二脒那秦通过内源性激活ACE2抑制AngⅡ-TGF-β1通路缓解小鼠肺纤维化

doi:10.11843/j.issn.0366-6964.2023.06.039

摘要/Abstract

摘要： 本试验通过博来霉素诱导建立小鼠肺纤维化损伤模型,探究二脒那秦(diminazene aceturate, DIZE)内源性激活血管紧张素转化酶2(angiotensin-converting enzyme 2, ACE2)对小鼠肺纤维化发生发展的缓解作用。将24只雄性ICR小鼠随机均分为对照组(CON组)、模型组(MOD组)和二脒那秦处理组(DIZE组)。除CON组外,其余2组小鼠一次性气管内注射博来霉素(5 mg·kg^-1)建立肺纤维化损伤模型。造模1 d后,DIZE组小鼠给予DIZE[15 mg·(kg·d)^-1]灌胃处理,CON组和MOD组小鼠同等剂量灌胃生理盐水。连续灌胃28 d,采血后,处死所有小鼠,取肺组织,进行如下试验:1)肺组织称重,计算小鼠肺系数;HE和Masson染色观察肺病理组织学变化;碱水解法检测肺组织中羟脯氨酸的含量;2)间接ELISA法检测血清中Ang Ⅱ和Ang 1-7含量;3)RT-qPCR及Western blot法检测肺组织中纤维化相关因子基因和蛋白等的表达水平。结果表明:1)与CON组相比,MOD组小鼠肺系数及羟脯氨酸含量极显著或显著升高(P<0.01 & P<0.05);肺组织出现明显病理变化,主要表现肺泡塌陷,炎性细胞浸润,肺泡间隔充血、增厚,有大量胶原纤维沉积。经DIZE处理后肺系数及羟脯氨酸含量显著或极显著降低(P<0.05 & P<0.01);肺纤维化程度明显减轻;2)与CON组相比,MOD组小鼠肺组织中ACE2蛋白表达下调,血清中Ang Ⅱ含量极显著升高(P<0.01),Ang 1-7含量极显著降低(P<0.01);DIZE处理逆转了以上变化;3)与CON组相比,MOD组小鼠肺组织中Col1a1和Col3a1基因表达极显著上调(P<0.01),α-SMA及TGF-β1基因和蛋白表达均极显著上调(P<0.01),上皮细胞的特征蛋白E-cadherin蛋白表达极显著下调(P<0.01);与MOD组相比,DIZE组缓解了以上变化。一次性气管内注射博来霉素,28 d后可成功诱导小鼠肺纤维化损伤,高水平的Ang Ⅱ 可能协同TGF-β1参与了小鼠的肺纤维化;DIZE可能通过内源性激活ACE2,降低Ang Ⅱ的高水平,对小鼠的肺纤维化具有一定的缓解作用。

关键词: 小鼠, 二脒那秦, 血管紧张素转化酶2, Ang Ⅱ-TGF-β1通路, 肺纤维化

Abstract: This study aims at investigating the alleviating effect of diazene aceturate (DIZE) on the development of mouse pulmonary fibrosis by endogenously activating Angiotensin-converting enzyme 2 (ACE2). Bleomycin induced pulmonary fibrosis injury model in mice was established for this aim. Twenty-four male ICR mice were randomly divided into control group (CON group), model group (MOD group) and DIZE group (DIZE group). Mice in the two groups other than CON group were injected with bleomycin (5 mg·kg^-1) intratracheal only once to establish pulmonary fibrosis injury model. One day after establishing the model, mice in DIZE group were given DIZE (15 mg·(kg·d)^-1) gavage, and mice in CON group and MOD group were given gavage with normal saline at the same dose. After continuous intragastric administration for 28 days, all mice were executed, serum and lung tissue were taken for the following tests: 1) Lung tissue was weighed and lung coefficient was calculated; HE and Masson staining were used to observe the pulmonary histological changes; the content of hydroxyproline in lung tissue was determined by alkaline hydrolysis; 2) The contents of Ang Ⅱ and Ang 1-7 in serum were determined by indirect ELISA; 3) The expression levels of fibrosis related factor genes and proteins in lung tissues were detected by qPCR and Western blot. Results showed that: 1) Compared with CON group, lung coefficient and hydroxyproline content in MOD group increased significantly (P<0.01 & P<0.05). The lung tissue showed obvious pathological changes, mainly manifested as alveolar collapse, inflammatory cell infiltration, alveolar septum congestion and thickening, and large amount of collagen fiber deposition. After DIZE treatment, the lung coefficient and hydroxyproline content decreased significantly (P<0.01 & P<0.05); The degree of pulmonary fibrosis reduced significantly; 2) Compared with CON group, the expression of ACE2 protein in lung tissue of MOD group was down-regulated, and the content of Ang Ⅱ in serum increased significantly (P<0.01), the content of Ang 1-7 decreased significantly (P<0.01); DIZE group showed a reverse on the changes happened in MOD group ; 3) Compared with CON group, the expressions of Col1a1 and Col3a1 genes in the lung tissues of MOD group were significantly up-regulated (P<0.01), α-SMA and TGF-β1 gene and protein expressions were significantly up-regulated (P<0.01), the expression of the characteristic protein E-cadherin in epithelial cells was significantly down-regulated (P<0.01); Compared with the MOD group, the DIZE group mitigated the above changes. After 28 days of one-time intratracheal injection of bleomycin, pulmonary fibrosis injury was successfully induced in mice. The production of Ang Ⅱ and the increased expression of TGF-β1 are the major factors of bleomycin-induced pulmonary fibrosis in mice. DIZE may reduce the high level of Ang Ⅱ by activating the expression of ACE2 and thus has a substantial relieving effect on pulmonary fibrosis in mice.

Key words: mice, diminazene aceturate, angiotensin converting enzyme, AngⅡ-TGF-β1 pathway, pulmonary fibrosis

中图分类号:

S852.2

曹西月, 纪晓霞, 张崇昊, 张亚峰, 陈雨涛, 张源淑. 二脒那秦通过内源性激活ACE2抑制AngⅡ-TGF-β1通路缓解小鼠肺纤维化[J]. 畜牧兽医学报, 2023, 54(6): 2631-2640.

CAO Xiyue, JI Xiaoxia, ZHANG Chonghao, ZHANG Yafeng, CHEN Yutao, ZHANG Yuanshu. Diminazene Aceturate Alleviates Pulmonary Fibrosis in Mice by Endogenous Activation of ACE2 to Inhibit AngⅡ-TGF-β1 Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2631-2640.

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