OBJECTIVE: To investigate the effect and mechanism of low doses of bisphenol A (BPA) alone or in combination with high fat diet (HFD) on glucose and lipid metabolism in mice. METHODS: Sixty C57BL/6J mice were randomly divided into five groups for exposure to BPA at different concentrations[1,10, 100,1 000 μg/(kg·d)]. After determining the optimal dose,another 60 C57BL/6J mice were randomly divided into 5 groups:control,BPA,BPA+HFD,HFD,BPA+Ginsenoside Rh1 (Rh1). The function of glucose and lipid metabolism in mice was evaluated by measuring liver triglyceride (TG) content and glucose tolerance. RESULTS: After exposure of 10 μg/(kg·d) BPA,morphological and biochemical determinations showed that these mice had the highest lipid accumulation. Compared with the control group, BPA exposure resulted in increased liver TG levels and decreased glucose tolerance (P < 0.05). Compared with HFD group,mice from the BPA + HFD group had increased liver TG content and decreased glucose tolerance (P < 0.05). Expression levels of peroxidosomal proliferator activated receptor γ (PPARγ), sterol regulatory element binding protein 1 (SREBP1), fatty acid synthase (FASN) and stearyl coenzyme A desaturase 1 (SCD-1), which are related to adipogenesis, were significantly increased (P < 0.05). In BPA exposed mice treated with Rh1, liver TG levels were significantly reduced compared with the BPA exposed mice (P < 0.05). CONSLUSION: BPA exposure induced glucose and lipid metabolism disorders in mice, and aggravate HFD-induced glucose and lipid metabolism disorders. BPA regulated a series of lipid metabolism-related genes to induce lipid accumulation, leading to the disorder of glucose and lipid metabolism. Inhibition of PPARγ expression with Rh1 significantly alleviated BPA-induced disorders of glucose and lipid metabolism. Therefore, increased PPARγ transcription could be a key reason of glucose and lipid metabolism disorder induced by BPA.

OBJECTIVE: To assess the potential developmental toxicity of lanthanum nitrate at the middle and advanced stage of gestation in post-implantation whole embryo culture and micromass test. METHODS: Rat embryos were randomly divided into 5 groups and cultured in immediately centrifuged serum,and were exposed to different doses of lanthanum nitrate (0,0.12,0.23,0.46 and 1 mmol/L). After 48 hours culture,yolk sac diameter (YSD),crown-rump length (CRL) and head length (HL) were measured, somites were counted, and the total morphological score (TMS) was calculated according to the Brown's Method to evaluated growth and functional development of embryos. Cytotoxicity in BALB/c 3T3 cells was tested by MTT. Toxicity of lanthanum nitrate in the early embryonic stage was evaluated using the prediction models of ECVAM. In addition,limb bud cells of embryos were isolated from matured SD rats and cultured in micro-mass culture for 5 days. These cultures were exposed to 0.03, 0.06, 0.13, 0.25, 0.5, 1, 2 and 3 mmol/L lanthanum nitrate. The 50% inhibition of cell viability and growth (IC₅₀) of limb bud cells was determined by neutral red staining, and the 50% inhibition of cells differentiation (ID₅₀) was measured by alcian blue staining. Toxicity of lanthanum nitrate in the middle and advanced stages of embryos was evaluated with the prediction model of ECVAM. RESULTS: The no observed adverse effect level (NOAEL) of lanthanum nitrate on embryo growth was 0.12 mmol/L. At the concentration of 0.46 mmol/L,lanthanum nitrate decreased the YSD,CRL and HL of embryos (P < 0.05),which showed that lanthanum nitrate significantly retarded growth of embryos. Besides, 0.46 mmol/L lanthanum nitrate induced developmental malformations and reduced the number of somites (P < 0.05). The NOAEL of lanthanum nitrate on the proliferation of limb bud was 1 mmol/L, the NOAEL on chondrocyte differentiation from limb bud cells was 0.25 mmol/L,the IC₅₀ and ID₅₀ for limb bud cells were 1.57 mmol/L (510.25 μg/mL) and 0.99 mmol/L (321.75 μg/mL),respectively. CONCLUSION: Lanthanum nitrate was evaluated as a weak embryotoxic chemical by whole embryo culture and as a nonembryotoxic chemical by micromass test.

