盐酸氨溴索治疗大鼠肺纤维化的作用研究及其对TGF-β/Smad/ERK信号转导通路和Th17/Treg细胞失衡的影响

doi:10.12092/j.issn.1009-2501.2018.11.001

中国临床药理学与治疗学 ›› 2018, Vol. 23 ›› Issue (11): 1201-1208.doi: 10.12092/j.issn.1009-2501.2018.11.001

• 基础研究 • 下一篇

盐酸氨溴索治疗大鼠肺纤维化的作用研究及其对TGF-β/Smad/ERK信号转导通路和Th17/Treg细胞失衡的影响

马远东¹，黄祖伟²，安培³，谢晓菲³，刘小彩¹，毛凌哲¹

¹温州康宁医院检验科，温州 325000，浙江；²宁波大学医学院附属医院呼吸科，宁波 315211，浙江；³宁波大学医学院药物研究重点实验室，宁波 315211，浙江

收稿日期:2018-07-22 修回日期:2018-09-25 出版日期:2018-11-26 发布日期:2018-11-22
作者简介:马远东，女，本科，主管检验师，研究方向：临床检验。 Tel: 15258625302 E-mail: ycm198002@163.com
基金资助:
浙江省自然科学基金（CR874750X489）

Effects of ambroxol hydrochloride on pulmonary fibrosis in rats and its effect on TGF-β/Smad/ERK signal transduction pathway and Th17/Treg cell imbalance

MA Yuandong¹, HUANG Zuwei², AN Pei ³, XIE Xiaofei ³, LIU Xiaocai ¹, MAO Lingzhe ¹

¹ Department of Laboratory Medicine, Wenzhou Kangning Hospital, Wenzhou 325000, Zhejiang, China; ² Department of Respiratory Medicine, the Affiliated Hospital of Medical School of Ningbo University, Ningbo 315211, Zhejiang, China; ³Key Laboratory of Medical School of Ningbo University, Ningbo 315211, Zhejiang, China

Received:2018-07-22 Revised:2018-09-25 Online:2018-11-26 Published:2018-11-22

摘要/Abstract

摘要：

目的: 分析盐酸氨溴索治疗大鼠肺纤维化的作用研究及其对TGF-β/Smad/ERK信号转导通路和Th17/Treg细胞失衡的影响。方法: 选取由温州康宁医院实验动物中心提供SPF级Wistar雄性大鼠30只，随机数字表法分成5组：正常组，模型组，氨溴索低、中、高剂量组，每组各10只。采用5 mg/kg博来霉素0.2 mL气管内灌注成功制备肺纤维化模型，术后第2天起氨溴索低、中、高剂量组大鼠腹腔内注射剂量为10、20、40 mg/kg盐酸氨溴索，正常组和模型组大鼠腹腔注射剂量为3.33 mL/kg生理盐水，连续注射14 d。HE染色和Masson染色观察各组大鼠组织病理变化，ELISA法检测大鼠成纤维细胞内转化生长因子β1（TGF-β1）含量，实时荧光定量PCR检测TGF-β1蛋白表达状况，Western blot检测成纤维细胞内ERK1/2、p-ERK和Smad3、Smad4、Smad7蛋白表达状况，流式细胞术法检测各组大鼠肺组织内Treg、Th17细胞表达状况。结果: 与正常组相比，模型组大鼠成纤维细胞内TGF-β1含量、TGF-β1基因表达量、ERK1/2、p-ERK和Smad3、Smad4蛋白表达、肺组织内Th17、Th17/Treg均升高（均P＜0.05）；与模型组相比，氨溴索中、高剂量组大鼠均降低（均P＜0.05）。而模型组大鼠成纤维细胞Smad7蛋白表达、肺组织内Treg表达比率较正常组均降低（均P＜0.05），氨溴索中、高剂量组大鼠较模型组升高（均P＜0.05）。结论: 盐酸氨溴索可维持肺纤维化大鼠肺组织Th17/Treg平衡，抑制大鼠TGF-β/Smad/ERK信号转导通路内主要细胞因子信号传递，进而起到抗肺纤维化的作用。

关键词: 肺纤维化, TGF-β/Smad/ERK信号转导通路, Th17/Treg细胞, 盐酸氨溴索

Abstract:

AIM: To study the effect of ambroxol hydrochloride on pulmonary fibrosis in rats and its effect on TGF-β/Smad/ERK signal transduction pathway and the imbalance of Th17/Treg cells. METHODS: Thirty male SPF-level Wistar male rats were selected from the Experimental Animal Center of our hospital and were randomly divided into five groups: normal group (10), model group (10), ambroxol low-dose group (10), ambroxol middle-dose(10) and ambroxol high-dose (10) goups. Pulmonary fibrosis model was prepared with 0.2 mL intratracheal perfusion of bleomycin (5 mg/kg). Rats in the low, medium and high dose groups were intraperitoneally injected with 10, 20, 40 mg/kg ambroxol hydrochloride from the first day after operation. Rats in the normal group and the model group were intraperitoneally injected with 3.33 mL/kg normal saline for 14 days. HE staining and Masson staining were performed to observe pathological changes. ELISA was applied to detect TGF-β1. The expression of TFG-β1 in fibroblasts was detected by real-time fluorescent quantitative PCR. The expressions of ERK1/2, p-ERK, Smad3, Smad4 and Smad7 in fibroblasts were detected by Western blot. The expressions of Treg and Th17 cells in the lung tissue of each group were detected by flow cytometry. RESULTS: Compared with normal group, content of TGF-β1 in the fibroblasts, expression of TGF-β1 gene in fibroblasts, expression of ERK1/2, p-ERK, Smad3 and Smad4 protein, and Th17 and Th17/Treg in the lung tissue of the model group were higher than those in the normal group (P<0.05), and those of the middle and high doses of ambroxol were lower than the model group (P<0.05). While the expression of Smad7 protein and Treg cell in the lung tissue in the model group was significantly higher than that in the normal group (P<0.05), and those of the middle and high doses of ambroxol were higher than the model group (P<0.05). CONCLUSION: Ambroxol hydrochloride has anti-pulmonary fibrosis effect by maintaining the balance of Th17/Treg in lung tissue of rats with pulmonary fibrosis and inhibiting the signal transduction of the major cytokines in the TGF-β/Smad/ERK signal transduction pathway in rats.

Key words: pulmonary fibrosis, TGF-β/Smad/ERK signal transduction pathway, Th17/Treg cells, ambroxol hydrochloride

中图分类号:

马远东，黄祖伟，安培，谢晓菲，刘小彩，毛凌哲. 盐酸氨溴索治疗大鼠肺纤维化的作用研究及其对TGF-β/Smad/ERK信号转导通路和Th17/Treg细胞失衡的影响[J]. 中国临床药理学与治疗学, 2018, 23(11): 1201-1208.

MA Yuandong, HUANG Zuwei, AN Pei, XIE Xiaofei, LIU Xiaocai, MAO Lingzhe. Effects of ambroxol hydrochloride on pulmonary fibrosis in rats and its effect on TGF-β/Smad/ERK signal transduction pathway and Th17/Treg cell imbalance[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2018, 23(11): 1201-1208.

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盐酸氨溴索治疗大鼠肺纤维化的作用研究及其对TGF-β/Smad/ERK信号转导通路和Th17/Treg细胞失衡的影响

Effects of ambroxol hydrochloride on pulmonary fibrosis in rats and its effect on TGF-β/Smad/ERK signal transduction pathway and Th17/Treg cell imbalance

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