Protective Effect And Mechanism Of Gallic Acid On ⁶⁰Co-γ Ray Radiation Damage

Accompanied by the advance of the human civilization and the science and technology,ionizing radiation has been widely used in many fields such as medicine and health and national security.The research of radiation protectants plays an important role in radioresearch^[1].The US food and drug administmicesion（FAD）has included radiation protection agents such as WR-2721,the Ex-RAD and GT3 as the national strategic reserve^[2].However,existing radiation protectants have some disadvantages such as strong toxic side effects and expensive prices,which limit their popularization in clinical practice.Therefore,we still need to make efforts to find safe,economical and efficient radiation protectants.The existing radiation protectants mainly including chemotherapeutics,cytokines,stem cells,plants and traditional Chinese medicine（TCM）^[3].In recent years,the TCM has become the focus of research of radiation protectan,because of it’s good curative effect,little side effects,good selectivity and wide range of sources and so on.Gallic acid（GA）is one of the main effective components of Chinese gall,which play good roles in antibacterial,antiviral and antioxidant.It has a variety of bioactivitys and pharmacological functions.Litemicesures showed that,gallic acid can selectively inhibit tumor cells and protect normal cells from dying^[4].It’s determined that TCM could be researched as a promising radioation protectant.We chose BALB/c and ICR mice and Human intestinal epithelial cell（HIEC）as experiment objects to study the protective effection of GA on mice and cells after ⁶⁰Co-?ray exposure in in vivo and in vitro level.Aim:To investigate the protective effect and primary mechanism of Gallic acid on ⁶⁰Co-?ray induced mice and HIEC damage.Methods:1 Animal experimentsMale ICR mice and BALB/c mice were exposed to ⁶⁰C_O-γray after GA treatment.By observing the changes of body weight,30d survival rate,number of splenic nodules,spleen index,peripheral blood picture and so on,the protective effect of GA on the irradiated mice was understood.The protective effect of GA on ⁶⁰Co-?ray induced oxidative damage in mice was studied by detecting the levels of SOD,GSH and MDA in the liver and kidney tissues of the irradiated mice.The expression of Bax,Bcl-2 and P53protein of spleen were detected by Western-Blot after ⁶⁰C_O-γray exposure and GA treatment.2 Cell experimentsHuman intestinal epithelial cell（HIEC）of the exponential phase were inoculated in petri dishes and were randomly divided into sham exposure group and exposure group.2.1 Cytotoxicity test:12h,24h and 48h after different concentration of GA treatment,HIEC proliferation rate was detected by cell counting Kit（CCK-8）to obtain the safe concentration and safe time of GA.2.2 HIEC cells were cocultured with 20?mol/l H₂O₂ after different concentration of GA treatment.HIEC proliferation rate was detected by cell counting Kit（CCK-8）.2.3 Four groups of cells were simultaneously exposed to 4Gy ⁶⁰Co-?ray after different concentration of GA treatment（0,1,10,20μmol/L）.HIEC proliferation rate was detected by cell counting Kit（CCK-8）.The Cell clone forming experiments were done by plate cloning.The levels of MDA,SOD and GSH in HIEC celles were measured according to kit instruction.Results:1 in vivo experiments1.1 GA improved the survial of 7.5Gy ⁶⁰Co-?ray irradiated miceCompared with 7.5Gy ⁶⁰Co-γray exposure group,GA can increase the body weight,improve the 30d survival rate and prolong the survival time of mice significantly（P﹤0.05）.1.2 GA promoted 8Gy ⁶⁰Co-γray irradiated mice hematopoietic stem cell damage repairCompared with 8Gy ⁶⁰Co-γray exposure group,the body weight,spleen weight,spleen coefficient and number of splenic nodules of GA treatment group increased significangly（P﹤0.05）.1.3 Gallic acid repaired 4Gy ⁶⁰Co-γray irradiated hematopoietic system injury in miceCompared with 4Gy ⁶⁰Co-γray irradiation exposure group,the number of RBC,WBC,LYM,and PLT of GA treatment group increased significantly（P﹤0.05）.2 in vitro experiments2.1 Cytotoxicity of GA to HIEC cellsGA can promote the growth of HIEC cells at 12h after GA treatment（P<0.05）,but GA has a toxic effect on the growth of HIEC cells at 24h and 48h after GA treatment.And the suitable concentrations of GA are 1,10 and 20μmol/L.2.2 GA increased the clone formation rate of ⁶⁰Co-γray irradiated HIEC cellsCompared with 4Gy ⁶⁰Co-γray irradiation exposure group,GA（1,10μmol/L）increased the clone formation rates of HIEC cell significantly（P<0.05）.2.3 GA increased the proliferation rate of ⁶⁰Co-γray irradiated HIEC cellsCompared with 4Gy ⁶⁰Co-γray irradiation exposure group,GA（1,10,20μmol/L）can increase the proliferation rate of HIEC cell.And 1,20μmol/L GA increased the proliferation rate of HIEC cell significantly（P<0.05）.3 Mechanism research3.1 GA inhibited oxidative stress reaction of liver and kidney in ⁶⁰Co-γray irradiated mice.Compared with 4Gy ⁶⁰Co-γray irradiation exposure group,SOD activity of liver and kidney in mice of GA group increased significantly（P﹤0.05）,MDA contents of liver and kidney in mice of GA group decreased significantly（P﹤0.05）.3.2 GA inhibited the oxidative stress level of ⁶⁰Co-γray irradiated HIEC cellsCompared with 4Gy ⁶⁰Co-γray irradiation exposure group,SOD activity of GA（1,10,20μmol/L）treatment group increased significantly（P<0.05）.GSH acitivity of20μmol/L GA treatment group increased significantly（P<0.05）.3.3 Gallic acid had protective effect on HIEC cell damage caused by H2O2 attackCompared with 4Gy ⁶⁰Co-γray irradiation exposure group,proliferation rate of HIEC cells in GA group（0.1,1,10,20μmol/L）increased significantly.3.4 GA inhibited the expression of apoptotic protein in spleen of ⁶⁰Co-γray irradiated miceCompared with 4Gy ⁶⁰Co-γray irradiation exposure group,GA can reduce the ratio of Bax/Bcl-2（P﹤0.05）,but the expression level of p53 has no change（P>0.05）.Conclusions:These results suggested that GA could has protective effect on ⁶⁰Co-γray irradiated mice and HIEC cells.The mechanism may be related to the protection of hemopoietic system,antioxidant,scavenging free radicals and inhibiting cell apoptosis.