Development of a new protocol for freeze-drying preservation of Pseudoalteromonas nigrifaciens and its protective effect on other marine bacteria

Background: Freeze-drying is known as one of the best methods to preserve bacterial strains. Protectant is the key factor affecting the survival rate of freeze-dried strains. In addition, salinity, bacterial suspension concentration, drying time, and other factors can also affect the survival rate of strains to varying degrees. At present, there are relatively few studies on freeze-drying preservation of marine bacteria. In the present study, we performed the freeze-drying protectant screening and optimized the preservation conditions for Pseudoalteromonas nigrifaciens, which is widely distributed in marine environment. The protective effects of the screened protectants were verified by 18 other marine bacterial strains. Results: The results indicated that the combination of 5.0% (w/v) lactose, 5.0% (w/v) mannitol, 5.0% (w/v) trehalose, 10.0% (w/v) skim milk powder, 0.5% (w/v) ascorbic acid and 0.5% (w/v) gelatin was the best choice for the preservation of P. nigrifaciens. The suggested salinity and concentration of initial cell suspension were 10 g/L NaCl and 1.0 × 109 CFU/mL, respectively. Furthermore, stationary-phase cells were the best choice for the freeze-drying process. The highest survival rate of P. nigrifaciens reached 52.8% when using 5­10% (w/v) skim milk as rehydration medium. Moreover, the other 18 marine strains belonging to Pseudoalteromonas, Vibrio, Photobacterium, Planomicrobium, Edwardsiella, Enterococcus, Bacillus, and Saccharomyces were freezedried under the abovementioned conditions. Their survival rates were 2.3­95.1%. Conclusion: Collectively, our results supported that the protectant mixture and parameters were beneficial for lyophilization of marine bacteria

Inhibitory effects of iridoid glycosides from Boschniakia rossica combined with 5-fluorouracil on human hepatoma HepG2 and SK-Hep1 cells epithelial-mesenchymal transition / 中草药

Objective: To study the inhibitory effect of iridoid glycosides from Boschniakia rossica (IGBR) combined with 5-Fu on epithelial-mesenchymal transition induced by TGF-β1 in human hepatoma SK-Hep1 and HepG2 cells, and compare the efficacy of drugs. Methods: The survival ability of HepG2 and SK-Hep1 cells was detected by MTT and the combination index (Q value) was calculated to judge the interaction of combined drugs. The EMT model of HepG2 and SK-Hep1 cells was established. The cell adhesion rate was detected by MTT. The expression of matrix metalloproteinase (MMP) MMP2, MMP7, MMP9, Snail, and Slug was detected by Western blotting. The localization and expression intensity of E-cadherin and Vimentin was detected by immunofluorescence. Results: MTT showed that compared with the control group, the 5-FU group, IGBR group and combination group cell survival ability were decreased (P < 0.05) at 48 h after administration; IGBR and 5-Fu had an additive or synergistic effect. Compared with the model group, the adhesion rate of 5-FU group, IGBR group and combination group was reduced (P < 0.05). Western blotting results showed that compared with the control group, the expression of MMP2, MMP7, MMP9, Snail, Slug were up-regulated (P < 0.05) in the model group. Compared with the model group, the expression of MMP2, MMP7, MMP9, Snail and Slug were down-regulated (P < 0.05) in 5-FU group, IGBR group and combination group. Compared with the control group, immunofluorescence showed that the E-cadherin fluorescence intensity was decreased in the model group, but the Vimentin fluorescence intensity was increased. Compared with the model group, the E-cadherin fluorescence intensity was increased in 5-FU group, IGBR group and combined group, but the Vimentin fluorescence intensity was decreased. Conclusion: IGBR and 5-Fu can inhibit human hepatoma EMT. The combined drugs have the combined effect on HepG2 cells and synergistic effect on SK-Hep1 cells. The therapeutic effect on SK-Hep1 cells is better than HepG2 cells.

Influence of miRNA-21 in radiosensilivity of breasl cancer cells and ils mechanism / 吉林大学学报(医学版)

Objective: To observe the influence of miRNA-21 in the radiosensitivity of the breast cancer cells, and to preliminarily explore its mechanism. Methods: The breast cancer T47D and MDA-MB-361 cells were irradiated with 0. 0. 2.5. and 5. 0 Gy y-ray. respectively; CCK8 assay was used to detect the survival rates of T47D and MDA-MB-361 cells and flow cytometry was used to analyze the cell cycle; real-time quantitative PCR was used to detect the expression levels of miRNA-21 in cells during 72 h. The T47D and MDA-MB-361 cells were transfected with anti-miRNA-21 sequence (anti-miRNA-21 group) and negative control sequence (negative control group)∗ respectively; the untransfected cells were set as blank control group. Real-time quantitative PCR was used to detect the expression levels of miRNA-21 in cells in various groups. After irradiation with 0. 0 and 5. 0 Gy y-ray. CCK8 assay was used to detect the survival rates, and flow cytometry was used to analyze the cell cycle. Results: After irradiation with 5. 0 Gy y-ray. the survival rate of T47D cells was significantly higher than that of the MDA-MB-361 cells (PC0.05). Compared with 0. 0 Gy radiation group, the percentages of T47D and MDA-MB-361 cells in G:

