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Blunt chest trauma with hemorrhagic shock frequently induces pulmonary inflammation that leads to acute lung injury (ALI). The present study aimed to explore the protective effects of dexmedetomidine (Dex) in blunt chest trauma and hemorrhagic shock-resuscitation (THSR)-induced ALI by mediating nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome formation in rats. An ALI model in rats induced by THSR was constructed and Dex was administered intraperitoneally (5 µg/kg/h) immediately after blunt chest trauma. Blood samples were collected for the determination of proinflammatory factor levels, and lung tissue specimens were harvested for wet/dry (W/D) weight ratio, hematoxylin and eosin staining, and transmission electron microscopy analyses. Additionally, malondialdehyde (MDA), superoxide dismutase (SOD), lactate dehydrogenase (LDH) and myeloperoxidase (MPO) activity were evaluated, and the expression of protein in lung tissues was examined via western blot analysis. Compared with the sham group, pathological alterations in the ALI group and the W/D ratios were significantly increased. MDA, LDH and MPO activity, and the levels of interleukin (IL)-1β, IL-18, IL-6 and tumor necrosis factor-α were significantly elevated. NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain and caspase-1 expression was significantly increased. Conversely, Dex treatment significantly reversed these changes. The present study demonstrated that by reducing inflammatory responses, Dex exerted protective effects against THSR-ALI in rats, potentially via the inhibition of NLRP3 signaling pathways.
Blunt chest trauma is commonly associated with a wide range of injuries, a number of which are life-threatening with high mortality and require immediate medical attention (
As an important mediator in HS and various types of ALI/ARDS (
Dexmedetomidine (Dex) is a short-acting, highly selective α-2 adrenoreceptor agonist that is extensively applied in clinical anesthesia and intensive care (
The present study aimed to test the hypothesis that Dex exerts protective effects on THSR-induced ALI in rats by alleviating the inflammatory response and inhibiting NLRP3 signaling pathways.
All laboratory and animal experiments were approved by the Medical Ethics Committee of Renmin Hospital of Wuhan University in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council (US) Committee (
Healthy adult male Sprague-Dawley rats (age, 8 weeks; weight, 200–220 g) were obtained from Vital River Laboratory Animal Technology Co., Ltd. (certificate no. SYXK 2014-0080) and maintained under specific pathogen-free conditions (12:12-h light/dark cycle, 20–24°C and 40–60% humidity) with food and water
A total of 40 rats were randomly divided into four equal groups: Sham, Dex, ALI and Dex + ALI groups. Rats in the ALI and Dex + ALI groups underwent THSR surgery, whereas those in the sham and Dex groups were subjected to sham surgery, including femoral arterial and venous cannulation, although neither blunt chest trauma nor HS-resuscitation were induced. In addition, rats in the Dex and Dex + ALI groups were administered with 5 µg/kg/h Dex for 30 min via intraperitoneal injection (cat. no. 14030332; Jiangsu Hengrui Medicine Co., Ltd.) immediately after the sham or blunt chest traumatic surgery. At 6 h after the surgery, all animals were sacrificed under anesthesia via intraperitoneal injection of 50 mg/kg pentobarbital sodium and exsanguination from the right carotid artery. At the same time, arterial blood, bronchoalveolar lavage fluid and lung tissue samples were collected. Lung tissues were snap-frozen in liquid nitrogen and stored at −80°C for subsequent analysis. All animal experiments were performed from 08:00 to 12:00.
The rat model of THSR-induced ALI adopted in the present study was described in mice by Seitz
In brief, the rats were provided free access to water and fasted for 12 h prior to surgery. After induction of anesthesia via intraperitoneal injection of pentobarbital sodium (2%; 50 mg/kg), sham or THSR surgery was performed according to the groupings. A polyethylene tube filled with heparinized saline was used for cannulation of the femoral artery and vein to monitor continuous invasive pressure [in the form of mean arterial pressure (MAP)] and heart rate using a monitor (IntelliVue MP20; Philips Healthcare), as well as to establish venous access. As previously described, blunt chest trauma was induced with a fixed 2.45-J chest impact by dropping a hollow cylindrical encased in a vertical stainless steel tube which was positioned onto a lexon platform from a definite height (
At 6 h following surgery, blood samples were collected from animals under anesthesia from the right femoral artery (0.5 ml/animal) and then immediately assayed with an i-STAT portable clinical analyzer (Abbott Point Of Care, Inc.; Abbott Pharmaceutical Co., Ltd.). Arterial partial pressure of oxygen (PaO2) was measured, and the oxygenation index [PaO2/fraction of inspired oxygen (FiO2)] was calculated.
