The American Journal of Chinese Medicine, Vol. 43, No. 2, 1–15 © 2015 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine DOI: 10.1142/S0192415X15500196
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Effects of the Total Saponins from Dioscorea nipponica on Immunoregulation in Aplastic Anemia Mice Yuliang Wang,* Tiangai Yan,† Lin Ma† and Baoshan Liu† *Department
of Clinical Laboratory Medicine, Tianjin First Central Hospital Key Laboratory for Critical Care Medicine of the Ministry of Health Tianjin 300192, China †Department
of Traditional Chinese Medicine Tianjin Medical University General Hospital, Tianjin 300052, China Published 18 March 2015
Abstract: Dioscorea nipponica Makino, a popular folk medicine, exerts anti-inflammation properties. The present study investigated the therapeutic effect of the total saponins from Dioscorea nipponica Makino (TSDN) on aplastic anemia (AA) and possible immune regulation mechanisms. Using a mouse model of AA, three different doses of TSDN were orally administrated for 14 consecutive days. We first demonstrated that TSDN was found to be effective in alleviating pancytopenia with a hypocellular bone marrow as compared with AA model group. Moreover, gastrogavage administration of a medium dose of TSDN was found to dramatically increase the percentage of CD4 þ cells in bone marrow nucleated cells (BMNC) and restore the CD4 þ /CD8 þ ratio. The pro-inflammatory cytokine concentrations of IL-2 and IFN- were significantly decreased, and anti-inflammatory cytokine IL-4 was significantly increased in culture supernatant of BMNC. Further investigations showed that TSDN obviously inhibited Fas–FasL-induced BMNC apoptosis as well as effectively suppressed intracellular apoptosis protein of caspase-3 and -8 expressions. Taken together, these findings suggested that TSDN could alleviate AA by elevating the CD4 þ /CD8 þ T-cell ratio, inhibiting inflammatory Th1-cytokines, and exerting anti-apoptosis effects. Keywords: Dioscorea nipponica; Saponins; Immune Regulation; Aplastic Anemia Mice; CD4 þ /CD8 þ Ratio; Pro-Inflammatory Cytokines.
Correspondence to: Dr. Baoshan Liu, Department of Traditional Chinese Medicine, Tianjin Medical University General Hospital, Heping District, Anshan Road 154, Tianjin 300052, China. Tel/Fax: (þ86) 22-6036-3599, E-mail: [email protected]
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Introduction Aplastic anemia (AA) is a bone marrow failure syndrome characterized by a peripheral blood pancytopenia with a hypocellular bone marrow (Young et al., 2013). Immune-mediated destruction of hematopoiesis has been identified as the important mechanism of AA. In most cases, an immune response dominated by oligoclonal expanded cytotoxic T-cells, which secrete pro-inflammatory cytokines such as TNF-, IFN-, and IL-6, targets hematopoietic stem and progenitor cells, inducing their death via apoptosis and hematopoietic failure (Chen et al., 2013; Scheinberg and Chen, 2013). Recently, numerous strategies have been applied in the treatment of AA, including immunosuppression and/or hematopoietic stem cell transplantation (Scheinberg, 2012; Young et al., 2013). Many lymphocytotoxic agents have been widely used, and however, some of them have potential adverse effects such as anaphylaxis fever, chest pain and diarrhea (Passweg and Aljurf, 2013). For hematopoietic stem cell transplantation, the type of allograft and the age of the patient are the most important factors affecting the outcome of treatment. These actualities make the transplantation less favorable when compared with immunosuppression as the age of AA patients increases (Schrezenmeier et al., 2007). In recent years, natural products from medicinal plants have attracted much attention as effective and safe alternative treatments for bone marrow failure (Gao and Chong, 2012). Steroidal saponins are widely spread throughout the plant kingdom, an example being Dioscorea nipponica Makino, which is a famous traditional Chinese medicinal plant that has diversified pharmacological efficacies (Lu et al., 2010). It has been widely used in China for relieving cough and asthma, eliminating rheumatic aches, alleviating pain, improving blood circulation, and having antilipemic and anticancer properties (Kim et al., 2011; Chien et al., 2012; Kwon et al., 2003; Lu et al., 2014). Recently, the total saponins from Dioscorea nipponica Makino (TSDN) was reported to exert a hepatoprotective effect against CCl4-induced liver damage in mice through the suppression of inflammation and apoptosis (Yu et al., 2014). However, no related experimental findings exist concerning the therapeutic effects of this plant on AA. The purpose of this study was to investigate whether TSDN administration could treat the AA mice effectively and explore the possible underlying immunomodulatory effect mechanisms. Materials and Methods Experimental Animals The pathogen free male ICR mice (18–23 g) were purchased from the Academy of Military Medical Science, Beijing, China (SPF Certificate No. SCXK2007-0004). The mice were used after one week of acclimatization. They were kept in a departmental animal housing in a well cross ventilated room at 24 C, relative humidity (44–56%), and a light–dark cycle of
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12 h during the experiments. All experimental procedures were approved by the Animal Care and Use Committee of Tianjin Medical University, and performed in strict accordance with the PR China Legislation Regarding the Use and Care of Laboratory Animals.
