CLINICAL STUDY TOPIC. Abstract

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1 Online Submissions: J Tradit Chin Med 1 August 15; (4): info@journaltcm.com ISSN JTCM. All rights reserved. CLINICAL STUDY TOPIC Efficacy and advantages of modified Traditional Chinese Medicine treatments based on "kidney reinforcing" for chronic aplastic anemia: a randomized controlled clinical trial Wu Dijiong, Shen Yiping, Ye Baodong, Fang Bingmu, Lin Shengyun, Chen Zhilu, Jiang Huifang, Feng Changwei, He Lüyuan, Gao Yanting, Liu Yonglin, Liu Yonghua, Zhu Jiajia, Wu Liqiang, Shao Keding, Zhou Yuhong aa Wu Dijiong, Shen Yiping, Ye Baodong, Lin Shengyun, Gao Yanting, Liu Yonglin, Zhu Jiajia, Wu Liqiang, Shao Keding, Zhou Yuhong, Department of Hematology, Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou 1, China Fang Bingmu, Liu Yonghua, Department of Hematology, People' s Hospital of Lishui City, Lishui, China Chen Zhilu, Jiang Huifang, Department of Hematology, Tongde Hospital of Zhejiang Province, Hangzhou 17, China Feng Changwei, Department of Hematology, Taizhou Hospital of Zhejiang Province, Taizhou 1, China He Lüyuan, Department of Hematology, Jinhua Hospital of Traditional Chinese Medicine, Jinhua 117, China Supported by Special Item of Important Disease of Zhejiang Province Traditional Chinese Medicine Sic-Tech Innovation Platform (Effect and Mechanism of Traditional Chinese Medicine on the Treatment of Aplastic Anemia and the Funding of Traditional Chinese Medicine Assessment Criterion, No. 9ZDJB1); Subject of Key Sic-Tech Innovation Team of Zhejiang Province (Clinical Study on Treatment of Chronic Aplastic Anemia by Tonifying Kidney and Promoting Blood Circulation, No. 11R94-); Special Research Funds for Traditional Chinese Medicine Industry (Effect of Traditional Chinese Medicine on the Treatment of Risk Factors of Chronic Aplastic Anemia, No. 1171); Special Research Funds for Traditional Chinese Medicine Industry (Clinical Study on the Diagnosis and Treatment of Aplastic Anemia Based on the Syndrome And Stage Differentiation, No. 1471); Zhejiang Provincial Traditional Chinese Medicine Administration Bureau Program (Establishment of Traditional Chinese Medicine Clinical Pathway on Aplastic Anemia, No. 1ZA9) Correspondence to: Prof. Shen Yiping, Department of Hematology, Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou 1, China. shenyp_@1. com; Prof. Ye Baodong, Department of Hematology, Zhejiang Provincial Hospital of Traditional Chinese Medicine, Hangzhou 1, China @1.com Telephone: Accepted: February, 1 Abstract OBJECTIVE: To compare the efficacy of modified treatments based on "kidney reinforcing" in the management of chronic aplastic anemia (CAA), and explore their advantages and specialties. METHODS: One hundred and eleven patients with CAA were randomly divided into three groups: kidney reinforcing alone (), "kidney reinforcing and Qi tonifying" (), and "kidney reinforcing and blood circulation invigorating" (). Normal and positive control groups were also formed. All patients were treated for months (two courses). Hemograms, Traditional Chinese Medicine (TCM) syndrome scores, and therapeutic effects were assessed, and changes in T-lymphocyte subsets, regulatory T cells and cytokines were detected. RESULTS: The and groups had lower TCM syndrome scores than the positive control group after months (P <.5). The group had a higher overall efficacy than the positive control group after months (P <.5), while platelet counts increased in the group after months (P <.5). CD+ T-lymphocyte ratios decreased only in the group, while CD + CD4 + CD Tlymphocytes increased only in the group after months (P <.5). Levels of interferon-γ, tumor necrosis fac- JTCM www. journaltcm. com 44 August 15, 1 Volume Issue 4

2 tor-α, interleukin (IL)- and IL- decreased and levels of IL-4 and IL-1 increased in all treated groups after months. Levels of IL- in the and groups were lower than those in the positive control group (P <.5). CONCLUSION: Treatments based on kidney reinforcing have a rebalancing effect on cytotoxic and T helper cells, and regulate expression of interferon-γ, IL-, IL- and IL-4. may be more effective in treating CAA, and may have an advantage in platelet recovery. 1 JTCM. This is an open access article under the CC BY-NC-ND license ( Key words: Anemia, aplastic; Reinforcing Qi strengthening kidney; Reinforcing kidney nourishing blood; Randomized controlled trial INTRODUCTION Aplastic anemia (AA) is a commonly encountered hematological disease that presents with bone marrow failure and pancytopenia. 