The Study Of Radioprotective Effect Of Fasudil On Radiation Damage Of Normal Human Fibroblasts And Its Mechanisms Of Action

Objectives:Ionizing radiation(IR)has been widely used in industry,agriculture,military and medical fields.However,while IR brings benefits to people’s lives,it also has toxic effects that endanger health,so it is important to prevent and treat IR-induced damage.Various drugs have been used as radioprotectants in clinical practice,but they still have disadvantages such as high toxicity,high price and serious side effects,therefore,the discovery of effective radioprotective agents has always been the focus of research in the field of radiology.Fasudil is a representative compound of Rho associated kinase inhibitors,which are clinically used to improve microcirculation in the treatment of cardiovascular and cerebrovascular diseases,and has been reported to be involved in cellular activities such as the process of adhesion during mitotic stages and adjustment of the cytoskeletal.Through the screening and identification of the radioprotective effects of 253 compounds in the chemical molecular library,we found that fasudil has a protective effect against radiation damage to normal human fibroblasts(HFs)and bronchial epithelial cells(16HBE);On this basis,the relationship between the radioprotective effect of fasudil and the damage repair mechanism of DNA double strand breaks(DSBs)by homologous recombination(HR)and non-homologous end joining(NHEJ)was investigated from the perspective of the damage repair mechanism of IR-induced DSBs.The purpose of this study is to discover novel safe and effective radiation damage protectants and to investigate the molecular mechanism of their radiation protection,aiming to provide a reliable preventive measures and experimental basis for protecting normal tissue cells from the toxic effects of ionizing radiation.Methods:1.Compounds was screened out and identified for its radioprotective effect in the chemical molecular libraryNormal 16HBE cells were pretreated for 24h with compounds from the molecular library at a concentration of 10μM before treatment with 6Gy irradiation dose,a customized chemical molecule library consisting of 253 compounds were screened by determining cell viability using CCK-8 assay and the clonogenic survival assay.First,the compound molecules were initially screened by the CCK-8 assay,and then we rescreened these molecules to verify the results by the CCK-8 assay and the clonogenic survival assay,and the potential radioprotectants were finally selected.2.Detection of radioprotective effect of fasudil on radiosensitive HFsThe CCK-8 assay was used to detect the cytotoxicity of different concentrations of fasudil on human fibroblasts;The radioprotective effect of fasudil on HFs was examined by measuring cell viability at different drug pretreatment times and and irradiation doses and time points after irradiation using CCK-8 assay and the clonogenic survival assay;Under the same treatment conditions,the radioprotective effect of fasudil on 16HBE normal cells was verified by the clonogenic survival assay;The cytotoxicity of the fasudil on human breast cancer cells(MCF7)was examined by the CCK-8 assay;The effect of the fasudil on the survival rate of MCF7 cells at different irradiation doses was examined by the clonogenic survival assay.3.Detection of the effect of fasudil on IR-induced DNA DSBs in HFsThe damage of cellular DSBs were detected by the neutral and alkaline comet assays;Protein expression and formation of protein foci of DNA DSBs marker yH2AX and 53BPlwere detected by the Western blotting analysis and cellular immunofluorescence assay,respectively.4.Detection of the effect of fasudil on the HR repair function of IR-induced DNA DSBsMCF7 cells stably expressing the homologous recombination substrate pDR-GFP were transfected with I-SceI nuclease to cause intracellular DNA double-strand breaks,and the cells with GFP expression were detected by flow cytometry to assess HR repair efficiency;After fasudil treatment,the cell cycle changes of MCF7 cells were detected by flow cytometry;the foci formation and protein expression levels of HR repair key proteins BRCA1 and Rad51 in HFs were detected by cellular immunofluorescence assay and Western blotting analysis,respectively.5.Detection of the effect of fasudil on the NHE J repair function of IR-induced DNA DSBsU2OS cells stably expressing the non-homologous end-joining substrate EJ5-GFP were transfected with I-SceI nuclease to cause intracellular DNA double-strand breaks,and the cells with GFP expression were detected by flow cytometry to assess NHEJ repair efficiency;The protein expression levels of NHEJ-associated proteins DNA-PKcs,Ku70 and Ku80 in HFs were detected by Western blotting analysis;The foci formation and phosphorylation expression levels of phosphorylated DNA-PKcs(pDNA-PKcs)in HFs were detected using cellular immunofluorescence and Western blotting analysis,respectively.Results:1.Fasudil was screened out and identified for its radioprotective effectEffect of 253 compounds in the chemical molecular library on radioresistance of 16HBE cells was preliminarily screened using the CCK-8 assay.The results showed that compared with the control group,the cell viability of the IR-treated alone group was 51.87%,according to which we thought that if the cell viability after the combination of IR and the compound was significantly higher than that of the IR-treated alone group and