| BACKGROUND AND OBJECTIVEThe (3-thalassemias are a genetically heterogeneous group of diseases resulting from decreasedβ-globin production and a subsequent imbalance in the a/β-globin chain ratio. It is very harmful to people’s health. Excess a-globin chains precipitate within red blood cells(RBC) resulting in hemolysis and ineffective erythropoiesis. The only means to definitively cure the disease is through allogeneic bone marrow transplantation, but it is not an option for most patients for many difficulties such as match limitations、operation risks and expensive costs. Gene correction therapy is the perfect scheme in principle, however, it is particular in its stringent transcriptional requirements:transgene expression has to be erythroid specific, differentiation stage specific and elevated. Drug-induced gene therapy has drawn attention of doctors and biologists as an alternative therapy. Enhancingγ-globin chain synthesis within the RBC reduces the a/p-globin chain imbalance and could potentially lead to an improvement in RBC survival and less anemia. Pharmacologic agents that increase in fetal hemoglobin(HbF), therefore, become a potential therapy for patients withβ-thalassemia. So far, the reported pharmaceutical chemicals with high therapeutic efficacy mainly contain three categories:(1)Cytotoxic agent, such as hydroxyurea (HU),cytosine arabinoside, vincristine(VCR), methotrexate(MTX), Cisplatin and Adriamycin derivatives, et al. (2)DNA methytransferase inhibitor, such as 5-azacytidine and decitabine,et al. (3) Histone deacetylase inhibitor, such as butyrate and its derivatives, apicidin, et al. Hydroxyurea and sodium butyrate are the popular candidates under research. HU is the only drug that was approved for treatment of sickle cell disease by United States Food and Drug Administration(FDA), it is effective for the mild and intermedia patients, but for the major patients the effect is inconsistent, and longtermusage may cause side effects such as distortion and cytotoxicity. Butyrate can induce fetal hemoglobin expression in thalassemia patients, while its disadvantages limit clinical application, such as short plasma half-life(5-10min), high oral dose, difficulty in synthesis,and high cost. There is an urgent need for more effective method in the development of new drugs and new treatment strategies to address these issues. The development of new drug is a time-consuming and costly process, thus the natural features of Chinese traditional medicine such as minor adverse reactions, drug-rich source, the right price, make it has good prospects for research and development, Element of the natural extract of Angelica, resveratrol have been found can effectively increase y-globin gene expression, but have not been used in clinic trial till now. During the process of new drugs’development, combined drugs therapy has become another research direction. Joint use of drugs with different mechanisms and targets synergistic can increase y globin gene expression, reduce treatment dose and cytotoxicity; Scholars have studied on HU combination with NaB or EPO in vivo and proved that combined drag use can generate synergistic induction of HbF, but the subjects, genotype, dose, sample size and other factors induced results are inconsistent. Most of the y-globin gene inducer are used in clinic currently are cytotoxic drugs, although combination therapy can reduce the dose therefore to reduce cell toxicity, but theβ-thalassemia needs a long-term treatment, the long-term efficacy and toxicity of these therapies still call for further exploring. We have screened out Astragalus root from the prescriptions which may therapy beta-thalassemia through serological method and contemporary molecular biology technology, we further proved that one of its active components, astragalus polysaccharide (APS) had the ability to improve gamma-globin gene expression and increase HbF synthesis with minor side effect. Astragalus, the dry root of Astragalus membranaceus Bge. var. mongholicus (Bge.)Hsiao or Astragalus membranaceus, is a traditional Chinese medicine for supplementing qi and benefiting blood. Astragalus was found to have the function of stimulating hematogenesis. Astragalus polysaccharide(APS), can induce colony-forming unit-erythroid (CFU-E) and burst-forming units-erythroid (BFU- E) expressions.Our former research have explored the best combination dose of APS and NaB. It was proved that the low combination dose can induce erythroid differentiation on K562 cells and up-regulated gamma-globin gene on both K562 cells and human erythroid progenitor cells,but whether the up-regulation of gamma-globin gene can enhance HbF synthesis is unknown.Our goal was to determine whether the combination administration of APS and NaB have synergistic effects on the induction of fetal hemoglobin in K562 cells and human erythroid progenitor cells. Our study first selected K562 cells as the study object, using benzidine staining technique to investigate the erythroid differentiation, then we selected cord blood as the research model which is rich of haemopoietic stem or progenitor cells, using trypan blue exclusion assay to detect cell inhibitory rates, using western blotting technique to investigate the fetal hemoglobin synthesis in K562 cells and human erythroid progenitor cells. The results of our study will provide the rationale foundation for low dose combined administration on clinical application, and search new ways to treat (3-thalassemia.METHOD1 Source materialsK562 cells were bought from Shanghai cell bank of the Chinese Academy of Sciences; Cord blood were taken from Nanfang hospital. 