1 AA can be divided into acute AA and chronic AA (CAA), according to the development and severity of the disease. CAA characteristics include long disease course, predisposition to relapse and refractoriness, and poor quality of life; these are ascribed to the maintenance of drug therapy and the persistent risk of infection and bleeding. It is reported that the incidence of AA is /1 per year in western countries, while in China, the rate is nearly triple (~7.4 / 1 ); CAA comprises almost % of these cases. In Traditional Chinese Medicine (TCM), AA is called "Suilao", which means exhaustion of marrow. The application of TCM in the treatment of AA began 5 years ago. Before the 19s, it was claimed that treatment of CAA should be based on "spleen (Pi)", and "fortify the spleen and replenish Qi" was emphasized, although limited effectiveness was achieved. After the 19s, the consensus was that treatment of CAA should be based on "kidney reinforcing", and the method of "kidney warming and essence replenishing" was recommended. To date, many studies have explored the mechanisms behind "kidney deficiency" in the development of AA, such as "Qi deficiency", "Yin deficiency", "blood stasis", and "stagnation of liver-qi", 4 yet no consensus has been achieved, and the actual efficacy is not well established. In our center, we treat CAA with the unification of disease and syndrome differentiation. Reinforcing Qi and activating blood is highlighted, followed by kidney reinforcing. 5, To clarify and compare the efficacy and advantages of modified TCM treatments based on kidney reinforcing with or without "Qi tonifying" or "blood activating", a multicenter randomized controlled trial was conducted. MATERIALS AND METHODS General data From April 1 to April 1, 15 patients with CAA visited Zhejiang Provincial Hospital of Traditional Chinese Medicine, People's Hospital of Lishui, Tongde Hospital of Zhejiang Province, Taizhou Hospital of Zhejiang Province, and Jinhua Hospital of Traditional Chinese Medicine. One hundred and eleven of these patients were enrolled in this study (1 patients were excluded for not meeting the inclusion criteria, and the other 1 declined to receive TCM treatment). Simple randomization was used following a computer-generated random number sequence. Participants and practitioners were not blinded to allocation. All 111 patients were randomly divided into three groups of 7: kidney reinforcing alone (), kidney reinforcing and Qi tonifying (), and kidney reinforcing and blood circulation invigorating (). Fifteen healthy people were enrolled in a normal control group, and another 1 patients without TCM treatment were enrolled as a positive control group. This study followed the Declaration of Helsinki and was approved by the Institution Committee of Ethics of Zhejiang Traditional Chinese Medicine Hospital. All the patients gave informed consent to treatment. Diagnostic standards Diagnostic standards in Western Medicine: AA was diagnosed according to the "Diagnosis and Treatment Standards of Hematology Disease (third edition)". 7 Patients with CAA present with: (a) slower progression than acute AA, and slight manifestation of anemia, infection and hemorrhage; (b) decreased hemoglobin, reticulocyte, neutrophil and platelet counts (but not to the same extent as that seen in acute AA); (c) decreased proliferation of two or three hematopoietic cell lines in the bone marrow, dysplasia at more than one site, significantly reduced numbers of megakaryocytes, and increased numbers of nonhematopoietic cells; and (d) a type of severe AA diagnosed upon confirmation of progressive disease. Kidney Yang deficiency syndrome differentiation in TCM: Patients presented with soreness and weakness in the waist and knees, cold body and limbs, pale complexion, poor appetite, diarrhea, plump tongue with whitish and slippery furring and bearing dental indentations on the margin, and a forceless deep pulse, which were diagnosed with kidney Yang deficiency according to "Guidelines for Diagnosis and Treatment of Common Internal Disease in Chinese Medicine". Standards for inclusion (a) Patients conformed to AA diagnostic criteria in Western Medicine and kidney Yang deficiency syndrome differentiation in TCM. (b) Patients with no other diseases present with pancytopenia, such as myelodysplastic syndrome, primary myelofibrosis, paroxys- JTCM www. journaltcm. com 45 August 15, 1 Volume Issue 4

3 mal nocturnal hemoglobinuria and leukemia. (c) Patients aged 14- years. (d) Patients voluntarily participating in the study and giving signed informed consent. Standards for exclusion (a) Patients who were pregnant or breastfeeding. (b) Patients with other serious diseases (cardiovascular and cerebrovascular diseases, liver and renal dysfunction, severe infection, and mental illness). (c) Patients presenting with other hematological disease or malignancy. (d) Patients presenting with complex TCM syndrome. Therapies Treatment based on TCM: (a) Patients in the group were treated with modified "Yougui decoction", which comprised Prepared Dihuang (Radix Rehmanniae) 15 g, Rougui (Cortex Cinnamomi Cassiae) g, Fuzi (Radix Aconiti Lateralis Preparata) 4 g, Yinyanghuo (Herba Epimedii Brevicornus) g, Xianmao (Rhizoma Curculiginis) g, Lujiaojiao (Colla Cornus Cervi) 1 g, Duzhong (Cortex Eucommiae) 1 g, Shanzhuyu (Fructus Macrocarpii) 15 g, Prepared Heshouwu (Radix Polygoni Multiflori) 1 g, Huangjing (Rhizoma Polygonati Sibirici) 15 g, Shanyao (Rhizoma Dioscoreae Oppositae) 15 g, Gouqizi (Fructus Lycii) 15 g, Chenpi (Pericarpium Citri Reticulatae), and Sharen (Fructus Amomi) g. (b) Patients in the group were treated with Yougui decoction combined with Huangqi (Radix Astragali Mongolici) 5 g, Dangshen (Radix Codonopsis) 1 g and Baizhu (Rhizoma