Sympathetic Stimulation Regulates Thrombopoiesis And Exerts Its Role On Platelet Recovery In Irradiation-injuried Mice

In recent years, a variety of injuries caused by ionizing radiation are gradually increasing along with the extensive use of nuclear power and nuclear technology in the field of military, medical treatment, energy, food processing, breeding, etc. As known, bone marrow serves as one of the main target organs after irradiation exposure since it is sensitive to ionizing radiation. Concequently, when the body is exposed to a certain dose of ionizing radiation, it will lead to hematopoietic dysfunction in bone marrow. Noticeably, the low level of platelets resulted from hematopoietic dysfunction is the main cause of bleeding, infection and even death in the body. Therefore, it is the key step for the treatment after radiation injury to investigate the regulation mechanism of thrombopoiesis, and to find effective countermeasures that can promote platelet recovery after radiation injury.Previous studies have shown that γ-ray can act on biological macromolecules directly, and then results in the breakage of DNA double strand, protease inactivation, as well as the modifications of cell membrane structure and permeability, which are able to affect cell normal function when the body is exposed to irradiation. In addition, ionizing radiation can also act on water molecules, and the decomposition products will further work on biological macromolecules, which will cause cell injury. Moreover, Cheng et al. had found that mekaryocytes could be phagocytized by neutrophilic cells after irradiation, which was named as "megakaryocytophagia" in our earlier researches on radiation injury. Therefore, it becomes a fact that the number of megakaryocytes in bone marrow and the level of peripheral platelets decrease significantly after irradiation. Aiming at this situation, studies are been focusing on the agent development that can elevate platelet level. Fortunately, an increasing number of studies found that thrombopoietin(TPO), the primary regulator of thrombopoiesis, as well as other cytokines could promote megakaryocytopoiesis by acting on multiple stages of thrombopoiesis, such as hematopoietic stem cell(HSC) proliferation and differentiation, megakaryocyte(MK) maturation and platelet release, etc. As a result, They can promote platelet recovery post radiation injury effectively. Yet, the administration of TPO in vivo is unable to shorten the period of platelet counts in low level, in addition, it can induce the production of neutralizing antibodies, which lead to certain restrictions in clinic application. Additionally, IL-11 is the only drug for low platelet level approved by FDA. Therefore, it is necessary to find new ways to promote platelet recovery after radiation injury.In the studies of thrombocytopenia caused by ionizing radiation, it is found that various internal and external factors play manifold roles on the body, which will lead to multiple synthetic adaptive responses through the action of internal environmental homeostatic axes including sympathetic-adrenal medulla system(SAS). Studies have showed that catecholamines including norepinephrine(NE) and epinephrine(EPI) released from SAS axis exert diverse roles through binding to their receptors in different tissues and cells. Speceifcally, it is indicated that the bone marrow is rich in sympathetic innervation, and NE and EPI released by the SAS axis play an important role in hematopoiesis after sympathetic stimulation.It has been demonstrated that NE and EPI can not only promote the proliferation of CD34+ cells, but also facilitate the migration of hematopoietic stem/progenitor cells. In addition, they also exert different roles on the progeny from HSCs, for instance, NE and EPI released from SAS can bind to different adrenergic receptor, which will be involved in the regulation of lymphopoiesis, erythropoiesis, as well as platelet activation. However, it is still unknown that whether sympathetic stimulation is involved in the regulation of thrombopoesis. Hence, it will not only help us to further understand the role of sympathetic function, as well as to reveal the regulation mechanisms of thrombopoiesis, but also provide new ideas for the treatment in platelet recovery after radiation injury by deeply investigate the roles of sympathetic stimulation on platelet production and activation.For a long time, focusing on the cause of low level of platelets after radiation injury, we have worked on the mechanism of thrombopoiesis and the drug development that can promote platelet generation. During the study, we found that peripheral platelet counts were at a high level in the stress state. Therefore, we first observed the changes of peripheral blood platelet levels in C57BL/6 mice after continuous noise and excessive exercise stress stimulation, and found that continuous stress stimulation could elevate platelet level. On this basis, we further confirmed that sympathetic stimulation is an important factor to promote platelet production in vivo by analyzing the changes of circulating platelets through adrenergic receptor blockade, β-hydroxy transfer enzyme defect(Dbh-/-), as well as TPO receptor deficient(C57BL/6J-Mplhlb219/J); Second, in order to analyze the mechanism of sympathetic stimulation on thrombopoiesis facilitation and platelet activation, we detected the effects of NE and EPI on