One-lung ventilation (OLV) is a special mode of mechanical ventilation for thoracic surgery; however, it inevitably produces some adverse effects while allowing both lungs to be ventilated separately and providing convenient conditions for surgery. In clinical anesthesia work, OLV-induced lung injury has received increasing attention, but the pathogenesis of OLV-induced lung injury has not been elucidated. In recent years, apoptosis plays an important role in lung tissue injury, and some studies have suggested the occurrence of alveolar epithelial cell apoptosis at the early stage of mechanical ventilation [1], while studies on apoptosis in lung tissue during OLV have been rarely reported. The aim of this experiment was to study the expression of apoptotic index and apoptosis execution factor Caspase-3 in lung tissue during OLV and to analyze the relationship between their changes and lung injury caused by OLV. Huang Bing, Department of Anesthesiology, Cancer Hospital, Guangxi Medical University
Materials and methods
Animal groups Forty-two healthy SD male rats, weighing 200-250 g (provided by the Experimental Animal Center of Guangxi Medical University). The rats were divided into blank control group (group C), double-lung ventilation control group (group B), and single-lung ventilation group (group A). group C was the spontaneous breathing group, and groups A and B were divided into three subgroups: group A1 (OLV 0.5h, resumption of ventilation 0.5h), group A2 (OLV 1h, resumption of ventilation 0.5h), group A3 (OLV 1.5h, resumption of ventilation 0.5h) and group B1 (double-lung ventilation 1h), group B2 (1.5h of double lung ventilation), and group B3 (2h of double lung ventilation). The rats were randomly assigned, six rats in each group, seven groups in total.
Model preparation Group A rats were anesthetized with ketamine 80mg/kg, and Simeon II 0.6~0.8ml/kg intramuscularly, tracheotomy was performed and a homemade tracheal tube was inserted to the main trachea, vecuronium bromide was given 2mg/kg, and when the respiration became faint, a ventilator (Jiangwan type I miniature animal ventilator) was connected for mechanical ventilation. Tidal volume (VT): 10 ml/kg, respiratory rate (RR): 60 times/min. The chest was opened at the fifth intercostal space on the left side, the left lung was exposed, the tracheal tube was inserted too deeply into the right bronchus, the ventilation parameters were adjusted VT: 5-6 ml/kg, RR: 80 times/min. In group B, after exposure of the left lung, both lungs were kept ventilated until the scheduled time.
Specimens were collected from group C rats after anaesthesia and were rapidly discharged from the carotid artery, while in groups A and B, the animals were discharged from the carotid artery immediately after ventilation to a predetermined time. The remaining tissues of the lower lobe of the left lung were taken and stored at -80℃ in an ultra-low temperature refrigerator for examination.
Detection items and methods The morphological and structural changes of lung tissues were observed under HE staining light microscopy; the expression of Caspase-3 protein in lung tissues was detected by immunoblotting, and the images of Western blot reaction protein bands after film scanning were analyzed by Image Lab gel image analysis system, and the grayscale values of the target bands were calculated for each group of target protein bands and the internal reference The ratio of the grayscale value of each target protein band to the grayscale value of the internal reference GAPDH protein band was calculated; the apoptotic index of lung tissue was detected by in situ end-labeling (TUNEL) staining (10 unduplicated fields were selected for each section, and the positive cells were stained with brownish yellow nuclei.
Statistical analysis Statistical analysis was performed by SPSS13.0 software package, and the measurement data were expressed as mean ± standard deviation (x(_)±s). One-way ANOVA was used for comparison among subgroups, and LSD-t test was used for two-way comparison between groups. The t-test was used to compare samples between corresponding subgroups; P < 0.05 was considered a statistically significant difference.
Results
The thickening of interstitium was obvious.
TUNEL staining No apoptotic cells were seen in the lung tissue of group C; a small number of apoptotic cells were seen in the lung tissue of group B at 2 h of ventilation in both lungs. The apoptotic index was significantly higher in the A3 group compared with the B3 group (P < 0.05).
Caspase-3 protein content in lung tissue Compared with group C, there was no significant change in Caspase-3 protein content in group A1 (P > 0.05), and the expression increased in groups A2 and A3 (P < 0.05), and with the extension of ventilation time, group C = group A1 < group A2 < group A3 (P < 0.05); compared with the subgroups of group B, the difference between groups A1 and B1 was not statistically Compared with the subgroups of group B, the differences between groups A1 and B1 were not statistically significant (P＞0.05), and the expression in groups A2 and A3 increased compared with the corresponding groups B2 and B3 (P＜0.05).
