| Primary immune thrombocytopenia(ITP)is a common autoimmune disease at home and abroad.Some patients often suffer from hemorrhage in the face,limbs.organs,etc.When the degree of hemorrhage is serious,it can threaten the life of the patients.In traditional Chinese medicine.ITP is divided into four types of syndromes:syndrome of the bleeding due to blood-heat,syndrome of blood stasis,syndrome of Yin-deficiency caused excessive Fire,and syndrome of qi failing to control blood.Among them,the syndrome of qi failing to control blood is the most common clinical syndrome type of ITP in traditional Chinese medicine.At present,the pathogenesis of ITP has not been clarified,and its treatment methods have not been unified.In order to solve these two problems,the corresponding combination of disease and syndrome animal model is the key means for in-depth research.Therefore,on the basis of previous research,this task studied the preparation method of ITP mouse model of qi failing to control blood syndrome,and preliminarily explored the pharmacodynamics of YQSX on this model.Objective:Referring to the clinical manifestations of diseases and syndromes in the syndrome of qi failing to control blood ITP patients,this task concentrated on establishiing an ITP mouse model of qi failing to control blood syndrome and making a dynamic study of the model to clarify the formation process of qi failing to control blood syndrome.In addition,the preliminary exploration on the pharmacodynamics of YQSX in this model were also did.To sum up,this study would provide new ideas and ways for the pathogenesis and treatment of ITP.Methods:1 The establishment and evaluation of syndrome of qi failing to control blood ITP mice modelThe mice model of ITP qi failing to control blood syndrome was established by intraperitoneal injection of anti-platelet serum(APS)combined with sleep deprivation.Balb/c mice were randomly divided into 3 weeks(normal group,model group),4 weeks(normal group,model group),5 weeks(normal group,model group),with 12 mice in each group.The mice in the three-week model group were deprived of sleep at the first week,and intraperitoneal injection of APS combined with sleep deprivation at the second and third weeks;the mice in the four-week model group were deprived of sleep at the first and second weeks,and intraperitoneal injection of APS combined with sleep deprivation at the third and fourth weeks;the mice in the fifth-week model group were deprived of sleep at the first,second and third weeks,and intraperitoneal injection of APS combined with sleep deprivation at the fourth and fifth weeks.At the end of the experiment,the optimal period and method for the establishment of the ITP mouse model of qi falling to control blood syndrome were determined by detecting platelet(PLT)count,bleeding,spleen index,T and B lymphocyte subsets of spleen and other related disease indicators and body mass,holding power,pulse amplitude,tongue and other related syndrome indicators in mice.2 The dynamic study of ITP mouse model of qi failing to control blood syndromeThe preliminary work of this study determined the optimal establishment period(3 weeks)and method(Sleep deprivation in the first week,intraperitoneal injection of APS combined with sleep deprivation in the second and third weeks)of the ITP mouse model of qi failing to control blood syndrome,and selected 3 time points(7d,10d,21d)for dynamic observation based on the characteristics of the model.Balb/c mice were randomly divided into the normal group(39 mice)and the model group(45 mice).After the 7th,10th and 21th day of the experiment,a batch of mice were taken for index detection,the mice were detected respectively for PLT count in peripheral blood of mice,bleeding grade,classification and counting of bone marrow megakaryocytes,spleen index,spleen pathology,T and B lymphocyte subsets of spleen,lung pathology and other related disease indicators,body mass,holding power,pulse amplitude and other related syndromes indicators,and PLT morphology,function,apoptosis and other related indicators.3 Preliminary study on the pharmacodynamics of YQSX in ITP mouse model of qi failing to control blood syndromeBalb/c mice were randomly divided into normal group,model group,YQSX high-dose group(15.22g/kg),YQSX low-dose group(7.61 g/kg)and prednisone acetate group,with 12 mice in each group.After 24 hours of the first intraperitoneal injection of APS,the drug was administered intragastrically for 12 days.The dosage volume of each group was 20ml/kg/d The normal group and the model group were given the same amount of normal saline.After the experiment,the mice were detected the peripheral blood PLT count,bleeding grade,spleen index.thymus index,T and B lymphocyte subsets of spleen and other related disease indicators,body mass,holding power,tongue and other related syndrome indicators and platelet activation and apoptosis related indicators.Results:1 The establishment and evaluation of ITP mouse model of qi failing to control blood syndrome(1)Disease indexesCompared with the normal group,the mice in the 3-week,4-week and 5-week model groups all had different degrees of bleeding.Peripheral blood PLT count was significantly decreased {P<0.01).Spleen index was significantly increased {P<0.01).The thymus index of mice in the 3-week and 4-week model group showed no significant change(P>0.05),while that in the 5-week model group decreased significantly(P<0.01),CD3 T cells.CD4 T cells,CD8+T cells and CD4+/CD8+were all significantly decreased(P<0.05-0.01).There was no significant change in CD19 B cells in the 3-week model group mice(P>0.05),and CD19+B cells were significantly decreased in the 4-week and 5-week model group(P<0.05).