OBJECTIVE: To study the effect of ferulic acid on damage of human umbilical vein endothelial cells (HUVECs) from their exposure to ionizing radiation METHODS: HUVECs were exposed to ⁶⁰Co gamma ray at the radiation doses of 4-16 Gy. Cells viability as determined by the MTS assay was examined 48 h after irradiation to select the appropriate irradiation dose. Expression levels of Thbd and γH2AX were examined by western blot assay with the total protein of HUVEC extracted at 1,3,6,12,24 and 48 h after irradiation. In addition,HUVEC received 0.001-1 μmol/L ferulic acid at 2 h before irradiation. Cell viability and expression of Thbd and γH2AX protein were determined at 48 hours after irradiation. RESULTS: Compared with the non-irradiated group,cell viability at 48 h after 10 Gy irradiation was decreased by 30% (P < 0.05). Thbd expression was reduced to about 0.6-fold (P < 0.05) at 1 h after irradiation. γH2AX was increased about 5 times (P < 0.05) at 48 h after irradiation. Addition of 0.1 to 1 μmol/L ferulic acid increased the viability of irradiated HUVEC and the protein expression of Thbd at 1 h after irradiation,and decreased the expression of γH2AX at 48 h after irradiation. CONCLUSION: Ferulic acid in the concentration of 0.1-1 μmol/L played a role in radiation protection since it promoted cell vitality and proliferation by increasing the expression of Thbd protein and decreasing the expression of γH2AX protein.

OBJECTIVE: To investigate the effect and mechanism of silencing of TPX2 gene expression by lentivirus on cell proliferation,migration and cell cycle in human cervical cancer HeLa cells. METHODS: Four lentivirus expression vectors carrying TPX2-shRNA1/2/3/4 which target TPX2 gene (named as LV-TPX2-shRNA-1/2/3/4) were constructed,while vector TPX2-NC-shRNA was used as the negative control. The five plasmids were separately transfected into 293T cells to prepare the recombinant lentivirus. The recombinant lentivirus carrying TPX2-shRNA1/2/3/4 and TPX2-NC-shRNA were infected into HeLa cells,respectively. Then,expression levels of TPX2 mRNA and protein in HeLa cells were determined by real-time fluorescent quantitative PCR and Western blotting,respectively. The effect of TPX2 gene silencing on the proliferation of HeLa cells was detected using the CCK-8 method. Cell migration and cell-cycle distribution were detected by transwell migration experiment and flow cytometry,respectively,in each group of HeLa cells. Changes of Ki-67,cyclin B2,P53 and Aurora-A expressions were detected by Western blotting. RESULTS: Compared with the negative control group,expressions of TPX2 mRNA and protein in HeLa cells were significantly inhibited by LV-TPX2-shRNA,especially LV-TPX2-shRNA-1 (P < 0.01). Subsequently,LV-TPX2-shRNA-1 was selected for follow-up experiments. Silencing TPX2 gene expression reduced the proliferation and migration(P < 0.05),increased the percentage of G2 and S phase cell in the cell cycle(P < 0.05),up-regulated the expression of P53,and down-regulated the expression of Ki-67,cyclin B2 and Aurora-A in HeLa cells (all P < 0.05). CONCLUSION: Our results show that silencing TPX2 gene expression reduced the proliferation and migration in HeLa cells,which may be related to changes in cell cycle distribution,up-regulating the expression of P53,and down-regulate the expression of Ki-67,cyclin B2 and Aurora-A in HeLa cells.

OBJECTIVE: To investigate the inhibitory effect of paclitaxel and SAHA on human cervical cancer cells. METHODS: Human cervical cancer (HeLa) cells were treated with paclitaxel (10 nmol/L),SAHA (10 μmol/L),or paclitaxel (10 nmol/L) + SAHA (10 μmol/L) for 24 hours or 48 hours. The inhibitory rates of HeLa cells were determined by MTT assay. p27 mRNA levels were determined by RT-PCR assay,protein levels of Ac-H4 were identified by Western blot and IC₅₀ of paclitaxel was calculated by SPSS 16.0. RESULTS: Results from the MTT assay show that the inhibition rates from exposure to the combination of paclitaxel and SAHA were significantly higher than those from the single treatment groups (P < 0.01). Similarly,RT-PCR assay show that the p27 mRNA levels from the combined treatments were significantly higher than from single treatments (P < 0.01). From combined treatments,the inhibitory rates were 54.27%±4.02% with 24 h treatment and 77.02%±3.86% with 48 h treatment,the mRNA level was 10.601±0.673,the protein level of Ac-H4 was 1.282±0.033,and IC₅₀ was reduced significantly. CONCLUSION: SAHA in combination with paclitaxel significantly inhibited HeLa cell proliferation by enhancing the level of histone acetylation and inducing the expression of tumor suppressor genes in vitro.

