Animals

C57BL/6 mice (male, 6–8 weeks) were purchased from the Animal Center of Central South University and housed under specific pathogen-free conditions. After a week of adaptive feeding, to observe the preventive effect of cyasterone, mice were divided into five groups randomly: control, CLP, CLP + L, CLP + M, and CLP + H group. Mice were injected intraperitoneally (i. p.) with cyasterone (1, 5, or 25 mg/kg/d) for 3 days. To verify the preventive effect of cyasterone, the mice were randomly divided into five groups: control, CLP, CLP + M, CLP + M + ML385, and CLP + M + LY294002. Mice were injected intraperitoneally (i. p.) ML385 (30 mg/kg/d, selleck, China) or LY294002 (5 mg/kg/d, selleck, China) for 3 h following administration intraperitoneally (i. p.) with cyasterone (5 mg/kg/d) for 3 days. To compare the drug effects of cyasterone and the positive drug dexamethasone, mice were divided into four groups randomly: control, CLP, CLP + M, and CLP + DEX. Mice were injected intraperitoneally (i. p.) with cyasterone (5 mg/kg/d) or dexamethasone (5 mg/kg/d) for 3 days [21, 22]. Cyasterone (Cat#: S9414, Selleck, China) was diluted with 0.9% saline, containing 40% PEG300 (Macklin, China), 5% dimethyl sulfoxide (DMSO) (Macklin, China), and 5% Tween80 (Macklin, China) (v/v/v). This study was conducted following the welfare and ethical principles of experimental animals. It was approved by the Laboratory Animal Welfare and Ethical Committee of Central South University (Approval No. CSU-2022-0095).

CLP model

Mice were fasted for 12 h prior to surgery. After anesthesia, the skin was disinfected, and the cecum was exposed by making a 1 cm incision toward the middle of the abdomen of the mice. The cecum was ligated with 4–0 braided silk thread through the midpoint between the root and the end of the cecum. A 21-gauge needle was inserted into the ligated cecum, and a small drop of intestinal contents was extruded to induce infection. Finally, the cecum was reset, and the incision was closed [23]. For the control group, the abdomen was opened, and then the incision was closed. 24 h later, lung tissue or bronchoalveolar lavage fluid (BALF) was obtained for subsequent experiments.

Histopathological evaluation

The right mid-lung of the mice was fixed, embedded in 4% paraformaldehyde neutral buffer overnight, cut into 4 µm sections, and stained with hematoxylin–eosin [24]. The severity of the injury was graded from 0 to 5: alveolar wall intact without thickening, no inflammatory infiltrate, no congestion, 0; alveolar wall thickening, slight inflammatory cell infiltration, 1; alveolar wall thickening, slight inflammatory cell infiltration, capillary dilation, 2; alveolar wall thickening significantly, inflammatory cell infiltration, interstitial congestion, 3; alveolar wall thickening, severe inflammatory cell infiltration, diffuse distribution, destruction of alveolar structure, necrosis and decompensation of bronchial mucosa epithelial cells, and solidification of lung tissue, 4; the alveolar wall thickening, severe inflammatory cell infiltration, diffuse distribution, destruction of alveolar structure, and solidification of lung tissue, 5. The inflammation score was measured independently by three pathologists blinded to the experiment.

Lung wet to dry (W/D) ratio

The left lung was removed and weighed on a precision electronic scale (BSA224S-CW; sartorius, Germany), then placed in an oven and baked at 56 ℃ for 48 h until a constant weight was obtained as dry weight. The W/D ratio was calculated to evaluate the degree of pulmonary edema.

M1 macrophage activation

The proportion of M1 macrophages in BALF was determined by flow cytometry. Cells were stained with PE-conjugated anti-mouse F4/80 (Cat# E-AB-F0995UD, Elabscience, China), APC-conjugated anti-mouse CD80 (Cat# E-AB-F0992E, Elabscience, China), and FITC-conjugated anti-mouse CD11b (Cat# E-AB-F1081C, Elabscience, China). Briefly, F4/80 is employed for labeling macrophages in BALF, while CD80 is utilized for marking M1-type macrophages.

