We synthesized 5-deoxy-rutaecarpine (5-DR) in our lab (Figure 1A). Lipopolysaccharide (Escherichia coli O55: B5, LPS) was purchased from Sigma Chemical Co. (St. Louis, MO, United States). RPMI-1640 cell culture medium was obtained from KeyGEN BioTECH (Nanjing, China). Fetal bovine serum (FBS) was purchased from Gibco (Waltham, MA, United States). Mouse IL-1β, IL-18, TNF-α, IL-6, and IL-10 (enzyme-linked immunosorbent assay) ELISA kits were obtained from Dakewe Biotech Co., Ltd. (Shenzhen, China). Mouse NO ELISA kit was obtained from Wuhan Saipei Biotechnology Co., Ltd. (Wuhan, China). pcDNA3.1-IKKβ plasmid was purchased from Genscript Co., Ltd. (Nanjing, China). Antibodies against p-IκBα, IκBα, p-p65, and p65 were purchased from Bioworld Technology, Inc. (Nanjing, China); Antibody against Caspase-1, Lamin B1, and GAPDH were purchased from HuaBio Biotech Co., Ltd. (Hangzhou, China). Antibody against NLRP3, ASC, IL-1β, and IKKβ was purchased from Cell Signaling Technology, Inc. (Boston, MA, United States). The other chemicals and reagents used were of analytical grade available.

Male C57/BL6 mice (6–8 weeks old) were obtained from Changsha Tianqin Biotechnology Co., Ltd. (Changsha, China). All animal experiments were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and the related ethical regulations of Changsha Medical University (IACUC approval no: D2023022). Mice were randomly divided into four groups: Saline group, LPS group, LPS+5-DR (10 mg/kg) group, and LPS+5-DR (30 mg/kg) group. 5-DR (dissolved in 0.5% CMC-Na) was administered once per day via gastric gavage according to the experimental design. On the 8th day, 2 h after gavage, the mice were treated by intratracheal instillation of LPS (5 mg/kg) (Luo et al., 2020; Wu et al., 2018; Qin et al., 2024; Lee C. et al., 2019). Mice in the Saline group were administered an equal volume of saline in the same schedule.

In the animal experiment, mice were anesthetized (3% isoflurane for induction, 1.5%–2% for maintenance) at 24 h post LPS stimulation, and sacrificed by cervical dislocation after blood collection from the eyeball. Mice death was confirmed by the absence of a heartbeat, lack of respiratory movement, and non-responsiveness to physical stimuli. The right lung of six mice from each group was used to calculate the lung wet/dry weight ratio, whereas the left lung was used to collect bronchoalveolar lavage fluid (BALF). The right lung of the other six mice in each group was utilized for histological study with Hematoxylin-eosin (H&E) staining, while the left lung was used for Western blotting.

Lungs from mice in all groups were removed and immediately fixed in 10% formalin. The fixed samples were embedded in paraffin, sectioned, and stained with H&E (Su et al., 2023). As previously described, the stained sections were examined blindly under a microscope to compute the lung injury score (Liu et al., 2016). Briefly, we conducted a semi-quantitative analysis of lung injury in mice according to the following criteria: (1) Alveolar hemorrhage; (2) Alveolar edema; (3) Alveolar and interstitial inflammation; (4) Alveolar wall thickening and/or hyaline membrane. Briefly, the histological changes were scored on a scale of 0–4 (0, normal; 1, mild; 2, moderate; 3, severe; 4, intense). Results were confirmed by an experienced and qualified pathologist.

Lungs of mice were obtained, and the wet weights were obtained immediately. Then, lungs were dried in an oven at 80°C for 48 h until achieving stable dry weights. The lung wet/dry weight ratio was calculated as the indicator of lung edema.

The lungs were lavaged with 0.5 mL of PBS four times. The BALF was centrifuged at 1,500 rpm for 10 min at 4°C. The supernatant was collected and kept at −80°C for subsequent analysis.

Lung tissue samples were sectioned into 1–2 mm³ cubes and fixed in 2.5% glutaraldehyde buffer at 4°C for 24 h. The samples were subsequently washed in PBS, post-fixed with 1% osmium tetroxide, dehydrated using a graded ethanol series, and embedded in epoxy resin. Ultrathin sections (70 nm) stained with uranyl acetate and lead citrate were analyzed with a transmission electron microscope (JEM1400, JEOL, Japan), images of each sample were captured at ×10000 magnifications.

Murine macrophage J774A.1 cells were obtained from ATCC and cultured in RPMI-1640 medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a 5% CO₂ humidified air.

