黄芪及其复方治疗小儿β地中海贫血的疗效及分子机制
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摘要
研究背景：我国尤其南方各省是p地中海贫血的高发区。p地贫患儿由于p珠蛋白链的合成障碍及α、p链的不平衡导致无效造血、慢性溶血性贫血及多种并发症。重型及部分中间型患儿常常需要依赖终身规律的输血,同时也需要长期的铁螯合治疗以减轻机体铁负荷。输血及铁螯合剂对于我国等发展中国家的患者均是沉重的经济负担,同时也存在输血相关性传染病、血管内皮损伤、骨关节痛等诸多副作用。地贫的基因治疗目前离临床应用还有很长一段距离,而干细胞移植由于干细胞来源、移植风险、费用等因素也很难普及。目前我国很多β地贫患儿均没有得到规律的治疗而长期处于低血红蛋白的状态从而严重影响到生长发育甚至危及到生命。对大量地贫患儿如何进行有效而又副作用小的治疗,是当今医学的难题,也是重要的社会问题。
药物基因诱导是p地贫的研究热点。其基本原理是通过药物诱导新生儿期后近乎关闭的γ珠蛋白基因重新高表达,合成胎儿血红蛋白(fetal hemoglobin,HbF)以在功能上替代正常的血红蛋白,同时也可结合过剩的α链以减轻红细胞破坏,从而改善贫血及减少并发症。Y珠蛋白基因诱导药物主要包括细胞毒药物、DNA甲基转移酶抑制剂、组蛋白脱乙酰基酶抑制剂、雷帕霉素靶点抑制剂、细胞因子等种类。但目前可真正应用于临床的药物还很少,而各种基因诱导的化学药物无论是羟基脲、5-氮胞苷、丁酸盐还是促红细胞生成素等均有各自明显的缺点,或是有骨髓抑制、潜在致癌等副作用,或是药效短暂,或是价格昂贵等,均严重限制了其临床应用。
中医药相对副作用较小,目前以中药治疗p地贫的研究多数是个案报道及一般性临床观察,规范深入的课题仅见益髓生血颗粒系列研究。但益髓生血颗粒为大复方,药味众多,难以进一步研究及发现其有效成分,有必要从单味中药或小复方发掘基因诱导药物。本课题组既往及新近的体外实验发现中药龟板、黄芪、党参等能使人类红系K562细胞或p地贫患者红系祖细胞的丫珠蛋白mRNA增加,HbF提升。与丁酸盐比较中药对细胞生长无抑制,诱导维持时间也显著较长。对单味黄芪的深入研究发现,其诱导HbF的主要有效成分在富含多糖的水溶部分,而黄芪多糖有类似作用。基于体外实验研究所显示的效果,结合中医中药理论,本研究首次选用单味黄芪以及由黄芪、党参、龟板组成的小复方中药对p地贫患儿开展随机对照双盲的前瞻性临床研究,以在体内验证中药的效果,客观评价其临床疗效及安全性。
本研究同时拟对中药在患者体内起效的分子机制进行探讨,验证本课题的中药在体内是否也能诱导γ珠蛋白基因的表达。p38丝裂原活化蛋白激酶(mitogen activated protein kinase, MAPK)是与γ珠蛋白的生成关系最为密切的信号分子之一。本课题组既往的研究表明,p38MAPK信号通路的激活是丁酸钠等药物诱导红系细胞γ珠蛋白基因表达的重要机制,涉及对转录因子的影响及表观遗传机制。本课题组其它博士生新近的研究也表明,龟板、黄芪在体外诱导γ基因也与p38MAPK的磷酸化有关。因而本研究也对患儿用药后体内p38MAPK的变化进行探讨,以从细胞信号通路角度分析中药的体内起效机制。
方法：1.患者分组与治疗方案：本课题为随机对照双盲的前瞻性临床试验。两家研究中心共有57例β地中海贫血患儿纳入研究,按中间型及重型随机分配进入复方组、黄芪组、安慰剂对照组。复方组患儿口服黄芪、党参及龟板颗粒,其中2-6岁患儿每日三种中药颗粒各1袋,6-12岁患儿每日各2袋,12-18岁患儿每日各3袋。黄芪组口服单味黄芪颗粒,其中2-6岁患儿每日3袋,6～12岁患儿每日6袋,12～18岁患儿每日9袋。对照组口服安慰剂颗粒,其中2-6岁患儿每日3袋,6-12岁患儿每日6袋,12～18岁患儿每日9袋。疗程均为12周。重型患儿在以上治疗基础上加用输血治疗,每隔4周输血一次。
2.评价疗效的指标：(1)治疗开始前及治疗满4周、8周、12周分别取患儿的手指末梢血检测血常规,观察血红蛋白(hemoglobin, Hb)、红细胞总数(redblood cell, RBC)、平均红细胞体积(mean corpuscular volume, MC V)、平均红细胞血红蛋白量(1mean corpuscular hemoglobin, MCH)、红细胞体积分布宽度(redblood cell volume distribution width, RDW)、(2)治疗开始前及治疗满12周后分别抽取患儿静脉血以碱性血红蛋白电泳检测HbF比例；采用碱性血红蛋白电泳结合比色法检测HbF含量。(3)治疗开始前及治疗满12周后分别抽取患儿静脉血计数外周血网织红细胞；采用比色法检测红细胞孵育渗透脆性。(4)对中间型患儿在治疗开始前及治疗满12周后腹部触诊检查肝肋下及脾肋下的大小。(5)在治疗开始前及治疗满4周、8周、12周分别测量体重、心率及血压一次。(6)参照中医证候分型标准,统计各组患儿气血两虚与肝肾亏虚四诊症状的改善率。
3.疗效标准：(1)显效：治疗后Hb上升≥3g/dL,同时伴有不同程度的HbF升高；(2)有效：治疗后Hb上升≥0.5g/dL但<3g/dL,同时伴有不同程度的HbF升高；(3)无效：治疗后Hb上升<0.5g/dL。疗程结束后统计各组的显效、有效及无效人数,计算显效率及总有效率。
4.安全性指标：如前述检测血常规,观察白细胞总数(White blood cell, WBC)、中性粒细胞总数(neutrophil, NEU)、血小板总数(platelet, PLT)。治疗开始前及治疗满12周分别检测肝肾功能指标,包括血清丙氨酸氨基转移酶、天门冬氨酸氨基转移酶、谷氨酸酰基转移酶、尿素氮、肌酐。疗程结束后统计各组患儿各项不良反应症状发生率及各项评价安全性的实验室指标。
5.分子生物学实验：课题同时以实时荧光定量RT-PCR、Western Blot方法研究患儿治疗前后α,β,Gγ和Aγ珠蛋白基因的表达水平以及p38MAPK细胞信号通路的激活。在治疗前及治疗满12周后,抽取中间型各组患儿外周血,采用不连续密度梯度离心法分离包含有核红细胞在内的单个核细胞。从Genbank查得各基因序列,设计并合成引物。用Trizol提取总RNA后进行逆转录反应,随后进行实时荧光定量PCR反应。根据标准曲线、各样本的Ct值并以内参照基因矫正得出各目的基因mRNA水平相对的拷贝数。另外提取蛋白,SDS-PAGE分离蛋白,蛋白质转膜,用p38MAPK和磷酸化p38MAPK抗体与蛋白质杂交,通过图像分析系统分析每个样p38MAPK磷酸化p38MAPK条带与内参照条带的积分光密度值比值。
6.统计学分析：以原始数据录入SPSS13.0软件建立数据库并进行统计学处理,对各指标进行组间比较及治疗前后比较.o P<0.05为差异有统计学意义。
结果：1.血常规指标：经黄芪及其复方中药颗粒12周的治疗,中间型p地贫患儿的Hb水平有显著提升,其中复方组Hb提升值为1.21±1.12g/dL,黄芪组为1.05±0.80g/dL,而安慰剂对照组则轻微下降了0.28±0.51g/dL,3组比较差异有统计学意义(F=10.209, P<0.001)。中间型复方组12周的Hb水平为8.91±1.07g/dL,黄芪组Hb水平为8.40±1.06g/dL,均显著高于对照组的7.30±1.07g/dL(分别P=0.001,P=0.017)。中间型复方组Hb在治疗前后不同时间水平间的差异具有显著性(F=8.773,P<0.001),黄芪组Hb前后差异也具有显著性(F=20.940, P<0.001),而对照组Hb在治疗前后则无显著性差异(P>0.05)。12周的治疗后,重型地贫患儿复方组Hb提升值为0.46±0.61g/dL,显著高于对照组的-0.55±0.66g/dL(P=0.033)。重型复方组12周的Hb水平为7.91±0.49/dL黄芪组Hb水平为7.74±1.15g/dL,均显著高于对照组的6.65±1.10g/dL(分别P=0.024,P=0.043)。中药治疗也能在一定程度上改善患儿的其他血常规指标包括RBC及MCH。
2.HbF水平：中间型复方组治疗后HbF比例较治疗前有显著提升(62.80%±22,85%vs.54.00%±20.63%,t=-2.910,P=0.016):黄芪组治疗后HbF比例也较治疗前显著提升(68.10%±16.63%vs.60.06%±16.29%,t=-4.103,P=0.001)。两组HbF含量在治疗后较治疗前也有显著增加(分别t=-3.896,P=0.003；t=-4.616,P=0.001)。中间型复方组、黄芪组治疗后HbF含量均显著高于对照组(分别P=0.001,P<0.001)。重型复方组治疗后HbF比例较治疗前也有显著提升(22.75±6.97%vs.20.23±7.06%,t=-3.424,P=0.014):黄芪组治疗后HbF比例同样比治疗前显著提升(29.01±12.50%vs.22.46±12.25%,t=-3.215,P=0.015)。两组HbF含量也有显著性增加(分别t=-3.156,P=0.020；t=-3.491,P=0.010)。无论在中间型患儿还是重型患儿中,安慰剂对照组的HbF比例及含量在治疗前后的差异均无统计学意义(P>0.05)。以上结果提示黄芪及其复方均能显著提升患儿的HbF水平。
