OBJECTIVE: To explore the role of cofilin for radiation damage via changes of microfilament morphology and cofilin phosphorylation which were induced by γ-ray in thymus lymphocytes of mice. METHODS: 36 mice were grouped randomly according to the time after radiation into control (0 h),3,6,9,12 and 24 h. Thymus lymphocytes were collected at the mentioned time points after exposure to 2 Gy γ-rays. Cofilin 1 in lymphocytes was detected by Western blot to make decision for the suitable time point. 24 mice were grouped randomly according to the dose of γ-ray,including 4 groups of control without radiation (0 Gy),2,6,10 Gy. Three hours after γ-ray exposure,lymphocytes were collected to observe some changes of microfilament morphology and cofilin 1 phosphorylation,including the expression of cofilin 1,p-cofilin,LIMK1,TESK2 and SSH2. RESULTS: mRNA and protein expression of cofilin were down regulated (P < 0.05),the time point of 3 h after γ-ray exposure was the suitable time for collecting thymus lymphocytes to test the expression of cofilin and its related proteins. The results show that expression of cofilin increased with increasing doses of radiation,especially the group of 10 Gy was significantly different (P < 0.05). On the contrary,expression of p-cofilin was reduced,especially the group of 10 Gy was significantly different (P < 0.05). The expression of LIMK1,TESK2 and SSH2 looked similar to controls(P > 0.05). CONCLUSION: The increased expression of SSH2 caused p-cofilin dephosphorylating at Ser3,indicating the quantity of cofilin 1 increased in cytoplasm to take part in microfilament assembly. As a result,movement ability of the lymphocytes was enhanced.

OBJECTIVE: To evaluate expression of ring finger protein 2(RNF2) and proliferating cell nuclear antigen(PCNA) in tumor tissue of patients with esophageal squamous cancer cells and their clinial significance. METHODS: By using immunohistochemical method,the expression of RNF2 and PCNA in tumor tissues and the tissues adjacent to tumors were detected in 64 cases of esophageal squamous cell cancer and in 30 cases of tissues adjacent to tumors. Relationships between their expression,and clinical pathology and prognosis were analyzed. The relationship between positive expression of RNF2 and postoperative 5-year survival rates of patients with esophageal cancers was also explored. RESULTS: RNF2 and PCNA proteins showed high expression in the tumor tissues. The positive expression rate of RNF2 was 54.69%(35/64) in the tumor tissues and 30.00%(9/30) in the adjacent tissues. The difference was statistically significant (χ²=5.000,P < 0.05). The positive expression rate of PCNA was 60.94%(39/64) in the tumor tissues and 46.67%(14/30) in the adjacent tissues. The difference was statistically significant (χ²=6.237,P < 0.05). The expressions of RNF2 and PCNA were closely related to the tumor size,TNM stage and lymph node metastasis (P < 0.05). However,no correlations were observed between the expression of RNF2 and other clinical features such as patient age,gender,and histological grade (P > 0.05). Among the esophageal cancer group,PCNA protein expression was significantly and positively correlated with RNF2 protein expression (r=0.494,P < 0.01). In addition,the survival time of patients with RNF2 positive expression was shorter(P < 0.01). CONCLUSION: RNF2 and PCNA expressions were found to be closely related and they also showed synergistic effects in the development of esophageal cancer.

OBJECTIVE: To investigate differential gene expression of supernatant proteins from Cronobacter sakazakii on cytopathy of HepG2 cells and to understand molecular interactions in pathopoiesis. METHODS: The CAYE culture medium was used to culture Enterobacter cloacae,and supernatant proteins were isolated and purified by the ammonium sulfate precipitation method. The supernatant proteins were added to the 1×10⁶/mL HepG2 hepatocyte culture at a final concentration of 1.6 μg/mL as the experimental group and distilled water was added as the control group. After 8 h of treatment,total RNA was extracted,the cDNA library was constructed and were sequenced using Illumina. The difference in gene expression between the two groups was analyzed. The function of differentially expressed genes was analyzed by the Gene Ontology (GO) enrichment analysis and Pathway analysis. Differential expression of RPS18 and RFC4 genes were validated using qPCR. RESULTS: High throughput sequencing results showed that there were 5 120 differentially expressed genes:2 155 were up-regulated and 2 965 were down-regulated. GO enrichment analyses showed that the differentially expressed genes were mainly involved in several biological processes,including mitosis,caryomitosis,replication,etc. Pathway analysis showed that the differentially expressed genes were mainly involved in DNA replication,protein transport,and arginine and proline metabolism. The results of qPCR and high-throughput sequencing were consistent. CONCLUSION: The supernatant proteins of Cronobacter sakazakii caused HepG2 liver cell damage which might be related to changes of ribosome and/or DNA replication,and repair with differential expression of RPS18 and RFC4 genes.

