On The Role And Compatibility Effect Of Yuzhang No.â…¡ Recipe To Aplastic Anemia Model Mouse Based On The PTEN/AKT/mTOR Signaling Pathway Of Marrow Cells

Objective Study on the role and compatibility effect of Yuzhang No.â…¡Recipe to Aplastic Anemia model mouse based on the action of PTEN/AKT/mTOR signaling pathway on marrow cells,in order to offer the evidence-based data supporting to guide the TCM treatment of Aplastic anemia.Methods 75SPF female BALB/c mice were randomly divided into the normal group（15） and the other group（60）.On the first day of the experiment,the 60 group mice were hypodermic injected by Acety lphenylhydrazine(100mg·kg^-1);On the second day,irradiated disposable body by X ray at the dose of 2.0Gy;from the 6th day to 12 th day,intraperitoneal injection of Cyclophosphamide(50.0mg·kg^-1).from the 13 th day,the 60 group were randomly divided into the normal group,The model group,the whole prescription group,soothing liver and invigorating spleen group andt onifying kidney group.Except the normal group,other mice were gavaged and Immobilized with diet control for 16 days.on the 28 th day,the blood samples were detected to measure the levels of RBC,WBC, HGB and PLT. Blood smears were treated by wright stain to observe blood cell morphology. Take the side of femur for bone marrow biopsy.Marrow cell suspension was separated for Westernblot and RT-PCR experiments at late stage.The proportion of CD45⁺CD117⁺cell in bone marrow mononuclear cells and the apoptosis rate of bone marrow Mononuclearcells were detected using FCM;Eexpression of PTEN, AKT, mTOR mRNA on the marrow cells were detected using RT-PCR;Expression of PTEN、p-PTEN、AKT、p-AKT、mTOR、p-mTOR、p-P70S6K、p-4EBP1 were detected using Western blot.Results1.After the treatment with Yuzhang No.â…¡Recipe, mice were more active, glossier in hair and showed better appetite, Conspicuously,they manifested improvement of anemia symptoms than mice in the model group（P<0.05）. The improvement of the general state in the whole prescription group was the most evident（P<0.01）.2.Compared with the normal group,the RBC,WBC,HGB and PLT significantly decreased（P<0.01） in peripheral blood of mice in the model group. Compared with the model group,the RBC,WBC,HGB and PLT in each treatment group significantly increased,The whole party group rebounded the fastest（P<0.05）,followed by the tonifying kidney group（P<0.05）.3.In the model group,the erythrocyte were scattered, and the numberof white blood cells reduced, and the degradation cells were common, while in the whole Party group, the permeability of the blood cells improved, the number of the nucleated cells increased while the-degradation of the cells decreased.Bone marrow smears:in the model group, fat droplets increased significantly.The nuclear proliferation as well as the number of erythroid, myeloid, megakaryocytic cells reduced, and non-hematopoietic cells appeared while in the treatment groups,fat drops in the number of bone marrow cells and non-hematopoietic cells reduced, while that of nucleated cells increased4.Compared with the normal group,the CD45⁺CD117⁺cells percentage of the model group Significantly decreased（P<0.01）.Compared with model group,the CD45⁺CD117⁺ cells percentage of each treatment group significantly increased,among which the whole party group rebounded thefastest（P<0.01）,Followed by tonifying kidney group（P<0.01）, harmonizing liver and spleen group（P>0.05）.5.Compared with the normal group, the early Apoptosis rate of the model group Significantly increased（P<0.01）.Compared with the model group,the early Apoptosis rate of all treatment group significantly decreased,among which whole party group decreased the fastest（P<0.01）,Followed by the tonifying kidney group（P<0.05）,harmonizing liver and spleen group（P<0.05）.6.By using RT-PCR,we found that,Compared with the normal group, the expression of AKT,mTOR mRNA of the model group Significantly decreased（P<0.01）.Compared with the model group,the expression of AKT,mTOR mRNA of each treatment group significantly increased,among which the whole Party group increased the fastest（P<0.01）, harmonizing liver and spleen group the slowest（P<0.05）.7.By using the Western Blot,we found that,Compared with the normal group, the expression of PTEN and p-PTEN of the model group and the treated group Significantly increased, the trend from high to low, respectively was the model group tonifying kidney group,harmonizing liver and spleen group,the whole Party group.Compared with the normal group, the expression of p-Akt、p-P70S6K（Ser371）、p-4E-BP1 of the model group and the treated group Significantly decreased,the activity of AKT,mTOR from low to high, respectively was the model group,tonifying kidney group,harmonizing liver and spleen group,the whole party group,the normal group.Conclusion:1.The Yuzhang No.â…¡Recipe can improve the marrow cells hematopoietic function of Aplastic anemia Model mouse;It can improve general state,increase the number of peripheral blood cells and nucleated cell of marrow, rectify the abnormal morphology of blood cells and pathological changes of bone marrow.2.The Yuzhang No.â…¡Recipe can improve the bone marrow CD45+CD117+cells percentage of Aplastic anemia Model mouse.The Yuzhang No.â…¡Recipe can decrease the bone marrow mononuclear cells early apoptosis rate ofAplastic anemia Model mouse.3.The Yuzhang No.â…¡Recipe can activate the activity of PTEN/AKT/mTOR signaling pathway, promote the proliferation of Bone marrow hematopoietic cells, and inhibiteits apoptosis to raise the number of bone marrow hematopoietic cells and help the recovery of hematopoietic function of bone marrow.4.PTEN/AKT/mTOR signaling pathway is one of the influence factors to inhibite its apoptosis to raise the number of bone marrow hematopoietic cells and help the recovery of hematopoietic function of bone marrow.5.Using two method simultaneously has better effect than independent use.regulating liver and spleen method has better effect than the tonifying kidney.