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Objective:To investigate the effect of miR-27b-3p on radiation resistance of breast cancer cells.Methods:The relative expression levels of miR-27b-3p in normal tissues and breast cancer tissues were analyzed through GEO database and verified by the qRT-PCR assay. Cloning formation, immunofluorescence, and EDU assay were used to assess the functions of miR-27b-3p on radioresistance of breast cancer cells. The luciferase reporter assay was used to verify whether miR-27b-3p directly targeted PLK2 mRNA. A rescue experiment was performed to identify the influence of PLK2 overexpression on miR-27b-3p regulated radioresistance.Results:The expression level of miR-27b-3p in both breast cancer tissues ( t=2.99, P<0.01) and breast cancer cells ( t=21.21, 32.88, P<0.05) was significantly higher than those in normal breast tissues and cells, especially, it was elevated in radioresistant MCF-7R cells ( t=25.63, P<0.05). Overexpression of miR-27b-3p enhanced the cloning efficiency of MCF-7 cells ( t=10.32, P<0.05), and had a protective effect on the proliferation of irradiated MCF-7 cells ( t=8.77, 8.26, 8.03, P<0.05). But interference of miR-27b-3p reduced the cloning efficiency and proliferation of MCF-7R cells ( t=40.00, P<0.05) after irradiation with different doses( t=8.54, 8.32, 8.23, P<0.05). Moreover, PLK2 was verified to be a direct target of miR-27b-3p, and overexpression of PLK2 inhibited miR-27b-3p-mediated radioresistance of breast cancer cells (MCF-7: t=9.66, P<0.05; MCF-7R: t=6.42, P<0.05). Conclusions:miR-27b-3p contributes to the radioresistance of breast cancer cells by targeting PLK2.
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Objective:To elucidate the change of whole genome expression profile for the effect of melatonin on radiation-induced intestinal injury in mice.Methods:C57BL/6J male mice were administrated with melatonin at 10 mg/kg body weight by intraperitoneal injection once a day for five consecutive days before abdominal irradiation with 14 Gy of γ-rays. Small intestines were harvested 3 d after radiation. GO annotation and KEGG pathway of the differential genes involved in small intestine were explored by DNA microarray analysis.Results:Compared with the control group, 584 differential genes were up-regulated and 538 differential genes were down-regulated for administration group pre-irradiation. The overlapping differential genes were selected from the irradiated mice and the administrated mice pre-irradiation. There were 324 up-regulated genes and 246 down-regulated genes unique to the administrated mice pre-irradiation. GO annotation analysis of the differential genes indicated that the top 15 significantly enriched biological processes for the administrated mice pre-irradiation mainly included autophagosome assembly (GO: 0000045), autophagosome organization (GO: 1905037) and regulation of acute inflammatory response (GO: 0002673). The genes ATG12, ATG16L2 and AMBRA1 were involved in autophagosome assembly and autophagosome organization. The genes C3, CPN1, CD55, CFP, CNR1, C1QA, C2 and CREB3L3 were involved in the regulation of acute inflammation response. KEGG pathway analysis of the differential genes involved indicated that the top 15 significantly enriched pathways for the administrated mice pre-irradiation mainly included O-glycan biosynthesis (hsa00512), glycosphingolipid biosynthesis (hsa00603), ECM-receptor interaction (hsa04512) and biosynthesis of unsaturated fatty acids (hsa01040). qRT-PCR verification showed that the expressions of ATG12 and ATG16L2 genes involved in autophagy for the administrated mice pre-irradiation increased significantly compared with the irradiated mice ( t=2.40, 4.35, P<0.05). Conclusions:The differential genes related with the biological process of autophagy, acute inflammatory response and the pathway of unsaturated fatty acid biosynthesis might be involved in the effect of melatonin on radiation-induced intestinal injury.
