Objective To investigate the synergistic protective effects of WR-2721 combined with lentinan and cytokines against radiation damage in mice, and to provide a new treatment for acute radiation injury. Methods Seventy Institute of Cancer Research mice were divided into seven groups: a control group, a model group, WR-2721 group, Lentinan & cytokine group, WR-2721 & Lentinan group, WR-2721 & cytokine group and WR-2721 & Lentinan & cytokine group. All groups except the control group were irradiated with ⁶⁰Co γ-rays at a dose rate of 0.8 Gy/min and a cumulative dose of 5.0 Gy. The mice were sacrificed by cervical dislocation 14 d after irradiation to measure their spleen index, thymus index, and serum levels of superoxide dismutase (SOD), malondialdehyde (MDA), interleukin-11 (IL-11), and tumor necrosis factor-alpha (TNF-α). Results For the mice treated with WR-2721, lentinan, and cytokines, the spleen index was 7.33 ± 2.84, the thymus index was 1.70 ± 0.30, the serum SOD level was 114.0 ± 8.3, the MDA level was 7.33 ± 1.16, the IL-11 level was 155.8 ± 49.4, and the TNF-α level was 174.0 ± 37.8. All these indicators except the spleen index in the combination group significantly differed from those of the model group (P < 0.05 or 0.01), indicating the combined treatment promoted recovery from radiation damage. Conclusion WR-2721 combined with lentinan and cytokines has significant synergistic protective effects, which is a promising treatment for acute radiation injury.

Objective To observe the effect of mitofusion 2 (MFN2) on the proliferation of prostate cancer cells and its molecular mechanism. Methods Lentivirus containing the MFN2 coding sequence (Lenti-MFN2) were used to infect the prostate cancer cell lines DU-145 and LNCaP, and the lentivirus containing the green fluorescent protein gene (Lenti-GFP) were defined as the control. Real-time quantitative PCR (qRT-PCR) and Western blot were used to detect the expression of MFN2 mRNA and protein in the infected cells. MTT assay and colony formation assay were used to detect the cell proliferation. Cell cycle distribution was measured by flow cytometry.Western blot was used to detect the expression of Ras,p-Raf and p-Erk1/2 proteins in infected cells. Results The expressions of MFN2 mRNA in DU-145 and LNCaP cells of Lenti-MFN2 group were 2.79±0.91 and 3.87±1.06, which were higher than those in Lenti-GFP group (1.02± 0.27 and 1.13±0.59),the differences were statistically significant(t=3.726,P=0.010;t=5.209,P =0.002). Compared with Lenti-GFP group, the expression of MFN2 protein in Lenti-MFN2 group was increased. The number of colonies formed in DU-145 and LNCaP cells of Lenti-MFN2 group was 147.42±32.91 and 130.26± 62.47, respectively, which was lower than that of the Lenti-GFP group (255.46±50.91 and 238.10±49.77), the differences were statistically significant (t =3.565, P=0.012; t =2.700, P=0.036). The cell cycle was arrested at G0/G1phase,and the expressions of Ras, p-Raf and p-Erk1/2 proteins were significantly decreased. Conclusion MFN2 can inhibit the proliferation of prostate cancer cells,and its mechanism may be related to the inhibition of activation of Ras-Raf1-Erk1/2 signaling pathway.