To investigate the effect of 5-DR on cell viability, J774A.1 cells were seeded in 96-well plates and cultured in a 37°C, 5% CO₂ incubator for 24 h. Then, they were incubated with 5-DR at different concentrations (0, 0.01, 0.1, 1, 5, 10, 25, 50, 100 μM) in the absence or presence of LPS (1 μg/mL) for 24 h (Luo et al., 2015), and the cell viability was tested by MTT assay.

To investigate the effect of 5-DR on inflammation, J774A.1 cells were seeded in 6-well plates and pretreated with different concentrations of 5-DR (0, 0.01, 0.1, 1 μM) for 20 h, then treated with LPS (1 μg/mL) for 4 h (Luo et al., 2020). The supernatants were collected for ELISA, and cell protein lysates were used for Western blotting.

For cell transfection-related assay, J774A.1 cells were seeded in 6-well plates and cultured in a 37°C, 5% CO₂ incubator for 24 h. Then the pcDNA3.1-IKKβ-overexpressing plasmid (4 μg/well) or pcDNA3.1 plasmid (4 μg/well) was transfected into J774A.1 cells using Lipofectamine 2000 reagent (Invitrogen, CA, Carlsbad, United States) according to the manufacturer’s instructions. After 4 h of transfection, the cells were treated with or without 5-DR (1 μM) for 20 h, then treated with LPS (1 μg/mL) for 4 h. The supernatants were collected for ELISA, and cell protein lysates were used for Western blotting.

Cells were cultured under indicated conditions, 20 μL of MTT solution (5 mg/mL in PBS) was added into each well and cultured for 4 h. The supernatants were removed, and the formazan crystals were dissolved in DMSO (150 µL/well). The absorbance was measured at 570 nm using a Model 1,500 Multiskan spectrum microplate Reader (Thermo, Waltham, MA, United States) (Song et al., 2023; Yao et al., 2024).

The concentrations of inflammatory cytokines (IL-1β, IL-18, TNF-α, IL-6, NO, and IL-10 in serum; IL-1β, IL-18, TNF-α, and IL-6 in BALF and cell supernatants) were detected by using ELISA kits according to the manufacturer’s instructions.

The NLRP3 promoter reporter vector was designed and constructed by Shanghai Sangon Biotech Co., Ltd. (Shang hai, China). The J774A.1 cells were transiently transfected with luciferase reporter plasmids, Renila control luciferase vector, pcDNA3.1-IKKβ-overexpressing plasmid or pcDNA3.1 plasmid. After 7 h of transfection, the cells were treated with or without 5-DR (1 μM) for 20 h, then treated with LPS (1 μg/mL) for 4 h. Dual Luciferase Reporter Assay System (Promega, United States) was used to measure luciferase activity (Li et al., 2024; Luo et al., 2024).

Protein lysates from J774A.1 cells and lung homogenates were prepared following standard protocols and the proteins from the cell culture supernatants were extracted using a Liquid sample protein extraction kit (Biao Leibo, Beijing, China) (Luo et al., 2024). All protein samples were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Burlington, MA, United States). Following by blocking with 5% bovine serum albumin (BSA, Beyotime) in TBST (Tris-buffered saline, 0.1% Tween 20) for 1 h at room temperature, the membranes were incubated with different antibodies overnight at 4°C. Membranes were washed three times and incubated with secondary antibodies for 1 h at room temperature. Protein bands were detected with an Ultra-sensitive ECL chemiluminescence kit (Beyotime) on a chemiluminescence imaging system (Tanon 5,200, Yuanpinghao Biotechnology, Beijing, China) and analyzed using ImageJ software. The dilutions of both primary and secondary antibodies were listed in Supplementary Table S1.

Data are presented as the means ± SEM. Statistical analysis was performed with one-way analysis of variance (ANOVA) using GraphPad Prism. For the results of survival experiments, the log-rank test was used. P values less than 0.05 (P < 0.05) were considered statistically significant.

