Human THP-1 cells were obtained from the Korea Cell Line Bank and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37˚C in a 5% CO₂ incubator. To induce macrophage-like differentiation, THP-1 monocytes were treated with 10 nM phorbol 12-myristate 13-acetate (Sigma-Aldrich) for 24 h (23). Differentiated cells were then stimulated with 5 ng/ml LPS (Sigma-Aldrich). This LPS concentration was established under conditions that did not saturate cytokine expression (24). YC (MedChem Express) was administered at varying concentrations for experimental analyses. Based on cytotoxicity tests and previous reports (22), YC showed no cytotoxicity up to 10 µM when used alone, but a reduction in cell viability was observed at 10 µM when co-treated with LPS. Possible saturation effects were noted at concentrations above 1 µM (data not shown). Therefore, in this report, concentrations below 1 µM were selected to avoid cytotoxicity and ensure accurate assessment of pharmacological activity. YC was dissolved in dimethyl sulfoxide (DMSO) and used at a final concentration of ≤0.1%; 0.1% DMSO treatment was used as a negative control, which did not affect cell activation. To assess JAK inhibition, cells were treated with 2.5, 5, or 10 µM filgotinib, while STAT3 inhibition was evaluated using 25, 50, or 100 µM S3I-201 (Axon Medchem) as comparative groups.

THP-1 cells were seeded at a density of 5x10⁴ cells/well in 96-well plates and differentiated into macrophages. Differentiated cells were treated with YC at concentrations ranging from 0.1 nM to 10 µM for 24 h, with or without co-treatment with LPS (5 ng/ml). Following incubation, a CCK-8 viability assay (Donginbio) was performed according to the manufacturer's instructions. The plates were incubated at 37˚C for 1 h, and absorbance was measured at 450 nm using an EMax Plus Microplate Reader (Molecular Devices).

Total RNA was extracted from differentiated and activated THP-1 cells using TRI reagent (Molecular Research Center), according to the manufacturer's protocol. RNA concentrations were adjusted to 1 µg per sample, and complementary DNA (cDNA) synthesis was performed using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega) and oligo(dT) primers. Conventional PCR was carried out using a PCR premix (Bioneer). PCR amplification was performed in a C1000 Touch Thermal Cycler (Bio-Rad Laboratories) under the following conditions: initial denaturation at 95˚C for 5 min; followed by 26 cycles of denaturation at 95˚C for 30 sec, annealing at 50-65˚C for 30 sec, and extension at 72˚C for 30 sec; with a final extension at 72˚C for 5 min and holding at 4˚C. The primer sequences used are listed in Table I.

THP-1 cells (5x10⁵ cells/ml) were seeded in 48-well plates for IL-6 measurement. After 24 h of LPS or YC treatment, the cell culture supernatants were collected by centrifugation at 10,000 x g for 5 min. The levels of IL-6 in the supernatants were quantified using an ELISA kit (BD Biosciences; cat. no. 555220), according to the manufacturer's instructions. Optical density was measured at 450 nm using an EMax Plus Microplate Reader (Molecular Devices), and cytokine concentrations were determined based on a standard curve.

For in vitro experiments, cells were lysed using RIPA buffer (LPS Solution) supplemented with a protease inhibitor cocktail (Roche) and phosphatase inhibitors 2 and 3 (Sigma-Aldrich) to prevent protein degradation. For in vivo experiments, tissues were lysed using NP-40 buffer. Protein concentrations were determined using the Bradford assay with reagents purchased from Takara (Takara Bio Inc.), following the standard protocol described by Bradford. Lysates were mixed with 5X loading buffer, heated at 100˚C for 10 min, and separated on a 10% Tris-glycine gel. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). To block nonspecific binding, membranes were incubated in 5% skim milk for 1 h, followed by washing with phosphate-buffered saline containing 0.05% Tween-20 (PBST; Duchefa Biochemie). Membranes were then incubated overnight at 4˚C with primary antibodies (listed in Table II). Detection was performed using horseradish peroxidase-conjugated secondary antibodies (Enzo Life Sciences), and signals were visualized using a chemiluminescent substrate (Cyanagen Srl, Bologna, Italy).

