表皮生长因子受体酪氨酸激酶抑制剂对大鼠尘肺纤维化干预的研究

Preliminary study of exon sequence in pneumoconiosis using high-throughput and intervention of EGFR-TKIs on silicosis rats

目的观察表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKIs)对二氧化硅(SiO2)所致大鼠肺纤维化的干预作用,探讨表皮生长因子受体(EGFR)信号通路在肺纤维化中的作用。方法SPF级健康雄性Wistar大鼠90只随机分为对照组、模型组、高剂量干预组、中剂量干预组和低剂量干预组,每组18 只。模型组和各剂量干预组大鼠用浓度50 mg/ml二氧化硅混悬液1 ml非气管暴露法缓慢注入气管染尘造模。对照组大鼠用同样方法注入等量灭菌生理盐水。建模后第1 天各剂量干预组大鼠用EGFR-TKIs灌胃干预,高、中、低剂量干预组分别给予EGFR-T KIs研磨粉混悬液13.59 6 75、3.375 mg/d,模型组和对照组分别给予等量生理盐水。建模后第3、 7、 14天各组分别处死6 只大鼠,HE染色观察肺组织病理学改变,Masson染色观察肺纤维化,免疫组化检测p-EGFR的表达,样本碱水解法测定肺匀浆径脯氨酸(HYP)含量,Western blot法检测肺组织肿瘤坏死因子-a(TGF-a)、 Ras磷酸化的细胞外信号调节激酶(p-Erk"2)、P[-3KCG 和蛋白激酶(Akt l)蛋白的含量。结果建模后第7、14天,HE染色和Masson染色可见大鼠肺纤维化程度随干预剂量的增加而降低,HYP 含量随干预剂量的增加而降低,差异均有统计学意义(P<0.05)。与模型组比较,各干预组肺组织TGF-a 、p-Erkl/2、 Aktl蛋白相对表达均下调,差异均有统计学意义(P <0.05),中、高剂量干预组Ras 、PI-3KCG蛋白相对表达下调,差异有统计学意义(P<0.05)。与低剂量干预组比较,中、高剂量干预组大鼠肺组织TGF-a 、Ras、 p-Erk"2蛋白相对表达下调,差异均有统计学意义(P <0.05)冲、高剂量干预组间比较,大鼠肺组织TG F-a、 Ras、 p-Erkl/2蛋白相对表达差异无统计学意义(Q0.05)。与低剂量干预组比较,高剂量干预组干预第7 、14 天大鼠肺组织PI-3KCG蛋白相对表达下调,差异有统计学意义(P<0.05 )。各剂量干预组间比较,大鼠肺组织Aktl相对表达水平差异均有统计学意义(P<0.05)。结论EG FR-TKIs可能通过抑制EGFR 相关的细胞内酪氨酸激酶活力及细胞内磷酸化过程,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-Erkl/2、 PI3K、 Aktl,从而减轻大鼠肺纤维化的改变。

Objective To observe the effect of epidermal growth factor receptor tyrosinase inhibitor (EGFR-TKIs) on silica (SiO2)-induced pulmonary fibrosis in rats, and to explore the role of epidermal growth factor receptor (EGFR) signaling pathway in pulmonary fibrosis. Methods A rat model of SiO2 silicosis was established (high, medium and low dose intervention groups, and the model group was infused with silica suspension). The control group was intratracheally injected with the same amount of sterile saline. The rats were intragastrically administered with different concentrations of EGFR-TKIs (13.5 mg/day, 6.75 mg/day, 3.375 mg/day). The model group and the control group were given the same amount of normal saline. Six rats in each group were euthanized on the 3rd, 7th, and 14th days after modeling. HE staining was used to observe the pathological of lung tissue. Masson staining to observe pulmonary fibrosis. Immunohistochemistry (IHC) expression of p-EGFR. Determination of hydroxyproline content by alkaline hydrolysis of the sample, Western Blot was used to detect the contents of TGF-a, Ras, p-Erkl/2, PI-3KCG and Aktl protein in lung tissue. Results The degree of fibrosis and hydroxyproline content in HE staining and Masson staining decreased with the increase of intervention dose, which was statistically significant (7(1, 14 d). Compared with the model group, the relative expressions of TGF-a, p-Erkl/2 and Aktl protein in lung tissue of each intervention group were statistically significant (P<0.05). The down-regulation of Ras and PI-3KCG protein in the high- and medium-dose intervention group was statistically significant (P<0.05), and there was no significant difference in the low-dose intervention group.Compared with the intervention group, the down-regulation of TGF-a, Ras, and p-Erkl/2 protein in the lung tissue was statistically different in the low-dose group (P<0.05). There was no significant difference between the dose and the medium dose intervention group (P>0.05). PI-3KCG was significantly different only in the high-dose intervention group and the low-dose intervention group on the 7th and 14th day (P<0.05), and there was no difference between the other groups (P >0.05). There was a statistically significant difference in Aktl between the dose intervention groups (P<0.05). Conclusion EGFRTKIs inhibits EGFR-related intracellular tyrosine kinase activity and intracellular phosphorylation, and inhibits the downstream signal transduction pathways of EGFR/Pl-3K/AKt and Ras/ERK/MAPK target protein Ras, p- Erkl/2, PI3K, Aktl, thereby alleviating changes in pulmonary fibrosis, this process is dose-dependent, and the high-dose and medium-dose intervention groups have a significant effect.