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Objective:To confirm the mechanism of Sedum alfredii extract (SafE) alleviating radiation injury in human small intestinal epithelial cells (HIEC-6). Methods:HIEC-6 cells were divided into 4 groups, including control group (Con), irradiation group (IR), SafE alone group (SafE) and SafE plus irradiation group (SafE+ IR). All of the SafE groups were treated with 0.02 g/ml (W/V) SafE for 24 h. Cell viability (CCK-8 method ) and intracellular ROS levels were investigated at 24 h after 2, 4, and 6 Gy irradiation. Samples were taken at 24 h after 4 Gy irradiation for transcriptome analysis, and the intracellular E3 ubiquitin ligase PRKN expression level was measured. The thickness of endoplasmic reticulum was detected at 24 h after 4 Gy irradiation using fluorescent dye.Results:SafE could maintain cell viability after irradiation ( t=2.94-10.40, P<0.05), and significantly reduced the level of ROS in the irradiated cells ( t=-13.29--4.53, P<0.05). PRKN was preliminarily verified to be the target gene of SafE that maintained PRKN transcript level and endoplasmic reticulum thickness after irradiation (IR group vs. Con group: t=-5.55, 3.27, P<0.05, SafE group vs. SafE+ IR group: P>0.05). Conclusion:SafE is effective in maintaining ER thickness and reducing cellular radiation damage and its target gene PRKN could be regulated by ionizing radiation.
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Objective:To investigate the effect of ionizing radiation on the N 6-methyladenine (m 6A) modification profile of circular RNA (circRNA) in mouse bone marrow cells and provide scientific basis for revealing the relationship between RNA epigenetic modification and hematopoietic radiation injury. Methods:A total of twenty four C57BL/6 J mice were randomly divided into two groups: the healthy control group ( n=12), and ionizing radiation group ( n=12) irradiated in total body with 4 Gy of 137Cs γ-rays. At 5 min after irradiation, mice were killed and bone marrow cells were collected from the femur. Total RNAs were extracted and the changes in circRNA m6A modification profiles were investigated by RNA immunoprecipitation-high-throughput sequencing (MeRIP-Seq) technology and bioinformatics analysis. The representative alterations of m 6A peaks were validated by MeRIP-PCR assay. Results:325 and 455 m 6A sites were identified on circRNAs in the healthy control group and ionizing radiation group (178 common sites, 147 specific sites in the healthy control group and 277 specific sites in ionizing radiation group), respectively. 1 275 and 1 017 deriving genes of m 6A-circRNAs were identified in the healthy control group and ionizing radiation group (767 common genes, 508 specific genes in the healthy control group and 250 specific genes in ionizing radiation group), respectively. Compared with the control healthy group, 414 (178) m 6A peaks was significantly up- (down-) regulated in the ionizing radiation group( P < 10 -10; fold-change cut-off > 5). Moreover, Gene Ontology (GO) assay revealed that the deriving genes of circRNAs with differentially methylated m 6A sites between two groups involves various functions including chromatin regulation, ciliary transition fiber and poly (A)-specific ribonuclease activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) assay revealed that the deriving genes of circRNAs with differentially methylated m 6A sites between two groups included numerous pathways such as platelet activation, Fc γ R-mediated phagocytosis and B cell receptor signaling pathway. Conclusions:Ionizing radiation triggers rapid alterations in the m 6A modification profile of circRNA in mouse bone marrow cells. The deriving genes of differentially methylated circRNAs are associated with a variety of functions and signaling pathways of hematopoietic radiobiology.
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Objective:To study the effect of fasting on 137Cs γ-ray radiation-induced intestinal injury in mice, and to explore the effect of fasting on fecal metabolites of mice through non-targeted metabolomics. Methods:C57BL/6 mice were divided into healthy control group, 9 Gy γ-ray whole body irradiation (WBI)/ 15 Gy γ-ray whole abdominal irradiation (WAI) group, fasting (24 h, 48 h, 72 h)+ 9 Gy WBI/ 15 Gy WAI group. After irradiation, the survival rate, spleen index and thymus index were calculated. C57BL/6 mice in non-target metabolism experiment were randomly divided into four groups: control group, fasting 24 h group, 15 Gy γ-ray WAI group, fasting 24 h + 15 Gy γ-ray WAI group, 6 mice in each group. After 15 Gy WAI, the feces of mice in each group were collected at 3.5 days for non-targeted metabolomics detection.Results:The median survival time of mice with 48 h and 24 h fasting before 9 Gy γ-ray irradiation was increased by 1 day and 4 days, and the survival rates of mice treated with 48 h and 24 h fasting before 15 Gy WAI were 16.67% and 25%, respectively. 15 Gy γ-ray WAI on mice with fasting for 24 h before irradiation could increase the body weight ( t=2.338, P=0.042) and spleen index ( t=2.289, P=0.045) at 3.5 days after irradiation. Through non-targeted metabonomic analysis, it was found that there were 30 differentially expressed metabolites in fecal samples of fasting and non-fasting mice subjected to WAI, and metabolic pathway enrichment analysis showed that there was an imbalance in the metabolic pathway of steroid biosynthesis. Conclusions:Fasting before irradiation can improve the survival rate of mice with intestinal radiation injury and change their intestinal metabolites, suggesting that pre-irradiation fasting or short-term dietary nutrition changes are involved in the regulation of intestinal radiation damage.
