【作者】 张华；

【导师】 肖伟；

【作者基本信息】 山东大学 ， 临床医学（呼吸内科）（专业学位）， 2018， 博士

【摘要】 目的观察不同剂量表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKIs)在不同时间点对染尘大鼠肺脏上皮间质转化(EMT)及其间质纤维化的干预作用,探讨表皮生长因子受体(EGFR)及其信号传导通路在矽肺性纤维化形成过程中的作用机制,为分子靶向药物防治尘肺纤维化提供理论依据。方法建立染尘Wistar大鼠动物模型,随机分为模型组、对照组、高剂量干预组、中剂量干预组、低剂量干预组。高、中、低剂量干预组分别给予不同剂量的EGFR-TKIs灌胃干预(高剂量组13.5mg/d、中剂量组6.75mg/d、低剂量组3.375mg/d)。分别在染尘3d、7d、14d,各组随机取6只大鼠将其安乐死。取材肺组织标本,进行大体肉眼观察和病理学染色,肺部炎症和纤维化半定量评分和肺匀浆羟脯氨酸(Hyp)含量测定,评估肺脏炎症和纤维化程度。免疫组织化学染色(ICH)观察和定量分析肺组织表皮生长因子受体(EGFR)及p-EGFR含量;ELISA方法检测支气管肺泡灌洗液(BALF)中转化生长因-α(TGF-α)含量;RT-PCR方法检测肺组织EGFR、E-钙粘蛋白(E-cadherin)、α-平滑肌激动蛋白(α-SMA)mRNA表达水平;Western Blot方法检测肺组织TGF-α、Ras、p-Erk1/2、PI-3KCG、Akt1 蛋白含量。结果1、应用非气管暴露法成功建立Wistar大鼠染尘(矽肺性纤维化)模型染尘不同时间点(3d、7d、14d)检测发现,模型组肺泡炎症评分与对照组相比均显著升高(4.0±0.120vs1.8±0.101,2.1±0.161vs1.0±0.104,1.5±0.135vs 0.5±0.184,均P<0.01);肺纤维化评分和羟脯氨酸含量在第7天开始升高,并随时间持续增高,染尘第7天、第14天与对照组比较,肺纤维化评分(1.3±0.164vs 0.7±0.131,3.5±0.263vs0.8±0.086,均P<0.01)和羟脯氨酸含量(1.24± 0.931vs 0.59±0.0735,1.49±0.0686vs0.64±0.05 88,P<0.01)都有统计学意义。本实验构建的染尘模型肺部炎症和纤维化特征和进展过程符合矽肺性纤维化体内模型特点。2、尘肺纤维化EGFR和EMT及其下游通路ICH肺组织EGFR及p-EGFR表达主要定位于气道上皮细胞和间质细胞的细胞膜或胞浆,模型组各时间点(3d、7d、14d)p-EGFR平均光密度随时间明显增加,与对照组比较有显著性差异(0.2692±0.0312vs0.0613±0.0301,0.5960±0.0641 vs0.0671±0.0348,0.7591±0.0532vs0.0544±0.0613,均P<0.01)。ELISA方法检测模型组BALF中TGF-α的含量在染尘第3天明显升高,各时间点(3d、7d、14d)与对照组相比,有显著性差异(69.964±7.92vs34.892±6.248,79.456±9.58vs 36.463 ±6.441,55.764±8.58 vs31.554±8.916,均P<0.01)。Western blot检测结果显示,染尘不同时间点(3d、7d、14d)模型组肺组织蛋白TGF-α相对表达量明显上调,与对照组比较,差异有统计学意义(P<0.0 1)。RT-PCR检测显示,模型组EGFRmRNA和间质表达物α-SMA mRNA的表达较对照组均明显上调(P<0.01),上皮表达物E-cadherin mRINA表达明显下降,在3d,7d,14d不同时间点差异显著(均P<0.01)。上述结果显示EMT与矽肺性纤维化发生、发展过程密切相关,EGFR和TGF-α表达增强,两者结合发挥其生物学效应。Western blot检测结果显示,染尘不同时间点模型组肺组织蛋白Ras、p-Erk1/2、PI-3KCG、Aktl相对表达量均明显上调,与对照组比较(3d、7d、14d),差异有统计学意义(P<0.01),提示EGFR下游的EGFR/PI-3K/AKt和Ras/ERK/MAPK通路参与此纤维化过程。3、EGFR-TKIs在矽肺性纤维化过程中的干预作用肺泡炎症评分,染尘第3天,高、中、低剂量干预组和模型相比较,有统计学意义(3.1±0.141vs4.0±0.120,3.3±0.075vs4.0±0.120,3.6±0.259vs s4.0±0.120,均P<0.01),且组间比较有统计学意义(P<0.05或P<0.01)。染尘第7天,高、中、低剂量干预组和模型相比较,有统计学意义(1.2±0.296vs2.1±0.161,1.5±0.213 vs2.1±0.161,1.8±0.316vs2.1±0.161,均P<0.05或P<0.01),组间比较有统计学意义(P<0.05或P<0.01)。染尘第3天、第7天随着干预剂量的增加,肺泡炎症明显减轻,高剂量干预组作用最明显。染尘第7天和第14天,与模型相比较,高、中、低剂量干预组随着干预剂量的增加,肺切片纤维化评分的增高明显下调,差异有统计学意义(7d:1.4±0.189 vs1.3±0.164,1.6±0.179vs1.3±0.164,1.7±0.250 vs1.3±0.164,均P<0.01;14d:2.4±0.101vs3.5±0.263,2.7±0.208 vs3.5±0.263,2.9±0.154 vs3.5±0.263,均P<0.01)。在染尘第7天,高剂量和低剂量干预组(1.4±0.189vs1.7±0.250)比较,高剂量和中剂量干预组间比较(1.4±0.189vs1.6±0.179),有统计学意义(P<0.01)。在染尘第14天,高剂量和低剂量干预组比较(2.4±0.101vs2.9±0.154,P<0.01),高剂量和中剂量干预组间比较(2.4±0.101 vs2.7±0.208,P<0.05),有统计学意义。羟脯氨酸的含量高、中、低干预组各时间点(7d,14d)与模型组相比较均降低,差异显著有统计学意义(7d:0.81±0.0955vs1.24±0.931,0.92±0.0833vs 1.24±0.931,1.10±0.0759vs1.24±0.931;14d:0.90±0.7350vs1.49±0.0686,1.09±0.0612 vs1.49±0.0686,1.30±0.0637vs1.49±0.0686;P<0.05 或 P<0.01)。