| [Background]Interstitial lung disease(ILD)represents a kind of disease characterized by pathological changes of diffuse pulmonary parenchyma,alveolar inflammation and interstitial fibrosis.About 35% ~ 45% ILD is associated with connective tissue disease(CTD),and the prevalence of CTD-ILD above to 60%.Pulmonary fibrosis is the final outcome of CTD-ILD,the survival and quality of life were delined once the occurrence of pulmonary fibrosis in patients with CTD.The pathogenesis of CTD associated pulmonary fibrosis remains unclear,which is involved in dysfunction of immune regulation and inflammation by a variety of immune cells,cytokines,alveolar epithelial cells and lung fibroblasts,inflammatory mediators,extracellular matrix and TGF-beta 1 signal transduction pathways and so on,but still cannot explain the development of the disease completely.With the development of oxidative stress and pulmonary fibrosis as well as CTD,the role of oxidative stress in the pathogenesis of CTD associated pulmonary fibrosis and its pathogenesis needs further investigation.BTB-cap 'n' collar(CNC)homolog 1(Bach1)belongs to a transcriptional repressor of the CNC family,which is involved in the regulation of oxidative stress by competitive inhibition of nuclear factor E related factor 2(Nrf2)who is regarded as the central regulator of the antioxidant response.Nrf2 stimulates antioxidants generation the oxidation resistance of the tissue and inhibit the progression of pulmonary fibrosis,suggesting that Bachl may be related to pulmonary fibrosis.Recently,there is still a lack of research on the correlation and mechanism between Bachl and CTD associated pulmonary fibrosis.Therefore,we choose Bachl as the target and silencing it by small RNA interference(si RNA)and use transforming growth factor beta 1(TGF-?1)induced mouse lung fibroblasts as cell model of fibrosis in vitro as well as bleomycin induced pulmonary fibrosis in mice as animal model of CTD associated pulmonary fibrosis in vivo in order to observe oxidant/antioxidant and fibrosis progression on TGF-?1 induced mouse lung fibroblasts and BLM induced pulmonary fibrosis in mice,thus exploring the pathogenesis and potential therapeutic targets of CTD associated pulmonary fibrosis.[Objective][Objective] Based on the relationship between oxidative stress and pulmonary fibrosis,also the regulation of Bachl on the oxidative,the study was designed to investigate the effect and mechanism of Bachl on bleomycin induced pulmonary fibrosis in mice,thus further explore the relationship between Bachl and CTD associated pulmonary fibrosis as well as the possibility of Bachl as a target for the treatment of CTD associated pulmonary fibrosis.[Methods]1.Construction of recombinant adenovirus with Bach1 si RNA Two of Bach1 si RNA adenovirus vector and an empty viral vector were constructed,and then were identified for sequence consistent with design by enzyme digestion and gene sequencing.The recombinant adenovirus were transfected into mouse lung fibroblasts,transfection efficiency was detected by fluorescence microscopy and flow cytometry as well as the expression of Bach1 m RNA and protein for silencing efficiency was detected by reverse transcriptase polymerase chain reaction(RT-PCR)and western blot.2.Cell experiment Mouse lung fibroblasts was cultured and inoculated as density of 1×105/m L and then were divided into f Ive groups: control group,TGF-?1 group,TGF-?1+empty vector group,TGF-?1+ Bach1#1 si RNA group and TGF-?1+ Bach1#2 si RNA group.Both cells starvation for 24 hour followed by culture medium containing 10% fetal bovine serum(FBS),blank control group cultured without treatment and TGF-?1 group was treated by 5ng/m L TGF-?1 for 72 h,TGF-?1+empty vector group was treated by 5ng/m L TGF-?1 for 24 h and 50 multiplicity of infection(MOI)vector empty for 48 h,TGF-?1+ Bach1#1 si RNA group was treated by 5ng/m L TGF-?1 for 24 h and 50 MOI Bach1#1 si RNA for 48 h and TGF-?1+ Bach1#2 si RNA group was treated by 5ng/m L TGF-?1 for 24 h and 50 MOI Bach1#2 si RNA transfecting for 48 h.Cell proliferation was analyzied by CCK8 detection;The m RNA and protein expression of Bach1,heme oxygenase 1(HO-1),glutathione peroxidase 1(GPx1)and nicotinamide adenine dinucleotide phosphate reductase 1(NQO1)were measured by RT-PCR and Western blot;The expression of factors involving in fibrosis progression including interleukin-6(IL-6)and collagen type?