RAW 264.7 cells were obtained from the Institute for Advanced Study of Central South University (Changsha, China). DMEM (Shanghai BasalMedia Technologies Co., Ltd.) supplemented with 10% FBS (Shanghai BasalMedia Technologies Co., Ltd.), 100 U/ml penicillin and 25 µg/ml amphotericin B (Hyclone^™; Cytiva) was used as the cell culture medium, with incubation conditions set at 37˚C and 5% CO₂ (21).

Cytotoxicity assays were performed using the Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions (NCM Biotech Co., Ltd.). Briefly, RAW 264.7 cells were seeded into 96-well plates at a density of 1.0x10⁴ cells/well for 24 h, followed by treatment with varying concentrations (0, 100, 200, 400, 800 or 1,600 µM) of SPD (≥99% gas chromatography grade; MilliporeSigma) for additional durations of 6, 12, 24 and 48 h at 37˚C in a 5% CO₂ incubator. Subsequently, for the cytotoxicity assay, 10 µl CCK-8 solution was added to each well and incubated for 1 h. Finally, the absorbance was measured at a wavelength of 450 nm and cell viability was calculated based on the measured optical density values.

RAW 264.7 cells were inoculated into a 6-well plate at a density of 4x10⁵ cells/well and cultured for 24 h until they adhered to the well walls. Pretreatment with varying concentrations (100, 200, 400 or 800) of SPD or vehicle control (complete DMEM) for 1 h was followed by stimulation with 1 µg/ml LPS 24 h at 37˚C in a 5% CO₂ incubator, and this cell treatment protocol was applied uniformly prior to all subsequent experiments in this study. Prior to antibody staining, cells were stained with Zombie NIR^™ Fixable Viability Kit (1:1,000; cat. no. 423105; BioLegend) for 15 min at room temperature in the dark to distinguish live/dead cells. Subsequently, the cells were gently harvested, washed twice with PBS and stained with CD86-phycoerythrin (1:100; clone GL-1; cat. no. 553692; BD Biosciences) and CD206-BUV395 (1:100; clone Y17-505; cat. no. 568817; BD Biosciences) for 30 min at 4˚C in the dark. Fluorescence intensity was assessed using the BD FACSCanto^™ II flow cytometer (BD Biosciences), and the collected data were analyzed using BD FACSDiva^™ software (Version 8.0; BD Biosciences).

Gating hierarchy was as follows: i) Forward scatter (FSC)/side scatter gating to exclude debris; ii) singlet gating (FSC-area vs. FSC-height); iii) live cell gating (Zombie NIR^™-negative); and iv) CD86/CD206 double staining gating. Compensation was performed using single-stained controls and fluorescence minus one control and isotype-matched antibodies (PE Rat IgG2a; 1:100; cat. no. 553930; and BUV395 Rat IgG2a; 1:100; cat. no. 563556; both BD Biosciences) were used to set the gating thresholds.

Total RNA was isolated from the cells using the Cell Total RNA Extraction Kit (cat. no. G3694; Wuhan Servicebio Technology Co., Ltd.) according to the manufacturer's instructions. The concentration of total RNA was quantified using a NanoDrop spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc.), whereas cDNA was synthesized using the PrimeScript^™ RT kit (Takara Bio, Inc.). Gene-specific primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and qPCR was carried out using the TB Green^® Premix Ex Taq^™ II kit as per the manufacturer's instructions (Wuhan Servicebio Technology Co., Ltd.). The primer sequences are listed in Table I. RNA amplification reactions were performed using the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Inc.) with an initial denaturation step at 95˚C for 30 sec, followed by 40 cycles of amplification (denaturation at 95˚C for 5 sec and annealing at 60˚C for 34 sec), and finally, a dissociation curve was generated. After amplification, the data were analyzed using the 2^-ΔΔCq method (22) with the mouse Gapdh gene as the reference (23).

