OBJECTIVE: To investigate effects of mitochondrial-targeted antioxidant Mito-TEMPO on cytotoxicity which was induced by nitrogen mustard(HN2) in the human bronchial epithelial cell line BEAS-2B cells. METHODS: BEAS-2B cells were divided into four groups:normal control,Mito-TEMPO control,HN2exposure and Mito-TEMPO intervention groups. The two control groups were treated with serum-free medium containing DMSO(0.1%) or Mito-TEMPO(100 μmol/L) for 26 h,respectively. The HN2 exposure group was pretreated with DMSO-containing serum-free medium for 2 h,and then with HN2(8 μmol/L) for 24 h. The Mito-TEMPO intervention group was pretreated with Mito-TEMPO(100 μmol/L) for 2 h,and then co-treated with HN2(8 μmol/L) and Mito-TEMPO(100 μmol/L) for 24 h. Cell viability was detected by CCK-8 method,LDH activity in cell culture medium supernatant was detected by rate method,cell morphology was observed by inverted microscope,cell apoptosis was detected by Annexin V/PI probe method using flow cytometry,and mitochondrial membrane potential was detected by JC-1 probe. UV spectrophotometry were used to detect thecontent of ATP in cell homogenate. MitoSOX,DCFH-DA and DHE probe were used to detect the level ofmitochondrial ROS(reactive oxygen species), intracellular H2O2and intracellular total ROS respectively.RT-PCR was used to detect the mRNA expression levels of inflammatory factor TNFα and IL-6. RESULTS: Compared with the nitrogen mustard exposure group,the cell viability of the Mito-TEMPO intervention groupdecreased by about 42.6%(P<0.05),and the LDH level in the cell supernatant increased by about 73.5%(P<0.05), accompanied by increase of abnormal cell morphology. Compared with the HN2 exposure group, theMito-TEMPO intervention group showed the level of mitochondrial ROS decreased by 53.6%(P<0.05),and thelevel of intracellular H2O2and total ROS increased by 171%(P<0.05) and 43.4%(P<0.05) respectively.Mito-TEMPO intervention had no significant effects on changes of apoptosis ratio, mitochondrial membranepotential, ATP content, TNF-α and IL-6 mRNA expression levels caused by HN2 exposure(P>0.05). CONCLUSION: 100 μmol/L Mito-TEMPO intervention significantly promoted cell damage in the HN2exposure BEAS-2B cell model, and the mechanisms might be related to increase of intracellular H2O2andtotal ROS levels.

OBJECTIVE: To evaluate toxicity from chronic fluoride and arsenic exposures to kidneys during pregnancy through adulthood in rats,and to explore the potential regulatory mechanisms. METHODS: Rats were divided into four groups:control (distilled water),fluoride exposure (100 mg/L sodium fluoride),arsenic exposure (75 mg/L sodium arsenate),and fluoride and arsenic combined exposure (100 mg/L sodium fluoride+75 mg/L sodium arsenic) groups.The methods of exposure were as follows:from the first day of pregnancy to the 21st day after birth,offspring rats were exposed to fluoride and arsenic toxicity through maternal uterus and breast milk respectively.After weaning,exposure through free drinking water up to 120 days through the same method of exposure as the parents.The kidney coefficient was calculated after weighing the fresh kidney tissues of the offspring male rats.Pathological changes of glomerulus and tubules were observed by Masson staining and transmission electron microscopy.Expression intensity and distribution of autophagy-related proteins in glomerulus and tubules were determined by immunofluorescence technique. RESULTS: Compared with the control group,the renal organ coefficient of the arsenic exposure group was significantly increased (P<0.01).The degrees of renal fibrosis were aggravated after alone and combined exposure of fluoride and arsenic,and renal fibrosis was the most obvious in the fluoride exposed group.Hyperplasia of mesangial cells and vacuolation of mitochondria in podocytes were found in all exposed group.Typical autophagosomes in podocytes were found in the arsenic exposed group.Compared with the control group,the expression levels of autophagy related proteins Beclin1,LC3 and p62 in renal tissues were significantly increased after alone and combined exposure of fluoride and arsenic (P<0.05).Fluorescence intensity and fluorescence area of autophagy-related proteins of glomerulus and renal tubules were also markedly increased (P<0.01). CONCLUSION: Alone and combined exposures to fluoride and arsenic from pregnancy to adulthood induced toxic damage to glomerulus and tubules in renal tissues.Moreover,autophagy of kidney was associated with increasing expressions of autophagy related proteins Beclin1,LC3 and p62.