Objects To study the protective effects of cimetidine against oxidative stress in rats induced by cumulative low-dose irradiation.Methods Sixty SD rats were randomly divided into 6 groups (10 each):normal control group,model control group,lentinan group [89mg/(kg.d)] and 3 dose groups of cimetidine.After oral administration,all the rats were exposed to γ-ray irradiation 8 hours/day for 12 days,and sacrificed on the 13th day.The activities of superoxide dismutase (SOD),glutathione peroxidase (GPx),catalase (CAT) and the content of malondialdehyde (MDA) in serum,liver,thymus and spleen were determined.By using the superoxide anion radical system,hydroxyl radical system,H2O2 radical system,oxidation system of linoleic acid induced by alkane radical system and diphenyl picryl hydrazinyl radical (DPPH) radical system,the antioxidation activities of cimetidine were detected.Results The activities of SOD in liver and thymus decreased significantly,the GPx activity in serum,liver and spleen decreased significantly and MDA level in serum,liver and spleen increased significantly after 0.3Gy cumulative ionizing radiation.Cimetidine enhanced the activities of antioxidant enzymes in serum and organs,and reduced the MDA level.In a certain concentration range,cimetidine had different scavenging effects onto these radical systems,and showed good performance in hydroxyl radical.Conclusion Cimetidine can effectively ameliorate the oxidative stress from low-dose cumulative irradiation by scavenging free radicals,increase the activity of antioxidant enzymes and reduce the content of lipid peroxidation products,thus presents a potential radio protective effect.

Objective To investigate the effect of microRN-206 (miR-206) on the expression of Cyclin-dependent kinase 4 (CDK4) and Cyclin G-associated protein kinase (GAK), and the growth of prostate cancer cells.Methods Prostate cancer cell lines DU-145 and PC-3 were transfected with miR-NC (the control group) or miR-206 (the experimental group).The expressions of CDK4 and GAK mRNA were detected by real-time quantitative PCR (qRT-PCR).The expressions of CDK4 and GAK protein were detected by Western blotting.Cell cycle distribution was detected by flow cytometry.EdU proliferation assay and colony forming assay were used to analyze the cell proliferation ability.Results In DU-145 and PC-3 cells, the expressions of CDK4 mRNA in miR-NC group were 1.00±0.09, 1.00±0.10, the expressions of GAK mRNA were 1.00±0.05, 1.00±0.06.The expressions of CDK4 mRNA in miR-206 group were significantly decreased in DU-145 (0.36±0.18;t=6.572, P=0.001) and PC-3 cell lines (0.43±0.17;t=5.794, P=0.001).The expressions of GAK mRNA were also significantly decreased in DU-145 (0.23±0.04;t=22.420, P<0.001) and PC-3 cell lines (0.32±0.08;t=14.500, P<0.001).Western blotting results were consistent with qRT-PCR results.The results of flow cytometry showed that compared with the miR-NC group of DU-145 and PC-3 cell lines, the percentage of cells in S phase (23.60%±5.68% vs.32.53%±4.52%, t=2.462, P=0.049;22.09%±4.35% vs.30.96%±4.86%, t=2.720, P=0.035) and G2-M phase (16.28%±7.12% vs.26.63%±4.33%, t=2.484, P=0.048;14.60%±1.62% vs.24.68%±7.13%, t=2.758, P=0.033) decreased after transfection of miR-206, and the percentage of cells in G0-G1 phase (60.13%±5.82% vs.40.84%±5.37%, t=4.872, P=0.003;63.31%±3.27% vs.44.36%±3.82%, t=7.533, P<0.001) increased.The results of EdU proliferation assay showed that the proliferation abilities were significantly attenuated after transfection of miR-206 (22.56±3.81 vs.38.90±8.51, t=3.503, P=0.013;25.12±6.42 vs.48.45±8.92, t=4.244, P=0.005).The results of colony formation experiments showed that the numbers of colonies formed by DU-145 and PC-3 in miR-NC group were 218.66±44.59 and 177.35±24.49, respectively.The numbers of colonies formed in miR-206 group were 125.38±32.80 (t=3.370, P=0.015) and 82.65±14.05 (t=6.708, P=0.001), suggesting that cell proliferation ability in miR-206 group was reduced.Conclusion miR-206 significantly inhibits the growth of prostate cancer cells by interfering with the expressions of CDK4 and GAK, suggesting that miR-206 may be a molecular targeted therapy tool for prostate cancer.