2 Cells were divided into 5 gorups:APS 2.5mg/mL+NaB 250μmol/L the combination group, APS 2.5mg/ml、NaB 250μmol/L single agent group, NaB 500μmol/L the positive control, untreated cells as the blank control.3 K562 cells cultureThe human leukemia K562 cells were cultured in a humidified atmosphere of 5% CO2/air in RPMI 1640 medium supplemented with 10% fetal bovine serum,100 units/ml penicillin,100 units/ml streptomycin and 2mmol/1glutamine.4 Two-phase liquid culture system to culture human erythroid progenitor cellsMononuclear cells were isolated from cord blood samples by Ficoll-Hypaque density gradient centrifugation and seeded in a-minimal essential medium and 10% conditioned medium from the 5637 bladder carcinoma cell line. Compounds were added on day 6 of phaseⅡand at day 14 culture was finished.5 Trypan blue exclusion assay was used to detect cell inhibitory rates.6 Benzidine staining assay was used to analyze erythroid differentiation of human K562 erytholeukemia cells induced with combination administration of APS and NaB.7 Western blotting was employed to measure the content of HbF induced with combination administration of APS and NaB.8 Statistical methodsAll data was analyzed with SPSS 13.0 statistical software. Unless otherwise stated, data was expressed as mean±S.D. The cell inhibitory rates and benzidine-positive cells were analyzed with Univariate. The other data was analyzed with One-way ANOVA followed by post hoc test. P<0.05 was considered to be significant.RESULTS1 The effect of cell inhibition of APS combined with NaB1.1 Cell inhibitory rates of combination use on K562 cellsAfter 96 hours of culture, the effect of the drugs on cell inhibitory rate and cell viability were (67.47±3.76)% and (91±1.3)%, compared with the ruler does NaB500μM (88.86±1.53)% and (74±2.4)%, Combination group showed greater decrease of growth inhibitory and cytotoxic effects (P<0.05).1.2 Cell inhibitory rates of combination use on human erythroid progenitor cellsAfter d6, there didn’t exsit significant differences of the inhibition ratio of the cells in the group APS+NaB (2.5 mg/mL+250μmol/L) (F=3.184, P=0.210), and the inhibition ratio touched the peaking[(36.84±1.27)%] at d10,and then started to decrease. At d10, there exsit significant difference of inhibition ratio of cells between different experiment groups(F=28.211, P=0.000), The inhibition ratio of the cells in group APS+NaB (2.5 mg/mL+250μmol/L) was less than group NaB 500μmol/L [(44.55±2.83)%(P<0.05)].2 APS with NaB were applied on K562 cells to induced erythroid differentiationThere were significant difference between each group in BZ%(F=7185.879. P=0.000), the BZ% was the highest in the experimental group; There were significant difference between each time point in BZ%(F=1519.977, P=0.000), at 72h and 96h reached the peak; It existed interaction effect between different groups and different time points, which showed the difference of BZ% in different groups was along with the changes of time point, at 96h the difference was the maximal.Benzidine staining showed APS2.5mg, NaB250μM combination can induce K562 cells to erythroid differentiation, the BZ% increased significantly during 48 to 144 hours, at 96 hours the BZ% reached the peak of (32.89±0.24)%, significantly increased comparing with the regular dose group NaB500μM (14.66±0.17)% (P <0.05); significantly increased comparing with low-dose monotherapy group APS 2.5mg (7.72±0.12)%, NaB 250μM (7.58±1.46)% (P<0.05). While the BZ% of the ruler dose group NaB500μM reach the peak at 72h and decline rapidly after 96h.3 APS with NaB were applied on K562 cells to induced fetal hemoglobin synthesisHbF levels are significantly increased in the combination group and the regular dose NaB than in the blank control(P<0.05). When compared with untreated K562 cells, the HbF expression levels increased to (1.69±0.14)、(1.67±0.10) fold and (1.82±0.16)、(1.57±0.08) fold respectively after 72h and 96h treated with 2.5mg/ml APS+250μmol/L NaB and 500μmol/L NaB (F=31.181, P=0.010; F=32.845, P=0.009).There are no significantly difference on HbF expression between the combination group and the regular NaB group.4 APS with NaB were applied on human erythroid progenitor cells to induced fetal hemoglobin synthesisHbF levels are significantly increased in the combination group and the regular dose NaB than in the blank control(P<0.05). When compared with untreated K562 cells, the HbF expression levels increased to (1.96±0.18) and (1.78±0.10) fold respectively after 12d treated with 2.5mg/ml APS+250μmol/L NaB and 500μmol/L NaB (F=34.674, P=0.008). There are no significantly difference on HbF expression between the combination group and the regular NaB group.Conclusion1 We demonstrated that low dose combination of APS and NaB have synergistic effects on the induction of fetal hemoglobin in K562 cells, as the same as the regular dose of NaB.2 We demonstrated that low dose combination of APS and NaB have synergistic effects on the induction of fetal hemoglobin in human erythroid progenitor cells, as the same as the regular dose of NaB.3 We further proved that combination administration of APS and NaB can induce K562 cells more effectively to erythroid differentiation than regular single-dose of NaB.4 Compared with the regular dose of NaB, the time of duration of K562 cells erythroid differentiation was more longer in combination group.5 Combination group showed greater decrease of growth inhibitory and cytotoxic effects when compared with the regular dose of NaB.6 Our deta provided the rationale foundation for low dose combined administration on clinical application. |
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