| BACKGROUND AND OBJECTIVEβ-thalassemia is a common monogenic disease also one of the hereditary diseases with highest morbidity. Its molecular pathogenesis has been found to be related with imbalance between α-globin peptide chain and β-globin peptide chain which to construct the hemoglobin. Mutations or deletions of-globin gene cause low synthesis of P-globin chains, resulting in free a-globin chains deposit on red blood cell surface, which leads to decline of erythrocyte deformability and increase of hematoclasis in spleen. It has been known that increase of y-globin expression is clinically beneficial in patients with β-thalassemia. Increasing y-globin expression decreases the imbalanceof non-a-/a-globin chains in β-thalassemia and subsequently promotes fetal hemoglobin (Hb F, a2y2) synthesis, substantially ameliorating the clinical symptoms of patients with this disorder.To date, the DNA methyltransferase inhibitors5Azacytidine (5-Aza) and Histone deacetylase (HDAC) inhibitors sodiumbutyrate(SB)andTriehostatin(TSA)have been applied for the Pharmaeological induction of HbF. Unfortunately, a number of factors, such as nodrug response, hematopoietic suppression, short half-live in vivo, limit the useful ness of these compounds.And it still has a long way to go for clinical use of above agents because of the potential carcinogenicity and their high cost.The challenge to the field of cell and molecular biology of hemoglobin switching is to develop a cure for β-thalassemia using the method discovered by nature. Many traditional Chinese herbal medicines have function of relieving symptoms of anaemia, decreasing the volume of blood transfusion and increasing Hb. A characteristic theorysystem and therapeutics of Chinese crude drug has developed and copious clinicalexperience has accumulated for β-thalassemia. The forte include abundant source, cheap, lower risk compare with Western medicine. A prescription made up of many Chinese crude drugs is difficult to definite the mechanism of action because of theinteraction among the constituents.Study on single-herbs is important to investigatethe valuable constituents and to develop new drugs. Research efforts focused on screening valuable Chinese crude drug and investigating the mechanism of action and toxicity of it that reactivate y-globin gene to elevate HbF for P-thalassemia.According to traditional Chinese medicine theory of "nourishing yin and supplementing blood", Plastrum Testudinis (PT) is an important element for most medicaments of nourishing yin and supplying blood. Plastrum Testudinis is also known as tortoise shell, the shell of Chinemys reevesii (Grauy), first shown in Sheng Nong’s Herbal Classic. It is mildly salty and sweet in taste, slightly cold and does to meridian of liver, kidney and heart."Yin" in traditional Chinese medicine refers to the material base of body fluid and pith. Pith can give birth to marrow which can be turned into blood. Blood depends on the body, fluid which can be made into blood.((Ling Shu·lcer)) says body fluid can be adjusted to create blood. For this reason, traditional Chinese medicine uses yin-nourishing medicines to treat blood deficiency. Active ingredients giving pharmacological effects are mainly gelatin, keratin, steroids compounds and trace elements of all kinds. Modern pharmacology studies find that various pharmacological effects of PT include energy metabolism adjustment, immunity enhancement, aging resistance, bone strengthening, development promotion and nervous system protection. But researches and reports on its induction γ globin gene expression and erythroid proliferation and differentiation are relatively sparse.For identification of novel HbF inducers, understanding the potential mechanisms that involved in y-globin gene reactivation is the focus of intense investigation, so far, however, the exact mechanisms is not yet fully understood. Traditionally, most Hb F inducing agents have been viewed as affecting y-globin gene expression by altering local promoter chromatin structure, or by regulating the expression via different cell signaling pathways, or by altering thekinetics of erythroid differentiation.p38MAPKs is one of the major MAPK pathways. Evidences indicate that p38MAPK may play a significant role in y-globin gene regulation.Erythroid