Research On The Anti-radiation Activity Of Resveratrol, Salidroside And Nitroxide Free Radical Structure Derivatives

Objective:To explore the protective effect of resveratrol?salidroside and nitroxide derivatives on radiation injuryMethods: The effect of resveratrol?salidroside and nitroxide derivatives on the proliferation of spleen cells of BALB/c mice by CCK-8 method.Structural derivatives with lower cytotoxicity were selected for subsequent experiments;DCFH-DA fluorescent probe was used to detect intracellular ROS changes;CCK-8 method and DCFH-DA fluorescent probe method were used to detect Jurkat T cells,the experimental conditions were explored,the screening system was established and the compounds were screened.PI staining was used to detect cell cycle;Annexin-V and PI double staining was used to detect apoptosis,structural derivatives that could alleviate radiation-induced apoptosis were screened;In order to explore the intrinsic mechanism of structural derivatives of alleviate radiation-induced apoptosis,Western blot was used to detect the expression of apoptosis related proteins and mitochondrial cytochrome c;JC-1 fluorescent probe incorporation method was used to detect the effect of the lead compound on radiation-induced mitochondrial membrane potential changes in Jurkat T cells;cytochrome c assay was used to evaluate the effect of the lead compound on radiation-induced mitochondria production and release of cytochrome c in Jurakt T cells.Results:(1)The radiosensitivity of spleen cells of BALB/c mice is relatively high.Jurkat T cells with moderate radiosensitivity are more suitable for this research,and find out the most suitable experimental conditions to establish a stable and effective screening system.(2)The CCK-8 experiment results show that compared with salidroside,the cytotoxicity of salidroside and nitroxide derivatives(CSP-NPS)is significantly higher than that of the salidroside.The cytotoxicity of CSP-NPS is not consistent with the original intention of transforming the structure.For resveratrol and nitroxide derivatives,CSP-NPR-1(monosubstituted),CSP-NPR-2(disubstituted),CSP-NPR-3(trisubstituted),compared to the resveratrol prototype,the cytotoxicity of the derivatives were significantly reduced,and the structural modification effectively improved the cytotoxicity of the monomer.(3)As can be seen from the ROS test results,intracellular ROS levels in irradiated Jurkat T cells with X-rays at a radiation dose of 6 Gy were significantly higher than those of untreated cells.Pretreatment of Jurkat cells with 100,10,and 1 ?M structural derivatives,the results at 24 h after irradiate showed that the prototypes of resveratrol,and nitroxide radicals could be only reduced intracellularly ROS at medium and high concentrations.But the prototype of salidroside can not reduce intracellular ROS levels,and its structure derivatives with nitroxide have no obvious effect on intracellular ROS level.The resveratrol and nitroxide derivatives can significantly reduce the level of intracellular ROS induced by radiation.Compared with the Amifostine(100 ?M),the effect of at a concentration of 10 ?M CSP-NPR-1,CSPNPR-1,CSP-NPR-2 and CSP-NPR-3 is more prominent.(4)The results of cell cycle detection showed that the Jurkat T cells were irradiated by radiation dose of 6 Gy had G2 phase block in the cell cycle significantly.Two series of structural derivatives pretreated cells could not reverse the cell cycle arrest induced by radiation.(5)Apoptosis test results showed that Jurkat T cells were irradiated with 6 Gy X-rays were pretreated with structural derivatives at concentration of 10 ?M,resveratrol and nitroxide radical derivatives can significantly reduce radiation-induced apoptosis(The control group VS experimental group(CSP-NPR-3):20.7% VS 7.28%)was more effective than the resveratrol prototype and the positive drug amifostine(100 ?M).(6)The results of Western blot showed that pretreated irradiated cells with 10 ?M resveratrol and nitroxide derivatives could increase the expression level of apoptosis inhibitory protein(cIAP-1).Decrease the expression of activated Caspase-9.Reduce the mitochondrial cytochrome-c release into the cytoplasm,in which the effect of CSP-NPR-2 is better than CSP-NPR-3.(7)The results of mitochondrial membrane potential assay showed that pretreated irradiated cells with structural derivatives at concentration of 10 ?M had no effect on the reduction of radiation-induced mitochondrial membrane potential.Conclusions : Comprehensive evaluation of the radioresistance effect of resveratrol,salidroside and nitroxide radicals series derivatives.it is found that resveratrol and nitroxide derivatives have lower cytotoxicity compared with resveratrol prototypes.The compounds CSP-NPR-2 and CSP-NPR-3 can significantly reduce the radiation-induced increase of intracellular ROS levels in Jurkat T cells,and can significantly reduce the radiation-induced apoptosis.Further explored and found that its mechanism of action is through the regulation of expression of apoptosis-related proteins Caspase-9,cIAP-1 and mitochondrial cytochrome c to play a radiation protection effect.In summary,resveratrol series compounds show promising research prospects.The pharmacokinetic study and pharmacodynamic evaluation of resveratrol series compounds can be further study in vivo.