疏风凉血补肾法对CITP骨髓巨核细胞分化成熟及TGF-β_1的调节
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摘要
目的:
慢性特发性血小板减少性紫癜(CITP)是临床较常见的出血性疾病,属祖国医学的“血证”、“肌衄”、“发斑”等范畴。以往认为该病是血小板免疫性破坏增多的临床综合征,免疫异常是其发病机制的核心,故又称为自身免疫性血小板减少性紫癜。随着近年来研究的不断深入,国内外均认识到免疫异常虽然是其发病的首要因素和主要机制,但并不是其全部的机制,应还有其他非免疫因素参与了CITP的发病。临床上应用强的松、长春新碱等调整CITP的免疫异常,或采用手术切脾减少血小板的免疫性破坏,并不能使所有CITP得以治愈,仍有部分病情迁延转为难治,且以上治疗方法毒副作用较大。导师陈志雄教授多年来运用疏风凉血补肾法(方)治疗CITP,疗效显著,无明显毒副作用,综合比较优于强的松等药物。本课题即主要通过动物实验和临床观察来进一步验证疏风凉血补肾法(方)的疗效,并通过对相关指标的对比观察来探讨该法(方)治疗CITP的疗效机制以及该法(方)疗效优于强的松的可能机制,同时也通过研究结果初步探讨CITP除免疫异常之外可能存在的其他发病机制。
方法:
1动物实验研究
将SPF级BALB/C小鼠共70只随机分为正常组10只、空白模型组12只、强的松组12只、疏风凉血补肾方高剂量组12只、疏风凉血补肾方中剂量组12只、疏风凉血补肾方低剂量组12只,除正常组外其余各组均参考杨宇飞法改进,分别在1、3、5、7、9、11、13天于小鼠腹腔注射1:4稀释的豚鼠抗小鼠血小板血清(APS),每次100μl,建立CITP小鼠模型。于造模第6天起按0.2ml/10g体重剂量每日1次、连续9天灌胃,灌胃药物分别为生理盐水、生理盐水、强的松溶液(0.5mg/ml,临床用量的10倍)、疏风凉血补肾方高浓度水煎浓缩液(2.6g/ml,临床用量的15倍)、疏风凉血补肾方中浓度水煎浓缩液(1.73g/ml,临床用量的10倍)、疏风凉血补肾方低浓度水煎浓缩液(0.87g/ml,临床用量的5倍),于造模第15天处死小鼠取材。观察一般情况,不良反应,死亡情况,血小板计数变化,骨髓巨核细胞总数及原幼巨、颗粒巨、产板巨、裸核巨数量比例变化,血清PAIgG及骨髓TGF-β_1水平变化情况。
2临床研究
将符合CITP诊断标准和纳入标准的50例患者随机分为疏风凉血补肾方组(A组)20例、疏风凉血补肾方加强的松组(B组)15例和以强的松为主的对照组(C组)15例,A组以疏风凉血补肾法(方)治疗,B组以疏风凉血补肾法(方)加强的松治疗(中药用法同A组,强的松用法同C组)。C组以强的松为主,加达那唑和升血小板胶囊辅助治疗,强的松按病情轻重决定用量,达那唑用量为0.2/次,3/日,升血小板胶囊用量为3粒/次,3/日。以上治疗均4周为一疗程,连续治疗3疗程后,观察不良反应,疗效,出血症状积分改善,血小板计数变化,骨髓巨核细胞总数及原幼巨、颗粒巨、产板巨、裸核巨数量比例变化,血清PAIgG及骨髓TGF-β_1水平变化情况。
结果:
1动物实验结果
1.1本实验以1、3、5、7、9、11、13天时间点于小鼠腹腔注射1:4稀释的APS,每次100μl,成功复制了CITP小鼠模型,在停止注射APS后模型维持时间为72小时左右,和文献结论相符。
1.2在提高CITP小鼠血小板计数方面,疏风凉血补肾法(方)高、中、低剂量组和强的松组均能提高CITP小鼠血小板计数,和模型组比较有极显著性差异(P＜0.01),但疏风凉血补肾法(方)高、中、低剂量组和强的松组间比较无显著性差异。
1.3在降低CITP小鼠骨髓巨核细胞总数方面,疏风凉血补肾法(方)高、中剂量组均能降低骨髓巨核细胞总数,和模型组比较有显著性差异(P＜0.05),而其组间比较无显著性差异;在提高产板巨核细胞比例上,疏风凉血补肾法(方)高、中剂量组产板巨核细胞比例上升明显,和模型组比较有极显著性差异(P＜0.01),而疏风凉血补肾法(方)低剂量组、强的松组和模型组比较只有显著性差异(P＜0.05)。
1.4在降低CITP小鼠升高的PAIgG值方面,疏风凉血补肾法(方)高、中、低剂量组、强的松组PAIgG值均下降明显,和模型组比较均有极显著性差异(P＜0.01),而其组间比较无显著性差异。
1.5在降低CITP小鼠升高的TGF-β_1值方面,疏风凉血补肾法(方)高、中、低剂量组TGF-β_1值降低明显,和模型组比较有显著性差异(P＜0.05),而其组间比较无显著性差异。强的松组TGF-β_1值有所下降,但和模型组比较无显著性差异。
2临床观察结果
2.1在临床疗效方面,A组总有效率值(90.0%)高于C组(73.3%),和B组(93.3%)相近,但经比较三组在总有效率上无显著性差异;在良效率方面,A组(55.0%)显著高于C组(20.0%)(P＜0.05),和B组(60.0%)相近。
2.2在降低出血症状积分方面,A、B两组均能有效降低出血症状积分,治疗前后比较有显著性差异(P＜0.05),而C组治疗前后出血症状积分比较无显著性差异。
2.3在提高血小板计数方面,A组治疗后血小板计数明显上升,和治疗前比有极显著性差异(P＜0.01),且其明显优于C组(P＜0.05),和B组疗效相似。
2.4在降低骨髓巨核细胞总数和提高产板巨比例方面,A组治疗后骨髓巨核细胞总数明显降低、产板巨比例明显上升,和治疗前较比有极显著性差异(P＜0.01),和B组疗效相似。而C组治疗后骨髓巨核细胞总数有所减低、产板巨比例有所上升,但治疗前后对比无显著性差异。
2.5在降低PAIgG值方面,A、B、C三组治疗后PAIgG值明显降低,和治疗前比较均有显著性差异(P＜0.05)。三组治疗后比较无显著性差异。
2.6在降低TGF-β_1值方面,A、B二组治疗后TGF-β_1值明显降低,和治疗前比差异显著(P＜0.05),C组TGF-β_1值有所下降,但和治疗前比无显著差异。A、B二组和C组治疗后比较均有显著性差异(P＜0.05),A、B二组治疗后比较无显著性差异。
结论:
1动物实验显示,疏风凉血补肾方三个剂量组均能有效降低CITP小鼠升高的PAIgG和TGF-β_1值,有效降低CITP小鼠骨髓巨核细胞总数和提高产板巨核细胞比例,从而有效促进骨髓巨核细胞分化成熟和提高血小板计数,其作用综合比较优于强的松组;疏风凉血补肾方三个剂量组间以高、中剂量组疗效最佳,但高剂量组可能出现剂量相关副作用,故中剂量(相当于临床常用量)组应为最佳剂量组。
2临床观察显示,疏风凉血补肾方能有效降低CITP患者升高的PAIgG和TGF-β_1值,有效促进骨髓巨核细胞分化成熟(表现在降低骨髓巨核细胞总数和提高产板巨核细胞比例上),有效提高CITP患者血小板计数,其作用综合比较优于以强的松为主的对照组;本实验中疏风凉血补肾方加强的松组和单纯的疏风凉血补肾方组疗效相近,考虑这和所入选的病例较多是经强的松类药物治疗无效而转诊我院有关,强的松对这部分患者的疗效已很有限,故强的松在该组治疗中未能起明显的增效作用。
3动物实验和临床观察均显示,疏风凉血补肾方对降低CITP升高的PAIgG和TGF-β_1值均有良好作用,而强的松在本研究中只对PAIgG值有较好的降低作用,而对TGF-β_1值无显著性降低作用,这可能就是疏风凉血补肾法疗效优于强的松的机理之一。