在 7d、14d 时间点干预组间比较:高剂量干预组和低剂量干预组比较,差异有统计学意义(0.81±0.0955vs 1.10±0.0759,0.90±0.735vs1.30±0.0637,均P<0.01);高剂量干预组和中剂量干预组比较,差异具有统计学意义(0.81±0.0955vs0.92±0.0833,0.90±0.735 vs1.09±0.0612,P<0.05或P<0.01);中剂量干预组和低剂量干预组比较,差异具有统计学意义(0.92±0.0833vs1.10±0.0759,1.09±0.0612 vs 1.30±0.0637,均P<0.01)。总之,在染尘第7天,第14天,纤维化指标肺羟脯氨酸的含量随EGFR-TKIs干预剂量的增加而降低,干预可减轻其纤维化发展,高剂量干预组作用最明显。对于染尘肺纤维化EMT过程EGFR-TKIs的干预效果,RT-PCR检测肺组织α-SMA mRNA结果显示其上调表达干预减缓。高、中、低剂量干预组分别在染尘3d、7d、14d时间点和模型组比较,差异都具有统计学意义(P<0.01)。在染尘第3天,第7天,第14天干预组间比较,高剂量干预组和低剂量干预组比较,都有统计学意义(均P<0.01);高剂量干预组和中剂量干预组比较,染尘第3天有差异(P<0.01),染尘第7天和第14天差异不明显(P>0.05);中剂量和低剂量干预组比较,都具有统计学意义(P<0.05或P<0.01)。RT-PCR检测肺组织E-cadherin mRNA,结果显示可使其下降的表达减缓。染尘第3天,第7天,高、中剂量干预组分别和模型组,差异具有统计学意义(P<0.0 1);低剂量干预组与模型组比较,差异不显著(P>0.05);染尘第14天,高、中、低剂量干预组分别同模型组比较,差异具有统计学意义(P<0.01)。干预组间比较,在染尘第3天,第7天,第14天,高剂量干预组和中剂量干预组比较,差异不明显(P>0.05)。以上结果显示,随着EGFR-TKIs干预剂量的增加,EMT过程,即肺组织α-SMAmRNA上升和E-cadherin mRNA下降的表达干预减缓,高剂量干预组和中剂量干预组作用最明显。EGFR-TKIs干预后,ICH肺组织p-EGFR平均积分光密度和RT-PCR检测EGFRmRNA增高的表达均显著受抑,与模型组相比较,各时间点(3d,7d,14d)高、中、低干预组都有显著性差异(均P<0.01)。高、中、低干预组组间在染尘3d、7d、14d时间点比较,高剂量干预组和低剂量干预组比较,有统计学意义(0.1]04± 0.0211vs0.20]5±0.0494,0.3207±0.0290 vs 0.4113 ±0.0316,0.4019±0.0307 vs0.5329±0.0465,均P<0.01);中剂量干预组与低剂量干预组比较,都有统计学差异(均P<0.05);而高剂量干预组和中剂量干预组比较,仅在3d、14d 时间点有统计学意义(0.1104±0.021 1vs 0.1558 ±0.0334,0.4019±0.0307vs0.4635 ±0.0322,均P<0.05),染尘第7天差异没有统计学意义(P>0.05)。EGFRmRNA在染尘第3天,第7天,第]4天,各干预剂量组间比较,差异均具有统计学意义(均P<0.01)。随着EGFR-TKIs干预剂量的增加,肺组织p-EGFR平均积分光密度和EGFRmRNA增高的表达均显著受抑,高剂量干预组和中剂量干预组作用最明显。经EGFR-TKIs的干预后,干预组各时间点的BALF中TGF-α浓度均低于模型组,高剂量干预组在染尘3d、7d、14d时间点与模型组相比较,差异显著具有统计学意义(46.204±4.395 vs69.964±7.92,54.1 99±5.492vs79.456±9.58,34.017±9.3911vs 55.764±8.58,均P<0.01);中剂量干预组在3d、7d、14d时间点与模型组相比较,差异显著具有统计学意义(均P<0.01);低剂量干预组和模型组比较,没有统计学意义(P>0.05)。高、中剂量干预组间比较,在3d、7d、14d时间点没有统计学意义(P>0.05)。和模型组比较,干预各组TGF-α蛋白相对表达水平上调数值下降,染尘第3天、第7天、第14天,高、中、低剂量干预组分别和模型组比较,差异具有统计学意义(P<0.01)。不同剂量干预组间比较,在染尘第3天,第7天,第14天,高剂量干预组和低剂量干预组比较,都有统计学意义(均P<0.01);高剂量干预组和中剂量干预组比较,TGF-α相对表达水平差异无统计学意义(P>0.05);中剂量和低剂量干预组比较差异都具有统计学意义(均P<0.01)。随着EGFR-TKIs干预剂量的增加,高、中、低剂量干预组BALF中TGF-α浓度和肺组织TGF-α蛋白相对表达上升明显减缓,高剂量和中剂量干预组作用最明显。经EGFR-TKIs干预后,Ras蛋白相对表达水平的上升下调,高、中剂量干预组分别在染尘3d、7d、14d时间点和模型组比较,差异具有统计学意义(P<0.01),低剂量干预组和模型组比较,差异不显著(P>0.05)。各时间点(3d,7d,14d)高剂量和中剂量组组间比较未见明显统计学意义(P>0.05)。干预后p-Erk1/2蛋白相对表达水平下调,高、中、低剂量干预组分别在3d、7d、14d时间点和模型组比较,差异具有统计学意义(P<0.05或P<0.01)。不同剂量干预组间比较,在染尘第3天、第7天、第14天,高剂量干预组和低剂量干预组比较,都有统计学意义(均P<0.01);中剂量和低剂量干预组比较,差异都具有统计学意义(均P<0.05);高剂量干预组和中剂量干预组比较,p-Erk1/2相对表达水平差异无统计学意义(P>0.05)。高、中、低剂量干预组随着EGFR-TKIs干预剂量的增加,肺组织Ras和TGF-α蛋白相对表达上升明显减缓,高剂量和中剂量干预组作用最明显。经EGFR-TKIs干预后,PI-3KCG相对表达的上升水平下调,与模型组相比较,低剂量干预组在染尘3d、7d、14d时间点没有统计学意义(P>0.05);中剂量干预组分别在染尘第7天、第14天的下调具有统计学意义(P<0.05)。高剂量干预组在第3天、第7天、第14天下调都具有统计学意义(P<0.05或P<0.01)。剂量干预组的组间比较,染尘第7天、第14天,高剂量组相对于中剂量组PI-3KCG相对表达上升水平下调不显著,没有统计学意义(P>0.05)。高剂量和中剂量干预组作用最明显。干预后,Akt1相对表达上升的水平下调,高、中、低剂量干预组分别在染尘3d、7d、14d时间点与模型组相比较,下调都具有统计学意义(P<0.01)。剂量干预组间比较,在染尘第3天、第7天、第14天,高剂量干预组和低剂量干预组比较,都有统计学意义(P<0.001);中剂量和低剂量干预组比较,差异都具有统计学意义(P<0.01或P<0.05);高剂量干预组和中剂量干预组比较,差异有统计学意义(P<0.05)。高剂量干预组作用最明显。以上结果提示EGFR-TKIs干预后,EGFR与配体TGF-α活化减轻,可抑制下游信号转导通路EGFR/PI-3K/AKt和Ras/ERK/MAPK的靶点蛋白Ras、p-Erk1/2、PI3K、Akt1,从而减少了矽尘肺组织损伤诱导的EMT,减轻肺纤维化的改变。这一过程与剂量有关,高剂量和中剂量干预组作用明显。结论实验结果证实矽肺性纤维化的形成发生与EMT有关,与EGFR、TGF-α、Ras、p-Erk1/2、PI3K、Akt1 关系密切。EGFR-TKIs 干预可减少与配体 TGF-α结合,抑制二氧化硅损伤所导致的EGFR的过度活化,从而阻遏下游信号转导通路靶点蛋白Ras、p-Erk1/2和PI-3KCG、Akt1的激活,减轻肺组织损伤诱导的EMT,减轻肺纤维化。此干预过程中与剂量有关,高剂量和中剂量干预组作用明显。EGFR和Ras/ERK/MAPK和PI-3K/AKt信号转导通路的靶点蛋白在矽肺性纤维化发病机制中都存在关联。研究提示通过干预EGFR-TKIs靶点抑制EGFR活化,进而延缓矽肺肺纤维化的EMT发生,可减轻病情发生及进展,为矽肺性纤维化的防治策略开拓新的思路。更多还原