different stages of thrombopoiesis by using a variety of in vitro methods, such as the proliferation and differentiation of CD34+ cells, the differentiation, proliferation, adhesion and migration of MKs, proplatelet formation, as well as platelet release and activation, etc. Finally, we duplicated the model of injured mice caused by radiation, and then investigated the effect of sympathetic stimulation on platelet recovery after radiation injury.The main results and conclusions were as follows:1、We duplicated the model of noise stress and extensive exercise stress, and found that platelet levels gradually increased when C57BL/6 mice subjected to continuous stress treatment, suggesting continuous stress exposure can elevate peripheral platelet level; we further confirmed that sympathetic stimulation exerted an important role in thrombopoiesis facilitation by analyzing the changes of circulating platelet counts in C57BL/6 mice that received a splenectomy, dopamine β-hydroxylase deficient(Dbh-/-) mice, as well as C57BL/6J-Mplhlb219/J mice after continuous stress exposure.2、In the early stages of megakaryocytopoiesis, flow cytometric analysis showed that NE and EPI could promote the proliferation of CD34+ cells, but unable to facilitate the commitment of CD34+ cells to megakaryocytic lineage, or the differentiation and proliferation of megakaryocytes.3、The results from fluorescence detection and transwell assays suggested that NE and EPI were able to promote megakaryocyte adhesion and migration in a dose-dependent manner; however, when α adrenoceptor was blocked, the adhesive and migrated MKs induced by NE and EPI significantly reduced, indicating that NE and EPI promote the adhesion and migration of MKs through alpha adrenoceptors.4、Western Blot analysis showed that NE and EPI treatment were able to enhance the phosphorylation of ERK1/2, however, α and α2 adrenoceptor blockade significantly inhibited the phosphorylation of ERK1/2, suggesting that NE and EPI activated ERK1/2 signal pathway via α2 adrenoceptor; furthermore, while α2 adrenoceptor and ERK1/2 signaling pathway blocked, the number of adhesive and migrated MKs induced by NE and EPI decreased significantly, further indicating that NE and EPI can promote the adhesion and migration of MKs.5、The results from confocal laser scanning microscope showed that NE and EPI could promote proplatelet formation from mature MKs in a dose-dependent manner, suggesting that NE and EPI play important roles in the late stages of megakaryocytopoiesis.6、NE and EPI were able to up-regulated the phosphorylation of RhoA in mature MKs; however, when RhoA GTPase activity inhibited, the pseudopods from proplatelet induced by NE and EPI extended longer to form the typical "beaded" structure, suggesting that the facilitation of proplatelet formation triggered by NE and EPI was closely related to RhoA GTPase activity.7 、 The blockade of α2 adrenergic receptor and ERK1/2 pathway inhibited the expression of p-RhoA that is elevated by NE and EPI treatment, and resulted in a significant reduction of the proplatelet number, which indicate that NE and EPI binding to α2 adrenoceptor, and then activating ERK1/2-RhoA pathway may be the mechanisms for the proplatelet formation facilitation trigger by NE and EPI treatment.8、Flow cytometric analysis showed that NE and EPI could promote platelet release from mature MKs, which further demonstrates that NE and EPI exert important roles in terminal stages of thrombopoiesis to promote platelet production.9、Western Blot and flow cytometry results indicated that NE and EPI binding to α2 adrenoceptor, and then activating ERK1/2 signal pathway, may be the mechanism of platelet activation induced by NE and EPI.10、In vivo studies showed that intraperitoneal administration of NE and EPI promoted the migration of MKs to the vascular niche, and then elevated peripheral platelet level in C57BL/6 mice, suggesting that NE and EPI can promote platelet production in vivo.11、We successfully duplicated the mouse model for severe thrombocytopenia caused by radiation injury(mice were whole-body irradiated with γ-ray at a single dose of 6.0 Gy), the changes of peripheral blood platelet number after NE and EPI treatment showed that the administration of NE and EPI in vivo significantly increased peripheral platelet counts post irradiation exposure. The results indicated that the sympathetic stimulation could promote platelet recovery in irradiated mice.12、We duplicated the mouse model for severe acute radiation injury following bone marrow transplantation(mice were whole-body irradiated withγ-ray at a single dose of 10.0 Gy receiving following bone marrow transplantation). The grafted mice were injected with NE and EPI while circulating platelet counts reached the lowest level, platelet recovery were significantly accelerated, and the bleeding tendency was significantly reduced, further suggesting that sympathetic stimulation can promote the platelet recovery in mice with radiation injury.In conclusion, we first discovered that sympathetic stimulation exerted an important role in thrombopoiesis, and then preliminary investigated its effect on platelet recovery post irradiation exposure. This study further enriches the understanding of sympathetic function, fills the gap in the field of sympathetic stimulation in hematopoiesis regulation, and provides new clues for the treatment after radiation injury as well.