转化生长因子β1/Smads通路在百草枯中毒所致上皮-间充质转变和肺纤维化中的作用研究

doi:10.3877/cma.j.issn.1674-6880.2018.01.001

目的

本研究通过观察百草枯诱导大鼠肺纤维化中转化生长因子β1（TGF-β1）/Smads信号通路的上皮-间充质转变（EMT）现象及相关蛋白的表达。

方法

将60只雄性大鼠分为对照组及百草枯组，每组各30只。百草枯组腹腔注射80 mg/kg百草枯建立中毒模型，对照组注射3 mL等渗NaCl溶液。分别在建模后1、3、7、14、21、28 d分批处死大鼠，比较两组大鼠肺组织湿干比重及羟脯氨酸水平，取左肺下叶予苏木素-伊红（HE）染色及Masson染色。评估肺组织病理学和肺纤维化程度，采用Western-blotting检测百草枯中毒后1、3、7、14、21、28 d肺组织中EMT相关指标和TGF-β1/Smads信号通路相关蛋白的表达。

结果

两组各时间点肺湿干比重及羟脯氨酸水平的比较，差异有统计学意义（F=9.772、22.541，P均< 0.001），且百草枯组湿干比重的比值在3、7、14、21、28 d均显著高于对照组（P均<0.05），而羟脯氨酸表达水平仅在14、21、28 d明显高于对照组（P均<0.05）。病理结果显示百草枯组大鼠中毒后1~7 d，炎症细胞浸润，肺泡间质增厚水肿，中毒后21 d和28 d可见肺泡壁明显增厚及胶原纤维明显增生，而对照组大鼠肺组织无明显改变。对照组与百草枯组各时间点TGF-β1、Smad2/3、p-Smad2/3、Smad4、p-Smad4、Smad7、p-Smad7、E-cad、α-平滑肌肌动蛋白（α-SMA）、Vimentin和成纤维细胞特异性蛋白1（FSP-1）表达水平的比较，差异均有统计学意义（F=10.685、7.381、7.878、10.743、14.575、17.791、33.200、14.453、10.849、25.415、26.263，P均< 0.001），且百草枯组TGF-β1、Smad2/3、p-Smad2/3、Smad4、p-Smad4、α-SMA、Vimentin及FSP-1表达水平逐渐升高，中毒后21 d达高峰（P均<0.05）。而Smad7、p-Smad7及E-cad表达水平逐渐降低，Smad7、p-Smad7水平在中毒后7 d达到谷底；E-cad表达水平在中毒后21 d达到谷底（P均<0.05）。

结论

TGF-β1/Smads信号通路可能通过调节EMT过程影响百草枯诱导的肺纤维化发生。

关键词: 转化生长因子β1, 百草枯, 中毒, 肺纤维化, 上皮细胞-间充质细胞转换, 大鼠

Objective

To observe the endothlial-mesenchymal transition (EMT) phenomenon and related protein expressions in transforming growth factor-β1 (TGF-β1)/Smads signaling pathway in lung tissues of the rats with paraquat-induced pulmonary fibrosis.

Methods

Sixty male SD rats were randomly divided into paraquat group (n=30) and control group (n=30). The paraquat poisoning model was established by intraperitoneally injecting with 80 mg/kg of paraquat saline solution in the paraquat group, while rats in the control group were injected with 3 mL of sterile saline. The rats were sacrificed in batches at 1, 3, 7, 14, 21, 28 d after modeling. The wet/dry ratio of left lung tissue and the expression of hydroxyproline were calculated between these two groups. The pathology and pulmonary fibrosis in the inferior lobe of left lung were assessed by hematoxylin-eosin (HE) staining and Masson staining. The expressions of related proteins in EMT and TGF-β1/Smads signaling pathway in lung tissues on days 1, 3, 7, 14, 21, 28 after paraquat poisoning were analyzed by Western-blotting.