Enzyme-linked immunosorbent assay (ELISA)

BALF was captured by 2 intratracheal injections of 0.8 ml of cooled phosphate buffered saline (PBS). BALF was centrifuged at 4 ℃ for 10 min at 1500 r/min, lysed in ACK lysis buffer for 5 min, washed twice with ice-cold PBS, and centrifuged for 5 min. Subsequently, the contents of tumor necrosis factor-alpha (TNF-α) and in-terleukin-1 beta (IL-1β) in BALF were measured using ELISA kits (Cat# TNF-α: 88–7324; IL-1β: 88–7013; Invitrogen, USA). The contents were assayed by comparison of the optical density (450 nm) with the standard curve [25].

Measurement of MPO, MDA, GSH and SOD levels

Lung tissues were lysed in extraction buffer, and all procedures were conducted strictly according to the instructions of superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH) and malondialdehyde (MDA) assay kits (Cat# SOD: A001-3-2; MPO: A044-1-1; GSH: A006-2-1; MDA: A003-1-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Cells culture

Primary murine peritoneal macrophages were isolated from C57BL/6 mice. Mice were injected with 3 ml of 3% thioglycolate (Sigma-Aldrich, St. Louis, MO, USA). 4 days later, peritoneal macrophages were obtained by intraperitoneal lavage with prechilled RPMI 1640 (Procell, Wuhan, China). Cells were collected and centrifuged at 1500 rpm for 10 min at 4 ℃, and the precipitate was resuspended in RPMI 1640. Cells were placed in 6-well plates (2 × 106 cells/well) for protein detection and ROS assessment, 12-well plates (1 × 106 cells/well) for RNA detection, and 24-well plates (0.5 × 106 cells/well) for immunofluorescence staining. After 2 h, RPMI 1640 containing 10% neonatal bovine serum (NBS, Sigma, USA) and 1% streptomycin/penicillin (Gibco, Waltham, MA, USA) were replaced, and non-adherent cells were removed. Cells were cultured in a humified CO2 incubator at 37 ℃. MLE-12 cells (provided by the State Key Laboratory of Genetics, Changsha) were cultured with 100 U/ml of streptomycin/penicillin and fetal bovine serum (10%) in DMEM (Gibco, USA) and arranged in an incubator containing 5% CO2 with a suitable temperature (37 ℃).

All cells were incubated with cyasterone (10 μM, 30 μM or 100 μM) for 24 h before being stimulated with or without LPS (100 ng/ml, from Escherichia coli O111: B4, Sigma-Aldrich, USA) according to different requirements.

CCK-8 assay

Cell viability was assayed with Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan). Primary murine peritoneal macrophages and MLE12 cells were incubated on 96-well plates and treated with different concentrations of cyasterone (1 μM, 3 μM, 10 μM, 30 μM, 100 μM, 200 μM). After 24 h, 10 μl CCK-8 reagent was added to each well and incubated at 37 ℃ for 1 h. OD values were measured by 450 nm enzyme assay.

Measurement of MDA, GSH and SOD levels

Primary peritoneal macrophages were lysed in extraction buffer, and all procedures were conducted strictly according to the instructions of superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH) and malondialdehyde (MDA) assay kits (Cat# SOD: A001-3-2; MPO: A044-1-1; GSH: A006-2-1; MDA: A003-1-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Measurement of ROS content

Primary peritoneal macrophages and MLE12 cells were incubated with 20 μM 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) (Thermo, D399) for 30 min at 37 ℃ in the dark and washed three times with pre-cooled PBS. Fluorescence microscopy (Nikon Ti-s, Tokyo, Japan) and flow cytometry (BD LSRFortessa, Franklin Lakes, NJ, USA) were performed to observe the production of ROS.

