Combined Use Of Astragalus Polysaccharide With Sodium Butyrate In The Induction Of Erythroid Differention And Globin Gene Expression On K562 Cells

BACKGROUND AND OBJECTIVEβ-thalassemia is one of the most common heritable hemolytic diseases that results in considerable morbidity and mortality throughout the world,and presently there is no safety,effective and easy curing method.Mutations or deletions ofβ-globin gene cause low synthesis ofβ-globin chains,and result in extra freeα-globin chains deposition on red blood cell surface,which leads to declining of erythrocyte deformability and increasing of hematoclasis in spleen.β-thalassemia is one of the most common monogenic diseases in Southern China.While the preferable therapy, bone marrow transplantation,has certain difficulty to be applied widely in clinic, drug-induced gene therapy has drawn attention of doctors and biologists as an alternative therapy.Agents as drug-inductor are used to modulate globin gene expression in the genetic switch,activate the non-αglobin gene function,and increase non-αglobin protein synthesis,or inhibiteα-globin gene function,to reduceα-globin synthesis,thus reduceαand non-αglobin's imbalance,and even restore the normalαandβglobin ratio,to achieve the purpose of alleviating clinical symptoms.Theγ-globin inducers can reactivate expression of nearly closedγ-globin gene, increase the production of fetal hemoglobin(HbF)(α2γ2) to substitute adult hemoglobin(HbA)(α2β2).Studies found that someγ-globin gene can be induced by conditioning agentα,ξ,β,ε,δ-globin gene expression and affect the balance betweenα-chain and non-αchain.In 1982,the application of 5-azacytidine as treatment ofβ-thalassemia patients with better efficacy was first reported abroad,but its short lasting time,adverse reactions,and long-term carcinogenicity restricted its further clinical use; Hydroxyurea was found subsequently as the treatment of sickle-cell anemia,the result is very good,but at least 25%of theβ-thalassemia patients had no response the treatment,and could be the trigger of bone marrow suppression after a long-term treatment;Sodium butyrate,although with less side effects,its short half-life, inconvenient application and cost also limits its clinical use.A number of factors make the clinical application of these drugs are subject to certain restrictions.Since 1980s,over the past two decades,there has been＞70 compounds ofγ-globin gene inducers had been proved to be effective in vitro and in vivo,but because of specificity,toxicity of some inducers,only a few of them were used in clinical,HU is the only drug that was approved for treatment ofβ-thalassemia by United States Food and Drug Administration(FDA),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 increaseγ-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 increaseγglobin gene expression,reduce treatment dose and cytotoxicity;Scholars have studied on HU combination with NaB or EPO in vivo and proved that combined drug use can generate synergistic induction of HbF,but the subjects,genotype,dose,sample size and other factors induced results are inconsistent,so there is no uniform treatment as clinical standards.Most of theγ-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.Our previous study found that Astragalus had the ability to improveγ-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.One of its active components,astragalus polysaccharide (APS),can induce colony-forming unit- erythroid(CFU-E) and burst-forming units-erythroid (BFU- E) expressions.Combining APS with NaB may have synergistic effects on induction of Hb and low toxicity.Our goal was to determine whether the APS plus NaB have synergistic effects on the induction of Hb production and globin gene expression in K562 cells.Effects of each group on K562 cells proliferative inhibition were determined by trypan blue exclusion assay;Benzidine staining,and RT-PCR technique were used to investigate the expression of hemoglobin cellular and mRNA level respectively.At last,this study will provide new strategy of clinical therapy forβ-thalassemia.METHODPart one The optimal drug combination of APS with NaB1.1 Effect of APS,NaB alone on inhibition and differentiation of benzidine-postive cells. K562 cells were divided into 2 groups:APS treated group 0～20mg/ml;NaB treated group 0～1000μmol/L.At 24-hour intervals,the cell number was counted by trypan blue exclusion assay and benzidine-positive cells were determined by benzidine staining at 96h.1.2 The optimal drug combinationThe selection criteria of:drug combination group In accordance with a single drug inhibition rate,half inhibitory concentration(IC50) and benzidine staining results,select the concentration which can induce erythroid differentiation,IC50 values of different concentrations of the whole joint.1)the K562 cells number were detected by trypan blue exclusion assay after treatment for 96h,Calculate