| BackgroundPrimary immune thrombocytopenia(ITP)is an acquired autoimmune disease characterized by increased platelet destruction and insufficient platelet production resulting in low platelet counts.Based on the basic theory of TCM and the principles of diagnosis and treatment,the spleen can control blood in the meridians and veins without escaping from the veins,so the etiology of ITP is closely related to the deficiency of spleen qi and the failure of the spleen to control blood.Thus,the basic principle of "treating ITP from the spleen" was determined,and the formula JPYQSX formula was composed by strengthening the spleen,benefiting qi,and regulating blood.The previous team has confirmed through clinical trials and basic experiments that the treatment of ITP from the spleen and the treatment of ITP with the formula of strengthening the spleen,benefiting qi,and regulating blood have clear effects in rapidly reducing bleeding,improving patients’ symptoms,raising platelets and regulating vasoactive substances.Although the pathogenesis of ITP is not yet clear,it is now believed that its occurrence is closely related to excessive platelet destruction mediated by humoral and cellular immunity,and that immune imbalance plays an important role in ITP.An increasing number of studies confirm that most autoimmune diseases have an imbalance between proand anti-inflammatory mechanisms.This is also true for ITP,which is mainly characterized by increased Th1/Th17 expression and decreased Th2/Treg expression,and defective Breg function.In this study,we investigated the effect of JPYQSX formula on the expression level of related cytokines in the spleen and blood by establishing mice model of ITP,and investigated the mechanism of JPYQSX formula in regulating the imbalance of cellular immunity,so as to elaborate the theoretical advantages of "spleen-based treatment" of ITP and provide experimental basis and guidance for the treatment of ITP in Chinese medicine.ObjectiveBy establishing ITP model mice and detecting the changes of cytokine levels in serum and spleen,we investigated the effect mechanism of JPYQSX formula in treating ITP.MethodThirty BALB/c mice were randomly divided into a normal control group(NC),model control group(MC),prednisone group(PDN),JPYQSX medium dose group(JP-M),and JPYQSX high dose group(JP-H),with six mice in each group.Except for the normal control group,mice in each group were injected with guinea pig anti-mouse platelet serum(GP-APS)to establish the ITP model.On day 8 after successful modeling,mice in the normal and model groups were given saline 0.1 ml/10 g volume by gavage,the prednisone group was given a concentration of 2 mg/ml prednisone solution 0.1 ml/10 g by gavage,the JPYQSX medium dose group was given 4.1 mg/g by gavage,and the high dose group was given 8.2 mg/g by gavage,and all groups were administered once a day for 8 days.The peripheral platelet,hemoglobin,leukocyte values,and platelet distribution width of mice were measured before and after the experiment using an automatic hematology analyzer.After the experiment,the expression levels of various cytokines(IFN-γ,IL-2,IL-4,IL-10,TGF-β,IL-27,IL-17A)in the serum and spleen of each group of mice were measured by enzyme-linked immunosorbent assay(ELISA).Result1.Peripheral blood cell counts:A.Platelet count:Before the start of the experiment,there was no significant difference in platelet counts among the experimental groups(P>0.05).On the 4th day of modeling,the platelet count in each group decreased compared with that in the NC group(P<0.01);on the 8th day of modeling,the platelet count decreased further and was significantly lower than that in the NC group(P<0.01);on the 4th day of administration,the platelet count in PDN group,JP-M group,and JP-H group increased significantly compared with that in MC group(P<0.01);on the 8th day of administration,the platelet count in each experimental group was still lower than that in the normal group(P<0.05).On the 8th day of administration,the platelet count in each experimental group was still lower than that in the normal group(P<0.05);however,compared with the MC group,the platelet count levels in the PDN,JP-M,and JP-H groups were comparable and remained high(P<0.01).B.Others:There was no significant difference in hemoglobin,leukocyte count,and platelet distribution width among different experimental groups at each time point(P>0.05).2.Cytokines:A.Compared with the NC group,all cytokine changes in the MC group were statistically significant(P<0.05).B.Spleen:Compared with the MC group,IFN-y expression in the spleen was decreased in the PDN,JP-M,and JP-H groups,but the differences were not statistically significant(P>0.05);TGF-β levels were increased in the PDN,JP-M,and JP-H groups(P<0.05 or P<0.01);IL-2 levels increased in the PDN,JP-M,and JP-H groups(P<0.05 or P<0.01);IL-4 increased most significantly in the JP-H group only(P<0.01);IL-10 expression increased in the PDN,JP-M,and JP-H groups(P<0.01);IL-27 expression slightly increased in the three groups(P>0.01).When compared between treatment groups,the JP-H group showed significant(P<0.01)elevated TGF-β and decreased IL-17A expression compared to the PDN group.C.Serum:Compared with the MC group,IFN-γ expression in serum was downregulated in the PDN and JP-H groups(P<0.01);TGF-βlevels were significantly increased in the JP-H group(P<0.01);IL-2 expression was increased in the PDN,JP-M,and JP-H groups(P<0.01);IL-2 expression was increased in the PDN,JP-M,and JP-H groups(P<0.01).IL-2 expression was elevated in the PDN,JP-M,and JP-H groups(P<0.01);IL-4 expression was upregulated in the PDN,JP-M,and JP-H groups(P<0.05 or P<0.01);IL-10,IL-27 and IL-17A expression levels were not significantly changed(P>0.05).IL-2 expression was significantly upregulated in the JP-M and JP-H groups compared with the PDN group(P<0.01).Conclusion1.JPYQSX formula can rapidly increase the peripheral platelet count in ITP model mice,and the time of increase is earlier than that of prednisone.JPYQSX formula had no obvious effect on peripheral hemoglobin,leukocyte count,and platelet distribution width in ITP model mice.2.JPYQSX formula can regulate IFN-γ,IL-2,IL-4,IL-10,TGF-β,IL-27,IL-17A levels in ITP model mice,which may be related to reversing Th1/Th2 and Th17/Treg immune imbalance and improving Breg function deficiency. |
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