differentiation in K562cells is augmented by butyrate and HU through of p38activation and ERK MAPK inactivation. Despite the various pharmacological properties of these compounds, HDAC inhibitors, cototoxic agents and DNMT inhibitors may induce y-globin gene expression via p38MAPK signaling. These studies indicate that different pharmacologic agents converge on the p38MAPK pathway to activate y-globin expression. Our previous study determine the role of p38MAPK signaling in y-globin gene indu-ction in K562cells treated with APS. And our previous study on high-expression phospho-p38cell models has also verified that direct activation of p38MAPK can induce y-globin gene expression.Gene expression is controlled not only by transcription factors, but also by chromatin remodeling and epigenetic modifications. Chromatin epigenetic modifications play important roles in the "open" and "close" of P-like globin genes during their developmental swiths. Some of y-globin gene inductors, such as DNMT inhibitors, HDAC inhibitors, short chain fatty acids, also are the inductors of epigenetic modifications. They can induce y-globin gene expression through DNA demethylation, or histone acetylation.Taking K562cells and human erythroid progenitor cells as cell model and using benzidine staining,Real time PCR and Western blotting technique,this study will elucidate the role of PT in y--globin gene expression and HbF synthesis and the role of p38MAPK signaling in y-globin gene induction treated with PT. We examined the changes of histone acetylation and/or phosphorylation at y-globin gene promoter regions in PT-treated cell models to elucidate the relation between p38MAPK activation, y-globin gene expression and epigenectic modifications at y-globin gene promoter in cell models treated with PT.The results of our study may provid novel evidences for the mechanisms of y-globin gene expression induced by PT, and rationale for the clinical application of PT. At last, this study will provide new molecular target for developing new drugs to treat β-thalassemia.METHODDrug preparation and cell culturePT granule was processed at the Materia Medica Center of Guangdong YiFang Pharmaceutical. K562cell line cultured in RPMI-1640medium containing10%fetal bovine serμm,100U/mL penicillin and100μg/mL streptomycin at37℃in a5%CO2hμmidified atmosphere. Using two-phase liquid culture system to culture human erythroid progenitor cells:Mononuclear cells were isolated from the cord blood or peripheral blood from patients with β-thalassemiasamples, cultured in two different culture systems in vitro. At sixth day of the second culturing phase, the drugs were added to the medium as this study need. After6days, culturing was stopped and cells were collected.(ChIP:After3days cells were collected)Trial grouping:K562cell model:K562cells induced with PT belonged to experimental group,K562cells treated with500μmol/LNaB for72h were used as the positive control group and untreated K562cells were seen as blank control cell model.human erythroid progenitor cell model:human erythroid progenitor cells induced with PT belonged to experimental group,human erythroid progenitor cells treated with500μmol/LNaB were used as the positive control group and untreated human erythroid progenitor cells were seen as blank control cell modelPT induced y-globin gene expression and HbF synthesis1.Benzidine staining assay was used to analyze erythroid differentiation of K562cells induced by PT and screen the optimal drug concentration2. α,βand y-globin gene expressions induced by PT were analyzed by Real time PCR.3. Western blotting was employed to measure the content of HbF.4. Trypan blue dye exclusion test was used to analyze the influence of PT on K562cells proliferation.Role of p38MAPK activation in PT-mediated y-globin gene expression1. phospho-p38/p38was analyzed by Western blotting.2. y-globin mRNA levels were measured by Real time PCR to depict the effect of p38MAPK activation.3. Western blotting was employed to measure the content of HbF to illuminate the relationship between phosphorylation level of p38and erythroid differentiation of human K562erytholeukemia cells treated with PT.PT induced y-globin gene expression through histone modification at γ-globin gene promoter regions via activation of p38MAPKReal