3.其他临床研究指标：中间型黄芪组、重型复方组治疗后的网织红细胞比治疗前均有显著性升高(分别t=-5.790,P<0.001；t=-3.195,P=0.019),中间型复方组治疗后的红细胞孵育渗透脆性也较治疗前有显著性改善(t=-2.331,P=0.042)。各组肝脾肋下大小在治疗前后无显著性差异。中间型黄芪组的体重、重型复方组的收缩压治疗后比治疗前均有显著性提升(分别F=5.330,P=0.017；F=8.551,P=0.001)。另外中药治疗对中医气血两虚症状及肝肾亏虚症状的改善均优于安慰剂。
4.临床疗效判定：中间型复方组患儿的总有效率为63.6%,黄芪组患儿的总有效率为61.5%,而对照组为9.1%,三者有显著性差异(x2=8.681,P=0.013)。重型复方组患儿的总有效率为42.9%,黄芪组患儿的总有效率为50.0%,而对照组为0%,三者也有显著性差异(x2=6.871,P=0.032)。综合以上结果,提示黄芪及其复方中药治疗小儿β地贫是具有一定疗效的,可基本排除安慰剂效应。
5.不同药物及不同类型患儿的疗效：对于以上大多数指标,复方组及黄芪组的差异无统计学意义,提示两种药物的疗效接近。中间型患儿的总有效率虽高于重型患儿,但按目前的样本量未发现两者有显著性差异。中药对不同表型、不同基因型、不同中医证候类型患儿的疗效虽有差别,但均未达到统计学意义。
6.安全性评价：服用单味黄芪的患儿鼻衄症状发生率相对较高(x2=7.697,P=0.021),其他可能与药物不良反应相关的症状发生率在中药与安慰剂之间无显著性差异。各组患儿的WBC、NEU、PLT及肝肾功能指标在疗程结束后的组间差异均无统计学意义(P>0.05)；治疗前后的差异也无统计学意义(P>0.05)。无一例患儿因不良反应而需要停药。
7.随访期指标：中间型及重型复方组及黄芪组患儿在随访期内输血次数均少于对照组,但差异未达到显著性(P>0.05)。
8.分子生物学实验结果：实时荧光定量RT-PCR结果显示,复方组、黄芪组治疗后的Gγ珠蛋白基因mRNA均显著高于治疗前水平(分别F=14.152,P=0.004:F=14.854,P=0.002),两组分别提升了1.65倍及1.53倍,同时也显著高于对照组(分别P=0.010,P=0.036),提示中药可在体内诱导患儿Gγ珠蛋白基因的转录表达。中药对患儿αβΑγ珠蛋白基因以及p38MAPK基因的IRNA水平无显著性影响。Western Blot结果显示,复方组、黄芪组的磷酸化p38MAPK蛋白水平在治疗后均显著高于治疗前(分别t=-4.234,P=0.002:t=-3.953,P=0.002),两组分别提升了2.3倍及2.21倍,同时也显著高于对照组(分别P=0.006,P=0.019)；而p38MAPK蛋白水平的前后差异与组间差异则无统计学意义。以上结果提示中药可在体内促进p38MAPK的磷酸化激活。
结论与展望：综合以上各部分结果,本研究的结论如下：
1.单味黄芪及黄芪、党参、龟板组成的小复方中药口服能显著提升β地中海贫血患儿的总血红蛋白水平及HbF水平,并能一定程度上改善患儿的RBC、MCH、网织红细胞等血液学指标及中医四诊症状。
2.本随机对照双盲的前瞻性临床研究表明,中间型患儿复方组及黄芪组的总有效率分别为63.6%及61.5%,重型患儿复方组及黄芪组的总有效率分别为42.9%及50.0%,均显著高于安慰剂对照组；中药对WBC、NEU、PLT及各项肝肾功能指标均无显著影响,提示黄芪及其复方中药治疗小儿β地贫是具有一定疗效且比较安全的。
3.黄芪及其复方中药口服均可在体内诱导G平珠蛋白基因的表达,Gγ基因mRNA水平分别提升1.53倍及1.65倍,是中药提升患儿HbF水平以及总血红蛋白水平的重要机制。
4.黄芪及其复方中药口服可以增强p地贫患儿体内p38MAPK的磷酸化,p-p38MAPK蛋白水平分别提升2.21倍及2.3倍,提示p38MAK信号通路的激活可能是中药在体内诱导γ珠蛋白基因表达的机制之一。
本研究对于开拓p地贫的临床治疗新手段具有重要的意义。我国是地贫的高发区,目前各种治疗方法如输血、铁螯合治疗、干细胞移植、基因治疗等均有明显的缺点。而各种珠蛋白基因诱导的化学药物在疗效与安全性方面也不尽如人意。本研究及课题组系列研究表明,黄芪及其复方治疗小儿p地贫不仅具有良好疗效,也无细胞毒性、肝肾毒性及骨髓抑制的副作用,颗粒剂型服用也比较方便,患者耐受性及依从性均较佳。可以预期,黄芪及其复方中药在今后p地贫的临床治疗上具有良好的应用前景。
中药大多存在多环节、多靶点的作用机制。本研究中药是否还有基因诱导或者改善贫血的其他机制,另外对地贫并发症如肝脾肿大、心衰、生长发育落后以及体内铁负荷水平的影响如何,还有其远期疗效、长期安全性、最佳疗程与用量等等,均需要今后更长期更多指标的研究来证实。本研究选用单味中药及小复方,有利于进一步研究与分离出有效成分,也有利于挖掘出传统中医治法理论的科学性。本研究及后续研究对于开发地贫新药以及研究人类血红蛋白的转换与诱导均有较强的科学价值,对我国南方各省等地贫高发区来讲具有重要的社会意义及广阔的应用前景。
Background:The prevalence of β-thalassemia is high in China, especially in southern provinces. The defects in the synthesis of β globin chains and imbalances of α and β globin chains in β-thalassemia patients cause ineffective erythropoiesis, chronic hemolytic anemia and many complications. Children with β-thalassemia major and some with thalassemia intermedia need regular lifelong blood transfusions and chelation therapy to reduce iron overload. Both transfusion and chelation therapy bring heavy economic burden for the patients in China or other developing countries. Furthermore, there are many side effects such as transfusion-transmitted infections, vein endothelium damage, arthralgia, etc. Gene therapy is not applied to the clinical treatment for patients with thalassemia yet. And it's difficult to popularize hematopoietic stem cell transplantation because of the problems about stem cell sources, transplantation risk or expenditure, etc. At the present time there are still lots of children affected by P-thalassemia and not received regular treatment in China. Their lasting low hemoglobin levels result in poor growth and development even life threat. How to treat the great amount of children with β-thalassemia effectively with low side effect is a difficult medical problem as well as important social problem.