OBJECTIVE: To investigate whether formaldehyde can induce endoplasmic reticulum stress in hepatocytes. METHODS: Hoechst 33258 was used for nuclear staining to observe morphological changes of hepatocytes in different formaldehyde concentration groups. The human hepatocellular carcinoma cell line HepG2 was treated with formaldehyde (FA) solution at 0.004 mmol/L,0.02 mmol/L and 0.1 mmol/L for 24 h and 48 h respectively. Expression of Bip and CANX mRNA in stress-related genes was detected by fluorescence quantitative PCR (qPCR). Western blot was used to detect the expression of stress-related immunoglobulin heavy chain binding protein (Bip) and connexin (CANX) in endoplasmic reticulum. RESULTS: Cells in the negative control group and the low dose group were significantly higher with Hoechst 33258 staining than in the control group (P < 0.05). Results of qPCR show that expression of Bip and CANX mRNA in each dose group was not significantly different from that in the negative control group. Results of Western blot show that there was no significant difference in the expression of Bip and CANX proteins among the different doses of FA group and the negative control group (P>0.05). Compared with the negative control group (P < 0.05), expression of Bip protein was increased in 0.02 mmol/L and 0.1 mmol/L FA group,the difference was statistically significant (P < 0.05). CONCLUSION: Formaldehyde exposure may induce endoplasmic reticulum stress of hepatocytes.

OBJECTIVE: To study the protective effects of Colla Corii Asini on respiratory system injury which was induced by artificial fine particulate matter (aPM2.5) in rats. METHODS: 96 SD rats(half male and half female)were randomly divided into four groups, control,model,Colla Corii Asini low dose (0.3 g/kg) and high dose (1.2 g/kg) groups. Rats in both model and Colla Corii Asini groups were exposed to aPM2.5 15 minutes per day of 7 days a week for 8 weeks using the aerosol exposure system. In contrast,rats in the control group were exposed to clear air. 1 h before daily exposure to aPM2.5,rats of Colla Corii Asini groups were given Colla Corii Asini by intragastric administration. During the experimental period,the EMKA animal lung function monitoring system was used to monitor the lung function of rats. Every two weeks,blood serum and bronchoalveolar lavage fluid (BALF),and lung tissues were collected to detect changes in biomarkers of oxidative injury. RESULTS: Compared with the control group,the tidal volume (TV),expiratory volume (EV) and minute volume (MV) of the model group were markedly reduced,while the inspiratory time (Ti),expiratory time (Te) and relaxation time (RT) were significantly increased.,However,treating rats with Colla Corii Asini before aPM2.5 exposure significantly prevented the decline of TV,MV,EV and the increase of Ti,RT,Te. The lung phagocyte counts of the model group increased significantly compared with the control group,while the situation of Colla Corii Asini high dose group achieved a certain degree of relief. Compared with the control group,the concentration of methane dicarboxylic aldehyde (MDA) in serum and BALF of the model group increased,and with the treatment of Colla Corii Asini,the MDA decreased. Compared with the control group,the acvtivity of glutathione peroxidase (GSH-Px) in serum and BALF of the model group decreased,and with the treatment of Colla Corii Asini,the the acvtivity of glutathione peroxidase (GSH-Px) increased. CONCLUSION: Treatment of the model rats with Colla Corii Asini prevented decline of lung function and reduced lung phagocyte counts which were caused by aPM2.5,It is possible that Colla Corii Asini can prevent oxidative damage caused by aPM2.5. Further study is needed to address the mechanism for interactions.

OBJECTIVE: To construct the CYP2E1 knock-out (KO) HEK293FT cell line using CRISPR/Cas9 system and explore its usefulness in chemical toxicity testing. METHODS: The sgRNA expression vector targeting CYP2E1 gene was transfected into HEK293FT cells. Single cells from the transfected cells were isolated through serial dilutions to get the HEK293FT CYP2E1 KO cell line. The cell line was tested for expression of cytotoxicity from N-(4-hydroxyphenyl)-acetamide(4-APAP) and 1,2-dichloroethane(1,2-DCE). RESULTS: Two HEK293FT CYP2E1 KO cell lines were obtained and they showed dramatic reduction in susceptibility to N-(4-hydroxyphenyl)-acetamide (4-APAP) and 1,2-dichloroethane (1,2-DCE) cytotoxicity. CONCLUSION: HEK293FT CYP2E1 KO cell lines were successfully constructed using a CRISPR/Cas9 system and they can be useful for studying the function of CYP2E1.