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Objective:To study the effect of emulsifier Tween-80 on radiation-induced bile acid enterohepatic circulation disturbance and the treatment strategy.Methods:Male C57BL/6 J mice were randomly divided into healthy control group, radiation-only group, radiation + Tween-80 group and radiation + Tween-80 + butyric acid group. The mice were exposed to total abdominal irradiation (TAI) using a specific steel lead chamber and γ-ray irradiator was used throughout the experiments. Mice in radiation+ Tween-80 group and radiation+ Tween-80+ butyric acid group were intragastrically administrated with Tween-80 for 7 d before irradiation, while healthy control group and radiation-only group were treated with sterile water. After irradiation, butyric acid was administrated to mice in radiation+ Tween-80+ butyric acid group until euthanasia, while healthy control group, radiation-only group and radiation+ Tween-80 group were treated with sterile water until euthanasia. Small intestine and fecal particles were collected 21 d after irradiation. The concentrations of bile acid in small intestinal and fecal samples were measured using enzyme linked immunosorbent assay (ELISA), the expression of TGR5 and JAM-A, as well as the ratio of IL-10/IL-12 in intestine were detected by quantitative real-time PCR (qRT-PCR). The expression levels of GPR43 in the colon were compared using immunohistochemistry (IHC).Results:Tween-80 pretreated mice exhibited lower concentration of bile acid in small intestine and higher level of bile acid in fecal sample after irradiation (7.92%, 7.99%, t=3.93, 2.94, P<0.05), the expression of TGR5, which mediating the biological function of bile acid, and it′s downstream JAM-A gene were down-regulated (20.93%, 9.91%, t=4.85, 5.14, P<0.05), the ratio of IL-10/IL-12 (indicator related to inhibition of inflammation) (4.59%, t=3.39, P<0.05) as well as the expression of GPR43 protein, a G-protein-coupled receptor for butyric acid, decreased in the colon of Tween-80-pretreated mice compared with the radiation-only group. ELISA assay revealed that butyric acid administration elevated bile acid level in small intestines (8.06%, t=9.25, P<0.05), but reduced that in feces (14.41%, t=4.71, P<0.05). In addition, TGR5 and JAM-A showed higher expression in the intestine of butyric acid-treated mice (19.35%, 32.71%, t=7.69, 19.23, P<0.05), as well as the ratio of IL-10/IL-12 (2.39%, 4.05%, t=3.38, 5.92, P<0.05) and the content of GPR43 protein in colon. Conclusions:Tween-80 deteriorates the disturbance of bile acid enterohepatic circulation induced by ionizing radiation in mice. Butyric acid administration erases the adverse effects of Tween-80.
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Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits tumor migration and invasion. Obtaining TIMP-2 protein is conducive to a comprehensive and in-depth study of its function and mechanism in tumorigenesis and development. We collected human TIMP-2 protein through prokaryotic expression in vitro. We expressed, purified and characterized human TIMP-2 protein. First, the human TIMP-2 gene was cloned from the cDNA obtained by reverse transcription of total RNA of human lung cancer A549 cells, and constructed to pET28a vector. The recombinant plasmid pET28a-TIMP-2 was transformed into Escherichia coli BL21(DE3) after restriction endonuclease digestion and sequencing analysis. The expression of TIMP-2 protein was induced by isopropyl-β-D-thiogalactoside (IPTG), and the expression conditions were optimized. After purification by nickel affinity column, the fusion protein His-TIMP-2 was identified by Western blotting method and its biological activity was detected by gelatin zymography. The fusion protein His-TIMP-2 existed in the form of inclusion body in E. coli. In a certain range, the concentration of IPTG had no significant effect on the expression amount of His-TIMP-2. But in this expression system, induction temperature and time were the key parameters, and the expression amount of His-TIMP-2 in E. coli increased with the increase of induction temperature. The purified and refolded fusion protein could effectively inhibit the activity of matrix metalloproteinases expressed by human lung cancer A549 cells. The acquisition of active fusion protein lays a foundation for further study of the function and mechanism of human TIMP-2, and is of great significance for tumor therapy.
