OBJECTIVE: To compare the induction of cytotoxicity,chromosome aberrations and DNA damage among iron oxide nanoparticles (IONPs) with different surface-modifications in A549 cells. METHODS: The effects of amine-modified (Amine-IONP) and polyethylene glycol-modified iron oxide nanoparticles (PEG-IONP) of the same particle size range (5 nm) on cell viability and cell cycle were compared in A549 cells;the content of intracellular reactive oxygen species (ROS),mitochondrial membrane potential (MMP) and endoplasmi reticulum stress (ERS) in cells treated with IONPs were analyzed using high content screening method;the in vitro cytokinesis micronucleus test and Comet assay were performed to evaluate their effects on chromosome and DNA integrity. RESULTS: The growth inhibitory rates of both IONPs to A549 cells were less than 20% after 48 h. Under the same concentration,PEG-IONP exhibited a more significant effect on the G0/G1 cell cycle arrest (P < 0.05):the initial concentration to significantly reduce the cell rate of S phase was 20 μg/mL,and the expression of p21 and p53 was up-regulated by a treatment of 320 μg/mL PEG-IONP for 24 h (P < 0.05). After a 48 h treatment,the initial concentrations to show significant effects of Amine-IONP on ROS,MMP and ER were 20,20 and 80 μg/mL,while for PEG-IONP they were 40, 40 and 160 μg/mL respectively. In addition,compared to PEG-IONP,Amine-IONP was able to induce the formation of micronuclei and Comet tail (the initial concentrations for Amine-IONP were 20 and 80 μg/mL;while PEG-IONP were 40 and 160 μg/mL respectively). After the pre-treatment with oxygen free radical scavengers N-acetylcysteine and butylated hydroxyanisole,both IONPs induced ROS and Comet DNA Tail were significantly inhibited (P < 0.05). CONCLUSION: Positively charged Amine-IONP was more effective in inducing oxidative stress and DNA damage in A549 cells;whereas,PEG-IONP with relatively weaker cytotoxicity and genotoxicity interfered with cell proliferation,and showed advantages in the development of tumor diagnostic reagents.

OBJECTIVE: To explore the influence of expression of human papilloma virus (HPV) on MNNG-induced malignant transformation of Het-1A normal esophageal epithelial cells,and to provide better understanding for pathogenesis of esophageal cancer. METHODS: Cells were divided into HPV (transferred with HPV lentivirus),control (transferred with empty vector),MNNG,and combined HPV and MNNG groups. HPV expression and empty carrier cells were exposed to MNNG (2 μmol/L) for 24 hours per generation. The morphology of these cells was observed at the same time. Functional tests were conducted in cells of the 10^th,20^th and 35^th generations. CCK-8 and cell cycle distribution detection assays were conducted to document changes of viability and proliferation activity. Proliferation indexes were used to monitor proliferation. Apoptosis was detected by Annexin V-APC/PI double staining. Invasion and migration ability of cells were detected by transwell assay. Soft agar colony forming experiments were performed. Nude mice tumorigenicity assay was conducted to detect malignantly transformation. RESULTS: Among the different cell groups,the MNNG and the combined HPV and MNNG groups had the most obvious change in morphology,with the formation of loose,slender and pseudopods. These observations were consistent with that from the CCK-8 and the cell cycle distribution assays. The proliferative activities for the MNNG and the combined HPV and MNNG groups were increased first,then inhibited and finally increased again (P < 0.05). Compared with the MNNG exposure group,the anti-apoptotic ability of the combined HPV and MNNG group was increased (P < 0.05). The results of invasion and migration tests showed that the number of transmembrane in cells of the combined HPV and MNNG group was significantly higher than that in the other groups (P < 0.05) which was followed by the MNNG exposure group. In the soft agar cloning experiment,the colony formation rate of the combined HPV and MNNG group was (30.8±0.2)% higher than that of the MNNG group (27.1±0.3)% (P < 0.05). Cells from the MNNG and the combined groups induced tumors in nude mice while the HPV and the control groups did not form any local mass. CONCLUSION: Combined exposure of HPV and MNNG caused malignant transformation of the Het-1A normal esophageal epithelial cells via significant enhancement of cell proliferation,anti-apoptosis,invasion,cell migration and non-anchorage independent growth. HPV expression could therefore participate in and promote the malignant transformation process.