Results

The wet/dry ratio and hydroxyproline both showed significant differences between two groups at different time points after modeling (F=9.772, 22.541, both P < 0.001). In the paraquat group, the wet/dry ratios at 3, 7, 14, 21, 28 d were much higher than those in the control group (all P<0.05), and the levels of hydroxyproline only increased at 14, 21, 28 d as compared with those in the control group (all P<0.05). Pathological findings indicated that inflammatory cell infiltrated, alveolar interstitium thickened in the paraquat group at 1, 3, 7 d, and alveolar walls thickened gradually and a large amount of collagen fibers proliferated at 21, 28 d after modeling. There was no significant change in lung tissue of the control group. The expressions of TGF-β1, Smad2/3, p-Smad2/3, Smad4, p-Smad4, Smad7, p-Smad7, E-cadherin (E-cad), α-smooth muscle actin (α-SMA), Vimentin and fibroblast specific protein-1 (FSP-1) between the two groups at each time point all showed significant differences (F=10.685, 7.381, 7.878, 10.743, 14.575, 17.791, 33.200, 14.453, 10.849, 25.415, 26.263, all P < 0.001). The expressions of TGF-β1, Smad2/3, p-Smad2/3, Smad4, p-Smad4, α-SMA, Vimentin and FSP-1 gradually increased and reached a peak at 21 d after paraquat poisoning, while the Smad7, p-Smad7 decreased and reached a bottom at 7 d in the paraquat group (all P<0.05). The expression of E-cad in the paraquat group reduced from 3 d to 21 d (all P<0.05).

Conclusion

The TGF-β1/Smads signaling pathway may affect the development of PQ-induced pulmonary fibrosis by regulating EMT process.

Key words: Transforming growth factor beta1, Paraquat, Poisoning, Pulmonary fibrosis, Epithelial-mesenchymal transition, Rats

表1 两组大鼠建模后各时间点肺组织肺湿干比重的变化（±s）

组别	只数	1d	3d	7 d	14 d	21 d	28 d
对照组	30	3.8 ± 0.3	4.0 ± 0.5	3.9 ± 0.4	4.2 ± 0.4	4.1 ± 0.7	3.9 ± 0.8
百草枯组	30	4.2 ± 0.9	5.9 ± 1.1^ab	6.2 ± 1.3^ab	6.8 ± 1.4^ab	7.3 ± 1.6^ab	7.1 ± 1.5^ab

表1 两组大鼠建模后各时间点肺组织肺湿干比重的变化（±s）

组别	只数	1d	3d	7 d	14 d	21 d	28 d
对照组	30	3.8 ± 0.3	4.0 ± 0.5	3.9 ± 0.4	4.2 ± 0.4	4.1 ± 0.7	3.9 ± 0.8
百草枯组	30	4.2 ± 0.9	5.9 ± 1.1^ab	6.2 ± 1.3^ab	6.8 ± 1.4^ab	7.3 ± 1.6^ab	7.1 ± 1.5^ab

图1 两组大鼠建模后不同时间点的病理表现

表2 两组大鼠不同时间点羟脯氨酸水平变化的变化（mg/L，±s）

组别	只数	1d	3d	7 d	14 d	21 d	28 d
对照组	30	2.47 ± 0.83	3.31 ± 0.72	2.80 ± 0.63	2.52 ± 0.73	3.21 ± 1.14	2.63 ± 1.24
百草枯组	30	2.73 ± 1.23	4.63 ± 0.85	5.24 ± 1.64	7.33 ± 1.33^ab	8.34 ± 0.97^ab	9.68 ± 1.22^ab

表2 两组大鼠不同时间点羟脯氨酸水平变化的变化（mg/L，±s）

组别	只数	1d	3d	7 d	14 d	21 d	28 d
对照组	30	2.47 ± 0.83	3.31 ± 0.72	2.80 ± 0.63	2.52 ± 0.73	3.21 ± 1.14	2.63 ± 1.24
百草枯组	30	2.73 ± 1.23	4.63 ± 0.85	5.24 ± 1.64	7.33 ± 1.33^ab	8.34 ± 0.97^ab	9.68 ± 1.22^ab

图2 Western-blotting法测两组大鼠各时间点TGF-β1/Smads通道相关蛋白表达的比较

图3 两组大鼠各时间点上皮-间充质转变相关蛋白表达的比较

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Transforming growth factor-β1/Smads signaling pathway involves the development of paraquat-induced pulmonary endothlial-mesenchymal transition and fibrosis

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Transforming growth factor-β1/Smads signaling pathway involves the development of paraquat-induced pulmonary endothlial-mesenchymal transition and fibrosis