| In clinic, primary or secondary thrombocytopenia, as well as hematopoietic injury of bone marrow caused by ionizing radiation, could result in a sharp decrease of platelet counts. Platelets play an essential role in hemostasis, wound healing, vascular repair, thrombosis and inflammatory reaction in physiological and pathological processes. Severe reduction of platelet counts will result in bleeding, infection, poor prognosis of patients, and even death. Currently, Therapeutics for thrombocytopenia are rare, and most of them have drawbacks, such as a slow effect, great side effects and high prices. In addition, oral administration is much safer compared to current medicines for increasing platelet counts through parenteral administration, moreover, diet-like therapies are more acceptable by patients. Hence, it may become a potential way to prevent radiation damage and a new therapeutic for thrombocytopenia that searching and developing special elements from flood which can promote thrombopoiesis and protect megakaryocytes against radiation.Megakaryocytes generate from hematopoietic stem cells in bone marrow, and subsequently release plateles followed by the process of proliferation, differentiation, polyploidy formation. Thus, it can directly influence platelet counts in vivo via the facilitation of megakaryocyte proliferation, differentiation and maturation, as well as the protection against the injury caused by different factors to megakaryocytes. Thrombopoietin(TPO) is the most important regulatory factor in thrombopoiesis, which can promote the proliferation, differentiation and mature of megakaryocytes by activating the relevant signal pathways. Some elements in food that have a similar function as TPO may regulate megakaryocytopoiesis and thrombopoiesis.Tannic acid(TA) is a polyphenol existing in fruits such as pomegranates, walnuts, grape seeds, peanuts, and emblica widely. It is recognized that the peanut skin and pomegranates can enrich the blood and increase platelets, however, it remains unknown whether TA could directly promote thrombopoiesis. Early studies showed that TA could activate ERK and p38 MAPK pathways and then affect differentiation and mature of some cells. Thus, it needs to explore whether it can regulate differentiation and mature of megakaryocytes. Moreover, TA has an antioxidant character based on its chemical structure, therefore, TA can exert its protective role when organs and tissues are injured by chemicals, as well as of animals are aging, again, TA can reduce DNA injuries caused by chemical poisons and radiation through suppressing oxidative stress. Above all, it is predicted that TA may potentially prevent the damage of megakaryocytes in bone marrow induced by radiation.In this study, we mainly discussed the effect TA on thrombopoiesis with or without radiation injury. The study was divided into two parts. The first part was discussed the role of TA on megakaryocyte proliferation, differentiation, mature as well as platelet production. We investigated whether TA could promote proliferation of megakaryocytes with M07 e cell line since it has the capacity of proliferation. Subsequently, HEL, Meg-01 cell lines and primary megakaryocytes derived from human CD34+ cells, featured with mature and capacity of differentiation of MKs, were used to analyze the ability of TA on the facilitation of megakaryocyte differentiation and polyploidy formation, and then to explored the relevant mechanisms. At last, TA was administrated into mice so as to further determine whether TA can promote thrombopoiesis in vivo. The second part was mainly focused on the protective effect of TA on megakaryocytes injuried by ionizing radiation and its relevant mechanisms: we first observed a protective effect of TA at different concentrations on M07 e cells irradiated by ionizing radiation at a dose of 10.0 Gy 60Coγ ray in vitro; and then detected the ROS level, mitochondrial membrane potential and apoptosis rate, which aimed to elucidate the protective mechanism of TA on megakaryocytes injuried by ionizing radiation. Thereafter, we studied the survival rate, peripheral platelet level, megakaryocyte number in bone marrow, and the colony changes when the irradiated mice were administrated with TA through different ways within 21 days, in order to validate whether TA could effectively prevent injury of megakaryocytes in bone marrow and thrombocytopenia induced by ionizing radiation.Major results and conclusion:1. CCK-8 assay showed that TA at different concentrations could not promote proliferation of M07 e cells, indicating that TA did not have the capacity of proliferation facilitation of megakaryocytic progenitors.2. Flow cytometry analysis suggested that TA facilitate megakaryocyte differentiation and mature since HEL, Meg-01 cell lines and primary MKs derived from human cord blood CD34 cells had a higher expression of CD42 b, a mature marker on the surface of MKs, moreover, polyploid cells also significantly increased after TA treatment.3. Western blot indicated that TA could activate ERK1/2 signal pathway, since ERK1/2 signal pathway inhibitor U0126 could suppress the facilitation of TA on megakaryocyte differentiation, suggesting that TA could stimulate the differentiation and mature of megakaryocytes by activating ERK1/2 signal pathway.4. The gavage was performed on BALB/c mice by treating them with 300mg/kg/d of TA. As a result, peripheral platelet level was elevated significantly, further proving that TA could promote thrombopoiesis.5. MTT method suggested that TA could dose-dependently increase the survival rate of M07 e cells when irradiated at a dose of 10.0 Gy Co60 γ ray; The results of flow cytometry analysis showed that TA at a certain concentration could considerably suppress the apoptosis of M07 e cells induced by ionizing radiation, indicating that TA was able to protect megakaryocytes against radiation.6. Fluorescence analysis showed that TA was able to noticeably reduce the overall ROS level of megakaryocytes when injured by radiation. It indicated that protection of TA against radiation might relate to its anti-oxidant capability.7. The fluorescence intensity from flow cytometry analysis showed that the fluorescence intensity of MKs exposed to radiation is lower than normal controls, while the fluorescence intensity of irradiated MKs that were treated with TA were significantly higher than those exposed to radiation only. It indicated that TA might suppress the apoptosis of megakaryocytes’ induced by radiation via stabilizing the mitochondrial membrane potential.8. We observed the effect of TA on the recovery of platelet level when mice were irradiated with Co60 γ ray at a dose of 5.0 Gy. As a result, the peripheral platelet level of mice after TA treatment was higher than those exposed to radiation only at day 9-21 after radiation, and on day 18, the platelet counts of mice treated with TA recovered to the normal level as those on day 0, while the platelet level in the group of radiation only was still in a slow recovery process; HE staining of bone marrow showed that the number of nucleated cells including megakaryocytes increased excessively in bone marrow in TA–treated group. Colony formation assay in vitro found that colony number was higher in mice that were given with TA before radiation than those exposed to radiation only, indicating that TA was able to effectively protect megakaryocytes against ionizing radiation, as well as promote a quick recovery of platelet level.9. TA was given by means of gavage at different time when mice irradiated, leading to a considerably increased survival rates of injured mice within 21 days after exposure to a severe radiation(7.0Gy Co60 γ ray). It further proved that TA could protect against radiation.In short, it was the first time to find that TA could play an important role in promoting megakaryocyte differentiation, mature and platelet release. Meantime, it was also found that TA was able to reduce the injury of megakaryocytes in bone marrow caused by ionizing radiation via an anti-oxidant way. It indicated that TA had a prominent therapeutic effect on thrombopenia, which deserved to be further investigated. |
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