Differentiated THP-1 cells were treated with YC (8, 40, and 200 nM), with or without LPS (5 ng/ml), for 2 h to investigate protein translocation. Cells were detached using Accutase solution (Innovative Cell Technologies), and nuclear and cytoplasmic protein fractions were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Inc.; cat. no. 78833), according to the manufacturer's instructions. Subsequently, the intracellular positions of phosphorylated STAT3 (p-STAT3) and total STAT3 were confirmed by western blotting analysis as described above.

Macrophage-like THP-1 cells were treated with varying concentrations of YC and LPS for 2 h, fixed onto slide glass, permeabilized with methanol, and blocked with 1% bovine serum albumin (BSA; bioWORLD). Fixed cells were incubated overnight at 4˚C with primary antibodies against p-STAT3 and STAT3 (both at 1:200 dilution; Cell Signaling Technology). Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:500 dilution; Life Technologies) was used for fluorescence detection. After washing, drying, and mounting with Roche mounting media, slides were examined at room temperature using a Zeiss LSM 510 Meta confocal microscope (Zeiss). Images were analyzed using Zeiss microscopy software (Zeiss).

Male C57BL/6N mice (6-8 weeks old, weighing 24-27 g) were purchased from Koatech and maintained under specific pathogen-free conditions with free access to food and water (25); 4-5 mice were housed per cage. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Research Institute of Bioscience and Biotechnology (KRIBB; approval no.: KRIBB-AEC-24265) and conducted in accordance with Korean national animal welfare regulations and the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

The mice were divided into four groups for the experiment: Control, LPS, YC (0.2 µg/kg) + LPS, and YC (5 µg/kg) + LPS. On day 0, the mice were administered YC intraperitoneally 30 min prior to LPS injection. Two doses of YC (0.2 and 5 µg/kg) were chosen based on previous studies investigating the toxicity and effects of YC on renal function (26,27). Thirty minutes later, the mice received an intraperitoneal injection of LPS (10 mg/kg; Sigma-Aldrich) dissolved in PBS (28,29), while the control group was given vehicle treatment. Blood samples (~500 µl per mouse) were collected via retro-orbital bleeding under 2-3% isoflurane anesthesia in accordance with protocols approved by the IACUC and international animal welfare guidelines. The mice were euthanized immediately after blood collection by cervical dislocation to minimize pain. This procedure was performed quickly and skillfully by trained personnel. Kidneys were promptly harvested, then either stored at -80˚C for later experiments or fixed in 10% formalin for subsequent analysis. Acute kidney injury (AKI) was confirmed within 48 h by an increase in baseline serum creatinine (sCr) of 2.3-fold according to the KDIGO guidelines (30).

Serum urea nitrogen and creatinine levels in the AKI mouse model were analyzed by KP&T as a commissioned service.

Total RNA was extracted from mouse kidney tissues using TRI reagent (Molecular Research Center) according to the manufacturer's instructions. RNA concentrations were normalized to 1 µg per sample for cDNA synthesis. qPCR was performed using the QuantStudio 3 Real-Time PCR System (Applied Biosystems) and PowerUp SYBR Green Master Mix (Applied Biosystems), according to the manufacturer's protocol. The thermal cycling conditions included an initial uracil-DNA glycosylase activation step at 50˚C for 2 min, followed by polymerase activation and initial denaturation at 95˚C for 10 min. This was followed by 50-60 cycles of denaturation at 95˚C for 15 sec and annealing/extension at 60˚C for 1 min. A melt curve analysis was conducted following amplification to confirm the specificity of the PCR products. Target gene expression levels were normalized to GAPDH expression and quantified using the comparative 2^-ΔΔCq method (31). Primer sequences used for quantitative PCR are listed in Table III.

For histological analysis, mouse kidneys were fixed in 10% formalin for 24 h, embedded in paraffin, and sectioned at a thickness of 4 µm. Tissue sections were stained with hematoxylin and eosin (H&E) for histopathological evaluation. For immunohistochemistry (IHC), the sections were permeabilized with 1% Triton X-100 solution (Sigma-Aldrich) and treated with peroxidase-blocking solution (Dako) to reduce nonspecific background staining. To block nonspecific binding, sections were incubated with 1% BSA (bioWORLD) for 10 min. After blocking, sections were incubated overnight at 4˚C with primary antibodies against p-STAT3 (1:200 dilution; Cell Signaling Technology) and p-JAK1 (1:200 dilution; Invitrogen; Thermo Fisher Scientific, Inc.). After washing, slides were incubated with a secondary antibody (1:500 dilution; Enzo Life Sciences) for 1 h at room temperature. The slides were counterstained with hematoxylin, followed by bluing using a bluing buffer, and then immersed in distilled water. Finally, slides were mounted with mounting solution (Sigma-Aldrich), and images were captured using a light microscope (Leica).