OBJECTIVE: To investigate the costimulatory molecule B7 homolog 4 (B7-H4) expression during progression of esophageal precancerous lesions in mice and to evaluate its clinical significance in esophageal squamous cell carcinoma formation. METHODS: 133 C57BL/6 mice were assigned to the control group (n=42) or 4NQO model group (n=91). 50 μg/mL 4-Nitroquinoline -1-oxide (4NQO) in drinking water was fed to the 4NQO model mice for 15 weeks. Hematoxylin eosin (HE) staining was used to evaluate changes in pathological stages of esophageal tissues. B7-H4 protein expression in esophageal tissues was detected by immunohistochemical staining and Western blot. B7-H4 protein expression in serum was detected by Western blot. RESULTS: From 16th to 28th week,esophageal showed visible pathological changes,such as incrassation,pimplings and lumps. Furthermore,HE staining showed that there was a significant and positive correlation between the esophageal pathological grade and the induction time of carcinogenesis,(r=0.55,P < 0.01). Additionally,B7-H4 protein expression in esophageal tissue was positively correlated with the pathological grade of precancerous lesions(r=0.57,P < 0.01). At 26 th and 28 th week, B7-H4 protein expression increased significantly compared with the control group(P < 0.05 or P < 0.01). Moreover, B7-H4 protein concentration in sera of mice of high grade intraepithelial neoplasia increased significantly compared with the normal mice,P < 0.05. CONCLUSION: B7-H4 expression in esophageal and sera increased gradually during the progression of esophageal precancerous lesions. Therefore,B7-H4 may be involved in the formation of esophageal precancerous conditions.

OBJECTIVE: To investigate effects of reactive oxygen species (ROS) scavenging on vitamin E succinate(VES)-induced endoplasmic reticulum stress response (ERS) in human gastric cancer SGC-7901 cells. MATERIALS: SGC-7901 human gastric cancer cells were treated with 1,5,10,20 mmol/L N-acetyl-L-cysteine (NAC) for 2 h and then with 20 μg/mL VES for an additional 12 h;ROS production was measured using confocal microscopy. In addition,cells were pretreated with 20 mmol/L NAC for 2 h and then with 20 μg/mL VES for an additional 12 h;there after,ROS production was measured using flow cytometry. In addition,cells were pretreated with 20 mmol/L NAC for 2 h and subsequently treated with 20 μg/mL VES for 15 h and 24 h,CHOP and GRP78 were determined by RT-PCR and Western blot. RESULTS: Pretreatment with NAC drastically decreased VES-induced ROS generation in SGC-7901 cells (P < 0.05). NAC significantly inhibited induction of CHOP and GRP78 mRNA and protein expression in VES-treated SGC-7901 cells (P < 0.05). CONCLUSION: ER stress response is an event downstream of the oxidative stress induced by VES in SGC-7901 cells.

OBJECTIVE: To evaluate the potential immunotoxicity of three traditional Chinese medicines:Mailuoning,Shengmai and Danxiangguanxin,in mice using the direct popliteal lymph node assay (D-PLNA) and reporter antigen popliteal lymph node assay (RA-PLNA). METHODS: In D-PLNA,mice were randomly divided into five groups:Mailuoning,Shengmai and Danxiangguanxin,normal saline control and diphenylhydantoin (DPH) positive control. In RA-PLNA,the above groups were combined administration with reporter antigens TNP-Ficoll or TNP-OVA. Mice were randomly assigned to their respective treatment groups,8 in each group and received a subcutaneous injection into left hind footpad with or without RAs in a final volume of 50 μL. At 7 days after injection,mice were sacrificed and popliteal lymph nodes were excised and then grinded to prepare single cell suspensions. Cell number was counted and cell index was calculated. Concentration of IgE and MCP-1 cell supernatant were measured by ELISA assay after cell suspension was cultured with stimulant in a 96-well plate. RESULTS: In D-PLNA,compared with the control group,Danxiangguanxin injection increased PLN cellularity significantly (P < 0.05). In RA-PLNA,each treatment group significantly increased PLN cellularity and the concentration of MCP-1 when co-injected with TNP-Ficoll(P < 0.05). When co-injected with TNP-OVA,Mailuoning stimulated significant secretion of IgE while Danxiangguanxin increased the concentration of IgE and MCP-1(P < 0.05). CONCLUSION: Mailuoning and Shengmai injection only caused positive reaction after RAs were added. This suggests they may contain low molecular weight compounds as hapten to induce type I hypersensitivity reaction. However,the composition of Danxiangguanxin is complex and may directly induce type I hypersensitivity reaction,and may also act as a neo-antigen or neo-epitope to elicit immune response.