The cells were resuspended in complete DMEM, seeded into 6-well plates and incubated for 24 h. After a 1 h pretreatment with SPD, LPS was added to the co-culture system. Cytokine levels in the cell supernatants were quantified using a ELISA kits (Mouse IL-6 ELISA kit, cat. no. EMC004.96; Mouse IL-10 ELISA kit, cat. no. EMC005.96; both Neobioscience Technology Co., Ltd.) according to the manufacturer's instructions.

The cells were seeded into 6-well plates and incubated for 24 h. Subsequently, the cells were pretreated with SPD for 1 h and then stimulated with LPS for another 24 h. Immediately after stimulation, total proteins were extracted on ice using RIPA Lysis Buffer (Wuhan Servicebio Technology Co., Ltd.). The protein concentration was determined using a BCA assay (BCA Protein Assay Kit; cat. no. P0012; Beyotime Biotechnology). Equal amounts of protein (20 µg per lane) were separated by 12% SDS-PAGE (cat. no. BL522A; Labgic Technology Co., Ltd.) and transferred onto PVDF membranes (cat. no. IPVH10100, Merckmillipore Co., Ltd.). The membranes were blocked with 5% skimmed milk in Tris-Buffered Saline with Tween-20 (TBST; cat. no. G0004-1L; Wuhan Servicebio Technology Co., Ltd.) for 1 h at room temperature. The membranes were then incubated with the following primary antibodies overnight at 4˚C: p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), phosphorylated (p)-p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.), IκBα (1:1,000; cat. no. 4814; Cell Signaling Technology, Inc.), p-IκBα (1:1,000; cat. no. 2859; Cell Signaling Technology, Inc.) and β-tubulin (1:1,000; cat. no. 10094-1-AP; Proteintech Group, Inc.). After washing with TBST, the membranes were incubated with the corresponding HRP-linked secondary antibodies for 1 h at room temperature, namely anti-rabbit IgG (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.) and anti-mouse IgG (1:5,000; cat. no. 7076; Cell Signaling Technology, Inc.). The protein bands were visualized using a super-sensitive ECL chemiluminescent substrate (cat. no. BL5965A; Labgic Technology Co., Ltd.) and detected with a chemiluminescence imaging system (ChemiDoc Imaging System, Bio-Rad Laboratories, Inc.). The gray values of the target bands were semi-quantified using ImageJ software version 1.53 (National Institutes of Health) to determine the relative protein expression levels and β-tubulin was used as an internal control.

Statistical analysis and data presentation were performed using SPSS 26.0 (IBM Corp.) and GraphPad Prism 8.0 (Dotmatics) software. Differences among multiple groups were evaluated using one-way ANOVA, followed by Tukey's multiple comparison test for pairwise comparisons. P<0.05 was considered to indicate a statistically significant difference.

To evaluate whether SPD treatment induced cytotoxicity, cells were incubated with different concentrations of SPD for 6, 12, 24 and 48 h. As shown in Fig. 1, treatment with 100 and 200 µM SPD for 6 h significantly enhanced RAW 264.7 cell viability (P<0.05). After 12 h of treatment, only 100 µM SPD significantly increased cell viability (P<0.001). After 24 h of 0-800 µM SPD treatment, RAW 264.7 macrophage viability increased significantly (P<0.001), however the difference in cell viability between different concentration subgroups (200, 400, 600 and 800 µM) was not significant (P>0.05). Treatment with 1,600 µM SPD induced cytotoxicity compared with that of the control group (DMEM) (P<0.001). After 48 h of treatment, none of the SPD concentrations improved cell viability. Therefore, SPD concentrations of 0-800 µM and a 24 h treatment duration were chosen for subsequent experimentation.