Table 1 Comparison of apoptosis index and Caspase-3 protein expression in rat lung tissue between groups (n=6,`x±s)
Detection index
Group C
Group A1
Group A2
Group A3
Group B1
Group B2
Group B3
AI(%)
0.12±0.08
0.13±0.08
6.17±0.75 ab
16.00±1.10 ab
0.15±0.10
0.18±0.08
4.83±0.75 a
Caspase-3
0.21±0.02
0.22±0.02
0.34±0.01ab
0.69±0.01ab
0.22±0.01
0.23±0.01
0.28±0.01a
aP<0.05 compared with group C; bP
DISCUSSION
OLV as a special mechanical ventilation is now widely used in thoracic surgery, but venous blood adulteration during OLV due to insufficient oxygenation of the blood in the non-ventilated side of the lung causes lung tissue hypoxia and leads to lung tissue cell injury as well as functional impairment, in addition, post-atrophic lung retension and excessive traction during retension ventilation can cause a series of reactions that can aggravate and The experimental results showed that with the prolongation of OLV, the lung tissues of rats gradually showed the destruction of alveolar structure, congestion and edema, and gradual thickening of the interstitial space, which is consistent with the results of the previous study [3].
OLV-induced lung injury involves a variety of complex injury mechanisms, including mechanical and biological injuries. Many studies in recent years have suggested that various factors are involved in the process of acute lung injury by inducing excessive apoptosis of lung tissue cells leading to structural destruction of alveoli and aggravating damage to alveolar capillary membranes [4]. In studies of mechanical ventilation, it is mainly believed that bronchial epithelial cells and alveolar epithelial cells promote apoptosis by activating the inflammatory cascade response of NF-κB due to the stimulation of mechanical traction [1]. In this experiment, a small number of apoptotic cells were seen in the lung tissue of the control group at 2 h of ventilation; while in the OLV group, with the prolongation of OLV time, a small number of apoptotic cells were seen in the inner flanks of the alveolar lumen of rats at 0.5 h of OLV 1h retension, and more apoptotic cells were found in the inner flanks of the alveolar lumen and the interstitium of rats at 0.5 h of retension after 1.5 h of OLV, and these changes were higher than those in the same time These changes were more pronounced in the lung tissue of rats in the two-lung ventilation group than in the same time. Krick and colleagues [5] found that induction of hypoxia-inducible factor (HIF-1) induced apoptosis in alveolar type II epithelial cells in rats exposed to hypoxia. During OLV, the non-ventilated side of the lung atrophies and the alveolar oxygen partial pressure decreases, making the non-ventilated test lung tissue hypoxic, which can lead to apoptosis of lung tissue cells. This decrease in alveolar oxygen partial pressure can cause an increase in pulmonary vascular resistance (HPV), which is a compensatory response of the pulmonary circulation to hypoxia, but while HPV plays these protective roles, it also decreases the amount of blood perfusion in lung tissues. In the OLV group, the rats resumed ventilation for 0.5 h after OLV for a predetermined period of time, and when ventilation was restored, the rapid restoration of oxygen supply to lung tissue could also activate PMN in lung tissue and activate oxidative stress, which could cause oxygen free radicals, tumor necrosis factor-α (TNF-α) and interleukin-lβ (LI-lβ) in blood and lung tissue. lβ (LI-lβ) and other inflammatory mediators are increased [6]. The results of a study by You et al [7] also showed that the level of oxidative stress in lung tissue was significantly increased during OLV in rabbits, and this trend was more pronounced with the prolongation of OLV time. However, large amounts of inflammatory factors such as oxygen free radicals, TNF-α and LI-lβ can lead to increased apoptosis of alveolar epithelial cells and vascular endothelial cells and result in dysfunction of the pulmonary vascular endothelial screen [8].An S et al [9] suggested that small intestine ischemia-reperfusion leads to type II epithelial cell apoptosis through upregulation of TNF-α thereby causing lung tissue injury. In the process of reopening after lung atrophy on the non-ventilated side, alveolar expansion is not always coordinated, and excessive stretching of the alveolar wall by interfacial tangential forces generated at the junction of expanded and atrophied alveoli can cause apoptosis of alveolar epithelial cells [10]. The increase of apoptotic cells in lung tissue with the prolongation of OLV time in the present experiment can lead to alveolar structural damage and aggravation of lung histopathological changes, which is consistent with the lung histopathological changes observed under light microscopy in the present experiment.
Caspase-3 is the most important apoptotic effector in the Caspase family, which is responsible for the cleavage of all or some of the key proteases during the execution phase of apoptosis, leading to DNA fragmentation and apoptotic cell death. Therefore, changes in Caspase-3 protein expression in rat lung tissue may also directly respond to apoptosis in lung tissue. To further understand the mechanism and pathways of action of OLV-induced apoptosis, we investigated the changes of Caspase-3 protein expression by immunoblotting. From the experimental results, the changes in Caspase-3 protein expression and the changes in apoptosis detected by TUNEL were generally consistent, thus confirming the results of the TUNEL assay.
In conclusion, with the prolongation of ventilation time, OLV can lead to changes in lung tissue structure and increase the expression of apoptosis and its apoptosis execution factor Caspase-3 protein, thus it is speculated that apoptosis may be an important factor of lung injury caused by OLV. However, further studies on the more specific mechanism of action are needed.
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