(Col?)were detected by enzyme linked immunosorbent assay(ELISA).The correlation between the expression level of Bach1 in mouse lung fibroblasts and the cell proliferation,expression level of antioxidant factor and factors involving in fibrosis progression were analyzed.3.Animal experiment A total 40 of 7-8 week-old C57BL/6 male and inbred mice were random Ly divided into four groups: a control group;bleomycin group;bleomycin + empty vector group and bleomycin + Bach1 si RNA group,10 in each group.5 mg/kg bleomycin was administered intratracheally into the mice of latter three groups,but the control animals were injected intratracheally with the same volume of sterile saline.Mice in empty vector and Bach1 si RNA group were injected empty vector or Bach1-si RNA,respectively,via tail vein one injection every two days,with a total dose of 1 × 109 pfu viruses per mouse after 2 weeks of bleomycin administration.At the designated time points,the mice were sacrificed for serum,bronchoalveolar lavage fluid(BALF)and lung tissue.The m RNA and protein expression of Bach1 and its downstream antioxidant factor heme oxygenase 1,glutathione peroxidase 1 and nicotinamide adenine dinucleotide phosphate reductase 1 in lung tissue of mice were detected by RT-PCR and Western blot;Expressions of IL-6 and collagen type?in serum and BALF were detected by ELISA;And the inflammation and fibrosis in lung tissue was measured by hematoxylin eosin(HE)and Masson staining.The correlation between the expression level of Bach1 in lung tissue,the expression of antioxidant factor and factors involving in fibrosis progression as well as alveolitis and fibrosis scores were analyzed.4.Statistical analysis The data were analyzed by SPSS 17.0 and the results were expressed as mean±standard deviation(X ±S).One-way analysis of variance(ANOVA)was performed for multiple group comparisons.The comparison within groups used LSD test when the equal variances assumed,but the Tamhane test was performed when equal variances not assumed.Pearson was used for correlation analysis of normal distribution,and spearman rank for correlation of abormal distribution.P?0.05 was accepted as statistically significant,P ?0.01 was considered as remarkably statistically significant and P>0.05 was accepted as no statistically significance.[Results]1.Successful construction of Bach1 si RNA adenovirus vector The sequencing of Bach1 si RNA was accordance with our designed ones;The results of fluorescence and flow cytometry showed that the transfection efficiency of two Bach1 si RNA on mouse lung fibroblasts reached above 90%;RT-PCR also show that silencing efficiency of two Bach1 si RNA after transfection for mouse lung fibroblasts reached above 70%,western blot found that the two Bach1 si RNA inhibited Bach1 protein expression and Bach1#1 si RNA is more obvious.2.Effects of Bach1 si RNA on TGF-?1 induced mouse lung fibroblasts 2.1 Effects of Bach1 si RNA on the proliferation of TGF-?1 induced mouse lung fibroblasts The results of CCK8 found that the proliferation of mouse lung fibroblasts is higher than the blank group after TGF-?1 treatment for 48 h and 72 h(0.50±0.03 vs 0.44±0.02 and 0.92±0.02 vs 0.65±0.01,P ?0.05),but after two Bach1 si RNA intervention for 48 h,the proliferation was decreased compared to TGF-?1 group(0.33±0.00 vs 0.50 ±0.03 and 0.35±0.05 vs 0.50 ±0.03,P ?0.01),the differences were statistically significant.2.2 Effects of Bach1 si