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

PART ? 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-?₁ 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 � 5.68?m,2.47 � 0.15?were significantly lower than those in the model group?84.43 � 6.19?m,2.83 � 0.24?and the isotype control group?83.21� 5.72?m,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-?₁?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-?₁?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-?₁,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-?₁ 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-?₁ may be involved in the pathogenesis of SSc,blocking IL-17 A can inhibit the expression of TGF-?₁,type I collagen m RNA,thereby reducing the SSc inflammation and fibrosis.PART ? 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-?₁ m RNA expression and enzyme-linked immunosorbent assay?ELISA?to assay culture supernatant IL-6,TGF-?₁levels.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-?₁ 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-?₁ 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-?₁ and type I collagen m RNA in the SSc mouse model,promote the secretion of IL-6 and TGF-?₁.2.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-?₁.