SHI was purchased from Yuanye Bio-Technology (B21703, Shanghai, China) and was solubilized in 0.1% (w/v) DMSO. LPS was obtained from Sigma (L4516, St. Louis, United States). Dexamethasone (DXM) was purchased from Chenxin Drug Store (H37021969, Jining, China).

Male C57BL/6 mice (8 weeks old) were obtained from Jihui Co., Ltd. (Shanghai, China). The mice were housed in a standard laboratory environment maintained a temperature of 24 $\pm$ 1 °C, humidity of 40–60%, and a 12 h light/dark cycle. The mice were allowed to acclimatize to these conditions for one week before the start of the experiment. All experimental procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and were based on established literature [8]. All efforts were made to minimize the number of animals used and their suffering. Each group consisted of three or more animals. For our study, anesthesia was induced in mice using inhaled isoflurane (3–4%) and maintained at 1.5–2% prior to euthanasia. The euthanasia method consisted of a two-step procedure compliant with the AVMA Guidelines (2020): first, a deep overdose with $\ge$5% isoflurane for over five minutes post-respiratory arrest, followed immediately by cervical dislocation as a confirmatory physical method to ensure death. All procedures were approved by our Institutional Animal Care and Use Committee (No.2023032). The mice were randomly assigned to five groups: control, LPS, LPS+DXM (5 mg/kg), LPS+SHI-L (50 mg/kg), and LPS+SHI-H (100 mg/kg). A murine sepsis model was induced by intraperitoneal injection of LPS. Drug administration was performed via intragastric gavage 2 hours before LPS injection, and again at 0, 2, and 12 hours post-injection. The mice were euthanized by inhalation of an overdose of anesthetic agent. Lung tissues were then collected and processed as follows: one portion was used to determine the wet-to-dry weight ratio, another portion was fixed in 4% paraformaldehyde was purchased from Servicebio (G1101, Wuhan, China), and the remaining samples were stored at –80 °C.

The lungs were removed from the sacrificed mice and weighed to obtain the wet weight (W). The lung tissues were then dried at 65 °C for 48 hours and weighed again to obtain the dry weight (D). The wet-to-dry weight (W/D) ratio was calculated to assess the degree of pulmonary edema.

Lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. After deparaffinization and rehydration, the sections were stained with hematoxylin and eosin (H&E). Pulmonary histopathological changes were observed under a CX53 microscope (Olympus, Tokyo, Japan). Lung injury scoring was performed by an experienced investigator blinded to the treatment groups (categorized as absent, mild, moderate, or severe with scores ranging from 0 to 3). The assessment was based on the presence of exudates, hyperemia, congestion, neutrophilic infiltration, intra-alveolar hemorrhage, debris, and cellular hyperplasia. Each of the following indicators was scored on a 0–4 scale: edema, atelectasis, necrosis, alveolar and interstitial inflammation, hemorrhage, and hyaline membrane formation. The scoring criteria were defined as follows: 0 = no injury; 1 = 25% injury; 2 = 50% injury; 3 = 75% injury; 4 = 100% injury. For each slide, the score of each injury indicator was evaluated in 10 randomly selected fields at 200$\times$ magnification [16].

Murine RAW264.7 macrophage cells were obtained from iCell (Shanghai, China) and were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% heat-inactivated fetal bovine serum. Cells were maintained in a 37 °C incubator with a humidified atmosphere of 5% CO₂ and 95% air. Subculturing was performed every 1–2 days, and only cells in the logarithmic growth phase were used for experiments. Cells were seeded into six-well or 96-well plates at a density of 5 $\times$ 10³ cells/mL and incubated for 24-hour before the culture medium was replaced. The RAW264.7 cells were divided into four groups: control, LPS, LPS+SHI-L (2 µg/mL), and LPS+SHI-H (4 µg/mL). Cells were pretreated with SHI at concentrations of 2.0 µg/mL (SHI-L) and 4.0 µg/mL (SHI-H) for 2 hours, then stimulated with LPS (5 µg/mL) for 24 hours. Cell supernatants and cells were then collected for subsequent experimental analyses. All cell lines were validated by STR profiling and tested negative for mycoplasma.