【Abstract】 ObjectivesTo observe the effect of different doses of epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKIs)on interstitial transformation(EMT)and fibrosis in the lung of rats at different time points,and to explore the mechanism of epidermal growth factor receptor(EGFR)and its signaling pathway in the formation of silicosis fibrosis.It provides a theoretical basis for molecular targeted drug prevention and treatment of pneumoconiosis fibrosis.MethodsThe animal model of Wistar Rats was established,which was randomly divided into model group,control group,high dose intervention group,middle dose intervention group and low dose intervention group.The high,medium and low dose intervention groups were given different doses of EGFR-TKIs perfusion intervention(high dose group 13.5mg/d,middle dose group 6.75mg/d,low dose group 3.375mg/d)_Respectively in the 3d,7d,14d,each group randomly took 6 rats to their euthanasia.The lung tissue specimens were taken for gross visual and pathological staining,lung inflammation and fibrosis semi-quantitative scoring and pulmonary homogenate hydroxyproline(HYP)assay to assess lung inflammation and fibrosis levels.Immunohistochemical staining(ICH)was used to observe and quantitatively analyze the content of epidermal growth factor receptor(EGFR)and p-EGFR in lung tissue,and the content of-α(TGF-α)in bronchoalveolar lavage fluid(BALF)was detected by Elisa method,and the EGFR in lung tissue was detected by RT-PCR.E-cadherin(E-cadherin)and alpha-smooth muscle inflammatory protein(a-SMA)mRNA expression level;Western Blot method was used to detect the contents of TGF-a,Ras,P-ERKL/2,PI-3KCG and Aktl in lung tissues.Results1、Silicosis fibrosis model successfully established by non-tracheal exposure in Wistar ratsAt different time points(3d,7d and 14d),the test of alveolar inflammation in the model group was found to be significantly higher than that in the control group(4.0±0.120vs1.8±0.101,2.1±0.161vs1.0±0.104,1.5±0.135vs0.5±0.184,P<0.01).P ulmonary fibrosis score and hydroxyproline content in the model group is higher in the seventh day,and over time and continues to increase,in dye dust 7 days,14 days,compared with the control group,pulmonary fibrosis score(1.3±0.164vs 0.710.131,3.5±10.263vs0.8±0.086,P<0.01)and hydroxyproline content(1.24±0,931vs0.59±0.0735,1.49±0.0686vs0.64±0.0588,P<0.01)there is statistical significance.The characteristics and progression of pulmonary inflammation and fibrosis