The W/D ratio is used as an index of the severity of pulmonary edema. The W/D ratio was determined by measuring the water content in the lungs 6 h after THSR challenge. The right middle lobe of the lungs was dissected from non-pulmonary tissues and then accurately weighed to determine wet weight using an electronic balance after the surface blood and water were wiped off. Afterward, the lungs were incubated for 72 h in an oven at 60°C and then reweighed to determine the dry weight. Finally, the W/D ratio was calculated.
Lung tissue samples were collected and fixed in 4% paraformaldehyde at 4°C for 48 h. Subsequently, the samples were dehydrated, embedded in paraffin and sectioned routinely (5 µm thickness). Finally, H&E staining was performed with the following steps at room temperature using H&E solution (Sigma-Aldrich; Merck KGaA): Firstly, paraffin sections were incubated at 60°C for 30 min, twice immersed in xylene for 15 min at room temperature and then treated with a descending ethanol series for 5 min each at room temperature. The sections were then treated with 0.5% hematoxylin for 1–5 min at room temperature and then rinsed in tap water for 1 min. Sections were incubated with PBS for 8 sec until a blue color was observed, and then the sections were washed using tap water for 1 min and then distilled water for 8 sec. Sections were then stained with 1% eosin for 3 min at room temperature and then washed with tap water. Then, sections were treated with an ascending ethanol series for 1 min each at room temperature.
Pathological alterations of the lung tissues were observed under a light microscope (magnification, ×200; BX51; Olympus Corporation). Simultaneously, lung injury scores were calculated by applying the histological scoring system designed for mice by Belperio
Fragments of the right middle lung tissues were cut into 1-mm slices and then fixed in 2.5% glutaraldehyde solution for 2 h at 0–4°C. Afterward, the specimens were rinsed with PBS three times, fixed with 1% osmic acid at room temperature for 1 h, rinsed with PBS three times and stained with 2% uranium acetate solution at room temperature for 30 min, and then dehydrated with dimethyl ketone. The specimens were embedded in Epon-812 at 60°C for 2 days, cut into ultrathin sections (60 nm), and stained at room temperature with 1% uranyl acetate for 30 min and lead citrate for 15 min. The sections were observed under a transmission electron microscope (magnification, ×10,000; Hitachi H-600; Hitachi, Ltd.).
MDA reflects the degree of lipid peroxidation, whereas SOD protects cells from superoxide damage (
The blood samples were immediately centrifuged at 1,500 × g for 10 min at 4°C after collection to obtain the serum. The supernatant fluid was harvested and assayed using ELISA kits (IL-1β, cat. no. RLB00; IL-18, cat. no. DY521-05; IL-6, cat. no. R6000B; TNF-α, cat. no. RTA00; all R&D Systems, Inc.) to measure the levels of proinflammatory cytokines in serum according to the manufacturer's protocols. The absorbance was measured at 450 nm using an ELISA reader (BioTek Instruments, Inc.).
