LPS and SPD were obtained from Sigma-Aldrich (Merck KGaA). Nigericin (cat. no. HY-100381) and chloroquine (CQ) (cat. no. HY-17589A) were provided by MedChemExpress. Antibodies against caspase-1 (cat. no. ab207802), GSDMD (cat. no. ab219800), and sequestosome 1 (SQSTM1)/p62 (cat. no. ab109012) were supplied by Abcam. The antibody against microtubule-associated protein light chain 3 (LC3) I/II (cat. no. 12741) was supplied by Cell Signaling Technology, Inc. The HRP-conjugated β-actin antibody (cat. no. ET1702-67) and goat anti-rabbit IgG-HRP antibody (cat. no. HA1001) were supplied by HUABIO. Calcein/PI Cell Viability and Cytotoxicity Assay Kit (cat. no. C2015M), RIPA lysis buffer (cat. no. P0013B), Cell Counting Kit-8 (CCK-8) (cat. no. C0038), BCA Protein Concentration Assay Kit (cat. no. P0010S) were obtained from Beyotime Institute of Biotechnology. The ECL chemiluminescent substrate (cat. no. RM02867) was obtained from ABclonal. The CheKineTM Micro Lactate Dehydrogenase (LDH) Assay Kit (cat. no. KTB1110) was provided by Abbkine Scientific Co., Ltd.

Male C57BL/6 mice (n=30), weighing 20-25 g and aged 8-10 weeks, were obtained from Jinan Pengyue Experimental Animal Breeding Center (Jinan, China). The mice were housed in a controlled environment with regulated temperature (22-24˚C) and humidity (50-60%), maintained on a 12-h light/dark cycle, and provided with a standard diet and tap water ad libitum. SPD was administered at doses of 10 mg/kg once daily, 10 mg/kg twice daily and 20 mg/kg once daily (10). Mice were pretreated with different concentrations of SPD for 3 days, followed by the induction of ALI via intratracheal instillation of LPS (5 mg/kg) for 24 h (11,12). Mice were anesthetized by an i.p. injection of 0.3% sodium pentobarbital (30 mg/kg), and then subjected to intratracheal instillation of LPS. After 24 h, mice were euthanized via an i.p. injection of an overdose of sodium pentobarbital (200 mg/kg). The present study was approved (approval no. KYDWLL-202212) by the Ethics Committee of Qilu Hospital of Shandong University (Qingdao, China). All animal experiments were conducted in accordance with the ARRIVE guidelines (13) and complied with the NIH Guide for the Care and Use of Laboratory Animals (14).

The collected BALF was centrifuged at 500 x g for 10 min at 4˚C. The protein concentration of the supernatant was determined using the BCA Protein Concentration Assay Kit.

The right lung lobe was fixed by soaking in 10% formalin buffer at room temperature for 48 h. It was then embedded in paraffin and sliced. The lung tissue sections (4 µm in thickness) were stained with hematoxylin for 2 min and eosin for 10 min at room temperature. Images were observed under a light microscope (Olympus Corporation).

Following the removal of the right lung, its wet weight was measured and then dried in an oven at 60˚C for ~48 h. The W/D was calculated by dividing the wet weight by the dry weight.

The HULEC-5a (cat. no. C1402) immortalized pulmonary microvascular endothelial cell line (cat. no. CVCL_0A11) was purchased from Wuhan SUNNCELL Biotechnology Co., Ltd. To establish the pyroptosis model of PMVECs, the cells were stimulated with LPS (1 µg/ml) for 4 h, followed by treatment with nigericin (NIG) (20 µM) for 1 h. In the SPD group, the cells were pretreated with SPD for 12 h prior to stimulation with LPS and NIG. In the LPS + NIG + SPD + CQ group the cells were pretreated with CQ (20 µM) for 2 h prior to stimulation with SPD. Cells were incubated with CCK-8 working solution for 2 h, followed by cell viability detection. The necrotic cells were detected using the Calcein/PI Cell Viability and Cytotoxicity Assay Kit, according to the manufacturer's instructions. The cells were observed using a fluorescence microscope (Nikon, Ti-E Live Cell Imaging System; Nikon Corporation). LDH concentrations in cell homogenates were measured using the CheKine^™ Micro Lactate Dehydrogenase (LDH) Assay Kit. After incubation, the cells were analyzed using an automatic microplate reader (SpectraMax i3x; Molecular Devices, LLC).

