Male C57BL/6 mice of this study, aged 6–8 weeks, weigh 18–22 g, were obtained from Chongqing Medical University. The entire study involved 15 mice. Each mouse was completely randomized into groups using a random number table. Before the experiment, the mice were kept free to eat and drink in specific pathogen free facility for at least 7 days. The Institutional Animal Care and Use Committee of Chongqing Medical University approved the procedures for the care and use of the mice. The First Affiliated Hospital of Chongqing Medical University Animal Ethics Committee approved the experimental scheme (reference number 2020-850).

Before performing CLP surgery, we administered inhalation anesthesia to the mice. In short, 5% isoflurane is used to induce anesthesia, and 2% isoflurane is used to maintain anesthesia. The operation method of CLP refers to previous research [21]. The cecum was exposed. Following a 5.0 mm ligation from the tip, puncture the cecal stump with a 22-gauge needle and squeeze out a small amount of intestinal contents. Sham operation control mice exposed cecal stump without ligation and puncture. After 24 h, all mice were euthanized via excessive carbon dioxide inhalation, and whole-blood and lung were collected for further analysis. Following ligation, 200 µL of PBS was administered into the right lung through the trachea and subsequently removed after 10 seconds as bronchoalveolar lavage fluid (BALF). Repeat the operation twice for the next analysis.

Mice were divided randomly by random number table into three groups (n = 5): (1) control; (2) CLP; and (3) CLP+Dex. Dexmedetomidine (SML0956, Sigma-aldrich, Darmstadt, Germany) was administered intraperitoneally at 50 µg/kg, 30 minutes post-CLP. The control mice intraperitoneally injected with the same amount of PBS. The dose of Dex was determined in previous studies [22, 23].

Lung tissue was fixed in paraformaldehyde (4%) for 24 hours (4 °C) and then embedded in paraffin. Then the slices were fixed on glass slides and stained with H&E (G1120, Solarbio, Beijing, China) to evaluate tissue damage with a photon microscope (Leica, Wetzlar, Germany). The lung-injury score results were obtained according to previously study of Mikawa et al. [24]. Alveolar congestion, hemorrhage, neutrophil infiltration, and transparent membrane formation were measured. Pathological damage was assessed using a 4-point scale: 0 = no or very slight damage, 1 = mild, 2 = moderate, 3 = serious, and 4 = very serious.

Apoptosis was evaluated by TUNEL assay, following the methodology of a previous study [25]. Following the in situ cell apoptosis detection kit instructions (G001-1, Jiancheng Biotechnology, Nanjing, China), the lung sample was sealed, fixed, and sliced into 4-µm-thick sections. Proteinase K was used to digest the tissue for 15 minutes at 37 °C after the endogenous peroxidase was blocked. The terminal deoxynucleotidyl transferase was diluted in a reaction buffer and applied to sections at 37 °C for 2 hours, followed by three 2-minute washes. Slice with a dilution of anti-digoxin antibody incubation at 37 °C for 30 minutes. Apoptotic cells were measured by 3,3^′-diaminobenzidine chromogenic after about 20 min. For statistical analysis, three images were captured, and TUNEL-labeled cells were measured as reported previously [26].

An earlier study reported on the collection of whole blood and the preparation of serum [27]. Serum and BALF were analyzed with ELISA kits for interleukin (IL)-6, IL-10, tumor necrosis factor-alpha (TNF-$\alpha$) and IL-17A (KMC0061, BMS614, BMS607-3, BMS6001, EBioscience, San Diego, CA, USA).