Atractylodis Macrocephalae) 15 g. (c) Patients in the group were treated with Yougui decoction combined with Danggui (Radix Angelicae Sinensis) g, Chishao (Radix Paeoniae Rubra) g, Taoren (Semen Persicae) g, Danshen (Radix Salviae Miltiorrhizae) 1 g and Jixueteng (Caulis Spatholobi) 1 g. Maintenance of Western Medicine: all patients enrolled continued with their Western Medicine treatment protocols: androgen, including stanozolol (4- mg/day tid per os), andriol (-1 mg/day per os), and others (such as danazol and testosterone propionate). The dose of cyclosporine A was - mg kg - 1 d - 1, and the serum concentration was maintained at 15-5 ng/l. Supporting treatment: patients received erythrocyte suspension transfusion when hemoglobin was < 75 g/l or if they had significant symptoms of anemia even when hemoglobin was just below g/l. Platelet transfusion and hemostatic treatment were administered when platelet count was < /L (with no bleeding risk factor) or <. 1 1 /L with bleeding risk factors. Anti-infective therapy was followed according to the site and severity of infection. Treatment period: each treatment course lasted months, and two courses were followed. Indexes and methods for observation Common index: gender, age, body height and weight, vital signs, history of present illness, history of past illness, and frequency of blood transfusion in the last months were recorded. Safety index: routine tests of blood, urine and stools, stool occult blood, abdominal ultrasound, electrocardiography, liver and renal function, and serum concentration of cyclosporine (if necessary) were assessed at the beginning and end of the trial. Curative effect index: (a) Hemogram. Patients were checked routinely for blood and reticulocytes every weeks. (b) Assessment of TCM syndrome. TCM syndrome scores of all patients were calculated according to the Scheme of Clinical Diagnosis and Treatment of Suilao, published by the State Administration of Traditional Chinese Medicine in 1. Symptoms of the major syndrome include pale face, dizziness, palpitations, and shortness of breath, while symptoms of the minor syndrome include feverish palms and soles, hectic fever and night sweating, dipsia, yellow urine, cold body, anorexia, and diarrhea. According to the severity (light, moderate or severe) of main and minor syndromes,,, 4 and points or, 1, and points were scored respectively during the statistical process. Efficacy evaluation criteria: criteria in Western Medicine. (a) Basically cured: disappearance of anemia and bleeding, with a hemoglobin level of 1 and 1 g/l in male and female patients, respectively, without recovery within 1 year. (b) Remission: disappearance of anemia and bleeding, with a hemoglobin level of 1 and 1 g/l in male and female patients, respectively. The white blood cell (WBC) count reaches /L with an improvement in platelet count. Disease stays stable or improved in the following months. (c) Improved: major improvement in the appearance of anemia and bleeding, transfusion independent, with a > g/l increase of hemoglobin. Disease stays stable for months. All three criteria should be assessed without transfusion in the last months. (d) Invalid: symptoms and hemogram have not met the definition of improved after therapy. Criteria in TCM. [Score Improving Rate (SIR) = (score before treatment score after treatment) / score before treatment 1% ] (a) Clinical recovery: disappearance or basically disappearance of TCM clinical symptoms and signs, with SIR 9%. (b) Notable efficacy: disappearance or basic disappearance of TCM clinical symptoms and signs, with SIR < 9% and 7%. (c) Efficacy: improvement of TCM clinical symptoms and signs, with SIR < 7% and %. (d) Invalid: no improvement in TCM clinical symptoms and signs, with SIR < %. Immune function detection: T-lymphocyte subsets, regulatory T cells (CD4 + CD5 + FoxP +), were detected before and after the trial, and cytokines [e.g., interleukin (IL)-, IL-4, IL-, IL-1, tumor necrosis factor (TNF)-α and interferon (IFN)-γ] were detected every months. All these indices were detected in the Department of Clinical Laboratory of Zhejiang Provincial Hospital of Traditional Chinese Medicine. T-lympho- JTCM www. journaltcm. com 4 August 15, 1 Volume Issue 4

4 cyte monoclonal antibodies were purchased from BD Pharmingen (San Diego, CA, USA) and the human cytokine fluorescent reagent kits were purchased from Millipore (Boston, MA, USA). Side effect observation: commonly encountered side effects ascribe to the application of immune suppression and androgen, including gastrointestinal adverse reactions, secondary hypertension, polytrichia, gingival hyperplasia, rash, liver and renal dysfunction, and hand tremor, were observed. Statistical analysis The compiled data were first entered into an Excel spreadsheet, and then read into SPSS version 15. (SPSS, Chicago, IL, USA) biometric statistical software for further analysis. Normal data were expressed as the mean (standard deviation), and non-normal data were expressed as the median (interquartile range). Variance and pairwise comparisons were used for normal data, whereas non-normal data were subjected to nonparametric