OBJECTIVE: To investigate effects from over-expression of DnaJ heat shock protein family member B6 isoform b (DNAJB6b) on invasive and migration abilities of colorectal cancer (CRC) cells. METHODS: Expressions of DNAJB6b in cultured CRC cell lines DLD-1 and HCT116 were knocked down with small interfering RNA (siRNA) and short hairpin RNA (shRNA),respectively.Control cells were transfected with negative control siRNA and shRNA.Changes in protein levels were detected by Western blot.Changes of DNAJB6a and DNAJB6b mRNA levels in CRC specimens were statistically analyzed using the GEO dataset.DLD-1 and HCT116 cells were treated with PI3K/mTOR dual inhibitor BEZ235 and control cells were treated with solvents.Then,transwell invasion and migration assays were performed and the number of transmembrane cells were counted.Constitutively activated AKT (myr-Akt) was transiently-transfected into DNAJB6b stably-depleted DLD-1 and HCT116 cells (Referred as DLD-1-shD6b,HCT116-shD6b in this paper) and the controls were transfected with the empty vector.The rescue assay were conducted,changes in protein expression levels were detected by Western blot,and levels of cell invasion and migration were evaluated by Transwell assays. RESULTS: Compared with adjacent normal tissues,mRNA expressions of DNAJB6b but not DNAJB6a were significantly up-regulated in the CRC tissues (P<0.05).DNAJB6b depletion markedly reduced the p-Akt (Ser473) protein levels in DLD-1 and HCT116 cells compared with the control groups.The numbers of transmembrane cells treated by BEZ235 were significantly reduced compared to only about 20% of the control group (P<0.01).Resultsfrom the rescue assay showed that enforced expression of exogenous myr-Akt in DLD-1-shD6b and HCT116-shD6b cells up-regulated the p-Akt (Ser473) levels and prominently reversed the decreased number of transmembrane cells caused by DNAJB6b knockdown (P<0.01). CONCLUSION: DNAJB6b was up-regulated in CRC tissues,and its overexpression enhanced the invasion and migration abilities of CRC cells by activating the AKT pathway.Our observations suggest that these abnormal expressions played an oncogenic role in the development of CRC.

OBJECTIVE: To investigate expressions of melanoma-associated antigen A family (MAGE-As)in peripheral blood and cancer tissues from breast cancer patients,and to analyze their relationships with clinical parameters. METHODS: Expression levels of MAGE-As mRNA in peripheral blood of 106 breast cancer patients and 30 healthy controls were detected by multi-nested PCR,and expression levels of MAGE-A1,A2,A3,A4 and A6 were identified by restriction endonuclease digestion method.Expressions of MAGE-As protein in paraffin-embedded tissues of cancer tissues were detected by immunohistochemistry.Correlations between expressions of MAGE-As mRNA in peripheral blood and of MAGE-As protein in corresponding tumor tissues were analyzed. RESULTS: Positive expression rate of MAGE-As mRNA in the blood samples from patients was 40.5%(43/106).Expression rates of MAGE-A1,A2,A3,A4 and A6 were 10.3%(11/106),19.8%(21/106),15.1%(16/106),13.2%(14/106) and 5.7%(6/106),respectively.The sequence of MAGE-As gene expressions was A2>A3>A4>A1>A6.The MAGE-As mRNA expression was positively correlated with age,tumor size,clinical stage and lymph node metastasis (P<0.05).Expressions of MAGE-As mRNA in blood samples of patients were positively correlated with expression of MAGE-As protein in corresponding tumor tissues (r=0.368,P<0.01).Positive expressions of MAGE-As subtypes (MAGE-A1,A2,A3,A4 and A6)were correlated with 5-year survival rates of breast cancer patients (P<0.01).MAGE-As expression and lymph node metastasis were independent prognostic factors for 5-year survival of these patients (all P<0.01). CONCLUSION: MAGE-As may be a specific biomarker for detection of circulating tumor cells in peripheral blood of breast cancer patients,and its expression was correlated with the prognosis of these patients.MAGE-As in peripheral blood of breast cancer patients may be useful as an important biomarker to monitor prognosis of breast cancer.