The apoptosis assay

Apoptosis assays were performed using the Annexin V-FITC Apoptosis Detection Kit (Cat# C1062L, Beyotime, China) according to the manufacturer's instructions. Briefly, MLE12 cells were washed twice with cold PBS and resuspended in PBS at a concentration of 1 × 106 cells/ml. Subsequently, cells were stained with 5 μl of FITC Annexin V and 5 μl of propidium iodide, and incubated at room temperature in the dark for 15 min. Finally, the samples were analyzed using flow cytometry (BD LSRFortessa, Franklin Lakes, NJ, USA), and the results were analyzed using Flowjo10 software.

Real-time quantitative polymerase chain reaction (Q-PCR)

Total RNA from lung tissues and cells was extracted with TRIzol (Thermo Fisher Scientific, USA) and reverse transcribed into cDNA using a reverse transcription kit (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s protocol. Q-PCR was performed using SYBR GREEN (Promega, USA) and Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA) Q-PCR. PCR conditions were as follows. 95 ℃ for 2 min, followed by 40 cycles of 95 ℃ for 3 s and 60 ℃ for 30 s, plus a 60–95 ℃ melting curve. Data were expressed in C.T. values normalized to β-actin, and the fold change between control and treated groups was determined using the 2-ΔΔCt method. The sequences are shown in Table 1.

Table 1 Primer sequences for qPCRWestern blot analysis

Lung tissue and cells were plunged in ice-cold RIPA lysis buffer with protease and phosphatase inhibitors (Abcam, Cambridge, UK). Per the manufacturer's instructions, protein concentrations were quantified using the BCA Protein Assay Kit (Beyotime Biotech, Shanghai, China). Briefly, protein samples were loaded and separated on SDS-PAGE gels, transferred to PVDF membranes (Bio-Rad, USA), and blocked with 5% (w/v) skimmed milk containing 0.1% PBS buffer Tween 20 (v/v) or 5% (w/v) protease free bovine serum Albumin (BSA) (Sigma-Aldrich, USA). Then the membranes were incubated with β-actin antibody (1:5000, Cat#: 81,115-1-RR, SAB), NLRP3 anti-body (1:2000, Cat#: 15,101, CST), pro-caspase-1/p10/p20 antibody (1:1000, Cat#: ab179515, Abcam),Heme Oxygenase 1 antibody (1:1000, Cat#: ab52947, Abcam), NQO1 antibody (1:3000, Cat#: ab80588, Abcam),p-GSK3β (S9) antibody (1:1000, Cat#: WL03518, WanleiBio), GSK3β antibody (1:300, Cat#: WL01456, WanleiBio), p-AKT (Ser473) antibody (1:500, Cat#: WLP001a, WanleiBio), AKT antibody (1:500, Cat#: WL0003b, WanleiBio)overnight at 4 ℃, and secondary antibodies (1: 5000, SAB) were labeled with horseradish peroxidase for 2 h at room temperature. After washing with PBST, bands were detected with Luminata™ Crescendo chemiluminescence horseradish peroxidase substrate (Millipore, USA) and scanned using GeneGnome XRQ imager (Syngene, UK).

Immunofluorescence

Primary peritoneal macrophages were fixed in 4% paraformaldehyde for 15 h, permeabilized with 0.5% (v/v) Triton X-100 for 20 min, blocked with goat serum (ZSGB Bio, China) for 30 min, and then incubated with Nrf2 antibody (1:100, Proteintech, China) overnight at 4 ℃. After washing with PBST, samples were incubated with fluorescent secondary antibody (1:100, Proteintech, China) in PBST for 1 h at room temperature, and DAPI was used to stain cell nuclei for 5 min and observed under the fluorescence microscope (Nikon Ti-s, Tokyo, Japan).

Statistical analysis

All experiments were independently repeated three times. All data were presented as means ± standard deviations and analyzed using GraphPad Prism 9.0. A one-way analysis of variance and Student-Newman-Kersee (SNK) tests were used to compare the groups. P value < 0.05 was defined as a statistically significant difference.