The animal experiments were performed based on the guidance for the Care and Use of Laboratory Animals (National Institutes of Health) (23). All studies were performed between June 2020 and February 2022 and were approved by the Animal Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiaotong University, School of Medicine, Shanghai, China (approval no. XHEC-NSFC-2020-038). For in vivo experiments, a total of 36 male mice (age, 4-6-weeks, Institute of Cancer Research) weighing 20-25 g were housed at 22-25˚C with 12/12-h day/night cycle, humidity of 50-60% and free access to food and water at specific-pathogen-free grade, were randomly divided into three groups: Control, LPS and LPS + Cit (n=12/group). In each group, one set of mice (n=6/group) was used for histological analysis, while a second set (n=6/group) was used to analyze bronchoalveolar lavage fluid (BALF). For the in vitro experiments, primary pulmonary endothelial cells were isolated from 20 additional male mice (age 3 weeks, weight, 15 g, Shanghai Jihui Laboratory Animal Care Co., Ltd.). These mice were assigned to five groups: Control, LPS, LPS + Cit, LPS + N-acetyl-L-cysteine (NAC; 5 mM) and LPS + Cit + ML385 (5 µM; n=4/group). Control mice were intraperitoneally (i.p.) injected with saline for 6 h. LPS group: Mice were i.p. injected with LPS (10 mg/kg; Sigma-Aldrich; Merck KGaA) for 6 h. LPS + Cit Cit (MedChemExpress) was i.p. injected at a dose of 1 g/kg once daily for 1 week, followed by i.p. injected with LPS for 6 h. Lung tissue was collected following treatment. All mice were humanely sacrificed by CO₂ inhalation (May 2021) using a gradual displacement of 30-70% of the chamber volume/min when they met the following criteria: Loss of body weight >20%, difficulty breathing and sharp decrease in body temperature (24). Absence of movement and breathing, as well as presence of cardiac arrest and pupil dilation for 5 min, was used to confirm death.

Left lung lobe samples were collected, fixed with 4% paraformaldehyde at room temperature for 1 day and lung tissue was cut into 5-µm thick slices, followed by staining with hematoxylin for 5-10 min at room temperature and eosin (Beyotime Institute of Biotechnology) for 1-2 min at room temperature. Images were recorded using a light microscope (Olympus Corporation). Lung morphometry and injury analysis were scored by two independent pathologists blinded to the treatment strategy based on inflammatory infiltration and thickness of the alveolar wall. A tissue injury scoring system was implemented as follows: 0, no injury; 1, slight; 2, mild; 3, moderate; 4, moderate-to-severe and 5, severe inflammatory injury (25).

Pulmonary edema index was estimated by calculating pulmonary W/D weight ratio. The fresh upper left lung lobe was isolated, weighed and dried at 65˚C for 48 h in an oven. The dry weight was determined and the W/D ratio calculated.

Following anesthesia with i.p. 100 mg/kg ketamine and 10 mg/kg xylazine, BALF was isolated from mice via a tracheal tube by infusing 1 ml chilled PBS three times. Following collection, mice were euthanized via CO₂ inhalation. BALF was centrifuged at 500 x g for 10 min at 4˚C and the cell precipitate was resuspended in 200 µl PBS. Cell counting was performed manually under a 40x magnification using a light microscope and total protein concentration in the BALF supernatant was determined using commercial kits (Enhanced BCA Protein Assay kit; cat no. P0009; Beyotime Institute of Biotechnology).

Lung tissue specimens (100 mg each) were homogenized in PBS containing a protease inhibitor cocktail (cat no. P9599; Sigma-Aldrich; Merck KGaA). Supernatant was obtained via centrifugation at 1,000 x g for 10 min at 4˚C. IL-1β and IL-18 concentration in the lung tissue was measured using commercial ELISA kits for IL-1β (Mouse IL-1β/IL-1F2 Quantikine ELISA; cat no. MLB00C; R&D System, Inc.) and IL-18 (Mouse IL-18/IL-1F4 ELISA; cat no. 7625; R&D Systems, Inc.) according to the manufacturer's instructions.

