The reagents used in the experiment were as follows: FeCl₃⋅6H₂O (aladdin, F102739-55 g), Capsaicin (MCE, 404864-50 mg), polyvinylpyrrolidone (PVP) (aladdin, P434440-250 g), lipopolysaccharide (LPS) (Sigma, L3129-10 MG), PBS (Gibco, 8123157), DMEM culture medium (Gibco, C11995500BT), Fetal bovine serum (Gibco, 30044333), M-CSF protein (MCE, HY-P7085), Color Pre-stained Protein Marker (Thermo, 26,619), Primary and secondary antibody dilution solution (BOSTER, 17K28C17), Pierce TM BCA protein Assay kit (Thermo Fisher Scientific, Prod # 23227), β-actin (Cell Signaling Technology, 4970S), Anti-Mannose Receptor (CD206) (abcam, ab125028), CD86 (Huabio, ET1606-50), JAK2 (Huabio, ET1607-35), p-JAK2 (Huabio, ET1607-34), STAT3 (Cell Signaling Technology, #12640), p-STAT3 (Cell Signaling Technology, #9145), Bcl-2 (Huabio, ET1602-53), Caspase (abcam, ab184787), Bax (Huabio, ET1603-34), F4/80 (Huabio, RT1212), anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, 7074S), anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology, 7076S), Secondary antibody (Thermo Fisher Scientific, Prod # A-21202), Secondary antibody (Thermo Fisher Scientific, Prod # A-31572), Reactive Oxygen Species Assay Kit (ROS, Beyotime, S0033S), SuleLumia ECL kit (Abbkine, K22020), Latex beads (Sigma, L3030), Calcein-AM Reagent (Dojindo, PJ671), Transwell permeable supports (Costar, United States).

Bone Marrow-Derived Macrophages (BMDM) were isolated from the femur and tibia of male C57BL/6 mice. The procedure was as follows: Flushed the marrow cavity with DMEM medium containing 10% FBS and added red cell lysate to it. Subsequently, the primary macrophages were cultured in 25 cm² bottles with 20 ng/mL macrophage colony-stimulating factor (100 ng/mL, abcam, United Kingdom, ab129146) for 7 days.

The BMDM were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco by Thermo Fisher Scientific, United States, C11995500BT) supplemented with 10% fetal bovine serum (FBS, Gibco by Thermo Fisher Scientific, United States, 30044333). Then the cells were cultured in 25 cm² bottles at 37°C in humidified containing 5% CO₂ and 95% air. Every 48 h to replace part of the culture medium.

Cell Counting Kit-8 (CCK-8, Beyotime, C0038) was used to assess the viability of BMDM cells. The BMDM cells (2 × 10⁴ cells/well) were inoculated into a 96-well plate. The cells were treated with different concentrations of CAP (capsaicin, MCE, United States) (ranging from 25 to 100 µM) and Fe-CAP (ranging from 7.5 to 30 μg/mL) and cultured for 0, 3, 6, and 9 h. After removing the old medium, 90 µL of fresh base medium and 10 µL of CCK-8 solution were added to per well. The cells were further incubated for 2 h, and the optical density was read at 450 nm using a microplate reader. To visually assess the effect of the above drug treatment, 5 µM calcein AM and 5 µM PI were added for 30 min and observed under a microscope. This experiment was repeated thrice.

BMDM cells were randomly divided into four groups: control group, LPS group, CAP + LPS group and Fe-CAP + LPS group. In the control group, the cells were cultured with basic medium (DMEM) for 8 h. In the LPS group, the cells were treated with LPS (1 μg/mL) for 6 h. In the CAP + LPS group, the cells were pretreated with CAP (50 µM) for 2 h, and then treated with LPS (1 μg/mL) for 6 h. In the Fe-CAP + LPS group, the cells were pretreated with Fe-CAP (15 μg/mL) for 2 h, and then treated with LPS (1 μg/mL) for 6 h. The cells were collected for protein extraction and immunofluorescence. The experiment was repeated three times.

