Effects Of Tetramethylpyrazine On Bone Marrow Injury And Renal Oxidative Stress In Immune-mediated Bone Marrow Failure Mice

Part 1 The effect of Tetramethylpyrazine on bone marrow of Immune-Mediated Bone Marrow Failure MiceObjectiveBy using irradiation and lymphocyte infusion to establish a suitable immune-mediated bone marrow failure model,to evaluate the effects of TMP on the bone marrow of immune-mediated BMF mice,to provides new therapeutic targets for AA.Methods and ResultsMethods The effect of TMP on BMF was evaluated in immune-mediated BMF mice which were induced by X-ray irradiation and allogeneic lymphocyte infusion.First of all,The mice were subjected to X-ray irradiation at the dose of 5.0 Gy,5.75 Gy,6.0 Gy,and then were infused with 0.2 ml of cell suspension containing 4�106 mixed lymphocyte cells to induce BMF model within 4 hours.General conditions and survival days of mice in each group were observed and recorded every day.Mice were sacrificed on the 14th day for blood cell count and bone marrow pathological examination.The results showed that all three doses of X-ray could induce the establishment of immune-mediated BMF mouse model,but the survival rate(80%)of the 5.0 Gy group was higher,and the degree of BMF was mild.Therefore,5.0 Gy X-ray was the appropriate dose for the establishment of immune-mediated BMF mouse model,and this dose of X-ray was selected for subsequent experimental studies.After establishing a suitable BMF model,the effect of TMP on BMF was explored.The mice in each group were treated with low(5 mg/ml),medium(10 mg/ml)and high(20 mg/ml)doses of TMP every day from the first day after modeling.On the 14th day,the mice were sacrificed,and peripheral blood was collected,as well as femoral and bone marrow rinsed fluid obtained.The effects of TMP on BMF were evaluated by blood cell counts,bone marrow nucleated cell counts,H&E staining,and immunofluorescence techniquesResults The results of blood cell and bone marrow nucleated cell counts showed that high-dose TMP significantly increased bone marrow nucleated cells and red blood cells,while low-dose TMP solution increased white blood cells and platelets.The results of bone marrow H&E staining and immunofluorescence showed that with the increase of TMP dose,bone marrow congestion was significantly reduced and nucleated cells increased,and the expression of Fas protein in bone marrow microenvironment was decreased.Conclusion5.0 Gy X-ray is the suitable dose for establishing immune-mediated BMF model.TMP can alleviate BMF,which might be linked to inhibiting the expression of apoptosis-related protein Fas,and further expands the theory of "removing stasis and producing newness"Part 2 The effect of Tetramethylpyrazine on renal oxidative stress in immune-mediated bone marrow failure miceObjectiveTo investigate the effect of TMP on renal oxidative stress in BMF mice and its potential mechanism,and to provide the evidents for the renal protection of TMP.Methods and ResultsMethods Immune-mediated BMF model was established by 5.0 Gy X-ray combined with mixed lymphocyte infusion.Mice were treated with low(5 mg/ml),medium(10 mg/ml)and high(20 mg/ml)doses of TMP every day.Peripheral blood was collected on the 14th day,and renal,femoral and bone marrow rinsed fluid were obtained.Bone marrow nucleated cell counts,bone marrow H&E staining and bood cell counts were used to determine whether the immune-mediated BMF model was successfully established.Renal oxidative stress injury and the effect of TMP on it were detected by renal H&E staining,oxidative stress response related indicators and immunohistochemistry.Results Bone marrow H&E staining showed that the bone marrow of the control group showed a significant decrease in nuclear cells,a large number of fat vacuoles in the bone marrow,and a significant decrease in blood cell counts,indicating that the immune-mediated BMF model was successfully established.Renal H&E staining showed mild tubules edema in the control group,and no abnormalities were observed after TMP treatment.In addition,the oxidative stress index MDA in the control group was increased,while the antioxidant stress index GSH,SOD and GSH-Px were decreased.After TMP treatment,MDA was decreased,GSH and SOD were increased.The result of immunohistochemistry showed that the expression of protein HIF-1?was down-regulated and protein VEGF was observed after TMP administration.ConclusionTMP can alleviate the oxidative stress of kidney tissues in the immune-mediated BMF model,which might be related to the regulation of HIF-1?/VEGF signaling.