OBJECTIVE: To investigate effects of chloroquine on cell proliferation and apoptosis of a hepatocellular carcinoma cell line,HepG2. METHODS: Cultured HepG2 cells were treated with various concentrations of chloroquine (0,10,20 and 40 μmol/L). Cell viability was determined by the MTT assay. Morphological changes in cell nuclei was detected using fluorescence microscope at 24 h after treatment. Cell apoptosis was detected by flow cytometry. Expression levels of Caspase-3,Bcl-2,Bax,P-P53,P38MAPK and P-P38MAPK were performed using Western blot analyses. RESULTS: After treatment with 10-40 μmol/L chloroquine for 24,48 and 72 h,proliferation of HepG2 cells was significantly inhibited compared with the control group (P < 0.05). In addition,treatment with 10,20 and 40 μmol/L led to a significant increase in nuclear concentration and shrinkage. Flow cytometry analyses showed that 10,20 and 40 μmol/L treatment significantly induced apoptosis (P < 0.05). Increased in protein expression of P-P53,P-P38MAPK,cleaved Caspase-3 and Bax was detected in the 20 and 40 μmol/L groups while Caspase-3 and P38MAPK did not change. Expression of Bcl-2 was decreased after treatment with 20 and 40 μmol/L. CONCLUSION: Chloroquine inhibited HepG2 cell growth and induced apoptosis. The underlying mechanism might involve the P38MAPK signaling pathway.

OBJECTIVE: To explore the inhibitory effect of the total alkaloids of Tougucao (TTAT) on bone metastasis of breast cancer cells in mice and to investigate the mechanisms. METHODS: Alkaloids contained in the extract was identified by chromogenic reaction. Female nude mice were randomly divided into 6 groups of 7 each:normal (routine feeding),model (only modeling),TTAT low concentration (100 mg/kg TTAT intraperitoneal injection),TTAT high concentration (500 mg/kg TTAT intraperitoneal injection),DOX (5 mg/kg DOX intraperitoneal injection),and ZOL group (0.4 mg/kg ZOL subcutaneous injection after neck). The human breast cancer MDA-MB-231 cells were injected into the tibia to establish the bone metastasis model of breast cancer. The other groups were administered the drugs once every other day for 6 weeks after tumor formation except the normal and the model groups. During the administration,the body weight and tumor volume of each group were measured regularly and the living activities of each group were observed closely. At the end of the experiment,all the nude mice were killed by cervical dislocations,and the right hind limbs were removed at the right hip joint. Imaging,HE staining and TRAP staining were used to observe and evaluate the effect of TTAT on the growth of tibia and tumor tissues of nude mice with tibial bone metastases from breast cancer. The effects of TTAT on the RANKL/OPG ratio and the expression of p-ERK,ERK,p-JNK and JNK in the tumor tissue of the tibial bone metastases of breast cancer were detected by Western blot. RESULTS: 3.28 g was extracted from Tougucao and three alkaloid chromogenic reagents showed positive results,indicating the presence of alkaloids. Compared with the model group,the quality of tumor in the TTAT group decreased significantly (P < 0.01) while imaging scores increased (P < 0.01). The HE staining showed that TTAT significantly inhibited the formation of tumor nests,reduced the nuclear division and protected metastases to the tibia. The TRAP staining results showed that the number of osteoclasts in the TTAT group decreased significantly (P < 0.05). Western blot results showed that TTAT significantly reduced the RANKL/OPG ratio in the tumor tissue of the tibial bone metastases (P < 0.05),and reduced the expression of p-ERK and p-JNK (P < 0.05). CONCLUSION: TTAT significantly inhibited the growth and tibial bone metastasis of breast cancer. The observed effect could be mediated via the OPG/RANK/RANKL signaling pathway.

OBJECTIVE: To find suitable concentration ranges for future urine metabolomics studies. METHODS: Morning urine samples from six volunteers were collected and mixed equally before samples were assigned to three groups. In control group,no preservative was used. In the sodium azide (NaN₃) group,NaN₃ was added to the urine at final concentrations of 0.1,1 and 10 mmol/L. In the boric acid group,boric acid was added with final concentration of 2,20 and 200 mmol/L. All samples were detected using the ultra-performance liquid chromatography tandem quadrupole time of flight mass spectrometry (UPLC/Q-TOF MS/MS) technique and data were analyzed by chemometrics method such as principal component analysis (PCA),partial least-squares discriminant analysis (PLS-DA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA) to compare with metabolic profiles in different treatments. RESULTS: The PLS-DA scores plot show that there was no obvious separate trend between the control group and the three NaN₃ groups. The same observed between the control group and the 2 mmol/L boric acid treatment group. However,significant differences in urine metabolites were observed between the 200 mmol/L boric acid and the control groups. CONCLUSION: NaN₃ at a concentration of 0.1-10 mmol/L and boric acid at a concentration of 2 mmol/L were found to be suitable for future urine metabolomics research based on the UPLC/Q-TOF MS/MS technology.

