| 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. |
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