RNA on the antioxidants expression of TGF-?1 induced mouse lung fibroblasts RT-PCR demonstrated that the expression of Bach1 m RNA in mouse lung fibroblasts is higher than the blank group after TGF-?1 treatment(2.13 ± 0.09 vs 1.00 ± 0.07,P?0.05),while the expressions of heme oxygenase 1 and glutathione peroxidase 1 m RNA were significantly decreased(0.40 ± 0.04 vs 1.00 ± 0.02 and 0.57 ± 0.11 vs 1.00 ± 0.26,P?0.05 and P?0.01)compared with the blank group;After Bach1 si RNA transfection,Bach1 m RNA expression was significantly decreased(0.15 ± 0.02 vs 2.13 ± 0.09,P?0.01),while heme oxygenase 1 and glutathione peroxidase 1 m RNA expression significantly increased significantly(3.30 ± 0.29 vs 0.40 ± 0.04 and 1.84 ± 0.23 vs 0.57 ± 0.11,P?0.05 and P?0.01)compared with the TGF-?1 group,the difference were statistically significant.The expression of nicotinamide adenine dinucleotide phosphate reductase 1 m RNA in TGF-?1 group and Bach1 si RNA group were not significantly different from blank group(0.78 vs 0.15 and 0.75±0.06 vs 1.00±0.29,P >0.05 respectively).Western blot showed that the TGF-?1 promoted the protein expression of Bach1 but the inhibited the protein expression of heme oxygenase 1 and glutathione peroxidase 1 in mouse lung fibroblasts;And after Bach1 si RNA intervention,Bach1 protein expression was inhibited but the expression of heme oxygenase 1 and glutathione peroxidase 1 protein were significantly increased compared with the TGF-?1 group.The expression of nicotinamide adenine dinucleotide phosphate reductase 1 protein in TGF-?1 group and Bach1 si RNA group were not significantly different from blank group.2.3 Effects of Bach1 si RNA on the expression of IL-6 and collagen type?in TGF-?1 induced mouse lung fibroblasts ELISA showed that the expression of factors involving in fibrosis progression such as IL-6 and collagen type?in cell supernatant of mouse lung fibroblasts was significantly increased than the blank group(33.62 ± 5.72 vs 20.29 ± 4.88 and 32.77±4.04 vs 23.40±2.36,both P?0.01);After Bach1 si RNA transfection,expression of IL-6 and collagen type?were significantly decreased compared with TGF-?1 group(11.11 ± 3.17 vs 33.62 ± 5.72,P ?0.01 and 25.52±2.16 vs 32.77±4.04,P?0.05),the difference were statistically significant.2.4 The relationship between the expression of Bach1 in mouse lung fibroblasts and cell proliferation,the expression of antioxidant factor and factors involving in fibrosis progression The results of correlation analysis showed that the level of Bach1 in mouse lung fibroblasts and cell proliferation showed positive correlation(r=0.40,P?0.01),also the expression of factors involving in fibrosis progression such as IL-6 and collagen type?were positively correlated(r=0.79 and r=0.57,P?0.01),but the level of Bach1 in MLF was negatively correlated with expression of antioxidant factor heme oxygenase 1 and glutathione peroxidase 1(r=-0.79 and r=-0.64,P?0.01),the differences were statistically significant and had no relationship between Bach1 and nicotinamide adenine dinucleotide phosphate reductase 1 expression,there had no significant differences(r=-0.13,P>0.05).3.Effects of Bach1 si RNA on BLM induced pulmonary fibrosis in mice 3.1 Effects of Bach1 si RNA on the antioxidants expression of lung tissue in bleomycin induced pulmonary fibrosis mice RT-PCR and Western blot found that the expression of Bach1 m RNA in lung tissue of mice in bleomycin group(2.34 ± 0.15 vs 1.00 ± 0.13,P?0.05)as well as protein expression were significantly increased,while the expression of heme oxygenase 1 and glutathione peroxidase 1 m RNA(0.54 ± 0.06 vs 1.00 ± 0.37,P?0.05 and 0.30 ± 0.02 vs 1.00 ± 0.15,P?0.01)as well as protein expression significantly decreased compared with the control group;After Bach1-si RNA treatment,Bach1 m RNA(0.32 ± 0.06 and 2.34 ± 0.15,P?0.01)and protein expression in lung tissues decreased significantly,while the expression of heme