OBJECTIVE: To screen for apoptosis-related lncRNAs and to investigate their regulatory effects on glycidyl methacrylate(GMA)-induced malignant transformation of human bronchial epithelial (16HBE) cells. METHODS: The 16HBE cells in the logarithmic growth phase were exposed to dimethyl sulfoxide (DMSO) as the solvent control group, and to 8 μg/mL GMA as the treatment group. Cells were subcultured after repeated exposure 3 times for 72 hours each time. Subsequently, cells at the 10th, 20th and 30th generations were collected. Based on cell transformation phenotypes of the cultured cells, they were divided into pre-, middleand late-transformation stages. Flow cytometry was used to detect expression of apoptosis and apoptosis-related lncRNAs were screened from the lncRNA microarrays of the three transformation stages. Bioinformatics database was used to find the target genes of the differentially expressed lncRNAs. Real-time quantification PCR was used to detect the relative expression of differential lncRNAs. RESULTS: Compared with the control group of the same (10th) generation, the apoptosis levels of the GMA-treated cells were significantly increased. The apoptosis levels of the 20th generation GMA-treated cells were significantly lower than the same generation control group (P < 0.05) and were not significantly different from the control group of the 30th generation. The results from the lncRNA microarrays show that lncRNA CFLAR-AS1 was down-regulated 22%, up-regulated 244% and down-regulated 30% in the 10 th, 20th and 30th generations after GMA-treatments, suggesting that the target mRNA CFLAR was up-regulated 27%, down-regulated 30.6% and down-regulated 31%. The results from qPCR verification of lncRNA CFLAR-AS1 were consistent with the results of the lncRNA microarrays which were down-regulated 42%, up-regulated 442% and down-regulated 9% in the 10th, 20th and 30th generations, respectively. The difference between the 10th and the 20th generations was statistically significant (P < 0.05). CONCLUSION: In the process of malignant transformation of 16HBE from repeated exposure to GMA, the pre-transformation apoptosis ratio increased and the mid-transformation apoptosis ratio decreased. During this period, lncRNA CFLAR-AS1 played a role in inhibiting apoptosis but the mechanism for this function needs further study.

OBJECTIVE: To investigate the effect of PM_2.5 exposure on expression of oncogenes and apoptosis genes in human renal epithelial cells (HK-2). METHODS: The CCK8 assay was used to obtain the half maximal inhibitory concentration (IC₅₀) of Taiyuan PM_2.5 on human renal epithelial (HK-2) cells. HK-2 cells were treated with PM_2.5 for 24 h. Four groups were set up:negative control group, low-dose PM_2.5 group (10 μg/mL PM_2.5), high-dose PM_2.5 group (50 μg/mL PM_2.5) and positive control group (Cr6+10 μmol/l). qPCR and Western blot were used to detect mRNA and proteins levels of apoptosis genes and oncogenes (c-myc, c-fos, p53, Caspase-8, Caspase-9 and Bcl-2). RESULTS: The IC₅₀ value of Taiyuan PM_2.5 on HK-2 cells was 95.98 μg/mL. After 24 hours of PM_2.5 exposure, qPCR results show that, compared with the control group, expressions of c-myc, c-fos, Caspase-8, Caspase-9 mRNAs were significantly increased in the high-dose and positive control groups (P < 0.01). However, expressions of p53 and Bcl-2 mRNAs were significantly decreased in these two groups (P < 0.01). Western blot data show that, compared with the control group, expressions of c-myc, c-fos, Caspase-8, Caspase-9 proteins were significantly increased in the high-dose and positive control groups (P < 0.01). However, expressions of p53 and Bcl-2 proteins were significantly decreased in these two groups (P < 0.05). CONCLUSION: PM_2.5 exposure to HK-2 cells increased the expression of oncogenes and apoptosis genes but decreased the expression of tumor suppressor and anti-apoptosis genes. Our results suggest that PM_2.5 exposure can promote oncogenesis.