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Humanos , Clonagem Molecular , Escherichia coli/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes , Inibidor Tecidual de Metaloproteinase-2/genéticaRESUMO
Objective To explore the effects of FOXO4 D-retro-inverso peptide (FOXO4-DRI)on the radiosensitivity of non-small cell lung cancer (NSCLC) cells.Methods To detect the effect of FOXO4-DRI on NSCLC cells,H460 and A549 human lung cancer cells were divided into four groups,including untreated control,FOXO4-DRI,γ-ray irradiation and FOXO4-DRI + γ-ray groups.A sigle dose rate of 0.99 Gy γ-rays was used for radiation.H460 cells were administered with 6 μmol/L FOXO4-DRI and A549 cells were adiminstered with 30 μmol/L FOXO4-DRI at 10 min before radiation.Cell viability and survival were detected by CCK-8 assay and colony formation assay,respectively.Cell migration was detected by wound healing assay.Apoptosis and cell cycle arrest were detected with flow cytometry.Results FOXO4-DRI inhibited growth of H460 and A549 cells (t =1.06-50.75,P＜ 0.05),and decreased cell mobility (t =33.37-139.10,P＜0.05) and colony formation (t =5.20-93.48,P＜0.05).FOXO4-DRI also increased apoptosis (t=2.95-42.00,P＜0.05) and caused a cell cycle arrest at G0/G1 phase accompanied with a decreased proportion of G2/M phase (t =3.50-3 1.59,P＜0.05).Furthermore,FOXO4-DRI increased radiosensitivity of both H460 cells and A549 cells (t =2.94-23.40,P＜0.05),caused a Further Decrease of radiation-mediated mobility (t =5.25,7.56,P＜0.05) and colony formation (t =8.43-34.00,P＜ 0.05) and a more increase of radiation-induced apoptosis (t =9.20-11.52,P ＜0.05).FOXO4-DRI also further decreased the proportion of G2/M phase cells but increased the proportion of S phase cells (t =3.85-17.62,P ＜ 0.05).Conclusion FOXO4-DRI increases radiosensitivity of NSCLC cells by inducing apoptosis and suppressing cell proliferation.
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Objective@#To explore the effects of FOXO4 D-retro-inverso peptide (FOXO4-DRI) on the radiosensitivity of non-small cell lung cancer (NSCLC) cells.@*Methods@#To detect the effect of FOXO4-DRI on NSCLC cells, H460 and A549 human lung cancer cells were divided into four groups, including untreated control, FOXO4-DRI, γ-ray irradiation and FOXO4-DRI + γ-ray groups. A sigle dose rate of 0.99 Gy γ-rays was used for radiation. H460 cells were administered with 6 μmol/L FOXO4-DRI and A549 cells were adiminstered with 30 μmol/L FOXO4-DRI at 10 min before radiation. Cell viability and survival were detected by CCK-8 assay and colony formation assay, respectively. Cell migration was detected by wound healing assay. Apoptosis and cell cycle arrest were detected with flow cytometry.@*Results@#FOXO4-DRI inhibited growth of H460 and A549 cells (t=1.06-50.75, P<0.05), and decreased cell mobility (t=33.37-139.10, P<0.05) and colony formation (t=5.20-93.48, P<0.05). FOXO4-DRI also increased apoptosis (t=2.95-42.00, P<0.05) and caused a cell cycle arrest at G0/G1 phase accompanied with a decreased proportion of G2/M phase (t=3.50-31.59, P<0.05). Furthermore, FOXO4-DRI increased radiosensitivity of both H460 cells and A549 cells (t=2.94-23.40, P<0.05), caused a Further Decrease of radiation-mediated mobility (t=5.25, 7.56, P<0.05) and colony formation (t=8.43-34.00, P<0.05) and a more increase of radiation-induced apoptosis (t=9.20-11.52, P<0.05). FOXO4-DRI also further decreased the proportion of G2/M phase cells but increased the proportion of S phase cells (t=3.85-17.62, P<0.05).@*Conclusion@#FOXO4-DRI increases radiosensitivity of NSCLC cells by inducing apoptosis and suppressing cell proliferation.