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Preparation of TSDN Dioscorea nipponica Makino was obtained from Beijing Tcmages Pharmaceutical Co., Ltd. (Beijing, China). D. nipponica extracts were prepared by initial condensation followed by lyophilization as described previously (Ho et al., 2011). The total saponins from airdried Dioscorea nipponica Makino extract were obtained according to the method of Yu et al. (2014). Determination of the marker compounds in the extract was performed by high-performance liquid chromatography (HPLC). Experimental Design ICR mice were randomly divided into a normal control group (n ¼ 15) and AA model group (n ¼ 75). The AA model was made according to the previous description (Chen et al., 2013). Briefly, the mice were irradiated with 2.0 Gy 60 Co and then treated with daily intraperitoneal injections of cyclophosphamide (Sigma-Aldrich, USA) at 50 mg/kg/ day and chloramphenicol (Sigma) at 62.5 mg/kg/day for the next three days. Then, the AA group was randomly divided into five groups: the low, medium, or high dose TSDN-treated groups, which received 37.44, 74.88 or 149.76 mg/kg/day TSDN, respectively; a positive control group treated with 23.5 mg/kg/day cyclosporine A (CsA) (Sigma); and an untreated model control group (model group). TSDN or CsA were administered by gastrogavage daily in each respective group for 14 consecutive days. The doses of TSDN were calculated according to the standard dose of total saponins extract used in the Pharmacology of Chinese Materia Medica (2000 Edition) for the effective and safe dosage of TSDN. Mice in the model group were given the same volume of saline. Each group contained 15 animals. On the 15th day, all mice were sacrificed under ether anesthesia. Bone marrow cells were extracted from bilateral femurs and tibiae, peripheral blood was obtained by retroorbital sinus bleeding using EDTA-coated blood collection tubes. Peripheral Hemogram and Bone Marrow Smear Study The hemoglobin (HGB) concentration, white blood cells (WBCs) and platelets (PLTs) counts were determined using a XT-1800iV Haematology Analyser (Sysmex, Kobe, Japan). Bone marrow smear was observed under light microscopy using routine Giemsa staining. Culture of Bone Marrow Nucleated Cells Bone marrow was flushed from the above-mentioned bones using a syringe containing RPMI-1640 media (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), and was mixed well with repeated pipetting. The bone marrow nucleated
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cells (BMNC) were isolated via density gradient centrifugation. The isolated BMNC were then depleted of RBCs using RBC-depletion buffer (BD-Biosciences, USA). Finally, the cells were incubated in RPMI-1640 media supplemented with FBS at 37 C in a 5% CO2 atmosphere for 24 hours.
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Flow Cytometric Analysis of BMNC Surface Receptor Expression mAbs for mouse CD3-FITC, CD4-PE/Cy5, CD8-PE, CD95 (Fas)-PE, and CD178 (FasL)PE, were obtained from BD Biosciences (BD Biosciences, USA) (Nisar et al., 2014). Cells were incubated with antibodies on ice for 30 min, washed twice with phosphatebuffered saline (PBS), resuspended, and phenotyped on flow cytometric analysis using the FACSCalibur flow cytometer (BD Biosciences). The ratio of CD4 þ /CD8 þ was calculated. Measurement of Cytokine Concentration in Culture Supernatant of BMNC BMNC suspensions were prepared as described above then adjusted to a concentration of 1 10 6 cells/ml. BMNC were cultured in RPMI-1640 media supplemented with 15% FBS at 37 C in 5% CO2 and saturated humidity. After 72 hours of cultivation, culture supernatant was harvested. The secretion levels of IL-2, IFN-, and IL-4 in culture supernatant of BMNC were measured with an ELISA kit (R&D Systems, USA) according to the manufacturer’s instructions. Light absorbance was measured using the EnSpire Multimode Plate Reader (PerkinElmer, USA), at 450 nm with a correction wavelength set at 570 nm. RT-PCR Assay for Relative Caspase-3 and -8 Gene Expression in BMNC Total RNA was extracted from BMNC with Trizol Reagent according to manufacturer’s instructions (Invitrogen). Reverse transcription to complementary DNA (cDNA) was undertaken with the use of random primer and Superscript RNase H-reverse transcriptase (Invitrogen). Polymerase chain reaction (PCR) was performed with the use of the ABI Prism 7500 Sequence Detector (Applied Biosystems) with a total reaction volume of 20 mL in 0.2 mL Microamp Optical Tubes and Strip Lids (Applied Biosystems) for a total of 40 cycles. The PCRs for caspase-3, -8, and β-actin were performed with the use of Table 1. Primer Sequences Used for RT-PCR Analysis Gene
Amplified Products Size
Sequence
Caspase-3
188 bp
Caspase-8
186 bp
β-Actin
183 bp
Sense: 5 0 -TGACTGGAAAGCCGAAACTCT-3 0 Antisense: 5 0 -CGACCCGTCCTTTGAATTTCT-3 0 Sense: 5 0 -AGATGACTTGAGCCTGCTTGA-3 0 Antisense: 5 0 -GGCAACTCTTCCCTTCCTTCA-3 0 Sense: 5 0 -GAGGCCCAGAGCAAGAGAGGT-3 0 Antisense: 5 0 -TTCACGGTTGGCCTTAGGGTT-3 0
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Universal PCR Mastermix (Applied Biosystems). Primers were synthesized with the sequences given in Table 1. The level of target molecule mRNA was expressed relative to that of the mRNA of β-actin.
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Western Blot Assay for Caspase-3 and -8 Protein Expression in BMNC Total protein was extracted from BMNC using a standard method. Western blot analysis was performed using primary antibodies against caspase-3, -8, and β-actin (Proteintech, USA) (Hu et al., 2013). The relative photographic densities were quantitated by scanning the photographic negatives using a gel documentation and analysis system (Alpha Innotech, USA). Statistical Analysis Data was presented as mean standard deviation (SD). The statistical analysis was performed by conducting one-way ANOVA using the SPSS software v.16.0 and Tukey in post hoc multiple comparisons was used to examine statistical significance (p < 0:05 and p < 0:01) between groups. Results TSDN Increased the Peripheral Hemogram and Bone Marrow Hyperplasia in AA Mice In order to confirm the recovery of the hematopoiesis effect of TSDN in the experimental AA group, a peripheral hemogram and bone marrow smear were determined following standard laboratory techniques. The results showed that the mean counts of WBCs and PLTs and the concentration of HGB were significantly lower in model group than in the normal control group. WBCs, PLTs, and HGB were all significantly higher in positive control group and TSDN-treated groups than in model group. Also note that, TSDN at the dose of 74.88 mg/kg considerably increased the peripheral hemogram, when compared with that of the low, high dose TSDN-treated groups, and positive control group (Fig. 1). The typical bone marrow architecture was observed in normal as well as in experimental groups through a bone marrow smear study. In AA (Fig. 2B), the marrow was filled with large fat cells (adipocytes) leaving scattered empty spaces in comparison with the normal marrow (Fig. 2A), which showed a densely packed distribution of cells. Bone marrow hyperplasia was significantly observed in medium dose TSDN-treated group compared to low, high dose TSDN-treated groups, and the positive control group (Fig. 2E). All these data indicated TSDN played potential role in alleviating AA. Immunomodulatory Effects of TSDN on T Lymphocyte Subset To evaluate the immunomodulatory effects of TSDN on the bone marrow immune system, percentages of T lymphocyte subsets in the BMNC were measured. As shown in Fig. 3, the
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Figure 1. The comparison of peripheral hemogram in each group. (A) The number of WBCs. (B) The number of PLTs. (C) The concentration of HGB. *p < 0:01 vs. normal control group, # p < 0:01 vs. model group, p < 0:01 vs. positive control group.
