Molecular Mechanisms Of Intervention Of Flavonoids Of Rosa Roxburghii Tratt On Radiation Damage Via Caspase-3/AIF

Background:Radiotherapy is one of the three methods for the clinical treatment of tumor,whose principle is that ray has an effect on the tumor tissue to induce apoptosis of tumor cells and inhibit the growth of tumor tissue to achieve therapeutic purposes;however,radiotherapy could induce tumor cell apoptosis.In addition,it also caused damage to human normal cells,affected the efficacy of radiotherapy,and even leaded to treatment interruption.FRT is a kind of flavonoid compounds which was extracted from the fruit of Rosa roxburghii Tratt.FRT has the better ability on scavening free radicals in vitro.Preliminary experiments showed that FRT could improve the survival rate and survival time in irradiation mice.The experiment used ⁶⁰Co gamma rays to make the mice and cell radiation damage model to study the radiation protection mechanisms of FRT.Objective:FRT was extracted and purified and also its active components were determined.The FRT radioprotective effect was evaluated through in vivo and in vitro experiments,and Caspase3 and AIF were used as the target molecules to explore the molecular mechanisms of radiation protection.Methods:The total flavonoids were extracted and separated from fruit of Rosa roxburghii Tratt by using 90%ethonal.The refined FRT was purified by using macroporous resin.Qualitative and quantitative determination of the main ingredients of FRT underwent via HPLC and LC-MS.Mice were randomly divided into four groups:（1）Normal,（2）6 Gy,（3）FRT 30mg/Kg,（4）FRT 60 mg/Kg.The mice was fed with FRT for 4 days before irradiation,the preparing mice radiation injuried model was conducted under ⁶⁰Co gamma irradiation.The effect of FRT on 30 days survival rate of mice with radiation injury was observed.The pathological changes of FRT in liver,spleen,thymus and testis,the formation rate of spleen nodules and the influence of sperm aberration rate were observed at different time after irradiation.MTT assay was used to detect the toxicity and protective effect of FRT on mouse thymocytes.The comet tail experimental was used to observed the peotective effect of FRT in irradiated cells DNA.The adiation protective effect of cell apoptosis was used by flow cytometry instrument and DNA ladder experiment.Hoechst was used in mice to detect the thymus apoptosis.Detecting the ROS content in irradiated cell was used by fluorescent probe.Caspase3 and AIF were used as target molecules to invest the FRT protective effect in signal pathway of apoptosis.Extracting the protein in different points could detect the expression of caspase8,caspase9,Caspase3,PARP-1 and AIF.Caspase3and PARP-1 was used as inhibitors cells could detect the effect of inhibitors in apoptosis,the expression of Caspase3,PARP-1 and AIF.RT-PCR was used to detect Caspase3,PARP-1 and AIF genes expression changes in cellsResult:（1）The separation,isolation and identification of FRTThe FRT which was extracted in 90%ethanol and purified through the macroporous resin has a high concentration and is in accordance with the standards of medicine;FRT contains catechin and quercetin.（2）The protective effect of FRT in irradiated miceFRT could improve the survival rate and survival time of mice and the SOD content after ⁶⁰Co 6 Gy irradiation,and also could decrease the MDA content in liver and blood cells and the lipid peroxidation after irradiation.FRT could reduce the damage of the spleen and increase the formation of endogenous after radiation.Giving mice FRT before radiation could reduce the astrophy of thymus,inhibit the apoptosis of thymus tissue and the thymus fibrosis.FRT could reduce the pathological changes in the liver and testis and the rate of sperm deformity.（3）The protective effect of FRT on irradiated thymocytesGiving the extracted FRT could improve the activity of thymus cells in mice with radiation damage and reduce the apoptosis of thymus cells.（4）The molecular mechanisms of FRT in radioprotectionFRT could remove excess intracellular ROS,reduce DNA damage and intracellular Ca²⁺to play a role in radioprotective agent.The PARP-1 cleavage and the activation of Caspase3-PARP1 signaling pathway were reduced through inhibiting of caspase8 and Caspase3 and caspase9 activation;Inhibiting nuclear translocation of AIF protein and reducing DNA damage could reduce cell apoptosis.Conclusions:FRT could significantly improve the survival rate and survival time in irradiated mice,and had a better protective effect on mice tissues and organs.FRT could effectively inhibit the radiation damage and apoptosis in murine thymocytes,increase the vitality of cells.FRT could protect the structure of DNA by reducing the intracellular excess of ROS in irradiated cells,and reduce apoptosis by inhibiting activation of Caspase3 and AIF signaling pathways.The expression of AIF m RNA could be inhibited by FRT,the expression of Caspase3 m RNA and PARP1 m RNA could be inhibited by FRT at the same time.FRT had the ability in anti-oxidation,anti-apoptosis by regulating the expression of the protein and m RNAin irradiated cells,which is expected to be widely applied in radiation protection agent.