TUNEL detection was used to examine apoptosis in lung tissue. Lung tissues were fixed and embedded as aforementioned. Paraffin-embedded 5-µm sections of lung tissue were rehydrated and antigen retrieval was performed by boiling in citrate buffer (pH=6.0). TUNEL staining was conducted at room temperature for 1 h according to the manufacturer's instructions (One Step TUNEL Apoptosis Assay kit; cat. no. C1090; Beyotime Institute of Biotechnology). Finally, nuclei were counterstained with DAPI (1 µg/ml) at room temperature for 2 h. The stained tissue was observed under a fluorescence microscope (magnification, x20; Nikon Corporation).

MLVECs were isolated as previously described (26). Briefly, peripheral and subpleural tissue was cut into small pieces (1-mm³) and tissue fragments were placed into 60-mm cell culture dishes. DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 20% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) was added as culture medium. Following 48 h incubation at 37˚C, the tissue was removed and MLVECs were cultured in DMEM supplemented with 10% FBS (Gibco) and 0.01% ampicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO₂. MLVECs were characterized by their cobblestone morphology and stained with factor VIII-related antigen (cat. no. sc-53466, 1:100, Santa Cruz Biotechnology, Inc.) at 4˚C for one night as previously described (27). Cells after 3-4 passages were used in subsequent experiments.

Lung tissue (5-µm sections) were deparaffinized in xylene and rehydrated with graded ethanol. For antigen retrieval, sections were boiled in citrate buffer (pH=6.0). Following treatment with 5% bovine serum albumin (BSA; cat. no. A8010; Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at room temperature, the slices were incubated with F4/80 antibody (1:300; cat. no. GB11027; Servicebio Technology Co., Ltd.) overnight at 4˚C followed by incubation with cyanine 3-labeled goat anti-rabbit secondary antibody (1:300; cat. no. GB21303, Servicebio Technology Co., Ltd.). MLVECs were fixed in 4% paraformaldehyde at room temperature for 10 min, treated with 5% BSA for 30 min at room temperature and incubated with anti-Nrf2 primary antibody (1:200; cat. no. bs-1074R; BIOSS Biotech Co., Ltd.) at 4˚C overnight followed by incubation with FITC-labeled goat anti-rabbit secondary antibody (1:300; cat. no. GB22303; Servicebio Technology Co., Ltd.). Cell nuclei were stained with DAPI (1 µg/ml, Beyotime Institute of Biotechnology) at room temperature for 2 h. Images were obtained using a Nikon fluorescence microscope (magnification, x20; Nikon Corporation).

The fluorescence probe 2',7'-dichlorofluorescein diacetate (DCFH-DA; cat. no. S0033S; Beyotime Institute of Biotechnology) was used to monitor the accumulation of intracellular ROS. Following treatment with LPS, LPS + Cit or LPS + Cit + ML385 for 6 h, MLVECs were incubated with DCFH-DA (10 µM) at 37˚C for 30 min, followed by washing twice with PBS. Images were collected using a Nikon fluorescence microscope (magnification, x20; Nikon Corporation).