To evaluate the impact of 5-DR on LPS-induced lung injury, the histological alterations in lung tissue were examined using H&E staining. As shown in Figure 1B, Histological examination of lung tissues from mice subjected to ALI induction by LPS revealed significant histopathological alterations characteristic of lung injury. These changes included alveolar congestion, hemorrhage, and infiltration of inflammatory cells, indicating the development of ALI. However, treatment with 5-DR (10, 30 mg/kg) mitigated these histopathological changes, preserving lung architecture and reducing inflammatory cell infiltration. Quantitative analysis of lung injury score also demonstrated treatment with 5-DR significantly attenuated lung injury (Figure 1C), Lung sections from mice treated with 5-DR exhibited decreased lung inflammation and alveolar wall destruction compared to the untreated ALI group. Calculating the wet-to-dry weight ratio of the lung showed that LPS-induced ALI increased lung water content, indicating pulmonary edema. However, treatment with 5-DR attenuated lung edema, as evidenced by a significant reduction in the wet-to-dry weight ratio compared to the untreated ALI group (Figure 1D). In addition, transmission electron microscopy was used to examine the ultrastructural changes of lung tissues (Supplementary Figure S1). In the LPS group, cytoplasmic vacuolation, disorganized and fragmented mitochondrial cristae, marked mitochondrial swelling and vacuolation were observed, indicating cellular stress and injury. When compared to the LPS group, the pathologic damage was significantly alleviated in the LPS+5-DR (30 mg/kg) group, with reduced cytoplasmic vacuolation, more integrated and manifested of morphology, reduced disorganization of mitochondrial cristae, and less swelling. These results demonstrated the protective effect of 5-DR against LPS-induced lung damage.

Inflammatory cytokines play a critical role in the occurrence and development of LPS-induced ALI (Kan et al., 2021; Feng et al., 2022; Liu et al., 2023). Understanding the effects of 5-DR on inflammatory cytokines in LPS-induced ALI is essential for elucidating its therapeutic potential in this condition. The results showed that 5-DR administration significantly suppressed the serum levels of pro-inflammatory cytokines IL-1β, IL-18, TNF-α, Nitrite/nitrate, and IL-6 in LPS-intoxicated mice, although had no significant effect on IL-10 level (Figures 2A–F). We further examined the effect of 5-DR on the BALF levels of IL-1β, IL-18, TNF-α, and IL-6. Results show that the BALF levels of IL-1β, IL-18, TNF-α, and IL-6 (Figures 2G–J) were significantly upregulated in response to LPS, while 5-DR (10, 30 mg/kg) administration could reduce inflammatory cytokine levels caused by LPS. By attenuating the production of inflammatory cytokines, 5-DR can effectively modulate the inflammatory response, thereby protecting lung tissue from excessive inflammation and damage.

Macrophages are the primary producers of proinflammatory/anti-inflammatory cytokines such as IL-1β, IL-18, TNF-α, and IL-6, which play a significant role in controlling the inflammatory process (Sun et al., 2019; Al-Qahtani et al., 2024; Li et al., 2023). We then evaluated the effects of 5-DR on cytokine expressions in LPS-stimulated J774A.1 macrophages, a murine macrophage cell line commonly used to study inflammation. Treatment with LPS significantly upregulated the expressions of IL-1β, IL-18, TNF-α, and IL-6 in J774A.1 cells compared to PBS-treated controls, consistent with the pro-inflammatory response induced by LPS stimulation in vivo. While pre-treatment with 5-DR (0.01, 0.1, 1 μM) dose-dependently attenuated the LPS-induced increase in inflammatory cytokine expressions (Figures 3A–D).

To exclude the possibility that the anti-inflammation effect of the test compounds on the J774A.1 cells was due to cytotoxicity, effects of 5-DR on the viability of J774A.1 cells in the presence or absence of LPS (1 μg/mL) was detected using an MTT assay. The results showed that although when LPS and 5-DR are used together, cells are more susceptible to additional stressors due to the signaling pathways activated by LPS (Yuan et al., 2009), 5-DR remains not affect the viability of J774A.1 cells at concentrations below 25 μM (Figure 3E).

The NLRP3 inflammasome is a multiprotein complex that plays a crucial role in the innate immune system by initiating an inflammatory response (Yao et al., 2024). It is activated in response to various danger signals, including microbial products like LPS, as well as endogenous danger signals associated with tissue damage or stress. The role of NLRP3-related inflammation in ALI has been well identified previously (Wang and Zhao, 2022; Liu et al., 2021). In our study, 5-DR treatment markedly suppressed the levels of IL-1β and IL-18 both in BALF and serum of LPS-challenged mice, which are the products of NLRP3 inflammasome activation (Figure 3). Then, we examined whether the anti-inflammatory role of 5-DR was related to the NLRP3 inflammasome. When activated, the NLRP3 inflammasome recruits the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) (Proell et al., 2013), which acts as a bridge to recruit pro-caspase-1, leading to its proximity-induced self-cleavage and activation. Caspase-1 is initially synthesized as an inactive zymogen (pro-caspase-1). Upon activation, it is cleaved into two subunits: p20 and p10 (Bolívar et al., 2019). Activated caspase-1 then processes pro-inflammatory cytokines such as IL-1β and IL-18 into their mature forms, which are secreted by the cell to propagate the inflammatory response (Zheng et al., 2020). Western blotting analysis revealed that ALI induction by LPS resulted in increased expression levels of NLRP3, cleaved caspase-1 p20, and mature IL-1β in lung tissues, indicating activation of the NLRP3 inflammasome and subsequent release of pro-inflammatory cytokines (Figures 4A, B). However, treatment with 5-DR significantly inhibited NLRP3 inflammasome activation, as evidenced by decreased expression levels of NLRP3, cleaved caspase-1 p20, and mature IL-1β in lung tissues compared to the LPS group. The above findings are sufficient to show that 5-DR suppressed NLRP3 inflammasome-related inflammation in ALI mice, thereby attenuating pulmonary inflammation and tissue damage.

