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Background Arsenic can be toxic to human by triggering oxidative stress, which is companied by epigenetic modifications. Objective To investigate the modification of N6-methyladenosine (m6A) in human embryonic lung fibroblasts (HELF) during oxidative stress induced by sodium arsenite (NaAsO2). Methods HELF cells were treated by designed concentrations of NaAsO2 (0, 2.5, 5, 10, and 20 μmol·L−1) for 48 h. Cell viability was detected by 3-(4,5-dimethylthia zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS) method; the activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) as well as the content of malondialdehyde (MDA) were detected with corresponding kits; the level of m6A methylation in total RNA was detected by enzyme-linked immunosorbent assay; the mRNA expressions of m6A modified enzymes were detected by real-time fluorescence quantitative PCR, including methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms' tumor 1-associated protein (WTAP), fat mass and obesity-associated protein (FTO), alkB family of Fe(II)/α-ketoglutarate-dependent dioxygenases 5 (ALKBH5), YTH domain containing protein 2 (YTHDC2), YTH domain family protein 2 (YTHDF2), and YTH domain family protein 3 (YTHDF3); the protein expressions of METTL3, FTO, YTHDC2, YTHDF3, and nuclear factor erythroid 2-related factor 2 (NRF2) were detected by Western blotting. The enrichment of m6A in NRF2 mRNA was detected by RNA methylated immunoprecipitation combined with real-time fluorescence quantitative PCR (MeRIP-qPCR). Results After the 0, 2.5, 5, 10, and 20 μmol·L−1 NaAsO2 treatment, the MTS results showed that compared with the control group, the cell viability of the 20 μmol·L−1 group decreased to 84% (P<0.05). The colorimetry results showed that compared with the control group, the activities of T-SOD in the 10 and 20 μmol·L−1 groups decreased (P<0.05); the activities of GSH-Px in the 2.5 and 10 μmol·L−1 groups decreased (P<0.05); the contents of MDA in the 10 and 20 μmol·L−1 groups increased. The results of enzyme-linked immunosorbent assay showed that the overall m6A methylation levels in the 0, 2.5, 5, 10, and 20 μmol·L−1 groups were (0.193 ± 0.023)%, (0.247 ± 0.021)%, (0.253 ± 0.006)%, (0.233 ± 0.006)%, and (0.262 ± 0.010)%, respectively, and compared with the control group, the m6A methylation levels in all the NaAsO2 treated groups increased (P<0.05). The real-time fluorescence quantitative PCR results showed that compared with the control group, the mRNA relative expression level of METTL3 decreased in the 2.5, 10, and 20 μmol·L−1 groups (P<0.05); the mRNA relative expression level of FTO decreased in the 20 μmol·L−1 group; the mRNA relative expression level of YTHDC2 increased in the 10 and 20 μmol·L−1 groups (P<0.05); the mRNA relative expression level of YTHDF3 increased in the 2.5, 10, and 20 μmol·L−1 groups (P<0.05). The Western blotting results showed that compared with the control group, the relative protein expression of METTL3 decreased in the 10 and 20 μmol·L−1 groups; the relative protein expression of FTO decreased in the 5 and 20 μmol·L−1 groups; the relative protein expression of YTHDC2 decreased in the 20 μmol·L−1 group (P<0.05); the relative nuclear protein expression of NRF2 decreased in the 10 and 20 μmol·L−1 groups (P<0.05). The MeRIP-qPCR results showed that m6A enrichment was significantly increased in the 20 μmol·L−1 NaAsO2 exposure group compared with the control group (P<0.05). After over-expression of FTO, the mRNA and protein relative expression levels of FTO and the relative expression level of nuclear protein of NRF2 in the FTO group were higher than those in the control group (P<0.05); the mRNA and protein relative expression levels of FTO in the NaAsO2 + FTO group and the nuclear protein expression level of NRF2 were higher than those in the NaAsO2 group (P<0.05). Conclusion In the process of oxidative stress induced by NaAsO2, m6A methylation level, m6A modified enzymes, m6A modification of NRF2 mRNA, and NRF2 expression could change in HELF cells.
