The Role Of TLR4/IRAK/NF-κB Signaling Pathway In The Sythnesis And Secretion Function Of Pulmonary Artery Smooth Muscle Cells Treatment With LPS In COPD Rats

objective: This study is focus to explore the relation between the synthesis and secretion function of PASMCs and TLR4 expression.And further to explore the role of the TLR4-IRAK-NF-κB signaling pathway in the synthesis and secretion function of PASMCs of COPD rats.The experiment provides a theoretical basis for the early intervention of COPD.Methods: In the first part:1.COPD rat model was established,PASMCs were digested,separated and purified and the cells were randomly divided into control group and LPS group.2.Western blotting was used to detect the expression level of TLR-4 in PASMCs treated with LPS in COPD rats with different time.3.ELISA was used to detect the levels of IFN-γ、IL-6 and PDGF-AB in PASMCs treated with LPS in COPD rats with different time.4.Correlation analysis was performed between TLR4 expression and the concentration of the secretion and synthesis factor in PASMCs treated with LPS in COPD rats.In the second part:To investigate the role of TLR4/IRAK/NF-κB signaling pathway in the synthesis and secretion function of PASMCs treated with LPS in COPD rats.1.Samples were randomly divided into control group,LPS group,TLR4 inhibitor group(TAK-242)and LPS + TLR4 inhibitor group;2.RT-PCR was used to detect the m RNA expression level of TLR4,and Western blotting was used to detect the proteins expression level of TLR-4,IRAK-1,IKBα,IKKαand NF-κB groups.3.The levels of IFN-γ,IL-6 and PDGF-AB in PASMCs supernatant in each group were detected by ELISA.4.Further to explore the influence of TLR4/IRAK1/NF-k B signaling pathway on the synthesis and secretion of PASMCs treated with LPS in COPD rats,PASMCs were treated by IRAK1/4 inhibitor and NF-κB inhibitor,randomly divided into control group,LPS group,IRAK1/4 inhibitor group,LPS+ IRAK1/4 inhibitor group,NF-κB inhibitor group and LPS + NF-κB inhibitor group.Western blotting was used to detect the proteins expression level of IRAK-1,IKBα,IKKαand NF-κB groups.The levels of IFN-γ 、 IL-6 and PDGF-AB in PASMCs supernatant in supernatant in each group were detected by ELISA.Correlation analysis was performed between these protein expressions in PASMCs and the concentration of PASMCs secretion and synthesis factors.Results: The first part: 1.Compared with control group,LPS induced PASMCs of COPD rats,TLR4 expression in LPS induced PASMCs of COPD rats was significantly increased at 12 hours,48 h peaked,72 h slightly decreased(P <0.05).And the expression level of IFN-γ,IL-6 and PDGF-AB increased with time points,the expression reached a peak at 48 h and maintained at 72h(P <0.05).2.The concentrations of IL-6,PDGF-AB and IFNin the supernatant of PASMCs were positively correlated with TLR4 expression.The second part: 1.Compared with control group,LPS upregulated the expression of TLR4、Phosphorylation IRAK1,NF-κB and the levels of IFN-γ,IL-6 and PDGF-AB.TLR4 inhibitor TAK-242,IRAK1/4 inhibitor and NF-κB inhibitor BAY117082 were capable of suppressing the effects of LPS.Conclusion: 1.There is a positive correlation between TLR4 expression and the concentration of secretion factors in PASMCs treated with LPS in COPD rats,it was positively correlated.2.TLR4/IRAK/NF-κB signaling pathway could regulate PASMCs synthesis and secretion function,and then may affect COPD pulmonary vascular remodeling.