Based on a previous study (32), the H&E-stained kidney slides were histologically graded for tubular injury. Histological evaluation was performed according to three criteria of kidney injury: tubular necrosis, tubular vacuolization, and tubular cast formation. Severity was graded on a scale from 0 to 3 as follows: 0 (damage in <5% of tubules), 1 (damage in 5-33% of tubules), 2 (damage in 34-66% of tubules), and 3 (damage in >66% of tubules). Twenty random fields per kidney sample were analyzed at 20x magnification.

Statistical analysis was performed using GraphPad Prism version 10.0 (GraphPad Software, Inc.). Data are presented as the mean ± standard deviation. One-way ANOVA followed by Tukey's or Dunnett's multiple comparisons test, or Kruskal-Wallis test followed by Dunn's multiple comparisons test were performed depending on data distribution. Two-way ANOVA followed by Šidák post hoc test was also performed for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference. Image processing and quantification were conducted using ImageJ software (National Institutes of Health).

YC, a diterpene compound (Fig. 1A), was evaluated for cytotoxicity by treating cells with various concentrations (0.1 nM-10 µM) for 24 h in the presence or absence of LPS (5 ng/ml). As shown in Fig. 1B, YC alone did not exhibit cytotoxicity up to 10 µM (left panel). However, co-treatment with LPS resulted in approximately a 30% reduction in cell viability at 10 µM (right panel). Since a saturation effect was observed at concentrations above 1 µM (data not shown), subsequent experiments were conducted using YC at concentrations below 1 µM to avoid both cytotoxic effects and potential saturation associated with higher concentrations.

To assess the effects of YC, we examined its impact on LPS-induced cytokine expression using RT-PCR and ELISA. THP-1 cells were treated with 5 ng/ml LPS and 200 nM YC. We found that TNF-α was initially expressed with LPS induction at 1 h; subsequently, TNF-α expression appeared to increase in the YC group at 4 h. IL-1β expression started at 2 h after treatment, and there was minimal variation in comparison to the LPS treatment group. However, the IL-6 expression was decreased by YC, following 4 h of administration, in contrast to the LPS treatment group (Fig. 1C).

To further validate these results, YC was administered at varying concentrations (8, 40, and 200 nM) at 4 h, at which time the presence of IL-6 was confirmed. RT-PCR analysis showed a dose-dependent decrease in IL-6 mRNA expression after LPS stimulation, but not in TNF-α and IL-1β expression, by YC (Fig. 1D). Moreover, to confirm the effect on IL-6 inhibition at the protein level, THP-1 cells were treated with 5 ng/ml LPS and 200 nM YC, and their effects on the cells were recorded over time (0, 6, 18, and 24 h). As with previous RT-PCR data, the expression of IL-6 induced by LPS treatment was significantly suppressed by YC (Fig. 1E). Additionally, ELISA results for IL-6 protein levels treated with various concentrations of YC confirmed dose-dependent inhibition after 24 h of treatment (Fig. 1F).

To determine how YC specifically regulates LPS-induced IL-6 expression, we analyzed LPS-related pathways using western blotting. The total protein levels of key signaling molecules, including NF-κB, MAPK, and JAK/STAT3, remained unchanged in LPS- and 200 nM YC-treated cells over 12 h. Phosphorylation of p65, p38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase was detected within 1 h of LPS treatment; however, YC co-treatment did not significantly affect the phosphorylation of these molecules. In contrast, phosphorylation of JAK1 and STAT3 was observed at 2 h of LPS treatment, and YC co-treatment effectively inhibited the phosphorylation of these proteins (Fig. 2A). The JAK/STAT pathway is a critical signaling cascade involved in IL-6 production (10). To further elucidate the concentration-dependent effects of YC, an additional experiment was conducted to assess its impact on the expression and phosphorylation levels of JAK1 and STAT3. The results confirmed that YC suppressed JAK1 and STAT3 phosphorylation in a concentration-dependent manner (Fig. 2B). These findings suggest that the YC-mediated inhibition of JAK1/STAT3 activation is a key mechanism underlying the suppression of IL-6 expression.