(2)Syndrome indexesThe mice hair color in the normal group were bright and white,and their stools were yellowish,brown and strip-like,however,mice in the 3-week,4-week and 5-week model groups had coarse,bristling and dark yellow hairs,squinting eyes,and dark brown or wet stools.Compared with the normal group,there were no significant changes in the body mass of mice in the 3-week,4-week and 5-week model groups(P>0.05).but the holding power was significantly decreased(P<0.01),The pulse amplitude of mice was significantly decreased in the 3-week and 5-week model group(P<0.01).while that in the 4-week model group was no significant change(P>0.05)There were no significant changes in the R,G,B and R/B values of mice in the model groups at 3,4,and 5 weeks(P>0.05),but R/G of the tongue surface of mice in the 3-week model group was significantly decreased(P<0.05),2 The dynamic study of ITP mouse model of qi failing to control blood syndrome(1)Disease indexesCompared with the normal group,there were no bleeding and no significant changes in spleen index in the model group mice at 7 days(P>0.05),but the mice in the model groups had bleeding in different degrees and different parts at 10 days and 21 days,and the spleen index was significantly increased(P<0.07).The PLT count of mice in model groups were decreased significantly at 7 days,10 days and 21days(P<0.01).The number of granular megakaryocytes,plate megakaryocytes and all the megakaryocytes of the model group mice were decreased significantly at 7 days(P<0.05),while the immature megakaryocytes of the model group mice were significantly decreased(P<0.05)and the number of granular megakaryocytes,naked megakaryocytes and all the megakaryocytes were significantly increased(P<0.05)at 10 days.When it was at 21 days,the number of immature megakaryocytes,granular megakaryocytes,naked megakaryocytes and all the megakaryocytes of the model group mice were increased significantly(P<0.05-0.01).The CD3+T cells of model group mice was significantly increased at 7 days(P<0.01),while that were significantly decreased at 10 days and 21 days(P<0.01).The CD4+T cells of model group mice were significantly decreased at 7 days and 10 days(P<0.01),while that was no significant change at 21 days(P>0.05).The CD8+T cells of model group mice was significantly increased at 7 days(P<0.01),while that had no significant differences at 10 days and 21 days(P>0.05).The CD19+B cells of model group mice were significantly increased at 7 days and 21 days(P<0.01),while that had no significant change at 10 days(P>0.05)(2)Syndrome indexesThe hair of the normal group mice was bright and smooth,and the stools were yellow-brown and strip-shaped,with good mental state,while the model group mice were dark and rough,with blinking and vertical hair,dark brown or wet stools at 7 days,10 days and 21 days.Compared with the normal group,the body mass,holding power and pulse amplitude of the model mice were significantly decreased(P<0.01)at 7 days,10 days and 21 days.(3)The related indexes of PLTCompared with the normal group,the TPO,TGF-?1,?-TG,PF4,ATP,ADP,CD62P,ROS-MFI of model group mice were significantly increased at 7 days,10 days and 21 days(P<0.05?0.01).The mitochondrial membrane potential of model group mice were significantly decreased at 7 days and 10 days(P<0.01),while that was increased significantly at 21 days(P<0.01).There was no significant change of PNA and PMA of model group mice at 7 days(P>0.05),but that were significantly decreased at 10 days(P<0.01),anymore,PNA was significantly increased(P<0.05)and PMA was significantly decreased at 21 days(P<0.01).3 Preliminary study on the pharmacodynamics of YQSX in ITP mouse model of qi failing to control blood syndrome(1)Disease indexesCompared with the normal group,hemorrhage was observed in model group mice,and PLT count in peripheral blood was decreased significantly(P<0.01).The spleen index was significantly increased(P<0.01)and there was no significant difference in thymus index(P>0.05).The CD3+T cells was decreased significantly(P<0.01)and the CD4+T cells,CD8+T cells,CD4+/CD8+,CD19+B cells did not change significantly(P>0.05).Compared with the model group,prednisone acetate group and YQSX high-dose group had different degrees of improvement in hemorrhage.PLT count in peripheral blood of YQSX high-dose group was increased significantly(P<0.05).The spleen index and thymus index of prednisone acetate group were decreased significantly(P<0.01).CD3+and CD8+T cells were significantly increased {P<0.01),while CD4+/CD8+was decreased significantly(P<0.01)and there was no significant change in CD4+T cells and CD19+B cells in the prednisone acetate group mice(P>0.05).(2)Syndrome indexesCompared with the normal group,the body mass of the model group mice did not change significantly(P>0.05),while the holding power was significantly decreased(P<0.01)and the R/G value was significantly decreased(P<0.05).Compared with the model group,there was no significant change of the body mass in prednisone acetate group,YQSX high and low-dose group(P>0.05).The holding power had no significant change of the prednisone acetate group(P>0.05),but that was significantly increased(P<0.05-0.01)in the high and low-dose group of YQSX.There was no significant change in the R/G value of the prednisone acetate group,the YQSX high and low-dose group(P>0.05).(3)The related indexes of PLTCompared with the normal group,there were no significant changes in CD62P and AV in the model group(P>0.05),but there was a significant increase trend.Compared with the model group,there was no significant change in CD62P in the prednisone acetate group and the YQSX low-dose group(P>0.05),while the YQSX high-dose group was significantly decreased(P>0.05).There was no significant change in AV in the high and low-dose of YQSX group(P>0.05),but there was a significant decrease trend.Conclusions:1 The optimal period of the establishment of ITP mouse model of qi failing to control blood syndrome was 3 weeks,and the method was sleep deprivation in the first week and intraperitoneal injection of APS combined with sleep deprivation in the second and third weeks.2 The pathological changes of ITP mouse model of qi failing to control blood syndrome were the dynamic process of qi deficiency first formed and then bleeding and then aggravated,which was consistent with the etiology and pathogenesis of qi failing to control blood in traditional Chinese medicine.3 YQSX can improve the holding power and PLT count in peripheral blood,and inhibit platelet activation in ITP mouse model of qi failing to control blood syndrome.It improves the main indicators of disease and syndrome of this model. |
PDF Full Text Request