oxygenase 1 and glutathione peroxidase 1 m RNA(1.94 ± 0.19 vs and 0.54 ± 0.06 and 2.07 ± 0.09 vs 0.30 ± 0.02,both P ?0.05),the differences were statistically significant and protein expression were increased compared with bleomycin group.There were no significant differences between the expression of nicotinamide adenine dinucleotide phosphate reductase 1 m RNA(0.80 ± 0.07 vs 1.00 ± 0.26 and 0.79 ± 0.07 vs 1.00 ± 0.26,both P > 0.05)and protein in lung tissue of mice in bleomycin group and Bach1 si RNA group compared with control group.3.2 Effects of Bach1 si RNA on the expression of IL-6 and collagen type?in serum and BALF of bleomycin induced pulmonary fibrosis in mice ELISA also demonstrated that the protein expression of IL-6(43.99 ± 5.10 vs 8.71 ± 2.42 and 11.69 ± 2.08 vs 2.15 ± 0.51,both P ?0.01)and collagen type?(82.22±6.46 vs 70.39±1.56,P?0.05 and 5.50±0.54 vs 2.53±0.12,P?0.01)in serum and BALF of bleomycin group increased compared with control group,the differences were remarkably statistically significant;But expression of IL-6(12.88 ± 2.93 vs 43.99 ± 5.10 and 1.83 ± 0.37 vs 11.69 ± 2.08,both P ?0.01)and collagen type?(72.55±2.73 vs 82.22±6.46,P?0.05 and 3.85±0.78 vs 5.50±0.54,P?0.01)in serum and BALF of Bach1-si RNA group decreased compared with bleomycin group,the differences were statistically significant.3.3 Effects of Bach1-si RNA on the pathological changes of bleomycin induced pulmonary fibrosis in mice CT and pulmonary pathological changes are accordance with characteristic of pulmonary fibrosis imaging and pathology after bleomycin intervention for 14 days.Nevertheless,there were milder inflammatory infiltrations and destruction of alveoli after Bach1 si RNA treatment for 14 days compared with bleomycin group.In the trichrome masson staining,Bach1 si RNA significantly reduced blue staining areas compared with bleomycin group.The the degree of alveolar inflammation and fibrosis significantly reduced in Bach1-si RNA group compared with bleomycin group(1.90±0.12 vs 3.30±0.25 and 2.80±0.37 vs 4.60±0.51,both P?0.01),the differences were statistically significant remarkably.3.4 The relationship between the expression of Bach1 in lung tissue of mice and antioxidant factors,factors involving in fibrosis progression and pathological changes The results of correlation analysis showed that the expression level of Bach1 in lung tissue of mice was negatively related to and expression of antioxidant factor heme oxygenase 1 and glutathione peroxidase 1(r=-0.69 and r=-0.56,P?0.01),but was positively related to IL-6(r=0.62 and r=0.65,P?0.01)and collagen type?(r=0.61,P?0.01 and r=0.54,P?0.05)expressions in the serum and BALF as well as alveolar inflammation and fibrosis score(r=0.35 and r=0.33,P?0.05),the differences were statistically significant.Moreover,Bach1 and nicotinamide adenine dinucleotide phosphate reductase 1 expression had no relationship,and there had no significant differences(r=-0.06,P>0.05).[Conclusion]1.Bach1 expression in of TGF-?1 induced mouse lung fibroblasts and the lung tissue of bleomycin induced pulmonary fibrosis increased significantly.2.Silencing Bach1 inhibits the proliferation of mouse lung fibroblasts and the expression of factors involving in fibrosis progression as well as degree of inflammation infiltration and fibrosis,but promotes the expression of antioxidant factors.3.The expression of Bach1 was negatively correlated with the expression of antioxidant factors,but was positively correlated with proliferation of mouse lung fibroblasts,expression of the factors involving in fibrosis progression as well as the alveolitis and fibrosis degrees.4.Bach1 is involved in the pathogenesis and progression of pulmonary fibrosis by regulating oxidative stress,and thus providing experiment basis for study on antioxidant pathogenesis and theraputic target for CTD associated pulmonary fibrosis. |
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