OBJECTIVE: To investigate effects of hydrogen peroxide,curcumin,quercetin and tert-butyl hydroquinone(tBHQ) on expressions of 84 oxidative stress-related genes. METHODS: HaCaT cells in logarithmic growth phase were used,and different concentrations of hydrogen peroxide,curcumin,quercetin and tert-butyl hydroquinone were applied.The concentrations were:hydrogen peroxide (0.5 and 1.25 mmol/L),curcumin (5 and 20μmol/L),quercetin (200 and 500μmol/L) and tert-butyl hydroquinone (10 and 50μmol/L) for 4 h and 24 h,respectively.Cytotoxicity of the test substances were evaluated by tetramethylthiazole blue (MTT).RNA samples were extracted and their qualities were evaluated.Then,expression levels of 84 oxidative stress-related genes in HaCaT cells were detected by quantitative real-time PCR (qPCR). RESULTS: MTT assay showed that the survival rate of HaCaT cells was less than 80% when the concentrations of hydrogen peroxide,curcumin and tert-butyl hydroquinone were>1 mmol/L,>10μmol/L and>50μmol/L,respectively.Quercetin did not show obvious cytotoxicity at the concentration of 1 000μmol/L.Resultsof qPCR showed that the four chemicals affected expressions of oxidative stress-related genes in HaCaT cells at different concentrations and at different times.The expression levels of some oxidative stress-related genes were increased,and otherswere decreased.Hydrogen peroxide increased expression of HMOX1,SPINK1 (1.25 mmol/L,4 h) but reduced that of CCL5 (1.25 mmol/L,24 h).Curcumin increased HMOX1,ALB (20μmol/L,4 h)expression but reduce that of GSR (20μmol/L,4 h) and other genes.Quercetin increased ALB,HMOX1 (500μmol/L,4 h) expressions but reduced expression of SEPP1 (500μmol/L,4 h).tert-butyl hydroquinone increased expression of EPX,HMOX1 (10μmol/L,4 h) but reduced that of AOX1 (10μmol/L,24 h),etc. CONCLUSION: Exposure to hydrogen peroxide,curcumin,quercetin and tert-butyl hydroquinone caused changes in expression of oxidative stress-related genes,especially increased expression the HMOX1 gene.Moreover,higher concentrations of the chemicals caused more changes than lower doses.

OBJECTIVE: To study effects of Chaihushugan powder on the JAK2/STAT3 signaling pathway in rats with early liver fibrosis. METHODS: Fifty SD rats were treated with CCl₄ and randomly divided into normal group (intraperitoneal injection of olive oil + intragastric administration of distilled water), model group(intraperitoneal injection of 40% CCl₄ olive oil solution+intragastric administration of distilled water),and low,medium and high dose groups of Chaihushugan powder (intraperitoneal injection of 40% CCl₄ olive oil solution+intragastric administration of 3.5, 6.3 and 12.6 g/kg crude drugs, respectively). After administration of Chaihushugan powder until the tenth week,general morphology was observed,and liver indices were measured.ALT and AST were determined by automatic biochemical analyzer. Changes of serum (HA,PCⅢ,Ⅳ-C) were detected by ELISA. Pathological changes of the hepatic fibrosis tissues in each group were observed by HE staining. mRNA expressions of JAK2 and STAT3 in liver tissues were detected by qPCR, and protein expressions of JAK2 and STAT3 in liver tissue were detected by Western blot. RESULTS: Compared with the normal group, levels of serum ALT,AST,HA,PCⅢ,Ⅳ-C in the model group were significantly increased(P<0.01). HE staining showed obvious edema of liver cells, with increased volume and inflammatory cell infiltration. Compared with the model group, different doses of Chaihushugan powder inhibited hepatocyte necrosis and inflammatory cell infiltration, and serum levels of ALT, AST, HA, PC Ⅲ ,and Ⅳ-C were significantly reduced (P<0.05 or P<0.01). The liver indices of rats in the middle and high dose groups of Chaihushugan powder decreased significantly (P<0.05). Each dose group of Chaihushugan powder inhibited the expression of JAK2, STAT3 mRNA and protein in liver tissues (P<0.05 or P<0.01). CONCLUSION: The protective effect of Chaihushugan powder on early hepatic fibrosis rats might be related to inhibition of the JAK2/STAT3 signaling pathway.