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Objective To screen radiotherapy resistance related microRNAs (miRNAs) in breast cancer and provide experimental basis for basic researches and clinical solutions of radiotherapy resistance in breast cancer patients.Methods The miRNA microarray dataset GSE107743 related to breast cancer radiotherapy patients was downloaded from the Gene Expression Omnibus (GEO).The GEO2R analysis tool was used to screen differentially expressed miRNAs in patients with local recurrence after radiotherapy.The target genes of differentially expressed miRNAs were predicted by the mirDIP database.GO enrichment analysis and KEGG pathway analysis on the target genes were performed by DAVID dataset.Finally,differential expression verification was performed in human breast cancer cell line MCF-7 by real-time fluorescent quantitative PCR.Results A total of 9 differentially expressed miRNAs related to radiotherapy resistance were screened by the GEO2R analysis tool,in which three miRNAs (hsa-miR-600,hsa-miR-525-3p and hsa-miR-591) were up-regulated and 6 miRNAs (hsa-miR-488-5p,hsa-miR-582-3p,hsa-miR-520h,hsa-miR-488-3p,hsa-miR-744-3p and hsa-miR-103b) were down-regulated.Target gene prediction results showed that there were 134 potential target genes in these nine differentially expressed miRNAs.These target genes were significantly enriched in related biological processes such as apoptosis and stem cell differentiation (all P＜0.05) and signal transduction pathways such as transforming growth factor-β and phosphatidylinositol 3-kinase-protein kinase B signaling pathway (all P＜0.05).The results of real-time PCR showed that the differential expression of six miRNAs,i.e.hsa-miR-600,hsa-miR-525-3p,hsa-miR-591,hsa-miR488-5p,hsa-miR-582-3p and hsa-miR-520h,was detected in the MCF-7 cells irradiated by 5 Gy 137Cs γ-rays,and this result was consistent with the results of GEO2R analysis.Conclusion The differentially expressed miRNAs screened from clinical samples of breast cancer patients with local recurrence using bioinformatics may be closely associated with the radiotherapy resistance of these patients.These miRNAs are expected to become new biomarkers for the therapy of radiotherapy resistance.
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Objective To investigate the protective effect of resveratrol on γ-ray radiation induced small intestinal injury in mice.Methods Sixty-four C57BL/6 male mice were divided into 7.2 Gy irradiation group,7.2 Gy irradiation+resveratrol group,15 Gy irradiation group,15 Gy irradiation+resveratrol group,17 Gy irradiation group,17 Gy irradiation +resveratrol group,resveratrol group and control group according to the method of radiation and administration.Radiation irradiation was performed with a single dose of 0.99 Gy/min.The control and single irradiation groups were given 14.4％ of ethanol solution by intragastric administration.The resveratrol group and irradiation+resveratrol groups were given a resveratrol mixed solution at a dose of 60 mg/kg.The 30-day survival rate of the mice was observed.The morphological changes of the small intestine at 96 hours after the irradiation,and the expression of the intestinal crypts at 4 hours after irradiation was examined.Results Compared with the single irradiation groups,resveratrol can significantly increase the 30-day survival rate of the mice after 7.2 Gy total body irradiation and 17 Gy abdominal irradiation (all P＜0.05).In addition,resveratrol can significantly improve the small intestinal crypt-villus structure damage in mice after 15 Gy abdominal irradiation,down-regulated the expression of p21 and Puma by 0.71 and 0.52 times,respectively (all P＜0.05),and up-regulated the expression of Nrf2,Ho1,iNos and Nqo1 by 3.03,1.62,3.94,and 4.13 times,respectively (all P＜0.05).Conclusion Resveratrol can effectively alleviate the radiation-induced small intestine injury in mice,which may be achieved by regulating anti-apoptotic and anti-oxidant gene expression.