tests, wherein the Kruskal-Wallis H test was used for pairwise comparisons and the Mann-Whitney U test was used for multiple comparisons. P <.5 was considered statistically significant and P <.1 was considered statistically very significant. RESULTS Features of patients During the observation (Figure 1), 17 of 111 patients were withdrawn: four in the group; six in the group; and seven in the group. Patients in the group (n = ) were aged 15- years (median age 9.5 years), and included 1 men and 1 women. Patients in the group (n = 1) were aged 1-7 years (median age. years), and included 11 men and women. Patients in the group (n = ) were aged years (median age 5.5 years), and included eight men and 17 women. The positive control group (n = 1) was aged years (median age 5.5 years), and included five men and 11 women. The normal control group (n = 15) was aged 4-5 years (median age 1. years), and included seven men and eight women. There was no significant difference in general data among the groups (P >.5) (Table 1). Comparison of hemograms There were no differences in the hemograms of patients in the four groups before treatment (P >.5). After months, the WBC counts increased significantly only in the group, from.9 ± /L to.7 ± /L (P <.5), which was higher than that in the positive control and groups (.5 ± /L and. ±. 1 9 /L, respectively) (P <.5). The level of hemoglobin (Hb) increased in all three TCM groups combined (, and ) (P <.5), whereas in the group, the difference was notable only when compared with Hb levels in the positive control group (P <.5). No differences were observed among all three TCM groups combined. Platelet counts did not increase in any group at the end of the first course of treatment. After months, the increase in WBC Assessed for eligibility (n = 15) Patients decline to receive TCM (n = 1) Excluded (n = 47) Not meeting inclusion criteria (n = 1) Decline to receive TCM (n = 1) Zhejiang Provincial Hospital of TCM (n = 45) People's Hospital of Lishui (n = ) Tongde Hospital of Zhejiang Province (n = 1) Taizhou Hospital of Zhejiang Province (n = ) Jinhua Hospital of TCM (n = 1) Randomized (n = 111) Maintenance of Western Medicine Compared group received maintenance of Western Medicine (n = 1) Allocated to group received kidney reinforcing alone (n = 7) Allocated to group received kidney reinforcing and Qi tonifying (n = 7) Allocated to group received kidney reinforcing and blood circulation invigorating (n = 7) Discontinued intervention for discontinued hospital visit (n = ) Discontinued intervention for discontinued hospital visit (n = 4) Discontinued intervention for discontinued hospital visit (n = ) Discontinued intervention for discontinued hospital visit (n = 7) Analysed (n = 1) Analysed (n = ) Analysed (n = 1) Analysed (n = ) Figure 1 Flow diagram of the progress through the phases of the randomized trial TCM: Traditional Chinese Medicine. JTCM www. journaltcm. com 47 August 15, 1 Volume Issue 4

5 Table 1 General clinical data of patients Male 1 (.4) 11 (5.5) 1 (4.) 5 (1.) Gender [n (%)] Normal control 7 (4.7) (5.) 1. (4-5) Notes: patients with chronic aplastic anemia (CAA) were randomly divided into three groups: "kidney reinforcing alone ()", "kidney reinforcing and Qi tonifying ()" and "kidney reinforcing and blood circulation invigorating ()". Patients in the TCM treated groups were treated with modified "Yougui decoction", with the addition of kidney Yin tonifying herbs in group, Qi tonifying herbs in group and blood circulation invigorating herbs in group. Normal and positive control groups were also formed. Patients in normal control group were treated without anything, and patients in positive control group were treated with western medicine alone (androgen with or without cyclosporine A for six months) respectively. All patients were treated for months (two courses). The general data of the five groups were compared with each other. Data are shown as case (%), and no significant differences were observed. Female 1 (.) (4.5) 17 (5.7) 11 (.) Age (years) 9.5 (15-). (1-7) 9. (15-74) 5.5 (17-59) count was still not obvious in all groups, except the group (P <.5). The level of Hb increased in all groups (P <.5), and it was higher when compared with that months ago only in the group (P <.5). Platelet counts increased significantly only in the group when compared with counts before treatment. No differences were observed among the three TCM groups combined (Table ). Comparison of therapeutic effect in Western Medicine After the first course of treatment ( months), overall efficacy (OE) was 45.5%, 1.%, 5.7% and.% in the,, and positive control groups, respectively. There was a significant difference between the and positive control groups (P <.5). No differences were observed among the other groups. The OE was 9.7%, 77.4%,.% and 5.%, respectively, in the,, and positive control groups after the second course of treatment ( months), and there was improvement in the and groups when compared with months previously (P <.5). The OE of the group was higher than that in the positive control group after months' treatment (P <.5). No differences were observed among the three TCM groups combined (Table ). Table Comparison of hemograms ( xˉ ± s) Time Treated with months Treated with months Treated with months WBC (1 9 /L).