OBJECTIVE: To investigate the role of ROS-mediated mitochondrial oxidative damage in isoniazid (INH)-induced hepatotoxicity in vitro and the protective effects from quercetin. METHODS: L-02 hepatocytes in cultures were randomly divided into five groups:isoniazid (10 mmol/L INH), INH + quercetin treatment (10 mmol/L INH and 50 μmol/L quercetin), glutathione (GSH) pretreated (20 mg/mL GSH, 10 mmol/L INH and 50 μmol/L quercetin), glutathione (20 mg/mL GSH) and negative control (equal volume of serum-free medium). After 24 hours of treatment, cell mitochondria were prepared by differential centrifugation, and fluorescent probes DCFH-DA and Rho-123 were used to detect mitochondrial ROS level and membrane potential, Contents of malondialdehyde (MDA) were determined using TBA colorimetry. Contents of protein carbonyl were determined using DPNH colorimetry. Contents of 8-hydroxydeoxyguanine nucleoside (8-OHdG) were determined using ELISA. RESULTS: Compared with the control group, INH-treated cells showed the followings:levels of ROS in mitochondria were elevated (P < 0.01), and membrane potentials were declined (P < 0.01). Compared with the INH group, the quercetin-treated cells showed the followings:mitochondrial ROS levels were reduced (P < 0.01) and membrane potentials were elevated (P < 0.05). Compared with the quercetin-treated group, the glutathione-pretreated cells showed the followings:levels of ROS were lower and the mitochondrial membrane potentials were higher (P < 0.05). Compared with the control group, contents of MDA, protein carbonyl and 8-OHdG were increased after the INH treatment (P < 0.01). Compared with isoniazid group, contents of MDA, protein carbonyl and 8-OHdG in quercetin treatment group were decreased (P < 0.01).Contents of MDA, protein carbonyl and 8-OHdG in the GSH pretreatment group were lower than those in quercetin treatment group (P < 0.05). CONCLUSION: Under the experimental conditions, INH induced oxidative damage of mitochondria in L-02 cells. Quercetin was shown to provide protective effect on INH-induced mitochondrial oxidative damage and the mechanism was related to its inhibition of ROS activities.

OBJECTIVE: To investigate protective potential of deer serum on cardiomyocyte injury which was induced by glucose deprivation. METHODS: Cardiomyocytes were cultured in vitro and were subjected to glucose deprivation for 18 h to induce myocardial cell injury. An objective was to observe whether deer serum would alleviate the injury by 6 h and 12 h preconditioning, using sheep serum as comparison. The cardiomyocyte cultures were divided into several groups:normal control, model, deer serum-treated (5、10 and 20 mg/mL) and sheep serum-treated (5, 10 and 20 mg/mL) groups. Effects of cell morphology, relative activity of myocardial cells, lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA) content, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activity were used as evaluation indexes. RESULTS: Compared with the model and the normal control groups, the 6 h pretreatment by both sera significantly increased the relative activities of cardiomyocytes at different concentrations (P < 0.01), but there was no significant difference between the deer and the sheep serum groups (P > 0.05). After pretreatment for 12 h with different concentrations of deer serum, the relative activities of myocardial cells were significantly increased over the model group (P < 0.01), but the increases were lower than that of the normal control group (P < 0.05). There was no statistical significance in activities after treatment with the different concentrations of sheep serum (P > 0.05). The relative activities of myocardial cells in the 20 mg/mL deer serum group was significantly higher than that in the 20 mg/mL sheep serum group (P < 0.01).On the other hand, the concentrations of LDH in the deer serum groups was significantly decreased compared with the model group (P < 0.01). Activities of GSH-Px in the 20 mg/mL deer serum group and in different concentrations (5, 10, 20 mg/mL) of the sheep serum groups were significantly increased (P < 0.01), and activities of SOD in the 10 mg/mL deer serum group was significantly increased (P < 0.01). CONCLUSION: Deer serum preconditioning had a protective effect against cardiomyocyte apoptosis which was induced by glucose deprivation. The mechanism may be related to protection against oxygen free radical damage and stabilization of cell membrane.