4动物实验和临床观察中,强的松组和以强的松为主的对照组在用药后PAIgG值均明显降低,而TGF-β_1值无显著性下降,这与目前的某些类似研究结论相似。以往认为TGF-β_1升高是由于CITP免疫异常状态时体内升高的PAIg导致血小板和巨核细胞遭受免疫性破坏TGF-β_1从中释放出来造成的,PAIg和TGF-β_1值呈正相关性,经治疗干预PAIg值降低后TGF-β_1值也应随之下降,而本研究及其他类似研究的结论与此观点有不一致性的地方。因此我们有理由推论:免疫异常并不是CITP时TGF-β_1升高的全部原因,免疫异常触发后的非免疫依赖因素也可引起TGF-β_1水平的增高,这也可能是CITP除免疫异常之外的其他重要发病机制之一。
综上所述,疏风凉血补肾法(方)对CITP有较好的临床疗效,其疗效机制之一可能就在于该法能有效降低PAIg水平及免疫异常和非免疫因素造成的TGF-β_1水平的升高,从而更有效的促进骨髓巨核细胞的分化成熟,这也可能是该法疗效优于强的松的关键原因所在。同时,免疫异常触发后的非免疫依赖因素导致的TGF-β_1的增高可能也是CITP除免疫异常之外的其他发病机制之一及CITP难治性的关键所在。对此有待更多的后续研究去加以验证。
Objective:CITP is a common hemorrhagic disease of blood system in clinics, it belong to "blood syndrome"、"muscle apostaxis"、" macula syndrome" etc,in TCM.This disease was thought as clinic syndrome due to the increased damage of platelet immune,and the abnormality of immune is the core mechanism of the disease,lt was also called autoimmune thrombocytopenic purpura.As the research going on in the recent years,the researchers both home and abroad realized that although the abnormality of immunity is the trigger and main mechanism,it is not the all,there must be some other non-immunity factors causing the occurrence of CITP.In clinics,we usually use medicine like prednisone,vincristine etc,to adjust the immune abnormality of CITP,or to decrease the immune damage of platelet by splenectomy,while both way fail to cure the CITP from root,still some of the diseases turn into more complicated and difficult ones.My tutor,Professor Zhixiong Chen has been using the prescription of expelling wind,cooling blood and invigorating kidney since long time ago to cure CITP and has achieved distinguished effect,which is much more than prednisone.My project is to do the further verification through animal test and clinic observation regarding the effect of the prescription of expelling wind,cooling blood and invigorating the kidney,it also want to probe the effective mechanism of curing CITP,as well as the possible mechanism regarding why the prescription is better than prednisone in effect by comparing some related indexes.The project will also detect some other possible pathogenic factors and mechanism beside the abnormality of immune by the experimental result.
Methods:
1 Animals empirical study
In the first experiment,70 BALB/C mice with SPF order were randomly divided into six groups(10 mice for normal group;12 mice per group respectively for blank group、prednisone group and high-dose/midst-dose/low-dose groups of decoction of expelling wind,cooling blood and invigorating the kidney).The latter five groups were improved with yangyufei way and were injected intraperitoneally APS(diluted with 1:4 proportion)100μl per time at every other day in 13 days and the CITP mice models were made.From the following sixth day on,these groups were administrated intragastrically with a single dose per day of experiment substance(administration quantity 0.2ml/10g)in the next 9 days,which were respectively saline;prednisone(0.5mg/ml,decuple of clinical dose);high-dose(2.6g/ml,fifteenfold of clinical dose) /midst-dose(1.73g/ml,decuple of clinical dose)/low-dose(0.87g/ml,quintuple of clinical dose)of the decoction.At the same time,the general state of health、the adverse reaction and the death amount were observed.Afterwards, all mice were sacrificed and the needed specimens were taken in order to observe the change of platelet countss,marrow megacaryocyte amount、platelet-produced megacaryocyte proportion、PAIgG in the serum and TGF-β_1level in the marrow.