characterization of the lymphocyte subsets revealed a significant reduction in the percentage of total T cells (CD3 þ ), CD4 þ T cells as well as a significant increase of CD8 þ T cells in AA model groups compared to the normal control group. When compared with the AA model group, the percentage of CD3 þ , CD4 þ T cells and the ratio of CD4 þ /CD8 þ increased significantly, and the percentage of CD8 þ T cells significantly decreased in the medium dose TSDN-treated group (Fig. 3). Inflammatory Suppression Effects of TSDN The expression levels of IL-2 and IFN- in culture supernatant of BMNC were significantly increased in model control group compared with those in normal control group and each treated group, whereas the expression levels of IL-2 and IFN- in the medium dose TSDN-treated group were significantly decreased compared with those in positive control
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(A)
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(C)
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Figure 2. Bone marrow smear in each group. (A) Giemsa staining of bone marrow smear showing densely packed cellular distribution in normal condition. (B) AA bone marrow smear exhibiting frequent appearance of large fat cells (adipocytes) with empty spaces. (C) Positive control bone marrow smear exhibiting relatively higher hyperplasia activity and visible nucleated cells. (D) Low dose TSDN-treated bone marrow smear exhibiting relatively higher hyperplasia activity. (E) Medium dose TSDN-treated bone marrow smear exhibiting considerably higher hyperplasia activity. (F) High dose TSDN-treated bone marrow smear exhibiting significantly higher hyperplasia activity (400 magnification).
group. The expression levels of IL-4 were significantly decreased in the model control group compared with those in normal control group and each treated group, whereas the expression levels of IL-4 in the medium dose TSDN-treated group were significantly increased compared with those in the positive control group (Fig. 4). Anti-Apoptosis Effects of TSDN Evaluation of the BMNC apoptosis was characterized by extracellular Fas/FasL and the intracellular apoptosis protein caspase-3, -8 assay. The expression levels of Fas and FasL were significantly increased in the AA model group compared with those in normal control group, whereas the expression levels of Fas and FasL in medium-, high-dose TSDN-treated groups were significantly decreased compared with those in model group (Fig. 5). Compared with normal control group, the mRNA and protein expression levels of intracellular apoptosis protein caspase-3 and -8 were significantly increased in the AA model group, which were obviously reversed by TSDN-treated groups (Figs. 6 and 7).
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(A)
(B) Figure 3. Effect of TSDN administration on T cells. (A) The representative experiment shows the percentages of CD3 þ , CD4 þ , and CD8 þ T cells by flow cytometry. (B) The comparison of the percentages of CD3 þ , CD4 þ , CD8 þ T cells and the ratio of CD4 þ /CD8 þ in each group. *p < 0:05 vs. normal control group, **p < 0:01 vs. normal control group, p < 0:05 vs. model group, # p < 0:01 vs. model group, £ p < 0:05 vs. positive control group.
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Figure 4. Anti-inflammatory effects on TSDN administration in culture supernatant of BMNCs. *p < 0:01 vs. normal control group, **p < 0:05 vs. normal control group, # p < 0:01 vs. model group, p < 0:05 vs. positive control group.
(A) Figure 5. Anti-apoptosis effects of TSDN administration on BMNCs. (A) The representative experiment shows the expression of Fas and FasL by flow cytometry. (B) The comparison of the expression levels of Fas and FasL in each group. *p < 0:01 vs. normal control group, **p < 0:05 vs. model group, # p < 0:01 vs. model group, p < 0:01 vs. positive control group.
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(B) Figure 5. (Continued )
Figure 6. Anti-apoptosis effects of TSDN on gene expression of intracellular apoptosis protein. *p < 0:05 vs. normal control group, # p < 0:05 vs. model group.
Discussion AA is characterized by hypocellular bone marrow resulting from hematopoietic stem cell damages. It results in pancytopenia with risk of severe anemia, major hemorrhage, and lifethreatening infection (Benson-Quarm et al., 2014). Current data have suggested that the pathophysiology of AA is largely mediated by immune dysfunction, in which impaired activation, differentiation and increased cellular apoptosis in the AA bone marrow play critical roles (Young et al., 2013). Therapies, including immunosuppression and/or bone marrow transplantation, have a clinical therapeutic effect in approximately 50% of AA patients. However, a large portion of patients may fail to respond to the treatments or relapse after the treatment (He et al., 2011). Recently, Dioscorea nipponica Makino, a Chinese medicinal plant, has received more attention as the saponins, the main bioactive compounds in this herb, have shown various
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Figure 7. Anti-apoptosis effects of TSDN on the expression of intracellular apoptosis protein. (A) The protein expression of caspase-3 in each group by western blot. (B) The protein expression of caspase-8 in each group by western blot. *p < 0:05 vs. normal control group, # p < 0:05 vs. model group.