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Objective:To study the inhibitory effect of overexpression of mitofusion 2 (Mfn2) protein on acute respiratory distress syndrome (ARDS) pulmonary fibrosis and its mechanism.Methods:Human embryo lung fibroblasts (HELF) were cultured in vitro, and digested and passaged when the adherent rate of HELF reached 80%, and then the cells in good condition were selected for experiment. The ARDS cell model was reproduced by 5 mg/L of lipopolysaccharide (LPS, LPS group); 75 mol/L adenovirus vector carrying mitofusion 2 (Adv-Mfn2) was transfected into HELF (Adv-Mfn2+LPS group); at the same time, blank control group (complete medium culture) and Adv-vector+LPS group were set as controls. The cell proliferation was observed by sulforhodamine B (SRB) method at 0, 12, 24, 36 and 48 hours. After Hoechst 33342 staining, the morphological changes were observed under confocal microscope. Western blotting was used to detect the protein expressions of Bcl-2 and caspase-3. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the gene expressions of Bcl-2 and caspase-3. Results:After LPS stimulation for 12-48 hours, the cell proliferation rates in the LPS group increased gradually, which were significantly higher than those in the blank control group [12 hours: (10.75±1.51)% vs. (0.73±1.22)%, 24 hours: (20.09±1.71)% vs. (1.15±1.12)%, 36 hours: (20.58±1.55)% vs. (1.20±1.12)%, 48 hours: (21.30±1.51)% vs. (1.23±1.10)%, all P < 0.01]. There was no statistically significant difference in the cell proliferation rate between the LPS group and the Adv-vector+LPS group. After overexpression of Mfn2, the cell proliferation rates at 12, 24, 36, 48 hours in the Adv-Mfn2+LPS group were (8.93±1.14)%, (10.52±1.24)%, (10.72±1.30)%, and (10.91±1.20)%, which were significantly lower than those in the LPS group (all P < 0.05). Confocal microscopy showed that some cells in the blank control group had nuclei of different sizes, and some nuclei fragmented or shrank to form apoptotic bodies. The nuclei of the cells in the LPS and Adv-vector+LPS groups were round or oval in size, and only a few apoptotic cells appeared. When Mfn2 was overexpressed, there were more apoptotic cells in the visual field in the Adv-Mfn2+LPS group than LPS group. Western blotting and RT-qPCR results showed that Bcl-2 expressions increased significantly after LPS stimulation in the LPS group as compared with the blank control group [Bcl-2 protein (Bcl-2/GAPDH): 0.68±0.01 vs. 0.29±0.01, Bcl-2 mRNA (2 -ΔΔCT): 2.23±0.34 vs. 1.00±0.00, both P < 0.01], and caspase-3 expressions decreased significantly [caspase-3 protein (caspase-3/GAPDH): 0.37±0.02 vs. 0.66±0.02, caspase-3 mRNA (2 -ΔΔCT): 0.31±0.05 vs. 1.00±0.00, both P < 0.01]. Compared with LPS group, the expressions of Bcl-2 after overexpression of Mfn2 in the Adv-Mfn2+LPS group were down-regulated [Bcl-2 protein (Bcl-2/GAPDH): 0.46±0.01 vs. 0.68±0.01, Bcl-2 mRNA (2 -ΔΔCT): 1.45±0.14 vs. 2.23±0.34, both P < 0.01], and the expressions of caspase-3 were up-regulated [caspase-3 protein (caspase-3/GAPDH): 0.54±0.02 vs. 0.37±0.02, caspase-3 mRNA (2 -ΔΔCT): 0.88±0.10 vs. 0.31±0.05, both P < 0.01]. Conclusion:Mfn2 protein is involved in ARDS pulmonary fibrosis, which may be related to mitochondrial mediated inhibition of cell proliferation.