Objective To investigate the effect of dsP21-555 transfection on the expression of tumor suppressor gene p21 in renal clear cell carcinoma cell lines ACHN and 786-O.Methods Renal clear cell carcinoma cells were transfected with dsControl and dsP21-555 with Lipofectamine 3000 respectively.Real-time quantitative PCR (RT-qPCR) and Western blotting were used to detect the expression of p21 mRNA and protein.Cell cycle distribution was detected by flow cytometry (FCM).Cell viability and proliferation were analyzed by cell viability assay (MTS method) and colony culture assay.Results In ACHN and 786-O cells, the expressions of p21 mRNA in dsP21-555 group (2.86±0.33, 1.96±0.35) were significantly higher than those in dsControl group (1.05±0.34, 1.01±0.14), which were increased to 2.72 times (t=7.640, P<0.001) and 1.95 times (t=5.058, P=0.002).Western blotting showed that the expressions of P21 protein were up-regulated in both renal cell lines, which was consistent with p21 mRNA up-regulation.The result of FCM showed that the cell cycle was blocked in G0-G1 phase (57.08%±5.66% vs.46.06%±4.60%, t=3.023, P=0.023;61.58%±6.23% vs.42.25%±6.08%, t=4.444, P=0.004) after transfection of dsP21-555 in renal clear cell carcinoma cells.MTS result showed that the vitality of both cell lines after transfection of dsP21-555 decreased compared with dsControl group, their absorbance values were 0.85±0.20 vs.1.27±0.13, t=3.410, P=0.014;1.04±0.25 vs.1.55±0.10, t=3.758, P=0.009.Colony culture experiments showed that the numbers of colonies formed by ACHN and 786-O in the dsControl group were 110.91±26.21 and 129.99±22.87 respectively, and the numbers of colonies formed in the dsP21-555 group were 59.37±14.23 (t=3.456, P=0.014) and 71.26±21.38 (t=3.745, P=0.010), indicating that the proliferation of cells in the dsP21-555 group was significantly reduced.Conclusion dsP21-555 can up-regulate the expression of p21 gene in renal clear cell carcinoma cells and inhibit the growth of carcinoma cells, suggesting that dsP21-555 may become a new gene therapy tool.

Objective To investigate the radioprotective effect of cimetidine on survival rate and hematopoietic system in acutely irradiated mice.Methods The total body irradiation doses were 6.0Gy and 8.0Gy respectively at 1.01Gy/min rate. Sixty healthy male C57BL/6 mice were randomly divided into control group, model group, positive-drug (523) group and cimetidine groups (33.3mg/kg, 100mg/kg and 300mg/kg). Each group had ten mice. The mice were given intragastric administration of cimetidine for 6d before the irradiation in cimetidine groups, and 523 was administered before irradiation once a day for one day in 523 group, and at 5h after irradiation, was given again. The 30d survival rate after 8.0Gy irradiation was recorded. The peripheral blood cells, bone marrow DNA content and frequency of micronucleated polychromatic erythrocytes (fMNPCE) were determined 30d after 6.0Gy irradiation.Results After 8.0Gy irradiation, all the mice died on 21th day in model control group. The survival rates in cimetidine groups were 50%, 20% and 30%, respectively. After 6.0Gy irradiation on 30th day, compared with control group, the peripheral white blood cells (WBC) and bone marrow DNA content were decreased significantly (P<0.01,P<0.05) in model group, and fMNPCE was increased significantly (P<0.05). Compared with model group, WBC was significantly increased in 300mg/kg cimetidine group (P<0.01). In cimetidine groups, the bone marrow DNA content was increased significantly after irradiation (P<0.01 orP<0.05), and the fMNPCE was decreased significantly (P<0.01 orP<0.05) and tended towards normal.Conclusion Cimetidine could improve 30d survival rate of acutely irradiated mice and has good protective effect on hematopoietic system.

