| BACKGROUND AND QBJECTIVEβ-thalassemia is one of the most common heritable hemolytic diseases that results in considerable morbidity and mortality throughout the world,and at present there is no safety,effective and easy curing method.Mutations or deletions ofβ-globin gene cause low synthesis ofβ-globin chains,resulting in freeα-globin chains deposit on red blood cell surface,which leads to decline of erythrocyte deformability and increase of hematoclasis in spleen.γ-globin inducers can reactive expression of nearly closedγ-globin gene,increase the production of fetal hemoglobin(HbF)(α2γ2) to instead of adult hemoglobin(HbA)(α2β2),improve the hemolysis in situ cause by cytoryctes and ameliorate clinical symptoms of patients.Since 5-azacytidine (5-AZAC) was found to have ability to treatβ-thalassemia,lots ofγ-globin inducers were found in last two decades,such as hydroxyurea(HU),erythropoietin(EPO), cytarabine,vincristine,trichostatin A(TSA),butyrate,decitabine and so on.However, HU is the only drug that was approved for treatment ofβ-thalassemia by United States Food and Drug Administration(FDA),even then there is 25%nonresponse rate in patients.So there is a great need of novel agents that can activateγ-globin gene.In last two decades,the biggest barrier for the development ofγ-globin inducers is poor understanding of its regulation mechanisms.Studies in recent years revealed that cellular signaling transduction,especially mitogen-activated protein kinases(MAPKs) pathway,played a significant role inγ-globin gene regulation,p38 MAPK,an important member of MAPKs,can be actived by many environmental stimulations such as protein kinase C,receptor tyrosine kinase,tumor necrosis factor,ultraviolet and drugs.After the cascade reactions of protein kinase are provoked,p38 MAPKs have an effort on many vital processes including cell transcription,protein synthesis, and cell surface receptor expression.It is reported that HU,TSA,MS-275, thalidomide and scriptaid increasedγ-globin mRNA levels in K562 cells and primary erythroid progenitors by activating p38MAPK cell signal pathway.Our previous study on high-expression phospho-p38 cell models has also verified that direct activation ofp38MAPK can induceγ-globin gene expression.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 stimuling haematogenesis.One of its active components,astragalus polysaccharide(APS),can induce colony-forming unit- erythroid(CFU-E) and burst-forming units- erythroid (BFU- E) expressions.Our previous study had also found that APS had the ability to improveγ-globin gene expression and increase HbF synthesis.However,the mechanism this process was still unknown.Our goal was to determine the role of p38 MAPK signaling inγ-globin gene induction in K562 cells treated with APS.Taking K562 cells as cell model and using RT-PCR, Western blotting and benzidine staining technique,this study will elucidate the relation between p38MAPK activation andγ-globin gene expression in K562 erytholeukemia cells treated with APS,provide new experiment data forγ-globin gene regulation mechanism and rationale for the clinical application of APS.At last,this study will provide new molecular target for developing new drugs to treatβ-thalassemia. METHOD1.Drug preparation and cells culture:APS was extracted in Ultrasonic device with ultrapure water after alcohol as solvent from Astragalus membranaceus Bge.var.mongolicus(Bge.)Hsiao,quantitated by phenol-concentrated sulfuric acid method and then filtered.K562 cells were chosen as the cell model;K562 cells induced with APS belonged to experimental group;K562 cells treated with 0.5mmol/L Na-buytrate(NaB) for 48h were used as the positive control group and untreated K562 cells were seen as blank control cell model.2.Phospho-p38/p38 was analyzed by Western blotting.2.1 Dosage effect:Total protein was isolated from K562 cells with APS(150mg/L, 300mg/L,450mg/L,respectively) and untreated K562 cells for 48 h to determine the level for p38 phosphorylation levels.2.2 Time effect:Total protein was isolated from K562 cells with 300mg/L APS from 3 to 72 hours respectively to determine the time course for p38 MAPK activation.2.3 Depressant effect of SB20358 on p38 phosphorylation:Total protein was isolated from K562 cells with 300mg/L APS alone or following pretreatment with the p38 kinase specific inhibitor SB203580(10μmol/L) for 1 hour.p38 MAPK activation was learned by Western blotting.3.Aγ-globin mRNA and Gγ-globin mRNA levels were measured by RT-PCR to depict the effect of p38 MAPK activation.4.Western