OBJECTIVE: To investigate the effect of a new type of organic selenium compound ethaselen (BBSKE) on growth and proliferation of a human tongue cancer CAL27 cell line. METHODS: In vitro cell assay MTT,Hoechst 33258 immunofluorescence staining,annexin V-FITC/PI staining flow cytometry,transwell chamber assay were performed to determine effects of 2,5,10,20 μmol/L (containing 1‰ DMSO) of BBSKE on CAL27 proliferation,and cell cycle and migration compared with solvent control and normal control groups. RESULTS: CAL27 cells treated with 5,10,and 20 μmol/L BBSKE had significantly reduced proliferation compared with untreated cultures (P < 0.01),and with further reduction in time. With the Hoechst 33258 immunofluorescence staining,the morphology of cells in the 5 and 10 μmol/L BBSKE groups was changed compared to the control group. Annexin V-FITC/PI staining flow cytometry results showed that the G2/M phase ratios in the 2,5,10,and 20 μmol/L BBSKE group were increased compared to the control group (P < 0.01),with blockage in the G2/M phase (P < 0.01). Results from the transwell chamber assay showed that the number of CAL27 cells exhibiting migration in the 2,5,10,and 20 μmol/L BBSKE groups were significantly lower than that in the control group (P < 0.01). The number of migrating cells in the 20 μmol/L BBSKE group was the least. In addition,the migration number and the concentration of drug were inversely proportional. CONCLUSION: BBSKE demonstrated significant inhibitory effect on CAL27 cells,induced cell apoptosis by arresting the cell cycle in the G2/M phase and inhibited cell migration. These observations suggest that BBSKE can be used as an alternative drug for chemotherapy in patients with TSCC.

OBJECTIVE: To explore the environmental risk of butachlor on aquatic organisms. METHODS: 0.4,0.8 and 1.2 mL/L concentrations of butachlor were prepared according to 24 h-LC₅₀. Carassius auratus were administered by intraperitoneal injection with 0.1 mL/g dose of each concentration for 24 h. PAGE method was used to detect the activity of EST and POD isozyme. RESULTS: EST and POD isozyme in the hearts of C. auratus were not expressed in the control group but was more than 50 times (in 1.2 mL/L) that of the control group. With increased concentrations of butachlor,the activity of EST isozyme in hepatopancreas gradient attenuated while the POD decreased followed by increased. The activities of EST isoenzymes in gill and kidney were 2 times and 3 times higher than those in the control group at 0.4 mL/L,respectively. In addition,the activities of EST and POD isoenzymes in gill and kidney disappeared at 0.8 mL/L and re-expressed at 1.2 mL/L. CONCLUSION: The concentration of butachlor and the activity of EST and POD isozyme in C. auratus show dose-effect relationship. Butachlor with medium concentration (0.8 mL/L) was at risk for EST and POD isozyme in gill and kidney of C. auratus.

OBJECTIVE: To investigate expression of the WFDC1 gene in hepatocellular tissues and cell lines and their clinicopathological values. METHODS: Expression of WFDC1 mRNA was detected using qPCR in tumor and adjacent tissues from patients with liver cancer,as well as in MC-LR-induced L02 malignantly transformed cells and in hepatocellular carcinoma cell lines. Expression in 331 cases of tumor tissues and 50 cases of adjacent tissues was analyzed based on the TCGA database. The relationship between expression of WFDC1 and survival and clinicopathological parameters was also studied. RESULTS: Results from the qPCR analyses show that expression of WFDC1 was significantly down-regulated in hepatocellular carcinoma tissues,MC-LR-induced L02 malignantly transformed cells and hepatocellular carcinoma cells (P < 0.05). The expression of WFDC1 in paracancerous tissues was significantly higher than that in tumor tissues in TCGA database (P < 0.01). Kaplan-Meier analysis show that patients with high expression of WFDC1 had longer survival time than those with low expression (Log-rank=4.80,P < 0.05). The results of ROC curve analysis show that the expression of WFDC1 was a specific and sensitive indicator of liver cancer (AUC=0.829). CONCLUSION: WFDC1 may be a tumor suppressor gene which can possibly provide reference value for diagnosis and survival prognosis of patients with liver cancer.

