Experimental Study Of ?-tocotrienol In The Therapy Of Hematopoietic Acute Radiation Syndrome Through Granulocyte Colony-stimulating Factor Induction

Nuclear attacks and large nuclear accidents can cause many people to be exposed to large doses of radiation.Acute radiation sickness?also known as acute radiation syndrome,ARS?is a systemic disease in which the body is damaged by large doses?>1 Gy?of ionizing radiation in a short period of time,such as hematopoietic andgastrointestinal injuries.According to the characteristics of irradiation dose size,pathology and clinical process,ARS is divided into bone marrow type,intestinal type and brain type,and the bone marrow type is divided into light,medium,heavy and extremely heavy four degrees.Hematopoietic stem cells and progenitor cells in hematopoietic tissues have high radiosensitive,and there is obvious damage soon after being exposed to radiation,resulting in reduction of whole blood cells.Granulocyte colony-stimulating factor?G-CSF?is a kind of hematopoietic factor,which can specifically stimulate the proliferation and differentiation of granulocyte progenitor cells,and maintain its function and survival.In 1991,the US FDA approved the recombinant human G-CSF?rh-G-CSF?for marketing.It is mainly used for the prevention and treatment of granulocytopenia and bone marrow hematopoietic disorders caused by tumor radiotherapy or chemotherapy.Numerous studies have shown that administration of rh-G-CSF after irradiation can significantly promote the recovery of peripheral blood leukocytes in large doses of irradiated mice and macaques,and improve the survival rate of lethal irradiated animals.rh-G-CSF is currently the drug of choice in clinical treatment of patients with radiation accidents.In 2018,the US FDA approved new indications for rh-G-CSF to treat acute radiation sickness.rh-G-CSF is mostly expressed by E.coli or CHO cells,and has a short half-life in vivo,and has side effects such as rash,flushing,and bone pain.Therefore,the use of small molecule compounds to promote the generation of endogenous G-CSF has attracted much attention from scholars at home and abroad.Vitamin E was discovered as early as the 1920s.It includes 8 compounds,including tocopherols and tocotrienols,namely tocopherols and tocotrienols in the?,?,?,and?configuration.In addition to promoting reproduction,vitamin E also has a variety of biological effects,such as antioxidant,inhibiting lipid peroxidation,reducing cholesterol synthesis,and anti-cancer effects.In recent years,studies have found that?-and?-tocotrienols and tocopherol succinate derivatives have strong anti-radioactive activity and induce endogenous G-CSF production.The survival rate of mice,and the anti-radioactivity of these compounds was found to be dependent on endogenous G-CSF production.Compared with tocopherols,tocotrienols have a stronger effect on promoting G-CSF production.Tocotrienols are a functional component of palm oil and rice bran oil,but they are low in content,many in variety,and difficult to separate.?-tocotrienol?DT3?is rich in carmine oil,single in species,and easy to obtain.Based on this,this study intends to apply ELISA,hematopoietic stem,analysis of progenitor cell surface markers,and bone marrow hematopoietic cell competition transplantation experiments to explore the dose-time effect of DT3-induced G-CSF production in mice and compare the increase in DT3 The specificity of G-CSF is to understand the effects of DT3 on peripheral blood cell formation,bone marrow hematopoiesis,and spleen extramedullary hematopoiesis in healthy mice.The therapeutic effect of DT3 on acute radiation sick mice and its mechanism were also studied.1. DT3 dose-and time-dependently induced endogenous G-CSF production in miceThe study found that DT3 100 mg kg^-1 can significantly promote G-CSF production in mice,and the serum G-CSF concentration can be maintained at a high level within 48hours after a single administration and during continuous administration.It was found that DT3 also increased the production of CXCL1 in mice to a certain extent,and that serum TPO and EPO in mice were slightly increased during administration.DT3 can significantly increase G-CSF content in mouse lymphoid-hematopoietic tissues such as thymus,spleen,lymph nodes,and bone marrow tissue,but has no significant effect on G-CSF production in other tissues and organs.2. DT3 promotes proliferation of mouse bone marrow hematopoietic stem progenitor cells and peripheral blood leukocytes and plateletsContinuous administration of DT3 for 5 days can significantly promote the mobilization and generation of peripheral blood leukocytes,neutrophils and monocytes in healthy