HMGB1-siRNA was obtained from Hippobiotec Co., Ltd. (Huzhou, China). For cell preparation, 1 $\times$ 10⁵ cells were inoculated in 400 µL of antibody free medium one day before transfection to ensure 50% cell confluence. For transfection, siRNA was diluted with 50 µL Opti-MEM to a final concentration of 50 nM and gently pipetted 3–5 times to mix. The transfection reagent, Lipofectamine 2000, was gently mixed by inversion before use. Then, 1.2 µL of Lipofectamine 2000 was diluted in 50 µL of Opti-MEM, gently pipetted (3–5 times) to mix thoroughly, and incubated at room temperature for 5 minutes. The transfection reagent mixture was combined with the siRNA diluent via gentle pipetting (3–5 times) and allowed to stand at room temperature for 20 minutes. The transfection complex was added to the 24-well cell plate, and the plate was gently shaken back and forth to mix evenly. Cell culture plates were incubated in a 5% CO₂ incubator at 37 °C for 48 hours. Fresh medium was replaced 4–6 hours after transfection. RAW264.7 cells were divided into the following groups: control, LPS, HMGB1-siRNA (knockdown), LPS+HMGB1-siRNA, LPS+SHI, and LPS+HMGB1-siRNA+SHI. Cells were first treated with SHI at a concentration of 2.0 µg/mL for 2 hours, followed by stimulation with LPS for 24 hours [8].

Total RNA was isolated from cells using the MolPure Cell/Tissue Total RNA Kit (Yeasen 19221ES50, Shanghai, China). Reverse transcription was performed using with the HiScript II Q RT SuperMix Kit (Vazyme R223-01, Nanjing, China). Quantitative real-time PCR (qRT-PCR) was then performed using SYBR qPCR Master Mix (Medicalbio MR0321, Suzhou, China) according to the manufacturer’s protocols. The 20 µL reaction system consisted of 1 µL cDNA template, 0.4 µL each of forward and reverse primers, 10 µL SYBR Premix Ex Taq, and 8.2 µL ddH₂O. $\beta$-actin was used as the internal reference gene, with primer sequences detailed in Table 1 (Sangon Biotech, Shanghai, China). Relative mRNA expression levels were calculated using the 2^{-Δ⁢Δ⁢CT} method.

The concentrations of IL-1$\beta$, IL-6, TNF-$\alpha$, IL-10, TGF-$\beta$1 and GM-CSF, were measured using commercial ELISA kits according to the manufacturer’s instruction (MultiSciences EK201, EK206, EK282, EK210, EK981, EK263, Hangzhou China). The concentrations of HMGB1 were determined using commercial ELISA kits (Qiaoyi JEN-018, Hefei, China) in accordance with the manufacturer’s instructions.

Proteins were extracted from RAW264.7 cells using radio immunoprecipitation assay (RIPA) buffer (Beyotime P0038, Shanghai, China), and their concentrations were measured with a BCA kit (Beyotime P0009, Shanghai, China). After denaturation, equal protein loads were resolved on 8–12% SDS-PAGE gels and electrotransferred to PVDF membranes. Following a 1-hour block in 3% bovine serum albumin (Servicebio GC305010, Wuhan, China), membranes were probed with specific primary antibodies overnight at 4 °C. Primary antibodies against HMGB1 (ZenBio R22773 1:3000, Chengdu, China), p-NF-$\kappa$B (ZenBio 310013 1:1000, Chengdu, China), NF-$\kappa$B (ZenBio 380172 1:1000, Chengdu, China), MyD88 (ZenBio 340629 1:1000, Chengdu, China), TLR4 (ZenBio 505258 1:1000, Chengdu, China), iNOS (ZenBio 340668 1:1000, Chengdu, China), Arg1 (Proteintech 16001-1-AP 1:10,000, Wuhan, China), and $\beta$-actin (ZenBio R380624 1:7500, Chengdu, China). Subsequently, membranes were washed, incubated with HRP-conjugated secondary antibodies (Zenbio 511203 1:7500, Chengdu, China) for 1 hours, and the signals were visualized via enhanced chemiluminescence.

Cells were fixed with 4% paraformaldehyde, then permeabilized and blocked with 3% BSA containing 0.1% Triton X-100. The cells were then incubated overnight at 4 °C with an anti-Arg1 primary antibody. After thorough washing, cells were incubated with an Alexa Fluor® 488-conjugated secondary antibody for 1 hour in the dark at room temperature. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for 10 seconds. Finally, samples were mounted with an anti-fade mounting medium (Beyotime, Shanghai, China) and visualized under a CX53 microscope (Olympus, Tokyo, Japan). Fluorescence intensity was quantified using ImageJ software 2.1.4.7 (National Institutes of Health, Bethesda, MD, USA), and representative images were captured.