±.9.5±.7.±.7.9±1.1.7±1.7 ac.±1.5 ac.5±1..±. b.±1..1±1..5±1. Hb (g/l).±4. 5.1±. a 9.5±5. a.4±5. 9.1±.1 ac 9.1±.4 cde 7.±5.5 7.±. a 91.±7. a 5.1±4.9 7.±.7 PLT (1 9 /L).9±. 9.9±. 4.5±..9± ± ± ±4. 5.± ±47. a 7.±9. 9.±7. Treated with months.±. 7.±. a.7±5.5 Notes: routine blood tests in the (kidney reinforcing alone), (kidney reinforcing and Qi tonifying), (kidney reinforcing and blood circulation invigorating) and positive control groups were performed before treatment and and months after. Patients in the TCM treated groups were treated with modified "Yougui decoction", with the addition of kidney Yin tonifying herbs in group, Qi tonifying herbs in group and blood circulation invigorating herbs in group. Patients in positive control group were treated with western medicine alone (androgen with or without cyclosporine A for six months). Hb: hemoglobin; PLT: platelet; WBC: white blood cell; TCM: Traditional Chinese Medicine. a P <.5, d P <.1, compared with that before treatment in the same group; b P <.5, compared with that in the group at the same time; c P <.5, compared with that in the positive control group at the same time; e P <.5, compared with that treated with months in the same group. JTCM www. journaltcm. com 4 August 15, 1 Volume Issue 4

6 Table Comparison of therapeutic effect in Western Medicine Course of treatment (months) Basically cure Remission 5 Improve 1 Invalid 1 Overall efficacy [n (%)] 15 (45.5) (9.7) a (1.) b (77.4) (5.7) 1 4 (.) ab (.) 5 (5.) Notes: therapeutic effect of Western Medicine in the (kidney reinforcing alone), (kidney reinforcing and Qi tonifying), (kidney reinforcing and blood circulation invigorating) and positive control groups was evaluated after treatment for and months. Patients in the TCM treated groups were treated with modified "Yougui decoction", with the addition of kidney Yin tonifying herbs in group, Qi tonifying herbs in group and blood circulation invigorating herbs in group. Patients in positive control group were treated with western medicine alone (androgen with or without cyclosporine A for six months). TCM: Traditional Chinese Medicine. a P <.5, compared with that after months' treatment in the same group; b P <.5, compared with that in the positive control group at the same time. Comparison of TCM syndrome score No differences were observed in the TCM syndrome score of each group (,, and positive control groups) before treatment (1. ± 4., 11.9 ±., 11.7 ± 4.4 and 11. ± 4.7, respectively, P >.5). All the TCM groups combined (, and ) showed improvement in TCM syndrome when treated for or months. All syndrome scores decreased significantly (P <.1) and more obviously after months (P <.5). In the positive control group, the score had a notable decrease only after the second course of treatment ( months). Additionally, the score was lower in the and groups when compared with that in the Table 4 Comparison of TCM syndrome score ( xˉ ± s) Time positive control group after months (P <.5), while no differences were observed among the other groups (P >.5) (Table 4, Figure ). Comparison of therapeutic effect in TCM OE was 57.%, 7.7%,.% and 5.% in the,, and positive control groups, respectively, after months' treatment. After the second course of treatment ( months), OE was 7.%, 7.1%,.% and.5%, respectively, in the four groups, and there was an improvement in the group when compared with months previously (P <.5). No significant differences were observed between the three TCM groups combined (Table 5). Syndrome score Treated with months Treated with months Treated with months 1.±4. 9.±. a.±. ab 11.9±. 7.±.9 a 5.7±. abc 11.7±4.4.4±.4 a 5.9±.1 abc 11.±4.7 1.±4. Treated with months 7.±.9 ab Notes: the TCM syndrome scores in the (kidney reinforcing alone), (kidney reinforcing and Qi tonifying), (kidney reinforcing and blood circulation invigorating) and positive control groups were evaluated before treatment and and months after. Patients in the TCM treated groups were treated with modified "Yougui decoction", with the addition of kidney Yin tonifying herbs in group, Qi tonifying herbs in group and blood circulation invigorating herbs in group. Patients in positive control group were treated with western medicine alone (androgen with or without cyclosporine A for six months). TCM: Traditional Chinese Medicine. a P <.1, compared with that before treatment in the same group; b P <.5, compared with that after months' treatment in the same group; c P <.5, compared with that in the positive control group at the same time. JTCM www. journaltcm. com 49 August 15, 1 Volume Issue 4