2 clinical research
50 CITP patients were randomly divided to three groups(including 20 patients administrated with the decoction of expelling wind,cooling blood and invigorating the kidney as A group;15 patients with the same decoction and prednisone as B group;15 patients with prednisone as C group.To more explain,B group' administration way referred to A and C group'.C group was constituted of prednisone,danazol and shengxuexiaoban capsule.The prednisone administration quantity was given according to the pathogenetic condition and danazol 0.2 each time and shengxuexiaoban capsule 3 granule each time,3 times per day.The above treatment was 4 weeks as one time of therapy.After 3 times of therapy,a series of observation were made as before.
Results:
1 animal experiment results
1.1 The experiment successfully made the CITP mouse model according to injecting intraperitoneally the APS(dilution proportion 1:4)with 100μl per time respectively at the first、third、fifth、seventh、ninth、eleventh、thirteenth day.The duration time of the CITP model was 72 hours and the result was the same as that of correlative data.
1.2 In the aspect of platelet counts of the CITP mice,the high-dose、midst-dose、low-dose decoction and prednisone groups could all improve platelet counts and P＜0.01 was considered significant compared with the control group.However,there was no significant difference among the four groups.
1.3 In the aspect of marrow megacaryocyte amount of the CITP mice,that amount of the high-dose、midst-dose and low-dose decoction groups could all be lowered and P＜0.05 was considered significant compared with the control group.For the platelet-produced megacaryocyte proportion,that of the high-dose and midst-dose decoction groups upgraded obviously and P＜0.01 was considered significant compared with the control group.But the low-dose decoction group and the prednisone group had significant difference compared with the control when P＜0.05 was considered.
1.4 In the aspect of PAIgG value of the CITP mice,the high-dose、midst-dose and low-dose decoction groups,along with the prednisone group,could all drop the value of PAIgG obviously and P＜0.01 was considered significant compared with the control group.However,there was no significant difference among the four groups.
1.5 In the aspect of TGF-β_1 value of the CITP mice,that of the high-dose、midst-dose and low-dose decoction groups could be degraded obviously and P＜0.05 was considered significant compared with the control group.However, there was no significant difference among the three groups.But the TGF-β_1 value of the prednisone group had decreased slightly and there was no signifycant difference compared with the control.