Research about P-thalassemia focuses on pharmacological gene induction. Its basic principle is reactivating the y-globin gene expression which is almost silent after newborn period by some drugs and then producing fetal hemoglobin (HbF) which can functionally compensate for the shortfall of adult Hb (HbA) synthesis and in β-thalassemia patients. In addition, the synthesized γ-chains can neutralize the excess unbalanced a-chains to reduce red blood cell damage and ameliorate anemia as well as many complications of β-thalassemia. The y-globin induction agents include cytotoxic agents, DNA methyl transferase (DNMT) inhibitors, histone deacetylase (HDAC) inhibitors, target of rapamycin (TOR) inhibitors and some cytokines, etc. But just a little induction agents can be applied to the clinical use for now. Most induction chemical agents including hydroxyurea,5-azacytidine, butyrate, erythropoietin, and so on have respective obvious disadvantages such as myelotoxicity. potential carcinogenesis, impermanent effect or high cost, etc, which badly restrict the clinical usefulness of these agents.
Chinese herbal medicine has little side-effect relatively. To date, most studies published on Chinese medicine therapy for β-thalassemia were case reports or clinical observations except for the series studies about "Yisuishengxue granule". But the formula of "Yisuishengxue granule" consists of11Chinese herbs so it's difficult to study or find the effective constituent inside this formula. There is great need to screen for natural y-globin inducer from single Chinese herb or little Chinese medicinal formula. Our previous and recent in vitro studies suggested that Tortoise plastron (Tortoise shell), Astragalus membranaceus(Radix Astragali) and Codonopsis pilosula(Radix Codonopsis) could enhance y-globin mRNA expression and HbF production either in human erythroid K562cell cultures or erythroid progenitors from patients with β-thalassemia. Compared with butyrate, the induction effect by these Chinese medicines lasted for longer time significantly and had no inhibitory effect on erythroid cells. Our subsequent experiment about Astragalus membranaceus indicated its valuable constituents were contained in hydro-soluble astragali polysaccharide and the further studies about single astragali polysaccharide indicated it had similar induction effect. According the in vitro effect of these herbs and the traditional Chinese medicine (TCM) theory, we designed and performed a random controlled double-blind prospective clinical study of single Astragalus membranaceus and the little Chinese medicinal formula consist of Astragalus membranaceus, Codonopsis pilosula and Tortoise plastron for treatment of β-thalassemia innovatively in order to determine their in vivo effect and evaluate their clinical efficacy and side effects objectively.
This study also investigated whether or not these herbs can induce y-globin gene expression in children with β-thalassemia to explore their in vivo molecular mechanisms. The activation of p38mitogen activated protein kinase (MAPK) signaling pathway.plays a role in the mechanism of action for y-globin synthesis. Our previous studies indicated that butyrate activate y-globin expression in erythroid cells via a p38MAPK-dependent mechanism that involves the affected transcription factor and epigenetics mechanisms. The experiment performed by other PhD students in our research team suggested the Astragalus membranaceus and Tortoise plastron's in vitro effect of y-globin induction due to the activation of p38MAPK pathway, too. Thus the role of p38MAPK in patients was also investigated to analyze the in vivo molecular mechanisms from the signaling pathway angle.
Methods:The present study was a random controlled double-blind prospective clinical trial involved57pediatric patients with (5-thalassemia from two institutions. The children were split into layers of thalassemia major or thalassemia intermedia and randomly assigned to formula medicine group, Astragalus membranaceus group or placebo control group. The children in formula medicine group were administrated oral Astragalus membranaceus, Codonopsis pilosula and Tortoise plastron granules. One, two or three bags of each kind of these Chinese medicine granules were for each child ranged from2to6years old, from6to12years old, or from12to18years old, respectively everyday. Three, six or nine bags of oral Astragalus membranaceus granules were administrated for each child in Astragalus membranaceus group ranged from2to6years old, from6to12years old, or from12to18years old, respectively everyday. And three, six or nine bags of oral placebo granules were administrated for each child in placebo control group ranged from2to6years old, from6to12years old, or from12to18years old, respectively everyday. The course of treatment was12weeks. The children with thalassemia major used the therapy above in combination with regular transfusion therapy at4weeks intervals.
Before the treatment and after treatment for4weeks,8weeks and12weeks, blood routine tests were performed in each participant studied to measure the parameters including hemoglobin (Hb), red blood cell (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and red blood cell volume distribution width (RDW). Before the treatment and after treatment for12weeks, the peripheral blood of the patients were derived to count the reticulocytes, determine the HbF proportions and contents by hemoglobin electrophoresis and colorimetry and determine the erythrocyte osmotic fragility by colorimetry method. The sizes of liver and spleen below ribs in each participant with thalassemia intermedia were measured by abdominal palpation before the treatment and after treatment for12weeks. The children's heart rates, weights and blood pressures were measured at baseline and every4weeks. According the TCM syndromes criterion, the improvement rates of "deficiency of Qi and blood"(TCM) symptoms and "weakness of liver and kidney"(TCM) symptoms in each group were assessed after the treatment.
The clinical efficacy criterion was as follows:1) good response:Hb increase no less than3g/dL after the treatment with HbF elevation in some degree;2) moderate response:Hb increase no less than0.5g/dL but less than3g/dL after the treatment with HbF elevation in some degree;3) no response:Hb increase less than0.5g/dL. Responses to the Chinese medicine were assessed and numbers of patients with good response, moderate response and no response were added up after12weeks of treatment then the good response rate and total effective rate were calculated.
Blood routine tests were performed as described before to measure the safety parameters including White blood cell (WBC). neutrophil (NEU) and platelet (PLT). Before the treatment and after treatment for12weeks, the hepatic and renal functions parameters including alanine aminotransferase, aspartate aminotransferase, glutamyl transferase, blood urea nitrogen and creatinine were determined. The side-effect symptoms were assessed after12weeks of treatment.
In addition, studies were performed that analyzed the expression levels of a-, β-, Cry-and Ay-globin genes and the role of the p38MAPK pathway in regulating HbF induction using real-time fluorescent quantitative RT-PCR and Western Blot methods. Before the treatment and after treatment for12weeks, mononuclear cells contains nucleated red blood cells were isolated from the peripheral blood of the participants with thalassemia intermedia by discontinuous density gradient centrifugation. According the sequences of each gene from Genbank, the primers were designed and synthesized. RNAs were isolated using Trizol then reverse transcription reactions and real-time fluorescent quantitative PCR reactions were carried out. The target genes mRNA levels were quantitated by the standard curves and the cycle threshold values of each sample and normalized to the levels of internal control gene. Furthermore, the total proteins of each sample were isolated and separated using SDS-PAGE method. Subsequently, the proteins were transferred to PVDF membranes and hybridization with p38MAPK and phosphorylation p38MAPK antibodies were carried out in next day. The integrated optical densities of p38MAPK, phosphorylation p38MAPK and internal control hybridization bars of each sample were analyzed by image software.
Databases were established and statistical analyses were performed using SPSS13.0. Statistically significant differences of each parameter between or within groups were performed finally. Differences were considered to be significant when P is less than0.05.
