Protection Of Vanillin Derivative VND3207 On Radiation Injury Induced By α Particles

Ionizing radiation is consisted with particles, which produced ionization directly or indirectly(or a mixture composed of particles).The injure on living organisms from ionizing radiation can cause damage directly by radiation energy in biological molecules,which lead to DNA, proteins damage, incident of apoptosis, organ failure, such as hematopoietic dysfunction, the number of peripheral white blood cells decreased, digestive or cardiovascular system function reduced ,immune system disorders, etc; ionizing radiation can also produced an indirect effect of radiation through the free radicals or reactive oxygen species (ROS)produced in water reaction, which can attack DNA, proteins, biofilm, and other important molecules and cause activation of transcription factors, this injure also lead to apoptosis, gene mutation, the incident of secondary tumors, such as leukemia, radiation-induced skin carcinoma.Radiation can be divided into ionizing radiation and non-ionizing radiation. Ionizing radiation include charged particles (αparticles,βparticles, protons, etc.) and non-charged particles (X-rays,γrays, neutrons, etc.); and non-ionizing radiation include light (visible, infrared, ultraviolet) and electromagnetic radiation (microwave, radio frequency, etc.). Based on great harm of radiation on the body, we should find effective drug against radiation injure. At present, the mechanism of radioprotective agents includes two aspects, one is to enhance DNA repair, inhibit the incident of apoptosis, such as growth factors, protease inhibitors, but these agents had toxic side effects and reduced the clinical value; the other mechanism is to resist oxidative stress, decrease ROS generation, such as sulfur compounds, phenols, etc., but these drug need high concentration and had low efficacy. Therefore, To search a non-toxic or low toxicity and efficient radioprotective agents is particularly important.Natural compound vanillin (3-methoxy-4-hydroxy -benzaldehyde), also known as vanillin or vanilla aldehyde, is a food-flavoring agent with antioxidant and anti-mutagenic activities. It has been shown that vanillin can reduced ROS, chromosome aberrations and cells micronucleus ,and it could also enhanced DNA repair induced by X-ray and ultraviolet .but its effect of radioprotection need high concentration and its activity is very low. So, our group synthesized derivative of vanillin named Syringaldehyde, (VND3207), which have a good effect of radioprotection. Preliminary tests have confirmed that VND3207 has more effectively in clearing free radicals and reducing the radiation-induced DNA damage than vanillin; Meanwhile, in experimental animals ,VND3207 could decreased genome damage and apoptosis of normal human cell induced byγ-ray. And,VND3207 increased the number of leukocytes ,and also reduced the apoptosis of spleen cells and MN formation. Based on the above theory and experiment, Apply model of radiation damage inducedα-particle to our study, We research VND3207 anti-radiation effects further and explore its mechanism in cellular, and molecular levels.(1) We established cell model ofα-particle damage from immunofluorescence assay, The results suggest that theγ-H2AX clusters induced byαparticles , compared with theγ-rays, have large damage area, high intensity fluorescence, long time persistence . Therefore, ImageJ image statistical analysis also display the appropriate conclusions; Meanwhile,immunoflourescence shows that p-Chk-2 andγ-H2AX have a common subcellular localization inα-particles radiation. It is Suggest that p-Chk-2 involved in theα-particle radiation on DNA damage repair response.(2) We detected the effect of vanillin derivatives VND3207 on normal cells by MTT assay. The results show that VND3207 presented no toxic at concertration as larger as100μmol/L on HFS cells. while compared with DMSO control group, VND3207has no significant changes in cell proliferation.(3) . The clonogenic survival assay shows that VND3207 can significantly enhance radioresistance of HFS cells toαparticles irradiation, no matter the cells are treated with VND3207 before irradiation or after irradiation. Meanwhile, VND3207 at 50 mol / L can enhance the anti-radiosensitive after different doses ofαparticles irradiation .And the radioprotection from VND3207 treated 2h before irradiation have more effective than from its immediately treated post-irradiation ,this results reflect VND3207 can scavenged free radicals and reduced DNA damage reported in previously issues, In addition, AnnexinⅤ-FACS showed VND3207 at 50μmol/L concentration can decreased the induction of apoptosis and display a good radioprotective effect. (4) DNA double-strand breaks induced by ionizing radiation is the most serious damage in the cells. Neutral single cell gel electrophoresis results showed that VND3207 can significantly decreased the rate of DNA double-strand induced by 2.0Gyαparticle, and there was a good drug dose-dependent effect . Meanwhile, also VND3207 at 50μmol / L can effectively reduced DNA damage detected at given times of repair after 2.0Gy ofαparticles irradiation. Micronucleus test showed that with protection of VND3207at 20μmol / L or above this concentration ,the micronucleus rate were effectively decreased induced by 2.0Gyαparticles ; Immunofluorescence and Western blot also displayed VND3207 effectively reduces the expression ofγ-H2AX.in 0.5Gyαparticles cells.(5)It is occurred that the body will start the DNA repair mechanism when the DNA damage. We detected the protein level of DNA repair in by Western blot.The result displayed an increased expression of p-DNA-PKcs (S2056) and non-significant expression of p-Chk-2 by VND3207 treatment at 5-100μmol / L concentration inαparticles-irradidiated cell. Meanwhile, The observation of lasre confocal mircroscope demostrated that VND3207 can effectively increased the nuclear translocation of NF-κB protein displayed resistance to apoptosis in theα-particles irradiated cells.In conclusion, VND3207 has no significant toxicity to normal cells, and effectively enhance radioresistance and incident of apoptosis in irradiated cells, At the same time, VND3207 increase ability to protect the radiation-induced DNA damage, maintenance of cell genomic stability and evaluated the expression of repair proteins. Therefore, VND3207 have developed as the value of anti-radiation drugs, and provide a theoretical basis in clinical treatment of radiation sickness.