in the stained dust model constructed in this experiment were consistent with the model characteristics of silicosis fibrosis.2、The pathogenesis of pneumoconiosis,EGFR and EMT and its downstream pathways.The expression of EGFR and p-EGFR in lung tissue was mainly localized in the cell membrane or cytoplasm of airway epithelial cells and pulmonary interstitial cells,The average optical density of p-EGFR increased significantly with time(3d,7d,14d)in the model group,and there was a significant difference compared with the control group(0.2692±0.0312 vs0.0613±0.0301,0.5960±0.0641vs0.0671±0.0348,0.7591±0.0532vs0.0544± 0.0613,P<0.01).The content of TGF-a of BALF with ELISA in the model group was significantly increased in the third day,and the time points(3d,7d,14d)were significantly different from those in the control group(69.964±7.92 vs 34.892±6.248,79.456±9.58vs36.463±6.441,55.764±8.58vs31.554±8.916,P<0.01).Co-mpared with the control group,there was a significant higher in the relative expression of TGF-a in lung tissue in the model group(3d,7d and 14d)(P<0.01).The expression of E-cadherin,a-SMA and EGFR mRNA was detected by RT-PCR.The expressions of EGFR mRNA and interstitial expression of a-SMAmRNA were up-regulated and the expression of E-cadherin mRNA in the model group was significantly lower than that in the control group at different time points(P<0.01).The above results show that EMT is closely related to the occurrence and development of silicosis fibrosis.The expression of EGFR and TGF-a is enhanced and their biological effects are combined.Western blot analysis showed that the relative expression of TGF-α,Ras,p-Erkl/2,PI-3KCG and Aktl protein in the lung tissue of the model group at different time points(3d,7d,14d)were significantly higher than those in the control group,the difference was statistically significant(P<0.01).The above results indicate that EGFR/PI-3k/AKt and Ras/ERX/MAPK pathways downstream of EGFR participate in this fibrosis process.3、The intervention of EGFR-TKIs in the process of silicosis fibrosisIn dye dust 3 days,alveolar inflammation score in the EGFR-TKIs with high,medium and low dose intervention group was statistically significant compared with the model(3.1±0.141vs4.0±0.120,3.3±0.075vs4.0±0.120,3.6±0.259vs s4.0±0.120,all P<0.01),and the comparison between groups was statistically significant(P<0.05 or P<0.01).In dye dust 7 days,alveolar inflammation score in the EGFR-TKIs with high,medium and low dose intervention group was statistically significant compared with the model(1.2±0.296vs2.1±0.161,1.5±0.213vs2.1±0.161,1.8±0.316vs2.1±0.161,P<0.05or P<0.01),and the comparison between groups was statistically significant(P<0.05 or P<0.01).In the 3rd and 7th days.of the model,the alveolar inflammation decreased significantly with the increase of intervention dose.High dose intervention was most obvious.On the 7th and 14th days of