OBJECTIVE:To detect the protein expression of Skp1(S-phase kinase-associated protein 1) in non-small cancer lung cancer (NSCLC) tissues and peripheral blood,and to explore its clinical significance.METHODS:Expressions of Skp1 protein in 34 NSCLC tissues and 34 adjacent tissues were detected by Western Blot.In addition,the expressions in 86 NSCLC tissues and 86 adjacent tissues were detected by tissue microarray and immunohistochemical staining method.The plasma expression level of Skp1 in 39 NSCLC patients and 27 healthy person was detected by ELISA (enzyme linked immunesorbent assay).The relationships between Skp1 expression with clinicopathological parameters and prognosis of NSCLC patients were analyzed.RESULTS:The expression level of Skp1 protein in NSCLC tissues was significantly higher than that in adjacent tissues by both Western blot and immunohistochemical staining methods (P < 0.01).The plasma levels in NSCLC patients were significantly higher than that in healthy person (P=0.003).The simple factors analyses show that over-expression of Skp1 protein in NSCLC tissues was significantly associated with poor prognosis in the middle to late cancer stages (Ⅱ+Ⅲ)(P=0.001),and the multiple factors Cox regression analysis showed that Skp1 protein could be used as the independent risk factors for the middle to late stages (Ⅱ+Ⅲ).The expression of Skp1 protein had no correlation with pathological type,clinical stage,pathological grade,lymph node metastasis and other factors (P > 0.05).CONCLUSION:Skp1 protein was over-expressed in NSCLC tissues and plasma from patients,and the over-expression of Skp1 protein in NSCLC tissues was significantly associated with poor prognosis in the middle to late cancer stages (Ⅱ+Ⅲ).Therefore,the over-expression can be considered for use as an independent risk factor for the middle to late stages among NSCLC patients.

OBJECTIVE:To investigate the influence of proanthocyanidin on amyloid β-peptide (25-35)(Aβ_25-35)-mediated tau hyperphosphorylation and p38 MAPK pathway signaling in SH-SY5Y cells.METHODS:SH-SY5Y cells were exposed to 1.0 μmol/L Aβ_25-35 to establish an Alzheimer's disease (AD) model in vitro.The study involved,a control group (cells and medium only),Aβ_25-35 intervention group,Aβ_25-35+different concentrations (0.1,1.0,2.5,5.0 μg/mL) of proanthocyanidin treatment group and p38 pathway blocker SB203580 group,Aβ_25-35+SB203580 group,Aβ_25-35+5.0 μg/mL proanthocyanidin intervention group.MTT assay was used to detect vitality of SH-SY5Y cells.Content of MDA in the cells was detected by TBA method and the total level of SOD was detected by WST-1 method.Expression of p-tau,tau,p38,p-p38 protein and p-tau,tau,p38,p-p38 levels after p38 pathway blocker were detected by Western blot analysis.RESULTS:Proanthocyanidin showed no obvious toxic effects on SH-SY5Y cells but,different concentrations of Aβ_25-35 decreased the survival rate of these cells.Compared with the control group,the level of SOD was decreased in 1.0 μmol/L Aβ_25-35 group,the content of MDA was increased (P < 0.05) and the expression of p-tau (Ser396) and p-p38 protein was also increased (P < 0.05).After the intervention with different concentrations of proanthocyanidin and p38 signaling pathway blocker,and compared with the 1.0 μmol/L Aβ_25-35 group,PC inhibited the expression levels of p-tau (Ser396) and p-p38 protein (P < 0.05),improved the vitality of SH-SY5Y cells and the total SOD level,Moreover,the MDA content was decreased (P < 0.05),and Aβ+blocker group down-regulated protein expression levels (P < 0.05).CONCLUSION:Proanthocyanidin inhibited hyper-phosphorylation of tau protein which was mediated by Aβ_25-35,improved the vitality of cells and reduced oxidative stress level.In addition,the inhibitory effect of proanthocyanidin on p38MAPK signaling pathway was of great significance to neuroprotection by inhibiting the hyperphosphorylation of tau protein.

