| Objective: Idiopathic thrombocytopenic purpura (ITP) is the most common hemorrhagenic disease in pediatrics and is a disorder characterized by thrombo-cytopenia resulting from abnormal destruction of platelets in the reticulo-endotheial system (RES) . Recently, it's believed that abnormal cell-mediated immunity make the core of the pathogenesis in ITP, include activation of autoreactive T cells, molecular mimic theory, epitope spread theory, wrong antigen-presenting, and destructed stability of immune system. A new class of antidiabetic drugs known as Thiazolidinediones (TZDs) have been identified as agonist of the nuclear receptor, peroxisome proliferator -activated receptor Y (PPAR- Y ). They stimulate the differentiation of adipocytes and inhibit the production of TNF-α, thereby playing a pivotal role of the insulin sensitizing and leading to improvement of glucose and lipid profiles in type 2 diabetic patient. It has been reported that PPAR- Y is also expressed on peripheral blood monocytes and tissue macrophages. Furthermore, TZDs showed inhibitory effect on the production of monocyte inflammatory cytokines and monocyte activity via PPAR- Y-independent pathway. Thus, as a member of Thiazolidinediones (TZDs) family, Rosiglitazone Maleate may be good candidate drugs to supress the phagocytic activity of RES or macrophages in ITP. In this study, we developed a mouse model with acute thrombocytopenia and observed the effects of Rosiglitazone on platele numbers, as well as phagocytic activity of macrophage in vivo using the ITP mouse model.Subjects and Methods: 1. Animals: Healthy BALB/c mice were bred in the animal facility of Zhengzhou university, Henan. Either sex , aged 8 to 10 weeks, weighting 19 to 21g, were used in experiments. All mice were nestled under a 12-hour light-dark cycle. Any experiments were performed in the morning. In addition, healthy male rabbit weighting 2.5 to 3.0kg were prepared for experiments. 2. Preparation of mice with thrombocytopenia: Washed platelets from BALB/c mice were given to rabbit by intravenous for preparation of rabbit-mouse antiplatelet antiserum(abundant with polyclonal antibody against mouse platelets) (APS). On the purpose to create mice with thrombocytopenia, lOOul APS(diluted in 1:4) were injected to BALB/c mice via tail vein. Those peripheral platelets count lower at the values between 150xl09/L and 250xl09/L within 24 hours is brought in further experiment as mouse model with thrombocytopenia. 3. In vivo experiments and methods: 32 BALB/c mice with thrombocytopenia were randomly separated into two groups which had 16 individuals respectively, characterized as trail group (A)and control group(B). Trail group animals were given Rosiglitazone by ways of oral-gastral irrigation, 0.5mg/Kg/d, totally 8 d persisted. While control group animals were given nothing but food and water. In addition, 32 normal mice were used as normal group(C) and average group(D).Mice in normal group were also given Rosiglitazone by ways of oral-gastral irrigation, 0.5mg/Kg/d, totally 8 d persisted. But average group only were used to find a normal value in macrophage phagocytic activity assay. 4. Test and assay: 0, 2,4,5, 6 and 8 days after the treatment, peripheral blood specimens were collected for hematocyte counts. Simultaneously, bleeding time after a 10-mm tail incision was detected at each time schedule. Furthermore, mice macrophage phagocytic activity assay were carrried out in each group at the end of the experiment as well. 5. Statistical analyses: All statistical tests were processed on the ground of SPSS13.0. The means of platelets or hematocyte counts were compared using an unpaired Student's t test at the 0.05 significance level.Results: 1. Platelets counts in mice administrated with APS via tail vein within the initial 12 hours: Platelets counts in mice administrated with APS via tail vein gradully decreased within the initial 12 hours. A relatively low value, 143.44+ 47.63xlO9/L> was observed at 12 hours after administration., which significantly lower than the value that before or later the time of administeration (P<0.001). 2. Effects of Rosiglitazone on the platelets counts in mice with thrombocytopenia: Administration of Rosiglitazone gradully increased platelet counts in trial group(A) compared to that in control group(B). A relatively upper value, 588.75 ± 92.15xlO9/L, was observed at day 8, which significantly greater than that before treatment and control group (F=70.564, P=0.000; t=7.685, P=0.000) . Platelet counts in normal group(C) exhibited no differ at each time schedule. At day 4, a slight increase was appered in control group. Such a increment reached at a relatively upper value, 391.25 + 73.38xlO9/L at day 8 and remained significantly low compared to that in normal group 0=18.690, P=0.000). 3. Effects of Rosiglitazone on the peripheral WBC and RBC: On the peripheral WBC and RBC counts, no significant differences was observed between trail group,control group and normal group (P>0.05) . However, there was a slight increased WBC counts after day 3 in trail group and control group. But it showed no great differences compared with normal group (P>0.05). 4. Effects of Rosiglitazone on the Bleeding time: Bleeding time in mice of trail group differed significantly from that of control group, reached a stable lower value of 6.63+1.20min, adjacent to the average of normal group, 5.50±1.32min . Those value in mice of control group persist a minute increase, reached at a highest point of 16.75 ± 1.73min. And then, it fall off to a lower point of 13.31 ± 1.54min , inferior to the highest. On the side of normal group, bleeding times exhibited no differ at each time schedule (P>0.05) . 5. Effects of Rosiglitazone on phagocytic activity of macrophage: Phagocytic percentage of macrophage differed greatly in A, B, C and D group(F=66.26, P=0.000). A relatively low value, 36.91 + 7.24%, appeared in trail group(A), which significantly lower than that in average group(D) (P=0.001) . Phagocytic percentage of macrophage were found increased in control group(B) (71.69 + 11.35%), compared with average group(D) and normal group(C) (P<0.01) .Except that, the value in normal group,26.06±7.58%, was significantly lower than that in average group(D) (P<0.01) .Conclusions: 1. It is feasible and inexpensive to achieve a mouse model with passive thrombocytopenia using a dose of APS injected by intravenous. 2. Rosigli-tazone can relax the destruction of platelets in RAS by evidently inhibiting phagocytic activity of macrophage in mouse model with passive thrombocytopenia. Thereby, it may be a brand new remedy among numerous therapies for thrombocytopenia. 3. Rosigli-tazone effect little or can hardly result in variance on peripheral WBC and RBC counts of normal mice. 4. That enabled macrophage enormously phagocytize abnormal platelets make the pivotal pathogenesis of passive thrombocytopenia mice model. |
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