the model,compared with the model,the increase of pulmonary fibrosis score in the EGFR-TKIs with high,medium and low dose intervention group was significantly reduced,and the difference was statistically significant(7d:1.4±0.189vsl.3±0.164,1.6±0.179vs1.3±0.164,1.7±0.250vs1.3± 0.164,P<0.01;14d:2.4±0.101vs3.5±0.263,2.7±0.208vs3.5±0.263,2.9±0.154vs3.5±0.263,P<0.01).On the 7th of the model,the comparison between high-dose and low-dose intervention group(1.4±0.189vs1.7±0.250),the comparison between high-dose and medium-dose intervention group(1.4±0.189vs1.6±0.179),was statistically significant(P<0.01).On the 14th of the model,the comparison between high-dose and low-dose intervention group(2.4±0.101vs2.9±0.154,P<0.01),the comparison between high-dose and medium-dose intervention group(2.4±0.101vs2.7±0.208,P<0.05),was statistically significant.The content of hydroxyproline in high,medium and low intervention groups(7d,14d)was lower than that of the model group,and the difference was statistically significant(7d:0.81±0.0955vs1.24±0.931,0.92±0.0833vs 1.24± 0.931,1.10±0.0759vsl.24±0.931;14d:0.90±0.7350vs1.49±0.0686,1.09±0.0612vs1.490.0686,1.30± 0.0637 vs1.49±0.0686;P<0.05 or P<0.01).Comparison between the dose group at the 7d and 14d time points of the model:the comparison between the high-dose intervention group and the low-dose intervention group was statistically significant(0.81±0.0955vs1.10±0.0759,0.90±0.735vsl.30±0.063 7,all P<0.01);The difference between high-dose intervention group and medium dose intervention group was statistically significant.(0.81±0.0955vs0.92±0.0833,0.90±0.735vsl.09±0.0612,P<0.05orP<0.01);The differe-nce was statistically significant between the medium dose intervention group and the low-dose intervention group(0.92±0.0833vs1.10±0.0759,1.09±0.0612vs1.30± 0.0637,all P<0.01).In summary,On the 7th and the 14th of the model,the content of the lung hydroxyproline decreased with the increase of EGFR-TKIs intervention dose,the intervention coulld reduce the development of fibrosis,and the high-dose interverntion group had the most obvious effect.For the intervention effect of EGFR-TKIs in the EMT process of pneumoconiosis,the up-regulated expression intervention was shown to be reduced by RT-PCR detection in lung tissue.On the 3th、7th and 14th days of the model,compared with the model,the increase of a-SMA mRNA in the EGFR-TKIs with high,medium and low dose intervention group was significantly reduced,and the difference was statistically significant(P<0.01).On the 3th,7th and 14th days of the model,compared with the intervention group,the comparison between.high-dose intervention group and low-dose intervention group was statistically significant(all P<0.01);On the 3th,there was a difference between the high-dose intervention group and the medium dose intervention group(P<0.01),and the difference between day 7 and day 14 was not obvious(P>0.05);Both