7 TCM syndrome score a ab group group group group s Figure TCM syndrome scores of the group, group, group and the positive control group were evaluated before and and months after treatment Patients in the TCM treated groups were treated with modified "Yougui decoction", with the addition of kidney Yin tonifying herbs in group, Qi tonifying herbs in group and blood circulation invigorating herbs in group. Patients in positive control group were treated with western medicine alone (androgen with or without cyclosporine A for six months). TCM: Traditional Chinese Medicine; : kidney reinforcing alone; : kidney reinforcing and Qi tonifying; : kidney reinforcing. a P <.1, compared with that before treatment in the same group; b P <.5, compared with that at months after treatment in the same group; c P <.5, compared with that in the positive control group at the same time. Comparison of T-lymphocyte subsets and regulatory T cells No differences in the ratio of T-lymphocyte subsets and regulatory T cells were observed among the three TCM groups combined and the positive control group. The ratio of CD+ T lymphocytes decreased only in the group after months of treatment, while CD + CD4 + CD T lymphocytes increased only in the group after months of treatment (P <.5). All the TCM groups combined had a decreased level of CD + CD4 CD + T lymphocytes (P <.1), while it increased in the positive control group after months of treatment (P <.1). The CD + CD4 + CD5 + FoxP+ regulatory T cell/cd+cd4+ ratio decreased in all treated groups after months, while no significant differences were observed (Table ). Comparison of cytokines Levels of IFN-γ, TNF-α, IL-, IL-4, IL- and IL-1 were higher in patients with CAA when compared a abc Table 5 Comparison of therapeutic effect of TCM Course of treatment (month) a abc Clinical recovery ab Treated with months Notable efficacy (.5) Notes: the therapeutic effects of TCM in the (kidney reinforcing alone), (kidney reinforcing and Qi tonifying), (kidney reinforcing and blood circulation invigorating) and positive control groups were evaluated after treatment for and months. Patients in the TCM treated groups were treated with modified "Yougui decoction", with the addition of kidney Yin tonifying herbs in group, Qi tonifying herbs in group and blood circulation invigorating herbs in group. Patients in positive control group were treated with western medicine alone (androgen with or without cyclosporine A for six months). TCM: Traditional Chinese Medicine. a P <.5, compared with that after months' treatment in the same group; b P <.5, compared with that in the positive control group at the same time. 5 7 Efficacy Invalid 14 with those in the normal control group, while no significant differences were observed except for IL- and IL-4 levels (P <.5). Cytokine expression in all treatment groups did not show a significant change after months (P >.5). After two courses of treatment ( months), the levels of IFN-γ, TNF-α, IL- and IL- decreased, with further increases in IL-4 and IL-1 in all treated groups. IFN-γ, IL- and IL- levels in the TCM combined groups (, and groups) were significantly lower than before treatment (P <.5 or P <.1), and IL-4 increased in all treated groups (P <.5). When compared with months previously, IFN-γ, IL- and IL- in the and groups and IL-4 in the, and positive control groups had changed after months (P <.5). In addition, the level of IL- in the and groups after months was lower than in the positive control group. No significant differences were observed between the three TCM groups combined (Table 7) Overall efficacy [n (%)] 19 (57.) (7.) 19 (7.7) 7 (7.1) ab 1 (.) 5 (.) a (5.) JTCM www. journaltcm. com 44 August 15, 1 Volume Issue 4

8 Table Comparison of T-lymphocyte subsets and regulatory T cells in different groups (%, xˉ ± s) s Time After months After months After months After months CD+ 77.4± ±.5 7.5±.4 75.±.9 a.±.1 1.±.5 75.±14.5.4±7.5 Normal reference value Notes: comparison of T-lymphocyte subsets and regulatory T cells in the (kidney reinforcing alone), (kidney reinforcing and Qi tonifying), (kidney reinforcing and blood circulation invigorating) and positive control groups was performed before and months after treatment. Patients in the TCM treated groups were treated with modified "Yougui decoction", with the addition of kidney Yin tonifying herbs in group, Qi tonifying herbs in group and blood circulation invigorating herbs in group. Patients in positive control group were treated with western medicine alone (androgen with or without cyclosporine A for six months). TCM: Traditional Chinese Medicine. a P <.5, b P <.1, compared with that before treatment in the same group. Table 7 Comparison of cytokines in different groups (pg/ml, xˉ ± s) Time IFN-γ 5.±4.4 TNF-α.1±4.9 Positive control Treated with months Treated with months Treated with months 4.4±..1±.19 a 4.±..±5. 1.9±.4 ab 4.14±. 4.±4.4.7±1.4 ab 5.51±.9 4.5±.9 Comparison of side effects Incidence of gastrointestinal adverse reaction, polytrichia, gingival hyperplasia, and rash ranged from 7.5% to.5% in the positive control group. The incidence was higher than for other side effects: secondary hypertension 1.5%, liver dysfunction 1.75%, renal dysfunction.5%, and hand tremor 1.75%. With TCM treatment, the incidence of gastrointestinal adverse reaction, polytrichia, and rash decreased in the According to TCM, long-term disease is always accom- CD+CD4+CD- 4.77±.54 4.±4.5.97± ±.1 4.4± ± ±.7 4.1±.1 5.± ±.1.7±9. 7.±7..1±..4± ± ±5. a 4.± ±9.1 IL-.±.91 c.±1.79 c 1.7±.4 a.9±. c.1±.9.4±.71 bd.