Results:After treatment of either Astragalus membranaceus granules or its medicinal formula granules for12weeks, the Hb levels in children with β-thalassemia intermedia elevated significantly. The mean Hb elevation value was1.21±1.12g/dL in the formula group and1.05±0.80g/dL in the Astragalus membranaceus group, as compared with-0.28±0.51g/dL in the placebo control group (F=10.209, P<0.001). After treatment of12weeks, the mean Hb levels of children with β-thalassemia intermedia both in the formula group and the Astragalus membranaceus group were higher significantly than that in the placebo group (8.91±1.07g/dL vs.7.30±1.07g/dL, P=0.001;8.40±1.06g/dL vs.7.30±1.07g/dL, P=0.017, respectively). There was significant differences over time with respect to Hb levels in children with β-thalassemia intermedia both in the formula group (F=8.773, P<0.001) and the Astragalus membranaceus group (F=20.940, P<0.001) in contrast to no significant difference over time in the placebo control group (P>0.05). The mean Hb elevation value of children with β-thalassemia major after treatment of12weeks was0.46±0.61g/dL in the formula group as compared with-0.55±0.66g/dL in the placebo control group (P=0.033). The post-treatment Hb levels in children with p-thalassemia major both in the formula group and the Astragalus membranaceus group were higher significantly than that in the placebo group (7.91±0.49g/dL vs.6.65±1.10g/dL, P=0.024;7.74±1.15g/dL vs.6.65±1.10g/dL, P=0.043, respectively). This therapy with Astragalus membranaceus or its formula medicine also improved other parameters of blood routine test including RBC and MCH in some degree.
Comparing initial and last values, mean HbF proportion of children with β-thalassemia intermedia in the formula group rose significantly (62.80%±22.85%vs.54.00%±20.63%, t=-2.910, P=0.016) and so did mean HbF proportion in the Astragalus membranaceus group (68.10%±16.63%vs.60.06%±16.29%, t=-4.103, P=0.001). Both Astragalus membranaceus and its formula medicine also raised HbF contents above baseline significantly in children with P-thalassemia intermedia (t=-3.896, P=0.003and t=-4.616, P=0.001, respectively). The post-treatment HbF contents in children with β-thalassemia intermedia either in formula group or Astragalus membranaceus group were significant higher compared with control (P=0.001and P<0.001, respectively). Mean HbF proportion of children with P-thalassemia major in the formula group rose above baseline significantly (22.75±6.97%vs.20.23±7.06%, t=-3.424, P=0.014) and so did in the Astragalus membranaceus group (29.01±12.50%vs.22.46±12.25%, t=-3.215, P=0.015).The HbF contents in both groups above also increased significantly after treatment (t=-3.156, P=0.020and t=-3.491, P=0.010, respectively). Pair-wise comparisons for baseline and post-treatment levels of either HbF proportions or HbF contents had no statistical significance in the placebo control groups (P>0.05). The results above demonstrated that both Astragalus membranaceus and its formula medicine induced an increase of HbF in the β-thalassemia patients studied.
A marked elevation of reticulocyte levels was observed either in the intermedia Astragalus membranaceus group or in the major formula medicine group (t=-5.790, P<0.001and t=-3.195, P=0.019, respectively). Significant improvement in erythrocyte osmotic fragility of children with β-thalassemia intermedia was documented after formula medicine therapy (t=-2.331, P=0.042). There was no significant difference with respect to the sizes of liver and spleen between before and after treatment in all groups. The weights of children with P-thalassemia intermedia in Astragalus membranaceus group and the systolic blood pressures of children with β-thalassemia major in formula group rose significantly over time (F=5.330, P=0.017and F=8.551, P=0.001, respectively). And these Chinese medicines ameliorate the "deficiency of Qi and blood"(TCM) symptoms and "weakness of liver and kidney"(TCM) symptoms markedly relative to control.
The total effective rate in children with β-thalassemia intermedia was63.6%in the formula medicine group and61.5%in the Astragalus membranaceus group which were higher significantly than that in the placebo control group (9.1%; x2=8.681, P=0.013). The total effective rate in children with β-thalassemia major was42.9%in the formula medicine group,50.0%in the Astragalus membranaceus group and0%in the placebo control group. There were significant difference among them, too (x2=6.871, P=0.032).According all the results above, it can be demonstrated that Astragalus membranaceus or its formula medicine is effective without placebo effect as a treatment for children with β-thalassemia.
Most of the parameters above were statistically identical between in formula medicine group and Astragalus membranaceus group, suggesting comparable efficacies of these two Chinese medicines for P-thalassemia treatment. Although the total effective rate in children with β-thalassemia intermedia was higher than that in children with p-thalassemia major, there was no statistical significance between them in the present sample size. And there was no statistical difference in the Chinese medicines'effect among children with different phenotypes, different genotypes or different TCM syndromes.
The incidence of epistaxis was relatively high in the children administrated Astragalus membranaceus granules (x2=7.697, P=0.021). There was no significant difference with respect to the incidences of other symptoms probably involved adverse reactions between children administrated Astragalus membranaceus and those administrated formula medicine. Furthermore, there was no statistical significance with respect to WBC, NEU, PLT and hepatic and renal functions parameters among groups or between before and after treatment in all groups. No one patient required discontinuation of treatment because of adverse effects.
The blood transfusion frequencies in follow-up period in the Chinese medicine groups were less but not significantly than those in the corresponding placebo groups (P>0.05)
The results of real-time fluorescent quantitative RT-PCR indicated that the difference of Gy-globin gene mRNA levels between before the treatment and after treatment for12weeks both in formula medicine group and Astragalus membranaceus group were significant (F=14.152, P=0.004and F=14.854, P=0.002, respectively). And the post-treatment levels of Gγ-globin gene mRNAs in both Chinese medicine groups were higher significantly than the control group (P=0.010and P=0.036, respectively). These results suggested that our Chinese medicines induced Gy-globin gene expression in vivo at the transcriptional level. The Chinese medicines had no marked effect on a-, β-and Ay-globin genes expression. We also observed significant increases of in vivo phosphorylation p38MAPK protein levels for both Astragalus membranaceus and formula medicine (t=-4.234, P=0.002and t=-3.953, P=0.002, respectively) compared with no increase for placebo, as revealed in western blot analysis. And significant differences were observed in post-treatment phosphorylation p38MAPK levels compared both Chinese medicine groups with the placebo group (P=0.006and P=0.019, respectively). But the Chinese medicines had no significant effect on the protein level of p38MAPK without phosphorylation. The results above suggested Astragalus membranaceus or its formula medicine could stimulate the in vivo phosphorylation activation of p38MAPK.
Conclusion and prospect:In the present study the major conclusions were as follows:
1) Both oral single Astragalus membranaceus and the little Chinese medicinal formula consist of Astragalus membranaceus, Codonopsis pilosula and Tortoise plastron can elevate total hemoglobin and HbF levels of children with β-thalassemia significantly. Furthermore, they can ameliorate the patients'symptoms of TCM four diagnostic methods and hematological parameters including RBC, MCH, reticulocyte and so on.
2) This random controlled double-blind prospective clinical trial suggested that the therapy with Astragalus membranaceus or its formula medicine is effective and safe for children with β-thalassemia.
3) Both oral Astragalus membranaceus and its formula medicine can induce the Gy-globin gene expression in vivo which constitutes an important mechanism that stimulate HbF synthesis then enhance total hemoglobin in patients with β-thalassemia.
4) Both oral Astragalus membranaceus and its formula medicine can activate the phosphorylation of p38MAPK in children with β-thalassemia and it suggests that the in vivo y-globin induction of these Chinese medicines may be at least in part regulated through a p38MAPK signaling pathway.
This study had great significance for developing a novel therapeutic approach to β-thalassemia. The prevalence of β-thalassemia is high in China, but the therapeutic methods for β-thalassemia including blood transfusion, chelation therapy, gene therapy, hematopoietic stem cell transplantation and so on have respective obvious disadvantages. And most chemical agents for globin induction are not satisfying in respect of either clinical efficacy or safety. The results of this study and other studies in our team indicated Astragalus membranaceus or its formula medicine has not only relatively good clinical efficacy for children with β-thalassemia but also no cytotoxicity, hepatic or renal toxicity, or myelotoxicity. The oral granules are convenient for patients with P-thalassemia and the patients'tolerance and compliance are good. Astragalus membranaceus and its formula medicine will be very promising in the treatment of β-thalassemia in the foreseeable future.
