)). Furthermore, more severe liver inflammation, collagen deposition, and expression of [alpha]-SMA were seen in the Model control group, respectively (Figures 4(g), 4(h), 4(i), and 4(j)), compared to those in the Normal control group. Ergosterol treatment ameliorated serum liver function, decreased liver/BW or spleen/BW, attenuated hepatic inflammation, decreased collagen deposition, and improved expression of [alpha]-SMA in Model group, which obviously indicated that ergosterol could protect against hepatic fibrosis in vivo.
Liver fibrosis represents chronic wound repair following liver injury . Upon liver injury, quiescent HSCs, the most relevant cell type for hepatic fibrogenesis, become active, convert into myofibroblast-like cells, and overproduce extracellular matrix (ECM) , which actually worsen the liver damage. The terminal outcome of liver fibrosis is the formation of nodules encapsulated by fibrillar scar matrix . Therefore, it is imperative that inhibition of HSCs activation is urgently needed.
Natural Cordyceps sinensis (Berk.) Sacc has been widely used in China for potentially vulnerable people with chronic diseases in all ages, and its use is strongly supported by clinical application. Nowadays, CMCS is widely used as the alternative of natural Cordyceps sinensis (Berk.) Sacc. , which has better curative effect and lightens the economic burden of patients in low income group. CMCS contains an array of active substances, which reveal the advantage of potential synergy between the various components. More and more research information has convinced its significant pharmacological properties. Some CMCS related nutraceuticals available in the market are considered to relieve the "stress for humans of living in technologically developed societies" by stimulating basic and secondary responses of the immune system .
To explore the antifibrotic effect on CMCS, we have investigated the role of CMCS and its active compounds in hepatic fibrosis in vivo and in vitro. In our study, we firstly confirmed earlier findings that the extractum of CMCS could alleviate levels of serum liver function in C[Cl.sub.4]-induced liver fibrosis mice in vivo. Then, we further analyzed the pathology of liver tissue to evaluate the efficacy of CMCS on treatment of liver fibrosis. It was obvious that CMCS could relieve the infiltration of inflammatory cells and collagen deposition. Besides, expression of [alpha]-SMA, the golden marker of activated HSC, was decreased by CMCS treatment. To observe the toxicity of CMCS, normal mice were administrated with the same dosage of CMCS in the meantime. The results of serum liver function and histopathological assessment indicated that treatment with CMCS for four weeks remained no hepatotoxicity for normal mice. These results suggested that CMCS had potential antifibrotic effect on C[Cl.sub.4]-induced liver fibrosis.
We previously made a quantitative analysis on the freeze drying powder of CMCS extractum by HPLC to detect the main active compounds. Subsequently, our TCM fingerprint revealed that CMCS mainly contained four active ingredients: adenosine, ergosterol, vernine, and uridine. Herein, we observed the toxicological and pharmacological studies on these four monomers on HSCs (LX-2 cell lines) in vitro in order to discover the main potential active substances in CMCS that played critical roles in resisting liver fibrosis. Moreover, the active antifibrotic substance should be with no hepatotoxicity. Hence, toxicological study of the four monomers was performed on hepatocytes (HL-7702 cell lines) in vitro simultaneously. The results of cytotoxicity and proliferation in HL-7702 and LX-2 implied that ergosterol had no hepatotoxicity on hepatocytes. In addition, it took effects in manner of dose-dependent inhibition on activated HSCs in vitro.
Lysosomes are involved in the cellular degradation of material either initially present in the cell or brought into the cell by phagocytosis or endocytosis . Lysosomal destabilization is critical for the organelle and living cells . In our study, we found ergosterol could decrease the level of permeability of the lysosomal membrane for HSCs at a low concentration, which revealed that ergosterol might be liable for damage on cell membrane of HSCs in vitro. Evidently, permeation of EdU and expression of [alpha]-SMA of HSCs stimulated by TGF-[beta] were repressed by ergosterol at a concentration of 20 in vitro, respectively. In nonmuscular cells, F-actin forms a system of fibers that provide mechanical support for several structures and, in association with myosin, assembles contractile units responsible for cellular movements. In our study, the expression of F-actin was increased in HSCs stimulated by TGF-[beta] while it was decreased after ergosterol intervention, which indicated that ergosterol might disrupt the cytoskeleton of HSCs.
