| Objective(s):This study intends to clarify the role of TLR4 membrane receptors in the development of pulmonary fibrosis from the perspective of immune inflammation and to comprehensively study the mechanism of TLR4 in pulmonary fibrosis.We studed whether TLR4 caused changes in IL-1? levels through the NF-?B signaling pathway,which ultimately affected the formation of pulmonary fibrosis.Methods:1.MRC-5 cells were cultured in DMEM medium containing 10%fetal bovine serum at 37? and 5%CO2.Cells in logarithmic growth phase were taken and passaged 1:2 using 0.25%pancreatin digestion method.TGF-?1(80ng/ml)was used to intervene in human lung fibroblast MRC-5 cells to construct an in vitro lung fibrosis model.Western blot,cellular immunofluorescence,and Q-PCR were used to analyze type ? collagen(Collagen-?,COL-?),type ? collagen(Collagen-?,COL-?),?-smooth muscel actin(?-SMA)protein and its mRNA expression to identify the model.Set NC group(normal group),TGF-?1 group(model control group),TAK-242+TGF-?1 group(TLR4 inhibitor intervention group),LPS+TGF-?1 group(TLR4 inducer intervention group),PDTC+TGF-?1 group(NF-?B inhibitor intervention group).1.1 In NC group and TGF-?1 group,the expression of TLR4 protein and mRNA in MRC-5 cells of each group was analyzed by Western blot,cellular immunofluorescence,and Q-PCR to study the expression of TLR4 during the fibrosis of MRC-5 cells induced by TGF-?1.1.2 In NC group,TGF-?1 group,TAK-242+TGF-?1 group,LPS+TGF-?1 group.Western blot,cellular immunofluorescence,and Q-PCR were used to analyze the COL-I,COL-?,and ?-SMA in MRC-5 cells of each group,to investigate whether the high expression of TLR4 during the fibrosis of MRC-5 cells induced by TGF-?1 was related to pulmonary fibrosis and the role of TLR4.1.3 InNC group,TGF-?1 group,TAK-242+TGF-?1 group,LPS+TGF-?1 group,PDTC+TGF-?1 group.Western blot,cellular immunofluorescence and Q-PCR were used to analyze the expression of COL-I,COL-?,?-SMA,TLR4,P65 protein and their mRNA in MRC-5 cells of each group,to investigate whether TLR4 plays a role in promoting pulmonary fibrosis by regulating NF-?B during the fibrosis of MRC-5 cells induced by TGF-?1.1.4 In NC group,TGF-?1 group,TAK-242+TGF-?1 group,LPS+TGF-?1 group,PDTC+TGF-?1 group.Q-PCR was used to analyze the expression of IL-1?mRNA in MRC-5 cells of each group,and to investigate whether TLR4/NF-?B could promote pulmonary fibrosis by regulating IL-1? during the fibrosis of MRC-5 cells induced by TGF-?1.1.5 In NC group,TGF-?1 group,TAK-242+TGF-?1 group,LPS+TGF-?1 group,PDTC+TGF-?1 group.EDU staining was used to analyze the proliferation of MRC-5 cells in each group,and the effects of intervention on the expression of TLR4 and NF-?B on the proliferation of MRC-5 cells induced by TGF-?1 were investigated.Results:1.The expression and function of TLR4 in MRC-5 cells induced by TGF-?1:Western blot detected the expression of TLR4 protein in MRC-5 cells induced by TGF-?1.The results showed that the expression of TLR4 increased,and the cell immunofluorescence,Q-PCR were consistent with the Western blot results.Western blot detected the expression of COL-?,COL-?,and ?