Synthesis And Radiation Protection Of N-oxalyl-D-Phenylalanine(NOFD)

Objective:HIF(Hypoxia inducible factor)is a transcription factor activated under hypoxia conditions,which is composed of a subunit and(3 subunit.The activity of HIF-1 a is regulated by oxygen concentration.Hypoxia inducible factor inhibitor(factor inhibiting hypoxia-inducible factor,FIH,also known as asparaginamide hydroxylase),is the main factor regulating HIF-1? transcription activity in vivo.Under hypoxia,FIH activity decreased,HIF-1 transcription activity is activated,and a huge hypoxic stress regulatory network is initiated.NOFD is a known as an FIH inhibitor.In order to study the role of NOFD inhibiting FIH in promoting HIF-1? transcription in radiation injury protection,NOFD was synthesized and then its radiation protection was systematically evaluated by using radiation injury animal model;Further exploring the mechanism of NOFD radiation protection was studied.Methods:The research was divided into three parts.1.Synthesis and structure identification of N-oxalyl-D-phenylalanine(NOFD)compound.2.Pharmacodynamic evaluation of radiation protection of NOFD.The overall protective effect of NOFD was evaluated by 30-day survival rate test.The radiation protective effect of NOFD on hematopoietic system injury was analyzed by bone marrow cell clone formation test,peripheral blood and bone marrow cell flow cytometry analysis and bone marrow cell?-H2AX and MitSox flow cytometry analysis.The radiation protective effect of NOFD on radiation-induced intestinal injury was evaluated by H&E staining,immunohistochemistry and immunofluorescence assay.3.Study on the mechanism of radiation protection of NOFD.The effects of NOFD on the expression of intracellular HIF protein and downstream target genes were evaluated by Western-blot and RT-PCR.The radiation protective effect of NOFD in vitro was evaluated by immunofluorescence assay,comet assay and apoptosis flow cytometry analysis.Results:1.NOFD was synthesized and the molecular structure of NOFD was determined by NMR and MS analysis.2.The pharmacological results of hematopoietic system showed that NOFD could improve the survival rate of irradiated mice,alleviate the phenomenon of bone marrow suppression after radiation,maintain the balance of peripheral blood system,increase the number of bone marrow cells in irradiated mice,and enhance the ability of self-renewal and proliferation of bone marrow cells in irradiated mice,and to reduce the degree of DNA damage and ROS aggregation of bone marrow cells after radiation.The results of intestinal injury showed that NOFD could effectively maintain the integrity of small intestinal tissue,increase the proliferation of small intestinal recess cells and reduce the proportion of apoptosis of small intestinal cells after radiation.3.The vitro experiments showed that NOFD could increase the expression of HIF protein in cells under normoxic conditions,stimulate the expression of downstream genes of HIF protein,reduce the proportion of DNA damage and apoptosis in cells after radiation,and decrease the aggregation of ROS.Conclusion:NOFD has a certain radiation protection effect.NOFD can increase the survival rate of mice after radiation,show protective effect against on radiation-induced damage to the hematopoietic system and intestines,and protect cells from ionizing radiation damage.