OBJECTIVE: To investigate the hepatotoxicity of 2, 4dichlorophenoxyacetic acid in young SD rats. METHODS: 64 young SD rats with SPF grade were randomly divided into three 2, 4-D inoculation groups (18.125, 36.250, 72.500 mg/kg) and a solvent control group (1% carboxymethyl cellulose). The rats were inoculated with the chemicals orally once a day for 28 days. After the exposure, their general behavior and weight changes were monitored, wet weights and viscera coefficients of the livers were calculated. Pathology of the livers were determined after their staining with hematoxylin-Eosin (HE). Activities of glutathione (GSH), total superoxide dismutase (T-SOD) and concentrations of malondialdehyde (MDA) were measured using the DTNB assay, and xanthine oxidase and TBA assays were conducted after liver samples were pretreated. Contents of cytochrome C, relative activities of Caspase-3 and Caspase-9 in liver tissues were determined by spectrophotometer. RESULTS: Compared with the control group, rats from the 72.500 mg/kg treatment group showed the following:body weight gains and liver wet weight were reduced significantly (P < 0.05);parts of the livers had bile duct hyperplasia which were accompanied with inflammatory cell infiltration;GSH activities in the liver homogenates were decreased (P < 0.05);and MDA concentrations were increased (P < 0.05). In rats from both the 36.250 mg/kg and 72.500 mg/kg treatment groups, the activities of TSOD were decreased (P < 0.05); cytochrome C concentrations were increased (P < 0.05); and the relative activities of caspase-3 and caspase-9 were increased (P < 0.05). CONCLUSION: Under the conditions of this investigation, 2, 4-D was shown to induce hepatotoxicity in young SD rats. The toxicity was probably mediated by changing mitochondrial membrane permeability and releasing apoptotic factor to induce hepatocyte apoptosis, thereby causing oxidative damage to hepatocytes.

OBJECTIVE: To investigate the induction of oxidative stress by arsenious acid solution As(Ⅲ) in human lung cancer A549 cells. METHODS: Cultured A549 cells were exposed to arsenious acid solution, As(Ⅲ), at doses of 0, 2.5, 5, 10 and 200 μg/mL for 24 h. Cell viabilities were determined using the CCK-8 assay. For cells exposed to 0, 2.5, 5 μg/mL doses for 24 h, superoxide dismutase (superoxide dismutase, SOD) in cell culture medium was detected using ELISA, and intracellular reactive oxygen species (ROS) were detected using fluorescent probe DCFHDA. RESULTS: As exposure concentration of As(Ⅲ) increased, A549 cell viabilities were gradually decreased (r=0.99, P < 0.05). The level of SOD in the 2.5, and 5 μg/mL exposure groups were (0.72±0.05), and (1.10±0.16) ng/mL, respectively, which were higher than that of the control group[(0.56 ±0.03) ng/mL] (P < 0.05). Relative fluorescence intensities of ROS were (169.31 ±6.13)%, and (242.60±2.35)% for the same two groups which were higher than that of the control group (100±2.80)%. CONCLUSION: As(Ⅲ) exposure caused significantly reduced cell survival rates, increased concentrations of SOD and ROS in A549 cells. Oxidative stress may therefore be an important mechanism for the toxicity of As(Ⅲ).

OBJECTIVE: To investigate effects of apigenin (API) on proliferation and apoptosis induction in the human gastric cancer SGC7901 cells. METHODS: SGC7901 cell cultures were organized into different groups:control and API-treated groups. The latter groups were treated with 20, 40, 60 and 80 μmol/L API for 12, 24 and 48 h. Cell proliferation rates were detected using the CCK-8 method, apoptosis rates by flow cytometry, and expression levels of death receptors DR4 and DR5 proteins in the TRAIL pathway by Western blot. Using RNAi technology, DR4 siRNA and DR5 siRNA were used to silence the expression of death receptors DR4 and DR5, respectively. Apoptosis rates after 24 h treatments for the control, API, DR4 siRNA + API and DR5 siRNA + API groups were determined using flow cytometry. RESULTS: The results indicated that API significantly inhibited proliferation rates of SGC7901 cells in a concentrationdependent way (r_{12 h}=-0.99, r_{24 h}=-0.88, r_{48 h}=-0.89, all P < 0.05). Apoptosis rates increased with increased API concentrations (r=0.96, P < 0.05). In addition, API treatment upregulated the expression levels of DR4 and DR5 proteins. When the expressions of DR4 and DR5 were silenced by their respectively siRNAs, the apoptosis rates for the DR4 siRNA + API and the DR5 siRNA + API groups were reduced compared with the API group (P < 0.05). CONCLUSION: Our results indicated that apigenin inhibited the proliferation rates and induced apoptosis in the human gastric cancer SGC-7901 cells. The observed changes might be mediated by apigenin-activation of the death receptors DR4 and DR5 proteins in the TRAIL pathway.