Effect of cryptotanshinone on ferroptosis-related gene expression in lung cancer cells / 中国药理学通报

Aim To investigate the effects of cryptotanshinone on cell viability Aim To investigate the effects of cryptotanshinone on cell viability and ferroptosis-related gene expression in A549 and A549/DDP cells, and to explore its possible mechanisms. Methods A549 and A549/DDP cells were treated with different concentrations of CTS. Cell viability was measured by CCK-8 assay. The mRNA levels of TFR1, DMT1, IREB2, HSPB1, VDAC2, VDAC3 and GPX4 were measured by qPCR, and the protein levels of TFR1 were measured by Western blot. Results The cell viability was down-regulated by CTS in A549 and A549/DDP cells, while the cisplatin-resistant A549/DDP cells were more susceptible to CTS. After CTS treatment, the mRNA levels of ferroptosis-related genes showed different degrees of change. The mRNA levels of HSPB1 and GPX4 increased in A 5 4 9 cells , of which the mRNA levels of IREB2, VDAC2 and VDAC3 were reduced and the mRNA levels of TFR1 and DMT1 exhibited no significant change. The mRNA levels of TFR1 and IREB2 increased in A549/DDP cells, while the mRNA levels of VDAC3 decreased, and the expression levels of DMT1, HSPB1, VDAC2 and GPX4 did not changesignificantly. Conclusions Cryptotanshinone may inhibit the proliferation of lung cancer A549 and A549/DDP cells, and affect the expression of ferroptosis-re-lated genes.

Effects of arsenic on HOXD10 gene expression / 中国职业医学

OBJECTIVE: To explore the effect of arsenic on the homeobox D10( HOXD10) gene expression in peripheral blood lymphocytes and human lung adenocarcinoma cell A549. METHODS: ⅰ) A total of 59 workers exposed to arsenic from a arsenic factory were selected as the exposure group and 17 local people without arsenic exposure were chosen as controls by using judgment sampling method. Hydride generation-cold hydrazine trapping-atomic absorption spectrometry was used to detect arsenic levels in urine of these 2 groups. The expression of HOXD10 in peripheral blood lymphocytes was detected by real-time fluorescent quantitative polymerase chain reaction( qRT-PCR).ⅱ) The A549 cells were treated with arsenic trioxide( As_2O_3) with concentration of 0. 0,0. 1,0. 5,1. 0 and 2. 0 μmol/L,and the survival rate of cells was examined by colorimetric assay. The expression of HOXD10 was detected by qRT-PCR. RESULTS: ⅰ) The levels of inorganic arsenic,methylarsonic acid,dimethyl arsenate,total arsenic in the urine,and the relative expression of HOXD10 mRNA in peripheral blood lymphocyte in exposure group were higher than that of the control group( P < 0. 05).ⅱ) As_2O_3 decreased the survival rate of A549 cells in a dose-dependent manner( P < 0. 01) and lead to a dose-dependent increase of HOXD10 mRNA expression( P < 0. 01). A549 cell survival rate and relative expression of HOXD10 mRNA showed a negative correlation,the correlation coefficient was-0. 777( P < 0. 01). CONCLUSION: Arsenic can up-regulate HOXD10 expression in the peripheral blood of occupational arsenic exposure individuals. As_2O_3 can inhibit the proliferation of A549 cells,which may be related to the up-regulation of HOXD10 expression.

Immune cytotoxicity of trichloroethylene and its mechanism in activated human T cells / 中国职业医学