time PCR based-chromatin immunoprecipitation (ChIP) was employed to determine the levels of acetylation of histone H3and H4(acH3and acH4), phosphorylation/acetylation of histone H3(ph/acH3) at Gγ-and Aγ-globin gene promoter regions.Statistical analysisData are presented as means±SD. Statistical analysis was performed by one-wayANOVA using SPSS18.0software. The least significant difference post hoc test(LSD or Dunnett’s T3) was used for ANOVA statistics. P values<0.05are considered statistically significan.RESULTSLEffect of PT on γ-globin gene expression and HbF synthesis in K562cells and human erythroid progenitor cells1.1Effects of PT on K562cells proliferationBenzidine staining result indicated PT induced K562cells erythroid differention time-and dose-dependently(P<0.05).And the optimal drug concentration was5mg/ml, the superlative percentages of Benzidine positive cells(BZ%) was11.72±0.68%after96h induced by5mg/ml PT.The peaking BZ%has significant difference with negative control (2.47±0.20%,P<0.05) and no difference with NaB (10.99±0.79%, P=0.292).That result proved PT can induced K562cells proliferation.1.2Effect of PT on α,β and γ-globin gene expression in K562cells and human erythroid progenitor cells1.2.1Effect of PT on α,βand γ-globin gene expression in K562cellsLevels of γ-globin mRNA rose up at24h and the maximum was6.47±0.94and8.18±1.68fold increased respectively at72h～96h in the presence of5mg/ml PT compared with that in untreated cells. Then it went to decline. There was significant difference in the levels of y-globin mRNA expression between K562cells treated5mg/ml PT and untreated K562cells (P<0.05). Levels of a-globin mRNA at72h,96h120h were0.63±0.02、0.62±0.02and0.50±0.01fold decrease respectively compared with that in untreated cells. There was significant difference in the levels of a-globin mRNA expression between K562cells treated5mg/ml PT and untreated K562cells (P<0.05).Levels of β-globin mRNA at72h,96h,120h were0.91+0.06,0.96±0.02and0.98±0.00fold compared with that in untreated cells (P>0.05).There was no significant difference in the levels of β-globin mRNA expression between K562cells treated5mg/ml PT and untreated K562cells. That result proved PT up regulated the levels of y-globin mRNA expression,down regulated the levels of a-globin mRNA expression and have no effect on the levels of β-globin mRNA expression.1.2.2Effect of PT on a,βand y-globin gene expression in human erythroid progenitor cellsLevels of y-globin mRNA was4.94±1.17fold increased respectively in human erythroid progenitor cells in the presence of5mg/ml PT. There was significant difference in the levels of y-globin mRNA expression between human erythroid progenitor cells treated5mg/ml PT and untreated cells (P<0.05).Levels of a-globin mRNA were0.72±0.06fold decrease respectively compared with that in untreated cells. There was significant difference in the levels of a-globin mRNA expression between human erythroid progenitor cells treated5mg/ml PT and untreated cells (P<0.05).Levels of β-globin mRNA were0.97±0.28fold compared with that in untreated cells. There was no significant difference in the levels of β-globin mRNA expression between human erythroid progenitor cells treated5mg/ml PT and untreated K562cells (P>0.05). PT down regulated the levels of a-globin mRNA expression and have no effect on the levels of β-globin mRNA expression. That result proved PT up regulated the levels of y-globin mRNA expression,down regulated the levels of a-globin mRNA expression and have no effect on the levels of β-globin mRNA expression.1.3PT induced HbF synthesis in K562cells and human erythroid progenitor cells13.1PT induced HbF synthesis in K562cellsHbF levels induced with PT were up regulated time-and dose-dependently in K562cells and the maximum was1.95±0.10fold、2.95±0.09fold increased respectively at72h-96h in the presence of5mg/ml PT compared with that in untreated cells. There was significant difference in the levels of HbF between K562cells treated5mg/ml PT and untreated K562cells (P<0.05). That result proved that PT induced HbF synthesis in K562cells.1.3.2PT induced HbF synthesis in human erythroid progenitor cellsHbF level was1.98±0.11fold increased in human erythroid progenitor cells in the presence of5mg/ml PT compared with that in untreated cells. There