白芍总苷对脂多糖诱导的小鼠急性肺损伤的保护作用及机制

Effect and mechanism of total glucosides of paeony protect mice from lipopolysaccharide-induced acute lung injury

摘要

目的  探讨白芍总苷（total glucosides of paeony, TGP）治疗急性肺损伤小鼠的效果及机制。方法  24只雌性C57小鼠采用数字表法随机分为4组：对照组、脂多糖（lipopolysaccharide, LPS）模型组、白芍总苷组（LPS+TGP）和地塞米松（dexamethasone, DEX）组(LPS+DEX)，每组6只。采用LPS（4 ng/kg）经鼻吸入，连续5 d建立小鼠急性肺损伤模型；LPS+TGP组、LPS+DEX组分别给予50 mg·kg^-1·d^-1 TGP和5 mg·kg^-1·d^-1 DEX灌胃；观察记录各组小鼠的体质量变化；取肺组织做苏木精-伊红(hematoxylin-eosin, HE)染色观察肺病理损伤情况；流式细胞术检测外周血、脾脏、肺脏组织树突状细胞（dendritic cell, DC）、中性粒细胞和巨噬细胞比例变化，肺脏组织M1型、M2型巨噬细胞的比例变化；酶联免疫吸附试验（enzyme linked immunosorbent assay, ELISA）分析支气管肺泡灌洗液中白细胞介素-1β（interleukin-1β, IL-1β）和白细胞介素-6（interleukin-6, IL-6）浓度；实时荧光定量聚合酶链反应（real time quantitative polymerase chain reaction, RT-qPCR）检测肺组织IL-1β、IL-6和肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）mRNA表达水平；蛋白印迹法（Western blotting）检测核转录因子-κB p65（nuclear transcription factor-κB p65, NF-κB p65）和磷酸化型核转录因子-κB p65（phospho-nuclear factor-κB p65, pNF-κB p65）蛋白含量。结果  TGP和DEX可明显改善LPS引起的小鼠体质量减轻；HE染色显示LPS组小鼠肺脏组织炎性细胞浸润、肺泡间隔破坏和肺泡间质增厚，LPS+TGP组、LPS+DEX组小鼠肺部病理改变程度较LPS组减轻。外周血流式细胞结果显示：与LPS组相比，LPS+TGP组、LPS+DEX组中性粒细胞比例明显降低，DC细胞和巨噬细胞比例无明显改变；脾脏组织流式细胞结果显示：与LPS组相比，LPS+TGP组、LPS+DEX组小鼠中性粒细胞、DC细胞和巨噬细胞比例均显著下降,肺组织流式细胞分析结果显示：与LPS组相比，LPS+TGP组、LPS+DEX组肺组织巨噬细胞比例无明显改变，M1型细胞比例显著下降，M2型巨噬细胞比例升高,中性粒细胞比例下降。ELISA结果显示：与LPS组相比，支气管肺泡灌洗液中IL-6、IL-1β水平明显下降。RT-qPCR结果显示：与LPS组相比，LPS+TGP组和LPS+DEX组肺组织IL-1β、IL-6和TNF-α mRNA表达水平显著下降；Western blotting结果显示：与LPS组相比，LPS+TGP组和LPS+DEX组NF-κB p65蛋白表达无明显改变，而pNF-κB p65蛋白表达较LPS组明显降低。结论  TGP改善LPS诱导的急性肺损伤，具体机制可能与其降低外周血、肺脏中性粒细胞和脾脏炎症细胞比例，抑制肺巨噬细胞向M1型极化，抑制NF-κB炎症信号通路激活和炎症因子释放有关。

Abstract

Objective  To investigate the effect and mechanism of total glucosides of paeony (TGP) in the treatment of acute lung injury in mice. Methods  Twenty-four female C57 mice were randomly divided into 4 groups： control group, lipopolysaccharide (LPS) model group, TGP group (LPS+TGP), and dexamethasone (DEX) group (LPS+DEX), with 6 mice in each group. Acute lung injury model was established by nasal inhalation of LPS (4 ng/kg) for 5 consecutive days. The LPS+TGP and LPS+DEX groups were given 50 mg·kg^-1·d^-1 TGP and 5 mg·kg^-1·d^-1 DEX by gavage, respectively. The changes in body weight of the mice in each group were observed and recorded. Lung tissue was taken for hematoxylin-eosin (HE) staining to observe lung pathological damage. Flow cytometry was used to detect the changes in the proportions of dendritic cells (DC), neutrophils, and macrophages in peripheral blood, spleen, and lung tissues, as well as the proportions of M1 and M2 macrophages in lung tissues. Enzyme linked immunosorbent assay (ELISA) was used to analyze the levels of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in bronchoalveolar lavage fluid. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of IL-1β, IL-6 and tumor necrosis factor-α (TNF-α) in lung tissue. Western blotting was used to detect the nuclear transcription factor-κB p65 (NF-κB p65) and phospho-NF-κB p65 (pNF-κB p65) protein content. Results  TGP and DEX significantly improved the weight loss caused by LPS. HE staining showed that the lung tissue of mice in the LPS group had inflammatory cell infiltration, alveolar septal destruction, and alveolar interstitial thickening, which were mitigated in the LPS+TGP and LPS+DEX groups. The results of flow cytometry of peripheral blood showed that, compared with the LPS group, the proportion of neutrophils in the LPS+TGP and LPS+DEX groups was significantly decreased, while the proportions of DC and macrophages did not change significantly. The results of flow cytometry of spleen tissues showed that, compared with the LPS group, the proportions of neutrophils, DC, and macrophages in the LPS+TGP and LPS+DEX groups were significantly decreased. The results of flow cytometry of lung tissues showed that, compared with the LPS group, the proportion of macrophages in the LPS+TGP and LPS+DEX groups did not change significantly, the proportion of M1 cells decreased significantly, the proportion of M2 macrophages increased, and the proportion of neutrophil is down. ELISA results showed that, compared with the LPS group, the levels of IL-6 and IL-1β in bronchoalveolar lavage fluid were significantly decreased； RT-qPCR results showed that, compared with the LPS group, the expression levels of IL-1β, IL-6 and TNF-α mRNA in lung tissues of the LPS+TGP and LPS+DEX groups were significantly reduced. Western blotting results showed that, the protein expression of NF-κB p65 did not change significantly in the LPS+TGP and LPS+DEX groups compared with the LPS group, and the protein content of pNF-κB p65 was significantly decreased. Conclusion  TGP can improve LPS-induced acute lung injury, which may be related to the reduction of the proportion of peripheral blood neutrophils, lung neutrophils and spleen inflammatory cells, the inhibition of M1 polarization of lung macrophages, the inhibition of NF-κB activation, and the reduction of inflammatory signaling pathway and inflammatory factor release. 

关键词

急性肺损伤 / 白芍总苷 / 炎症 / 巨噬细胞

Key words

acute lung injury / total glucosides of paeony / inflammation / macrophage