OBJECTIVE: To investigate the value of P16/Ki-67 double staining combined with DNA ploidy analysis in the atypical squamous cells of undetermined significance (ASCUS) shunt. METHODS: The valid cytological samples from 115 patients diagnosed as ASCUS in Changning Maternity and Child Healthcare Hospital of Shanghai,from December 2016 to August 2018,were collected from LCT test. P16/Ki-67 double staining and DNA ploidy analysis were tested at the same time. The gold standard was the pathological biopsy results of colposcopy. RESULTS: Pathological examination showed that there were 45 cases of cervical intraepithelial neoplasia 2 or above (CIN2+),including 20 cases of CIN2,23 cases of CIN3 and 2 cases of squamous cell carcinoma. Among the 115 patients,the sensitivity,specificity and diagnostic coincidence rate of CIN2+ by P16/Ki-67 double staining were 77.8%,77.1% and 77.4%,respectively;the sensitivity,specificity and diagnostic coincidence rate of CIN2+ by DNA ploidy analysis were 71.1%,34.3%,48.7%,respectively,and the P16/Ki-67 double staining test results were negative in the DNA ploidy negative cell samples. DNA ploidy analysis combined with P16/Ki-67 double staining detection found that the sensitivity, specificity, and diagnostic coincidence rate of CIN2+ were 92.9%,71.4%,86.3%,respectively,of which the diagnostic coincidence rate was significantly higher than that of DNA ploidy analysis alone (P < 0.05). CONCLUSION: P16/Ki-67 double staining has higher sensitivity and specificity than DNA ploidy analysis for CIN2+. In addition,combination of the two methods effectively improved the accuracy for the shunt diagnosis of ASCUS.

OBJECTIVE: To examine the effect of HMGB1 silencing by siRNA on cell proliferation, survival ability, and cell apoptosis of human esophageal squamous cell carcinoma, KYSE30, after X-ray radiation. METHODS: There were three study groups. The HMGB1 siRNA group:siRNA based on the sequences of the HMGB1 mRNA were synthesized and were transfected into the cultured KYSE30 cells. The negative control group:a negative siRNA was synthesized and transfected. The blank control group:no transfection was conducted. Expression of HMGB1 at the mRNA and protein levels was determined using quantitative real-time PCR (qPCR) and Western blot, respectively. The effect of HMGB1 knockdown on the proliferation,survival ability,cell apoptosis rate and the expression of cell apoptosis-related proteins of tumor cells were examined by MTS, clonal formation, flow cytometry and Western blot assays, respectively. RESULTS: Both mRNA and protein expression of HMGB1 were significantly reduced after silencing the HMGB1 gene (P < 0.01). MTS data demonstrated that HMGB1 knockdown,followed by irradiation,significantly inhibited the proliferation of KYSE30 cells compared with the negative control and the blank control groups (P < 0.05). Data from the clonal formation assay revealed that the values of D₀ were 2.57 Gy,2.54 Gy,1.55 Gy; the values of D_q were 1.69 Gy,1.65 Gy,1.30 Gy;the values of SF₂ were 0.27,0.27,0.13;the values of N were 1.93,1.91,2.31 in the blank control,negative control and HMGB1 siRNA groups,respectively (all P < 0.01). The apoptosis rate of tumor cells in the HMGB1 siRNA group after irradiation was markedly increased, followed by the downregulation of bcl-2 expression and the upregulation of bax, caspase-3 expression compared with negative control and blank control groups (P < 0.01). CONCLUSION: SiRNA interference technology inhibited the expression of HMGB1 gene in KYSE30 cells,reduced the proliferation and viability ability of cells, induced cell apoptosis, and increased radiosensitivity after X-ray exposure, which may be associated with regulating the expression of bcl-2,bax and caspase-3.

OBJECTIVE: This study aimed to explore mechanisms of RBM24 in inhibition of proliferation in nasopharyngeal carcinoma cells. METHODS: Cell models with RBM24 knocked down were built via RBM24 siRNA transfection into immortalized nasopharyngeal epithelial cells N5, NP69, and nasopharyngeal carcinoma cells CNE1. After the cell models was built,CCK-8 assay was performed to evaluate the effect of transfection on cell proliferation each day from day 1 to 5. Real-time PCR was carried out to evaluate the expression of HOTAIR. RESULTS: Successful modeling of RBM24 knocked down were confirmed by real-time PCR. The knockdown significantly induced proliferation of CNE1 (5.11±0.03) and N5 (2.09±0.18),compared with the control group (4.53±0.05 and 1.73±0.12, respectively). It also upregulated HOTAIR expression in CNE1 (67.54±1.87) and N5 (7.81±1.90), compared with the control group (1.00±0.21 and 1.00±0.19, respectively, all with P < 0.05). But it had no obvious effect on the proliferation and HOTAIR expression in NP69 (all with P > 0.05). CONCLUSION: RBM24 inhibited cell proliferation of nasopharyngeal carcinoma cell CNE1 and immortalized nasopharyngeal epithelial cells N5,through downregulation of HOTAIR expression.