the inhibitory rates of cells.Interaction between HU and NaB.APS and NaB were assessed with q value,where q＞1.15,0.85≤q≤1.15,and q＜0.85 indicated synergistic,additive,and antagonistic effects respectively.2) benzidine-positive cells was determined by benzidine stainingPart two APS,NaB and APS with NaB were applied on K562 cells to induced erythroid differentiation and globin gene expression.2.1 K562 cells were divided into 5gorups:APS2.5mg/mL+NaB250μmol/L the combination group,NaB250μmol/L single agent group,NaB500μmol/Lthe ruler dose group,K562 untreated group.2.2 At 24-hours intervals,benzidine-positive cells was determined by benzidine staining at 144h.2.3 The expression of globin mRNA were measure by RT-PCR to depict the effect of combination therapy.3.Statistical analysis:All data was analyzed with SPSS13.0 statistical software. Unless otherwise stated,data was expressed as mean±S.D.The inhibitory rates,the benzidine-positive cells,and the amount of mRNA were analyzed with Univariate.The other data was analyzed with One-way ANOVA followed by post hoe test.P＜0.05 was considered to be significant.ResultsChapter one The optimal drug combination of APS with NaB1 Trypan blue exclusion assay result indicated:APS and NaB alone inhibited cell proliferation time dose-dependently(P＜0.05).2 Benzidine staining result indicated APS and NaB alone induced K562 cells erythroid differentiation time dose-dependently(P＜0.05).3 Trypan blue exclusion assay and Benzidine staining results indicated that APS2.5mg with NaB100μM,APS2.5mg with NaB250μM were the optimal combination.After 96 hours of culture,the effect of the drug(s) on Benzidine positive cells(BZ%),cell proliferation rate and cell viability were (18.88±1.98)%,(46.24±2.97)%,(93±1.6)%and(35.59±2.23)%,(67.47±3.76)%,(90±1.2)%,respectively,compared with the ruler does NaB500μM(16.27±1.09)%,(88.86±1.53)%,(74±2.4)%,Combination group showed greater increase of Benzidine positive cells and decrease of growth inhibitory and cytotoxic effects(P＜0.05).Part two APS combination with NaB on K562 cells to induced erythroid differentiation and globin gene expression1 APS combination with NaB on K562 cells to induced erythroid differentiation and globin gene expressionBenzidine staining showed APS2.5mg,NaB250μM combination can induce K562 cells to erythroid differentiation,the BZ%increased significantly during 48 to144 hours,at 96 hours the BZ%reached the peak of(32.89±0.24)%,significantly increased comparing with the rule dose group NaB500μM(14.66±0.17)%(P＜0.05);significantly increased comparing with low-dose monotherapy group APS2.5mg(7.72±0.12)%,NaB250μM(7.58±1.46)%(P＜0.05).RT-PCR results showed that,APS+NaB group up-regulated theγglobin,the expression levels of Gγand Aγ-mRNA up-regulated 2.40±0.02 folds,1.61±0.01 folds respectively,comparing with the ruler dose groups NaB500μM(2.08±0.02 folds),(1.31±0.02 folds) and comparing with low-dose monotherapy group APS2.5mg(1.25±0.01-folds),(1.19±0.02-fold),NaB250μM(1.26±0.01-folds), (1.18±0.01 folds),there was no significant difference(P＜0.05).The expression levels ofα-mRNA up-regulated 1.16±0.02 folds,comparing with NaB500μM(1.14±0.02-folds)(P＞0.05),there was no significant difference;and comparing with APS2.5mg(1.07±0.04-folds),NaB250μM(1.02±0.13-folds),there were significantly difference(P＜0.05).Comparing with NaB500μM(1.25±0.03-folds) and APS2.5mg(1.11±0.01-folds) NaB250μM(1.16±0.02-fold),β-mRNA expression of 1.73±0.03-fold increase significantly(P＜0.05).Comparing with NaB500μM(1.53±0.06 folds) and APS2.5mg(1.02±0.02-folds), NaB250μM(1.28±0.02-folds),ε-mRNA expression of 1.40±0.04-fold increase(P＜0.05).The expression levels ofξ-mRNA up-regulated 1.18±0.03-folds,comparing with NaB500μM(1.16±0.03 folds) and APS2.5mg(1.11±0.02-folds) there were no significant difference(P＞0.05);comparing with NaB250μM(1.08±0.06 folds),there were difference significantly(P＜0.05).Changes inδ-mRNA was no significant difference(P＞0.05),compared with the other group without significantly different.Conclusion1 We first demonstrated that the optimal combination was APS2.5mg/ml+ NaB100μmol/L,APS2.5mg/ml+NaB250μmol/L in the induction of erythroid differentiation,up-regulatedγ-mRNA expression and decreased growth inhibitory and cytotoxic.effect2 We first demonstrated that APS+NaB up-regulated Gγ,Aγ,β,ε-mRNA expression obviously,but theα-gene cluster ofξ,α-mRNA were up-regulated a little.3 Experimental proof of the first APS+NaB combined low-dose NaB than conventional single-dose medication more effectively K562 cells induced to erythroid differentiation,and enhanceγ-globin gene expression and reduce cell growth inhibition and cytotoxicity.4 The experimental results ofr the combination therapy to induction ofγ-globin gene expression offered a new scientific evidence and ideas,and the new combination therapy for clinical program design and implementation to provide an experimental basis.