OBJECTIVE:To assess the associations between polymorphism of DNA methyltransferase 3B-149C > T (DNMT3B-149C > T) and cancer risk.METHODS:We evaluated and extracted the relevant literatures published from 1990 to 2017 based on Newcastle-Ottawa Scale (NOS).All genotype models of DNMT3B-149C > T were compared,including CC and TT,CT and TT,CC+CT and TT,and their distillations in case and control groups were comprehensively analyzed.Q-test and I2 value were used to analyze the heterogeneity among the studies.Begg and Egger regressions were used to verify the evaluation of publication bias,and Review Manager 5.0 and STATA version 12.0 software were used for Meta-analysis.The correlation between polymorphism of DNMT3B-149C > T and cancer risk was calculated by using the odds ratio (OR) and 95% confidence interval (95% CI).RESULTS:7 612 cancer cases and 9 679 healthy controls in the literature were analyzed.According to subgroup analysis of different cancer types,the DNMT3B-149C>T polymorphism was associated with lung cancer[OR=1.51,95%CI(1.08,2.12),P=0.02].CONCLUSION:DNMT3B-149C > T polymorphism may be a susceptibility factor for lung cancer.

OBJECTIVE:To investigate the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alone or in combination with high fat diet (HFD) on glucose and lipid metabolism in mice.METHODS:60 C57BL/6J mice were randomly divided into 4 control and treatment groups.All mice were evaluated for glucose and lipid metabolic functions through measurement of body weight,weights of liver and adipose tissues,glucose tolerance,and insulin tolerance,determined mitochondrial membrane potential and ROS level using specific probes,and measured the mRNA expression of Nrf2,antioxidant enzymes and inflammatory factors.RESULTS:TCDD or HFD alone could induce significant glucose and lipid metabolic disorders,as evidenced by increased adipose weight,increased blood glucose level,impaired tolerance of glucose and insulin.In addition,TCDD aggravated HFD-induced impairment of glucose and lipid metabolism.In liver,TCDD or HFD alone resulted in hyperpolarization of mitochondrial membrane potential and increased levels of ROS.TCDD and HFD induced a more significant mitochondrial dysfunction and increase in ROS.In adipose tissues,TCDD alone or in combination with HFD increased ROS level and perturbed HFD-induced ROS increase.In addition,TCDD decreased the mRNA expression of Nrf2,GCLc,and SOD2.In combination with HFD,the effect of TCDD on the mRNA expression of Nrf2,GCLc,and SOD2 was significantly enhanced.Either TCDD or HFD increased the mRNA expression of IL-1α,IL-6,and MCP-1.TCDD aggravated the HFD-induced increase of these inflammatory factors.CONSLUSION:TCDD and HFD induced glucose and lipid metabolic disorders in mice.In addition,they functioned cooperatively to affect glucose metabolic parameters,oxidative stress indexes,and expression of inflammatory factors In fact,the effects of TCDD in combination with HFD were stronger than the additive effects from each acting alone.The decrease of Nrf2,the downstream effects on antioxidant enzymes and the increase of inflammatory factors may be a key mechanism responsible for the observed cooperative effects.

OBJECTIVE:Using bioinformatics method,the data of pancreatic cancer in Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA) database were analyzed to evaluate the differential expression and prognostic value of lncRNA and mRNA in pancreatic cancers.METHODS:Firstly,we annotated the data of pancreatic cancers into GEO database to extract the information on mRNA and lncRNA.By intersecting the mRNA and lncRNA in the chip,we obtained the differentially expressed mRNA and lncRNA.Then,genetic analyses of the differentially expressed mRNA and lncRNA were carried out.At the same time,survival analysis was carried out with the relevant data of pancreatic cancers in the TCGA database.RESULTS:With the gene analyses,we obtained 1 147 differentially expressed mRNAs and 336 differentially expressed IncRNAs,which were reliability related to pancreatic cancer.Through gene ontology analyses and Kyoto encyclopedia of genes and genomes analyses,networks on lncRNA-mRNA co-expression,lncRNA-mRNA-pathways,signal pathways and protein interactions were constructed.The results show that mRNAs and lncRNAs had an important impact on the occurrence and development of pancreatic cancers.Survival analysis revealed that 12 lncRNAs were associated with the prognosis of pancreatic cancer,namely ATP1A1-AS1,CBR3-AS1,CTD-3080P12.3,FAM66D,FAM87A,FLJ38576,LINC00476,LINC00574,LINC01554,PY2,LINC00857,OVOL1-AS1.CONCLUSION:Several LncRNA demonstrated a significant association with the occurrence,development and prognosis of pancreatic cancers.Therefore,they are expected to provide new therapeutic targets and molecular markers for targeted therapy as well as prognosis evaluation of pancreatic cancers.