2 Results of clinical observation
2.1 With regard to the therapeutic effect,the total effective rate of A group was 90.0%,which was better than that of C group(73.3%)and close to that of B group(93.3%).But there was no significant difference among the three groups.Regarding the fine efficiency,when P＜0.05 was considered,that of A group(55.0%)excelled C group(20.0%)significantly and close to that of B group(60.0%).
2.2 With regard to the integration of hemorrhage symptoms,A group and B group could effectively cut down the integration of hemorrhage symptoms.And there was significant difference between pre-therapy and post-therapy when P＜0.05 was considered.But that of C group had no significant difference between pre-therapy and post-therapy.
2.3 With regard to platelet counts,that of A group was raised obviously after therapy and there was significant difference compared with that before therapy when P＜0.05 was considered.But the result of A group was similar to that of B group.
2.4 With regard to the marrow megacaryocyte amount and the platelet-produced megacaryocyte proportion,the former of A group was effectively decreased and the latter of A group was increased greatly.And there was significant difference between pre-therapy and post-therapy when P＜0.01 was considered. But the treatment of A group was similar to that of B group.Though the results of C group had some change,there was no significant difference between pre-therapy and post-therapy.
2.5 With regard to the PAIgG value of the CITP mice,that of the A,B,C group all dropped greatly and P＜0.05 was considered significant compared with that before therapy.However,there was no significant difference among the three groups.
2.6 With regard to TGF-β_1 value of the CITP mice,that of A and B groups degraded obviously and P＜0.05 was considered significant compared with that before therapy.But the TGF-β_1 value of C group had decreased slightly and there was no significant difference compared with the pre-therapy.The results of A and B groups were significant deviation compared with that of C group when P＜0.05 was considered.And there was no significant difference after treatment between A group and B group.
Conclusion:
1 The results of animal experiment displayed that the three different dose groups of the expelling wind,cooling blood and invigorating the kidney decoction could effectively degrade the value of PAIgG and TGF-β_1,which became abnormally upgrade induced by CITP,also cut down the marrow megacaryo-cyte amount and raise the platelet-produced megacaryocyte proportion,then facilitating the maturity of the marrow megacaryocyte amount and enhancing the platelet counts.The combination effect of the decoction groups excelled that of the prednisone group.The experimental results of the high-dose and midst-dose groups were better that the low-dose group.Considering the high-dose group' latent adverse reaction,the midst-dose group(corresponding to the usual clinical dose)was the optimum.
2 The results of clinical observation showed that the expelling wind, cooling blood and invigorating the kidney decoction could effectively degrade the value of PAIgG and TGF-β_1,which became abnormally upgrade induced by CITP,could facilitate the maturity of the marrow megacaryocyte amount and enhance the platelet counts.The combination effect of the decoction groups excelled that of the prednisone group.In the study,the curative effect of the decoction and prednisone group were close to that of the simple decoction group.Considering the selected patients being transfer treatment cases due to those invalid to prednisone treatment,this result could accounts for prednisone' s failed synergistic effect.
3 The results of animal study and clinical observation showed that the expelling wind,cooling blood and invigorating the kidney decoction did well to balance the value of PAIgG and TGF-β_1 for the CITP case and the prednisone just could reduce the value of PAIgG and had no effective to TGF-β_1,which could well explain that the curative effect of the decoction was better than that of the prednisone.
4 The results of animal study and clinical observation displayed that the value of PAIgG was lowered obviously after administration for the prednisone group and the group in which prednisone was the main component and there was no significant decrease on the value of TGF-β_1.That investigation was similar to the results of the current correlative studies.The previous research presumed that the TGF-β_1 was overfull released due to the destroyed platelet or megacaryocyte which induced by the elevated PAIgG in CITP abnormality immunological environment and there was positive correlation between the PAIgG value and the TGF-β_1 value.After therapy,the value of TGF-β_1 descended following the decreased PAIgG.But our study and other parallel researches have some discordance with the previous view.For this reason,we can deduce that immunological abnormality is not the entire agent that accounts for the elevated TGF-β_1 value.The immune-independent factors triggered by immuneological abnormality can also provoke the enhanced TGF-β_1,which may become one of the important pathogenesies besides the immunological abnormality factor.
In a word,the expelling wind,cooling blood and invigorating the kidney decoction has good clinical effect on CITP.One of it's mechanism,the key point superior to prednisone,maybe lies in balancing PAIgG and TGF-β_1 effectively,then facilitating the cell differentiation maturity of the marrow megacaryocyte.Meanwhile,the increased TGF-β_1 induced by the immuneindependent factors owing to immunological abnormality maybe becomes one of the important pathogenesies besides the immunological abnormality factor, also probably being as the refractory key to CITP,which need more research to make verification.