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Royal jelly (RJ) from honeybee has been widely used as a health promotion supplement. The major royal jelly proteins (MRJPs) have been identified as the functional component of RJ. However, the question of whether MRJPs have anti-senescence activity for human cells remains. Human embryonic lung fibroblast (HFL-I) cells were cultured in media containing no MRJPs (A), MRJPs at 0.1 mg/ml (B), 0.2 mg/ml (C), or 0.3 mg/ml (D), or bovine serum albumin (BSA) at 0.2 mg/ml (E). The mean population doubling levels of cells in media B, C, D, and E were increased by 12.4%, 31.2%, 24.0%, and 10.4%, respectively, compared with that in medium A. The cells in medium C also exhibited the highest relative proliferation activity, the lowest senescence, and the longest telomeres. Moreover, MRJPs up-regulated the expression of superoxide dismutase-1 (SOD1) and down-regulated the expression of mammalian target of rapamycin (MTOR), catenin beta like-1 (CTNNB1), and tumor protein p53 (TP53). Raman spectra analysis showed that there were two unique bands related to DNA synthesis materials, amide carbonyl group vibrations and aromatic hydrogens. These results suggest that MRJPs possess anti-senescence activity for the HFL-I cell line, and provide new knowledge illustrating the molecular mechanism of MRJPs as anti-senescence factors.
Subject(s)
Animals , Cattle , Humans , Bees , Cell Line , Cell Proliferation , Cellular Senescence/drug effects , Culture Media , Dose-Response Relationship, Drug , Fatty Acids/chemistry , Fibroblasts/drug effects , Insect Proteins/chemistry , Lung/drug effects , Serum Albumin/metabolism , Spectrum Analysis, Raman , Superoxide Dismutase-1/metabolism , TOR Serine-Threonine Kinases/metabolism , Tumor Suppressor Protein p53/metabolism , beta Catenin/metabolismABSTRACT
Objective To investigate the effect of Salvia miltiorrhiza injection on proliferation , apoptosis and expression of hydroxyproline in human embryonic lung fibroblast cells. Methods TGF-β1 was administered to induce the proliferation in human embryonic lung fibroblast cells; the effect of Salvia miltiorrhiza injection on the number of human embryonic lung fibroblast cells was detected by MTT method; ki67 expression by immunocytochemical method;cell apoptosis by flow cytometry and the expression of hydroxyproline by colorimetry method. Results TGF-β1(0, 2.5, 5, 10, 20, 40 μg/L)could up-regulate cell number of human embryonic lung fibroblast cells in a dose-dependent manner , while the OD value of human embryo lung fibroblasts cells declined pretreated with Salvia miltiorrhiza injection in a dose-dependent manner and Salvia miltiorrhiza injection could induce the apoptosis and down regulated hydroxyproline expression in human embryo lung fibroblasts cells. The results of flow cytometry indicated that cell apoptosis increased after treated with Salvia miltiorrhiza injection when compared with TGF-β1 group (P < 0.05). Meanwhile, Salvia miltiorrhiza injection could down regulate the expression of hydroxyproline (P < 0.01). Conclusions Salvia miltiorrhiza injection can target human embryonic lung fibroblast cells , play a potent role in the airway remodeling through the promotion of its apoptosis and down regulate the expression of hydroxyproline.
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OBJECTIVE: To investigate the effect of beryllium sulfate( BeSO_4) on apoptosis of human embryonic lung fibroblast( MRC-5 cell). METHODS: MRC-5 cells were cultured in vitro and randomly divided into 6 groups: a control group,a low-,medium- and high-dose BeSO_4 group,an antagonist group,and an activator group. The former 4 groups were given final concentrations of 0,1,10 and 100 μmol / L of BeSO_4,respectively. The combined treatment of BeSO_4and2-aminoethoxydiphenyl borate( 10 μmol / L final concentration) was used in the antagonist group. The combined treatment of BeSO_4 and inositol triphosphate( IP3)( 10 μmol / L final concentration) was used in the activator group. After 24 and48 hours of culture,the cells were harvested. The apoptosis of MRC-5 cells was detected by flow cytometry. The intracellular calcium ion( Ca~(2+)) was detected using laser scanning confocal microscope. Quantitative real-time polymerase chain reaction was used to detect the relative expression of IP_3RⅢ and B-cell lymphoma-2( BCL-2) mRNA and the protein expression of IP_3RⅢ and IP3 were detected by enzyme-linked immunosorbent assay. RESULTS: The apoptosis rates of cells in the 3 BeSO_4 dose groups at the time points of 24 and 48 hours were lower than those in the control group at the same time points( P < 0. 