OBJECTIVE:To investigate the effects of nano-TiO₂ on expression of inflammatory cytokines and phosphorylation of JAK2/STAT3 in IEC-6 cells.METHODS:IEC-6 cells in cultures were treated with different doses (0.0,5.0,10.0,15.0,20.0 μg/mL) of nano-TiO₂ for 24 h.MTT assay was used to detect cell viability.The levels of inflammatory cytokines (IL-1β,IL-6 and TNF-α) were determined by ELISA assay.Protein expression and phosphorylated levels of JAK2 and STAT3 were measured by Western blot and the gray values of protein bands were assessed by Image J software.RESULTS:Compared with the control group,viability of IEC-6 cells was barely affected by nano-TiO₂.From the treatments with all doses of nano-TiO₂,IL-6 and TNF-α were highly secreted (P < 0.05),and significant secretion of IL-1β with the exception from the lowest dose of TiO₂,(P < 0.05).Western bolt results indicate the levels of p-JAK2 and p-STAT3 were increased after exposure of nano-TiO₂ and had a positive relationship with expressions of secreted inflammatory factors (r_JAK2,IL-1β=0.561,P < 0.05;r_JAK2,IL-6=0.711,P < 0.01;r_JAK2,TNF-α=0.675,P < 0.01;r_STAT3,IL-1β=0.782,P < 0.01;r_STAT3,IL-6=0.532,P < 0.05;r_STAT3,TNF-α=0.668,P < 0.01).The gray values of phosphorylated JAK2 and STAT3 increased when IEC-6 cells were exposed to 15.0 and 20.0 μg/mL nano-TiO₂(P < 0.05).CONCLUSION:Nano-TiO₂ exposure caused significant perturbation in expression of inflammatory cytokines and phosphorylation of JAK2 and STAT3 in IEC-6 cells.

OBJECTIVES:Use of two methods to compare the cytotoxicity of CuO nanoparticles on brain microvascular endothelial cells (BMECs) in neonatal rats.METHODS:Growth curves of BMECs were determined by Real-Time Cell Analysis (RTCA) which was used to determine the optimal number of cells to be inoculated and the optimal exposure time for the toxicity experiment.Cells were treated with CuO-NPs at 1.5,0.75,0.38,0.19,0.09 mg/mL,and the culture medium without cell was used as control (0 mg/mL).The cytotoxicity of CuO-NPs was analyzed by two methods:RTCA and Resazurin.Correlations of the two methods were determined by comparing the IC₅₀ values obtained.RESULTS:The growth curves of BMECs show that 2.5×10³ cells/well was the optimal inoculation concentration,and 40 h after inoculation was the best exposure time.RTCA results show that BMECs were transiently unstable at 2-3 h after exposure to 1.5,0.75,and 0.38 mg/mL CuO-NPs,followed by progressive death.The cell viability of BMECs at the 1.5 and 0.75 mg/mL groups began to decrease at about 2 h after exposure,and all cells died at 4 h.The cell viability was measured by the Resazurin method at 3,12,24,36,and 48 h after exposure from the same doses.The Resazurin method yielded similar results to RTCA,ie,the 1.5 and 0.75 mg/mL groups had the greatest cytotoxicity,and the cytotoxicity of CuO-NPs on BMECs showed a dose-dependent trend.Results from correlation analyses show that IC₅₀ obtained by the two methods has a good correlation,and correlation coefficient was 0.999,P=0.000.Paired t test results show the difference was not statistically significant (t=1.125,P=0.323).From the calculation of R² value of IC₅₀,R² value of RTCA fitting curve is higher than R² value of the Resazurin method Logistic regression coefficient.CONCLUSION:CuO-NPs demonstrated dose-dependent cytotoxic effects on BMECs in vitro.The results of cytotoxicity of BMECs as determined by the RTCA and Resazurin assays were related,but results obtained by the RTCA method appear to be more accurate.Therefore,RTCA may be more suitable for the research of cytotoxicity of CuO-NPs.

