Immune Regulation Mechanism Research Of Niujiao Dihuang Decoction Through Mediating CaN-NFAT2Signal Pathway Intervention Of Primary Immune Thrombocytopenia

Objective:to study the immune pathological state of ITP rat model and explore the influence of Niujiao Dihuang Decoction on Fxop3in peripheral blood CD4+CD25+regulatory T cell of ITP rat model through CaN-NFAT signaling passway, expounding immune regulatory mechanism of promoting and stabilizing platelet number.Methods:30SD rats were randomly divided into experimental group, model group and normal control group, each group10only.The experimental group were given Niujiao Dihuang Decoction intervention, model group and normal control group were given saline intervention at the seventh day of modeling. Rats of each group were killed after modeling4W, detecting platelet number in peripheral blood by automatic platelet analyzer, detecting megacaryocyte number in bone marrow by morphology, detecting IL-10and TGF-β1concentration in serum by ELISA, detecting CD4+CD25+regulatory T cell in peripheral blood by flow cytometry, detecting Foxp3mRNA in peripheral blood mononuclear cell by RT-PCR, and detecting CaN、NFAT2protein in peripheral blood mononuclear cell by western blot, in order to evaluate immune pathology of ITP rat model and intervention mechanism of Niujiao Dihuang Decoction.Results:(1)Compared with normal control rat, skin purpura and hemorrhage symptom of digestive track of model group ITP rats are more significant, there was statistically significant difference between the two groups (10VS0,4VS0, P<0.05);Platelet number in peripheral blood and megacaryocyte number in bone marrow of model group ITP rats decrease, there was statistically significant difference between the two groups (664±296VS957±163×109/L,234±114VS1235±177, P<0.05); IL-10and TGF-β1concentration in serum of model group ITP rats reduce, there was statistically significant difference between the two groups (270.78±88.08VS434.52±29.71pg/ml,163.04±51.94VS422.13±75.49pg/ml, P<0.05);Peripheral blood CD4+T lymphocyte of model group ITP rats increase and CD4+CD25+regulatory T cell of model group ITP rats decrease, there was statistically significant difference between the two groups (12.54±2.39VS2.85±0.87%,0.48±0.33VS5.96±0.89%, P<0.05);Foxp3mRNA, CaN, NFAT2expression in peripheral blood mononuclear cells of model group ITP rats decrease, there was statistically significant difference between the two groups (0.52±0.26VS2.65±0.64(E-3),0.37±0.15VS0.83±0.08,0.28±0.06VS0.82±0.07, P<0.05).(2) Compared with model group, peripheral blood platelet count and bone marrow megakaryocyte number of experimental group ITP rats increase, and purpura improve by Niujiao Dihuang Decoction, there was statistically significant difference between the two groups (869±161vs664±296×109/L,789±92VS234±114,10VS2, P<0.05); IL-10and TGF-β1concentration in serum of experimental group ITP rats increase by Niujiao Dihuang Decoction, there was statistically significant difference between the two groups (328.41±56.88vs270.78±88.08pg/ml,221.08±61.94VS163.04±51.94pg/ml, P<0.05); Peripheral blood CD4+T lymphocyte of experimental group ITP rats decrease and CD4+CD25+regulatory T cell of experimental group ITP rats iecrease by Niujiao Dihuang Decoction, there was statistically significant difference between the two groups(7.41±0.61vs12.54±2.39%,2.99±0.58vs0.48±0.33%, p<0.05); Foxp3mRNA, CaN, NFAT2expression in peripheral blood mononuclear cells of model group ITP rats "increase, there was statistically significant difference between the two groups (1.72±0.61VS0.52±0.26(E-3),0.61±0.14VS0.37±0.15,0.50±0.08VS0.28±0.06, P<0.05).But compared with normal control group, platelet count、megacaryocyte count、serum IL-10and TGF-β1concentration、CD4+T lymphocytes and CD4+CD25+regulatory T cell count, Foxp3mRNA and CaN, NFAT2expression of experimental group ITP rats do not reach normal levels, the differences has statistical significance (869±161vs957±163×109/L,789±92vs1235±177,328.41±56.88VS434.52±29.71pg/ml,221.08±61.94VS422.13± 75.49pg/ml,7.41±0.61VS2.85±0.87%,2.99±0.58VS5.96±0.89%,1.72±0.61VS2.65±0.64(E-3),0.61±0.14Vs0.83±0.08,0.50±0.08VS0.82±0.07, P<0.05).Conclusion:(1)ITP model rat in passive immune status is easy to cause purpura symptom by APS modeling methods and peripheral blood CD4+CD25+regulatory T cell number and Foxp3expression decrease, consistent with the pathological mechanism of ITP immune tolerance imbalance.(2)Niujiao Dihuang Decoction can promote CD4+CD25+regulatory T cell proliferation of ITP rats, increase the secretion of inhibitory cytokines IL-10and TGF-β1, reduce the destruction of CD4+T lymphocytes on their platelets, thus improving immune disorder state of ITP rats.(3) Niujiao Dihuang Decoction can enhance the transcription level of Foxp3in CD4+CD25+regulatory T cell by immune regulation signal CaN-NFAT2, promote the proliferation of CD4+CD25+regulatory T cell in ITP rats, so that ITP rats can recovery autoimmune tolerance balance, essentially lifting and stabling platelet count.