Rehydration and dewaxing of lung paraffin slices were performed. Triethylenediamine tetraacetic acid (GC202001, Servicebio, Wuhan, China) was used to retrieve antigens. Then sections were incubated with 5% BSA. The sections were incubated with anti-rat caspase-11 (1:100, NB120-10454, NOVUS, Littleton, CO, USA) or anti-mouse receptor for advanced glycation end products (RAGE, 1:100, ab37647, Abcam, Cambridge, UK) for 12 hours at 4 °C. Slices were incubated in the dark (room temperature) using either a FITC-labeled green secondary antibody (1:200, GB22403, Servicebio, Wuhan, China) for RAGE or a Cy3-labeled red secondary antibody (1:200, Servicebio, GB21302) for caspase-11. DAPI was applied to detected the nuclei. The immunofluorescence methods for inducible nitric oxide synthase (iNOS) and CD206 in RAW264.7 cells followed previously published protocols, with the primary antibodies substituted by rabbit anti-mouse CD206 antibody (1:100, 24595, Cell Signaling Technology, Danvers, MA, USA) and rat anti-mouse iNOS monoclonal antibody (1:1000, 740025T, Thermo Fisher Scientific, Waltham, MA, USA). FITC-goat anti-rat IgG (1:100, SA00003-11, proteintech, Wuhan, China) was used for iNOS, and goat anti-rabbit IgG-Cy3 (1:100, SA00009-2, proteintech, Wuhan, China) was used for CD206. Finally, the images were collected by confocal microscope (VS200-BU-L, OLYMPUS, Tokyo, Japan).

Total and nuclear protein samples in lung tissue and RAW264.7 cells were prepared using diluted RIPA Lysis Buffer and a nuclear extraction kit (P0013B, Beyotime, Shanghai, China). The prepared samples were transferred to polyvinylidene fluoride (PVDF) membrane after separation on sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE, 10–12%). After being washed and blocked at room temperature for 2 hours, the membranes were incubated with primary antibodies at 4 °C overnight: (RAGE, 1:100, ab37647, Abcam, Cambridge, UK), NF-$\kappa$B p-p65 (1:1000, ab76302, Abcam, Cambridge, UK), IL-1$\beta$ (1:1000, ab234437, Abcam, Cambridge, UK), gasdermin-D (1:1000, ab209845, Abcam, Cambridge, UK), iNOS (1:1000, ab202417, Abcam, Cambridge, UK), Arg-1 (1:5000, ab233548, Abcam, Cambridge, UK), caspase-11 (1:1000, NB120-10454, NOVUS, Littleton, CO, USA), GAPDH (1:50,000, A19056, ABclonal, Wuhan, China), $\beta$-actin (1:80,000, AC026, ABclonal, Wuhan, China), and histone H3 (1:1000, AF0863, Affinity, Jiangsu, China). The membrane was incubated with the secondary antibody, Goat Anti-Rabbit IgG (H+L) HRP (1:3000, S0001, Affinity, Jiangsu, China) or Goat anti-rat IgG (H+L) HRP (1:3000, S0009, Affinity, Jiangsu, China) at 37 °C for 1 hour after cleaning. Finally, the membrane was measured by ECL method (Immobilon® ECL UltraPlus Western HRP Substrate, #WBULP, Millipore, Billerica, MA, USA) using gel imaging systems software (Fusion, Vilber, Paris, French) was used to detect blot strength.

qRT-PCR experiments based on the description of the previous research [28]. In short, total mRNA samples were prepared from the lung tissue and RAW264.7 cells according to the RNAiso Plus reagent instructions. cDNA was synthesized using a Takara Technology cDNA synthesis kit. qRT-PCR was conducted using the TB Green™–Premix Ex Taq™ II reagent kit (RR820A, Takara, Kyoto, Japan) on a Bio-Rad CFX96 system (CFX96, Bio-Rad, Hercules, CA, USA) to evaluate the mRNA expression levels of RAGE, caspase-11, iNOS, Arg-1, IL-1$\beta$, and IL-6. The primer sequences are listed as follows (Tsingke-biology). Rage: sense 5^′-CTTGCTCTATGGGGAGCTGTA-3^′, antisense 5^′-CATCGACAATTCCAGTGGCTG-3^′; caspase-11: sense 5^′-CCTGAAGAGTTCACAAGGCTT-3^′, antisense 5^′-CCTTTCGTGTAGGGCCATTG-3^′; Inos: sense 5^′-CAGCTGGGCTGTACAAACCTT-3^′, antisense 5^′-CATTGGAAGTGAAGCGGTTCG-3^′; Arg-1: sense 5^′-GAACACGGCAGTGGCTTTAAC-3^′, antisense 5^′-TGCTTAGCTCTGTCTGCTTTGC-3^′; Il-1$\beta$: sense 5^′-GCAACTGTTCCTGAACTCAACT-3^′, antisense 5^′-ATCTTTTGGGGTCCGTCAACT-3^′; Il-6: sense 5^′-CAACGATGATGCACTTGCAGA-3^′, antisense 5^′-GTGACTCCAGCTTATCTCTTGGT-3^′; $\beta$-actin: sense 5^′-CCACCATGTACCCAGGCATT-3^′, antisense 5^′-CAGCTCAGTAACAGTCCGCC-3^′. The 2^{-Δ⁢Δ⁢Ct} method was applied to detected the mRNA expression levels.

