血复生浸膏结合辨证治疗对重型再障患者外周血microRNA、淋巴细胞亚群及细胞因子的影响

摘要

Objective:To study the effects of Xuefusheng extractum on peripheral blood microRNA,lymphocyte subsets and cytokines in severe aplastic anemia(SAA) patients.Methods:60 Patients were randomly divided into the observation group(n =30) and the control group(n =30).The observation group was treated with TCM treatment plus basic treatment;whereas,the control group was only given basic treatment.TCM treatment included administration of Xuefusheng extractum combined with treatment based on syndrome differentiation.On the basis of Xuefusheng extractum,modified Zuogui pill,Zhibai Dihuang pill and Xijiao Dihuang decoction were also administrated to the patients with Kidney-yin deficiency;modified Yougui pill was also given to the patients with Kidney-yang deficiency;modified Zuogui pill and Yougui pill were also applied to the patients with deficiency of both Kidney-yin and Kidney-yang;and modified Qingwen Baidu decoction and Xijiao Dihuang decoction were also given to the patients with excessive toxic-heat.The basic treatment included the applications of cyclosporine,eleven acid testosterone,blood transfusion,and other support treatment.Both groups were treated for six months.Expressions of miRNA-155-5p and miRNA-1260b in peripheral blood mononuclear cells(PBMNC) were detected with RT-PCR before and after the treatment,levels of IFN-γ,IL-12p70,TNF-α and TGF-β1 were tested with ELISA method and flow cytometry before and after the treatment.Results:The total effective rate of the observation group was higher than that of the control group(63.3％ Vs 36.7％,P =0.039).Before the treatment,expressions of miRNA-155-5p and miRNA-1260b,Tc proportion,plasma levels of IFN-γ,IL-12p70 and TNF-oα were significantly higher in CAA patients compared to those in normal people (P ＜ 0.01);whereas,Th proportion,Th/Tc ratio and TGF-β1 level were lower than those of normal people(P ＜0.01).After six-month treatment,in the observation group,expression of miRNA-1260b,levels of IFN-γand TNF-oα were significantly decreased (P ＜0.01);expression of miRNA-155-5p,Tc proportion and IL-12p70 were also decreased (P ＜ 0.05);TGF-β1 level were significantly increased(P ＜0.01).Th proportion,Th/Tc ratio and NK proportion were also increased(P ＜0.05).However,these indicators had not yet reached the normal level.The indexes of the treatment group changed more significantly than those of the control group.Conclusion:The up-regulation of miRNA-155-5p and miRNA-1260b,cellular immune hyperfunction and disorder of hematopoiesis regulating factors play important roles in the immune abnormalities in SAA patients.Therapeutic effect of treating SAA with Xuefusheng extractum and treatment based on syndrome differentiation is superior to that of simple western medicine treatment.The therapeutic mechanism includes down-regulation of miRNA-155-5p and miRNA-1260b,improving lymphocyte subsets imbalance and disorder of cytokines,and alleviating abnormal hyperfunction of cellular immune response and hematopoietic suppression.%目的:研究血复生浸膏结合辨证治疗重型再障(SAA)的疗效及对外周血microRNA、淋巴细胞亚群、细胞因子的影响.方法:再障患者均来源于南京中医药大学附属医院血液科,分为治疗组30例和对照组30例,治疗组应用中医治疗+基础治疗,对照组仅用基础治疗;中医治疗为血复生浸膏结合辨证分型随证加减,肾阴虚型患者服用血复生浸膏合左归丸、知柏地黄丸、犀角地黄汤加减,肾阳虚型患者服用血复生浸膏合右归丸加减,肾阴阳两虚型患者服用血复生浸膏合左归丸、右归丸加减,热毒壅盛型患者服用血复生浸膏合清瘟败毒饮、犀角地黄汤加减;基础治疗为环孢素、十一酸睾酮、输血等对症支持治疗,两组均治疗6个月.采用实时定量PCR(realtime PCR)方法检测SAA患者治疗前后外周血单个核细胞(peripheral blood mononuclear cell,PBMNC) miRNA-155-5p、miRNA-1260b表达情况,采用流式细胞术、酶联免疫吸附法(ELISA)检测CAA患者治疗前后淋巴细胞亚群及血浆IFN-γIL-12p70、TNF-α、TGF-β1水平.结果:治疗组总有效率高于对照组(63.3％ Vs 36.7％,P=0.039);治疗前SAA患者PBMNC miRNA-155-5 p、miRNA-1260b表达、Tc比例、血浆IFN-γ、IL-12p70、TNF-α水平均明显高于正常对照组(P＜0.01),而Th比例、Th/Tc比值、TGF-β1水平则低于正常对照组(P＜0.01);治疗组患者治疗6个月后miRNA-1260b表达、IFN-γ、TNF-α水平均明显下降(P＜0.01),miRNA-155-5p、Tc比例、IL-12p70亦有所下降(P＜0.05),TGF-β1水平明显升高(P＜0.01),而Th比例、Th/Tc比值、NK比例也有所升高(P＜0.05),但上述指标尚未达到正常水平,治疗组上述指标变化较对照组更明显.结论:miRNA-155-5p、miRNA-1260b表达上调、细胞免疫功能亢进、造血调控因子紊乱在SAA免疫异常的发病过程中起到关键作用;血复生浸膏结合辨证治疗重型再障疗效优于单纯西药治疗组,其作用机理包括下调miRNA-155-5p、miRNA-1260b表达,改善淋巴细胞亚群失衡,调节细胞因子紊乱,进而减轻异常亢进的细胞免疫,解除造血抑制.