The Effect Of Serotonin On Megakaryopoiesis/proplatelets Formation And On The Regulation Of Thrombopoietin Production From BM MSCs

Background:Megakaryopoiesis can be divided into two parts:first,the proliferation and differentiation from hemopoietic stem cells to mature megakaryocytes;second,the process from mature megakaryocytes to proplatelets and the terminal platelets release.Thrombopoietin(TPO)is a major cytokine for megakaryopoiesis and platelet production.In addition,bone marrow stromal cells(MSCs)also involve in megakaryopoiesis and thrombopoiesis.Therefore,the regulation of megakaryopoiesis can be separated into two components:the direct effect on megakaryocytes and the indirect effect on the production of TPO from MSCs.Our previous work confirmed that serotonin(5-HT)promoted the proliferation of megakaryocytes and hemopoietic stem cells and reduced the apoptosis of them.However,the mechanisms remain indefinitive.Although it is clear that various platelet contents could regulate the production of TPO in MSCs,the effect of 5-HT,98%of which is in dense granule is poorly understood.Method:(1)Identification of 5-HT2 receptors expressed on megakaryocytes and MSCs by Q-PCR,Flow Cytometry,Western Blot or Immunofluorescence.(2)To detect the effect and the signal pathway of 5-HT on the proliferation and the apoptosis of megakaryocytes and MSCs by MTT/CCK-8 and Flow Cytometry,respectively.(3)To classify the megakaryocytes in different stages by their morphology after treatment with 5-HT or 5-HT2 receptor inhibitor or TPO.(4)To explorer the relationship of the level of 5-HT and TPO in a traumatic stress mice model.(5)TPO mRNA and protein in MSCs was measured by Q-PCR and WB.(6)Western Blot were used to detect the sigmal pathway involving in the effect of 5-HT.Results:(1)5-HT2A,2B,2C receptors were detected in the surface of megakaryocytes and MSCs.(2)5-HT promoted the growth of megakaryocytes and MSCs in a dose-dependent manner.200 nM 5-HT have the maximal effect.(3)After treatment of 200 nM 5-HT,the phosphorylation of Akt was upregulated,the expression of AnnexinV/PI and active Caspase-3 were reduced,the proportion of JC-1 monomer was increased.The results of 5-HT were similar with those of TPO,but the former could be inhibited by Ketanserin,the selective inhibitor of 5-HT2 receptor.(4)Treated with 5-HT,the proportion of mature proplatelets-megakaryocytes and platelet-producing megakaryocytes was increased significantly,and the apoptosis megakaryocytes was reduced.(5)In a traumatic stress mice model,both of 5-HT and TPO were increased,but the increasing of TPO is posterior to 5-HT.After added LX1606,the synthesis inhibitor of 5-HT,the concentration of 5-HT was reduced markedly,as well as TPO.(6)The expression of TPO mRNA and the production of TPO protein were increased as compared with the control.(7)5-HT stimulated the AKT,ERK and STAT3 signal pathway,the former two could by blocked by Ketanserin,but the last one could’t.Conclusion:5-HT promotes megakaryopoiesis from two aspects:One is the direct effect on megakaryocytes.It can promote the proliferation and reduce the apoptosis of megakaryocytes and accelerate the maturation of megakaryocytes.Another is the indirect effect on megakaryopoiesis by promoting the production of TPO from bone marrow MSCs,which is a paracrine secretion to influence the megakaryopoiesis.5-HT can be a new cytokine to make the expansion of megakaryocytes and hemopoietic stem cells and to promote the production of platelets in vitro.Moreover,it may be a piece of evidence that the nervous system involves in the regulation of the hematopoietic system by neurotransmitter.