medium and low dose intervention groups were statistically significant(P<0.05 or P<0.01).E-cadherin mRNA of lung tissue was detected by RT-PCR.On the 3th and 7th days of the model,compared with the model,the increase of E-cadherin mRNA in the EGFR-TKIs with high and medium dose intervention group was significantly significant,and the difference was statistically significant(P<0.01),There was no significant difference between the low-dose intervention group and the model group(P>0.05);On the 14th days of the model,compared with the model,the increase of E-cadherin mRNA with high,medium and low dose intervention group was significantly significant,and the difference was statistically significant(P<0.01);On the 3th,7th and 14th days of the model,compared with the intervention group,the comparison between high-dose intervention group and medium-dose intervention group was no significant difference(P<0.05).In summary,with the increase of EGFR-TKIs intervention dose,the EMT process,the increase of the lung tissue a-SMA and the decrease of E-cadherin mRNA were slowed down,and the intervention of the high-dose group and the medium dose group was the most obvious.After EGFR-TKIs intervention,the mean integrated optical density of p-EGFR in ICH lung tissue and the expression of EGFR mRNA detected by RT-PCR were significantly inhibited.Compared with the model group,there was a significant difference in the high,middle,and low intervention groups at each time point(3d,7d,14d)(all P<0.01).The groups in the high,middle,and low intervention groups were compared at the time points of 3d,7d,and 14d.There was significant difference between the high-dose intervention group and the low-dose intervention group(0.1104±0.0211vs0.2015±0.0494,0.3207±0.0290vs0.4113±0.0316,0.4019±0.0307 vs0.5329±0.0465,all P<0.01).The difference between the high-dose intervention group and the middle-dose intervention group was statistically significant only at the 3d and 14d time points(0.1104±0.0211vs 0.1558±0.0334,0.4019±0.0307vs0.4635±0.0322,all P<0.05),and there was no statistically significant difference at the 7th day(P>0.05).In the medium-dose intervention group and the low-dose intervention group,there was a statistically significant difference at 3d,7d,and 14d(all P<0.05).The data of EGFR mRNA on the 3rd,7th,and 14th days were statistically significant(all P<0.01).With the increase of EGFR-TKIs intervention dose,the mean integrated optical density of p-EGFR and the increase of EGFR mRNA expression in lung tissue were significantly inhibited,and the effect was most obvious in high-dose and middle-dose intervention groups.After EGFR-TKIs intervention,the concentration of TGF-a in the BALF of the intervention group was lower than that of the model group at each time point.The high-dose intervention group had statistically significant differences at 3 days,7 days,and 14 days with the model