OBJECTIVE: To study the effect of particulate matter 2.5(PM_2.5) exposure on expression of oncogenes and apoptosis-related genes in normal hepatocytes (L02 cells). METHODS: L02 cells were exposed to PM_2.5 (10 and 50 μg/mL), as the experimental group, to 10 μmol/L Cr⁶⁺ as the positive control group, and nothing as the negative control group,for 24 h. After the treatment,mRNA expression of oncogenes including promoting oncogenes including c-myc, c-fos and suppressing oncogenes p53 and promoting apoptosis-related genes including Caspase-3, Caspase-8 and suppressing apoptosis gene Bcl-2 were detected by fluorescent quantitative real-time PCR(qPCR),the protein expression of oncogenes and apoptosis genes were detected with Western blot. RESULTS: The qPCR showed that compared with the negative control group, significantly different mRNA expressions in the PM_2.5 10 μg/mL,50 μg/mL and positive control groups were:c-myc gene increased by 69.5%,118.0%,51.1%,c-fos gene increased by 50.3%,64.4%,23.3%;p53 gene decreased by 4.0%,22.0%,18.0%;Caspase-3 gene increased by 31.0%,30.5% and 42.0%;Caspase-8 expression level increased by 25.3%,40.3% and 64.5%,Bcl-2 gene expression level decreased by 40.5%,46.9% and 41.4%, respectively (P < 0.05). Western blot analyses showed that expression levels of c-myc protein increased by 32.2%, 49.3%,70.6%;c-fos protein increased by 11.3%,50.2%,45.2%;p53 protein decreased by 17.5%,40.0%, 42.5%;Caspase-3 protein increased by 23.1%,33.9%,43.1%;Caspase-8 protein increased by 31.4%,52.1%, 80.0%; Bcl-2 protein decreased by 14.3%, 23.3%, 36.9%, respectively, (P < 0.05). CONCLUSION: PM_2.5 exposure increased expression of promoting oncogenes and promoting apoptosis genes as well as decreased expression of suppressing oncogenes and suppressing apoptosis genes in L02 hepatocytes. The results indicate that PM_2.5 exposure can promote and activate malignant transformation and apoptosis of L02 hepatocytes.

OBJECTIVE: To establish a subcutaneously transplanted tumor model of human lung cancer in nude mice and to evaluate effect of ketogenic diet on growth of the subcutaneously transplanted tumor. METHODS: Human lung cancer A549 cells were subcutaneously injected into the right lower limb of 10 female BALB/c nude mice. After establishing the transplanted tumor model, the nude mice were randomly divided into a standard diet group (standard diet,SD group) and a ketogenic diet group (ketogenic diet,KD group). Body mass,tumor volume,blood glucose and blood ketone concentration of nude mice were measured on the same day,and then every 5 days. On the 30th day after inoculation,the nude mice were killed,and blood lipids were determined from blood samples taken from their eyes. The transplanted tumor tissue was stripped, and the protein expression level of 4-HNE in the transplanted tumor tissue of nude mice was detected by Western blot and immunohistochemistry. RESULTS: From the 15th day after inoculation, the blood ketone concentration in the KD group was higher than that in the SD group, and the blood glucose concentration in the SD group was lower (P < 0.05). From the 20th day after inoculation,the body weight and tumor volume of the KD nude mice were significantly smaller than those in the SD group (P < 0.05). On the 30th day after inoculation, the concentration of total cholesterol (CHOL), high-density lipoprotein cholesterol (HDL-C),and low-density lipoprotein cholesterol (LDL-C) in the KD nude mice were higher than those in SD group,and the concentration of triglyceride (TG) were lower than SD. The difference between the two groups was statistically significant (P < 0.05). Western blot results showed that the relative expression level of 4-HNE in the KD group (1.45±0.10) was significantly higher than that in the SD group (1.00±0.03) (P < 0.05). Immunohistochemical results showed that the expression level of 4-HNE in the KD group (4.40±0.89) was significantly higher than that in the SD group (2.40±0.89) (P < 0.05). CONCLUSION: KD diet inhibited the growth of subcutaneously transplanted human lung cancer in nude mice,and its mechanism may be related to the enhancement of oxidative stress of tumor cells.

