The effect of BTB and cap'n'collar proteinhomology 1 siRNA on the expression of antioxidant factors of lung fibroblasts in transforming growth factor-β1-induced interstitial fibrosis in mouse
10.3760/cma.j.issn.1007-7480.2017.04.003
- VernacularTitle:BTB-cap'n'collar同源异构体1-siRNA对转化生长因子-β1诱导的小鼠肺成纤维细胞中抗氧化因子表达的影响
- Author:
Yuan LIU
;
Yi ZHENG
- Keywords:
BTB and cap'n'collar protein homology 1;
Mice;
Lung;
Fibroblasts;
Oxidative stress
- From:
Chinese Journal of Rheumatology
2017;21(4):225-230
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effects of adenovirus vectors for BTB and cap'n'collar protein homology 1 (Bach1) small interfering RNA (siRNA) on antioxidanffactors and fibrosis related cytokines in lung fibroblasts (MLF) in transforming growth factor (TGF)-β1 induced mouse.Methods Bach1 siRNA recombinant adenovirus vectors and blankadenovirus vectorwere constructed,then the MLF cells were incubated with TGF-β1 (5 ng/ml) for 24 h and infected with blankvector and successful constructed Bach1 siRNAs.The messenger RNA (mRNA) and protein expression of Bach1,heme oxygenase (HO)-1,glutathione peroxidase 1 (GPx1) and nicotinamide-adenine dinucleotide phosphate:quinone oxidoreductase-1 (NQO1) in cell supernatants were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.Changes of fibrosis-related cytokines including TGF-β1 and interleukin (IL)-6 levels in cell supernatants were determined by enzyme-linked immunosorbent assay (ELISA).One-way analysis of variance (ANOVA) was performed for multiple group comparisons and LSD test was used to compare the two groups.Results Bach1 mRNA (2.127±0.089) and protein expression increased significantly after TGF-31 stimulation compared with blank group (1.000±0.067,t=-21.77,P＜0.01),as well as the expression of fibrosis-related cytokines TGF-β1 (52±6) and IL-6 (34±6) in cell supernatants increased significantly after TGF-β1 stimulation compared with blank group (26±4,t=-11.11,P＜0.01 and 20±5,t=-5.32,P＜0.01),but the mRNA expression of HO-1 and GPx1 (0.403±0.040 and 0.567±0.112) also the protein expression decreased significantly compared with mRNA (1.000±0.181,t=25.57,P＜0.01 and 1.000±0.212,t=6.68,P＜0.05) and protein expression in blank group.Follow the Bach1 siRNA treatment,Bach1 mRNA (0.153±0.015) and protein levels were significantly downregulated compared with mRNA (2.129±0.089 and 1.973±0.035,F=1835.95,P＜0.01) and protein expression of TGF-β1 and blank vector group,as well as TGF-β1 (26±3) and IL-6 (11±3) expression in cell supernatant were significantly inhibited compared with TGF-31 (52±6 and 34±6) and blankvector group (49±5 and 33±6) (F=22.25,P＜0.01 and F=28.38,P＜0.01).But the mRNA levels of HO-1 (3.303±0.294) and GPx1 (1.840±0.231) in MLF were promoted significantly compared with TGF-β1 (0.403±0.040 and 0.567±0.112) and blank vector group (0.353±0.057 and 0.667±0.090) (F=53.90,P＜0.01 and F=526.25,P＜0.01).Conclusion Silencing Bach1 rescues TGF-β1 induced reduction of antioxidants and increasethefibrosis in MLF cells.The study offers an experimental basis to explore pathogenesis of oxidative stress and antioxidant therapy for connective tissue disease related inter-stitial lung disease.
