Effect Of Method Of Expelling Wind, Cooling Blood And Invigorating Kidney On The Marrow Megacaryocyte Differentiation Maturity And TGF-β₁ In CITP

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-β₁level in the marrow.2 clinical research50 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 results1.1 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-β₁ 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-β₁ value of the prednisone group had decreased slightly and there was no signifycant difference compared with the control.2 Results of clinical observation2.1 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-β₁ 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-β₁ 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-β₁,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-β₁,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-β₁ for the CITP case and the prednisone just could reduce the value of PAIgG and had no effective to TGF-β₁,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-β₁.That investigation was similar to the results of the current correlative studies.The previous research presumed that the TGF-β₁ 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-β₁ value.After therapy,the value of TGF-β₁ 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-β₁ value.The immune-independent factors triggered by immuneological abnormality can also provoke the enhanced TGF-β₁,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-β₁ effectively,then facilitating the cell differentiation maturity of the marrow megacaryocyte.Meanwhile,the increased TGF-β₁ 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.