| Objective: to establish a mouse fibrosis model stimulated by TGF-β1 to simulate human pulmonary fibrosis,and to explore the effects of different concentrations of ERK1/2 signal pathway blocker U0126 on pulmonary fibrosis and TGF-β1/ Smad3 pathway in the process of phenotypic transformation of mouse lung fibroblasts to myofibroblasts induced by 10 ng TGF-β1,and to understand the possible interaction of multiple pathways in the process of pulmonary fibrosis.In order to enrich the study of the pathogenesis of idiopathic pulmonary fibrosis at the cellular and molecular level,and provide more theoretical basis for anti-fibrosis therapy.Methods: mouse lung fibroblasts(L929 cell line)cultured in vitro were used to establish a mouse model of pulmonary fibrosis stimulated by exogenous active TGF-β1at the cellular level.The experimental groups were randomly divided into the following5 groups: blank control group,10ng/ml TGF-β1 group(model group),10ng/ml TGF-β1+2.5u M U0126 inhibitor group(low dose inhibitor group),10ng/ml TGF-β1+5u M U0126 inhibitor group(medium dose inhibitor group),and 10ng/ml TGF-β1+10u M U0126 inhibitor group(high dose inhibitor group).The cells of the above experimental groups were cultured for 24 hours,and the cells were collected.The transcription levels of fibrosis phenotypic markers α-smooth muscle actin(α-SMA),type I collagen and upstream molecule EGFR of ERK1/2 in L929 cells were detected by real-time fluorescence quantitative reverse transcription PCR(RT-q PCR).Western blotting technique(Western Blot)was used to detect the protein phosphorylation levels of ERK1/2 and Smad3.Collect test data and make statistical analysis.Results:(1)compared with the blank control group,the relative expressions of type I collagen(1.049±0.0159)and α-SMA m RNA(1.143±0.1293)in the model group stimulated by exogenous 10ng/m L TGF-β1 were up-regulated at the transcriptional level,and the relative expressions of type I collagen and α-SMA were 1.859 ±0.0243 and 2.074 ±0.0105(P<0.05).(2)compared with the model group,the expressions of type I collagen and α-SMA in low dose inhibitor group,medium dose inhibitor group and high dose inhibitor group were all down-regulated at the transcriptional level.The relative expressions of type I collagen and α-SMA in low dose inhibitor group were 1.609 ±0.0079 and 1.840 ±0.0877,while those in medium dose inhibitor group were 1.284 ±0.0016 and 1.512 ±0.1120,the relative expressions of type I collagen and α-SMA in high dose inhibitor group were1.497 ±0.0032 and 1.651 ±0.0979,there was significant difference between each two groups(P<0.05);In addition,the expression of type I collagen and α-SMA m RNA in the middle dose inhibitor group was significantly lower than that in the low dose inhibitor group(P<0.05),but there was no significant difference in the expression of type I collagen and α-SMA m RNA between the high dose inhibitor group and the middle dose inhibitor group(P>0.05);Compared with the control group,there was no significant difference in the expression of EGFR m RNA between the model group and the control group(P>0.05).(3)compared with the blank control group,the phosphorylation expression of ERK1/2 and Smad3 protein in the model group was up-regulated,and there was significant difference between the model group and the model group(P<0.05);Compared with the model group,the phosphorylation expression of ERK1/2 and Smad3 protein in low-dose inhibitor group,middle-dose inhibitor group and high-dose inhibitor group were all down-regulated,and there was significant difference between each two groups(P<0.05).Conclusion:(1)TGF-β1 plays a positive regulatory role in the phenotypic transformation of mouse lung fibroblasts to myofibroblasts,and the regulatory mechanism may be related to its mediating ERK1/2 signal pathway and Smad3 pathway.(2)TGF-β1 mediated ERK1/2 pathway in pulmonary fibrosis may not pass through its upstream signaling molecule EGFR but directly affect the ERK1/2 level.(3)U0126 antagonist can simultaneously inhibit TGF-β1-mediated ERK1/2 signal pathway and Smad3 pathway in the process of pulmonary fibrosis,and the degree of inhibition of fibrotic response may be related to the dose of U0126.In view of the blocking effect of U0126 on multiple pathways,it provides more evidence for the treatment of pulmonary fibrosis.(4)the occurrence mechanism of IPF may be related to the network response of multiple signal pathways,that is,the phenomenon of "crosstalk" between the so-called signal pathways. |
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