BMDM cells were treated based on the aforementioned groups. Subsequently, RNA extraction (Invitrogen TRIzol), purification (Thermo Scientific NanoDrop 2000; Thermo Scientific, Waltham, Massachusetts, United States), and library preparation (NEBNext Ultra II RNA library Prep Kit for Illumina) were conducted. The samples were sequenced using next-generation sequencing (NGS) on the Illumina platform and analyzed through RNA-seq using PANOMIX. The differential expression analysis of the two groups was performed using DESeq software (version 1.20.0). Gene Ontology (GO) enrichment analysis was carried out using topGO, with a significance level set at P < 0.05. Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was executed using clusterProfiler software (version 3.4.4), focusing on pathways with a P-value <0.05. Differential splicing events were analyzed using rMATS software (version 3.2.5), concentrating on the five primary alternative splicing event types: SE, RI, MXE, A5SS, and A3SS.

After the cells were treated according to the above group, they were incubated with red Latex beads (Sigma-Aldrich, L3030) for 1 h. The fluorescent microspheres that were not engulfed were removed by washing with PBS three times. Then it was fixed with 4% paraformaldehyde and washed with PBS thrice. The images were captured at 200× magnification. The experiment was carried out at least three times.

BMDM were treated as described above. ROS levels in BMDM were detected using the Reactive Oxygen Species Assay Kit (ROS, Beyotime, S0033S). The specific steps were carried out according to the instructions. Fluorescence intensity analysis was performed using ImageJ software. The experiment was repeated three times.

BMDM were seeded in a 24-well plate. Transwell chambers (Costar, United States) with an 8.0 µm pore size were used to evaluate the effect of drug on BMDM migration. First, 200 μL cell suspension (10⁴ cells) was added into the insert. Second, the cells were treated as described earlier. Third, 200 μL of serum-free medium was added to the upper cavity, and 500 μL of complete medium was added to the lower cavity. The cells were then incubated in an incubator at 37°C and 5% CO₂ for 24 h. The old medium was discarded, and the inserts were carefully removed. The cells were then fixed with methanol and stained with 0.5% crystal violet. Cells that did not cross the membrane were gently erased with a wet cotton swab. The migrated cells were randomly captured under the microscope and counted using ImageJ software. The experiment was carried out at least three times.

The use and handling of mice used for sepsis complied with the ethical guidelines outlined in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The use and experimental program of animals were approved by the ethics committee at No. 326, 2023 of Sichuan Provincial People’s Hospital. Every possible measure was taken to minimize the number of mice used and alleviate any potential suffering.

Male C57 mice aged 6–8 weeks and weighing 18–22 g (Table 1) were procured from Byrness Weil biotech Ltd. Corporation. The mice received a standard laboratory diet and access to free water. The mice were randomly divided into four groups: sham, LPS (10 mg/kg, i. p.), CAP (8 mg/kg, i. p.) + LPS (10 mg/kg, i. p.) and Fe-CAP (8 mg/kg, i. p.) + LPS (10 mg/kg, i. p.). In the CAP + LPS group, mice were injected with CAP (8 mg/kg, i. p.) every 24 h followed by LPS at 96 h. Similarly, in the Fe-CAP + LPS group, mice received Fe-CAP (8 mg/kg, ig) every 24 h prior to LPS injection at 96 h. The mice were euthanized 12 h after LPS injection.

BMDM were treated as described above. The left lung from mice was stored in liquid nitrogen at −80°C. Lysis buffer containing phosphatase and protease inhibitors (Pierce, Thermo Fisher Scientific Corporation, Bloomingdale, Illinois, United States) was added to the lung tissue and cells. The cell fragments from different groups were collected by scraping with a rubber spatula and centrifuged at 15,000 rpm for 15 min to collect the supernatant. The lung tissue were homogenized in an animal tissue grinder at 4°C and 80 Hz for 10 min, and the supernatant was collected. Protein concentration in cells and tissues was detected with the BCA protein detection kit (BCA, Beyotime, P0010). The protein sample were heated to 100°C for 10 min and stored at −80°C.