OBJECTIVE: To explore the roles of miR-21 in regulating migration and invasion of triplenegative breast cancer MDA-MB-231 cells via targeting PDCD4. METHODS: Quantitative real-time PCR (qPCR) was used to detect the expression of miR-21 and PDCD4 mRNA in MDA-MB-231 and MCF-10A cells. MDA-MB-231 cells were divided into five groups,including blank control,miR-21 mimics,miR-21 inhibitor, negative mimics and negative inhibitor. Western blotting was used to detect PDCD4 protein expression, and luciferase reporter gene assay was used to detect whether miR-21 was targeting PDCD4. Migration and invasion of MDA-MB-231 cells were detected using Transwell chambers. RESULTS: miR-21 and PDCD4 mRNA in MDA-MB-231 cells was significantly higher and lower than that of MCF-10A cells, respectively (P < 0.01). The overexpression or inhibition of miR-21 were concurrent with the up-or downregulation of PDCD4 expression. Luciferase reporter gene assays showed that PDCD4 was a direct target of miR-21. Transwell assays demonstrated that modulating miR-21 with mimics or inhibitors changed the migration and invasion of MDA-MB-231 cells. CONCLUSION: Migration and invasion of MDA-MB-231 cells were regulated by miR-21 through targeting PDCD4. Regulating the expression of miR-21 could be a novel target for inhibiting these invasive activities in triple-negative breast cancers.

OBJECTIVE: To explore the effect of c-fos gene silencing on expression of oncogenes and apoptosis-related genes in human bronchial epithelial (HBE) cells which were exposed to PM_2.5. METHODS: According to the c-fos gene mRNA sequence provided by GenBank,interfering sequences were designed and synthesized,and the recombinant lentiviral vector was transfected into HBE cells. Real-time quantitative PCR (qPCR) and Western blot were used to identify the silencing of c-fos gene. HBE cells and c-fos gene-silenced cells were exposed to PM_2.5 suspension with a concentration of 50 μg/mL for 24 h,and a non-exposed control group was established. The mRNA levels and protein levels of oncogenes including c-myc,k-ras,c-fos,p53 and apoptosis-related genes including Caspase-3,Caspase-8 were detected by qPCR and Western blot. RESULTS: In HBE cells transfected with c-fos-shRNA lentivirus,qPCR and Western blot analyses revealed that expression of c-fos gene mRNA was 70.1% lower,and expression of c-fos protein was 53.7% lower than that of control group HBE cells. After HBE cells were treated with PM_2.5, mRNA expression of c-myc, k-ras, c-fos, Caspase-3,and Caspase-8 were increased by 30.46%,27.17%,and 50.78%,71.91%,74.16%,respectively, compared with the untreated HBE cells,p53 mRNA expression level decreased by 12.27% (P < 0.05). After the c-fos gene silenced cells were treated with PM_2.5, mRNA expression of c-myc, k-ras, Caspase-3, and Caspase-8 decreased by 13.08%, 5.39%, 25.03%, 21.37%, respectively, and p53 mRNA decreased by 53.39%,compared with the untreated group. In comparison with normal HBE cells,mRNA expression of c-myc,k-ras,p53,c-fos,Caspase-3,and Caspase-8 were significantly reduced(P < 0.05). Furthermore,protein level of c-myc also showed a decreasing trend after c-fos gene-silenced cells were exposed to PM_2.5,compared with that of normal HBE cells. CONCLUSION: c-fos gene-silenced cells were successfully constructed. PM_2.5 promoted expression of oncogenes and apoptotic genes in HBE cells. On the other hand,c-fos gene silencing inhibited the expression of oncogenes and apoptotic genes after the PM_2.5 treatment in HBE cells.