OBJECTIVE: To investigate expressions of HDJ1 in lung adenocarcinoma tissues and cell lines,and to reveal the role of HDJ1 in invasive ability of lung adenocarcinoma. METHODS: Immunohistochemistry method was used to detect expressions of HDJ1 in lung adenocarcinoma tissues and adjacent normal tissues. Western blot method was applied to determine expressions of HDJ1 in human lung adenocarcinoma cell lines (A549,H1299,H292) and normal lung epithelial cell line (Bease-2B). pMagic 7.1-HDJ1-KD lentivirus was transfected into the A549 cell line to construct a HDJ1 down-expressed cell line,and pMagic 7.1-NC lentivirus was transfected into the same cells as the control. The invasion ability of HDJ1 down-expressed group and control group was analyzed using the Transwell and the Wound Healing assays. RESULTS: The expression levels of HDJ1 in lung adenocarcinoma tissues were significantly higher than those in the adjacent normal tissues. HDJ1 was over-expressed in the A549 cell line. Compared with the control group,the Wound Healing assay results show that,the migration and healing ability of the HDJ1 down-expressed A549 cells were decreased. At 48 h after transfection,both the invasion and migration abilities of the HDJ1 down-expressed A549 cells were lower than that of control cells (P < 0.05). CONCLUSION: Heat shock protein HDJ1 were highly expressed in the lung adenocarcinoma tissues and cell lines. In addition,HDJ1 promoted the invasion and migration ability of lung adenocarcinoma cells.

OBJECTIVE: To investigate the effects of isoimperatorin on expression of tyrosinase and β-actin in cultured human epidermal melanocytes. METHODS: Human epidermal melanocytes were separated,purified and cultured in vitro. These cultures were randomly divided into control and isoimperatorin groups. Cells were cultured with M-254 medium. For the isoimperatorin group,25 μmol/L isoimperatorin in M-254 was added to the cultures. After 48 h of incubation,the morphology of melanocytes was observed under an inverted microscope. Melanin content was assayed by using the NaOH method. The methods of qPCR,Western blot and immunofluorescent were used to analyze the expression of tyrosinase and β-actin. RESULTS: After treatment with 25 μmol/L isoimperatorin for 48 h,dendrites of melanocytes were slightly elongated compared with the untreated cells. In addition,the treatment reduced melanin content (P < 0.01). The results of qPCR,Western blot and immunofluorescence assay consistently indicated that isoimperatorin reduced tyrosinase mRNA and protein,and increased β-actin mRNA and protein. CONCLUSION: Cultured human epidermal melanocytes which were treated with 25 μmol/L isoimperatorin had reduced content of melanin. The observed effect was associated with inhibition of tyrosinase mRNA and protein,and with increased β-actin mRNA and protein.

OBJECTIVE: To study protective effect of nicotiflorin from Nymphaea candida on D-Gal-induced liver injury in mice. METHODS: Sixty healthy adult mice were randomly divided into six groups:control,model,dimethyl diphenyl bicarboxylate (DDB,150 mg/kg) and nicotiflorin (25,50,100 mg/kg) groups. Chemicals were intragastrically administrated for seven days. One hour after the last administration,mice in all except the control group were intraperitoneally injection with D-Gal (800 mg/kg,0.02 mL/g). All mice were sacrificed at the 8th hour,blood was collected from the eyeball,and serum ALT,AST,IL-1β,IL-6,TNF-α and INF-γ were determined. Mice were killed cervical dislocation,the quality of the body after the autopsy,the liver,spleen,the quality of the organ coefficient,the liver homogenate were placed in -20 freezer. The left hepatic lobes at 0.5 cm from the edge of tissues were fixed with 10% formalin,dehydrated,transparentized,paraffined and embedding slices were observes under light microscope for pathological changes. RESULTS: Compared with the model group,nicotiflorin significantly reduced serum ALT and AST activity of rats with acute liver injury (P < 0.01) at doses greater than 50 mg/kg, significantly improved liver homogenate of SOD and GSH activity at higher than 50 mg/kg (P < 0.05),significantly decreased MDA and NO content in the liver (P < 0.05),IL-1,IL-6,reduced inflammation and promoted the level of INF-γ and TNF-α in serum of mice (P < 0.05),reduced liver and spleen index (P < 0.05) at a dose of more than 50 mg/kg. Liver pathological examinations showed that,compared with the model group,nicotiflorin significantly reduced the degree of liver cell injury. Inflammatory cell infiltration was significantly decreased,including high,medium and low dose group compared to the extent of the damage and repair. CONCLUSION: The protective effect of D-Gal on acute liver injury in mice was related to the promotion of antioxidant enzymes and the expression of proinflammatory cytokines.

