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.