group(46.204 14.395vs69.9641±7.92,54.199±5.492 vs79.456 ±9.58,34.017±9.391 vs 55.764±8.58,all P<0.01);At the 3d,7d,and 14d time points,the difference between the middle-dose intervention group and the model group was statistically significant(all P<0.01).There was no significant difference between the low-dose intervention group and the model group(P>0.05);There was no statistically significant difference between the high and middle dose intervention groups at 3d,7d,and 14d(P>0.05).Compared with the model group,the relative expression level of TGF-a protein in each group was decreased.On the 3d,7th,and 14th days,the high-,medium-,and low-dose intervention groups were significantly different from the model group(P<0.01).Comparison between different doses of intervention groups:On the 3rd,7th,and 14th days,there was a statistically significant difference between the high-dose intervention group and the low-dose intervention group(P<0.01);There was no significant difference in the relative expression levels of TGF-a between the high-dose intervention group and the middle-dose intervention group(P>0.05).The differences between the middle-dose and low-dose intervention groups were statistically significant(P<0.01).With the increase in EGFR-TKIs intervention dose,the expression of TGF-a in BALF and the relative expression of TGF-a in lung tissue were significantly slowed down in the high,medium and low dose intervention groups.The effect of high-and medium-dose intervention group was the most obvious.After the intervention of EGFR-TKIs,the relative expression level of Ras protein was down-regulated.On the 3rd.,7th.,and 14th days,the high-and middle-dose intervention groups were significantly different from the model group(P<0.01).There was no significant difference between the low-dose intervention group and the model group(P>0.05).There was no significant difference between the high-dose and middle-dose groups at each time point(3d,7d,14d)(P>0.05).The relative expression level of p-Erkl/2 protein was down-regulated after intervention.Compared with the model group,on the 3rd,7th,and 14th days,the high,medium,and low dose intervention groups were compared with the model group,the difference was statistically significant(P<0.05orP<0.01).Comparison between different doses of intervention groups,On the 3rd,7th,and 14th days,there was a statistically significant difference between the high-dose intervention group and the low-dose intervention group(P<0.01).The differences between the medium and low dose intervention groups were statistically significant(all P<0.05).There was no significant difference in the relative expression level of p-Erkl/2 between the high-dose intervention group and the middle-dose intervention group(P>0.05).With the increase of EGFR-TKIs intervention dose,the relative expression of Ras and TGF-a protein in lung tissue increased significantly in