目的 探讨宣肺败毒方（Xuanfei Baidu Formula，XFBD）来源小RNA（small RNA，sRNA）通过靶向血管紧张素转换酶（angiotensin-converting enzyme，ACE）对脂多糖（lipopolysaccharides，LPS）诱导小鼠急性肺损伤（acute lung injury，ALI）的保护作用及机制。方法 采用改良CTAB法提取XFBD的sRNA并建立文库，预测筛选靶向ACE的sRNA。通过双荧光素酶报告系统验证其靶向性，并在人肺微血管内皮细胞（human pulmonary microvascular endothelial cells，HPMEC）中筛选可抑制ACE表达的sRNA。采用ELISA、Western blotting和qRT-PCR检测sRNA对血管紧张素Ⅱ（angiotensin Ⅱ，AngⅡ）生成、核因子-κB抑制蛋白α（inhibitor of nuclear factor-κB α，IκBα）及炎症因子表达的影响。建立LPS诱导的ALI小鼠模型，设置对照组、模型组、卡托普利（10 mg/kg）组、XFBD（9.2 g/kg）组、NC-sRNA（10 nmol/只）组、ACE-sRNA-1（10 nmol/只）组和ACE-sRNA-26（10 nmol/只）组，每组6只。采用苏木素-伊红（hematoxylin-eosin，HE）染色和Micro-CT评估肺组织病理与影像学变化；检测外周血中白细胞数、中性粒细胞数及支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）中总蛋白浓度、白细胞、中性粒细胞、淋巴细胞数量；免疫组化法检测肺组织中上皮型钙黏蛋白（vascular endothelial-cadherin，VE-cadherin）和细胞间黏附分子-1（intercellular adhesion molecule-1，ICAM-1）表达；Western blotting和qRT-PCR检测肺组织ACE- Ang Ⅱ-Ang Ⅱ 1型受体（AngⅡ type 1 receptor，AT1R）通路、IκBα及炎症因子表达；ELISA检测血清中Ang II和炎症因子水平。结果 从XFBD中筛选出50条潜在靶向ACE的sRNA，经验证26条具有靶向性，其中12条显著抑制HPMEC细胞中ACE表达（P＜0.05、0.01、0.001）。7条sRNA显著抑制Ang Ⅱ生成，以ACE-sRNA-1/26作用最显著（P＜0.001），并可抑制IκBα蛋白及炎症因子表达（P＜0.01、0.001）。动物实验中，模型组小鼠肺损伤严重（P＜0.001）；与模型组比较，ACE-sRNA-1和ACE-sRNA-26显著改善小鼠肺损伤（P＜0.05、0.01、0.001），上调肺组织VE-cadherin表达（P＜0.001），下调肺组织ICAM-1、ACE、AT1R、IκBα和炎症因子表达（P＜0.05、0.01、0.001），同时降低血清Ang II和炎症因子水平（P＜0.05、0.01、0.001）。结论 XFBD来源的ACE-sRNA-1和ACE-sRNA-26能够靶向抑制ACE表达及活性，减轻肺血管内皮炎症和屏障损伤，改善LPS诱导的小鼠ALI，其机制可能与调控ACE-Ang Ⅱ-AT1R通路有关。

Objective To investigate the protective effect and mechanism of small RNAs (sRNA) derived from Xuanfei Baidu Formula (宣肺败毒方, XFBD) targeting angiotensin-converting enzyme (ACE) in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Methods The modified CTAB method was employed to extract sRNA from XFBD and a library was constructed, followed by prediction and screening of sRNAs targeting ACE. The targeting specificity was verified using a dual-luciferase reporter system, and sRNAs capable of suppressing ACE expression were screened in human pulmonary microvascular endothelial cells (HPMEC). The effects of sRNA on angiotensin Ⅱ (Ang II) generation, inhibitor of inhibitor of nuclear factor-κB α (IκBα) and inflammatory cytokine expressions were examined by ELISA, Western blotting and qRT-PCR. An LPS-induced ALI mice model was established, control group, model group, captopril (10 mg/kg) group, XFBD (9.2 g/kg) group, NC-sRNA (10 nmol/animal) group, ACE-sRNA-1 (10 nmol/animal) group and ACE-sRNA-26 (10 nmol/animal) group were set up, with six mice in each group. Hematoxylin-eosin (HE) staining and Micro CT were used to evaluate the pathological and imaging changes of lung tissue; The number of white blood cells and neutrophils in peripheral blood, as well as the total protein concentration, white blood cells, neutrophils and lymphocytes numbers in bronchoalveolar lavage fluid (BALF) were detected; Immunohistochemistry was used to detect the expressions of vascular endothelial-cadherin (VE-cadherin) and intercellular adhesion molecule-1 (ICAM-1) in lung tissue; Western blotting and qRT-PCR were used to detect the expressions of ACE-Ang Ⅱ-Ang Ⅱ type 1 receptor (AT1R) pathway, IκBα and inflammatory factors in lung tissue; ELISA was used to detect the levels of Ang II and inflammatory factors in serum. Results A total of 50 potential ACE-targeting sRNAs were screened from XFBD, with 26 sRNAs validated to exhibit targeting effects, among which 12 sRNAs significantly inhibited ACE expression in HPMEC cells (P < 0.05, 0.01, 0.001). Seven sRNAs significantly suppressed Ang II generation, with ACE-sRNA-1/26 demonstrating the most potent effect (P < 0.001), inhibited IκBα protein and inflammatory factor expressions (P < 0.01, 0.001). In animal experiments, mice in model group had severe lung injury (P < 0.001); Compared with model group, ACE-sRNA-1 and ACE-sRNA-26 significantly improved lung injury in mice (P < 0.05, 0.01, 0.001), up-regulated VE-cadherin expression in lung tissue (P < 0.001), down-regulated ICAM-1, ACE, AT1R, IκBα and inflammatory factor expressions in lung tissue (P < 0.05, 0.01, 0.001), and reduced Ang II and inflammatory factor levels in serum (P < 0.05, 0.01, 0.001). Conclusion ACE-sRNA-1 and ACE-sRNA-26 derived from XFBD could target the inhibition of ACE expression and activity, alleviate pulmonary endothelial inflammation and barrier damage, and improve LPS-induced ALI in mice. The mechanism may be related to the regulation of ACE-Ang Ⅱ-AT1R pathway.

R285.5

天津市教委科研计划项目（2023KJ139）