Under anesthesia, whole blood samples were collected 24 hours post-CLP procedure, and PBMCs were isolated using Ficoll Histopaque (17544602, Cytiva, Logan, UT, USA) through density gradient centrifugation.

RAW 264.7 macrophage cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China. All cells were validated by STR profiling and tested negative for mycoplasma. After counting 10⁵ cells, all were cultured in 90% high glucose DMEM with 10% heat-activated FBS (Gibco, Carlsbad, CA, USA) in a humidified environment at 5% CO₂ and 37 °C. We began with 10⁵ cells per group and cultured them until reaching 80% confluence in a six-well plate. Three groups were established: a control group, an lipopolysaccharide (LPS)-stimulated group (500 ng/mL), and an LPS+Dex group (10 µM). The dosage of LPS and Dex were determined in previous research [29, 30]. The cells were collected for analysis after 24 hours.

Blank controls of harvested PBMCs and RAW264.7 cells underwent the same procedure as the samples, excluding flow-cytometry antibody staining. We used the fluorescent antibody of anti-CD11b-PerCP/Cyanine5.5 (101228, BioLegend, San Diego, CA, USA) and anti-F4/80-FITC (BioLegend, 123108) to test the macrophages in PBMCs. Fluorescent antibody staining was performed on cultured RAW264.7 cells and isolated PBMCs to assess the proportions of macrophage phenotypes (M1 CD86+; M2 CD206+). CD86 and CD206 were stained with anti-CD86-PE (105008, BioLegend, San Diego, CA, USA) and anti-CD206-APC (BioLegend, 141708), respectively, following the manufacturer’s instructions. A minimum of 10⁵ cells were collected and analyzed using flow cytometer (DxFLEX, Beckman Coulter, Brea, CA, USA) and FlowJo V10 (BD Biosciences, Franklin Lakes, NJ, USA).

Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA) and SPSS 21.0 (IBM-SPSS Statistics, Chicago, IL, USA). A p-value $\le$ 0.05 was deemed statistically significant. Results are expressed as mean (SD). Normality was assessed using the Shapiro-Wilk test. The Brown-Forsythe test and Bartlett’s test were utilized to confirm the homogeneity of variance assumption. One-way ANOVA was used to compare groups before Dunnett’s multiple comparison test.

The role of Dex in the CLP mice model was assessed by measuring lung damage through histological examination after H&E staining. Compared with the control group, the lung injury score in CLP group was significantly higher (p 0.0001, Fig. 1A), due to severe congestion, hemorrhage, and neutrophil infiltration (Fig. 1A). However, lung injury severity was reduced by treatment with Dex (Fig. 1A). To assess lung injury severity, we conducted a TUNEL assay to measure apoptosis in lung tissue. The proportion of TUNEL-positive cells in the lungs of the CLP group was also significantly higher than control group (p 0.0001, Fig. 1B), but Dex treatment decreased the proportion of these cells (Fig. 1B). Inflammation is a distinctive feature of acute lung injury. Elevated levels of inflammatory cytokines are sensitive indicators of ALI [31]. We detected the expression levels of cytokines in BALF and serum. The CLP challenge increased IL-6, TNF-$\alpha$, and IL-17A levels in both BALF and serum, while Dex treatment reduced these pro-inflammatory cytokines (Fig. 1C). The release of IL-10 in CLP+Dex group was significantly more than that in CLP group (p 0.001, Fig. 1C).