Data are expressed as mean $\pm$ SEM. All experiments were independently repeated at least three times. Statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, Boston, MA, USA). For comparisons among three or more groups, one-way analysis of variance (ANOVA) was used followed by Tukey’s honestly significant difference (HSD) post hoc test for pairwise comparisons when variances were equal.

Our findings indicate that SHI can mitigate ALI in LPS-induced septic mice. Histopathological changes were evaluated via H&E staining. As shown in Fig. 1A, LPS challenge induced characteristic pathological features including inflammatory cell infiltration, pulmonary edema, and alveolar wall thickening. Treatment with SHI (100 mg/kg) or DXM markedly attenuated these changes, whereas SHI at 50 mg/kg showed a lesser effect (Fig. 1A). Additionally, SHI significantly improved the lung injury score (Fig. 1B). The LPS group exhibited a significantly elevated wet-to-dry weight ratio compared to the control group (p 0.01). In contrast, treatment with SHI and DXM markedly reduced the pulmonary wet-to-dry weight ratio relative to the LPS group (p 0.01) (Fig. 1C).

Fig. 1.

SHI ameliorated LPS-induced ALI in vivo. (A) The histopathological alteration by H&E staining. Scale bar: 100 μm. (B) The lung injury score. (C) The wet-to-dry ratio of the lungs. Compared with the control group, ##p 0.01, compared with the LPS group, **p 0.01. SHI, Shionone; LPS, lipopolysaccharide; ALI, acute lung injury; H&E, hematoxylin and eosin; DXM, dexamethasone; L, low; H, high.

LPS stimulation significantly increased serum levels of IL-1$\beta$, IL-6, and TNF-$\alpha$ (p 0.05). Both SHI and DXM treatments significantly decreased these serum levels (p 0.05) (Fig. 2A–C). Compared to the LPS group, the relative levels of GM-CSF, IL-10, and TGF-$\beta$1 in the serum of SHI and DXM groups were significantly increased (p 0.05) (Fig. 2D–F). A higher dose SHI (100 mg/kg) exhibited a stronger effect than a lower dose SHI (50 mg/kg). Compared to the control group, LPS stimulation increased the expression of IL-1$\beta$, IL-6, TNF-$\alpha$, GM-CSF, IL-10, and TGF-$\beta$1 (p 0.05) (Fig. 2).

Fig. 2.

SHI inhibited the expression of serum inflammatory factors in ALI models. (A–F) IL-1$\beta$, IL-6, TNF-$\alpha$, GM-CSF, IL-10 and TGF-$\beta$1 expression in the serum. Compared with the control group, #p 0.05, ##p 0.01, compared with the LPS group, *p 0.05, **p 0.01.

The mRNA levels of IL-1$\beta$, IL-6, and TNF-$\alpha$ in lung tissue of mice stimulated by LPS were significantly increased (p 0.05). Both SHI and DXM interventions significantly reduced these mRNA levels (p 0.05) (Fig. 3A–C). Compared with the LPS group, the relative mRNA levels of GM-CSF, IL-10, and TGF-$\beta$1 in lung tissues of SHI and DXM groups were significantly increased (p 0.05) (Fig. 3D–F). A higher dose of SHI (100 mg/kg) appeared to have a stronger effect than a lower dose of SHI (50 mg/kg). Compared with the control group, LPS stimulation increased the mRNA expressions of IL-1$\beta$, IL-6, TNF-$\alpha$, GM-CSF, IL-10, and TGF-$\beta$1 (p 0.05) (Fig. 3).

Fig. 3.

SHI inhibited the expression of inflammatory factors in ALI model lung tissue. (A–F) IL-1$\beta$, IL-6, TNF-$\alpha$, GM-CSF, IL-10 and TGF-$\beta$1 expression in the lung tissues by mRNA. Compared with the control group, ##p 0.01, compared with the LPS group, *p 0.05, **p 0.01.

The expression of the polarization markers iNOS and Arg1 mRNA was examined in the lung tissues of mice stimulated by LPS for 24 hours. The results showed that the iNOS mRNA level in ALI lung tissues significantly increased after LPS stimulation for 24 hours (p 0.01). Compared to the LPS group, pretreatment with low-dose inhibited the iNOS mRNA level in ALI lung tissues (p 0.05). Moreover, high-dose SHI group was greater than that of low-dose SHI group (Fig. 4A). In addition, after LPS stimulation for 24 hours, the mRNA level of Arg1 in ALI lung tissues increased (p 0.01), and pretreatment with high-dose SHI enhanced Arg1 more than the low-dose SHI group (p 0.05) (Fig. 4B). After LPS stimulation for 24 hours, HMGB1 mRNA level was significantly increased (p 0.01). Compared to the LPS group, both DXM and SHI down-regulated HMGB1 levels with the high-dose SHI group showing a greater effect than the low-dose group (p 0.01) (Fig. 4C). Similarly, the expression level of HMGB1 in the serum of the mice showed the same trend (Fig. 4D).