OBJECTIVE: To study the effect of Herba Saginae Japonicae (HSJ) on differentiation of human chronic myeloid leukemia cells (K562). METHODS: K652 cells were organized into experimental groups (with treatment of 250, 125, 62.5 μg/mL HSJ) and a negative control group. Cell proliferation was detected using the MTT assay. Cell morphology was observed after their staining by Wright-Giemsa. Their differentiation ability was determined by the NBT reduction assay. Their expression rates of CD11b, CD14, CD41a and CD42b positive cells were determined using flow cytometry. Western blot method was used to detect their expression of GATA1 and PU.1 proteins. RESULTS: Compared with the negative control group, HSJ exposure caused inhibition of proliferation of the K562 cells. Morphologically, treated cells showed lobulated nuclei and some showed obvious cell differentiation features. The different doses of treated cells showed significantly increased NBT reduction rates (P < 0.05);CD11b, CD14, CD41a and CD42b positive rates (P < 0.05) and GATA1 and PU.1 protein relative contents (P < 0.05) compared with the negative controls (P < 0.05). CONCLUSION: HSJ induced the K562 chronic myeloid leukemia cells to mature in the direction of differentiation.

OBJECTIVE: To study cellular changes during induction of differentiation of human acute promyelocytic leukemia cells (NB4) by an alcohol extract from Herba Saginae Japonicae (HSJ). METHODS: NB4 cells in culture were were organized into four groups:HSJ low dose (30 μg/mL), HSJ middle dose (60 μg/mL), HSJ high dose (120 μg/mL) and negative control groups. Cells in the experimental groups were treated with HSJ for 48 h and their cellular morphology was observed by transmission electron microscopy. nitrotriazole nitrogen (NBT) reduction test was used to detect the differentiation ability of NB4 cells. The distribution of NB4 cell cycles and expression rates of CD14 and CD11b cells were detected using flow cytometry. Expression of c-fos mRNA was detected using quantitative real-time PCR(qPCR). Expression of cfos protein was detected using the Western blot method. RESULTS: Compared with the control group, the nuclei of HSJ-treated cells, in the three different doses, were reduced and changed into kidney-shape, and the cytoplasm was vacuolated. The NBT test showed that the reduction rate of the treated cells were significantly higher than that in the control group (P < 0.05). The proportions of G2 phase cells in the treated groups were significantly higher than that in the control group, while the proportion of S phase cells were significantly lower (P < 0.05). The expression rates of CD14 and CD11b positive cells in the treated groups were significantly higher than that in the control group (P < 0.05). In addition, expression rates of c-fos mRNA and c-fos proteins in the middleand the high-dose groups were significantly higher than that in the control group (P < 0.05) but no difference for the low-dose group. CONCLUSION: HSJ may induce NB4 cells to differentiate into mature granulocytes.

OBJECTIVE: To investigate influence of age and gender on spontaneous formation of nucleoplasmic bridges (NPB) in peripheral blood lymphocyte from in healthy Chinese subjects. METHODS: A total of 98 healthy adults (52 male and 46 female) aged 2068 years (40.7 ±12.6 years) were enrolled. The subjects were divided into different age groups:20-29 (22 subjects), 30-39 (23 subjects), 40-49 (25 subjects) and over 50 (28 subjects). At 40 h of their lymphocyte cultures in the cytokinesis-block cytome assay, cytochalasin-B (10 μg/mL) was added and cultures were harvested at 68 h. The presence of NPB, micronucleus (MN) and nuclear bud (NBD) in their peripheral blood lymphocytes were determined by analyzing 1 000 binucleated cells in each sample. RESULTS: The overall NPB spontaneous rate was 0.56‰, but the rates were non-significantly higher in males than in females. In addition, there was no statistically significant difference in male subjects among different age groups. However, the highest NPB spontaneous rate in females was found in 40-49 age group which was significantly higher than that in the 20-29 age group (U=2.31, P < 0.05). The MN spontaneous rates in females were higher than that in males (U=4.40, P < 0.05) and the highest rates in both male and female subjects were found in the 40-49 age groups. There was no statistical difference of NBD spontaneous rates between genders and among different age groups. CONCLUSION: NPB spontaneous rate in healthy population was very low which was not affected by gender but had increased trend within the 40-49 age group.