exceeded 80%,it indicated that the compound has radioprotective effect,and thus 51 compounds were initially screened out.The screened compounds in the primary screen were further validated using the CCK-8 assay in the rescreening,and the compounds with cell viability below 88%after combination treatment were removed,and 30 compounds were obtained again.After rescreening,30 compounds were validated again by the clonogenic survival assay,and it was observed that 13 compounds could significantly increase the radiation survival compared with the IR-treated alone group(P<0.01).The 13 compounds included dexamethasone and dexamethasone acetate,which are known to have radioprotective effects,thus suggesting the validity and reliability of the screening results of this study.Among the 13 compounds,the cell survival rate was higher in the fasudil-treated alone group than those in the other compounds-treated alone groups,the survival rate of the combination of IR and fasudil group was also significantly higher than those of other combination groups,and the results were similar to those of dexamethasone and dexamethasone acetate.The results suggest that fasudil is a potential radioprotectant.2.Fasudil has radioprotective effect on radiosensitive HFsFirst,10μM fasudil was selected as the radioprotective dose for this study by cytotoxicity experiments of fasudil on radiosensitive HFs.The results of the CCK-8 assay showed that the cell viability of HFs in IR-treated alone group was significantly decreased compared with the control group.After the HFs were pretreatment with fasudil for 24h before IR at doses of 2,4,6,8,and 10Gy,cell viability in the combination group was significantly increased compared to the IR-only group(P<0.01),suggesting the radioprotective effect of fasudil on HFs.The results of clonogenic survival assay showed that the cell viability in the combination group was significantly higher than that in the IR-only group after the HFs were pretreatment with fasudil for 0,6,24h before 2,4,6Gy IR treatment,verifying the radioprotective effect of fasudil on HFs.The radioprotective effect of fasudil on 16HBE normal cells was also confirmed by cell clonogenic survival assay under the same treatment conditions.Furthermore,fasudil alone did not affect the survival and radiosensitivity of the tumor cell line MCF7(P>0.05).3.Fasudil reduces IR-induced accumulation of DSBs in HFsThe results of the neutral comet assay and the alkaline comet assay both showed that compared with the IR treatment alone,the length of the comet tail in the fasudil pretreatment group did not change significantly at 0min and 30min after IR treatment(P>0.05),but at 2h、6h and 24h after IR treatment,the comet tail length in the combination of fasudil and IR group was significantly lower than that in the IR alone group(P<0.01).The results of immunofluorescence assay to detect the foci formation of DNA DSBs markers γH2AX and 53BP1 protein showed that the percentage of γH2AX and 53BP1 foci-positive cells was significantly increased in the IR-treated alone group compared with the control group(P<0.01),and the percentage of γH2AX and 53BP1 foci-positive cells was significantly decreased at 24h after IR treatment compared with 6h after IR treatment.If HFs were pretreated with fasudil for 24h before IR treatment,fasudil significantly reduced the percentage of IR-induced γH2AX and 53BP1 foci-positive cells at both 6h and 24h after IR treatment(P<0.01).Meanwhile,the protein expression levels of γH2AX and 53BP1 showed the same changing trend as their foci formation.4.Fasudil increases the precise HR function of DNA DSBsTreatment of MCF7 cells expressing HR substrates with 10μM fasudil significantly increased the cellular HR efficiency by 45.24%(P<0.05),but fasudil had no effect on the S and G2 phases of the cell cycle.The percentages of HR repair-related proteins Rad51 and BRCA1 foci-positive cells in the IR alone group were significantly higher than those in the control group(P<0.01).If HF cells were pretreated with fasudil for 24h before IR treatment,and 6h and 24h after IR treatment,fasudil significantly increased the percentage of Rad51 and BRCA1 focipositive cells(P<0.01).In addition,the protein expression levels of Rad51 and BRCA1 had the same changing trend as their protein foci formation.5.Fasudil reduces error-prone NHEJ function of DNA DSBsTreatment of U2OS cells expressing NHEJ substrates with 10μM fasudil significantly reduced cellular NHEJ efficiency by 63.88%(P<0.01).Western blotting results showed that compared with the IR-treated alone group,the protein levels of Ku70,Ku80,DNA-PKcs and protein levels of pDNA-PKcs were significantly decreased in cells pretreated with fasudil at 24h post-IR treatment.Compared with the control group,the percentage of pDNA-PKcs focipositive cells in the IR-treated alone group was significantly increased(P<0.01),the percentage of pDNA-PKcs foci-positive cells was significantly decreased in the combination of IR and fasudil group compared with the IR-treated alone group(P<0.01).Conclusions:1.Fasudil was screeed and identified as a novel normal cell radioprotector,through screening a repository of chemical molecules.2.Fasudil significantly enhances the tolerance of normal cells to radiation and reduce DNA double strand breaks damage.3.Fasudil exert its radioprotective effects by specifically promoting precise HR repair mechanism of DSBs and inhibiting NHEJ repair pathways of DSBs that can be prone to errors.