OBJECTIVE: To culture human granulosa cells in vitro,detect the cytotoxic effect of CuInS₂/ZnS quantum dots and analyze its molecular mechanism. METHODS: Hyaluronidase was used to remove granulosa cells outside of human superovulation,and primary cultures were carried out. Cells were treated with medium (10 μg/mL) and high (100 μg/mL) CuInS₂/ZnS quantum dot doses. Changes of cell viability after treatment were detected by the CCK-8 method and the median lethal dose (IC₅₀) was calculated. Apoptosis was detected by flow cytometry with the Annexin V/PI double staining method;and release and oxidation of sex hormones were detected by kits stress changes. RESULTS: Human granulosa cells were successfully cultured in vitro. The IC₅₀of CuInS₂/ZnS quantum dots was 66.72 μg/mL. Compared with the PBS control group,cell viability was significantly inhibited and apoptosis was induced in the high- dose group (P<0.01). In the two treatment groups,release of progesterone (P) and estradiol (E2) were obviously increased (P<0.05 or P<0.01),levels of reactive oxygen species (ROS) in granulosa cells were significantly elevated (P<0.05 or P<0.01),and malondialdehyde (MDA),total antioxidative capacity (T-AOC) and total superoxide dismutase (T-SOD) activity were significantly decreased (P<0.01) compare with the control group. In addition, intracellular glutathione(GSH) activity was remarkedly increased in the high-dose treated group (P<0.01). CONCLUSION: Human granulosa cell lines were successfully cultured in vitro,and CuInS₂/ZnS quantum dots above 10 μg/mL induced oxidative damage by invading granulosa cells and promoted apoptosis. Our results provide valuable information for the biosafety assessment of CuInS₂/ZnS quantum dots in clinical applications.

OBJECTIVE： To investigate mechanisms of morin in lipopolysaccharide (LPS)-induced acute lung injuries (ALI). METHODS： The ALI model was established by in vivo experiments in male C57BL/6 mice which were divided into four groups：control (saline)，LPS-treated ALI，LPS+morin (25 mg/kg)，and LPS+morin (50 mg/kg) groups. LPS (6 mg/kg) solution was instilled into the airways of mice through the trachea to stimulate bronchial and lung tissues after 3 days of intraperitoneal injection of morin solution at a concentration of 25 mg/kg or 50 mg/kg. Both lungs were harvested from each mouse. Some lung tissues were stained with HE and the others for measurements of ratio of lung wet mass to dry mass，and histopathological observations were conducted. Real-time quantitative PCR (qPCR) and Western blot were used to detect expressions of oxidative stress signaling pathway-related genes (NOX1，NOX4，Nrf2 and HO-1) in lung tissues. RESULTS： Compared with the control group，HE staining and the ratio of lung wet mass to dry mass showed that airway instillation of LPS successfully established a mouse model of acute lung injury，and morin can effectively alleviate the LPS-induced injury. The results of qPCR and Western blot experiments showed that expressions of Nrf2 and HO-1 were up-regulated in the lung tissue of the LPS-stimulated ALI model group (P<00.01)，compared with the control group. Morin treatment significantly reduced the expressions of NOX1 and NOX4 (P<00.05 or P<00.01). Furthermore， and morin treatment up-regulated expressions of Nrf2 and HO-1 to inhibit the occurrence of oxidative stress (P<00.05 or P<00.01)，compared with the ALI model group. CONCLUSION： Oxidative stress was involved with the occurrence and development of ALI. In addition，the Nrf2-HO-1 signaling pathway reduced the expression of NOX1 and NOX4 through the activation of morin，and inhibit oxidative stress，thereby slowing the alveolar edema and lung tissue fibrosis caused by LPS.