It has been generally assumed that most Chinese medicines have mechanisms at multiple levels or multiple targets. The present preliminary observations suggest the need for evaluating larger number of patients in long-term, large collaborative trials with more parameters to study other mechanisms of these Chinese medicines about globin gene induction or ameliorate anemia, their influence on iron load level or β-thalassemia complications such as hepatosplenomegaly, heart failure, and poor growth and development as well as their late results, long-term safety, optimal dose and duration of therapy, etc. Compared with traditional big Chinese medicinal formula, the single Chinese herb and little Chinese medicinal formula in this study are relatively fit for study and isolation of effective constituent and discovery of the scientific values of TCM treatment theories. The present and subsequent studies in our team have great scientific interest about developing new drug for β-thalassemia and studying the hemoglobin switching and induction. In addition, they have important social meaning and practical prospect in the areas where the prevalence of β-thalassemia is high, particularly the southern provinces in China.
药物基因诱导是p地贫的研究热点。其基本原理是通过药物诱导新生儿期后近乎关闭的γ珠蛋白基因重新高表达,合成胎儿血红蛋白(fetal hemoglobin,HbF)以在功能上替代正常的血红蛋白,同时也可结合过剩的α链以减轻红细胞破坏,从而改善贫血及减少并发症。Y珠蛋白基因诱导药物主要包括细胞毒药物、DNA甲基转移酶抑制剂、组蛋白脱乙酰基酶抑制剂、雷帕霉素靶点抑制剂、细胞因子等种类。但目前可真正应用于临床的药物还很少,而各种基因诱导的化学药物无论是羟基脲、5-氮胞苷、丁酸盐还是促红细胞生成素等均有各自明显的缺点,或是有骨髓抑制、潜在致癌等副作用,或是药效短暂,或是价格昂贵等,均严重限制了其临床应用。
中医药相对副作用较小,目前以中药治疗p地贫的研究多数是个案报道及一般性临床观察,规范深入的课题仅见益髓生血颗粒系列研究。但益髓生血颗粒为大复方,药味众多,难以进一步研究及发现其有效成分,有必要从单味中药或小复方发掘基因诱导药物。本课题组既往及新近的体外实验发现中药龟板、黄芪、党参等能使人类红系K562细胞或p地贫患者红系祖细胞的丫珠蛋白mRNA增加,HbF提升。与丁酸盐比较中药对细胞生长无抑制,诱导维持时间也显著较长。对单味黄芪的深入研究发现,其诱导HbF的主要有效成分在富含多糖的水溶部分,而黄芪多糖有类似作用。基于体外实验研究所显示的效果,结合中医中药理论,本研究首次选用单味黄芪以及由黄芪、党参、龟板组成的小复方中药对p地贫患儿开展随机对照双盲的前瞻性临床研究,以在体内验证中药的效果,客观评价其临床疗效及安全性。
本研究同时拟对中药在患者体内起效的分子机制进行探讨,验证本课题的中药在体内是否也能诱导γ珠蛋白基因的表达。p38丝裂原活化蛋白激酶(mitogen activated protein kinase, MAPK)是与γ珠蛋白的生成关系最为密切的信号分子之一。本课题组既往的研究表明,p38MAPK信号通路的激活是丁酸钠等药物诱导红系细胞γ珠蛋白基因表达的重要机制,涉及对转录因子的影响及表观遗传机制。本课题组其它博士生新近的研究也表明,龟板、黄芪在体外诱导γ基因也与p38MAPK的磷酸化有关。因而本研究也对患儿用药后体内p38MAPK的变化进行探讨,以从细胞信号通路角度分析中药的体内起效机制。
方法：1.患者分组与治疗方案：本课题为随机对照双盲的前瞻性临床试验。两家研究中心共有57例β地中海贫血患儿纳入研究,按中间型及重型随机分配进入复方组、黄芪组、安慰剂对照组。复方组患儿口服黄芪、党参及龟板颗粒,其中2-6岁患儿每日三种中药颗粒各1袋,6-12岁患儿每日各2袋,12-18岁患儿每日各3袋。黄芪组口服单味黄芪颗粒,其中2-6岁患儿每日3袋,6～12岁患儿每日6袋,12～18岁患儿每日9袋。对照组口服安慰剂颗粒,其中2-6岁患儿每日3袋,6-12岁患儿每日6袋,12～18岁患儿每日9袋。疗程均为12周。重型患儿在以上治疗基础上加用输血治疗,每隔4周输血一次。
2.评价疗效的指标：(1)治疗开始前及治疗满4周、8周、12周分别取患儿的手指末梢血检测血常规,观察血红蛋白(hemoglobin, Hb)、红细胞总数(redblood cell, RBC)、平均红细胞体积(mean corpuscular volume, MC V)、平均红细胞血红蛋白量(1mean corpuscular hemoglobin, MCH)、红细胞体积分布宽度(redblood cell volume distribution width, RDW)、(2)治疗开始前及治疗满12周后分别抽取患儿静脉血以碱性血红蛋白电泳检测HbF比例；采用碱性血红蛋白电泳结合比色法检测HbF含量。(3)治疗开始前及治疗满12周后分别抽取患儿静脉血计数外周血网织红细胞；采用比色法检测红细胞孵育渗透脆性。(4)对中间型患儿在治疗开始前及治疗满12周后腹部触诊检查肝肋下及脾肋下的大小。(5)在治疗开始前及治疗满4周、8周、12周分别测量体重、心率及血压一次。(6)参照中医证候分型标准,统计各组患儿气血两虚与肝肾亏虚四诊症状的改善率。
3.疗效标准：(1)显效：治疗后Hb上升≥3g/dL,同时伴有不同程度的HbF升高；(2)有效：治疗后Hb上升≥0.5g/dL但<3g/dL,同时伴有不同程度的HbF升高；(3)无效：治疗后Hb上升<0.5g/dL。疗程结束后统计各组的显效、有效及无效人数,计算显效率及总有效率。
4.安全性指标：如前述检测血常规,观察白细胞总数(White blood cell, WBC)、中性粒细胞总数(neutrophil, NEU)、血小板总数(platelet, PLT)。治疗开始前及治疗满12周分别检测肝肾功能指标,包括血清丙氨酸氨基转移酶、天门冬氨酸氨基转移酶、谷氨酸酰基转移酶、尿素氮、肌酐。疗程结束后统计各组患儿各项不良反应症状发生率及各项评价安全性的实验室指标。
5.分子生物学实验：课题同时以实时荧光定量RT-PCR、Western Blot方法研究患儿治疗前后α,β,Gγ和Aγ珠蛋白基因的表达水平以及p38MAPK细胞信号通路的激活。在治疗前及治疗满12周后,抽取中间型各组患儿外周血,采用不连续密度梯度离心法分离包含有核红细胞在内的单个核细胞。从Genbank查得各基因序列,设计并合成引物。用Trizol提取总RNA后进行逆转录反应,随后进行实时荧光定量PCR反应。根据标准曲线、各样本的Ct值并以内参照基因矫正得出各目的基因mRNA水平相对的拷贝数。另外提取蛋白,SDS-PAGE分离蛋白,蛋白质转膜,用p38MAPK和磷酸化p38MAPK抗体与蛋白质杂交,通过图像分析系统分析每个样p38MAPK磷酸化p38MAPK条带与内参照条带的积分光密度值比值。
6.统计学分析：以原始数据录入SPSS13.0软件建立数据库并进行统计学处理,对各指标进行组间比较及治疗前后比较.o P<0.05为差异有统计学意义。
结果：1.血常规指标：经黄芪及其复方中药颗粒12周的治疗,中间型p地贫患儿的Hb水平有显著提升,其中复方组Hb提升值为1.21±1.12g/dL,黄芪组为1.05±0.80g/dL,而安慰剂对照组则轻微下降了0.28±0.51g/dL,3组比较差异有统计学意义(F=10.209, P<0.001)。中间型复方组12周的Hb水平为8.91±1.07g/dL,黄芪组Hb水平为8.40±1.06g/dL,均显著高于对照组的7.30±1.07g/dL(分别P=0.001,P=0.017)。中间型复方组Hb在治疗前后不同时间水平间的差异具有显著性(F=8.773,P<0.001),黄芪组Hb前后差异也具有显著性(F=20.940, P<0.001),而对照组Hb在治疗前后则无显著性差异(P>0.05)。12周的治疗后,重型地贫患儿复方组Hb提升值为0.46±0.61g/dL,显著高于对照组的-0.55±0.66g/dL(P=0.033)。重型复方组12周的Hb水平为7.91±0.49/dL黄芪组Hb水平为7.74±1.15g/dL,均显著高于对照组的6.65±1.10g/dL(分别P=0.024,P=0.043)。中药治疗也能在一定程度上改善患儿的其他血常规指标包括RBC及MCH。