SDS-PAGE was used to separate proteins with different molecular weights. The isolated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% milk at room temperature for 1 h. Subsequently, the membrane was incubated with primary antibody (1:1,000) and secondary antibody (1:5,000) overnight at 4°C and for 1 h at room temperature, respectively. SuleLumia ECL kit (Abbkine, United States, K22020) was employed for imaging. Band intensity was quantified using ImageJ software. Specific details of the antibodies used were shown in Table 2. The experiment was carried out at least three times.

In brief, BMDM were treated as described previously. First, BMDM cells were fixed in 4% paraformaldehyde at room temperature for 15 min. Second, it was blocked with 10% donkey serum for 1 h and washed with 1 × PBS three times for 5 min each time. Third, the cells were incubated with the corresponding primary and secondary antibodies at 4°C overnight and at room temperature for 1 h, respectively. DAPI was added and incubated for 10 min. Then, the cells were washed with 1 × PBS three times. The images were taken under a microscope at 200× magnification. The experiment was carried out at least three times.

The right lung of mice was used for double immunofluorescence. Twelve hours after injection of LPS, mice were anesthetized with 3% pentobarbital sodium (30 mg/kg), then infused with 2% paraformaldehyde and killed. After gradient dehydration, the lung tissue was embedded with OCT and sectioned on a 4 µm thickness microtome (Model: 2,165; Leica, Bensheim, Germany). Sections were then incubated with the corresponding primary and secondary antibodies at 4°C overnight and at room temperature for 1 h. Add DAPI to the slices and incubated for 10 min before washing with PBS for three times. The images were taken under a microscope with a magnification of 400×. The experiment was carried out at least three times.

Following 12 h of LPS injection, mice were anesthetized with 3% pentobarbital sodium (30 mg/kg). The right lung tissues were carefully excised, fixed with 4% paraformaldehyde, paraffin embedded, and sectioned at a thickness of 4 µm. The sections were stained conventionally with hematoxylin-eosin (H&E). Lung injury score (LIS) was quantified by blind method.

ApopTag^® In Situ Apoptosis assay Kit was used to assess apoptosis. The kit used dUTP nickel end labeling (TUNEL) staining mediated by terminal deoxynucleotide transferase (TdT). The tissue sections were then observed under a fluorescence microscope (Olympus Corporation). This method can accurately detect and evaluate apoptosis in tissue samples. ImageJ soft was used to analyse the result.

Statistical analysis was performed using GraphPad Prism 8.0 Software (GraphPad Software, San Diego, CA). After homogeneity of variance assessment, one-way analysis of variance (ANOVA) followed by Tukey test for multivariate comparison was used to determine the statistical significance of the differences between groups. P < 0.05 was considered statistically significant.

The flow chart of the whole experiment was shown in Figure 1A. CCK-8 assay was used to detect the cytotoxicity of CAP and Fe-CAP on BMDM cells. BMDM were exposed to concentrations ranging from 25 to 100 µM of CAP (Figure 1B–1) and 7.5–30 μg/mL of Fe-CAP (Figure 1B–2) for 9 h. The results indicated no significant alteration in cell viability following the treatments within the 9-h. Additionally, the cytotoxic effects of the various treatments were observed by live/dead cell staining under a microscope (Figure 1C), and the results showed that the cells in both CAP and Fe-CAP groups appeared green, which was consistent with the results of CCK-8 assay. Subsequently, we performed in vitro assays using CAP (50 µM) or Fe-CAP (15 μg/mL) for 8 h, and LPS (1 μg/mL) for 6 h, respectively.

The flow chart of the cell experiment was shown in Figure 2A. We investigated phenotypic markers of macrophage by Western blotting assay and immunofluorescence methods.

CD206 and CD86 are phenotypic markers of M2 macrophages and M1 macrophages, respectively. Western Blot results showed that the expression of CD206 (Figure 2B–2, 2B–3) in BMDM of Fe-CAP NPs + LPS group notably increased compared with LPS group. Interestingly, capsaicin alone also increased CD206 expression, but not as effectively as Fe-CAP NPs. Similarly, the expression of CD86 (Figure 3A–2) was increased in LPS group and significantly decreased after Fe-CAP pretreatment.