OBJECTIVE: Serum pharmacology was used to explore anti-gastric cancer effects of licorice and dried ginger decoction (LDGD) in vitro,and its anti-gastric cancer mechanism was discussed based on network pharmacology and molecular docking. METHODS: After intragastric administrations of LDGD in different proportions to mice,serum samples were extracted by taking blood from abdominal aorta.Effects of the drug containing serum on activities of human gastric cancer cells AGS and human umbilical vein endothelial cells HUVEC in vitro were observed using the MTT method.The "component target" network of LDGD against the gastric cancer cells was constructed by network pharmacology.Key targets of LDGD against the cancer cells were selected by constructing protein-protein interaction network (PPI).Possible biological functions and signal pathways of LDGD against the cancer cells were explored by the GO function and KEGG pathway enrichment analyses.The binding of 8 pharmacodynamic components in LDGD and key anti-gastric cancer targets reported in the literature were analyzed by molecular docking. RESULTS: In the serum pharmacology experiment,30% medicated serum with different proportions of LDGD had significant inhibitory effects on cell viability of AGS but had no significant inhibitory effect on HUVEC.A total of 101 active components of LDGD were obtained,and the key targets of LDGD in the gastric cancer cells were VEGFA,TNF-α,CASP3 and MYC.Bioinformatics enrichment analyses show that LDGD anti-gastric cancer effects may mainly regulate apoptosis signal,oxidative stress and reactive oxygen species response,as well as influence on TNF,p53 and other signal pathways.The results of molecular docking show that glycyrrhizic acid and liquiritin had strong affinity with VEGFA,and 6-gingerol had strong affinity with TNF-α. CONCLUSION: LDGD showed certain antitumor activities on gastric cancer cells.Through network pharmacology and molecular docking,the antitumor activities of LDGD involved regulating oxidative stress and acting on TNF,p53 and other signal pathways.In addition,glycyrrhizic OBJECTIVE: To study effects of Fuzi (aconite) extracts on proliferation and radiosensitization of human lung cancer A549 cells. METHODS: The cells were cultured and were divided into 4 groups:control,Fuzi extract,X-ray-irradiated,and Fuzi plus X-ray-irradiated groups. The control group was given culture medium,the Fuzi extract group was treated with IC₂₀30 μg/mL of Fuzi extract,the radiation group was given X-ray-irradiated,and the Fuzi plus X-ray-irradiated group were first treated with the Fuzi extract and then X-ray. After treatments,cell proliferation was detected by CCK-8 assay and colony formation assay was used to measure cell survival. Single-hit multi-target model was used to fit the survival curve and to calculate the sensitive enhancement ratio (SER). Apoptosis rates were detected by flow cytometry,and protein expression levels of Bax and Bcl-2 were detected by Western blot. RESULTS: Fuzi extract concentrations(<20 μg/mL) enhanced viability of A549 cells but increasing concentrations (>30 μg/mL) inhibited growth of the cells in a dose-dependent manner. The shoulder at the beginning of the survival curve in the Fuzi plus X-ray-irradiated group was slightly narrower, and the D₀values in the X-ray-irradiated and Fuzi plus X-ray-irradiated group were 1.83 Gy,1.48 Gy,and the SER was 1.24,respectively. The apoptosis rates were significantly increased in the Fuzi plus X-ray-irradiated group and were higher than that in both the Fuzi and X-ray irradiation groups (P<0.05). The results of Western blot show that protein expressions of Bax were increased but were decreased for Bcl-2 in the Fuzi extract versus the X-ray-irradiated groups. Bax and Bcl-2 protein levels were changed more significantly in the Fuzi plus X-ray-irradiated than the other groups (P<0.01). CONCLUSION: Fuzi extract at 30 μg/mL inhibited proliferation and enhanced radiosensitizing effects on human lung cancer A549 cells,and the mechanism may be related to the induction of apoptosis.

OBJECTIVE: To study effects of Fuzi (aconite) extracts on proliferation and radiosensitization of human lung cancer A549 cells. METHODS: The cells were cultured and were divided into 4 groups:control,Fuzi extract,X-ray-irradiated,and Fuzi plus X-ray-irradiated groups. The control group was given culture medium,the Fuzi extract group was treated with IC₂₀ 30 μg/mL of Fuzi extract,the radiation group was given X-ray-irradiated,and the Fuzi plus X-ray-irradiated group were first treated with the Fuzi extract and then X-ray. After treatments,cell proliferation was detected by CCK-8 assay and colony formation assay was used to measure cell survival. Single-hit multi-target model was used to fit the survival curve and to calculate the sensitive enhancement ratio (SER). Apoptosis rates were detected by flow cytometry,and protein expression levels of Bax and Bcl-2 were detected by Western blot. RESULTS: Fuzi extract concentrations(<20 μg/mL) enhanced viability of A549 cells but increasing concentrations (>30 μg/mL) inhibited growth of the cells in a dose-dependent manner. The shoulder at the beginning of the survival curve in the Fuzi plus X-ray-irradiated group was slightly narrower, and the D₀values in the X-ray-irradiated and Fuzi plus X-ray-irradiated group were 1.83 Gy,1.48 Gy,and the SER was 1.24,respectively. The apoptosis rates were significantly increased in the Fuzi plus X-ray-irradiated group and were higher than that in both the Fuzi and X-ray irradiation groups (P<0.05). The results of Western blot show that protein expressions of Bax were increased but were decreased for Bcl-2 in the Fuzi extract versus the X-ray-irradiated groups. Bax and Bcl-2 protein levels were changed more significantly in the Fuzi plus X-ray-irradiated than the other groups (P<0.01). CONCLUSION: Fuzi extract at 30 μg/mL inhibited proliferation and enhanced radiosensitizing effects on human lung cancer A549 cells,and the mechanism may be related to the induction of apoptosis.