Fig. 4.

SHI affects the polarization and inhibits HMGB1 expression in ALI model lung tissue. (A,B) mRNA expression of Arg1 and iNOS in lung tissues. (C,D) HMGB1 mRNA expression in lung tissues and serum level. Compared with the control group, #p 0.05, ##p 0.01; compared with the LPS group, *p 0.05, **p 0.01.

In this study, RAW264.7 cells were stimulated with lipopolysaccharide (LPS) as a model, and then treated with low (2 µg/mL) or high (4 µg/mL) doses of shionone (SHI). Shi proliferation was assessed using CCK-8 assays. Compared to the control group, LPS group was significantly reduced RAW264.7 cell proliferation (p 0.01). However, SHI treatment, at both low (SHI-L) and high (SHI-H) doses, significantly increased cell proliferation compared to the LPS group (p 0.05) (Fig. 5A). Cell morphology was observed via inverted microscopy (Fig. 5B). To investigate the effects of SHI on M1 and M2 macrophage polarization markers, iNOS and Arg1 mRNA expression in RAW264.7 cells was measured after 24 hours of LPS stimulation. LPS stimulation significantly increased iNOS mRNA levels (p 0.01). Pretreatment with 2 µg/mL SHI significantly inhibited iNOS mRNA expression (p 0.05), and pretreatment with 4 µg/mL SHI further inhibited iNOS mRNA levels (p 0.05), as shown in Fig. 5D. Similarly, LPS stimulation significantly increased Arg1 mRNA levels after 24 h (p 0.01). Pretreatment with 2 µg/mL SHI significantly increased Arg1 mRNA expression (p 0.05), and pretreatment with 4 µg/mL SHI further increased Arg1 mRNA levels (p 0.01), as shown in Fig. 5C. Immunofluorescence results indicated that SHI (2 µg/mL and 4 µg/mL) effectively blocked iNOS expression compared to the LPS group (p 0.05) (Fig. 5F,G). Additionally, 4 µg/mL SHI enhanced Arg1 expression to a greater extent than 2 µg/mL SHI (p 0.05) (Fig. 5E,H). Western blot analysis revealed that LPS stimulation significantly increased iNOS protein levels in RAW264.7 cells after 24 h (p 0.01). Pretreatment with 2 µg/mL SHI for 2 hours down-regulated iNOS protein levels (p 0.05), and pretreatment with 4 µg/mL SHI for 2 hours significantly down-regulated iNOS protein levels (p 0.01). Concurrently, Arg1 protein levels increased after LPS stimulation (p 0.01). Pretreatment with 2 µg/mL SHI increased Arg1 protein levels (p 0.05), and pretreatment with 4 µg/mL SHI significantly increased Arg1 protein level (p 0.01) (Fig. 5I–K). The effect of SHI on lipopolysaccharide-induced inflammatory cytokines in RAW264.7 cells was also assessed by measuring the expression of TNF-$\alpha$, IL-6, IL-1$\beta$, GM-CSF, IL-10 and TGF-$\beta$1, LPS stimulation significantly increased were measured. Results showed that levels of TNF-$\alpha$, IL-6 and IL-1$\beta$ significantly increased after LPS stimulation for 24 h. SHI pretreatment significantly inhibited the levels of TNF-$\alpha$, IL-6, and IL-1$\beta$, and increased the levels of GM-CSF, IL-10, and TGF-$\beta$1 (p 0.05), suggesting that SHI can promote macrophage function and reduce inflammation (Fig. 5L–Q).

Fig. 5.

The effect of SHI on macrophage polarization. (A) Cell Proliferation after 24 hours (CCK-8 assay). (B) Microscopic images. Scale bar: 100 μm. (C,D) Arg1 and iNOS mRNA expression in RAW264.7 cells. (E–H) Arg1 and iNOS protein expression in RAW264.7 cells by Immunofluorescence. Scale bar: 50 μm. (I–K) iNOS and Arg1 protein expression in RAW264.7 cells by Western Blot. (L–Q) TNF-$\alpha$, IL-1$\beta$, IL-6, TGF-$\beta$1, GM-CSF and IL-10 expression cell culture in the supernatant. Compared with the control group, #p 0.05, ##p 0.01, compared with the LPS group, *p 0.05, **p 0.01.