The lung tissue samples were homogenized with RIPA lysis buffer (25 mM Tris-HCl, pH 7.6, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with 1% PMSF. Lysates were sonicated and centrifuged at 3,000 × g at 4°C for 10 min, and then the homogenate supernatant was centrifuged again at 10,000 × g at 4°C for 10 min to obtain the final lung homogenate supernatant, which was used to detect proteins. The concentrations of the proteins were measured using a bicinchoninic acid protein assay kit. On the basis of the protein concentrations, an equal quantity of protein (30 µg) was loaded into each well and then separated via 10% SDS-PAGE. The proteins were subsequently electrotransferred to PVDF membranes. The membranes were blocked with 5% fat-free milk at room temperature for 1 h and then incubated with different primary polyclonal antibodies (rabbit anti-rat) at 4°C overnight. The primary polyclonal antibodies used in the present study were NLRP3 (1:1,000; cat. no. ab214185; Abcam), ASC (1:1,000; cat. no. D2W8U; Cell Signaling Technology, Inc.), caspase-1 (1:300; cat. no. ab1872; Abcam) and GAPDH (1:1,000; cat. no. sc-137179; Santa Cruz Biotechnology, Inc.). Afterward, the membranes were incubated with secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG; 1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature. The immunoreactive protein bands were visualized by enhanced chemiluminescence (cat. no. 32132; Thermo Fisher Scientific, Inc.) with an Odyssey color infrared laser scan-imaging instrument (LI-COR Biosciences). The quantities of the target proteins were analyzed using Image Lab software (version 5.2.1; National Institutes of Health) and reported as the densitometric ratios between the target protein and GAPDH, which was used as a loading control.
All quantitative data are presented as the mean ± SD (n=10/group). Multiple comparisons were performed for statistical analysis via one-way ANOVA by using GraphPad Prism Software 7.0 (GraphPad Software, Inc.). Bonferroni post hoc test was used to test differences between individual means when the F-statistic was significant. P<0.05 was considered to indicate a statistically significant difference.
The results of arterial blood gas analysis revealed that the levels of PaO2 significantly decreased in the ALI group 6 h after THSR procedure (
The lung W/D ratios in the ALI group were significantly increased compared with those in the sham group. Dex treatment significantly reversed the increase induced by THSR (
H&E staining of the lung sections in the sham group showed parenchymal microscopic findings of normal lungs: The alveolar structure of the sham group was intact, the alveolar wall was smooth and the pulmonary interstitial showed no evident exudation (
Lung injury scores were calculated to indicate the lung pathological alterations based on the observations of lung damage parameters under a light microscope. The ALI group reported significantly higher lung injury scores than the sham group. Conversely, the Dex + ALI group exhibited significantly reduced lung injury scores compared with the ALI group (
Transmission electron microscopy was used to examine the ultrastructural changes in lung tissues. Electron microscopy of the lung tissues in the sham or Dex group demonstrated clear edges of cells, nuclear membranes and certain osmiophilic lamellar bodies, whereas the ALI group showed significant pathological alterations and decreased number of lamellar bodies. Moreover, the Dex + ALI group exhibited a substantial attenuation of THSR-induced pulmonary injury compared with the ALI group (
Oxidative stress indices (MPO, SOD, LDH and MDA) were selected to determine ALI severity and explore the mechanisms of THSR-induced ALI. As presented in
The serum expression levels of the proinflammatory cytokines IL-1β, IL-18, IL-6 and TNF-α in the ALI group were significantly increased compared with those in the sham group. Conversely, Dex treatment resulted in a significant decrease in their expression levels compared with the ALI group (
Western blot analysis was performed to determine NLRP3, ASC and caspase-1 protein expression levels (
In a previous study, a rat model of ALI was successfully established using THSR (
Severe THSR damage often triggers systemic inflammatory response syndrome and multiple organ dysfunction syndrome (
As the core of innate immunity, NLRP3 forms an oligomeric molecular complex called an inflammasome, which responds to harmful stimuli, such as pathogen-associated molecular patterns and danger-associated molecular patterns (
An increasing number of studies have reported that Dex exerts protective effects on the lungs (
However, the present study contains several limitations. The regulatory mechanism of NLRP3 inflammasome activation and the role of Dex in this mechanism remain unclear. Moreover, the question of whether Dex directly interacts with the NLRP3 inflammasome or via other indirect mechanisms that trigger signaling remains unanswered. Furthermore, there remain unresolved issues regarding whether the interaction was through the α2 adrenergic receptor pathway and whether other drugs aside from Dex could exert similar protective effects. If so, whether these effects were different, and whether the NLRP3 inflammasome plays the same role in ALI attenuation by different drugs, require further verification. Further studies are required to address these issues and questions.
The present study demonstrated that Dex inhibits NLRP3 inflammasome signaling pathways and alleviates THSR-induced ALI in rats. This pathomechanism needs to be further analyzed in mechanistic and dose-related studies.
Not applicable.
The present study was supported by grants from the National Natural Science Foundation of China (grant nos. 81901952 and 81970722).