OBJECTIVE: To explore the immune cytotoxicity effect and its mechanism of trichloroethylene( TCE) on activated human T cells. METHODS: a) Different concentrations of TCE( 0. 32,0. 63,1. 25,2. 50,5. 00,10. 00 mmol / L)were used to treat activated T cells [activated with cluster of differentiation( CD) 3 and CD28] respectively. Dimethyl sulfoxide( DMSO) was used in the solvent group and the control group used no TCE or DMSO. The survival rate of activated T cells was calculated using CCK-8 assay after being cultured for 24 hours. b) Different concentrations of TCE( 0. 00,2. 50,5. 00 mmol/L) were used to treat activated T cells. The apoptosis of cells was detected using flow cytometry. c) Different concentrations of TCE( 0. 00,0. 32,0. 63,1. 25,2. 50,5. 00 mmol / L) were used to treat activated T cells and the level of cytokines as interleukin( IL)-2 and IL-6 in cell culture supernatant was detected using enzyme linked immunosorbent assay after culturing for 24 hours. d) The control group and TCE treatment group of activated T cells were treated with 0. 00 and 5. 00 mmol / L TCE respectively. Cells were collected after culturing 0,30,60 and 120 minutes. Western Blot was used to detect the protein expression of signal transducers and activators of transcription3( STAT3) and phospho-STAT3( p-STAT3). RESULTS: a) After 24-hour-exposure to TCE,the activated T cell survival rate of 10. 00 mmol / L TCE treatment group were significantly lower than that in the control group and DMSO group( P <0. 05). b) There were no significant differences in cell apoptosis of activated T cells after treatment with 0. 00,2. 50 and5. 00 mmol / L TCE( P > 0. 05). c) In groups treated with different concentrations of TCE( 0. 32,0. 63,1. 25,2. 50,5. 00 mmol / L),the level of IL-2 and IL-6 in the cell culture supernatant of activated T cells were significantly higher than that in the control group( P < 0. 05). With the increasing of TCE exposure doses,the levels of IL-2 and IL-6 significantly increased( P < 0. 01) with dose-effect relationship. Compared with the control group,the levels of IL-17 A,interferongamma and transforming growth factor-beta in cell culture supernatant of activated T cells of the TCE treatment groups were no significant differences( P > 0. 05). d) The expression of p-STAT3 protein was low in the control group at different times. The expression of p-STAT3 protein in TCE treatment group was low at 0 minute,but increased at 30,60,120 minutes. The expression of p-STAT3 protein in TCE treatment group was higher than that in the control group at different time points. The levels of STAT3 total protein in TCE treatment group and the control group were similar at different time points,and were higher than the p-STAT3 proteins. CONCLUSION: TCE at 5. 00 mmol / L had no observed toxic effect on activated T cells. High doses of TCE( ≥10. 00 mmol / L) showed cytotoxic damages to activated T cells,and low doses of TCE( ≤5. 00 mmol / L) could stimulate activated T cells to secrete IL-2 and IL-6. Treatment of TCE at 5. 00 mmol / L on activated T cells could up-regulated the level of p-STAT3.

Immune cytotoxic effect of trichloroethylene in Jurkat T cells / 中国职业医学

OBJECTIVE: To explore the immune cytotoxic effect and the maximum non-effect dose of trichloroethylene( TCE) on Jurkat T cells in vitro. METHODS: i) Naive and activated Jurkat T cells were treated with different concentrations of TCE( 0. 10, 0. 50, 1. 00, 2. 00, 5. 00, 10. 00 mmol / L). Phorbol-12-myristate-13-acetate and ionomycin were used as agonist. No TCE was used in the control group and dimethyl sulfoxide( DMSO) was used as the solvent group. The morphology of Jurkat T cells was observed using a light microscope and the survival rate of Jurkat T cells was investigated using CCK-8 essay after cells were cultured for 24,48 and 72 hours. ii) Nave and activated Jurkat T cells were treated with different concentrations of TCE( 0. 00,0. 02,0. 20,2. 00 mmol / L). The apoptosis of cells was detected using flow cytometry and the level of interleukin-2( IL-2) in supernatant was detected using enzyme linked immunosorbent assay after cells were cultured for 24,48 and 72 hours. RESULTS: i) Cytotoxic effect was observed after cells were exposed to 10. 00 mmol / L TCE for 24 hours. Cells dispersed,cell volume diminished,cell membrane ruptured,cytoplasm condensed and increased outflow of intercellular organelles. The effect of interaction between exposure dose and exposure time was statistically significant on cell survival rate( P < 0. 01). Compared with the control and DMSO groups at the same time points,there were no significant differences in the 0. 10,0. 50,1. 00 and 2. 00 mmol / L TCE treatment groups in cell survival rates in three different time points( P > 0. 05),while the cell survival rates of 5. 00 and 10. 00 mmol / L TCE treatment groups were significantly decreased( P < 0. 01). ii) When TCE concentration was 0. 00-2. 00 mmol / L,there were no significant differences in the main effect of exposure dose and interactions of between exposure dose and cell type or exposure time on cell apoptosis rate( P > 0. 05). Compared with the same time points and groups of naive Jurkat T cells,the levels of IL-2 of activated Jurkat T cells were significantly increased( P < 0. 01). In the three different time points,the level of IL-2 of activated Jurkat T cells increased in accordance with the TCE exposure dose,showing a dose-effect relationship( P < 0. 01). The level of IL-2 of activated Jurkat T cells increased in accordance with TCE exposure time,showing a time-effect relationship( P < 0. 01). CONCLUSION:s TCE at the level of 2. 00 mmol / L had no observed effect in Jurkat T cells. High doses of TCE( ≥5. 00 mmol / L) showed cytotoxic damages to naive and activated Jurkat T cells and low doses of TCE( ≤2. 00 mmol / L) could stimulate activated Jurkat T cells secrete IL-2 in a dosedependent and time-dependent manner.