| With the development of nuclear science,the nuclear industry and the wide application of nuclear technology,people are increasingly exposed to radiation in production,life,medical and scientific research activities.Ionizing radiation(IR)can cause various injuries such as bone marrow injury and intestinal injury to the body.For those who may be threatened by irradiation or those who receive radiation therapy,the use of radioprotectors can significantly reduce the late radiation injuries and improve the cure rate.However,there is still no effective radioprotectors,so the research and development of radioprotectors has been a hot topic in the field of radiation medicine.Genistein is a phytoestrogenic compound with strong anti-oxidation effect,which can reduce radiation-induced free radical levels.It can also exert ionizing radiation protection by inhibiting apoptosis,regulating cell cycle and gene expression,and promoting DNA damage repair.It has a protective effect and has good drug-forming properties.The chemically synthesized Genistein Bio-300 has been registered and developed in theUSFDA and has entered clinical trial research.However,the research on the development of genistein as a protective agent for ionizing radiation is relatively lagging in China.In the early stage,our department obtained the Chinese invention patent by extracting sophoricoside from the Chinese herbal medicine fructus sophorae and then digesting sophoricoside into genistein via acidolysis,so as to obtain the pharmaceutical grade fructus sophorae genistein(FSGen)monomer(purity of more than 99%),rate of recovery is 0.54%,and the cost per gram is less than 1 yuan,making genistein possible as a drug substance application.The treatment of osteoporosis with FSGen has obtained the national drug clinical research approval,and the results of the phase I clinical trial have proved the safety of the drug substance.However,the process also has the steps of increasing the degreasing and recovering alcohol by the Sophoricoside,and the technical defects such as chloroform reflux degreasing to increase the cost and pollute the environment.However,the process has technical defects such as an increase in the steps of degreasing and recovering alcohol,and chloroform reflux degreasing increases the cost and pollutes the environment.In view of the important role of FSGen in the protection of ionizing radiation injuries and the research basis of our department in the early stage,our research intends to improve the process of extracting FSGen from fructus sophorae on the basis of the previous period,optimize the steps,save costs,comply with environmental protection principles,and initially explore the function and mechanism of FSGen on ionizing radiation injuries protection.To accomplish the above research goals,we conducted the following scientific work:1.Based on the previous extraction process,we simplified the steps,omitted the alcohol extraction and organic solvent degreasing steps,and carried out three batches of extraction experiments to collect FSGen,high purity liquid chromatography(HPLC)to detect the purity of samples,and infrared spectroscopy(IR),ultraviolet spectroscopy(UV),nuclear magnetic resonance(NMR)spectroscopy and mass spectrometry(MS)were used to analyze the structure of samples.2.Flow cytometry and western blotting were used to detect the inhibitory effect of FSGen on apoptosis of mice bone marrow mononuclear cells(BMCs)induced by ionizing radiation.H&E staining,immunohistochemical staining and immunofluorescence staining were used to observe the protetive effect of FSGen on bone marrow injury caused by ionizing radiation.3.Studied the radiation protective effects of FSGen on rat intestinal epithelial cells(IEC-6)by means of CCK-8 cell viability assay,flow cytometry,western blotting,single cell gel electrophoresis(SCGE),cellular senescence assay,cellular reactive oxygen species(ROS)assay and cellular immunofluorescence staining;studied the protection effect of FSGen on small intestinal tissue injury and inflammation of mice induced via ionizing radiation by H&E staining,immunohistochemical staining,immunofluorescence staining,enzyme-linked immunosorbent assay(ELISA).4.Studied the significance of DNA damage repair genes Rassf1 a and Ercc1 in FSGen ionizing radiation protection by means of transcriptomics sequencing,Quantitative real time PCR(qRT-PCR),RNA interference assay(RNAi),CCK-8 cell viability assay,western blotting and immunohistochemical staining.We have obtained the following conclusions through the above research work:The improved FSGen extraction process yielded a high purity bulk drug and was identified by structure Identification as 5,7,4’-trihydroxyisoflavone.The process after simplifying steps is conducive to cost saving,environmental protection,and suitable for mass production.High concentration of FSGen is toxic to BMCs.Low concentration of FSGen can inhibit the apoptosis of BMCs caused by ionizing radiation.FSGen can protect the bone marrow structure of mice after whole body radiation,reduce the apoptosis of bone marrow tissue cells,and protect hematopoietic cells.The effect of FSGen on the viability of IEC-6 cells was dose-dependent.High concentration reduced viability when low concentration increased viability.FSGen pretreatment could improve the viability of IEC-6 cells after X-ray irradiation,reduce apoptosis and the degree of DNA damage,relieve cellular senescence and reduce intracellular ROS.Pre-administration of FSGen canalso reduce the destruction of small intestine tissue caused by ionizing radiation,reduce the degree of inflammation and apoptosis,alleviate the degree of oxidative injury,and improve the survival rate of mice.The protection of radiation damage by FSGen is mainly through the DNA damage repair pathway.FSGen can exert the effect of DNA damage repair by regulating the expression of Rassf1 a and Ercc1.This research improved the FSGen extraction process,systematically studied the protective effect of FSGen on bone marrow and small intestine injury caused by ionizing radiation,discussed the molecular mechanism of protective effect of FSGen,and laid the foundation for further development of FSGen as a radioprotector.The foundation has also accumulated information for the development of radioprotectors with independent intellectual property rights in China. |
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