±.5 c.11±.1.91±1. ab.4±.11 c.1±1.95 c CD+CD4-CD+, and groups (P <.1), and the development of liver dysfunction and hand tremor also decreased in the group (P <.5). No notable differences in the incidence of side effects were observed among the three TCM groups combined (Table ). DISCUSSION.± ±9.9 b 9.9±7. 9.9±7.4 b.±9..1±.4 b.±1.5 4.±9. b IL-4.77±.4 c.±.4 c.9±. ac.74±.5 c.7±. e.9±.47 abc.79±.4 c.77±.5 c 1.±.7 abc.±.55 c.±.9 c CD+CD4+CD5+ FoxP+/CD+CD4+.±4. IL-.1±4..±1.9 1.±1. a.51±5.1.±. 1.1±.1 abe.4±4.77.±. 1.1±1. abe 4.1±..± ±1..4±4. 1.5±1..±.7 1.5±1..±.7 1.1±.7 IL-1.4± ± ±..± ± ±7..5± ± ± ±1.1.5±1. Treated with months.11±.1 a 4.±.1 1.1±1.4.99±.41 abc 1.±.11 a 1.5±14. Norma -.± ± ±1.5.4±.4 1.4±.14 7.±.71 control Notes: comparison of cytokines in the (kidney reinforcing alone), (kidney reinforcing and Qi tonifying), (kidney reinforcing and blood circulation invigorating), positive control and normal control groups was performed before and and months after treatment. Patients in the TCM treated groups were treated with modified "Yougui decoction", with the addition of kidney Yin tonifying herbs in group, Qi tonifying herbs in group and blood circulation invigorating herbs in group. Patients in positive control group were treated with western medicine alone (androgen with or without cyclosporine A for six months). IFN: interferon; IL: interleukin; TNF: tumor necrosis factor; TCM: Traditional Chinese Medicine. a P <.5, d P <.1, compared with that before treatment in the same group; b P <.5, compared with that after months' treatment in the same group; c P <.5, compared with that in the normal control group; e P <.5, compared with that in the positive control group at the same time. JTCM www. journaltcm. com 441 August 15, 1 Volume Issue 4

9 Table Comparison of side effects in different groups [n (%)] Side effect Gastrointestinal reaction Secondary hypertension Polytrichia Gingival hyperplasia Rash Liver dysfunction Renal dysfunction group 5 (15.) a (9.1) (4.) b 7 (1.) 5 (15.) b (.1) 1 (.) group 4 (1.9) b Hand tremor 1 (.) 1 (.) () a (1.75) Notes: side effects in the (kidney reinforcing alone), (kidney reinforcing and Qi tonifying), (kidney reinforcing and blood circulation invigorating) and positive control groups were evaluated in the course of the treatment. Patients in the TCM treated groups were treated with modified "Yougui decoction", with the addition of kidney Yin tonifying herbs in group, Qi tonifying herbs in group and blood circulation invigorating herbs in group. Patients in positive control group were treated with western medicine alone (androgen with or without cyclosporine A for six months). TCM: Traditional Chinese Medicine. a P <.5, b P <.5, compared with that in the positive control group. (9.7) (19.4) b (19.4) 5 (1.1) b 1 (.) () group 4 (1.) b (.7) 7 (.) b 7 (.) 4 (1.) b () a () group (5) (1.5) 1 (.5) (7.5) 9 (4.) (1.75) 1 (.5) panied by deficiency syndrome and stasis syndrome, which challenges the therapeutic effects of treatment. Qi works with the assistance of Yang; CAA patients with kidney Yang deficiency also suffer from Qi deficiency. 9 Attributed to the stasis of blood in marrow, the production of "fresh blood" is always impaired as well. Besides kidney reinforcing, Qi tonifying and blood activating have already been applied in clinical practice, 1,11 while no control study has been conducted and no further mechanism explored. We found that improvement of WBC and Hb counts was quicker in the group than the and groups combined when compared with the positive control group, while platelet count only increased in the group. The recovery of hemograms was consistent with the therapeutic effect in Western Medicine. Patients in the group had a 1.% OE in months, which was higher than in the positive control group (.%, P <.5). After months, all groups showed improved OE, while only in the group, the index was significantly higher than in the positive control group (% vs 5%, P <.5). In addition, the TCM syndrome scores in the and groups also decreased significantly when compared with the positive control group after months, while the therapeutic effect in TCM improved significantly only in the group. Our results showed that the treatment based on kidney reinforcing and Qi tonifying achieved a quicker and greater therapeutic effect in CAA, although no differences were observed among the different TCM groups combined. The treatment based on kidney reinforcing and blood circulation invigorating may have a better effect on platelet recovery when compared with other TCM groups. It is confirmed that immune disorders play a crucial role in the onset and development of AA. The imbalance between cytotoxic and T helper (Th) cells leads to hyperactivity of cytotoxic T cells, which secrete cytokines that inhibit the process of hematopoiesis and induce apoptosis of stem cells, such as TNF-α and IFN-γ. 1,1 According to TCM theory, we know that with "vital Qi" inside, no "evil" comes near. Treatment by strengthening vital Qi may work by modulating the immune function, which may also favor recovery from CAA. The results showed that the treatment based on TCM had a better effect on decreasing the ratio of cytotoxic T cells than the positive control group (P <.1), and may have had an advantage in increasing the ratio of Th cells (P <.5). The regulation of T cells may not be the therapeutic target of TCM in CAA, for no prominent differences were observed among the groups after months. In our study, patients with CAA had significantly higher levels of IL- and IL-4 when compared with those in the normal control group. IL- is secreted by CD4+ Th1 cells, which may promote the proliferation of cytotoxic T cells, while IL-4 is secreted by CD4+ Th cells and causes downregulation. 