2.HbF水平：中间型复方组治疗后HbF比例较治疗前有显著提升(62.80%±22,85%vs.54.00%±20.63%,t=-2.910,P=0.016):黄芪组治疗后HbF比例也较治疗前显著提升(68.10%±16.63%vs.60.06%±16.29%,t=-4.103,P=0.001)。两组HbF含量在治疗后较治疗前也有显著增加(分别t=-3.896,P=0.003；t=-4.616,P=0.001)。中间型复方组、黄芪组治疗后HbF含量均显著高于对照组(分别P=0.001,P<0.001)。重型复方组治疗后HbF比例较治疗前也有显著提升(22.75±6.97%vs.20.23±7.06%,t=-3.424,P=0.014):黄芪组治疗后HbF比例同样比治疗前显著提升(29.01±12.50%vs.22.46±12.25%,t=-3.215,P=0.015)。两组HbF含量也有显著性增加(分别t=-3.156,P=0.020；t=-3.491,P=0.010)。无论在中间型患儿还是重型患儿中,安慰剂对照组的HbF比例及含量在治疗前后的差异均无统计学意义(P>0.05)。以上结果提示黄芪及其复方均能显著提升患儿的HbF水平。
3.其他临床研究指标：中间型黄芪组、重型复方组治疗后的网织红细胞比治疗前均有显著性升高(分别t=-5.790,P<0.001；t=-3.195,P=0.019),中间型复方组治疗后的红细胞孵育渗透脆性也较治疗前有显著性改善(t=-2.331,P=0.042)。各组肝脾肋下大小在治疗前后无显著性差异。中间型黄芪组的体重、重型复方组的收缩压治疗后比治疗前均有显著性提升(分别F=5.330,P=0.017；F=8.551,P=0.001)。另外中药治疗对中医气血两虚症状及肝肾亏虚症状的改善均优于安慰剂。
4.临床疗效判定：中间型复方组患儿的总有效率为63.6%,黄芪组患儿的总有效率为61.5%,而对照组为9.1%,三者有显著性差异(x2=8.681,P=0.013)。重型复方组患儿的总有效率为42.9%,黄芪组患儿的总有效率为50.0%,而对照组为0%,三者也有显著性差异(x2=6.871,P=0.032)。综合以上结果,提示黄芪及其复方中药治疗小儿β地贫是具有一定疗效的,可基本排除安慰剂效应。
5.不同药物及不同类型患儿的疗效：对于以上大多数指标,复方组及黄芪组的差异无统计学意义,提示两种药物的疗效接近。中间型患儿的总有效率虽高于重型患儿,但按目前的样本量未发现两者有显著性差异。中药对不同表型、不同基因型、不同中医证候类型患儿的疗效虽有差别,但均未达到统计学意义。
6.安全性评价：服用单味黄芪的患儿鼻衄症状发生率相对较高(x2=7.697,P=0.021),其他可能与药物不良反应相关的症状发生率在中药与安慰剂之间无显著性差异。各组患儿的WBC、NEU、PLT及肝肾功能指标在疗程结束后的组间差异均无统计学意义(P>0.05)；治疗前后的差异也无统计学意义(P>0.05)。无一例患儿因不良反应而需要停药。
7.随访期指标：中间型及重型复方组及黄芪组患儿在随访期内输血次数均少于对照组,但差异未达到显著性(P>0.05)。
8.分子生物学实验结果：实时荧光定量RT-PCR结果显示,复方组、黄芪组治疗后的Gγ珠蛋白基因mRNA均显著高于治疗前水平(分别F=14.152,P=0.004:F=14.854,P=0.002),两组分别提升了1.65倍及1.53倍,同时也显著高于对照组(分别P=0.010,P=0.036),提示中药可在体内诱导患儿Gγ珠蛋白基因的转录表达。中药对患儿αβΑγ珠蛋白基因以及p38MAPK基因的IRNA水平无显著性影响。Western Blot结果显示,复方组、黄芪组的磷酸化p38MAPK蛋白水平在治疗后均显著高于治疗前(分别t=-4.234,P=0.002:t=-3.953,P=0.002),两组分别提升了2.3倍及2.21倍,同时也显著高于对照组(分别P=0.006,P=0.019)；而p38MAPK蛋白水平的前后差异与组间差异则无统计学意义。以上结果提示中药可在体内促进p38MAPK的磷酸化激活。
结论与展望：综合以上各部分结果,本研究的结论如下：
1.单味黄芪及黄芪、党参、龟板组成的小复方中药口服能显著提升β地中海贫血患儿的总血红蛋白水平及HbF水平,并能一定程度上改善患儿的RBC、MCH、网织红细胞等血液学指标及中医四诊症状。
2.本随机对照双盲的前瞻性临床研究表明,中间型患儿复方组及黄芪组的总有效率分别为63.6%及61.5%,重型患儿复方组及黄芪组的总有效率分别为42.9%及50.0%,均显著高于安慰剂对照组；中药对WBC、NEU、PLT及各项肝肾功能指标均无显著影响,提示黄芪及其复方中药治疗小儿β地贫是具有一定疗效且比较安全的。
3.黄芪及其复方中药口服均可在体内诱导G平珠蛋白基因的表达,Gγ基因mRNA水平分别提升1.53倍及1.65倍,是中药提升患儿HbF水平以及总血红蛋白水平的重要机制。
4.黄芪及其复方中药口服可以增强p地贫患儿体内p38MAPK的磷酸化,p-p38MAPK蛋白水平分别提升2.21倍及2.3倍,提示p38MAK信号通路的激活可能是中药在体内诱导γ珠蛋白基因表达的机制之一。
本研究对于开拓p地贫的临床治疗新手段具有重要的意义。我国是地贫的高发区,目前各种治疗方法如输血、铁螯合治疗、干细胞移植、基因治疗等均有明显的缺点。而各种珠蛋白基因诱导的化学药物在疗效与安全性方面也不尽如人意。本研究及课题组系列研究表明,黄芪及其复方治疗小儿p地贫不仅具有良好疗效,也无细胞毒性、肝肾毒性及骨髓抑制的副作用,颗粒剂型服用也比较方便,患者耐受性及依从性均较佳。可以预期,黄芪及其复方中药在今后p地贫的临床治疗上具有良好的应用前景。
中药大多存在多环节、多靶点的作用机制。本研究中药是否还有基因诱导或者改善贫血的其他机制,另外对地贫并发症如肝脾肿大、心衰、生长发育落后以及体内铁负荷水平的影响如何,还有其远期疗效、长期安全性、最佳疗程与用量等等,均需要今后更长期更多指标的研究来证实。本研究选用单味中药及小复方,有利于进一步研究与分离出有效成分,也有利于挖掘出传统中医治法理论的科学性。本研究及后续研究对于开发地贫新药以及研究人类血红蛋白的转换与诱导均有较强的科学价值,对我国南方各省等地贫高发区来讲具有重要的社会意义及广阔的应用前景。
Background:The prevalence of β-thalassemia is high in China, especially in southern provinces. The defects in the synthesis of β globin chains and imbalances of α and β globin chains in β-thalassemia patients cause ineffective erythropoiesis, chronic hemolytic anemia and many complications. Children with β-thalassemia major and some with thalassemia intermedia need regular lifelong blood transfusions and chelation therapy to reduce iron overload. Both transfusion and chelation therapy bring heavy economic burden for the patients in China or other developing countries. Furthermore, there are many side effects such as transfusion-transmitted infections, vein endothelium damage, arthralgia, etc. Gene therapy is not applied to the clinical treatment for patients with thalassemia yet. And it's difficult to popularize hematopoietic stem cell transplantation because of the problems about stem cell sources, transplantation risk or expenditure, etc. At the present time there are still lots of children affected by P-thalassemia and not received regular treatment in China. Their lasting low hemoglobin levels result in poor growth and development even life threat. How to treat the great amount of children with β-thalassemia effectively with low side effect is a difficult medical problem as well as important social problem.