mice.Peripheral blood platelets increased slightly during and after administration,while lymphocytes and red blood cells increased.There is a transient decrease.Bone marrow nucleated cells did not increase significantly in the control group within 28 days after DT3 administration,but the proportion and absolute number of bone marrow hematopoietic stem progenitor cells?LSK?were significantly higher than those in the control group within 3 to 28 days after the start of administration,and the proportion of long-term hematopoietic stem cells The number and number increased significantly early after administration,while the proportion and number of short-term hematopoietic stem cells increased significantly later in administration.3. DT3 mobilizes mouse bone marrow hematopoietic stem cells to enhance extramedullary hematopoiesis in the spleenExtramedullary hematopoiesis of the spleen can reflect the activity of bone marrow hematopoiesis.The study found that the spleen weight began to increase on the 3rd day after the start of DT3 administration,and the spleen index increased,all reached a peak on the 14th day,then began to decrease,and was close to normal levels on the 28th day.With the increase of the spleen index,the proportion and number of spleen hematopoietic stem cells?LSK?and progenitor cells?LK?also increased significantly,both peaked on the 10th day,then began to decrease,and were close to normal levels on the 28th day.During and within 28 days of administration,the proportion and number of long-term hematopoietic stem cells,short-term hematopoietic stem cells,and pluripotent progenitor cells were significantly higher than those of the control group,and the peak times were located on the 10th,14th,and 21st days after administration,respectively.The number of spleen nucleated cells was also significantly higher than that of the control group at 14and 21 days after administration.Hematopoietic cell competition transplantation was used to analyze the ability of bone marrow and spleen hematopoietic stem cells to rebuild on the 10th day after administration.It was found that the ability of bone marrow hematopoietic cells to rebuild after DT3 administration was significantly lower than that of the control group,while the ability of spleen hematopoietic cells to rebuild was significantly higher.In the control group,it was shown that DT3 mobilized bone marrow hematopoietic stem cells to enter the spleen,thereby enhancing the extramedullary hematopoiesis of the spleen.4. DT3 administration after irradiation can promote the reconstruction of bone marrowhematopoietic function and the recovery of peripheral blood cells in sub-lethal irradiation miceThe optimal time course and dose effect of DT3 in the treatment of bone marrow-type acute radiation sickness were studied on a 7Gy irradiation mouse model.It was found that DT3 was administered at a dose of 100 mg kg^-1,2 hours after irradiation,and continued administration for 5 days.The effect on the recovery of white blood cells,red blood cells,and platelets from irradiated mice was most worrying.When DT3 was increased to 200 mg kg^-1,it promoted Hematopoietic recovery was not further enhanced.On the 10th day after irradiation,the number and function of bone marrow hematopoietic stem cells in the irradiated mice were measured.It was found that the number and number of nucleated cells,the percentage of hematopoietic stem cells?LSK?and hematopoietic progenitor cells?LK?in the bone marrow of the DT3 administration group were significantly higher than those in the control group.Among them,the proportion and number of long-term hematopoietic stem cells and pluripotent progenitor cells were significantly higher than those in the control group.Hematopoietic cell competition transplantation experiments showed that the bone marrow cells of the DT3 administration group had significantly higher hematopoietic reconstitution ability than the control group.In summary,DT3 can induce endogenous G-CSF production in mice in a dose-and time-dependent manner.DT3 short-term administration can increase the number of bone marrow hematopoietic stem and progenitor cells in healthy mice,promote peripheral blood leukocytes and platelets,and mobilize bone marrow hematopoietic stem cells into the spleen,thereby enhancing extramedullary hematopoiesis in the spleen.It was found that short-term administration of DT3 after irradiation can promote the reconstruction of bone marrow hematopoietic function and the recovery of peripheral blood cells in sublethal irradiated mice.DT3 has small side effects,is easy to obtain,and has potential application prospects in the treatment of acute radiation sickness.