In 2011, Professor Cheng Huagang's team from the Affiliated Hospital of Zunyi Medical College published an article entitled "Study on the Effect of Cevelestat Sodium on Rats with Inhalation Lung Injury Secondary to Brain Injury" in the Journal of Chongqing Medical University. The purpose of this study was to investigate the effect of nebulized inhalation of cevelestat sodium on rats with lung injury secondary to brain injury and its mechanism.
Animal models were used in the study, and rats were divided into six groups and received different treatments. The results showed that nebulized inhalation of cevilastat sodium significantly improved lung histomorphology and reduced the levels of neutrophil elastase (NE) and interleukin-8 (IL-8). This study showed that nebulized inhalation of cevelestat sodium could improve lung tissue injury and reduce the levels of neutrophil elastase (NE) and interleukin-8 (IL-8) in rats with inhalation lung injury secondary to brain injury. This study contributes to the understanding of the therapeutic mechanisms of lung injury secondary to brain injury, providing new clues for future treatments.
Objective: To investigate the effect of nebulized inhalation of Sivelestat sodiom on rats with lung injury secondary to brain injury, and to explore its mechanism from the perspective of inflammation.
Methods: A total of 60 SD rats were randomly divided into 6 groups, normal group (A), aspiration group (B), brain injury aspiration group (C), normal saline nebulized inhalation group (D), dexamethasone nebulized inhalation group (E), and cevelestat sodium nebulized inhalation group (F), 10 rats in each group lavage fluid (BALF) and interleukin-8 (IL-8) contents, and the concentration of albumin in BALF was determined by immunotransmission turbidimetry. Light microscopy was used to observe the pathological changes of lung tissue.
Outcome:
1. In terms of lung tissue morphology, the improvement was most obvious in the Xiwei group, followed by the dexamethasone group;
2. The NE content in BALF was measured in rats, and the Xiwei and dexamethasone groups were lower than those in the aspiration, brain injury aspiration and normal saline groups (P<0.05), and the Xiwei group was the most obvious.
3. The content of IL-8 in rat BALF was measured and the content of IL-8 in the SVI and dexamethasone groups was lower than that in the brain injury aspiration group and the saline nebulization inhalation group (P<0.05), but there was no significant difference between the two groups (P>0.05).
Conclusion: The improvement effect of cevelestat sodium nebulized inhalation on the lung tissue of rats with inhalation lung injury secondary to brain injury is better than that of dexamethasone, and its mechanism of action may be related to the inhibition of the production and activity of NE, the alleviation of the direct damage of NE to lung tissue, and the inhibition of lung tissue damage caused by the circulatory response of inflammatory network in the lungs.
After brain injury (BI), due to coma, vomiting, and decreased or absent gag reflex, oropharyngeal secretions or gastric contents are prone to inhalation into the respiratory tract, causing acute inhalation lung injury and even developing respiratory distress syndrome, which is one of the important reasons for the high mortality and disability rate after head injury [1]. It is thought that poly-morphonuclear leukocyte (PMN) aggregates and releases a variety of proteases, especially neutrophil elastase (NE), which plays an important role in the pathogenesis of acute lung injury (ALI) [2]. Sivelestat sodiom is an NE inhibitor that potently inhibits NE locally and alleviates lung tissue damage[3]. At present, most of the studies are mainly based on systemic administration, and due to the low concentration of local drugs, the dose is increased accordingly, which enhances the toxicity and side effects of the drug and increases the economic burden of patients. Nebulized inhalation can make the drug penetrate deep into the terminal bronchi and alveoli, absorb quickly, and have fewer side effects than systemic drugs, but the effect of nebulized inhalation of cevelestat sodium on inhaled lung injury is not very clear. Therefore, this study established a rat BI aspiration model to explore the effect and mechanism of nebulized inhalation of cevelestat sodium on inhalation lung injury secondary to BI, in order to provide an experimental basis for the implementation of convenient, economical and non-invasive treatment regimens for patients with BI aspiration.
1. General condition
When the BI model was made, the rat had apnea immediately after injury, and resumed breathing after 2s~5min by itself or with manual assistance, followed by shortness of breath, cyanosis of the face and limbs, convulsions, hemiplegia and coma all over the body. When the aspiration model was done, after the gastric juice dripped into the trachea, the rats showed symptoms such as choking, irritability, and even bloody foamy discharge from the mouth and nose. After the model was successfully made, 4 rats died, and a total of 56 rats were statistically processed.
2. Lung tissue was observed by HE staining light microscopy
The lung tissue of group A was normal, and the interstitial blood vessels were dilated and congested, the alveolar septum was thickened, some alveoli collapsed, and inflammatory cells were infiltrated (see Fig. 1).