OBJECTIVE:To investigate the effect of di (2-ethylhexyl) phthalate (DEHP) on the reproductive capacity of nematode Caenorhabditis elegans(C.elegans).METHODS:C.elegans cultures were organized into several groups:control (K solution) and three DEHP exposure groups:low (0.1 mg/L),medium (1.0 mg/L) and high (10.0 mg/L).Reproductive capacities were evaluated based on brood size,generation time and egg-laying rate/h.Dianamidophenylindole (DAPI) staining was used to observe the number of germline cells in the mitotic zone and the meiotic region of nematodes.Using transgenic MD701 nematodes,apoptotic cells in the pachytene zone were observed with the aid of fluorescence microscopy.RESULTS:Compared with control group,brood sizes of C.elegans were significantly decreased in the 1.0 mg/L and 10.0 mg/L exposed groups (P < 0.05),but had no significant effect on generation time (P>0.05).With the increase of exposure dose,the egg-laying rates were significantly decreased (P < 0.01) and showed a dose-effect relationship.In addition,the total number of germline cells and the number of germline cells in the mitotic region were significantly decreased in the 1.0 mg/L (P < 0.01) and 10.0 mg/L (P < 0.01) exposed group.The number of germline cells in the meiotic region also showed a downward trend.Compared with the control group,the germline cell numbers in the meiotic region were decreased significantly at 10.0 mg/L exposure concentration group (P < 0.01).With the increase of the dose,the germ cells showing apoptosis in the pachytene zone were also significantly increased (P < 0.01).CONCLUSION:DEHP caused arrest in mitotic proliferation and reduction in the number of germ cells in the meiotic zone.The observed toxicity was related to the increased number of apoptosis caused by DEHP in the pachytene of C.elegans.

OBJECTIVE:To explore the effects of paraquat (PQ) on blood-brain barrier and cognitive,learning and memory-related functions in rats.METHODS:SPF grade SD male rats were randomly divided into three groups:control (normal saline),low dose PQ exposure (1 mg/kg) and high dose exposure (10 mg/kg) groups.There were 15 rats per group and PQ was injected intraperitoneally for 6 weeks at 2 times a week.After the exposure,5 rats in each group were randomly selected for the Evans blue brain penetration test to detect the permeability of the blood-brain barrier.The remaining 10 rats in each group were subjected to behavioral experiments for their cognitive,learning,and memory abilities,including Morris water maze test,platform test,and shuttle box test.RESULTS:Compared with the control group,the content of Evans blue increased in the brain tissue of each dose group,the difference was statistically significant (P < 0.05).Morris water maze results show that the trajectories of rats in the 1 and 10 mg/kg PQ groups were more complex than those in the control group.With the increase of training days,the escape latency of each group was gradually shortened.The shortening trend of the 10 mg/kg PQ group was significantly lower than that of the control and the low dose groups (P < 0.05).During the test period,the number of crossings in the 1 and 10 mg/kg PQ group was significantly reduced compared with the control group (P < 0.05).The results of the platform test show that the memory latency of the 1 and 10 mg/kg PQ group was significantly lower than that of the control group,and the number of errors was significantly higher than that of the control group,and the number of errors in the 10 mg/kg PQ group was significantly higher than that in the 1 mg/kg PQ group (P < 0.05).The results of the shuttle box experiment show that the number of conditional responses in the two dose groups was significantly lower than that in the control group,and the number of errors was significantly higher than that in the control group (P < 0.05).CONCLUSION:Both 1 and 10 mg/kg doses of PQ damaged the blood-brain barrier and increased its permeability which in turn reduced cognitive,learning and memory functions.

OBJECTIVE:To investigate the kidney protective effects of lycium barbarum polysaccharide (LBP) in alcohol-induced liver injury in mice.METHODS:60 male mice were randomly divided into five groups,control,model and treatment with different concentrations of LBP (75,150,300 mg/kg) groups.Alcoholic liver injury model was induced by 50% ethanol for 7 days.At 16 h after the last treatment,serum and kidney samples were obtained for the determination of biochemical markers.The concentrations of GLU,BUN and CREA in serum were determined by automatic biochemical analyzer,and the levels of superoxide dismutase (SOD),glutathione (GSH),malondialdehyde (MDA) and inflammatory factor TNF-α,IL-1β in kidneys were measured by commercial kits.RESULTS:Compared with the control group,the weights of mice in the model group were decreased significantly (P < 0.01) but,the kidney indexes were increased significantly (P < 0.01),indicating that alcohol caused organ damage in mice.Compared with the model group,the average weight of middle dose group was increased significantly (P < 0.05),and the kidney indexes for the three dose groups were decreased significantly (P < 0.05).The contents of GLU in the medium and high dose groups (7.63、7.82 mmol/L) were decreased significantly (P < 0.05).In addition,the contents of BUN and CREA in high dose group were significantly decreased (P < 0.05),the activities of SOD and contents of GSH were increased significantly (P < 0.01),and the contents of MDA were decreased significantly (P < 0.05) in all of treatment groups.The contents of TNF-α were reduced significantly (P < 0.05),the contents of IL-1β were reduced significantly (P < 0.05) only in high dose group compared with model group.CONCLUSION:Our study demonstrates that lycium barbarum polysaccharide have kidney protective effect on alcohol-induced alcoholic liver injury.