Immunofluorescence results demonstrated that Fe-CAP NPs effectively increased the fluorescence intensity of CD206 (Figure 2B–1) and decreased the fluorescence intensity of CD86 (Figure 3A–1). The above results indicated that Fe-CAP NPs could promote BMDM polarization to M2 type and inhibit BMDM polarization to M1 type after LPS treatment.

Compared to the control, phagocytosis (Figure 3B) of BMDM in LPS group was increased, while that in Fe-CAP + LPS group was decreased.

Transwell migration assay (Figure 4C) showed that compared with the control group, the migration of BMDM in the LPS group was notably increased, while the migration of macrophages in the Fe-CAP + LPS group was significantly decreased.

To further understand how Fe-CAP ameliorated ALI, RNA sequencing was conducted using the Illumina platform to investigate the pathway of Fe-CAP on LPS-treated macrophages polarization. The RNA sequencing results shown in the figure were all obtained from a comparison between the LPS group and the Fe-CAP + LPS group. The volcano plot (Figure 5A) indicated that 15 genes were upregulated and 18 genes were downregulated. GO (Figure 5B) and KEGG (Figure 5C) enrichment analyses were used to identify pathways associated with macrophage polarization. A heat map was plotted, and cluster analyses were performed (Figure 5D). The results suggested that inhibition of JAK2/STAT3 activation may be one of the mechanisms by which Fe-CAP regulated the polarization of macrophages.

Therefore, we used Western Blot to detect the activation of JAK2 and STAT3. The results demonstrated that LPS stimulation led to significant increase in the phosphorylated forms of JAK2 (Figures 5E–1, 5E–2) and STAT3 (Figures 5E–1, 5E–2). However, pretreatment with Fe-CAP partially reversed these protein changes, showing a more pronounced effect compared to CAP alone. These results indicated that pretreatment with Fe-CAP could promote M2 macrophage polarization by inhibiting the activation of JAK2 and STAT3 signaling pathways.

The flow chart of the cell experiment was shown in Figure 4A. The results indicated that macrophages showed higher fluorescence after LPS treatment. The effect of scavenging ROS (Figure 4B) with CAP alone was not obvious. However, Fe-CAP NPs were effective in scavenging ROS.

Western Blot was used to assess apoptosis of macrophages in different groups. The results showed that the expression of proapoptotic protein Bax (Figures 5E–1, 5E–2) and Cleaved caspase-3 (Figures 5E–1, 5E–2) in BMDM significantly increased in the LPS group, while decreased in Fe-CAP NPs + LPS group. Conversely, the expression of anti-apoptosis protein Bcl-2 (Figures 5E–1, 5E–2) decreased in LPS group, which was reversed by pretreatment with Fe-CAP NPs.

The flow chart of animal experiments was shown in Figure 6A. Hematoxylin and eosin (H&E) staining was conducted on lung tissues of each group (Figures 6C–1). Lung sections revealed substantial pathological alterations in the LPS group, characterized by thickening of alveolar septum, interstitial hemorrhage, pronounced alveolar collapse, massive infiltration of interstitial lymphocytes and neutrophils, and high injury score (LIS) (Figures 6C–2). However, treatment with Fe-CAP lead to reduction in lung tissue inflammation and damage, surpassing the efficacy of CAP treatment.

TUNEL assay (Figure 6B) was used to evaluate the apoptosis rate in mouse lung tissue from different groups, which was increased by LPS. Nevertheless, pretreatment with Fe-CAP NPs mitigated the effects of LPS and notably reduced apoptosis.

Western Blot results indicated that the expression of CD206 (Figures 7B–2) in lung tissue of Fe-CAP NPs + LPS group increased compared with the LPS group. Notably, capsaicin alone also upregulated CD206 expression, but not as effectively as Fe-CAP NPs. Similarly, the expression of M1 macrophage marker CD86 (Figures 7A–2) was increased in LPS group and significantly decreased after Fe-CAP pretreatment.

The results of immunofluorescence indicated that Fe-CAP NPs effectively increased the fluorescence intensity of CD206 (Figures 7B–1) and decreased the fluorescence intensity of CD86 (Figures 7A–1).