M1-type macrophages express high levels of CD86, whereas M2-type macrophages express CD206(24). The proportions of M1 and M2 cells after LPS-induced polarization of RAW 264.7 cells were ascertained using flow cytometry. CD86^-CD206^- represents M0 macrophages, CD86⁺CD206^- represents M1 macrophages and CD86^-CD206⁺ represents M2 macrophages. The findings revealed that SPD treatment diminished macrophage CD86 molecule levels and shifted macrophage polarization from a pro-inflammatory M1 phenotype towards an anti-inflammatory M2 phenotype after 24 h; this effect was most notable in response to 800 µM SPD (Fig. 2A-E). The results revealed that LPS stimulation significantly increased the percentage of M1 macrophages and decreased the percentage of M0 or M2 macrophages compared with the control. Co-treatment with SPD dose-dependently reversed this effect. Specifically, increasing SPD concentrations (200, 400 and 800 µM) gradually decreased the M1 percentage while concurrently increasing the M2 percentage. The percentage of M0 macrophages remained largely unchanged across SPD groups. These data indicate that SPD can redirect LPS-induced M1 polarization toward an M2 phenotype in a dose-dependent manner (P<0.05; Fig. 2F-H).

RT-qPCR was performed to assess the ability of LPS to suppress the expression of inflammatory cytokines. LPS significantly elevated the mRNA expression levels of Tnfα, Il6, Il1b and nitric oxide synthase (Nos2) compared with those in the control group (Fig. 3A-D). The mRNA levels of Tnfα, Il6, Il1b and Nos2 then gradually diminished with increasing SPD concentrations, thereby suggesting a significant dose-dependent effect (P<0.01). Relative to those in the control group, LPS significantly elevated the mRNA expression levels of arginase 1 (Arg1) and Il10. Compared with those in the LPS group, the mRNA expression levels of arginase 1 (Arg1) and Il10 were significantly increased in the 200 and 400 µM SPD groups (P<0.01), and peaked after treatment with 800 µM SPD (Fig. 3E and F). Furthermore, SPD treatment reduced the mRNA expression levels of Tnfα, Il6, Il1b and Nos2 at all tested SPD concentrations. These results implied that SPD inhibited autoinflammation.

A major function of M1-polarized macrophages is the production of pro-inflammatory cytokines. Initially, IL-6 secreted by RAW 264.7 cells into the conditioned medium was examined using ELISA. LPS notably stimulated IL-6 secretion by macrophages compared with that in the control group (Fig. 4A). This suggested that RAW 264.7 cells were activated by LPS stimulation, thereby inducing an inflammatory response. In addition, SPD markedly reduced IL-6 production in a dose-dependent manner compared with that in the LPS group. LPS stimulation led to an elevation in IL-10 levels compared with those in the control group. IL-10 was not significantly induced at the lower SPD concentration (200 µM) but was significantly enhanced compared with that in the LPS group at higher concentrations (400 and 800 µM) (Fig. 4B). These results indicated that SPD may promote anti-inflammatory effects by both inhibiting IL-6 and inducing IL-10 secretion.

NF-κB is a key target pathway activated by inflammation (25). Therefore, NF-κB activation was investigated through western blotting to determine whether SPD inhibited its activation (Fig. 5A). The levels of the NF-κB pathway-associated proteins p65, p-p65, IκBα and p-IκBα were analyzed after 24 h of treatment with LPS and SPD. The expression levels of p-p65 and p-IκBα were significantly increased in macrophages after 24 h of LPS stimulation compared with that in the control group (Fig. 5B and C). Notably, in cultured RAW 264.7 cells, three SPD concentrations (200, 400 and 800 µM) inhibited the LPS-induced increase in p-p65 and p-IκBα protein expression after 24 h of incubation in a dose-dependent manner. Treatment with all tested SPD concentrations for 24 h markedly inhibited the activation of both p-p65 and p-IκBα proteins. These results suggested that the mechanism underlying the anti-inflammatory effect of SPD may involve inhibition of NF-κB activation, which in turn inhibits the expression of inflammatory genes.