“扶正”“祛邪”中药醇提物调控骨髓增生异常综合征细胞组蛋白乙酰化及TLR2表达诱导凋亡的体外研究

Effects of Ethanol-extractions of Compound Chinese Medicine Formula of “Supporting Right” and “Dispelling Evil” on Apoptosis,Acetylation,TLR2 Activation in Bone Marrow Mono-nuclear cells from Myelodysplastic Syndromes Patients in Vitro

目的：探讨＂扶正＂＂祛邪＂中药复方醇提物对骨髓增生异常综合征（MDS）患者骨髓单个核细胞（BMMNCs）组蛋白去乙酰化酶1（HDAC1）及Toll样受体2（TLR2）表达的干预作用,分析其对细胞凋亡水平的影响。方法：收集MDS患者15例（正虚组10例、瘀毒组5例）,正常组3例的BMMNCs进行体外培养。采用MTT法检测细胞毒性,流式细胞仪检测细胞凋亡水平,real-time PCR、Western Blot分别检测中药醇提物干预后HDAC1、TLR2的mRNA及蛋白水平。结果：MDS患者HDAC1、TLR2基因及蛋白水平均高于正常人（P〈0.05）,正虚型患者与瘀毒型患者组间未发现明显差异（P〉0.05）;扶正、祛邪药对HADC1、TLR2和凋亡水平均有影响,但扶正药与祛邪药组间未发现明显差异（P〉0.05）。结论：组蛋白乙酰化相关表观遗传学异常和先天免疫失调均与MDS发病有关,有效扶正与祛邪中药复方醇提物对正虚、瘀毒型MDS细胞的组蛋白乙酰化和TLR2表达水平均具有调控作用,提示中药可能通过降低HDAC1水平调控TLR2表达,最终影响细胞凋亡。

Objective： Myelodysplastic syndromes（ MDS） are clonal bone-marrow stem-cell disorders,characterized by ineffective haemopoiesis leading to peripheral blood cytopenias. The present study was to asses the effects of Chinces medicine formula of supporting right and dispelling evil on bone marrow mono-nuclear cells（ BMMNCs） from MDS patients. Methods： BMMNCs were aspirated from 10 lower risk MDS patients and 5 higher risk MDS patients and 3 healthy controls. Cells were treated with formula of supporting right or dispelling evil,or vorinostat（ SAHA） as positive control for 48 h. Cell proliferation was measured by an 3-（ 4,5 dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（ MTT） assay. Apoptosis was detected by flow cytometry using annexin V-fluorecein isothiocyanate/propidium iodide double-staining. The mRNA and protein expression levels of histone deacetylase 1（ HDAC1） and toll like receptor 2（ TLR2） were detected by real-time quantitative PCR and Western blot. Result： After treatment with formula of supporting right or with formula of dispelling evil at concentraions from 0. 0625 mg/m L to 2. 0 mg/m L in 48 hours,or with SAHA at concentraions from 3. 125 μM to 10 μM in 48 hours. The proliferation of both lower risk and higher risk MDS BMMNCs were inhibited. The apoptotic cells significantly increased in both lower risk and higher risk MDS BMMNCs byformula of supporting right or dispelling evil treatment（ P 〈0. 05）,and the expression levels of HDAC1,TLR2 mRNA and its protein were alternated by formula of supporting right or dispelling evil treatment. Conclusions： Formula of supporting right or dispelling evil could inhibit cell proliferation and induce cell apoptosis via histone acetylation mediated immunoregulation in both lower risk and higher risk MDS. The data suggests that epigenetic and immunological alternation may be one of the important therapeutic mechanisms of ethanol-extractions of compound Chinese medicine in treating MDS.