pharmacological activities (Yoshikawa et al., 2007; Ho et al., 2011; Wang et al., 2012; Lv et al., 2013) including relieving cough and asthma, eliminating rheumatic aches, alleviating pain, improving blood circulation, and having antilipemic and anticancer capabilities by enhancing anti-oxidant, antivirus, suppressing inflammation and apoptosis, and anti metastasis mechanisms. In the present study, we demonstrate that TSDN administration can promote the recovery of hematopoiesis in the AA model mice. The scanty peripheral hemogram is the evidence of bone marrow failure characterized by damaged stem cell population that has been termed as “residual injury” (Pugsley et al., 1978). The characteristics of “residual injury” are: it is permanent, there is impairment of stem cell proliferation leading to moderate marrow failure and complete failure of stem cell proliferation may ensue leading to severe marrow failure and death (Morley et al., 1975). The results of our study showed that the peripheral blood pancytopenia includes neutropenia, thrombocytopenia, declined HGB level, signifies hypoplastic marrow failure in the mouse model of AA (Zhao et al., 2013). At around 2 weeks after gastrogavage of TSDN, the peripheral blood started showing the hypoplastic marrow failure reversal, which was significantly evident on the 15th day after medium dose TSDN treatment (time of our experiment). Bone marrow smear study revealed a typical AA bone marrow feature with frequent presence of large fat cells (adipocytes) with empty spaces, when compared with the normal healthy marrow. Bone marrow hyperplasia after medium dose TSDN treatment was significantly observed which is consistent with peripheral hemogram. AA is an immune-mediated disease. The disorder of immune system especially T cells plays an important role in the development of AA. Therefore, immune reconstitution is very important in the treatment of AA (Weston et al., 2013). Our results showed that the
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oral administration of TSDN preparation caused a significant increase in the percentages of CD3 þ , T-helper cells (CD4 þ Th cells) and a decrease in the percentage of T-cytotoxic/ suppressor cells (CD8 þ Tc cells) in AA mice BMNC, respectively. Accordingly, TSDN treatment has resulted in a rapid recovery of the ratio of CD4 þ /CD8 þ under immunosuppressed conditions. Moreover, CD3 þ , CD4 þ T cells and CD4 þ /CD8 þ in AA mice even returned to normal after consecutive treatment with medium dose TSDN, indicative of immune restorative activity of this preparation (Zhang and Liu, 2011). Activated T cells were differentiated into CD4 þ Th1/Th2 cells and CD8 þ cells. All those cells play crucial roles in AA (Sheng et al., 2014). On one hand, Th1 cells can secrete a variety of immune molecules including IFN-, IL-2 directly. On the other hand, Th1 cells can promote the activation of NK cells, CD8 þ cells, and macrophage cells which could secrete a variety of cytokines, including IFN-, TNF-, and IL-2, and mediate apoptosis (Chen et al., 2014). The results of our study showed that the effector cells within the lymphocyte subset are activated cytotoxic T cells bearing a Th1 profile, expressing and secreting IL-2 and IFN- in the mouse model of AA, which would provide a favorable immunologic milieu for bone marrow destruction. As a result, bone marrow cells are significantly impaired and lose the capacity to proliferate and differentiate. The present study showed that the pro-inflammatory Th1 cytokines IL-2 and IFN- were significantly down-regulated, as well as anti-inflammatory Th2 cytokine IL-4 was significantly upregulated 14 days after treatment with TSDN. Apoptosis or programmed cell death is the most discussed pathophysiologic mechanism associated with AA progression (Chatterjee et al., 2010). The fine equilibrium between the differentiation and apoptosis of normal hematopoietic cells is altered in AA. The increased bone marrow cellular apoptosis leads to the increased occurrences of AA. The interaction of death receptor protein Fas and FasL has been shown to be a key regulator of apoptosis and major activation-induced cell death (AICD) (Wang et al., 2005). In the present study, we found that Fas and FasL played a major role in upregulated BMNC apoptosis in AA and were subsequently suppressed by TSDN treatment. One possibility to explain bone marrow cellular apoptosis in AA was that there was an increase in the levels of pro-inflammtory cytokine secreted by the stromal microenvironment that potentially induced apoptosis by acting as FasL to Fas (Omokaro et al., 2009). Another possibility was that there was an immune-mediated destruction of BMNC by contact-mediated transmembrane signaling that was shown to involve the surface molecule Fas on target BMNC and its ligand FasL on cytotoxic T lymphocytes (CTLs), which were also able to kill up-regulated Fas expression bone marrow cells by a Fasbased mechanism (Wang et al., 2010). Caspase-3 and -8 cascades resulting in cell apoptosis were significantly reversed by TSDN. Based on these findings, we suggest that the recovery of hematopoiesis effect of TSDN against AA may be through the suppression of apoptosis. Taken together, these findings suggested that TSDN could efficiently alleviate AA through regulating the differentiation of T cell subsets, inhibiting inflammatory Th1cytokines, and anti-apoptosis.
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Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (Nos. 30873349, 81173415, and 81470982) and Tianjin Research Program of Application Foundation and Advanced Technology (No. 11JCYBJC12200).
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Morley, A., K. Trainor and J. Blake. A primary stem cell lesion in experimental chronic hypoplastic marrow failure. Blood. 45: 681–688, 1975. Nisar, A., N. Akhtar, A. Hassan, T. Banday, B. Wani and M.A. Zargar. Effect of Ajuga bracteosa on systemic T-cell immunity in Balb/C mice: Dual Th1/Th2 immunostimulatory effects. Am. J. Chin. Med. 42: 375–392, 2014. Omokaro, S.O., M.J. Desierto, M.A. Eckhaus, F.M. Ellison, J. Chen and N.S. Young. Lymphocytes with aberrant expression of Fas or Fas ligand attenuate immune bone marrow failure in a mouse model. J. Immunol. 182: 3414–3422, 2009. Passweg, J.R. and M. Aljurf. Treatment and hematopoietic SCT in aplastic anemia. Bone. Marrow. Transplant. 48: 161, 2013. Pugsley, C.A., I.J. Forbes and A.A. Morley. Immunologic abnormalities in an animal model of chronic hypoplastic marrow failure induced by busulfan. Blood. 51: 601–610, 1978. Scheinberg, P. Aplastic anemia: Therapeutic updates in immunosuppression and transplantation. Hematology. Am. Soc. Hematol. Educ. Program. 2012: 292–300, 2012. Scheinberg, P. and J. Chen. Aplastic anemia: What have we learned from animal models and from the clinic. Semin. Hematol. 50: 156–164, 2013. Schrezenmeier, H., J.R. Passweg, J.C. Marsh, A. Bacigalupo, C.N. Bredeson, E. Bullorsky, B.M. Camitta, R.E. Champlin, R.P. Gale, M. Fuhrer, J.P. Klein, A. Locasciulli, R. Oneto, A.V. Schattenberg, G. Socie and M. Eapen. Worse outcome and more chronic GVHD with peripheral blood progenitor cells than bone marrow in HLA-matched sibling donor transplants for young patients with severe acquired aplastic anemia. Blood. 110: 1397–1400, 2007. Sheng, W., C. Liu, R. Fu, H. Wang, W. Qu, E. Ruan, G. Wang, H. Liu, Y. Wu, J. Song, L. Xing, J. Guan, L. Li, H. Liu and Z. Shao. Abnormalities of quantities and functions of linker for activations of T cells in severe aplastic anemia. Eur. J. Haematol. 93: 214–223, 2014. Wang, T., R.C. Choi, J. Li, C.W. Bi, W. Ran, X. Chen, T.T. Dong, K. Bi and K.W. Tsim. Trillin, a steroidal saponin isolated from the rhizomes of Dioscorea nipponica, exerts protective effects against hyperlipidemia and oxidative stress. J. Ethnopharmacol. 