OBJECTIVE: To assess the relation of idiopathic steroid-sensitive nephrotic syndrome (SSNS) and ACE gene polymorphism in children. METHODS: To search from the database of PubMed,Cochrane Library,EMBASE,Science Citation Index,Wiley Online Library and Google Scholar,and obtained literatures according to the selection criteria,extracted relevant data and performed statistical analysis by Review Manager 5.1. RESULTS: A total of 10 publications including 1 617 subjects were screened out (524 SSNS children and 1 093 normal people). In the SSNS children,the frequency of D allele,DD genotype and Ⅱ genotype were higher than that in controls but the difference was not statistically significant. CONCLUSION: Our data did not show a polymorphism in the ACE gene was related to idiopathic SSNS.

OBJECTIVE: This experiment mainly studied the toxicological effect of p-hydroxylcinnamal-dehyde(CMSP) extracted from the Cochinchina momordica seeds (CMS),on mice and provided a basis for clinical use of this drug. METHODS: 60 Kunming mice (4-6 weeks old) were randomly divided into three groups:control,200 mg/kg treatment and 400 mg/kg treatment groups. CMSP was administered by intraperitoneal injection. The general behavior,routine blood test,clinical biochemical test and teratospermia of the mice were evaluated. Furthermore,the pathologic changes of the vital organs were detected by immunohistochemical assay. Finally,the micronucleus formation in bone marrow cells was determined. RESULTS: There were no observed abnormal activities,blood test and teratospermia in CMSP treated-mice during the periods. However,the higher ALT and AST concentrations in serum were observed in the mice treated with 400 mg/kg CMSP. There were no abnormal changes of renal function,myocardial enzyme spectrum and pathologic changes of the vital organs. Moreover,there was no increase of micronucleus rate in bone marrow cells of treated mice. CONCLUSION: In our experiment,CMSP may have little toxic and side effects,so it may be considered for use in clinical treatments. However,it is also necessary to fully consider its possible toxicity to liver functions.

OBJECTIVE: To identify differentially expressed genes in gliomas which were related to their prognosis. METHODS: Raw data of the GEO (Gene Expression Omnibus) database GSE15824 involving normal and glioma tissues were analyzed using R language,and bioinformatics analysis tools (DAVID,String,Cytoscape,oncomine,and GEPIA). Differentially expressed genes were subjected to biological function,protein interaction analysis,and screening for prognostic markers of gliomas. RESULTS: A total of 648 differentially expressed mRNAs, including 471 up-regulated mRNAs and 177 down-regulated mRNAs were screened. Biological functions of these differential genes were mainly enriched in the activation of T cells,and regulation of adhesion of leukocytes and of cell migration. The KEGG signaling pathway was mainly enriched in PI3K/Akt,MAPK,and calcium signaling pathways. Formyl peptide receptor 1 was highly expressed in gliomas,and the expression level was negatively correlated with the prognosis of gliomas (Log-rank P < 0.01). CONCLUSION: Through the analysis of gene expression microarray analysis,FPR1 was highly expressed in gliomas and was related to patients' survival. The observations provide new ideas for further molecular investigations.

OBJECTIVE: To investigate the role of ubiquitin-conjugating enzyme E2 A (UBE2A) in cisplatin-induced stress response in HeLa cells. METHODS: HeLa cells were transiently transfected with UBE2A-specific siRNA,qPCR was then used to measure the expression level of UBE2A mRNA,and cell viability was measured using the CCK-8 assay after cisplatin treatment. RESULTS: Two different transcripts for UBE2A were identified in HeLa cells:U-1 and U-2,in which U-2 lacks a partial sequence of exon 4 compared to U-1,and both were up-regulated by cisplatin treatment. Expression of the two transcripts could be inhibited by siRNA,with an inhibition ratio of 27.43% for U-1 and 15.85% for U-2 compared to the control group,respectively. The inhibition of their expression in HeLa cells resulted in decreased cell viability after cisplatin treatment,with a 14.46% decrease for U-1 and 19.20% for U-2 compared with the cisplatin treatment group,respectively. CONCLUSION: UBE2A might be involved in cisplatin-induced stress response, via inhibition of UBE2A expression and increased sensitivity to the cytotoxic effect of cisplatin.

OBJECTIVE: Using TCGA data mining to explore lncRNAs signature with prognosis of lung squamous cell carcinomas. METHODS: Both the clinical and the RNAseq data of lung squamous cell carcinoma patients were extracted from TCGA transcriptome database. Association between lncRNAs and prognosis of lung squamous cell carcinoma were mined using LASSO Cox regression. Then,the lncRNA signature was constructed based on the coefficient of LASSO Cox regression model. The impact of the lncRNA signature expression level on prognosis of the patients were evaluated uisng Cox regression. RESULTS: 322 differentially expressed lncRNAs were screened out in tumor and adjacent tissues. A signature was constructed by LASSO Cox regression which included six lncRNAs as KTN1-AS1,FAM83A-AS1,AF131217.1,RP11-108M12.3,CTD-2555C10.3 and AC068831.16. When the patients were divided into high and low expression groups (based on the median of signature),the risk of death was 2.14 times higher in the high expression group patients than that in low expression group (HR=2.14,95%CI 1.50-3.04) (P < 0.01). When some clinical characteristics were added into the prediction model,the Harrell's C statistic of the prediction model was elevated to 0.69 (95%CI 0.64-0.75). CONCLUSION: The IncRNA signatures:KTN1-AS1,FAM83A-AS1,AF131217.1,RP11-108M12.3,CTD-2555C10.3 and AC068831.16 were associated with prognosis of lung squamous cell carcinoma based on the TCGA database analysis. Furthermore,expression levels of the lncRNA signature was predictive of the prognosis.

