补中益气汤基于ACE2-Ang(1-7)-Mas轴减轻CIH诱发的肺脏炎症损伤

Buzhong Yiqitang Reduces CIH-induced Pulmonary Inflammatory Injury by ACE2-Ang(1-7)-Mas Axis

目的:探讨补中益气汤对慢性间歇性低氧(CIH)小鼠肺脏炎症的干预作用,并初步阐明其作用机制。方法:将40只健康的6~8周龄雄性C57BL/6小鼠随机分成:常氧组、模型组(CIH)和补中益气汤低、中、高剂量组。常氧组暴露于常氧环境中,模型组与补中益气汤低、中、高剂量组暴露于间歇性低氧环境中。其中,补中益气汤低、中、高剂量每日放入低氧舱前30 min给予补中益气汤灌胃(低、中、高剂量分别为8.1、16.2、32.4 g·kg^(-1)·d-1),而模型组与常氧组给予同等体积生理盐水。造模5周后,应用EMKA型动物肺功能仪检测小鼠肺功能,肺功能检测完毕后取材。应用苏木素-伊红(HE)染色观察各组小鼠肺脏的病理学改变;酶联免疫吸附测定法(ELISA)检测血清中白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)和肺组织中血管紧张素Ⅱ(AngⅡ)、血管紧张素(1-7)[Ang(1-7)]的含量;蛋白免疫印迹法(Western blot)及免疫组化检测IL-6、IL-8、TNF-α、血管紧张素转化酶2(ACE2)、线粒体组装受体(Mas)蛋白的表达。结果:与常氧组比较,模型组小鼠肺功能出现明显异常(P<0.05,P<0.01),肺组织出现肺泡壁增厚、炎性细胞浸润等改变;血清中IL-6、IL-8、TNF-α和肺组织中AngⅡ的含量显著上升(P<0.01),Ang(1-7)含量显著下降(P<0.01),IL-6、IL-8、TNF-α蛋白表达显著升高、ACE2、Mas蛋白表达明显下降(P<0.05,P<0.01)。与模型组比较,中药组小鼠肺功能有所改善(P<0.05,P<0.01),肺脏HE染色可见肺泡壁厚度大致正常,炎性细胞浸润减少;免疫组化和Western blot检测炎症相关蛋白表达明显下降(P<0.05,P<0.01),ACE2、Mas蛋白表达明显升高(P<0.05,P<0.01)。结论:补中益气汤可通过调节ACE2-Ang(1-7)-Mas轴抑制炎症反应改善CIH暴露后小鼠的肺脏损伤。

Objective:To investigate the intervention effect of Buzhong Yiqitang(BZYQT)on pulmonary inflammation in mice induced by chronic intermittent hypoxia(CIH)and preliminarily elucidate its mechanism.Method:Forty healthy male C57BL/6 mice aged 6-8 weeks were randomly divided into the following groups:normoxia group,model group(exposed to CIH),and low-,medium-,and high-dose BZYQT groups.The normoxia group was exposed to a normoxic environment,while the model group and the low-,medium-,and high-dose BZYQT groups were exposed to intermittent hypoxia.In the BZYQT groups,the BZYQT(8.1,16.2,32.4 g·kg^(-1)·d-1)was administered orally 30 min before placing the mice in the hypoxic chamber,while the model group and the normoxia group received an equivalent volume of normal saline.After five weeks of modeling,pulmonary function of the mice was measured using an EMKA animal lung function analyzer,and lung tissue samples were collected after the pulmonary function tests.Hematoxylin-eosin(HE)staining was performed to observe the histopathological changes in the lung tissue of each group.Enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of interleukin-6(IL-6),interleukin-8(IL-8),tumor necrosis factor-α(TNF-α)in the serum,as well as angiotensinⅡ(AngⅡ)and angiotensin-(1-7)[Ang(1-7)]in lung tissue.Western blot and immunohistochemistry were used to detect the protein expression of IL-6,IL-8,TNF-α,angiotensin-converting enzyme 2(ACE2),and mitochondrial assembly receptor(Mas).Result:Compared with the normoxia group,the model group showed significant abnormalities in lung function(P<0.05,P<0.01),lung tissue changes,such as thickening of alveolar walls and inflammatory cell infiltration,increased levels of IL-6,IL-8,TNF-αin the serum and AngⅡin lung tissue(P<0.01),decreased level of Ang(1-7)(P<0.01),increased protein expression of IL-6,IL-8,and TNF-α,and decreased protein expression of ACE2 and Mas(P<0.05,P<0.01).Compared with the model group,the BZYQT groups showed improvement in lung function(P<0.05,P<0.01),and HE staining of lung tissue showed approximately normal alveolar wall thickness and reduced inflammatory cell infiltration.Immunohistochemistry and Western blot analysis showed a significant decrease in the expression of inflammatory-related proteins(P<0.05,P<0.01),and a significant increase in ACE2 and Mas protein expression(P<0.05,P<0.01).Conclusion:BZYQT can improve lung injury in mice exposed to CIH by regulating the ACE2-Ang(1-7)-Mas axis to inhibit inflammatory responses.