-SMA protein in TRC-?1-induced MRC-5 cells with TLR4 inducers and inhibitors.The results showed that when TLR4 expression was inhibited,TGF-?1-induced collagen secretion and transformation of MRC-5 cells could be inhibited.When TLR4 expression was induced,TGF-?1-induced MRC-5 collagen secretion and transformation could be promoted.Q-PCR and Western blot results were consistent.The results showed that the expression of TLR4 increased during the fibrosis of lung fibroblasts,which promoted the secretion and transformation of collagen during cell fibrosis.2.The mechanism of TLR4 regulates TGF-?1-induced fibrosis in MRC-5 cells:Western blotting was used to detect the expression of TLR4 and NF-?B protein in MRC-5 cells induced by TGF-?1 intervene by TLR4 inducer,TLR4 inhibitor and NF-?B inhibitor.The results showed that when TGF-?1-induced MRC-5 cells inhibited and induced TLR4 protein expression,NF-?B protein was simultaneously suppressed and induced expression,The results show that when TGF-?1-induced MRC-5 cells suppress and induce TLR4 protein expression,NF-?B protein is simultaneously suppressed and induced expression.When the expression of NF-?B protein was inhibited,the expression of NF-?B protein was suppressed,but there was no significant change in the expression of TLR4 protein.The results of cellular immunofluorescence,Q-PCR and Western blot were consistent.The results indicate that TLR4 may play a role in promoting TGF-?1-induced collagen secretion and transformation of MRC-5 cells by regulating the downstream NF-?B signaling molecule.To further inhibit the expression of NF-?B protein,Western blot detected the expression of COL-?,COL-? and ?-SMA protein in MRC-5 cells induced by TGF-?1 intervened by NF-?B inhibitor,the results showed that when inhibited the NF-?B expression,it can inhibit the secretion and transformation of collagen of MRC-5 cells induced by TGF-?1,and the results of cellular immunofluorescence,Q-PCR and Western blot are consistent.The results of cellular immunofluorescence,Q-PCR and Western blot are consistent.The results show that TLR4 can promote the secretion and transformation of collagen during the fibrosis of MRC-5 cells induced by TGF-?1 through the key signaling molecule NF-?B in the inflammatory pathway.3.TLR4-NF-?B signaling pathway regulates MRC-5 cell fibrosis:To detect the level of IL-1? mRNA in MRC-5 cells induced by TGF-?1 intervened by TLR4 inducer,TLR4 inhibitor and NF-?B inhibitor by Q-PCR.The results showed that when the expression of TLR4 and NF-?B was induced,the level of IL-1?mRNA increased,and when the expression of TLR4 and NF-?B was suppressed,the level of IL-1?mRNA decreased.The results indicate that TLR4/NF-?B may play a role in promoting the transformation,proliferation and collagen secretion of TGF-?1 induced lung fibrosis in MRC-5 cells by regulating IL-1?.4.Effect of intervention on TLR4-NF-?B signaling pathway on the proliferation of MRC-5 cells induced by TGF-?1:EDU staining method was used to detect the number of cells in the proliferation state in TGF-?1-induced MRC-5 cells intervened by TLR4 inducer,TLR4 inhibitor and NF-?B inhibitor.The results showed that the trend of cell proliferation increased when the expression of TLR4 and NF-?B was induced,and decreased significantly when the expression of TLR4 and NF-?B was suppressed.The results show that when the TLR4-NF-?B signaling pathway is activated,it can promote the proliferation of MRC-5 cells.After inhibiting the TLR4-NF-?B signaling pathway,cell proliferation is significantly restricted.Conclusion(s):1.TLR4 is highly expressed during the fibrosis process of MRC-5 cells induced by TGF-?1,and promotes the transformation,proliferation and collagen secretion of lung fibrosis induced by TGF-?1.2.TLR4 can promote the transformation,proliferation and collagen secretion of MRC-5 cells induced by TGF-?1 by regulating the key signaling molecule NF-?B in the inflammatory pathway.3.TLR4/NF-?B plays a role in promoting the transformation,proliferation and collagen secretion of MRC-5 cells induced by TGF-?1 through the regulation of IL-1?. |
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