OBJECTIVE: To study the adverse effect of nano-SiO₂ particles on reproductive system in male SD rats. METHODS: Male rats were treated with nano-SiO₂ particles or saline and then executed 4 weeks later. Testes were isolated for the extraction of protein. 2D-DIGE coupled with MALDI-TOF-MS was used to identify nano-SiO₂ induced abnormal expression of proteins. Two proteins were verified by Western blot analysis. RESULTS: Nano-SiO₂ induced abnormal expression of 9 proteins in rat testes (GRP78,HSP7C,KNT1,HSP72,ALBU,STIP1,PDIA1,ALDR and GDIR1). PDIA1 and HSP7C were validated by Western blot analysis,and the results were accordance with DIGE analysis. CONCLUSION: Our findings partially supported our early observation regarding seminiferous tubule dysgenesis in rat. In addition,this study revealed technological advancement in reproductive toxicology of SiO₂.

OBJECTIVE: To explore effects of PM2.5 exposure on expression of oxidative stress and DNA methylation genes during zebrafish embryo development. METHODS: Samples of PM2.5 were collected by large-volume samplers on fiber filters. Then,the particle materials were collected by ultrasonicing and freeze-drying. Zebrafish embryos were exposed to PM2.5 particle materials in the concentrations of 0,5 and 20 μg/mL. After extraction of total RNA,relative expression of oxidative stress genes (sod1 and ogg1) and DNA methylation genes (tet1 and dnmt1) was detected by qPCR. RESULTS: 2-6 h after PM2.5 exposure,expression of sod1,tet1 and dnmt1 were increased significantly with a dose-dependent relationship (r=0.98,0.98,0.99,P < 0.05,respectively);2-6 h after PM2.5 exposure,expression of ogg1 was increased significantly in the 5 μg/mL group (P < 0.05);24-48 h after PM2.5 exposure,expression of sod1,ogg1,tet1 and dnmt1 were decreased significantly compared with those in early stage (2-6 h). CONCLUSION: Expression of oxidative stress genes (sod1 and ogg1) and DNA methylation genes (tet1 and dnmt1) could be affected from exposure to PM2.5,especially during early embryo development.

OBJECTIVE: To investigate the effect of long-term benzopyrene (BaP) exposure on expression of epithelial growth factor receptor(EGFR) in SD rat liver. METHODS: 36 healthy SD female rats were randomly divided into exposure and control groups with 18 rats per group. Rats in the exposure groups were injected with 0.2 mL of 10 mg/mL corn-benzopyrene through chest punctured,once per 2 weeks for 2 months. Rats in the control group were injected with 0.2 mL corn oil. All rats were observed the survival status and body mass changes for one year. Immunohistochemical,immunofluorescence and Western blot assays were used to detect expression of EGFR protein in livers and HE staining for pathological effects. RESULTS: HE staining showed significant fragmentation necrosis and inflammatory infiltration after benzopyrene exposure. Immunohistochemistry and immunofluorescence showed that expression of EGFR in the liver of the exposure group was significantly higher than that of the control group. Based on Western blot results (0.452±0.095,0.915±0.253), expression level of EGFR protein in the exposed group was significantly higher than that in the control group (P < 0.05). CONCLUSION: BaP induced hepatic inflammation and EGFR overexpression,suggesting that the two effects were closely related.

OBJECTIVE: To establish a transiently expressed HBx via hydrodynamic gene delivery in mice. METHODS: Two experimental groups of CD-1 male mice (4 mice per group) were injected with pcDNA-Flag-HBx plasmid or pcDNA empty vector via their tail veins. Mice were then sacrificed and their liver tissues were collected for extraction of RNA and protein. RESULTS: HBx was detected by PCR and Western blotting at the RNA and protein levels,respectively. Transiently expressed HBx effectively regulated the expression of typical target genes,small heterodimer partner (SHP) and cytochrome P450 7A1(Cyp7a1) of farnesoid X receptor signaling,as shown by real-time PCR analyses. CONCLUSION: A transient expression model of HBx via hydrodynamic gene delivery in mice was established.

OBJECTIVE: To establish a method for isolation,cultivation and preservation of human urine-derived stem cells(hUSCs). METHODS: hUSCs were isolated from fresh urine samples from adult healthy volunteers. Survival rate of hUSCs after cryopreservation and resuscitation was detected by trypan blue staining to determine the best formula for freezing medium. Proliferation of hUSCs was determined by MTT assay. Stem cell surface marks were detected by flow cytometry. RESULTS: 6/10 hUSCs were successfully isolated and cultured from urine. The survival rate of hUSCs in the freezing medium-DMSO:FBS:hUSCs medium=1:4:5-was the highest based on trypan blue staining. MTT assay showed that the growth curve of hUSCs was S-shaped showing good proliferation activity. Mesenchymal stem cell surface marker CD44 and CD90 were detected positively using flow cytometry. CONCLUSION: A method for isolation,cultivation and preservation of human urine-derived stem cells was successfully established.