Research about P-thalassemia focuses on pharmacological gene induction. Its basic principle is reactivating the y-globin gene expression which is almost silent after newborn period by some drugs and then producing fetal hemoglobin (HbF) which can functionally compensate for the shortfall of adult Hb (HbA) synthesis and in β-thalassemia patients. In addition, the synthesized γ-chains can neutralize the excess unbalanced a-chains to reduce red blood cell damage and ameliorate anemia as well as many complications of β-thalassemia. The y-globin induction agents include cytotoxic agents, DNA methyl transferase (DNMT) inhibitors, histone deacetylase (HDAC) inhibitors, target of rapamycin (TOR) inhibitors and some cytokines, etc. But just a little induction agents can be applied to the clinical use for now. Most induction chemical agents including hydroxyurea,5-azacytidine, butyrate, erythropoietin, and so on have respective obvious disadvantages such as myelotoxicity. potential carcinogenesis, impermanent effect or high cost, etc, which badly restrict the clinical usefulness of these agents.
Chinese herbal medicine has little side-effect relatively. To date, most studies published on Chinese medicine therapy for β-thalassemia were case reports or clinical observations except for the series studies about "Yisuishengxue granule". But the formula of "Yisuishengxue granule" consists of11Chinese herbs so it's difficult to study or find the effective constituent inside this formula. There is great need to screen for natural y-globin inducer from single Chinese herb or little Chinese medicinal formula. Our previous and recent in vitro studies suggested that Tortoise plastron (Tortoise shell), Astragalus membranaceus(Radix Astragali) and Codonopsis pilosula(Radix Codonopsis) could enhance y-globin mRNA expression and HbF production either in human erythroid K562cell cultures or erythroid progenitors from patients with β-thalassemia. Compared with butyrate, the induction effect by these Chinese medicines lasted for longer time significantly and had no inhibitory effect on erythroid cells. Our subsequent experiment about Astragalus membranaceus indicated its valuable constituents were contained in hydro-soluble astragali polysaccharide and the further studies about single astragali polysaccharide indicated it had similar induction effect. According the in vitro effect of these herbs and the traditional Chinese medicine (TCM) theory, we designed and performed a random controlled double-blind prospective clinical study of single Astragalus membranaceus and the little Chinese medicinal formula consist of Astragalus membranaceus, Codonopsis pilosula and Tortoise plastron for treatment of β-thalassemia innovatively in order to determine their in vivo effect and evaluate their clinical efficacy and side effects objectively.
This study also investigated whether or not these herbs can induce y-globin gene expression in children with β-thalassemia to explore their in vivo molecular mechanisms. The activation of p38mitogen activated protein kinase (MAPK) signaling pathway.plays a role in the mechanism of action for y-globin synthesis. Our previous studies indicated that butyrate activate y-globin expression in erythroid cells via a p38MAPK-dependent mechanism that involves the affected transcription factor and epigenetics mechanisms. The experiment performed by other PhD students in our research team suggested the Astragalus membranaceus and Tortoise plastron's in vitro effect of y-globin induction due to the activation of p38MAPK pathway, too. Thus the role of p38MAPK in patients was also investigated to analyze the in vivo molecular mechanisms from the signaling pathway angle.
Methods:The present study was a random controlled double-blind prospective clinical trial involved57pediatric patients with (5-thalassemia from two institutions. The children were split into layers of thalassemia major or thalassemia intermedia and randomly assigned to formula medicine group, Astragalus membranaceus group or placebo control group. The children in formula medicine group were administrated oral Astragalus membranaceus, Codonopsis pilosula and Tortoise plastron granules. One, two or three bags of each kind of these Chinese medicine granules were for each child ranged from2to6years old, from6to12years old, or from12to18years old, respectively everyday. Three, six or nine bags of oral Astragalus membranaceus granules were administrated for each child in Astragalus membranaceus group ranged from2to6years old, from6to12years old, or from12to18years old, respectively everyday. And three, six or nine bags of oral placebo granules were administrated for each child in placebo control group ranged from2to6years old, from6to12years old, or from12to18years old, respectively everyday. The course of treatment was12weeks. The children with thalassemia major used the therapy above in combination with regular transfusion therapy at4weeks intervals.
Before the treatment and after treatment for4weeks,8weeks and12weeks, blood routine tests were performed in each participant studied to measure the parameters including hemoglobin (Hb), red blood cell (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and red blood cell volume distribution width (RDW). Before the treatment and after treatment for12weeks, the peripheral blood of the patients were derived to count the reticulocytes, determine the HbF proportions and contents by hemoglobin electrophoresis and colorimetry and determine the erythrocyte osmotic fragility by colorimetry method. The sizes of liver and spleen below ribs in each participant with thalassemia intermedia were measured by abdominal palpation before the treatment and after treatment for12weeks. The children's heart rates, weights and blood pressures were measured at baseline and every4weeks. According the TCM syndromes criterion, the improvement rates of "deficiency of Qi and blood"(TCM) symptoms and "weakness of liver and kidney"(TCM) symptoms in each group were assessed after the treatment.
The clinical efficacy criterion was as follows:1) good response:Hb increase no less than3g/dL after the treatment with HbF elevation in some degree;2) moderate response:Hb increase no less than0.5g/dL but less than3g/dL after the treatment with HbF elevation in some degree;3) no response:Hb increase less than0.5g/dL. Responses to the Chinese medicine were assessed and numbers of patients with good response, moderate response and no response were added up after12weeks of treatment then the good response rate and total effective rate were calculated.
Blood routine tests were performed as described before to measure the safety parameters including White blood cell (WBC). neutrophil (NEU) and platelet (PLT). Before the treatment and after treatment for12weeks, the hepatic and renal functions parameters including alanine aminotransferase, aspartate aminotransferase, glutamyl transferase, blood urea nitrogen and creatinine were determined. The side-effect symptoms were assessed after12weeks of treatment.
In addition, studies were performed that analyzed the expression levels of a-, β-, Cry-and Ay-globin genes and the role of the p38MAPK pathway in regulating HbF induction using real-time fluorescent quantitative RT-PCR and Western Blot methods. Before the treatment and after treatment for12weeks, mononuclear cells contains nucleated red blood cells were isolated from the peripheral blood of the participants with thalassemia intermedia by discontinuous density gradient centrifugation. According the sequences of each gene from Genbank, the primers were designed and synthesized. RNAs were isolated using Trizol then reverse transcription reactions and real-time fluorescent quantitative PCR reactions were carried out. The target genes mRNA levels were quantitated by the standard curves and the cycle threshold values of each sample and normalized to the levels of internal control gene. Furthermore, the total proteins of each sample were isolated and separated using SDS-PAGE method. Subsequently, the proteins were transferred to PVDF membranes and hybridization with p38MAPK and phosphorylation p38MAPK antibodies were carried out in next day. The integrated optical densities of p38MAPK, phosphorylation p38MAPK and internal control hybridization bars of each sample were analyzed by image software.
Databases were established and statistical analyses were performed using SPSS13.0. Statistically significant differences of each parameter between or within groups were performed finally. Differences were considered to be significant when P is less than0.05.
Results:After treatment of either Astragalus membranaceus granules or its medicinal formula granules for12weeks, the Hb levels in children with β-thalassemia intermedia elevated significantly. The mean Hb elevation value was1.21±1.12g/dL in the formula group and1.05±0.80g/dL in the Astragalus membranaceus group, as compared with-0.28±0.51g/dL in the placebo control group (F=10.209, P<0.001). After treatment of12weeks, the mean Hb levels of children with β-thalassemia intermedia both in the formula group and the Astragalus membranaceus group were higher significantly than that in the placebo group (8.91±1.07g/dL vs.7.30±1.07g/dL, P=0.001;8.40±1.06g/dL vs.7.30±1.07g/dL, P=0.017, respectively). There was significant differences over time with respect to Hb levels in children with β-thalassemia intermedia both in the formula group (F=8.773, P<0.001) and the Astragalus membranaceus group (F=20.940, P<0.001) in contrast to no significant difference over time in the placebo control group (P>0.05). The mean Hb elevation value of children with β-thalassemia major after treatment of12weeks was0.46±0.61g/dL in the formula group as compared with-0.55±0.66g/dL in the placebo control group (P=0.033). The post-treatment Hb levels in children with p-thalassemia major both in the formula group and the Astragalus membranaceus group were higher significantly than that in the placebo group (7.91±0.49g/dL vs.6.65±1.10g/dL, P=0.024;7.74±1.15g/dL vs.6.65±1.10g/dL, P=0.043, respectively). This therapy with Astragalus membranaceus or its formula medicine also improved other parameters of blood routine test including RBC and MCH in some degree.