To investigate whether YC prevents STAT3 nuclear translocation, we analyzed cytoplasmic and nuclear fractions of THP-1 cells treated with 5 ng/ml LPS and various concentrations of YC. Protein immunoblotting revealed that activated STAT3 translocated to the nucleus in response to LPS stimulation. However, in cells treated with YC, phosphorylated STAT3 levels were reduced in both the cytoplasmic and nuclear fractions in a dose-dependent manner (Fig. 3A). To further confirm these findings, confocal microscopy was performed to visualize STAT3 localization. In unstimulated cells, STAT3 was primarily localized in the cytoplasm. Following LPS treatment, phosphorylated STAT3 accumulated in the nucleus. However, YC treatment effectively blocked STAT3 phosphorylation, leading to a significant reduction in nuclear translocation (Fig. 3B and C).

To evaluate the efficacy of YC in suppressing LPS-induced IL-6 expression via inhibition of JAK1 and STAT3, we compared its effects with those of known inhibitors. Specifically, filgotinib, a selective JAK1 inhibitor, and S3I-201, a STAT3 inhibitor, were used for comparison. The results showed that LPS stimulation significantly increased IL-6 expression in THP-1 cells. However, treatment with filgotinib, S3I-201, or YC reduced IL-6 expression in a dose-dependent manner (Fig. 4A-C). In particular, YC exhibited similar inhibitory effects at markedly lower concentrations than those inhibitors. These findings suggest that LPS-induced IL-6 expression in THP-1-derived macrophages is regulated via inhibition of JAK and STAT3, and that YC is a potential therapeutic agent for various diseases associated with IL-6 and the JAK1/STAT3 pathway.

Through the above experiments, we found that YC inhibits the expression of LPS-induced IL-6 in human macrophages. Therefore, we hypothesized that YC could alleviate tubular damage caused by macrophages in an AKI disease model, a frequent complication of LPS-induced sepsis (33). AKI results in an abrupt deterioration in renal function (11). To evaluate the efficacy of YC in an LPS-induced AKI mouse model, we identified renal function markers and evaluated histopathological changes. As shown in Fig. 5A and B, YC treatment at 5 µg/kg slightly reduced sCr levels, though no significant decrease in blood urea nitrogen was observed. These findings suggest that YC confers partial protection against kidney dysfunction (34). To evaluate the impact of YC on kidney tubular damage, histological changes in H&E-stained kidney tissues were examined following LPS and YC treatment (32). Compared to kidneys treated with LPS alone, YC treatment alleviated renal pathological damage, including tubular necrosis, vacuolization, and cast formation (Fig. 5C and D).

The mRNA expression levels of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), well-established biomarkers of renal injury (35), were analyzed by RT-qPCR. The results showed that LPS-induced kidney injury led to a marked increase in NGAL and KIM-1 expression, whereas YC treatment reduced their mRNA levels in a dose-dependent manner (Fig. 5E and F). These results confirm that LPS effectively induces septic AKI and that YC treatment mitigates renal injury.

Inflammation significantly contributes to renal injury in sepsis, with IL-6 serving as a valuable prognostic marker for AKI (36). Therefore, we assessed IL-6 expression in kidney tissues using RT-qPCR. As shown in Fig. 5G, LPS administration significantly increased IL-6 mRNA levels in kidney tissue, whereas YC treatment suppressed IL-6 expression.

To determine whether the regulation of the JAK1/STAT3 axis in macrophages by YC also applies to renal tissues, we investigated the expression of JAK1/STAT3 in renal tissues in the AKI mouse model by western blotting and IHC. As shown in Fig. 5H, YC pretreatment effectively reduced JAK1 and STAT3 phosphorylation in LPS-treated kidneys. IHC analysis further confirmed that JAK1/STAT3 phosphorylation was significantly elevated in LPS-treated kidneys compared to normal controls, whereas YC treatment attenuated JAK1 activation and restored STAT3 activation to basal levels (Fig. 5D). These findings suggest that YC exerts prophylactic effects against septic AKI by inhibiting JAK1/STAT3 signaling and IL-6 expression.