慢性特发性血小板减少性紫癜(CITP)是临床较常见的出血性疾病,属祖国医学的“血证”、“肌衄”、“发斑”等范畴。以往认为该病是血小板免疫性破坏增多的临床综合征,免疫异常是其发病机制的核心,故又称为自身免疫性血小板减少性紫癜。随着近年来研究的不断深入,国内外均认识到免疫异常虽然是其发病的首要因素和主要机制,但并不是其全部的机制,应还有其他非免疫因素参与了CITP的发病。临床上应用强的松、长春新碱等调整CITP的免疫异常,或采用手术切脾减少血小板的免疫性破坏,并不能使所有CITP得以治愈,仍有部分病情迁延转为难治,且以上治疗方法毒副作用较大。导师陈志雄教授多年来运用疏风凉血补肾法(方)治疗CITP,疗效显著,无明显毒副作用,综合比较优于强的松等药物。本课题即主要通过动物实验和临床观察来进一步验证疏风凉血补肾法(方)的疗效,并通过对相关指标的对比观察来探讨该法(方)治疗CITP的疗效机制以及该法(方)疗效优于强的松的可能机制,同时也通过研究结果初步探讨CITP除免疫异常之外可能存在的其他发病机制。
方法:
1动物实验研究
将SPF级BALB/C小鼠共70只随机分为正常组10只、空白模型组12只、强的松组12只、疏风凉血补肾方高剂量组12只、疏风凉血补肾方中剂量组12只、疏风凉血补肾方低剂量组12只,除正常组外其余各组均参考杨宇飞法改进,分别在1、3、5、7、9、11、13天于小鼠腹腔注射1:4稀释的豚鼠抗小鼠血小板血清(APS),每次100μl,建立CITP小鼠模型。于造模第6天起按0.2ml/10g体重剂量每日1次、连续9天灌胃,灌胃药物分别为生理盐水、生理盐水、强的松溶液(0.5mg/ml,临床用量的10倍)、疏风凉血补肾方高浓度水煎浓缩液(2.6g/ml,临床用量的15倍)、疏风凉血补肾方中浓度水煎浓缩液(1.73g/ml,临床用量的10倍)、疏风凉血补肾方低浓度水煎浓缩液(0.87g/ml,临床用量的5倍),于造模第15天处死小鼠取材。观察一般情况,不良反应,死亡情况,血小板计数变化,骨髓巨核细胞总数及原幼巨、颗粒巨、产板巨、裸核巨数量比例变化,血清PAIgG及骨髓TGF-β_1水平变化情况。
2临床研究
将符合CITP诊断标准和纳入标准的50例患者随机分为疏风凉血补肾方组(A组)20例、疏风凉血补肾方加强的松组(B组)15例和以强的松为主的对照组(C组)15例,A组以疏风凉血补肾法(方)治疗,B组以疏风凉血补肾法(方)加强的松治疗(中药用法同A组,强的松用法同C组)。C组以强的松为主,加达那唑和升血小板胶囊辅助治疗,强的松按病情轻重决定用量,达那唑用量为0.2/次,3/日,升血小板胶囊用量为3粒/次,3/日。以上治疗均4周为一疗程,连续治疗3疗程后,观察不良反应,疗效,出血症状积分改善,血小板计数变化,骨髓巨核细胞总数及原幼巨、颗粒巨、产板巨、裸核巨数量比例变化,血清PAIgG及骨髓TGF-β_1水平变化情况。
结果:
1动物实验结果
1.1本实验以1、3、5、7、9、11、13天时间点于小鼠腹腔注射1:4稀释的APS,每次100μl,成功复制了CITP小鼠模型,在停止注射APS后模型维持时间为72小时左右,和文献结论相符。
1.2在提高CITP小鼠血小板计数方面,疏风凉血补肾法(方)高、中、低剂量组和强的松组均能提高CITP小鼠血小板计数,和模型组比较有极显著性差异(P＜0.01),但疏风凉血补肾法(方)高、中、低剂量组和强的松组间比较无显著性差异。
1.3在降低CITP小鼠骨髓巨核细胞总数方面,疏风凉血补肾法(方)高、中剂量组均能降低骨髓巨核细胞总数,和模型组比较有显著性差异(P＜0.05),而其组间比较无显著性差异;在提高产板巨核细胞比例上,疏风凉血补肾法(方)高、中剂量组产板巨核细胞比例上升明显,和模型组比较有极显著性差异(P＜0.01),而疏风凉血补肾法(方)低剂量组、强的松组和模型组比较只有显著性差异(P＜0.05)。
1.4在降低CITP小鼠升高的PAIgG值方面,疏风凉血补肾法(方)高、中、低剂量组、强的松组PAIgG值均下降明显,和模型组比较均有极显著性差异(P＜0.01),而其组间比较无显著性差异。
1.5在降低CITP小鼠升高的TGF-β_1值方面,疏风凉血补肾法(方)高、中、低剂量组TGF-β_1值降低明显,和模型组比较有显著性差异(P＜0.05),而其组间比较无显著性差异。强的松组TGF-β_1值有所下降,但和模型组比较无显著性差异。
2临床观察结果
2.1在临床疗效方面,A组总有效率值(90.0%)高于C组(73.3%),和B组(93.3%)相近,但经比较三组在总有效率上无显著性差异;在良效率方面,A组(55.0%)显著高于C组(20.0%)(P＜0.05),和B组(60.0%)相近。
2.2在降低出血症状积分方面,A、B两组均能有效降低出血症状积分,治疗前后比较有显著性差异(P＜0.05),而C组治疗前后出血症状积分比较无显著性差异。
2.3在提高血小板计数方面,A组治疗后血小板计数明显上升,和治疗前比有极显著性差异(P＜0.01),且其明显优于C组(P＜0.05),和B组疗效相似。
2.4在降低骨髓巨核细胞总数和提高产板巨比例方面,A组治疗后骨髓巨核细胞总数明显降低、产板巨比例明显上升,和治疗前较比有极显著性差异(P＜0.01),和B组疗效相似。而C组治疗后骨髓巨核细胞总数有所减低、产板巨比例有所上升,但治疗前后对比无显著性差异。
2.5在降低PAIgG值方面,A、B、C三组治疗后PAIgG值明显降低,和治疗前比较均有显著性差异(P＜0.05)。三组治疗后比较无显著性差异。
2.6在降低TGF-β_1值方面,A、B二组治疗后TGF-β_1值明显降低,和治疗前比差异显著(P＜0.05),C组TGF-β_1值有所下降,但和治疗前比无显著差异。A、B二组和C组治疗后比较均有显著性差异(P＜0.05),A、B二组治疗后比较无显著性差异。
结论:
1动物实验显示,疏风凉血补肾方三个剂量组均能有效降低CITP小鼠升高的PAIgG和TGF-β_1值,有效降低CITP小鼠骨髓巨核细胞总数和提高产板巨核细胞比例,从而有效促进骨髓巨核细胞分化成熟和提高血小板计数,其作用综合比较优于强的松组;疏风凉血补肾方三个剂量组间以高、中剂量组疗效最佳,但高剂量组可能出现剂量相关副作用,故中剂量(相当于临床常用量)组应为最佳剂量组。