OBJECTIVE: To investigate effect of verbascoside on recombinant platelet-derived growth factor (rrPDGF)-BB-mediated migration,collagen deposition and fibrosis in rat hepatic stellate cells (HSC). METHODS: HSC cell cultures were divided into:rrPDGF-BB+ verbascoside (1.5,3,6 mg/L) group[recombinant rat PDGF-BB (rrPDGF-BB) pretreatment for 24 h,no verbascoside intervention],rrPDGF-BB group (rrPDGF-BB pretreatment for 24 h,no verbascoside intervention),control group (no rrPDGF-BB pretreatment,no verbascoside intervention). Scratch test was used to detect cell migration ability,ELISA was used to detect content of type I collagen (Col-I),Western blot was used to detect fibrotic HSC activation marker α-SMA protein and apoptosis effector molecule caspase-3,and expression level of ERK1/2 and Akt signaling pathway-related proteins. RESULTS: Compared with the control group,rrPDGF-BB group promoted the migration of HSC (P < 0.01),verbascoside (1.5,3.0,6.0 mg/L) group significantly inhibited the migration of HSC (P < 0.01). Moreover,inhibition of HSC migration by verbascoside was dose-dependent (r=0.894,P=0.038). With increasing test substance concentrations,Col-I secretion showed a decreasing trend. Among them,the effect of inhibiting collagen production was the most obvious in the 6 mg/L verbascoside group (P < 0.01). Verbascoside (1.5,3.0,6.0 mg/L) inhibited expression of α-SMA protein,activated activities of caspase-3,and significantly reduced expression of ERK1/2,P-ERK1/2,Akt and P-Akt proteins. Moreover, expression of the determination protein was also shown to have a dose-effect relationship among the various dose groups of verbascoside (0.826 < r < 0.997,P < 0.01). CONCLUSION: Verbascoside inhibited the activation,migration and fibrosis of HSC which was mediated by rrPDGF-BB. The evidence suggests that activation of caspase-3 by verbascoside blocked ERK1/2 and the Akt signaling pathway,and inhibited ECM excessive deposition and collagen formation in HSC.

OBJECTIVE: To investigate effects of sub-chronic exposure to diethylhexyl phthalate(DEHP) on livers of male rats and protective effect of selenium on the livers. METHODS: Seventy-seven male rats were randomly divided into 7 groups:control group (feeding basal feed),3 DEHP-treatment groups (diluted doses of 300,600 and 900 mg/kg,respectively),and 3 Selenium intervention groups (1 mg/kg yeast selenium was added to the corresponding dose of the exposure group). After 8 weeks of exposure,the rats were weighed,blood samples were collected for biochemical detection and the excised liver was weighed after 16 hours fasting. RESULTS: Compared with the control group,the mean weight of livers and coefficient of the liver were significantly increased in each of the exposed groups and the intervention group (P < 0.01). In addition,the quality of the 600 mg/kg treated group was significantly reduced (P < 0.05);the 900 mg/kg treated group was significantly lower in the body weight gain index (P < 0.01),and significantly lower than the control group in the total food intake and utilization index (P < 0.05);Compared with the treated group,the Se+300 mg/kg intervention group showed a significant decrease in weight gain (P < 0.05). The results of biochemical indicators showed that,compared with the control group,the 600 mg/kg treated group showed a significant increase in TP (P < 0.01);the 900 mg/kg treated group showed a significant increase in all five indicators (P < 0.01);The Se+300 mg/kg intervention group significantly increased the ALT index (P < 0.01),and significantly increased the ALT and AST indexes (P < 0.01). The Se+600 mg/kg intervention group significantly increased the ALT index (P < 0.05). There was a significant increase in AST index (P < 0.01). The Se+900 mg/kg intervention group showed significantly increased AST,TP and GLB (P < 0.01). Compared with the treated group,the intervention group was lower than the corresponding exposure group,and the Se+900mg/kg intervention group showed a significant decrease in TP and GLB indicators (P < 0.01). CONCLUSION: Sub-chronic exposure of DEHP affected body weight and growth,and liver function as well as caused liver swelling. The intervention of selenium effectively alleviated the toxic effect of DEHP on the liver.