Comparing initial and last values, mean HbF proportion of children with β-thalassemia intermedia in the formula group rose significantly (62.80%±22.85%vs.54.00%±20.63%, t=-2.910, P=0.016) and so did mean HbF proportion in the Astragalus membranaceus group (68.10%±16.63%vs.60.06%±16.29%, t=-4.103, P=0.001). Both Astragalus membranaceus and its formula medicine also raised HbF contents above baseline significantly in children with P-thalassemia intermedia (t=-3.896, P=0.003and t=-4.616, P=0.001, respectively). The post-treatment HbF contents in children with β-thalassemia intermedia either in formula group or Astragalus membranaceus group were significant higher compared with control (P=0.001and P<0.001, respectively). Mean HbF proportion of children with P-thalassemia major in the formula group rose above baseline significantly (22.75±6.97%vs.20.23±7.06%, t=-3.424, P=0.014) and so did in the Astragalus membranaceus group (29.01±12.50%vs.22.46±12.25%, t=-3.215, P=0.015).The HbF contents in both groups above also increased significantly after treatment (t=-3.156, P=0.020and t=-3.491, P=0.010, respectively). Pair-wise comparisons for baseline and post-treatment levels of either HbF proportions or HbF contents had no statistical significance in the placebo control groups (P>0.05). The results above demonstrated that both Astragalus membranaceus and its formula medicine induced an increase of HbF in the β-thalassemia patients studied.
A marked elevation of reticulocyte levels was observed either in the intermedia Astragalus membranaceus group or in the major formula medicine group (t=-5.790, P<0.001and t=-3.195, P=0.019, respectively). Significant improvement in erythrocyte osmotic fragility of children with β-thalassemia intermedia was documented after formula medicine therapy (t=-2.331, P=0.042). There was no significant difference with respect to the sizes of liver and spleen between before and after treatment in all groups. The weights of children with P-thalassemia intermedia in Astragalus membranaceus group and the systolic blood pressures of children with β-thalassemia major in formula group rose significantly over time (F=5.330, P=0.017and F=8.551, P=0.001, respectively). And these Chinese medicines ameliorate the "deficiency of Qi and blood"(TCM) symptoms and "weakness of liver and kidney"(TCM) symptoms markedly relative to control.
The total effective rate in children with β-thalassemia intermedia was63.6%in the formula medicine group and61.5%in the Astragalus membranaceus group which were higher significantly than that in the placebo control group (9.1%; x2=8.681, P=0.013). The total effective rate in children with β-thalassemia major was42.9%in the formula medicine group,50.0%in the Astragalus membranaceus group and0%in the placebo control group. There were significant difference among them, too (x2=6.871, P=0.032).According all the results above, it can be demonstrated that Astragalus membranaceus or its formula medicine is effective without placebo effect as a treatment for children with β-thalassemia.
Most of the parameters above were statistically identical between in formula medicine group and Astragalus membranaceus group, suggesting comparable efficacies of these two Chinese medicines for P-thalassemia treatment. Although the total effective rate in children with β-thalassemia intermedia was higher than that in children with p-thalassemia major, there was no statistical significance between them in the present sample size. And there was no statistical difference in the Chinese medicines'effect among children with different phenotypes, different genotypes or different TCM syndromes.
The incidence of epistaxis was relatively high in the children administrated Astragalus membranaceus granules (x2=7.697, P=0.021). There was no significant difference with respect to the incidences of other symptoms probably involved adverse reactions between children administrated Astragalus membranaceus and those administrated formula medicine. Furthermore, there was no statistical significance with respect to WBC, NEU, PLT and hepatic and renal functions parameters among groups or between before and after treatment in all groups. No one patient required discontinuation of treatment because of adverse effects.
The blood transfusion frequencies in follow-up period in the Chinese medicine groups were less but not significantly than those in the corresponding placebo groups (P>0.05)
The results of real-time fluorescent quantitative RT-PCR indicated that the difference of Gy-globin gene mRNA levels between before the treatment and after treatment for12weeks both in formula medicine group and Astragalus membranaceus group were significant (F=14.152, P=0.004and F=14.854, P=0.002, respectively). And the post-treatment levels of Gγ-globin gene mRNAs in both Chinese medicine groups were higher significantly than the control group (P=0.010and P=0.036, respectively). These results suggested that our Chinese medicines induced Gy-globin gene expression in vivo at the transcriptional level. The Chinese medicines had no marked effect on a-, β-and Ay-globin genes expression. We also observed significant increases of in vivo phosphorylation p38MAPK protein levels for both Astragalus membranaceus and formula medicine (t=-4.234, P=0.002and t=-3.953, P=0.002, respectively) compared with no increase for placebo, as revealed in western blot analysis. And significant differences were observed in post-treatment phosphorylation p38MAPK levels compared both Chinese medicine groups with the placebo group (P=0.006and P=0.019, respectively). But the Chinese medicines had no significant effect on the protein level of p38MAPK without phosphorylation. The results above suggested Astragalus membranaceus or its formula medicine could stimulate the in vivo phosphorylation activation of p38MAPK.
Conclusion and prospect:In the present study the major conclusions were as follows:
1) Both oral single Astragalus membranaceus and the little Chinese medicinal formula consist of Astragalus membranaceus, Codonopsis pilosula and Tortoise plastron can elevate total hemoglobin and HbF levels of children with β-thalassemia significantly. Furthermore, they can ameliorate the patients'symptoms of TCM four diagnostic methods and hematological parameters including RBC, MCH, reticulocyte and so on.
2) This random controlled double-blind prospective clinical trial suggested that the therapy with Astragalus membranaceus or its formula medicine is effective and safe for children with β-thalassemia.
3) Both oral Astragalus membranaceus and its formula medicine can induce the Gy-globin gene expression in vivo which constitutes an important mechanism that stimulate HbF synthesis then enhance total hemoglobin in patients with β-thalassemia.
4) Both oral Astragalus membranaceus and its formula medicine can activate the phosphorylation of p38MAPK in children with β-thalassemia and it suggests that the in vivo y-globin induction of these Chinese medicines may be at least in part regulated through a p38MAPK signaling pathway.
This study had great significance for developing a novel therapeutic approach to β-thalassemia. The prevalence of β-thalassemia is high in China, but the therapeutic methods for β-thalassemia including blood transfusion, chelation therapy, gene therapy, hematopoietic stem cell transplantation and so on have respective obvious disadvantages. And most chemical agents for globin induction are not satisfying in respect of either clinical efficacy or safety. The results of this study and other studies in our team indicated Astragalus membranaceus or its formula medicine has not only relatively good clinical efficacy for children with β-thalassemia but also no cytotoxicity, hepatic or renal toxicity, or myelotoxicity. The oral granules are convenient for patients with P-thalassemia and the patients'tolerance and compliance are good. Astragalus membranaceus and its formula medicine will be very promising in the treatment of β-thalassemia in the foreseeable future.
It has been generally assumed that most Chinese medicines have mechanisms at multiple levels or multiple targets. The present preliminary observations suggest the need for evaluating larger number of patients in long-term, large collaborative trials with more parameters to study other mechanisms of these Chinese medicines about globin gene induction or ameliorate anemia, their influence on iron load level or β-thalassemia complications such as hepatosplenomegaly, heart failure, and poor growth and development as well as their late results, long-term safety, optimal dose and duration of therapy, etc. Compared with traditional big Chinese medicinal formula, the single Chinese herb and little Chinese medicinal formula in this study are relatively fit for study and isolation of effective constituent and discovery of the scientific values of TCM treatment theories. The present and subsequent studies in our team have great scientific interest about developing new drug for β-thalassemia and studying the hemoglobin switching and induction. In addition, they have important social meaning and practical prospect in the areas where the prevalence of β-thalassemia is high, particularly the southern provinces in China.
引文
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