OBJECTIVE: To mind databases for genome variations of anaplastic thyroid cancers and for screening targeted drugs that may be effective on anaplastic thyroid cancers. METHODS: The cBioPortal database for anaplastic thyroid cancer was used to search for the genes with mutations and copy number variations, and to calculate their genetic variation frequencies. The data were also used to draw survival curves and to identify gene variants that were closely related to patients' survival, to analyze protein interactions, to perform GO enrichment using the STRING database, and to screen potential targeted drugs using the CARE software. RESULTS: The database show that TP53, PI3KCA, ARID2 were the genes with the highest frequencies of genetic variation in anaplastic thyroid cancer. In addition, missense mutations in the TP53 gene were related to the decline in patient survival. GO enrichment analyses show that TP53 participated in the pathogenesis of anaplastic thyroid cancer by regulating biological processes such as DNA damage repair and cell cycle arrest. For the patients with the missense mutations in the TP53 gene, they were sensitive to treatment with Sorafenib, Lapatinib, Ruxotinib and other drugs, but were resistant to Ponatinib and other drugs. CONCLUSION: Missense mutations in the TP53 gene were the key gene variations which affected prognosis of patients with anaplastic thyroid cancer. In addition, Sorafenib and Ruxotinib might be candidate drugs which targeted patients with the missense mutations in the TP53 gene.

OBJECTIVE: To evaluate the in vitro cytotoxic effects of tongue depressors with different materials and their scientific bases for toxicity. METHODS: The L929 cell line was cultured and organized into different groups:blank control, negative control, positive control and treatment groups. The latter group was treated with tongue depressors of the three materials with 25%, 50%, 75% and 100% extraction concentrations. After 24 h of treatment, all cell cultures were evaluated for their morphology under a microscope, viability using the MTT method and other cytotoxic effects. RESULTS: The results showed that cells in the positive control group and in the group which was treated with the 100% extract from wooden tongue depressors were completely destroyed. On the other hand, cells in the group which was treated with 100% extract from bamboo tongue depressor grew normally. Approximately 20% of the cells in the group which was treated with 100% extract from stainless steel tongue depressor showed abnormal morphologies:round with shrinkage, poor adherence to the wall and lacking intracellular particles. Results from the MTT assay show that the cell viability for the mentioned two groups which indicated toxicity were 89.1% (bamboo) and 81.2% (stainless steel). According to criteria of cytotoxicity, exposure to extracts from wooden tongue depressors caused potential cytotoxicity. CONCLUSION: Our results indicate that tongue depressors which were made of certain materials caused cytotoxic effects in vitro. Our data should be useful in strengthening safety guidelines for use of these tongue depressors.

OBJECTIVE: To develop a human cardiomyocyte model for cardiotoxicity drug evaluation, and to evaluate its potential for screening new cardiotoxicity drugs. METHODS: Human cardiomyocytes in cultures were treated with tamoxifen (0.33, 1, 3 μmol/mL) and haloperidol (0.11, 0.33, 1 μmol/mL) for 48 h. At 1, 2, 3, 6, 9, 12, 18, 24, 30, 36, 42 and 48 h after the initiation of treatments realtime cell analyses and highcontent cell imaging technology were used to monitor changes of cardiac myocyte pulse frequency, amplitude, regularity and mitochondrial membrane potential before and after administration. The data were used to evaluate the usefulness of this model as an in vitro cardiotoxicity assay. RESULTS: After tamoxifen and haloperidol were co-incubated with human cardiomyocytes, the FPD and systolic frequencies reduced to 0 after 3 h treatment with 3μmol/mL tamoxifen but recovered after 18 h treatment. Results from the other treatment concentrations were similar to those in the control group. Both FPD and systolic frequency of cardiomyocytes were concentration-dependent on fluperidol. The higher the concentrations, the higher were the values of FPD (Max), and the smaller were the values of systolic frequency. After 24 h, complete fluid exchange was conducted and all cardiomyocytes treated with different concentrations recovered. CONCLUSION: Our data suggest that human cardiomyocytes can be used to establish a feasible model for evaluating cardiotoxicity in vitro. In addition, real-time cell analysis and high-content cell imaging can be used to evaluate cardiotoxicity of drugs.