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Objective To investigate the in vivo radioprotective effect of adenosine triphosphate (ATP) on radiation damage induced by high dose γ-ray ionizing radiation(IR) in mice.Methods Specific pathogen-free C57BL/6 female mice were randomly divided into IR group and IR+ATP group by body weight,with 10 mice in each group.All the mice were treated with a 8 Gy one-time and high-dose whole body γ-ray irradiation.Within 6 h after irradiation,mice were injected intramuscular injection of 150 pl sodium chloride solution (9 g/L) for IR group,and 150 μl ATP solution (6 mg/kg) for IR+ATP group,respectively.The drug was administered once a day until the death of the animal.The mean survival days,survival rate,body weight and major organ coefficients in both groups were measured.Results The average survival days of mice in IR group and IR +ATP group were 6.5 d and 9.6 d,respectively.The survival rate of the mice in IR+ATP group was higher than that in IR group (P＜0.01).The body weight values of the mice in IR+ATP group was higher than that in IR group on the after the 4th day post-irradiation,and the differences were statistically significant (all P＜0.05).Except for heart and stomach,the organ coefficients of liver,spleen,lung,and kidney in IR +ATP mice were higher than those in IR group,and the differences were statistically significant (all P＜0.05).Conclusion ATP has certain radiation protection effect,and it can reduce the radiation damage of mice induced by high-dose (8 Gy) γ-ray IR so as to increase the survival rate.
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Objective To investigate the protective effect of 3,3′-diindolylmethane (DIM) on radiation-induced injury in mouse hematopoietic system. Methods Thirty C57BL/6 mice were randomly divided into control group, 2 Gy irradiation group and 2 Gy irradiation+DIM group (n=10 for each group). Mice of control group received sham irradiation, and the other two groups accepted 2 Gy 137Cs γ-ray total body irradiation. Mice in 2 Gy irradiation +DIM group were intraperitoneally injected 75 mg/kg DIM 30 min before irradiation. Mice of other two groups were treated with reference solution. After 7 d and 15 d of 2 Gy irradiation, the peripheral blood samples were collected to count the number of bone marrow nuclear cells (BMNCs). The level of reactive oxygen species(ROS) was measured by DCFH-DA. The levels of colony forming units-granulocyte-macrophage (CFU-GM) were also detected. Results The numbers of white blood cell (WBC), platelet count (PLT), BMNCs and CFU-GM were significantly decreased and the ROS level of bone marrow cells increased significantly in the irradiated group than those of control group (P<0.05). Compared to 2 Gy irradiation group, the numbers of WBC, PLT, BMNCs and CFU-GM were significantly increased in 2 Gy irradiation +DIM group, and the level of ROS was decreased significantly (P<0.05). Conclusion DIM has a protective effect on hematopoietic cells following radiation-induced injury, which may be related with the decreased ROS level.
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Objective To observe the protective effect of anthocyanin on irradiation induced bone marrow c-kit positive cell injury, and further explore its possible mechanism. Methods Mouse bone marrow c-kit positive cells were collected by cell sorting method. There were 2 groups: control group and anthocyanin group, which were sub-divided into three groups and received 0 Gy, 1 Gy and 4 Gy irradiation respectively. The control group was added 700μL cell suspension and an equal volume of serum-free hematopoietic stem/progenitor cell culture medium. The 2 × 10-5 mol/L anthocyanin was co-cultured with mouse bone marrow c-kit positive cells of anthocyanin group half an hour before irradiation exposure, then cells were cultured for 18 hours under the conventional culture conditions (37℃,5%CO2). Mouse c-kit positive cell viability was measured by bioluminescence, and which was reflected by relative light units (RLU). The ability of colony-forming units was reflected by CFU-GM. The reactive oxygen species (ROS) level and mean fluorescence intensity (MFI) ofγ-H2AX were detected by flow cytometry. Results Compared to un-irradiated control group, the cell viability and the number of CFU-GM were decreased significantly, while the ROS level and MFI ofγ-H2AX were increased in c-kit positive cells irradiated with 1 Gy and 4 Gy (P<0.05). Compared to 1 Gy and 4 Gy irradiation groups, c-kit positive cell viability and the number of CFU-GM were increased, the ROS level and MFI of γ-H2AX were decreased in anthocyanin group (P < 0.05). Conclusion Anthocyanin exhibits a promising protective effect on radiation-induced bone marrow c-kit positive cell injury, which may be related to the alleviating ROS and DNA damage in bone marrow cells.
