探讨中性粒细胞归巢及胞外诱捕网在大鼠呼吸机相关性肺炎（VAP）中的发病机制及针灸治疗作用。

将30只Sprague-Dawley大鼠分为对照组、模型组、针灸组、针灸+ CXCR4抑制剂组、针灸+中性粒细胞胞外诱捕网（NETs）抑制剂组，每组6只。对照组大鼠仅予气管暴露，保留自主呼吸；模型组大鼠麻醉后暴露气管并向气管中注入肺炎克雷伯杆菌；72 h后行经口气管插管接小动物呼吸机，每30分钟行2 ~ 3次大潮气量通气，每天连续通气5 h，持续2 d；针灸组大鼠于造模成功30 min后行针灸治疗；针灸+ CXCR4抑制剂组大鼠于第1天注射菌液前0.5 h按体质量以2.5 mg/kg的剂量腹腔注射CXCR4抑制剂；针灸+ NETs抑制剂组大鼠于机械通气前0.5 ~ 1.0 h按体质量0.8 μM/290 ~ 340 g的剂量鞘内注射NETs抑制剂。采用流式细胞术检测大鼠肺组织中性粒细胞变化趋势，苏木素-伊红（HE）染色观察肺组织病理变化，免疫组织化学染色法检测肺组织瓜氨酸化组蛋白H3（Cit-H3）、髓过氧化物酶（MPO）表达水平。

对照组、模型组、针灸组和针灸+ CXCR4抑制剂组大鼠CD11b + CD62L表达水平比较，差异有统计学意义（F = 25.150，P<0.001）。进一步两两比较发现，与对照组比较，模型组CD62L + CD11b表达水平明显升高（P<0.05）；与模型组比较，针灸+ CXCR4抑制剂组表达水平显著下降（P<0.05）。HE染色结果显示，对照组大鼠肺组织未见明显充血、出血、水肿及中性粒细胞浸润；模型组肺组织明显水肿，充血及出血明显，大量炎症细胞浸润；针灸组肺泡及气道黏膜充血、水肿减轻，炎症细胞浸润减少；针灸+ NETs抑制剂组肺泡及气道黏膜充血、水肿明显减轻，炎症细胞浸润明显减少。对照组、模型组、针灸组和针灸+ NETs抑制剂组大鼠肺损伤评分、Cit-H3和MPO表达水平比较，差异均有统计学意义（F = 51.723、227.370、570.097，P均<0.001）。进一步两两比较发现，与对照组比较，模型组Cit-H3、MPO表达水平均显著升高，肺组织炎症浸润更严重（P均<0.05）；与模型组比较，针灸组、针灸+ NETs抑制剂组中Cit-H3、MPO表达水平均显著降低，肺组织炎症浸润明显减轻且针灸+ NETs抑制剂组更为显著（P均<0.05）。

VAP大鼠模型中存在中性粒细胞归巢及NETs过度形成的现象；针灸对中性粒细胞归巢的抑制不明显，但可通过调控Cit-H3及MPO的表达抑制NETs的过度形成从而改善炎症反应；同时，针灸联用CXCR4抑制剂可明显改善中性粒细胞归巢，联用NETs抑制剂对NETs的调控效果更为显著，对肺组织炎症反应改善作用更强。

To explore the pathogenesis and acupuncture treatment effects of neutrophil homing and extracellular trapping net in rats with ventilator-associated pneumonia (VAP).

Thirty Sprague-Dawley rats were divided into a control group, a model group, an acupuncture group, an acupuncture + CXCR4 inhibitor group, and an acupuncture + neutrophil extracellular traps (NETs) inhibitor group, with six rats in each group. Rats in the control group were only exposed to the trachea and kept spontaneous breathing. In the model group, the trachea was exposed and Klebsiella pneumoniae was injected into the trachea after anesthesia. After 72 hours, the rats were intubated and connected with a small animal ventilator. High tidal volume ventilation was performed 2-3 times every 30 minutes for five hours daily for two days. Rats in the acupuncture group were treated with acupuncture after 30 minutes of successful modeling. Rats in the acupuncture + CXCR4 inhibitor group were intraperitoneally injected with CXCR4 inhibitor at a dose of 2.5 mg/kg based on body weight half an hour before bacterial injection on the first day. Rats in the acupuncture + NETs inhibitor group received intrathecal injection of NETs inhibitor at a dose of 0.8 μM/290-340 g based on body weight in 0.5-1.0 h before mechanical ventilation. Flow cytometry was used to detect the neutrophil trends in lung tissue, hematoxylin-eosin (HE) staining to observe the lung tissue pathology, and immunohistochemical staining to observe the citrullinated histone H3 (Cit-H3) and myeloperoxidase (MPO) expression levels.

The expression level of CD11b + CD62L in the control group, model group, acupuncture group and acupuncture + CXCR4 inhibitor group was significantly different (F = 25.150, P < 0.001). Further pairwise comparison showed that the expression level of CD62L + CD11b in the model group was significantly higher than that in the control group (P < 0.05). Compared with the model group, the expression level of CD62L + CD11b in the acupuncture + CXCR4 inhibitor group was significantly decreased (P < 0.05). HE staining showed that there was no obvious congestion, hemorrhage, edema, and neutrophil infiltration in the lung tissue of rats in the control group. Rats in the model group had obvious edema, hyperemia, and hemorrhage, with a large number of infiltrating inflammatory cells. The congestion and edema of alveoli and airway mucosa were reduced, and the infiltration of inflammatory cells was decreased in the acupuncture group. The congestion and edema of alveoli and airway mucosa and the infiltration of inflammatory cells were significantly reduced in the acupuncture + NETs inhibitor group. The lung injury score and the expression levels of Cit-H3 and MPO in the control group, model group, acupuncture group and acupuncture + NETs inhibitor group were compared, and their differences were statistically significant (F = 51.723, 227.370, 570.097; all P < 0.001). Further pairwise comparison showed that compared with the control group, the expression levels of Cit-H3 and MPO in the model group were significantly increased, and the inflammatory infiltration of lung tissue was more severe (all P < 0.05). Compared with the model group, the expression levels of Cit-H3 and MPO in the acupuncture group and acupuncture + NETs inhibitor group were significantly decreased, and the inflammatory infiltration in lung tissue was significantly reduced, especially in the acupuncture + NETs inhibitor group (all P < 0.05).

There are neutrophil homing and excessive formation of NETs in rats with VAP. Acupuncture has no obvious inhibitory effect on neutrophil homing, but it can inhibit the excessive formation of NETs by regulating the expression of Cit-H3 and MPO, thereby reducing the inflammatory response. At the same time, acupuncture combined with CXCR4 inhibitor can significantly improve the homing of neutrophils, and their combination of NETs inhibitor has a more significant regulatory effect on NETs and a stronger improvement effect on the inflammatory response of lung tissue.