was significant difference in the levels of HbF between human erythroid progenitor cells treated5mg/ml PT and untreated cells (P<0.05). That result proved that PT induced HbF synthesis in human erythroid progenitor cells.1.4Effect of PT on the cell proliferation inhibitionTrypan blue exclusion assay result indicated:PT inhibited cell proliferation time-and dose-dependently and PT has no significant side effect on proliferation function such as NaB in K562cells. The proliferation of K562cells inducing by PT and NaB48h (11.91±0.40%and55.17±3.94%),72h(16.87±2.95%and64.57±3.75),96h(22.47±0.70%and75.87±3.51%), There was significant difference between K562cells treated PT or NaB (P<0.05)2. Role of p38MAPK activation in PT-mediated y-globin gene expression 2.1Effect of PT on p38phosphorylation in K562cells:2.1.1Dosage effect:p38phosphorylation were induced in K562cells treated with2.5mg/ml,5mg/ml and10mg/ml PT respectively, and the results range were from4.29±0.43folds to5.12±0.55folds (F=47.549, P=0.000);5mg/ml PT was found to be the best inducing dose (P=0.000) There was significant difference in the levels of Phospho-p38between K562cells treated5mg/ml PT and untreated K562cells (P<0.05)2.1.2Time course:Phospho-p38level began to rise at12h after addition of PT, the peak located between60h and72h, and then it went to decline (F=97.252, P=0.000). There is no significance of the peak levels of Phospho-p38between K562cells treated with PT or NaB (6.32±0.47folds, P=0.260).That result proved that PT induced p38phosphorylation in K562cells.2.2Effect of PT on p38phosphoralation,y-globin gene expression and HbF synthesis2.2.1Relationship between phosphorylation level of p38and y-globin gene expression in K562cells treated with PTResults of Real time PCR showed that Levels of y-mRNA in K562cells treated with PT was5.28±0.67fold increase (P=0.000) compared with that in untreated cells. Levels of γ-mRNA in K562cells treated with NaB was5.48±0.80fold increase(P=0.000). That result proved that as phosphorylation level of p38rising up, Levels of y-mRNA rose up.2.2.2Relationship between phosphorylation level of p38and HbF synthesis in K562cells treated with PT:Levels of HbF in K562cells treated with PT was2.31±0.09fold increased (P=0.000) compared with that in untreated cells. Levels of HbF in K562cells treated with NaB was2.36±0.03fold increased (P=0.000) compared with that in untreated cells. That result proved that as phosphorylation level of p38rising up, Levels of HbF rose up.2.3SB203580(SB)inhibited p38phosphoralation,y-globin gene expression and HbF synthesis induced by PT2.3.1SB203580(SB)inhibited p38phosphoralationinduced by PT.p38phosphoralation was decreased in K562cells pretreated with SB, a p38kinase inhibitor, for1h before induced with PT. Comparing with untreated K562cells, The increased folds decreased to2.17±0.05folds from5.10±0.36folds in cells with PT alone, and the inhibition ratio was57.27%.When it came to NaB, the increased folds decreased to1.46±0.03folds pretreated with SB from5.25±0.12folds in cells with NaB alone, and the inhibition ratio was71.38%. There was significant difference in the levels of p-p38among the various K562cell models (F=567.388, P=0.000)2.3.2SB203580(SB)inhibited y-globin gene expression induced in K562cells treated with PTComparing with untreated K562cells, The increased folds decreased to2.70±0.37folds from5.28±0.67folds in cells with PT alone, and the inhibition ratio was48.8%.When it came to NaB, the increased folds decreased to2.26±0.36folds pretreated with SB from5.48±0.80folds in cells with NaB alone, and the inhibition ratio was58.7%. There was significant difference in the levels of p-p38among the various K562cell models (F=79.184, P=0.000).2.3.3SB203580(SB)inhibited HbF synthesis in K562cells treated with PT:Comparing with untreated K562cells, The increased folds.decreased tol.67±0.11folds from2.31±0.09folds in cells with PT alone, and the inhibition ratio was27.45%.When it came to NaB, the increased folds decreased tol.29±0.13folds pretreated with SB from2.36±0.03folds in cells with NaB alone, and the inhibition ratio was45.73%. There was significant difference in the levels of p-p38among the various K562cell models (F=25.145, P=0.000).3. PT induced y-globin gene expression through histone modification at