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Objective To investigate the protective effect of indole-3-carbinol (I3C) on radiation-induced mouse bone marrow hematopoietic cell injury and the involved mechanisms. Methods (1) The bone marrow nuclear cells (BMNCs) from CD45.1 subtype of C57BL/6J mice were collected by a density gradient centrifugation method. The BMNCs were pretreated with a series doses of I3C (0 mol/L, 10-8 mol/L-10-3 mol/L) and then exposed with radiation of 137Csγ-ray (doses of irradiation were 0 Gy, 1 Gy and 4 Gy). After 18-hour culturing, the bioluminescence method was used to detect the cell viability. (2) These cells were divided into control group and 10-6 mol/L I3C group. Both groups were received the irradiation (0 Gy, 1 Gy and 4 Gy) and inoculated into the methylcellulose semi-solid culture medium to incubate 7 days, the colony forming unit-granulocyte monocytes (CFU-GM) were observed. (3) Twenty-four CD45.2 subtype mice used as the receptor were exposed with 8 Gy radiation. The CD45.1 BMNCs were divided into control group, 4 Gy irradiation group, 4 Gy irradiation and 10-6 mol/L I3C group. Donor cells were harvested from C57BL/6J (CD45.1) mice after they received various treatments, and were then mixed with competitive BMNCs from C57BL/6J (CD45.2) mice. The mixed cells were transplanted into recipient mice (8 mice/group). Flow cytometry was used to analyze the proportion of donor cells in peripheral blood of receptor. (4) The cells were divided into control group, 10-6 mol/L I3C group, 1 Gy irradiation group, 1 Gy irradiation with 10-6 mol/L I3C group. After 24-hour culturing, Western blot assay was used to detect the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1). Results (1) I3C showed a significant cytotoxic effect on the BMNCs when its concentration was above 10-4 mol/L. 10-7-10-6 mol/L I3C could reduce the radiation injury of BMNCs under the same dose of irradiation. Therefore, 10-6 mol/L I3C was chosen for subsequent experiments. (2) The CFU-GM was significantly higher in 10-6 mol/L I3C group than that of control group (P<0.05). (3) Results of flow cytometry showed that the proportion of donor cells in receptor was significantly higher in 4 Gy irradiation group than that of control group, which decreased the engraftment capability of irradiated HSCs (P<0.05), although the engraftment capability of irradiated HSCs improved after 10-6 mol/L I3C treatment. (4) I3C significantly enhanced the increased protein expression of Nrf2 and HO-1 caused by radiation (P<0.05). Conclusion I3C has a protective effect on hematopoietic cells following radiation-induced injury, which may be related with activating the Nrf2/HO-1 signal pathway.
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Objective To investigate the protective effect of theaflavins on thymus injury caused by total body irradiation (TBI).Methods Twenty-five C57BL/6 mice were randomly divided into 5 groups:control group,4 Gy TBI group,4 Gy TBI + 25 mg/kg theaflavins group,4 Gy TBI + 50 mg/kg theaflavins group and 4 Gy TBI + 100 mg/kg theaflavins group.Thymus index and total number of thymocytes were detected at the 14th d post-irradiation to determine the optimal dose of theaflavins.According to this optimal dose,32 C57BL/6 mice were randomly divided into 4 groups:control group,theaflavins group,4 Gy TBI group and 4 Gy TBI + theaflavins group.Thymus histomorphology,CD4CD8 T cell subsets,and reactive oxygen species (ROS) in thymocytes were examined at the 14th d post-irradiation.Results The irradiated thymus exhibited decreased thymus index and total number of thymocytes (P ＜ 0.05),aberrant histomorphology and T cell subsets (P ＜ 0.05),and increased ROS level in thymocytes (P ＜ 0.05).Compared with 4 Gy TBI group,the thymus index and total number of thymocytes were significantly increased in 4 Gy TBI + 50 mg/kg theaflavins group (P ＜ 0.05).The total number of thymocytes was significantly higher in 4 Gy TBI + 50 mg/kg theaflavins group than that in 4 Gy TBI + 25 mg/kg theaflavins group (P ＜ 0.05).Therefore,50 mg/kg theaflavins was chosen as the optimal dose for subsequent experiments.Moreover,the aberrant histomorphology of irradiated thymus was alleviated by theaflavins.A decline in the percentage of CD4-CD8-T cells and an elevation of CD4+CD8-and CD4+CD8+ T cells were found in irradiated mice administered with theaflavins (P ＜ 0.05).Compared with 4 Gy TBI group,the ROS level was significantly decreased in 4 Gy TBI + theaflavins group (P ＜ 0.05).Conclusion Theaflavins exhibits a protective effect on radiation-induced thymus injury.