Objective To investigate the regulatory effect of miRNA-370(miR-370)on the expression of tumor suppressor gene p21 in renal cell carcinoma cell lines ACHN and 786-O and its effect on cell growth. Methods RCC cells were transfected with dsRNA known lack homology to human genes (control group) and miR-370 (experimental group) by Lipofectamine 3000 respectively. Real-time fluorescence quantitative polynucleotide chain reaction (RT-qPCR) and Western blot were used to detect the expression of p21 mRNA and protein. The cell cycle distribution was identified by flow cytometry (FCM). Cell viability and proliferation ability were measured by cell viability assay (MTS) and colony culture assay. Results The expression of p21 mRNA in ACTN and 786-O cells in control group was 1.04±0.33, 1.04±0.31, respectively. The expression of p21 mRNA in experimental group was significantly increased by 3.68±0.62 (t=7.535, P<0.001), 3.15±0.29 (t=9.975, P<0.001). Western blot further demonstrated that the increased expression of p21 protein in both renal cell lines was consistent with the upregulation of p21 mRNA level. FCM results showed that the cell cycle of more cells was blocked in G0-G1phase after transfection of miR-370.MTS results showed that after transfection of miR-370,the number of colonies formed by ACHN and 786-O cells in the control group was 113±30 and 106±27 respectively. The number of colonies formed by experimental group was significantly reduced by 53±17 (t=2.982, P=0.041) and 50±16 (t=3.089, P=0.037). Conclusion miR-370 can significantly up-regulate the expression of tumor suppressor gene p21 in renal cell carcinoma and inhibit the growth of renal cell carcinoma.

Objective:To prepare old tea buccal tablets using wet granulation method and optimize the preparation technology. Methods:The amount of each adjuvant was studied by single factor experiments, and the formula of the buccal tablets was optimized by the orthogonal experiments using taste and disintegration time as indices. Results:The optimal formula was composed of old tea ex-tract 30 g,mannitol 60g,PEG6000 20 g,aspartame 10 g,citric acid 10 g and menthol crystal 1 g. All the tested indices including ap-pearance, hardness and disintegration time met the requirements described in Chinese pharmacopeia. Conclusion: The preparation technology is reasonable and feasible for the industrial production.

Objective:To establish the quality standard for Sipunculus nudus polysaccharide. Methods:The water content, igni-tion residues and heavy metals in Sipunculus nudus polysaccharide were determined. Authrone-sulfuric acid colorimetry was used to de-termine the polysaccharide content. Results: The water content in the polysaccharide should not exceed 5. 0%, ignition residues should not exceed 1. 0%, and the content of heavy metals should not exceed 20 ppm. The polysaccharide content should exceed 80%( glucose) . Conclusion:The method is accurate and simple, and can be effectively used in the quality control of polysaccharide in Sipunculus nudus Linnaeu.

Objective To investigate the combined biological effects of low dose radiation,carbon monoxide,benzene and noise on rats.Methods Sixteen male SD rats were randomly divided into experiment group and control group.The experiment group was exposed to carbon monoxide,benzene,low dose radiation and noise daily,the control group was in common environment.Peripheral blood,organ index,and marrow DNA content were detected.Two-dimensional electrophoresis (2-DE) was performed on serum protein analysis.Differential expressed proteins were identified by a matrix assisted laser desorption/ionization time of flight mass spectrometry (MAIDI-TOF-MS).Results Compared to control group,the liver index,spleen index,thymus index,leukocytes,platelets count,and marrow DNA content of the experiment group were decreased significantly (t =2.732,4.141,3.053,2.211,2.668,11.592,P ＜0.05).12 altered proteins were detected and through identification,3 proteins were definite in terms of serum amyloid A-4 protein (SAA4),trichoplein keratin filament-binding protein (TCHP) and tubulin alpha-4A chain (TUBA4A).Conclusions The hematopoietic system and immune system of rats are damaged significantly with the changes of several serum protein expressions by the combined exposure of low dose radiation,carbon monoxide,benzene and noise.This study may provide new information for the mechanism of the combination effects.