Fig. 1 Inflammatory cell infiltration (HE×200) rats were found to have thickened alveolar septa and partial alveolar collapse after aspiration
In the C and D2 groups, there was interstitial vasodilation and congestion, alveolar collapse, obvious inflammatory cell infiltration, interalveolar telangiectasia and congestion with a large number of inflammatory cell infiltration and fibrinous exudation, and bronchiole erythrocytes and serous exudation (Figs. 2 and 3).
Fig. 2 In the BI aspiration group (HE×200), the alveolar septum was disturbed, with interalveolar telangiectasia and congestion with a large number of inflammatory cell infiltration, and obvious exudation of red blood cells and serous fluid from the alveolar cavity and bronchioles
Fig.3 Normal saline group (HE×200) Alveolar septum disorder, interalveolar telangiectasia and congestion with a large number of inflammatory cells infiltrating and fibrinous exudation, and red blood cells in the alveolar cavity and bronchioles
Alveolar septum widening, partial alveolar fusion, telangiectasia and congestion, and leukocyte infiltration were also seen in group E, but the degree was milder than that in groups C and D (Fig. 4).
Fig.4 The alveolar structure of the dexamethasone group (HE×200) was basically present, with some alveolar fusion, some alveolar septal telangiectasia and hyperemia, and inflammatory cell infiltration
The alveolar structure of group F was basically present, the local interalveolar capillaries were dilated and congested, and some red blood cells were seen in the bronchiole lumen, the accumulation range was small, and the cellulose exudation was not obvious (see Fig. 5).
Fig.5 The alveolar structure of the cevelestat sodium group (HE×200) was basically present, and the local interalveolar capillaries were dilated and congested, the accumulation range was small, and the cellulose exudation was not obvious
3. Changes of NE, IL-8 and albumin in BALF in rat aspiration by BI
The contents of NE and albumin in BALF were the lowest in group A, followed by group F, and the differences were statistically significant by one-way ANOVA and Bonferroni test, while there was no significant difference in IL-8 concentration in groups E and F2. See Table 1.
Table 1 Comparison of NE, IL-8 and albumin contents in BALF of various rats (μg/x±s)
1. Mechanism of action between inflammation and inhalation lung injury secondary to BI
The pathogenesis of inhalation lung injury secondary to BI is complex, and the related mechanism is not well understood. From the perspective of inflammation, severe trauma, infection, shock, etc. can constitute a first blow to the body, so that the body's inflammatory cells and immune cells are in an active state, and if the disease continues to develop or is affected by other damaging factors again, it constitutes a second blow, causing the inflammatory cells in the excited state to release a large number of inflammatory mediators, causing an excessively uncontrolled inflammatory response, which in turn leads to the occurrence of ALI [6]. This study showed that the contents of NE, IL-8 and albumin in BALF in group C were significantly higher than those in groups A and B2, and the lung tissue showed serious pathological damage, indicating that BI aspiration can cause excessive inflammation in the lungs and cause lung injury. The mechanism may be related to the "second blow" theory, which causes sympathetic nerve excitation, systemic vasoconstriction, tissue ischemia and hypoxia, neutrophil activation, and mediates the production of a variety of inflammatory mediators due to the stress response and increased intracranial pressure after head injury [7]. Aspiration poses a second blow to lung tissue, neutrophils are further activated, producing a variety of inflammatory factors and proteolytic enzymes, especially NE, which directly degrades a variety of protein components of the extracellular matrix, destroys the tight junctions between cells, increases vascular permeability, and induces endothelial cells to produce chemotaxis cytokines, such as IL-8, IL-6, etc., and IL-8 is a chemokine of the C-X-C family, which in turn chemotactic and activates PMN, which in turn releases more NE, forming a network circulation response and aggravating lung injury.
2. Effect and mechanism of cevelestat sodium on rats with inhalation lung injury secondary to BI
Cevelestat sodium is an NE inhibitor that has been shown to effectively inhibit elastase and alleviate lung tissue damage in both in vivo and in vivo experiments [8]. The lesion site of acute inhalation lung injury is mainly in the lung interstitium, and inhibiting the activation of lung inflammatory factors, PMN, and reducing the direct effect of inflammation on lung tissue are important ways to improve the efficacy [9]. Nebulized inhalation administration can directly reach the alveoli, avoid first-pass effects, reduce drug dosage, and alleviate adverse reactions, and is economical and noninvasive [10]. The results of this study showed that the content of NE, albumin and lung tissue pathological damage in BALF in the cevelestat sodium group were lower than those in the normal saline control group and dexamethasone group, indicating that the nebulized inhalation of cevelestat sodium could inhibit NE and alleviate lung injury, which was mainly manifested in reducing vascular endothelial cell damage, improving vascular permeability, inhibiting collagen fiber proliferation and inflammatory cell infiltration, and reducing pulmonary edema. The mechanism of action may be that cevelestat sodium directly inhibits the amount and activity of NE, reduces the direct damage of NE to lung tissue, and reduces the PMN-mediated inflammatory network circulation, thereby playing a therapeutic role in ALI.