OBJECTIVE: To explore pathogenic characteristics and risk of human papillomavirus-52 (HPV-52) infection in cervical squamous cell lesions in Shantou,China. METHODS: 37 700 cases of HPV typing and thin-layer liquid-based cytology (LCT),and 3 924 cases of cervical biopsy were collected in the Shantou Central Hospital from 2015 to 2018. HPV was detected by using the medical nucleic acid molecule rapid hybridization genotype kit which contained 15 high-risk HPV (HPV-16,-18,-31,-33,-35,-39, -45,-51,-52,-53,-56,-58,-59,-66,and -68) and 6 low-risk HPV (HPV-6,-11,-42,-43,-44, CP8304). Cytology examination was classified according to the TBS (the Bethesda system) classification standard (2014 version),and was divided into negative for intraepithelial lesion or malignancy (NILM),atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), atypical squamous cells without exclusion of HSIL (ASC-H),high grade squamous intraepithelial lesion(HSIL) and squamous cell carcinoma (SCC). The database was established by SPSS 19.0. The rate comparison of the counted data was performed by using frequency distribution. The risk assessment was performed by using a chi-square test 2×2 list. RESULTS: The total infection rate of HPV-52 in 37 700 patients was 3.55% (1 338/37 700). There were 882 cases of HPV-52 single infection and 456 cases of HPV-52 multiple infection, including 313 cases of double infection and 143 cases of triple and above.The three most common genotype of HPV-52 multiple infection were HPV-58/52 (12.46%,39/313),HPV-53/52 (11.18%,35/313),HPV-16/52 (11.18%,35/313). The most common genotype was HPV-16/52 in ASC-US and above lesions,especially in HSIL. Risk analysis of cervical biopsy showed that HPV-52/16 increased the risk of HSIL/SCC,compared with HPV-52 single infection (P < 0.01,OR=8.27),and increased the risk from HSIL to SCC (P=0.01,OR=12.65). Multiple infections of HPV-52/16 increased the risk from HSIL to SCC,compared with multiple infections that included HPV-52 but not HPV-16 (P < 0.01,OR=3.36),and increased the risk from HSIL to SCC (P=0.03, OR=1.29). CONCLUSION: HPV-52 was the most common genotype in Shantou area but the patients suffered mainly HSIL lesions. On the other hand, infection with HPV-16 increased the risk of precancerous and cancerous lesions.

OBJECTIVE: The purpose of this study is to explore the enrichment and migration of strontium in different tissues of pepper,and to provide basic data for the radiation risk assessment of edible plants, vegetables and other foods after nuclear accident. METHODS: In this study, the stable nuclide strontium-88 instead of the radioactive nuclide strontium-90 was used to plant pepper in greenhouses. In the experiment, 159.33 mg/kg soil strontium was used as the background control, and five soil strontium concentration gradients were set as 1.5, 2, 2.5, 3 and 3.5 times the control. The contents of strontium in pepper and soil were determined by ICP-AES. The transfer coefficient (TF) of strontium in the stem,leaf and fruit, and the concentration coefficient (CR) of strontium in the root, stem, leaf and fruit were calculated. RESULTS: The content of strontium in different tissues of pepper increased with increasing strontium concentrations in soil. The order was C_root > C_leaf > C_stem > C_fruit. The order of CR values were C_Rroot > C_Rleaf > C_Rstem > C_Rfruit, and the order of TF values were TF_leaf > TF_stem > TF_fruit. CONCLUSION: The content of strontium in the edible parts of pepper is the lowest,therefore the pepper fruit is relative safe for consumption. The non-edible parts of the pepper, roots and leaves, had strong enrichment ability, the pepper leaves and stems had strong migration ability. Therefore,they can be used as a candidate plant for radionuclide pollution investigations.

OBJECTIVE: To observe the toxic effects from gavage of di(2-ethylhexyl) adipate (DEHA) on the general toxicity,mating behavior of parent and the fetal growth in rats. METHODS: 120 adult SPF SD rats were randomly divided into 4 groups according to body weight,namely control group (corn oil) and low,medium and high doses[28,170 and 1 080 mg/(kg·d) respectively]. In the DEHA group,30 animals in each group, with a female to male ratio of 2:1. The test substance was given by intragastric administration once a day. Male rats were exposed to the DEHA for 10 weeks and female rats were exposed to the DEHA for 2 weeks. Cages were closed. After the exposure,the body weight,related organ mass and organ coefficients of the parent rats were measured and pathological examinations were conducted to determine the blood biochemical indexes of the parent rats. The number of successful mating days,the number of pregnant animals,the number of litters per litter and the weight of litters were recorded. RESULTS: Among the parent rats,the final body mass of the male rats in the high-dose group decreased compared with the control group (P < 0.01),and the testicular organ coefficient increased compared with the control group (P < 0.05). The weight and liver and kidney organ coefficients increased compared with the control group (P < 0.01),and the kidney weight increased compared with the control group (P < 0.05). The levels of aspartate aminotransferase (AST) and creatinine (creatinine, CREA) in the high-dose group were higher than those in the control group (P < 0.05);the AST levels in the low-dose group were compared with the control decreased (P < 0.05); parental female rat globulin (GLO) and total cholesterol (TC) levels in the high-dose group were increased compared with the control group (P < 0.05). The weight of fetal body in the high-dose group was reduced (P < 0.01). Microscopic analyses showed no obvious pathological abnormalities. CONCLUSION: DEHA caused limited body weight gain and had toxicity in the liver and kidneys in parental rats,and might affect growth and development failure of fetuses.