Lung tissue or MLVECs were homogenized in cold RIPA buffer (cat. no. P0013; Beyotime Institute of Biotechnology) containing proteinase and phosphatase inhibitor cocktail (Roche Diagnostics) by a mechanical homogenizer. Following centrifugation at 1,000 x g and 4˚C for 10 min, the supernatant was collected. The total protein was quantified using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). Protein (40 µg/lane) was separated by SDS-PAGE using 10 or 12.5% SDS-PAGE Gel Fast Preparation kits (cat. no. PG212, cat. no. PG213; Epizyme, Inc.) and transferred onto PVDF membranes. Subsequently, the membranes were blocked with 5% skimmed milk at room temperature for 2 h. Membranes were incubated with primary antibodies against NLRP3 (cat. no. 15101S, 1:1,000; Cell Signaling Technology, Inc.), GSDMD (1:500; cat. no. ab155233; Abcam), caspase-1 (cat. no. A0964; 1:1,000; ABclonal Biotech Co., Ltd.), Bcl-2 (cat. no. ab32124; 1:1,000; Abcam), Bax (cat. no. ab182733; 1:1000, Abcam) and β-actin loading control (cat. no. A1978, 1:3,000; Sigma-Aldrich; Merck KGaA) overnight at 4˚C. Following incubation with primary antibodies, the membranes were incubated with secondary HRP-conjugated Affinipure Goat Anti-Rabbit (1:5,000; cat. no. SA00001-2; ProteinTech Group, Inc.) and anti-Mouse IgG (1:5,000; cat. no. SA00001-1; ProteinTech Group, Inc.) for 1 h at room temperature. An enhanced chemiluminescence solution (cat. no. BL523A; Hefei Labgic Technology Co., Ltd.) was used to visualize the immunoblots. Finally, the protein bands were semi-quantified using ImageJ software (V1.4.3.67; National Institutes of Health) and normalized to β-actin.

TRIzol^® reagent (Thermo Fisher Scientific, Inc.) was used to extract total RNA from lung tissue and MLVECs, according to the manufacturer's protocol. Total RNA was reverse-transcribed into cDNA using the One Step PrimeScript^™ RT-PCR kit (cat. no. RR064A; Takara Biomedical Technology Co., Ltd.), according to the manufacturer's protocol. qPCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.), according to the manufacturer's protocol. The amplification was performed using a Step One Plus system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the following thermocycling conditions: Initial denaturation at 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec, 60˚C for 30 sec and 72˚C for 30 sec and final extension at 72˚C for 5 min. The following primer pairs were used: NLRP3 forward, 5'-TCTGACCTCTGTGCTCAAAACCAAC-3' and reverse, 5'-TGAGGTGAGGCTGCAGTTGTTAAT-3'; ASC forward, 5'-ACTCATTGCCAGGGTCACAGAAGTG-3' and reverse, 5'-GCTTCCTCATCTTGTCTTGGCTGGT-3'; Nrf2 forward, 5'-CAGTCTTCACCACCCCTGAT-3' and reverse, 5'-CAGTGAGGGGATCGATGAGT-3'; HO-1 forward, 5'-AGAGGCTAAGACCGCCTTCC-3' and reverse, 5'-TCTGACGAAGTGACGCCATC-3' and β-actin forward, 5'-TGTATGCCTCTGGTCGTAC and reverse, 3'-TGATGTCACGCACGATTTCC-3'. The relative mRNA expression levels were quantified using the 2^-ΔΔCq method and normalized to internal reference gene β-actin (28).

Statistical analysis was performed using SPSS Statistics V22.0 (IBM Corp.). Data are presented as the mean ± standard error of the mean. All experiments were performed at least three times. One-way ANOVA followed by Bonferroni's post hoc test was used to compare multiple groups. Lung injury score was evaluated using Kruskal-Wallis followed by Dunn's test. P<0.05 was considered to indicate a statistically significant difference.

To study the effect of Cit on LPS-induced lung injury, total cell count and protein concentration in BALF of mice were measured to determine changes in lung vascular permeability. Total cell count and protein concentration in BALF were significantly greater in the LPS group than in the control group, indicating notable lung injury caused by treatment with LPS (P<0.01; Fig. 1A and B). On the other hand, the effect of LPS treatment was partially inhibited by pretreatment with Cit (Fig. 1A and B). In addition, the increase in the lung W/D ratio induced by LPS was significantly attenuated by pretreatment with Cit (Fig. 1C).

The effect of pretreatment with Cit on lung injury induced by LPS was investigated by histopathological analysis. The lung tissue in the LPS group showed a lung injury score significantly greater than that in the control, showing distinct pathological injury, including neutrophil accumulation, interstitial edema and alveolar wall damage (Fig. 2A and B). Lung injury score in the LPS + Cit group was significantly lower than that in the LPS group (Fig. 2B) but greater than that in the control (Fig. 2B). Mice in the LPS group showed severe infiltration of F4/80⁺ macrophages, while there was a decreasing tendency in LPS + Cit mice (Fig. 2C), indicating the anti-inflammatory effect of Cit.