Fig. 1.

Dex treats lung injury in CLP mice. (A) H&E staining scores and representative pictures (Scale bar = 100 µm, 50 µm). (B) The TUNEL-positive cells in the lung (Scale bar = 100 µm, 50 µm). Five random fields (200$\times$) were evaluated for positive cells. (C) Expression of cytokines in BALF and serum. (D) Nuclear p-p65 protein levels in mouse lung tissues. Use at least three independent experiment to calculate the mean (SD). n = 5. ns, no significance, *p 0.05, ***p 0.005, ****p 0.0001. CLP, cecal ligation puncture; BALF, bronchoalveolar lavage fluid; Dex, Dexmedetomidine; IL, interleukin; TNF, tumor necrosis factor.

NF-$\kappa$B significantly regulates inflammation by modulating pro-inflammatory cytokine production [32]. To evaluate Dex’s effect on NF-$\kappa$B, the nuclear protein of mouse lung tissue was extracted and detected. The results suggested that Dex treatment significantly reduced CLP- mediated NF-$\kappa$B (p65) nuclear translocation (p 0.01; Fig. 1D).

ALI involves pyroptosis, a form of programmed cell death. Recent study indicates that the lethality of the CLP mice is primarily dependent on caspase-11 activation, with the mechanism involving coordinated activities of the RAGE and caspase-11/Gasdermin-D (GSDMD) pathways [33]. To determine if Dex mitigates CLP-induced lung injury by affecting expression of RAGE and caspase-11-dependent pyroptosis, we assessed pyroptosis-related protein and mRNA levels in lung tissue. qRT-PCR analysis revealed significantly elevated mRNA levels of Rage and caspase-11 in the CLP group exhibited higher levels than control group, but this difference was reduced with Dex treatment (Fig. 2A). Western blot analysis revealed that Dex treatment reduced the protein levels of cleaved-caspase-11, cleaved-GSDMD, and IL-1$\beta$. (Fig. 2B,C). Immunofluorescence images revealed elevated levels of RAGE (green) and caspase-11 (red) in CLP than in controls (p 0.0001, Fig. 2D), and Dex treatment reduced RAGE and caspase-11 expression (Fig. 2D).

Fig. 2.

Dex inhibits RAGE/caspase-11 expression in the lung of CLP mice. (A) The lung mRNA levels of RAGE and caspase-11. (B) The RAGE protein levels in lung detected by western blotting. (C) Western blot detected the level of pyroptosis-related proteins. (D) Immunofluorescence assays of RAGE and caspase-11 (Scale bar = 50 µm, 20 µm). Use at least three independent experiment to calculate the mean (SD). n = 5. *p 0.05, **p 0.01, ***p 0.005, ****p 0.0001. RAGE, receptor for advanced glycation end products; IL, interleukin.

Given the importance of M1 and M2 polarization in ALI development, a study was conducted to determine if Dex’s anti-inflammatory effects influence this polarization. Flow cytometry’ results founded that Dex treatment reduced the proportion of M1 (F4/80+CD86+) (p 0.01, Fig. 3A), and elevated the proportion of M2 (F4/80+CD206+) (p 0.05, Fig. 3A), relative to the CLP group. The Western blot results founded that Dex reduced the expression of iNOS (p 0.01, Fig. 3B), which is the M1 macrophage biomarker. More importantly, Dex increased the expression of Arg-1 (p 0.05, Fig. 3B), which is the recognized marker of M2 macrophages. We then used qRT-PCR to test the expression levels of macrophage polarization biomarkers. The qRT-PCR experiments confirmed the western blot assay results, demonstrating that Dex decreased iNOS mRNA levels while increasing Arg-1 mRNA levels (Fig. 3C).