139: 214–220, 2012. Wang, Y., Y. Liu, R. Han, Q. Li, Z. Yao, W. Niu, Y. Yuan, Z. Tang, Z. Zhu and Z. Shen. Monitoring of CD95 and CD38 expression in peripheral blood T lymphocytes during active human cytomegalovirus infection after orthotopic liver transplantation. J. Gastroenterol. Hepatol. 25: 138–142, 2010. Wang, Y.L., Y.Y. Zhang, G. Li, Z.Q. Tang, Y.L. Zhou, Z.J. Zhu and Z. Yao. Correlation of CD95 and soluble CD95 expression with acute rejection status of liver transplantation. World. J. Gastroenterol. 11: 1700–1704, 2005. Weston, W., V. Gupta, R. Adkins and R. Jurecic. New therapeutic approaches for protecting hematopoietic stem cells in aplastic anemia. Immunol. Res. 57: 34–43, 2013. Yoshikawa, M., F. Xu, T. Morikawa, Y. Pongpiriyadacha, S. Nakamura, Y. Asao, A. Kumahara and H. Matsuda. Medicinal flowers. XII.(1) New spirostane-type steroid saponins with antidiabetogenic activity from Borassus flabellifer. Chem. Pharm. Bull (Tokyo) 55: 308–316, 2007. Young, M.E., V. Potter, A.G. Kulasekararaj, G.J. Mufti and J.C. Marsh. Haematopoietic stem cell transplantation for acquired aplastic anaemia. Curr. Opin. Hematol. 20: 515–520, 2013. Young, N.S. Current concepts in the pathophysiology and treatment of aplastic anemia. Hematology. Am. Soc. Hematol. Educ. Program. 2013: 76–81, 2013. Young, N.S., P. Scheinberg and R.T. Calado. Aplastic anemia. Curr. Opin. Hematol. 15: 162–168, 2008. Yu, H., L. Zheng, L. Yin, L. Xu, Y. Qi, X. Han, Y. Xu, K. Liu and J. Peng. Protective effects of the total saponins from Dioscorea nipponica Makino against carbon tetrachloride-induced liver injury in mice through suppression of apoptosis and inflammation. Int. Immunopharmacol. 19: 233–244, 2014.
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Zhang, W.F. and B.S. Liu. The effect of Dioscorea nipponica Makino on bone marrow T-lymphocyte subsets in mice with aplastic anemia. Journal of Tianjin University of Traditional Chinese Medicine 30: 160–162, 2011. Zhao, S., K. Mei, L. Qian, Y. Yang, W. Liu, Y. Huang, C. Zhang, X. Sun, C. Liu, B. Li, F. Gao, J. Cai and J. Ni. Therapeutic effects of hydrogen-rich solution on aplastic anemia in vivo. Cell. Physiol. Biochem. 32: 549–560, 2013.
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Effects of the Total Saponins from Dioscorea nipponica on Immunoregulation in Aplastic Anemia Mice Yuliang Wang,* Tiangai Yan,† Lin Ma† and Baoshan Liu† *Department
of Clinical Laboratory Medicine, Tianjin First Central Hospital Key Laboratory for Critical Care Medicine of the Ministry of Health Tianjin 300192, China †Department
of Traditional Chinese Medicine Tianjin Medical University General Hospital, Tianjin 300052, China Published 18 March 2015
Abstract: Dioscorea nipponica Makino, a popular folk medicine, exerts anti-inflammation properties. The present study investigated the therapeutic effect of the total saponins from Dioscorea nipponica Makino (TSDN) on aplastic anemia (AA) and possible immune regulation mechanisms. Using a mouse model of AA, three different doses of TSDN were orally administrated for 14 consecutive days. We first demonstrated that TSDN was found to be effective in alleviating pancytopenia with a hypocellular bone marrow as compared with AA model group. Moreover, gastrogavage administration of a medium dose of TSDN was found to dramatically increase the percentage of CD4 þ cells in bone marrow nucleated cells (BMNC) and restore the CD4 þ /CD8 þ ratio. The pro-inflammatory cytokine concentrations of IL-2 and IFN- were significantly decreased, and anti-inflammatory cytokine IL-4 was significantly increased in culture supernatant of BMNC. Further investigations showed that TSDN obviously inhibited Fas–FasL-induced BMNC apoptosis as well as effectively suppressed intracellular apoptosis protein of caspase-3 and -8 expressions. Taken together, these findings suggested that TSDN could alleviate AA by elevating the CD4 þ /CD8 þ T-cell ratio, inhibiting inflammatory Th1-cytokines, and exerting anti-apoptosis effects. Keywords: Dioscorea nipponica; Saponins; Immune Regulation; Aplastic Anemia Mice; CD4 þ /CD8 þ Ratio; Pro-Inflammatory Cytokines.
Correspondence to: Dr. Baoshan Liu, Department of Traditional Chinese Medicine, Tianjin Medical University General Hospital, Heping District, Anshan Road 154, Tianjin 300052, China. Tel/Fax: (þ86) 22-6036-3599, E-mail: [email protected]
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Introduction Aplastic anemia (AA) is a bone marrow failure syndrome characterized by a peripheral blood pancytopenia with a hypocellular bone marrow (Young et al., 2013). Immune-mediated destruction of hematopoiesis has been identified as the important mechanism of AA. In most cases, an immune response dominated by oligoclonal expanded cytotoxic T-cells, which secrete pro-inflammatory cytokines such as TNF-, IFN-, and IL-6, targets hematopoietic stem and progenitor cells, inducing their death via apoptosis and hematopoietic failure (Chen et al., 2013; Scheinberg and Chen, 2013). Recently, numerous strategies have been applied in the treatment of AA, including immunosuppression and/or hematopoietic stem cell transplantation (Scheinberg, 2012; Young et al., 2013). Many lymphocytotoxic agents have been widely used, and however, some of them have potential adverse effects such as anaphylaxis fever, chest pain and diarrhea (Passweg and Aljurf, 2013). For hematopoietic stem cell transplantation, the type of allograft and the age of the patient are the most important factors affecting the outcome of treatment. These actualities make the transplantation less favorable when compared with immunosuppression as the age of AA patients increases (Schrezenmeier et al., 2007). In recent years, natural products from medicinal plants have attracted much attention as effective and safe alternative treatments for bone marrow failure (Gao and Chong, 2012). Steroidal saponins are widely spread throughout the plant kingdom, an example being Dioscorea nipponica Makino, which is a famous traditional Chinese medicinal plant that has diversified pharmacological efficacies (Lu et al., 2010). It has been widely used in China for relieving cough and asthma, eliminating rheumatic aches, alleviating pain, improving blood circulation, and having antilipemic and anticancer properties (Kim et al., 2011; Chien et al., 2012; Kwon et al., 2003; Lu et al., 2014). Recently, the total saponins from Dioscorea nipponica Makino (TSDN) was reported to exert a hepatoprotective effect against CCl4-induced liver damage in mice through the suppression of inflammation and apoptosis (Yu et al., 2014). However, no related experimental findings exist concerning the therapeutic effects of this plant on AA. The purpose of this study was to investigate whether TSDN administration could treat the AA mice effectively and explore the possible underlying immunomodulatory effect mechanisms. Materials and Methods Experimental Animals The pathogen free male ICR mice (18–23 g) were purchased from the Academy of Military Medical Science, Beijing, China (SPF Certificate No. SCXK2007-0004). The mice were used after one week of acclimatization. They were kept in a departmental animal housing in a well cross ventilated room at 24 C, relative humidity (44–56%), and a light–dark cycle of
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12 h during the experiments. All experimental procedures were approved by the Animal Care and Use Committee of Tianjin Medical University, and performed in strict accordance with the PR China Legislation Regarding the Use and Care of Laboratory Animals.