To investigate whether SHI mediates macrophage polarization by regulating the HMGB1, p-NF-$\kappa$B, and NF-$\kappa$B signaling pathways, we examined the effect of SHI on these pathways. The results showed that SHI dose-dependently reduce the HMGB1 mRNA level in LPS-induced macrophages (p 0.05) (Fig. 6A). Additionally, SHI dose-dependently reduced the phosphorylation levels of HMGB1, MyD88 and p-NF-$\kappa$B in LPS-induced macrophages (p 0.05) (Fig. 6B–E).

Fig. 6.

The effect of SHI on HMGB1/NF-$\kappa$B signaling in RAW264.7 cells. (A) Effect of SHI on the HMGB1 mRNA in LPS-induced RAW264.7 cells. (B–E) Protein expression of p-NF-$\kappa$B, NF-$\kappa$B, MyD88 and HMGB1 in cells of each group. Compared with the blank group, #p 0.05, ##p 0.01, compared with the model group, *p 0.05, **p 0.01.

To further investigate the role of SHI in HMGB1-mediated polarization of macrophages, we used HMGB1-siRNA to reduce HMGB1 expression in cells. We then detected the mRNA expression and protein levels of the polarization markers iNOS and Arg1 in RAW264.7 cells after 24 h of LPS stimulation. As shown in Fig. 7A–E, knocking down HMGB1 (LPS+HMGB1-siRNA group) significantly inhibited the expression of iNOS (p 0.05) and enhanced the expression of Arg1 (p 0.05). As shown in Fig. 7F–K, knocking down HMGB1 significantly inhibited the expression of IL-1$\beta$, IL-6, TNF-$\alpha$, GM-CSF, IL-10 and TGF-$\beta$1 expression in the supernatant.

Fig. 7.

SHI affects macrophage polarization through HMGB1. (A,B) iNOS and Arg1 mRNA expression in the RAW264.7 cells. (C–E) iNOS and Arg1 protein expression in the RAW264.7 by WB. (F–K) IL-1$\beta$, IL-6, TNF-$\alpha$, GM-CSF, IL-10 and TGF-$\beta$1 expression in the supernatant. Compared with the LPS group, #p 0.05, ##p 0.01, compared with the LPS+HMGB1-siRNA+SHI group, *p 0.05, **p 0.01. Groups: control group, LPS group, HMGB1-siRNA group, LPS+HMGB1-siRNA group, LPS+SHI group, LPS+HMGB1-siRNA+SHI group.

We detected the expression of HMGB1, TLR4, MyD88, NF-$\kappa$B and p-NF-$\kappa$B proteins in macrophages in HMGB1-SiRNA knockdown cell lines using ELISA, IF and WB. The results showed that knocking down HMGB1 (LPS+HMGB1-siRNA group) inhibited the HMGB1-mediated TLR4/NF-$\kappa$B signaling pathway (p 0.05), and the results were the similar to those of the LPS+SHI group (p 0.05) (Fig. 8A). In addition, the two treatments showed a synergistic effect (p 0.05), confirming that SHI affected the polarization of macrophages through the regulation of the TLR4/NF-$\kappa$B pathway by HMGB1 (Fig. 8B,C). We detected the levels of related pathway proteins in RAW264.7 cells. As shown in Fig. 8E–G, HMGB1 knocked down can significantly inhibited the protein expression levels of P-NF-$\kappa$B, MyD88 and HMGB1 like SHI intervention (p 0.05), and the two have a synergistic effect (p 0.05). We detected the levels of related pathway proteins in RAW264.7 cells. As shown in Fig. 8D–G, HMGB1 knocked down can significantly inhibit the protein expression levels of p-NF-$\kappa$B, MyD88 and HMGB1 similar to SHI intervention (p 0.05), and the two had have a synergistic effect (p 0.05).

Fig. 8.

SHI affects macrophage polarization through the regulation of the TLR4/NF-$\kappa$B pathway by HMGB1. (A) HMGB1 expression in the supernatant. (B,C) TLR4 expression in RAW264.7 cells by IF. Scale bar: 50 μm. (D–G) p-NF-$\kappa$B,NF-$\kappa$B, HMGB1, MyD88 expression in the RAW264.7 by WB. Compared with the LPS group, #p 0.05, ##p 0.01, compared with the LPS+HMGB1-siRNA+SHI group, *p 0.05, **p 0.01. Group: control group, LPS group, HMGB1-siRNA group, LPS+HMGB1-siRNA group, LPS+SHI group, LPS+HMGB1-siRNA+SHI group.