It is widely accepted that LPS stimulation triggers the activation of the NLRP3 inflammasome in macrophages (Gurung et al., 2015; Zhang et al., 2020). We next investigated whether 5-DR directly inhibited NLRP3 inflammasome activation in LPS-stimulated J774A.1 murine macrophages. Treatment with LPS significantly induced the activation of the NLRP3 inflammasome in J774A.1 cells, as evidenced by increased cleavage of caspase-1 and maturation of IL-1β compared to untreated controls. However, pre-treatment with 5-DR decreased the cleavage of IL-1β by LPS in a dose-dependent manner, and decreased the levels of NLRP3, cleaved caspase-1 p10, and cleaved caspase-1 p20 (Figures 5A, B). Overall, these results demonstrated that 5-DR effectively suppressed the activation of the NLRP3 inflammasome in LPS-stimulated macrophages.

In the context of LPS-triggered NLRP3 inflammasome activation, nuclear factor kappa B (NF-κB) serves as a crucial priming signal (Kelley et al., 2019). NF-κB activation leads to the transcriptional upregulation of NLRP3, pro-IL-1β, and pro-IL-18, thereby priming the cell for subsequent inflammasome activation (Xu et al., 2020; Yu and Cheng, 2023). Understanding the intricate crosstalk between NF-κB signaling and the NLRP3 inflammasome pathway is essential for unraveling the mechanisms of 5-DR. We then investigated if the anti-inflammatory properties of 5-DR relied on its capacity to block NF-κB by evaluating the impact of 5-DR on NF-κB signaling. Western blotting research showed that ALI induction by LPS resulted in heightened NF-κB activation in lung tissues, demonstrated by higher levels of phosphorylated NF-κB p65 (p-p65) and phosphorylated IκBα (p-IκBα) subunit. Treatment with 5-DR inhibited NF-κB activation by reducing p-p65 and p-IκBα levels relative to the untreated ALI group (Figure 6). 5-DR treatment also markedly inhibited the LPS-induced phosphorylation of IκBα and the p65 subunit in J774A.1 cells, which indicated that 5-DR may inhibit NF-κB signaling (Supplementary Figure S2). In addition, 5-DR significantly inhibited the translocation of p65 subunit from the cytoplasm to the nucleus, potentially explaining the inactivation of NF-κB signaling by 5-DR.

The impact of NF-κB signaling on the suppression of 5-DR-mediated NLRP3 inflammasome was assessed in LPS-stimulated J774A.1 cells with and without activation of NF-κB signaling. J774A.1 cells were transfected with pcDNA3.1-IKKβ to simulate the activation of the NF-κB pathway, followed by treatment with LPS. Overexpression of IKKβ significantly increased the levels of p-IκBα and p-p65, indicating activation of the NF-κB signaling pathway (Supplementary Figure S3; Figures 7A, B). 5-DR treatment significantly reduced LPS-induced IκBα and p65 phosphorylation, attenuated NLRP3 inflammasome activation, and reduced IL-1β secretion in J774A.1 cells, while overexpression of IKKβ reversed the downregulation of NLRP3, cleavage of caspase-1, and decrease in IL-1β induced by 5-DR (Figures 7A–C). Subsequently, to explore whether 5-DR could regulate NLRP3 promoter activity in J774A.1 cells, we transfected J774A.1 cells with a luciferase reporter plasmid containing NLRP3 promoter region. Our results demonstrated that 5-DR treatment decreased NLRP3 promoter activity. However, overexpression of IKKβ could diminish the inhibition of NLRP3 promoter activity caused by 5-DR (Figure 7E). In summary, these findings showed that 5-DR inhibit NLRP3 though the upstream NF-κB pathway and then transcriptionally decreased the activity of the NLRP3 promoter.