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Preparation of TSDN Dioscorea nipponica Makino was obtained from Beijing Tcmages Pharmaceutical Co., Ltd. (Beijing, China). D. nipponica extracts were prepared by initial condensation followed by lyophilization as described previously (Ho et al., 2011). The total saponins from airdried Dioscorea nipponica Makino extract were obtained according to the method of Yu et al. (2014). Determination of the marker compounds in the extract was performed by high-performance liquid chromatography (HPLC). Experimental Design ICR mice were randomly divided into a normal control group (n ¼ 15) and AA model group (n ¼ 75). The AA model was made according to the previous description (Chen et al., 2013). Briefly, the mice were irradiated with 2.0 Gy 60 Co and then treated with daily intraperitoneal injections of cyclophosphamide (Sigma-Aldrich, USA) at 50 mg/kg/ day and chloramphenicol (Sigma) at 62.5 mg/kg/day for the next three days. Then, the AA group was randomly divided into five groups: the low, medium, or high dose TSDN-treated groups, which received 37.44, 74.88 or 149.76 mg/kg/day TSDN, respectively; a positive control group treated with 23.5 mg/kg/day cyclosporine A (CsA) (Sigma); and an untreated model control group (model group). TSDN or CsA were administered by gastrogavage daily in each respective group for 14 consecutive days. The doses of TSDN were calculated according to the standard dose of total saponins extract used in the Pharmacology of Chinese Materia Medica (2000 Edition) for the effective and safe dosage of TSDN. Mice in the model group were given the same volume of saline. Each group contained 15 animals. On the 15th day, all mice were sacrificed under ether anesthesia. Bone marrow cells were extracted from bilateral femurs and tibiae, peripheral blood was obtained by retroorbital sinus bleeding using EDTA-coated blood collection tubes. Peripheral Hemogram and Bone Marrow Smear Study The hemoglobin (HGB) concentration, white blood cells (WBCs) and platelets (PLTs) counts were determined using a XT-1800iV Haematology Analyser (Sysmex, Kobe, Japan). Bone marrow smear was observed under light microscopy using routine Giemsa staining. Culture of Bone Marrow Nucleated Cells Bone marrow was flushed from the above-mentioned bones using a syringe containing RPMI-1640 media (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), and was mixed well with repeated pipetting. The bone marrow nucleated
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cells (BMNC) were isolated via density gradient centrifugation. The isolated BMNC were then depleted of RBCs using RBC-depletion buffer (BD-Biosciences, USA). Finally, the cells were incubated in RPMI-1640 media supplemented with FBS at 37 C in a 5% CO2 atmosphere for 24 hours.
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Flow Cytometric Analysis of BMNC Surface Receptor Expression mAbs for mouse CD3-FITC, CD4-PE/Cy5, CD8-PE, CD95 (Fas)-PE, and CD178 (FasL)PE, were obtained from BD Biosciences (BD Biosciences, USA) (Nisar et al., 2014). Cells were incubated with antibodies on ice for 30 min, washed twice with phosphatebuffered saline (PBS), resuspended, and phenotyped on flow cytometric analysis using the FACSCalibur flow cytometer (BD Biosciences). The ratio of CD4 þ /CD8 þ was calculated. Measurement of Cytokine Concentration in Culture Supernatant of BMNC BMNC suspensions were prepared as described above then adjusted to a concentration of 1 10 6 cells/ml. BMNC were cultured in RPMI-1640 media supplemented with 15% FBS at 37 C in 5% CO2 and saturated humidity. After 72 hours of cultivation, culture supernatant was harvested. The secretion levels of IL-2, IFN-, and IL-4 in culture supernatant of BMNC were measured with an ELISA kit (R&D Systems, USA) according to the manufacturer’s instructions. Light absorbance was measured using the EnSpire Multimode Plate Reader (PerkinElmer, USA), at 450 nm with a correction wavelength set at 570 nm. RT-PCR Assay for Relative Caspase-3 and -8 Gene Expression in BMNC Total RNA was extracted from BMNC with Trizol Reagent according to manufacturer’s instructions (Invitrogen). Reverse transcription to complementary DNA (cDNA) was undertaken with the use of random primer and Superscript RNase H-reverse transcriptase (Invitrogen). Polymerase chain reaction (PCR) was performed with the use of the ABI Prism 7500 Sequence Detector (Applied Biosystems) with a total reaction volume of 20 mL in 0.2 mL Microamp Optical Tubes and Strip Lids (Applied Biosystems) for a total of 40 cycles. The PCRs for caspase-3, -8, and β-actin were performed with the use of Table 1. Primer Sequences Used for RT-PCR Analysis Gene
Amplified Products Size
Sequence
Caspase-3
188 bp
Caspase-8
186 bp
β-Actin
183 bp
Sense: 5 0 -TGACTGGAAAGCCGAAACTCT-3 0 Antisense: 5 0 -CGACCCGTCCTTTGAATTTCT-3 0 Sense: 5 0 -AGATGACTTGAGCCTGCTTGA-3 0 Antisense: 5 0 -GGCAACTCTTCCCTTCCTTCA-3 0 Sense: 5 0 -GAGGCCCAGAGCAAGAGAGGT-3 0 Antisense: 5 0 -TTCACGGTTGGCCTTAGGGTT-3 0
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Universal PCR Mastermix (Applied Biosystems). Primers were synthesized with the sequences given in Table 1. The level of target molecule mRNA was expressed relative to that of the mRNA of β-actin.