05). The apoptosis rate of the antagonist group was lower than those in medium-dose BeSO_4 group and control group at the same time points( P < 0. 05). At the time point of 48 hours,the apoptosis rate of the activator group was lower than that of control group( P < 0. 05) and higher than that of the medium-dose BeSO_4group( P < 0. 05). As for the Ca~(2+)concentration at time point of 24 hours,the low-dose BeSO_4 group was lower than the control group( P < 0. 05),and the high-dose BeSO_4 group was higher than the control group( P < 0. 05). The Ca~(2+)concentrations at time point of 48 hours in the medium- and high-dose BeSO_4 groups were lower than that in the control group( P < 0. 05). Compared with the medium-dose BeSO_4 group and control group at time points of 24 and 48 hours,the Ca~(2+)concentrations in the antagonist group decreased( P < 0. 05),while thoes of the activator group increased( P < 0. 05). The expression of BCL-2and IP_3RⅢmRNA in the 3 BeSO_4 groups,the activator and antagonist group were higher than those of the control group( P <0. 05). The expression of IP3 R Ⅲ protein at the time point of 24 hours in the medium-dose BeSO_4 group,the activator group and the antagonist group were lower than that of control group( P < 0. 05). The expression of IP_3RⅢ protein at the time point of 48 hours,the high-dose BeSO_4 group was lower than the control group( P < 0. 05); the activator and antagonist groups were higher than the medium-dose BeSO_4group( P < 0. 05). The expression of IP3 protein in the lowand medium-dose BeSO_4 groups and the activator group were higher than that in the control group( P < 0. 05). The expression of IP3 protein in activator group was higher than the medium-dose group( P < 0. 05). CONCLUSION: BeSO_4 might change the Ca~(2+)concentration and inhibite the apoptosis of MRC-5 cell through regulating the IP3 R / Ca~(2+)pathway,IP3 can improve the decrease of Ca~(2+)concentration in MRC-5 cells induced by BeSO_4.
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AIM:To investigate the effects of thalidomide ( THD) on the activation of connective tissue growth factor ( CTGF) gene promoter induced by transforming growth factor β1 ( TGF-β1 ) in human embryonic lung fibroblasts ( HELF) .METHODS:DNA sequence of CTGF gene promoter was cloned into luciferase reporter gene vector to construct the recombinant eukaryotic expression vector pGL 3-CTGFP, and the recombinant vector was transfected into HELF cell line.The effects of TGF-β1 and THD on the activation of CTGF gene promoter were detected by dual-luciferase analysis . RESULTS:TGF-β1 increased the reporter gene activity dose-dependently (P<0.05), with a plateau at 5 μg/L being 2.16 folds as high as the control .TGF-β1-induced increase in the reporter gene activity was also time-dependent ( P<0.05).After exposure to TGF-β1(5 μg/L), the level of luciferase activity reached its peak at 12 h and was 2.52 folds as high as the control .THD significantly inhibited TGF-β1-induced increase in the reporter gene activity in a dose-dependent manner , but its basal activity was not changed .CONCLUSION: TGF-β1 stimulates the transcriptional activity of CTGF gene promoter in HELF cells in a dose-and time-dependent manner , while THD may inhibit the effects dose-dependently .
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Objective To study the effect of Qingfei oral liquid contained serum on the transforming growth factor-?1 (TGF-?1) and platelet-derived growth factor-BB (PDGF-BB) mRNA gene expression of human embryonic lung fibroblast cells infected by adenovirus (ADV) type 3I and 7b. Method TGF-?1 and PDGF-BB mRNA expression of human embryonic lung fibroblast cells infected by ADV type 3I and 7b were determined by in situ hybridization before and after treated with Qingfei oral liquid contained serum. Results ADV could up-regulate TGF-?1 and PDGF-BB mRNA of human embryonic lung fibroblast cells (P
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Objective To study the effect of adenovirus type 3I,7b on the expressions of mRNA and protein of transforming growth factor-beta 1(TGF-?1) in human embryonic lung fibroblast cells.Method The expression of mRNA and protein of TGF-?1 were determined in human embryonic lung fibroblast cells before and after being infected by adenovirus type 3I,7b and in normal fibroblast cells with enzyme-linked immunosorbent assay and in situ hybridization.Results The mRNA and protein of TGF-?1 expression in human embryonic lung fibroblast cells increased siginificantly after being infected by adenovirous type 3I,7b compared with those in normal fibroblast cells(Pa0.05).Conclusion Lung fibroblast cells and TGF-?1 may play some roles in pathophysiological processes of viral pneumonia.