In the following studies in vivo, ergosterol, like CMCS, could ameliorate serum liver function, attenuate hepatic inflammation, decrease collagen deposition, and improve the expression of [alpha]-SMA in mouse model of liver fibrosis, respectively, which obviously confirmed that ergosterol could protect liver against hepatic fibrosis in vivo. This finding was conformed to that of experiments in vivo.
Our results demonstrated that CMCS and ergosterol attenuate hepatic fibrosis induced by C[Cl.sub.4] in mice. We confirmed the pharmacological effects of CMCS on inhibiting the active HSCs. We further defined that the antifibrotic effect of ergosterol was associated with the restriction of proliferation, wreck of cytoskeleton, and activation of HSCs. Our study is the first to report that ergosterol is the active compound of CMCS on antiliver fibrosis. However, as a therapeutic agent, the accurate effect of CMCS and ergosterol's effect on restraining liver fibrosis still need to be investigated. Further studies and other animal models of liver fibrosis are necessary to elucidate the precise mechanisms of their antifibrosis effects.
CMCS is effective in alleviating liver fibrosis induced by C[Cl.sub.4] in vivo and ergosterol might be the efficacious antifibrotic substance of CMCS in vivo and in vitro, which might provide alternative treatment options for hepatic fibrosis.
Abbreviations CMCS: Cultured mycelium Cordyceps sinensis C[Cl.sub.4]: Carbon tetrachloride ALT: Alanine aminotransferase AST: Aspartate aminotransferase Alb: Albumin T.Bil: Total bilirubin Hyp: Hydroxyproline H&E: Hematoxylin and eosin [alpha]-SMA: Alpha smooth muscle actin HPLC: High performance liquid chromatography CCK8: Cell Counting Kit-8 MTT: Thiazolyl Blue Tetrazolium Blue HSCs: Hepatic stellate cells DMEM: Dulbecco's modified Eagle's medium FBS: Fetal bovine serum DMSO: Dimethyl sulfoxide BSA: Bovine plasma albumin PBS: Phosphate-buffer saline DAB: 3,3'-Diamino Benzedrine DAPI: 4',6-Diamidino-2-phenylindole TGF-[beta]: Transforming growth factor-beta BW: Body weight.
Conflict of Interests
The authors declare no conflict of interests.
Chenghai Liu and Qinglan Wang designed the project; Yuan Peng, Li Shen, and Tao Yang performed experiments; Yuan Peng and Li Shen analyzed the data; Yuan Peng, Yanyan Tao, and Zulong Liu prepared the paper; Chenghai Liu made critical revision for paper. Yuan Peng and Yanyan Tao equally contributed as the first author.
This work was supported by the following Grants: National Natural Science Foundation of China (nos. 81173405, 81102701, and 81270053), National Science and Technology Major Project (2014ZX1005001), National Key New Drugs Creation Project, Innovative Drug Research and Development Technology Platform (no. 2012ZX09303009-001), ZYSNXD-CC-YJXYY, E-Institute (E03008), and Innovative Research Team in Universities of Shanghai Municipal Education Commission.
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Yuan Peng, (1) Yanyan Tao, (1,2) Qinglan Wang, (3) Li Shen, (1,2) Tao Yang, (1) Zulong Liu, (1) and Chenghai Liu (1,2,3)
(1) Institute of Liver Diseases, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, 528 Zhangheng Road, Pudong New Area, Shanghai 201203, China
(2) Shanghai Clinical Key Laboratory of Traditional Chinese Medicine, Shanghai 201203, China
(3) E-Institute of TCM Internal Medicine, Shanghai Municipal Education Commission, Shanghai 201203, China
Correspondence should be addressed to Chenghai Liu; email@example.com
Received 9 July 2014; Accepted 9 September 2014; Published 15 October 2014
Academic Editor: Jae Youl Cho
Table 1: I[C.sub.50] values ([mu]M) of active compounds in CMCS (a). Active compounds Cell line Adenosine Ergosterol Vernine Uridine HL-7702 nc 37.10 ([+ or -]2.07) nc nc LX-2 nc 55.51 ([+ or -]7.13) nc nc (a) Values are means of at least three experiments; standard deviation is given in parentheses (nc = no toxic).
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|Title Annotation:||Research Article|
|Author:||Peng, Yuan; Tao, Yanyan; Wang, Qinglan; Shen, Li; Yang, Tao; Liu, Zulong; Liu, Chenghai|
|Publication:||Evidence - Based Complementary and Alternative Medicine|
|Date:||Jan 1, 2014|
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