blotting was employed to measure the content of HbF to illuminate the relationship between phosphorylation level of p38 and erythroid differentiation of human K562 erytholeukemia cells treated with APS.5.Bbenzidine staining assay was used to analyze the relationship between phosphorrylation level of p38 and erythroid differentiation of human K562 erytholeukemia cells induced with APS.6.Statistical methods:The data are reported as the mean±S.SPSS 11.5 for Windows software was used to analysis the raw data by One-Way ANOVA.The Least-significant-Difference test was used to measure differences in samples for two group comparisons.Probability of less than 0.05 was considered significant.RESULTS1.Effect of APS on p38 phosphorylation in K562 cells:1.1 Phospho-p38 levels of K562 cells induced with APS were up-regulated and was 5.18±0.13folds(P=0.000) comparing with untreated K562 cells.1.2 Dosage effect:p38 phosphorylation were induced in K562 cells treated with 150mg/L,300mg/L and 450mg/L APS respectively,and the results range were from 3.24±0.20 folds to 5.18±0.13 folds(F=305.146,P=0.000;300mg/L was found to be the best inducing dose(P＜0.001).1.3 Time course:Phospho-p38 level began to rise at 6h after addition of APS,the peak located between 48h and 60h,and then it went to decline(F=399.72,P=0.000). There is no statistical significance of the peak levels of Phospho-p38 between K562 cells treated with APS or NaB(4.25±0.13 folds,P=0.076).1.4 SB203580(SB)inhibited p38 phosphoralation induced by APS:p38 phosphoralation was decreased in K562 cells pretreated with SB,a p38 kinase inhibitor,for 1h before induced with APS.Comparing with untreated K562 cells,The increased folds decreased to 1.59±0.12 from 4.10±0.09 in cells with APS alone (F=933.503,P=0.000),and the inhibition ratio was 80.97%.When it came to NaB, the increased folds decreased to 1.45±0.04 pretreated with SB from 4.25±0.13 in cells with APS alone,and the inhibition ratio was 86.15%.2.Relationship between phosphorylation level of p38 andγ-globin gene expression in K562 cells treated with APS: 2.1 Increased of Phospho-p38 in K562 cells treated with APS inducedγ-globin gene expression.2.1.1 Results of RT-PCR showed that Aγ-globin and Gγ-globin mRNA levels raised to 4.37±0.35 folds(F=134.602,P=0.000) and 5.23±0.28 folds(F=505.453,P= 0.000) respectively after 48h treated with APS.In NaB group,the data became to 4.50±0.14folds(P=0.000) and 6.52±0.25 fold(P=0.000),respectively.2.1.2 Results of HbF by Western blotting displayed that 300mg/L APS significantly increased HbF levels by 6.41-fold and the increaser treated with 0.5mmol/L NaB was 7.10±0.10(F=736.199,P=0.000).2.1.3 Results of benzidine staining assay displayed that APS induced HbF synthesis in K562 cells(F=67.278,P=0.000) to 15.67%,and the data of NaB group was 19.60%.There were significant differences comparing with untreated K562 cells (3.47%,P＜0.001).2.2 SB inhibitedγ-globin gene expression in K562 cells treated with APS:2.2.1 In APS-mediated K562 cells pretreated with SB for 1h,Aγ- and Gγ-globin gene mRNA diminished to 2.28±0.09 folds and 1.75±0.19 fols(P＜0.004),and the inhibition ratio were 62.02%and 82.27%,respectively.In cells pretreated with SB and induced with NaB alone,the increased folds were cut down from 4.50±0.14 and 6.52±0.25 to 2.61±0.30 and 1.58±0.10,and the inhibition ratio were 54.00%and 89.50%,respectively.2.2.2 After pretreated with SB,HbF synthesis induced by APS or NaB alone reduced to 1.84±0.15 folds and 1.58±0.27 folds(P≤0.004),and the inhibition ratio were 84.47 %and 90.49%,respectively.2.2.3After pretreated with SB,BZ%in K562 cells treated with APS or NaB were 3.93 %(P=0.733) and 4.00%(P=0.697),and the inhibition ratio were 96.23%and 96.71 %,respectively. CONCLUSION1.We demonstrated that APS can induce p38 phosphorylation in K562 cells for the first time,and there were dosage effect and time course in this enhancement.The best inducing dosage is 300 mg/L;Phospho-p38 level begin to rise at 6h,and the peak locates between 48h and 60h,then it goes to drawdown.Activation of p38 MAPK can be inhibited by SB.2.We justified for the first time that p38MAPK cell signaling played an important role in the activation ofγ-globin expression induced by APS in K562 cells.3.Our data provided other evidences for illuminating the role of p38 MAPK cell signaling in drug-mediatedγ-globin gene expression as well as for clinical application of APS,and provided new target for developing novel drugs to treatβ-thalassemia. |
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