3. Effect and mechanism of dexamethasone on rats with inhalation lung injury secondary to BI
High-dose glucocorticoid pulse therapy has been used for the treatment of ALI at home and abroad [11]. Studies have shown that dexamethasone can reduce alveolar-capillary permeability, reduce exudation, reduce interleukin, tumor necrosis factor, etc. However, high doses of corticosteroids can increase the incidence of infection and other complications of corticosteroids [11]. The results of this study showed that there was no significant difference between the IL-8 content in the BALF of the dexamethasone group and the cevelestat sodium, and the NE content and albumin content in the BALF were lower than those in the saline control group and higher than those in the cevelestat sodium group, indicating that dexamethasone had a certain effect on inhibiting inflammation and lung injury, but the effect was not as good as that of cevelestat sodium, which may be due to the fact that dexamethasone is an effective glucocorticoid with a wide range of anti-inflammatory effects and alleviating lung damage. In summary, a series of inflammatory reactions occurred in the lungs of rats after BI aspiration, and NE was one of the important reasons for the occurrence of inflammation, nebulized inhalation of cevelestat sodium and dexamethasone could inhibit the production of inflammatory mediators such as NE and IL-8, balance the inflammatory response in the lungs, and reduce the damage of lung tissue, but the effect of cevelestat sodium was significantly better than that of dexamethasone. Due to the complex and changeable conditions of patients with inhalation lung injury secondary to clinical BI, the clinical treatment effect of cevelestat sodium nebulization needs to be further studied.
1. Animal grouping
There were 60 healthy male SD rats weighing 325~360g (provided by the Laboratory Animal Center of Chongqing Daping Hospital, Third Military Medical University). They were randomly divided into 6 groups. Group A: normal group, group B: simple aspiration group, group C: BI aspiration group, D normal saline control group, group E: dexamethasone nebulized inhalation group, group F: cevelestat sodium nebulized inhalation group.
2. Method of administration
Groups D, E and F were given NS10ml, dexamethasone (6.25mg/kg) + NS10ml, cevelestat sodium (1.25mg/kg) + NS10ml nebulized inhalation for 3 times/d for 20min each time, the treatment time was 3 days, and the rats were sacrificed after blood was taken through the carotid artery at the end of treatment, and the required specimens were taken. Administration method: The rat was placed in a 30cm×15cm×20cm homemade glass box, and different solutions were given for atomization and inhalation according to the group. The nebulizer is a PARITurboBOY nebulizer produced by Braim in Germany, with a total output rate (TOR) of 500mg/min and a fog particle diameter of <5μm.
3. Build a model
The modified Marmarou method [4] was used to place the head of the normally awake rat under the guide catheter of the BI device, and the rat was slid down from a height of 50 cm through the guide tube with a 100g percussion hammer. After successful BI modeling, 10% chloral hydrate (0.33mg/kg) was given intraperitoneal injection anesthesia. The rat was fixed on the operating table supine, the neck was routinely prepared and disinfected, the superficial skin was incised in a sagittal manner, the trachea was separated, the head was tilted upwards at a 30° angle, and gastric juice (0.4ml/kg) was dripped into the trachea. The general condition of the rats was observed, the neck skin was sutured, returned to the cage, and observed and kept warm. Source of gastric juice: Collect the early morning gastric juice of nasogastric feeding patients and let it stand for 30min. Rats that died before the termination of the experiment were not included in the scope of this experiment.
4. Morphological observation of lung tissue
Part of the lung tissue was stored in 10% formaldehyde for light microscopy.
5. Determination of NE, interleukin-8 (IL-8) and albumin content in bronchoalveolar lavage fluid (BALF).
Referring to the method of the literature[5], 5~6ml of BAIF was obtained from each rat. Enzyme-linked immunosorbent assay (ELISA) was used to determine the contents of NE and IL-8 in BALF, and the concentration of albumin in BALF was determined by immunotransmission turbidimetry.
6. Statistical processing
The obtained data were analyzed using SPSS17.0 software. The continuous data were expressed as x±s, one-way ANOVA was used for comparison between the means of multiple samples, and the Bonferroni test was used for comparison of the means of two samples. The difference of P<0.05 was statistically significant.
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