All data generated or analyzed during this study are included in this published article.
TQM and MY drafted the manuscript. TQM and QK analyzed and interpreted the data and all manuscript figures. MY and QH performed the transmission electron microscopy and lung injury score analysis. ZYX and XJW conceived and designed the study, reviewed the data and revised the manuscript. All authors read and approved the final manuscript.
Ethical approval was provided by the Medical Ethics Committee of Renmin Hospital of Wuhan University. All surgical procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council (US) Committee.
Not applicable.
The authors declare that they have no competing interests.
acute lung injury
dexmedetomidine
blunt chest trauma and hemorrhagic shock-resuscitation
nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing protein 3
apoptosis-associated speck-like protein containing a caspase recruitment domain
wet/dry
malondialdehyde
superoxide dismutase
lactate dehydrogenase
myeloperoxidase
acute respiratory distress syndrome
interleukin
tumor necrosis factor
hemorrhagic shock
mean arterial pressure
ventilator-induced lung injury
Dex attenuates the effects of THSR-induced ALI on arterial blood gas. Dex increases the levels of (A) PaO2 and (B) PaO2/FiO2, and decreased (C) lung W/D ratios in a rat model of THSR-induced ALI. Data are presented as the mean ± SD (n=10). *P<0.05 vs. sham; #P<0.05 vs. ALI. ALI, acute lung injury; Dex, dexmedetomidine; PaO2, partial pressure of oxygen; FiO2, fraction of inspired oxygen; W/D, wet/dry; THSR, blunt chest trauma and hemorrhagic shock-resuscitation.
Dex attenuates lung pathological alterations in blunt chest trauma and hemorrhagic shock-resuscitation-induced ALI. (A) Microscopic presentations of lung tissues by H&E staining (magnification, ×400). (B) Detection of lung injury measured by the scoring system of lung injury. Data are presented as the mean ± SD (n=10). *P<0.05 vs. sham; #P<0.05 vs. ALI. ALI, acute lung injury; Dex, dexmedetomidine.
Dex attenuates pathological alterations to the lungs in THSR-induced ALI. Lung pathology was evaluated in a rat model of THSR-induced ALI via transmission electron microscopy (magnification, ×10,000). ALI, acute lung injury; Dex, dexmedetomidine; THSR, blunt chest trauma and hemorrhagic shock-resuscitation; Lb, lamellar body; Nu, nucleus.
Effects of Dex treatment on oxidative stress markers in a rat model of THSR-induced ALI. (A) Level of MDA and activity of (B) SOD, (C) MPO and (D) LDH in THSR-induced ALI. Data are presented as the mean ± SD (n=10). *P<0.05 vs. sham; #P<0.05 vs. ALI. ALI, acute lung injury; Dex, dexmedetomidine; THSR, blunt chest trauma and hemorrhagic shock-resuscitation; MDA, malondialdehyde; SOD, superoxide dismutase; LDH, lactate dehydrogenase; MPO, myeloperoxidase.
Effects of Dex treatment on cytokine levels in a rat model of THSR-induced ALI. (A) IL-1β, (B) IL-18, (C) IL-6 and (D) TNF-α levels in the serum of THSR-induced ALI rats. Data are presented as the mean ± SD (n=10). *P<0.05 vs. sham; #P<0.05 vs. ALI. ALI, acute lung injury; Dex, dexmedetomidine; THSR, blunt chest trauma and hemorrhagic shock-resuscitation; IL, interleukin; TNF, tumor necrosis factor.
Dex treatment inhibits the activation of NLRP3 signaling pathways in a rat model of THSR-induced ALI. (A) Protein expression levels of NLRP3, ASC and caspase-1 in the lung tissue of THSR-induced ALI rats were determined via western blot analysis. GAPDH was used as a loading control. (B) Protein expression was quantified and expressed as densitometric ratios between the target protein and GAPDH. Data are presented as the mean ± SD (n=10). *P<0.05 vs. sham; #P<0.05 vs. ALI. ALI, acute lung injury; Dex, dexmedetomidine; THSR, blunt chest trauma and hemorrhagic shock-resuscitation; NLRP3, nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing protein 3; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain.