Effect Of IL-17A On Fibrosis Of Skin And Lung In Systemic Sclerosis Mouse Model

PART Ⅰ Effects of anti-17 A monoclonal antibody on skin and pulmonary fibrosis in mice with systemic sclerosisObjective:To study the expression of interleukin-17 A in skin and lung tissues of mice with systemic sclerosis（SSc）and to explore the effect of anti-IL-17 A monoclonal antibody on fibrosis of SSc mouse model.Methods:Twenty-four female BALB/c mice were randomly divided into 4 groups,6in each group.Including the normal control group（mice were injected subcutaneously with phosphate buffer（PBS））,model group（mice were injected subcutaneously with bleomycin（BLM））,antibody group（BLM+IL-17 A monoclonal antibody）,isotype control group（BLM+isotype control）.The pathological changes,inflammatory and fibrotic scores of the skin and lungs were detected.Immunohistochemistry was used to detect the levels of IL-17 A in the skin and lungs of the mice.Real-time fluorescent quantitative PCR（RT-PCR）was used to detect the levels of IL-17 A,TGF-β₁ and type I collagen mRNA in the skin and lungs of the mice.Result:1.The murine skin thickness and the skin inflammation score of antibody group（61.08 ± 5.68μm,2.47 ± 0.15）were significantly lower than those in the model group（84.43 ± 6.19μm,2.83 ± 0.24）and the isotype control group（83.21± 5.72μm,2.85 ± 0.19）（P <0.05）.2.The murine lung fibrosis and inflammation score of antibody group（2.53±0.18、1.87±0.12）were significantly lower than those in the model group（3.07±0.24、2.83±0.24）and the isotype control group（3.03±0.29、2.28±0.16）（P <0.05）.3.Immunohistochemistry to detect the expression of IL-17 A in skin of mice,the antibody group（4.74±1.50）was significantly higher than the normal control group（1.32 ± 0.85）and was significantly lower than the model group（9.95±3.06）and the isotype control group（10.02±2.00）（P <0.05）.4.Immunohistochemistry to detect the expression of IL-17 A in lung tissue of mice,the antibody group（36.64±9.45）was significantly higher than the normal group（17.77±9.76）and was significantly lower than the model group（59.53±15.40）and the isotype control group（61.46±16.91）（P <0.05）.5.The expression of IL-17A（3.00±1.20）,TGF-β₁（3.20±1.27）and type I collagen（3.30±0.88）m RNA in the skin of mice in the antibody group were significantly lower than that in the model group（6.00±2.19,5.73±2.29,5.70±2.25）and isotype control group（5.98±2.50,5.25±1.75,5.27±2.12）（P<0.05）.6.The expression of IL-17A（1.57 ± 0.39）,TGF-β₁（1.83±0.75）and type I collagen（1.50±0.58）m RNA in the lung tissue of mice in the antibody group were significantly lower than that in the model group（2.44±0.55,3.04±0.84,2.40±0.73）and isotype control group（2.43±1.09,2.91±1.0,2.32±0.85）（P<0.05）.7.The expression of IL-17 A in the skin of mice was positively correlated with skin inflammation score,skin thickness,skin TGF-β₁,type I collagen m RNA expression.8.The expression of IL-17 A in lung tissue of mice was positively correlated with lung inflammation score,lung fibrosis score,lung TGF-β₁ and type I collagen m RNA expression.Conclusion:1.The expression of IL-17 A in the skin and lung tissue of SSc mouse model is increased,and the level of IL-17 A is positively correlated with the degree of inflammation and fibrosis.2.IL-17 A and TGF-β₁ may be involved in the pathogenesis of SSc,blocking IL-17 A can inhibit the expression of TGF-β₁,type I collagen m RNA,thereby reducing the SSc inflammation and fibrosis.PART Ⅱ Effects of IL-17 A on the secretion of pulmonary fibroblasts in mice with systemic sclerosis in vitroObjective:To observe the effect of IL-17 A and its monoclonal antibody on the secretion of pulmonary fibroblasts（FB）in SSc mouse model,and to further explore the role of IL-17 A in the formation of SSc fibrosis.Methods:Primary culture of FB was performed in SSc mouse model lung tissue,and observed the morphology through the inverted microscope.The cultured FBs were divided into three groups,including Control group（FB + DMEM medium）,Stimulation group（FB + IL-17A）,Blocking group（FB +anti-IL-17 A monoclonal antibody）.Cultured for 48 hours,RT-PCR to detect the expression of type I collagen,TGF-β₁ m RNA expression and enzyme-linked immunosorbent assay（ELISA）to assay culture supernatant IL-6,TGF-β₁levels.Result:1.The cultured lung tissue blocks were observed using by the inverted microscope in black shading area and adherent tightly,and the individual cells swam out about in 4-6 days.The cells were spindle mainly and intensive gradually,and can be passaged in 11-20 days.2.The expression of TGF-β₁ and type I collagen m RNA in the blocking group（18.73±5.52,342.66±78.39）were significantly lower than the stimulation group（57.17±12.40,556.52±116.04）（P <0.05）,and were significantly higher than the control group（3.53±0.94,216.49±18.33）（P <0.05）.3.The levels of IL-6 and TGF-β₁ in the supernatant in the blocking group（3009.13±198.76pg/ml,292.56±54.20pg/ml）were significantly lower than those in the control group（3812.54±92.03pg/ml,527.38±41.00pg/ml）（P <0.05）,and were significantly higher than the control group（2561.28±283.70pg/ml,217.05±55.08pg/ml）（P <0.05）.Conclusion:1.IL-17 A can promote the expression of FB TGF-β₁ and type I collagen m RNA in the SSc mouse model,promote the secretion of IL-6 and TGF-β₁.2.Anti-IL-17 A monoclonal antibody may inhibit fibrosis of skin and lung of SSc mice by inhibiting the production of FB type I collagen,and the secretion of IL-6 and TGF-β₁.