14 Increases in IL-4 levels may be the inverse feedback of the body to stop the excursion of Th1 cells in CAA. TCM had a role in decreasing the levels of IFN-γ, IL- and IL-, as well as promoting secretion of IL-4 (P <.5), which had a positive correlation with the treatment course. IL- decreased significantly in the and groups when compared with the positive control group, indicating that IL- may be the target of and in the treatment of CAA. Gastrointestinal adverse reaction, polytrichia, gingival hyperplasia, and rash are the most common side effects of taking cyclosporine or androgens, which are also the factors that affect patient compliance in clinical practice. 15,1 Our study indicated that the treatment of TCM had a role in decreasing the incidence of gastrointestinal adverse reaction, polytrichia, and rash, and treatment with kidney reinforcing and blood circulation invigorating may also contribute to the reduction of liver dysfunction and hand tremor. JTCM www. journaltcm. com 44 August 15, 1 Volume Issue 4

10 Our results lead us to the conclusion that a combination of TCM therapies has a positive role in CAA, which can exert an effect on rebalancing the cytotoxic and Th cells, and regulate the expression of IFN-γ, IL-, IL-4 and IL-. Among the three different TCM principles of treatment, kidney reinforcing and Qi tonifying may have a better treatment profile, while treatment with kidney reinforcing and blood circulation invigorating may have an advantage in the recovery of platelet levels. The results remind us that treatment with kidney reinforcing, Qi tonifying, and blood circulation invigorating should be considered in clinical practice, especially the combination of the first two, and more research is needed to confirm and explore the underlying mechanisms of the treatments. REFERENCES 1 Young NS. Current concepts in the pathophysiology and treatment of aplastic anemia. Hematology Am Soc Hematol Educ Program 1; 1: 7-1. Yang CL, Zhang XB. Aplastic anemia incidence survey across the country. Zhong Guo Yi Xue Ke Xue Yuan Xue Bao 199; 14(1): -1. Wu DJ, Ye BD, Ji CH, et al. Research status of clinical practice guidelines for aplastic anemia and amendment and thinking of Traditional Chinese Medicine guidelines. Zhong Yi Za Zhi 14; 55(4): Zhang Y, Zhang X, Ye BD, et al. Overview of Traditional Chinese Medicine on the treatment of aplastic anemia. Zhong Yi Za Zhi 14; 55(1): 7-75, 5. 5 Wu DJ, Zhou YH. The experience of doctor zhou on the treatment of chronic aplastic anemia. Zhong Yi Za Zhi 1; 54(4): -4. Zhu JJ, Zhou YH. Comparing two methods of the clinical study on treatment of chronic aplastic anemia: invigorating the kidney only and invigorating the kidney and Qi both. Zhejiang Zhong Yi Yao Da Xue Xue Bao 1; (1): Zhang ZN, Shen T. Diagnosis and Treatment Standards of Hematology Disease. rd ed. Beijing: Science Press, : 19-. China Association of Traditional Chinese Medicine. Guidelines for Diagnosis and Treatment of Common Internal Disease in Chinese Medicine, Disease of Modern Medicine. Beijing: China Press of Traditional Chinese Medicine, : Chen Z, Lin SY, Zhou YH, et al. Analysis of clinical features of Traditional Chinese Medicine symptoms and syndromes of patients withchronic aplastic anemia. Zhong Guo Zhong Xi Yi Jie He Za Zhi 14; 4(1): Xiang Y, Yu JW, Cheng YB, et al. Study on composing prescription laws of treating aplastic anemia by Chinese medicine using applying data mining technique. Zhong Guo Zhong Xi Yi Jie He Za Zhi 1; (7): Wang SQ, Tang YX, Li JH. Clinical study on treatment of chronic aplastic anemia assisted by TCM for reinforcing shen and activating blood circulation to dredge channels. Zhong Guo Zhong Xi Yi Jie He Za Zhi ; (4): Găman A, Găman G, Bold A. Acquired aplastic anemia: correlation between etiology, pathophysiology, bone marrow histology and prognosis factors. Rom J Morphol Embryol 9; 5(4): Jiang BL, Li JP, Li WQ, et al. Role of CD(+) T cells and their secreted cytokines in the pathogenesis of aplastic anemia. Zhong Guo Shi Yan Xue Ye Xue Za Zhi 14; (): Wu DJ, Ye BD, Hu ZP, et al. Bone marrow angiogenesis in patients presenting with differential Chinese medicine syndrome: correlation with the clinico-pathological features of aplastic anemia. Chin J Integr Med 1; 19(1): Jaime-Pérez JC1, Colunga-Pedraza PR, Gómez-Ramírez CD, et al. Danazol as first-line therapy for aplastic anemia. Ann Hematol 11; 9(5): Agarwal MB, Jijina F, Shah S, et al. Safety and efficacy of indigenous equine antithymocyte globulin along with cyclosporine in subjects with acquired aplastic anemia. Indian J Hematol Blood Transfus 15; 1(): JTCM www. journaltcm. com 44 August 15, 1 Volume Issue 4