Lung tissue homogenate and cell lysate were prepared in RIPA lysis buffer. The protein concentration of the extracted samples was determined using a BCA assay kit. Equal protein amounts (20 µg) were analyzed using 12% SDS-PAGE electrophoresis, followed by transfer to a PVDF membrane. The membrane was blocked with 5% non-fat milk at room temperature for 2 h and then incubated overnight at 4˚C with primary antibodies against caspase-1 (1:1,000), GSDMD (1:1,000), SQSTM1/p62 (1:10,000), LC3 I/II (1:1,000) and β-actin (1:2,000). The membrane was then incubated with the secondary antibody (1:10,000) at room temperature for 60 min and then developed using ECL chemiluminescent substrate. Densitometric analysis was performed using ImageJ (version 1.54; National Institutes of Health).

All data are presented as the mean ± standard error of the mean. The experiments were repeated 3 times. Unpaired Student's t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Post hoc Tukey's HSD test was applied following ANOVA to identify specific group differences. All statistical analyses were performed using GraphPad Prism (version 9.0.0, GraphPad Software; Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. S1, SPD at 10 mg/kg twice daily significantly alleviated lung injury. Consequently, this dosing regimen (10 mg/kg twice daily) was selected for all subsequent experiments. Notably, mice treated with LPS exhibited significant lung damage, including diffuse alveolar and interstitial edema, when compared with the control group as revealed in Fig. 1A. SPD pretreatment significantly reduced alveolar and interstitial edema and inflammatory cell infiltration. Lung W/D and protein levels in BALF were also measured (Fig. 1B and C) to assess pulmonary vascular endothelial permeability. SPD pretreatment significantly reduced the lung W/D and protein in BALF. These findings indicated that SPD pretreatment effectively reduces LPS-induced lung damage.

To explore whether SPD alleviates lung damage through its effect on pyroptosis, the expression levels of pyroptosis-related proteins were detected in lung tissue. As shown in Fig. 2A and B, LPS administration significantly increased the levels of cleaved caspase-1 and the gasdermin D N-terminal (GSDMD^NT) in lung tissue, as compared with the control group. By contrast, SPD pretreatment decreased the levels of these proteins. This suggested that SPD reduces LPS-induced lung damage by inhibiting pyroptosis in lung tissue.

A concentration-dependent NIG screening (5, 10, 20, 30 and 40 µM) was performed on LPS-primed (1 µg/ml, 4 h) PMVECs (15-17). As shown in Fig. S2A, 20 µM NIG was identified as the optimal concentration, balancing pyroptosis induction with preserved assay sensitivity, and was thus employed in further studies. Following SPD pretreatment (20, 40, 60, 80, 100 and 200 µM) and sequential LPS/NIG challenge, CCK-8 analysis (Fig. S2B) revealed maximal viability enhancement at 40 µM. Absence of additional benefit at higher concentrations (80-200 µM) established 40 µM as the optimal concentration for subsequent experimental applications.

To investigate the effect of SPD on endothelial cell pyroptosis, cells were treated with LPS and NIG. Pyroptotic cells form membrane pores that permit extracellular dyes, such as propidium iodide (PI), to enter and stain the nucleus (18). As revealed in Fig. 3A and B, a significant increase in the percentage of PI-positive cells was observed following stimulation with LPS and NIG. In addition, the stimulation with LPS and NIG led to a significant increase in the release of LDH from endothelial cells (Fig. 3C). Following stimulation with LPS and NIG, the expression levels of cleaved caspase-1 and GSDMD^NT increased in endothelial cells (Fig. 3D and E). However, SPD reduced all LPS- and NIG-induced effects.

SPD promotes autophagy, which can inhibit pyroptosis through different mechanisms. It was examined whether SPD restores autophagy in LPS- and NIG-treated endothelial cells. This was performed by measuring the levels of autophagic proteins LC3-II and p62. As shown in Fig. 4A and B, the levels of autophagy markers LC3-II and p62 increased in LPS- and NIG-treated endothelial cells. Following SPD treatment, the levels of LC3-II and p62 in endothelial cells affected by LPS and NIG decreased. Notably, the addition of CQ to the LPS + NIG + SPD group further elevated LC3-II and p62 levels, suggesting that the protective effect of SPD was attenuated by CQ-mediated autophagy blockade. These results indicated that LPS and NIG inhibited autophagic flux in PMVECs, while SPD successfully restored this disrupted process.

To better understand this process, the autophagy inhibitor CQ was used to block autophagic flux. As revealed in Fig. 5, the LPS + NIG + SPD + CQ group had more PI staining-positive cells and higher LDH release than the LPS + NIG + SPD group. In addition, this group exhibited increased levels of cleaved caspase-1 and GSDMD^NT. These findings indicated that by restoring autophagic flux, SPD mitigates pyroptosis in LPS- and NIG-treated PMVECs.