high-,medium-,and low-dose intervention groups,and the effect was most obvious in high-and middle-dose intervention groups.After EGFR-TKIs intervention,the relative increase of PI-3KCG expression was down-regulated.Compared with the model group,the low-dose intervention group had no statistical significance at any time period(P>0.05);The mid-dose intervention group had a statistically significant down-regulation on the 7th and 14th days(P<0.05);The high-dose intervention group had statistical significance on the 3rd day,the 7th day,and the 14th day(P<0.05 or P<0.01).Group comparisons between dose intervention groups,on the 7th and 14th day,the relative increase of PI-3KCG expression in the high dose group relative to the middle dose group was not significantly down-regulated(P>0.05).The effects of the high-and middle-dose intervention groups were most pronounced.The groups in the dose intervention group were compared with each other.On the 3rd,7th and 14th days,there was significant difference between the high-dose intervention group and the low-dose intervention group(P<0.01);The difference between the middle-dose group and the low-dose group was statistically significant(P<0.01 or P<0.05);There was a significant difference in the relative expression level of Aktl protein between the high-dose intervention group and the middle-dose intervention group(P<0.05).High-dose intervention group has the most obvious effect.The above results suggest that the activation of EGFR and ligand TGF-a is reduced,and the downstream signal transduction pathways(EGFR/PI-3K/AKt and Ras/ERK/MAPK)are inhibited after EGFR-TKIs intervention.Thus,the EMT induced by the damage of the lung tissue of the silica dust is reduced,and the change of pulmonary fibrosis is reduced.This process is related to the dose,and the effect of the high-dose and middle-dose intervention groups is obvious.ConclusionThe experimental results confirm that the formation of silicosis is related to EMT,which is closely associated with EGFR,TGF-a,Ras,P-ERKL/2,PI3K and AKT1.EGFR-TKIs intervention can reduce the binding of ligand TGF-a,inhibit the excessive activation of EGFR caused by silica damage,thus deterring the activation of the target protein RAS,P-ERKL/2 and PI-3KCG,AKT1 in the downstream signal transduction pathway,reducing the EMT induced by lung tissue injury,relieves fibrosis of the lungs.The intervention is dose-related,high-dose and medium-dose intervention group have significant effects.The target protein of EGFR and ras/erk/mapk and PI-3K/AKT signal transduction pathway are correlated in the pathogenesis of silicosis fibrosis.The study suggests that by intervening EGFR-TKIS target to inhibit EGFR activation,and then delaying the EMT of pulmonary fibrosis of silicosis,it can alleviate the occurrence and progress of the disease,and develop new ideas for prevention and treatment of silicosis fibrosis.更多还原