y-globin gene promoter regions via activation of p38MAPK3.1Analysis of acH3acH4and ph/acH3at Gy-and Ay-globin gene promoter regions3.1.1The levels of acH3or acH4and ph/acH3at Gy-globin gene, Ay-globin gene, or necdin genepromoter regions in each group of K562cell and human erythroid progenitor cell models.In each group of K562cell models, hyperacetylation of histone H3and H4and high levels of ph/acH3was found at Gγ-and Ay-globin gene promoters, while hypoacetylation of H3and H4and low levels of ph/acH3was detected at necdin gene promoter,the negative control (P<0.05).3.1.2PT increases the levels of acH3acH4and ph/acH3at Gy-and Ay-globin gene promoter regions in K562cells and human erythroid progenitor cellsIn K562(PT) cells, the levels of acH3acH4and ph/acH3at Gγ-and Aγ-globin gene promoters were significantly higher than that in K562cells, and there was a4.56-fold,3.57-fold or2.81-fold increase in Gγ-acH3Gγ-acH4or Gγ-ph/acH3(P<0.05), respectively, a2.04-fold,1.81-fold or2.74-fold increase in Ay-acH3or Aγ-acH4or Aγ-ph/acH3(P<0.05),respectively, in K562(PT) cells. In human erythroid progenitor cells, the levels of acH3,acH4and ph/acH3at Gγ-and Aγ-globin gene promoters in (PT) group were significantly higher than that in untreated cells, and there was a3.02-fold1.68-fold or1.91-fold increase in Gγ-acH3Gγ-acH4or Gγ-ph/acH3(P<0.05), a1.66-fold1.62-fold or1.72-fold increase in Aγ-acH3Ay-acH4or Aγ-ph/acH3(P<0.05),respectively. 3.2p38MAPK inhibitior SB203580decreases the levels of acH3acH4and ph/acH3at Gy-and Ay-globin gene promoter regions in K562cells and human erythroid progenitor cells induced by PTIn K562cells, the experiment of SB203580inhibition revealed that the levels of acH3,acH4and ph/acH3at Gγ-and Ay-globin gene promoters in K562(PT+SB) cells were decreased,in comparison with K562(PT) cells (48.87%,54.82%57.23%, and79.10%,64.83%,54.93%levels,respectively). In human erythroid progenitor cells,the experiment of SB203580inhibition revealed that the levels of acH3,acH4and ph/acH3at Gy-and Ay-globin gene promoters in (PT+SB) group cells were decreased,incomparisonwith(PT)groupcells(56.94%,76.18%,67.67%and63.28%,77.43%,69.65%levels,respectively).3.3Relationship between activation p38MAPK, acetylation and phosphorylation of histone at y-globin gene gene promoter regions,y-globin gene expression and HbF synthesis in cells model treated with PT:p38MAPK activation increases the levels of acH3acH4and ph/acH3at y-globin gene promoter regions and the levels of y-mRNA and HbF in cells model treated with PT.This effect can be inhibited by p38MAPKinhibitor SB203580.So the results indicate that PT induced y-globin gene expression through epigenectic modification at y-globin-gene promoter regions via activation of p38MAPK.Conclusions1. We further demonstrated that Plastrum Testudinis had ability to activate y globin gene expression in K562cells and human erythroid progenitor cells and there were dosage effect and time course in this process. The best inducing concentration is5mg/ml.The levels of y-globin mRNA and begin to rise at24h and and the peaks locate between72h and96h. PT down regulated the levels of a-globin mRNA expression and have no effect on the levels of β-globin mRNA expression. 2. We demonstrated that Plastrum Testudinis can induce p38phosphorylation in K562cells for the first time, and there were dosage effect and time course in this enhancement. The best inducing dosage is5mg/ml; Phospho-p38level begin to rise at12h, and the peak locates between60h and72h, then it goes to drawdown. Meanwhile effect of PT on γ-globin expression and HbF synthesis inhibited as inactivation p38MAPK by p38MAPK inhibitors SB203580. That result justified for the first time that p38MAPK cell signaling played a significant role role in the activation of γ-globin expression induced by Plastrum Testudinis in K562cells.3. We first indicate that p38MAPK activation increases the levels of acH3acH4and ph/acH3at γ-globin gene promoter regions and the levels of γ-mRNA and HbF in cells model treated with PT.This effect can be inhibited by p38MAPKinhibitor SB203580.So the results indicate that PT induced γ-globin gene expression through epigenectic modification at γ-globin gene promoter regions via activation of p38MAPK. |
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