OBJECTIVE: This study aimed to investigate the clinical significance in expression of Mda-7/IL-24 and C-myb in diffuse large B-cell lymphoma (DLBCL) tissues. METHODS: Expression of Mda-7/IL-24 and C-myb in 72 tissues from DLBCL patients and 36 from normal lymphonodus tissues were assayed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The relationship of Mda-7/IL-24 and C-myb expression and age,sex,clinical stage,bone marrow infiltration or not,Ecog scores,IPI index and survival was analyzed,and survival curves were prepared. RESULTS: The results show that compared with normal lymphonodus tissues,expression of Mda-7/IL-24 mRNA was higher,whereas expression of C-myb mRNA was lower. The expression of Mda-7/IL-24 mRNA was negatively correlated with C-myb (r=-0.43,P < 0.01). Low expression of Mda-7/IL-24 or high expression of C-myb were positively correlated with malignant grade,bone morrow metastases,IPI scores and shorter survival period (all P < 0.05). CONCLUSION: In summary,there was a negative correlation between Mda-7/IL-24 and C-myb expression in DLBCL tissues. The low expression of Mda-7/IL-24 and high expression of C-myb could be used as predictors for poor clinical conditions and prognosis of DLBCL.

OBJECTIVE: To explore whether the intraperitoneal administration of Cionbufagin has an analgesic effect on cancer-induced bone pain (BICP) rats,and to assess whether the inhibition of astrocytic and microglia activation is involved in the analgesic effects of Cionbufagin. METHODS: A total of 48 adult female Sprague-Dawley (SD) rats were divided into the following groups with 12 rats per group:Control,Sham,CIBP,and Cionbufagin. The groups of CIBP and Cionbufagin rats were prepared by injection of 20 μL Walker 256 breast cancer cell suspension into the tibial bone marrow cavity. For the sham-operated group,20 μL PBS was used to replace the tumor cells injected into the tibia. Cionbufagin was injected intraperitoneally on the seventh day post-operation and once daily for 7 days whereas normal saline was used for the other groups. Mechanical withdrawal threshold and radiant heat threshold of hind paws of rats were measured to evaluate analgesic effects of Cionbufagin. Moreover,tibia was detected by X-ray assay on the seventh day after operation. Immunofluorescence staining was used to determine the expression of spinal astrocytes and microglial activation. Enzyme-linked immunosorbent assay (ELISA) was used to detect expression of pro-inflammatory cytokines[interleukin (IL)-1β and tumor necrosis factor (TNF)-α]. RESULTS: After operation,mechanical pain and thermal pain threshold decreased in the BICP group,and the differences were statistically significant compared with the control and sham groups on the 7th day (P < 0.01). After injected with Cinobufagin,the pain threshold of the Cinobufagin group was increased and the difference was statistically significant compared with the CIBP group (P < 0.01). X-ray assay showed tibia were obviously broken,suggesting that model was successful. The activation of microglia and astrocytes in the spinal cord in BICP group were higher than that of the sham group (P < 0.05),the activation of microglia and astrocytes of the Cinobufagin group was significantly lower than that of the CIBP group (P < 0.05). The concentration of TNF-α and IL-1β in peripheral blood and spinal cord in BICP group were increased when compared with the sham rats,but there were low on the Cionbufagin group when compared with the BICP group (P < 0.05). CONCLUSION: The study showed that Cionbufagin has an analgesic effect on CIBP rats,the analgesic effects were mainly mediated through inhibition of activation of microglia and astrocytes in the spinal cord.

OBJECTIVE: To investigate effect from exposure to fine particles on development of zebrafish embryos. METHODS: Zebrafish embryos were exposed to different concentrations (0,5,20,80,320 and 800 μg/mL) of fine particles for 96 h. Their survival and hatchability were observed under stereoscopic microscope at 0,2,4,6,8,16,24,48,72,96 h after the exposures. RESULTS: Compared with the control group,increased embryo mortality was observed in each of the treatment groups at 48 h after exposure,especially in the 80,320,800 μg/mL groups. Embryo hatchability was decreased with increased fine particle concentrations. Embryos did not hatched well in the 80 μg/mL group,and all embryos died in the 320,800 μg/mL groups at 48 h after exposure. Median lethal dose (LD₅₀) of fine particles to zebrafish embryos was calculated to be (56.6±2.83) μg/mL. CONCLUSION: Exposure to fine particles caused death and hatching failure in zebrafish embryos.