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Western Blot Assay for Caspase-3 and -8 Protein Expression in BMNC Total protein was extracted from BMNC using a standard method. Western blot analysis was performed using primary antibodies against caspase-3, -8, and β-actin (Proteintech, USA) (Hu et al., 2013). The relative photographic densities were quantitated by scanning the photographic negatives using a gel documentation and analysis system (Alpha Innotech, USA). Statistical Analysis Data was presented as mean standard deviation (SD). The statistical analysis was performed by conducting one-way ANOVA using the SPSS software v.16.0 and Tukey in post hoc multiple comparisons was used to examine statistical significance (p < 0:05 and p < 0:01) between groups. Results TSDN Increased the Peripheral Hemogram and Bone Marrow Hyperplasia in AA Mice In order to confirm the recovery of the hematopoiesis effect of TSDN in the experimental AA group, a peripheral hemogram and bone marrow smear were determined following standard laboratory techniques. The results showed that the mean counts of WBCs and PLTs and the concentration of HGB were significantly lower in model group than in the normal control group. WBCs, PLTs, and HGB were all significantly higher in positive control group and TSDN-treated groups than in model group. Also note that, TSDN at the dose of 74.88 mg/kg considerably increased the peripheral hemogram, when compared with that of the low, high dose TSDN-treated groups, and positive control group (Fig. 1). The typical bone marrow architecture was observed in normal as well as in experimental groups through a bone marrow smear study. In AA (Fig. 2B), the marrow was filled with large fat cells (adipocytes) leaving scattered empty spaces in comparison with the normal marrow (Fig. 2A), which showed a densely packed distribution of cells. Bone marrow hyperplasia was significantly observed in medium dose TSDN-treated group compared to low, high dose TSDN-treated groups, and the positive control group (Fig. 2E). All these data indicated TSDN played potential role in alleviating AA. Immunomodulatory Effects of TSDN on T Lymphocyte Subset To evaluate the immunomodulatory effects of TSDN on the bone marrow immune system, percentages of T lymphocyte subsets in the BMNC were measured. As shown in Fig. 3, the
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Figure 1. The comparison of peripheral hemogram in each group. (A) The number of WBCs. (B) The number of PLTs. (C) The concentration of HGB. *p < 0:01 vs. normal control group, # p < 0:01 vs. model group, p < 0:01 vs. positive control group.
characterization of the lymphocyte subsets revealed a significant reduction in the percentage of total T cells (CD3 þ ), CD4 þ T cells as well as a significant increase of CD8 þ T cells in AA model groups compared to the normal control group. When compared with the AA model group, the percentage of CD3 þ , CD4 þ T cells and the ratio of CD4 þ /CD8 þ increased significantly, and the percentage of CD8 þ T cells significantly decreased in the medium dose TSDN-treated group (Fig. 3). Inflammatory Suppression Effects of TSDN The expression levels of IL-2 and IFN- in culture supernatant of BMNC were significantly increased in model control group compared with those in normal control group and each treated group, whereas the expression levels of IL-2 and IFN- in the medium dose TSDN-treated group were significantly decreased compared with those in positive control
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(A)
(D)
(B)
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(C)
(F)
Figure 2. Bone marrow smear in each group. (A) Giemsa staining of bone marrow smear showing densely packed cellular distribution in normal condition. (B) AA bone marrow smear exhibiting frequent appearance of large fat cells (adipocytes) with empty spaces. (C) Positive control bone marrow smear exhibiting relatively higher hyperplasia activity and visible nucleated cells. (D) Low dose TSDN-treated bone marrow smear exhibiting relatively higher hyperplasia activity. (E) Medium dose TSDN-treated bone marrow smear exhibiting considerably higher hyperplasia activity. (F) High dose TSDN-treated bone marrow smear exhibiting significantly higher hyperplasia activity (400 magnification).
group. The expression levels of IL-4 were significantly decreased in the model control group compared with those in normal control group and each treated group, whereas the expression levels of IL-4 in the medium dose TSDN-treated group were significantly increased compared with those in the positive control group (Fig. 4). Anti-Apoptosis Effects of TSDN Evaluation of the BMNC apoptosis was characterized by extracellular Fas/FasL and the intracellular apoptosis protein caspase-3, -8 assay. The expression levels of Fas and FasL were significantly increased in the AA model group compared with those in normal control group, whereas the expression levels of Fas and FasL in medium-, high-dose TSDN-treated groups were significantly decreased compared with those in model group (Fig. 5). Compared with normal control group, the mRNA and protein expression levels of intracellular apoptosis protein caspase-3 and -8 were significantly increased in the AA model group, which were obviously reversed by TSDN-treated groups (Figs. 6 and 7).
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(A)
(B) Figure 3. Effect of TSDN administration on T cells. (A) The representative experiment shows the percentages of CD3 þ , CD4 þ , and CD8 þ T cells by flow cytometry. (B) The comparison of the percentages of CD3 þ , CD4 þ , CD8 þ T cells and the ratio of CD4 þ /CD8 þ in each group. *p < 0:05 vs. normal control group, **p < 0:01 vs. normal control group, p < 0:05 vs. model group, # p < 0:01 vs. model group, £ p < 0:05 vs. positive control group.
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Figure 4. Anti-inflammatory effects on TSDN administration in culture supernatant of BMNCs. *p < 0:01 vs. normal control group, **p < 0:05 vs. normal control group, # p < 0:01 vs. model group, p < 0:05 vs. positive control group.
(A) Figure 5. Anti-apoptosis effects of TSDN administration on BMNCs. (A) The representative experiment shows the expression of Fas and FasL by flow cytometry. (B) The comparison of the expression levels of Fas and FasL in each group. *p < 0:01 vs. normal control group, **p < 0:05 vs. model group, # p < 0:01 vs. model group, p < 0:01 vs. positive control group.