2临床观察显示,疏风凉血补肾方能有效降低CITP患者升高的PAIgG和TGF-β_1值,有效促进骨髓巨核细胞分化成熟(表现在降低骨髓巨核细胞总数和提高产板巨核细胞比例上),有效提高CITP患者血小板计数,其作用综合比较优于以强的松为主的对照组;本实验中疏风凉血补肾方加强的松组和单纯的疏风凉血补肾方组疗效相近,考虑这和所入选的病例较多是经强的松类药物治疗无效而转诊我院有关,强的松对这部分患者的疗效已很有限,故强的松在该组治疗中未能起明显的增效作用。
3动物实验和临床观察均显示,疏风凉血补肾方对降低CITP升高的PAIgG和TGF-β_1值均有良好作用,而强的松在本研究中只对PAIgG值有较好的降低作用,而对TGF-β_1值无显著性降低作用,这可能就是疏风凉血补肾法疗效优于强的松的机理之一。
4动物实验和临床观察中,强的松组和以强的松为主的对照组在用药后PAIgG值均明显降低,而TGF-β_1值无显著性下降,这与目前的某些类似研究结论相似。以往认为TGF-β_1升高是由于CITP免疫异常状态时体内升高的PAIg导致血小板和巨核细胞遭受免疫性破坏TGF-β_1从中释放出来造成的,PAIg和TGF-β_1值呈正相关性,经治疗干预PAIg值降低后TGF-β_1值也应随之下降,而本研究及其他类似研究的结论与此观点有不一致性的地方。因此我们有理由推论:免疫异常并不是CITP时TGF-β_1升高的全部原因,免疫异常触发后的非免疫依赖因素也可引起TGF-β_1水平的增高,这也可能是CITP除免疫异常之外的其他重要发病机制之一。
综上所述,疏风凉血补肾法(方)对CITP有较好的临床疗效,其疗效机制之一可能就在于该法能有效降低PAIg水平及免疫异常和非免疫因素造成的TGF-β_1水平的升高,从而更有效的促进骨髓巨核细胞的分化成熟,这也可能是该法疗效优于强的松的关键原因所在。同时,免疫异常触发后的非免疫依赖因素导致的TGF-β_1的增高可能也是CITP除免疫异常之外的其他发病机制之一及CITP难治性的关键所在。对此有待更多的后续研究去加以验证。
Objective:CITP is a common hemorrhagic disease of blood system in clinics, it belong to "blood syndrome"、"muscle apostaxis"、" macula syndrome" etc,in TCM.This disease was thought as clinic syndrome due to the increased damage of platelet immune,and the abnormality of immune is the core mechanism of the disease,lt was also called autoimmune thrombocytopenic purpura.As the research going on in the recent years,the researchers both home and abroad realized that although the abnormality of immunity is the trigger and main mechanism,it is not the all,there must be some other non-immunity factors causing the occurrence of CITP.In clinics,we usually use medicine like prednisone,vincristine etc,to adjust the immune abnormality of CITP,or to decrease the immune damage of platelet by splenectomy,while both way fail to cure the CITP from root,still some of the diseases turn into more complicated and difficult ones.My tutor,Professor Zhixiong Chen has been using the prescription of expelling wind,cooling blood and invigorating kidney since long time ago to cure CITP and has achieved distinguished effect,which is much more than prednisone.My project is to do the further verification through animal test and clinic observation regarding the effect of the prescription of expelling wind,cooling blood and invigorating the kidney,it also want to probe the effective mechanism of curing CITP,as well as the possible mechanism regarding why the prescription is better than prednisone in effect by comparing some related indexes.The project will also detect some other possible pathogenic factors and mechanism beside the abnormality of immune by the experimental result.