Fig. 3.

Dex inhibited M1-type and promoted M2-type polarization in CLP-induced mouse model. (A) PBMCs were isolation and stained with F4/80-FITC/CD86-PE (M1) and F4/80-FITC/CD206-APC (M2), flow cytometry was used to test macrophage subset expression levels. (B,C) The western blot and qRT-PCR was used to test iNOS (M1) and Arg-1 (M2) expressed in the lung upon CLP-induced mouse model. Use at least three independent experiment to calculate the mean (SD). n = 5. ns, no significance, *p 0.05, **p 0.01, ***p 0.005, ****p 0.0001. PBMCs, peripheral blood mononuclear cells; iNOS, inducible nitric oxide synthase; qRT-PCR, quantitative real-time PCR; M1, classical activation phenotype; M2, alternate activation phenotype; Arg-1, Arginase 1.

In RAW264.7, qRT-PCR results demonstrated that the LPS+Dex group exhibited significantly lower Inos mRNA levels compared to the LPS group (p 0.0001, Fig. 4A), and increased mRNA levels of Arg-1 (Fig. 4A). Western blot analysis revealed that the LPS+Dex group exhibited reduced iNOS protein expression and increased Arg-1 protein expression compared to the LPS group (Fig. 4B). Flow cytometry results showed that the LPS+Dex group had fewer M1 (CD86+) and more M2 (CD206+) compared to the LPS group (Fig. 4C). The images of immunofluorescence of RAW264.7 indicated that LPS challenge induced the high intensity of iNOS and suppressed the intensity of CD206 (Fig. 4D). Dex intervention reduced iNOS fluorescence intensity and enhanced CD206 fluorescence intensity in RAW264.7 (Fig. 4D).

Fig. 4.

Dex inhibited M1-and promoted M2-type polarization in LPS-Induced RAW264.7. (A,B) The qRT-PCR and western blot was used to test iNOS (M1) and Arg-1(M2) expressed in RAW264.7. (C) Flow cytometry was used to test macrophage subset expression levels of CD86-PE and CD206-APC in RAW264.7. (D) Immunofluorescence intensity of iNOS (green) and CD206 (red) in RAW264.7 (Scale bar = 20 µm). Use at least three independent experiment to calculate the mean (SD). n = 5. ns, no significance, *p 0.05, **p 0.01, ***p 0.005, ****p 0.0001. iNOS, inducible nitric oxide synthase. LPS, ipopolysaccharide.

Macrophages play a major role in tissue homeostasis [34]. A recent study has indicated that caspase-11-induced macrophage pyroptosis is critical for the development of sepsis [35]. Over-activation of pyroptosis would lead to uncontrolled inflammation, and may aggravate the severity of ALI. In our in vitro experiments, immunofluorescence images showed that RAGE and caspase-11 intensities in the LPS group were higher than in controls, but were decreased in the LPS+Dex group (p 0.0001, Fig. 5B). Western blot analysis demonstrated that Dex decreased LPS-induced expression of cleaved-caspase-11, RAGE, cleaved-GSDMD, and IL-1$\beta$ (Fig. 5C,D). qRT-PCR results founded that Dex inhibited the mRNA levels of IL-1$\beta$ and IL-6 (Fig. 5A).

Fig. 5.

Dex suppressed RAGE expression and caspase-11-mediated pyroptosis in RAW264.7. (A) The IL-1$\beta$ and IL-6 mRNA levels in RAW264.7. (B) Immunofluorescence intensity of RAGE (green) and caspase-11 (red) in RAW264.7 (Scale bar = 20 µm). (C) RAGE protein levels detected by western blot. (D) Western blot detected the level of pyroptosis-related proteins. Use at least three independent experiment to calculate the mean (SD). n = 5. *p 0.05, **p 0.01, ****p 0.0001. IL, interleukin.