Yang et al (29) showed that NLRP3 inflammasome activation participates in LPS-stimulated lung injury. In the present study, NLRP3 and ASC mRNA levels, as well as NLRP3 and caspase-1 p20 cleavage fragment protein expression levels in the LPS group were significantly greater than those in the control (Fig. 3A and B). In the LPS + Cit group, levels were significantly lower than those in the LPS group (Fig. 3A and B).

The assembled NLRP3 inflammasome leads to the release of the pro-inflammatory cytokines IL-1β and IL-18, as well development of cell pyroptosis (30). IL-1β and IL-18 levels were significantly increased in the LPS group compared with the control (Fig. 4A-C). N-terminal region of GSDMD (GSDMD-N) expression relative to the full length GSDMD (GSDMD-FL) was upregulated in the LPS group compared with the control (Fig. 4C), while in the LPS + Cit group, pretreatment with Cit significantly attenuated the effect of LPS on IL-1β and IL-18 production, as well as GSDMD-N expression (Fig. 4C). Furthermore, levels of Bcl-2, an anti-apoptotic protein, significantly decreased in the LPS group compared with the control (Fig. 4D), while the Bcl-2 expression in the LPS + Cit group was greater than in the LPS group (Fig. 4D) and comparable with the control. Consistently, levels of Bax and TUNEL-positive cells (indicative of apoptosis) were greater in the LPS group than in the control and LPS + Cit groups, while levels of these markers in LPS + Cit group were comparable with control (Fig. 4D and E). These results indicated that Cit pretreatment ameliorated LPS-induced pulmonary pyroptosis and apoptosis.

ROS participate in ALI by triggering inflammatory reactions (31). In the present study, NAC was used to inhibit activation of ROS in MLVECs to demonstrate the effect of ROS on NLRP3 activation and subsequent cell death. NAC intervention inhibited expression of the NLRP3 inflammasome and cleaved caspase-1 (Fig. 5A). Levels of TUNEL positive cells, apoptotic protein Bax and anti-apoptotic protein Bcl-2 were also reversed following inhibition of ROS (Fig. 5A-C). These findings demonstrated that the ROS scavenger NAC may decrease activation of the NLRP3 inflammasome and relieve endothelial pyroptotic and apoptotic cell death in LPS-treated MLVECs.

The present study investigated the potential mechanism underlying the protective effects of Cit. Pretreatment with Cit decreased accumulation of intracellular ROS induced by LPS (Fig. 6A). Furthermore, Cit significantly attenuated the increase in mRNA levels of NLRP3, Nrf2 and HO-1 (the primary target gene of Nrf2) induced by LPS (Fig. 6B). Immunofluorescent staining of Nrf2 showed that LPS decreased expression of Nrf2 in cytoplasm and Cit pretreatment reversed the effect of LPS (Fig. 6C).

A specific inhibitor of Nrf2 was used to determine if the protective effect of Cit against LPS-treated MLVECs was mediated by the Nrf2 signaling pathway. In MLVECs, the effect of Cit on LPS-treated MLVECs was abrogated by Nrf2 inhibitor ML385 (5 µM), Cit pretreatment did not attenuate intracellular ROS levels induced by LPS (Fig. 7A). Inhibition of Nrf2 also blocked the protective effect of Cit against LPS-induced NLRP3 activation and pyroptosis-associated protein Casp-1(p20) and GSDMD-N (Fig. 7B). The protective effect of Cit on LPS-induced cell apoptosis was also abolished by ML385 (Fig. 7C and D). Together, these results indicated that Nrf2 was involved in the anti-pyroptotic and anti-apoptotic effect of Cit in LPS-stimulated endothelial dysfunction.