Methods:
1 Animals empirical study
In the first experiment,70 BALB/C mice with SPF order were randomly divided into six groups(10 mice for normal group;12 mice per group respectively for blank group、prednisone group and high-dose/midst-dose/low-dose groups of decoction of expelling wind,cooling blood and invigorating the kidney).The latter five groups were improved with yangyufei way and were injected intraperitoneally APS(diluted with 1:4 proportion)100μl per time at every other day in 13 days and the CITP mice models were made.From the following sixth day on,these groups were administrated intragastrically with a single dose per day of experiment substance(administration quantity 0.2ml/10g)in the next 9 days,which were respectively saline;prednisone(0.5mg/ml,decuple of clinical dose);high-dose(2.6g/ml,fifteenfold of clinical dose) /midst-dose(1.73g/ml,decuple of clinical dose)/low-dose(0.87g/ml,quintuple of clinical dose)of the decoction.At the same time,the general state of health、the adverse reaction and the death amount were observed.Afterwards, all mice were sacrificed and the needed specimens were taken in order to observe the change of platelet countss,marrow megacaryocyte amount、platelet-produced megacaryocyte proportion、PAIgG in the serum and TGF-β_1level in the marrow.
2 clinical research
50 CITP patients were randomly divided to three groups(including 20 patients administrated with the decoction of expelling wind,cooling blood and invigorating the kidney as A group;15 patients with the same decoction and prednisone as B group;15 patients with prednisone as C group.To more explain,B group' administration way referred to A and C group'.C group was constituted of prednisone,danazol and shengxuexiaoban capsule.The prednisone administration quantity was given according to the pathogenetic condition and danazol 0.2 each time and shengxuexiaoban capsule 3 granule each time,3 times per day.The above treatment was 4 weeks as one time of therapy.After 3 times of therapy,a series of observation were made as before.
Results:
1 animal experiment results
1.1 The experiment successfully made the CITP mouse model according to injecting intraperitoneally the APS(dilution proportion 1:4)with 100μl per time respectively at the first、third、fifth、seventh、ninth、eleventh、thirteenth day.The duration time of the CITP model was 72 hours and the result was the same as that of correlative data.
1.2 In the aspect of platelet counts of the CITP mice,the high-dose、midst-dose、low-dose decoction and prednisone groups could all improve platelet counts and P＜0.01 was considered significant compared with the control group.However,there was no significant difference among the four groups.
1.3 In the aspect of marrow megacaryocyte amount of the CITP mice,that amount of the high-dose、midst-dose and low-dose decoction groups could all be lowered and P＜0.05 was considered significant compared with the control group.For the platelet-produced megacaryocyte proportion,that of the high-dose and midst-dose decoction groups upgraded obviously and P＜0.01 was considered significant compared with the control group.But the low-dose decoction group and the prednisone group had significant difference compared with the control when P＜0.05 was considered.
1.4 In the aspect of PAIgG value of the CITP mice,the high-dose、midst-dose and low-dose decoction groups,along with the prednisone group,could all drop the value of PAIgG obviously and P＜0.01 was considered significant compared with the control group.However,there was no significant difference among the four groups.
1.5 In the aspect of TGF-β_1 value of the CITP mice,that of the high-dose、midst-dose and low-dose decoction groups could be degraded obviously and P＜0.05 was considered significant compared with the control group.However, there was no significant difference among the three groups.But the TGF-β_1 value of the prednisone group had decreased slightly and there was no signifycant difference compared with the control.