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(B) Figure 5. (Continued )
Figure 6. Anti-apoptosis effects of TSDN on gene expression of intracellular apoptosis protein. *p < 0:05 vs. normal control group, # p < 0:05 vs. model group.
Discussion AA is characterized by hypocellular bone marrow resulting from hematopoietic stem cell damages. It results in pancytopenia with risk of severe anemia, major hemorrhage, and lifethreatening infection (Benson-Quarm et al., 2014). Current data have suggested that the pathophysiology of AA is largely mediated by immune dysfunction, in which impaired activation, differentiation and increased cellular apoptosis in the AA bone marrow play critical roles (Young et al., 2013). Therapies, including immunosuppression and/or bone marrow transplantation, have a clinical therapeutic effect in approximately 50% of AA patients. However, a large portion of patients may fail to respond to the treatments or relapse after the treatment (He et al., 2011). Recently, Dioscorea nipponica Makino, a Chinese medicinal plant, has received more attention as the saponins, the main bioactive compounds in this herb, have shown various
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Figure 7. Anti-apoptosis effects of TSDN on the expression of intracellular apoptosis protein. (A) The protein expression of caspase-3 in each group by western blot. (B) The protein expression of caspase-8 in each group by western blot. *p < 0:05 vs. normal control group, # p < 0:05 vs. model group.
pharmacological activities (Yoshikawa et al., 2007; Ho et al., 2011; Wang et al., 2012; Lv et al., 2013) including relieving cough and asthma, eliminating rheumatic aches, alleviating pain, improving blood circulation, and having antilipemic and anticancer capabilities by enhancing anti-oxidant, antivirus, suppressing inflammation and apoptosis, and anti metastasis mechanisms. In the present study, we demonstrate that TSDN administration can promote the recovery of hematopoiesis in the AA model mice. The scanty peripheral hemogram is the evidence of bone marrow failure characterized by damaged stem cell population that has been termed as “residual injury” (Pugsley et al., 1978). The characteristics of “residual injury” are: it is permanent, there is impairment of stem cell proliferation leading to moderate marrow failure and complete failure of stem cell proliferation may ensue leading to severe marrow failure and death (Morley et al., 1975). The results of our study showed that the peripheral blood pancytopenia includes neutropenia, thrombocytopenia, declined HGB level, signifies hypoplastic marrow failure in the mouse model of AA (Zhao et al., 2013). At around 2 weeks after gastrogavage of TSDN, the peripheral blood started showing the hypoplastic marrow failure reversal, which was significantly evident on the 15th day after medium dose TSDN treatment (time of our experiment). Bone marrow smear study revealed a typical AA bone marrow feature with frequent presence of large fat cells (adipocytes) with empty spaces, when compared with the normal healthy marrow. Bone marrow hyperplasia after medium dose TSDN treatment was significantly observed which is consistent with peripheral hemogram. AA is an immune-mediated disease. The disorder of immune system especially T cells plays an important role in the development of AA. Therefore, immune reconstitution is very important in the treatment of AA (Weston et al., 2013). Our results showed that the
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oral administration of TSDN preparation caused a significant increase in the percentages of CD3 þ , T-helper cells (CD4 þ Th cells) and a decrease in the percentage of T-cytotoxic/ suppressor cells (CD8 þ Tc cells) in AA mice BMNC, respectively. Accordingly, TSDN treatment has resulted in a rapid recovery of the ratio of CD4 þ /CD8 þ under immunosuppressed conditions. Moreover, CD3 þ , CD4 þ T cells and CD4 þ /CD8 þ in AA mice even returned to normal after consecutive treatment with medium dose TSDN, indicative of immune restorative activity of this preparation (Zhang and Liu, 2011). Activated T cells were differentiated into CD4 þ Th1/Th2 cells and CD8 þ cells. All those cells play crucial roles in AA (Sheng et al., 2014). On one hand, Th1 cells can secrete a variety of immune molecules including IFN-, IL-2 directly. On the other hand, Th1 cells can promote the activation of NK cells, CD8 þ cells, and macrophage cells which could secrete a variety of cytokines, including IFN-, TNF-, and IL-2, and mediate apoptosis (Chen et al., 2014). The results of our study showed that the effector cells within the lymphocyte subset are activated cytotoxic T cells bearing a Th1 profile, expressing and secreting IL-2 and IFN- in the mouse model of AA, which would provide a favorable immunologic milieu for bone marrow destruction. As a result, bone marrow cells are significantly impaired and lose the capacity to proliferate and differentiate. The present study showed that the pro-inflammatory Th1 cytokines IL-2 and IFN- were significantly down-regulated, as well as anti-inflammatory Th2 cytokine IL-4 was significantly upregulated 14 days after treatment with TSDN. Apoptosis or programmed cell death is the most discussed pathophysiologic mechanism associated with AA progression (Chatterjee et al., 2010). The fine equilibrium between the differentiation and apoptosis of normal hematopoietic cells is altered in AA. The increased bone marrow cellular apoptosis leads to the increased occurrences of AA. The interaction of death receptor protein Fas and FasL has been shown to be a key regulator of apoptosis and major activation-induced cell death (AICD) (Wang et al., 2005). In the present study, we found that Fas and FasL played a major role in upregulated BMNC apoptosis in AA and were subsequently suppressed by TSDN treatment. One possibility to explain bone marrow cellular apoptosis in AA was that there was an increase in the levels of pro-inflammtory cytokine secreted by the stromal microenvironment that potentially induced apoptosis by acting as FasL to Fas (Omokaro et al., 2009). Another possibility was that there was an immune-mediated destruction of BMNC by contact-mediated transmembrane signaling that was shown to involve the surface molecule Fas on target BMNC and its ligand FasL on cytotoxic T lymphocytes (CTLs), which were also able to kill up-regulated Fas expression bone marrow cells by a Fasbased mechanism (Wang et al., 2010). Caspase-3 and -8 cascades resulting in cell apoptosis were significantly reversed by TSDN. Based on these findings, we suggest that the recovery of hematopoiesis effect of TSDN against AA may be through the suppression of apoptosis. Taken together, these findings suggested that TSDN could efficiently alleviate AA through regulating the differentiation of T cell subsets, inhibiting inflammatory Th1cytokines, and anti-apoptosis.
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Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (Nos. 30873349, 81173415, and 81470982) and Tianjin Research Program of Application Foundation and Advanced Technology (No. 11JCYBJC12200).
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