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Objective To investigate the protective effect of hydrogen-rich water (HRW) on radiation-induced hematopoietic stem and progenitor cells (HSPCs) injury.Methods Totally 32 C57BL/6 mice were randomly divided into four groups with 8 mice in each group,including control,HRW,radiation and radiation + HRW.Mice in HRW and radiation + HRW groups received 0.5 ml hydrogen-rich water per day by intragastric administration 5 min before irradiation until 7 d post-irradiation.Mice in other groups received 0.5 ml distilled water.Mice in radiation and radiation + HRW group were irradiated with 2 Gy of total body irradiation.Bone marrow cells were isolated at 15 d post-irradiation,and LSK cells were examined for the percentage of hematopoietic stem and progenitor cells,the ability of colony formation and reconstitution,reactive oxygen species (ROS) levels and cell apoptosis.Results Compared with radiation group,the percentages of hematopoietic progenitor cells and LSK cells,colony number of bone marrow cells were significantly increased in radiation + HRW group (t =-4.935,-7.898,5.488,P ＜ 0.05).An elevation of donor chimerism was also found in recipient mice administered HRW after competitive bone marrow transplantation (t =-12.769,P ＜ 0.05).Compared with radiation group,the ROS levels and cell apoptosis in LSK cells were significantly decreased (t =4.380,3.954,P ＜ 0.05).Conclusions Hydrogen-rich water exhibited a protective effect on radiation-induced HSPCs injury.
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Objective To study the correlation of cleft lip and palate transmembrane 1 like(CLPTM1L)expression and radiosensitivity of lung cancer cells.Methods Thiazolyl blue tetrazolium bromide(MTT) and cell colony formation assays were used to determine cell growth and survival.Western Blot assay was employed to measure protein expression.Results The results demonstrated a negative correlation between the CLPTM1L expression level and radiosensitivity of lung cancer cells.A lower radiosensitivity in lung cancer cells containing high level of CLPTM1L expression,and vice versa.Enforced expression of CLPTM1L resulted in a significant reduction of radiosensitivity in lung cancer cells irradiated with γ-rays.On the contrary,a marked elevation of radiosensitivity was observed in lung cancer cells transfected with CLPTM1L siRNA.Conclusions CLPTM1L may be a novel target gene in mediating radiosensitivity of lung cancer cells.
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Objective To study the radioprotective effect of indole-3-carbinol (I3C) acid condensation products.Methods Cell colony formation assay was used to determine cell survival rate,and Western Blot assay was employed to measure protein expression.Results Seven kinds of the I3C acid condensation products showed different radioprotective effect on normal fibrous epithelial cells 184A1,among which 24 h pre-treatment of CTET (1 μmol/L),LTET (1 μmol/L),HI-IM (1 μmol/L) and 3,3'-diindoly methane (DIM) (0.3 μmol/L) showed significant increase of cell survival rate following irradiation with γ-ray,and the difficence was statistically significant (P＜0.05,P＜0.01).However,CT (1 μmol/L),LTr-1 (1 μmol/L) and ICZ (1 μmol/L) showed no effect on cell survival rate caused by radiation (P＞0.05).Furthermore,CTET,LTET,HI-IM and DIM activated the phosphorylation of ATM,BRCA1 and NBS1 proteins.HI-IM significantly decreased radiation-caused cell death and apoptosis.Conclusions CTET,LTET,HI-IM,and DIM can significantly reduce the radiosensitivity in 184A1 cells,and the mechanism may be related to the regulation of DNA damage and the repair of protein phosphorylation.
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Objective To estimate the averaged excess relative risk(ERR) in Chinese population based on the radiogenic cancer risk of leukemia in Japanese atomic bomb survivor cohort,and to discuss proper method suitable for risk transfer between populations.Methods Based on BEIR Ⅶ radiogenic cancer model and population transfer model,and the 2009 Chinese leukemia baseline rates given in 2012 Chinese Cancer Registry Annual Report,comparison was made of population incidences in seveal countries to adjust the weighting factors.Results The ERR of three subtypes of leukemia as a whole was obtained,and the weighting factors for risk transfer model was assumed.The additive factor for male was 0.2,and the multiplicative factor was 0.8,while the additive factor for female was 0.15,and the multiplicative factor was 0.85.Conclusions For the risk transfer between populations,weighting factor was adjusted as a whole to obtain the ERR value for estimating the risk to Chinese population.The risk transfer method suitable for Chinese population was obtained by using the incidence rate available for Chinese population to directly transfer radiation-induced leukemia risk to Chinese from Japanese.