2 Results of clinical observation
2.1 With regard to the therapeutic effect,the total effective rate of A group was 90.0%,which was better than that of C group(73.3%)and close to that of B group(93.3%).But there was no significant difference among the three groups.Regarding the fine efficiency,when P＜0.05 was considered,that of A group(55.0%)excelled C group(20.0%)significantly and close to that of B group(60.0%).
2.2 With regard to the integration of hemorrhage symptoms,A group and B group could effectively cut down the integration of hemorrhage symptoms.And there was significant difference between pre-therapy and post-therapy when P＜0.05 was considered.But that of C group had no significant difference between pre-therapy and post-therapy.
2.3 With regard to platelet counts,that of A group was raised obviously after therapy and there was significant difference compared with that before therapy when P＜0.05 was considered.But the result of A group was similar to that of B group.
2.4 With regard to the marrow megacaryocyte amount and the platelet-produced megacaryocyte proportion,the former of A group was effectively decreased and the latter of A group was increased greatly.And there was significant difference between pre-therapy and post-therapy when P＜0.01 was considered. But the treatment of A group was similar to that of B group.Though the results of C group had some change,there was no significant difference between pre-therapy and post-therapy.
2.5 With regard to the PAIgG value of the CITP mice,that of the A,B,C group all dropped greatly and P＜0.05 was considered significant compared with that before therapy.However,there was no significant difference among the three groups.
2.6 With regard to TGF-β_1 value of the CITP mice,that of A and B groups degraded obviously and P＜0.05 was considered significant compared with that before therapy.But the TGF-β_1 value of C group had decreased slightly and there was no significant difference compared with the pre-therapy.The results of A and B groups were significant deviation compared with that of C group when P＜0.05 was considered.And there was no significant difference after treatment between A group and B group.
Conclusion:
1 The results of animal experiment displayed that the three different dose groups of the expelling wind,cooling blood and invigorating the kidney decoction could effectively degrade the value of PAIgG and TGF-β_1,which became abnormally upgrade induced by CITP,also cut down the marrow megacaryo-cyte amount and raise the platelet-produced megacaryocyte proportion,then facilitating the maturity of the marrow megacaryocyte amount and enhancing the platelet counts.The combination effect of the decoction groups excelled that of the prednisone group.The experimental results of the high-dose and midst-dose groups were better that the low-dose group.Considering the high-dose group' latent adverse reaction,the midst-dose group(corresponding to the usual clinical dose)was the optimum.
2 The results of clinical observation showed that the expelling wind, cooling blood and invigorating the kidney decoction could effectively degrade the value of PAIgG and TGF-β_1,which became abnormally upgrade induced by CITP,could facilitate the maturity of the marrow megacaryocyte amount and enhance the platelet counts.The combination effect of the decoction groups excelled that of the prednisone group.In the study,the curative effect of the decoction and prednisone group were close to that of the simple decoction group.Considering the selected patients being transfer treatment cases due to those invalid to prednisone treatment,this result could accounts for prednisone' s failed synergistic effect.
3 The results of animal study and clinical observation showed that the expelling wind,cooling blood and invigorating the kidney decoction did well to balance the value of PAIgG and TGF-β_1 for the CITP case and the prednisone just could reduce the value of PAIgG and had no effective to TGF-β_1,which could well explain that the curative effect of the decoction was better than that of the prednisone.
4 The results of animal study and clinical observation displayed that the value of PAIgG was lowered obviously after administration for the prednisone group and the group in which prednisone was the main component and there was no significant decrease on the value of TGF-β_1.That investigation was similar to the results of the current correlative studies.The previous research presumed that the TGF-β_1 was overfull released due to the destroyed platelet or megacaryocyte which induced by the elevated PAIgG in CITP abnormality immunological environment and there was positive correlation between the PAIgG value and the TGF-β_1 value.After therapy,the value of TGF-β_1 descended following the decreased PAIgG.But our study and other parallel researches have some discordance with the previous view.For this reason,we can deduce that immunological abnormality is not the entire agent that accounts for the elevated TGF-β_1 value.The immune-independent factors triggered by immuneological abnormality can also provoke the enhanced TGF-β_1,which may become one of the important pathogenesies besides the immunological abnormality factor.
In a word,the expelling wind,cooling blood and invigorating the kidney decoction has good clinical effect on CITP.One of it's mechanism,the key point superior to prednisone,maybe lies in balancing PAIgG and TGF-β_1 effectively,then facilitating the cell differentiation maturity of the marrow megacaryocyte.Meanwhile,the increased TGF-β_1 induced by the immuneindependent factors owing to immunological abnormality maybe becomes one of the important pathogenesies besides the immunological abnormality factor, also probably being as the refractory key to CITP,which need more research to make verification.
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