.0% purity, batch no. 20170206; Fig. 1A; Dalian Fusheng Natural Medicine Development Co., Ltd.) was dissolved in DMSO to a concentration of 10 g/ml as a stock solution, which was stored at −20°C for subsequent use. CP (batch no. 170905) was obtained from Jiangsu Haosen Co. at a concentration of 100 mM. SIRT1 (13161-1-AP, 1:1,000), NLRP3 (27458-1-AP, 1:1,000), p65 (80979-10RR, 1:1,000) and phosphorylated (p-)p65 (82335-1-RR, 1:1,000) were from Proteintech. Macrophage migration inhibitory factor (MIF) (ab7207, 1:2,000), p53 (ab32049, 1:2,000), p-p53 (ab33889, 1:1000), P-gp (ab170904, 1:1,000), OCT2 (ab179808, 1:5,000), vascular cell adhesion molecule 1(VCAM1, ab134047, 1:2,000) and intercellular adhesion molecule 1(ICAM1, ab282575, 1:1,000) were from Abcam. Caspase-3 (YM8058, 1:1,000) was from Immunoway, and Caspase-9 (cat. no. AF1264, 1:1,000) was from Beyotime (Table SI). EX527 was from Selleck. Anti-rabbit IgG (A0208, 1:1,000), Anti-mouse IgG (A0216, 1:1,000) and GAPDH (AF0006, 1:1,000) were obtained from Beyotime Biotechnology. The Alexa Fluor 488-goat anti-rabbit secondary antibody (abs20025, 1:100) was acquired from Absin Biotechnology Company.

LLC and human renal tubular (HK-2) cells were obtained from the School of Pharmacy, Jilin University (Changchun, China). RPMI-1640 medium, DMEM/F12, penicillin, streptomycin and fetal bovine serum were purchased from Absin (Shanghai) Biotechnology Co., Ltd. LLC cells were cultivated in RPMI-1640 medium containing 10% fetal bovine serum, 100 µg/ml streptomycin and 100 U/l penicillin. Cells were kept at 37°C in a 5% CO₂ humidified atmosphere. HK-2 cells were cultured in DMEM/F12 under the same conditions. Cell cultures were passaged every 2-3 days and passages 3-8 were utilized for subsequent experiments.

LLC cells were treated with CP (100, 200, 400, 800 and 1,600 nM), Rg3 (10, 20, 40 and 80 µg/ml) or CP (100 nM) + Rg3 (20, 40 and 80 µg/ml) for 4, 12, 24 and 48 h at 37°C. HK-2 cells were treated with CP (10 µM), Rg3 (80 µg/ml), Ex527 (30 µM), CP (10 µM) + Rg3 (80 µg/ml) or CP (10 µM) Ex527 (30 µM) at 37°C.

Cell viability was assessed using the MTT assay (Sigma-Aldrich; Merck KGaA). Briefly, cells were seeded in 96-well plates at 1×10⁴ cells/well. A total of 10 µl 5 mg/ml MTT solution was added to each well for 4 h. The supernatant was discarded, and 150 µl DMSO was added to each well to dissolve the formazan crystals. Absorbance was measured at 490 nm using a Microplate Reader (Agilent Technologies). Each experiment condition was replicated six times, and cell viability was calculated relative to the untreated control group.

The LLC cells were seeded (without serum) in 6-well plates at 5×10⁴ cells/well and cultured to 100% confluence. Sterile pipette tips were used to create 100 µm wounds. Cells were treated with CP (100 nM) in the presence or absence of Rg3 (20, 40 and 80 µg/ml) for 24 h at 37°C. Olympus CKX41 light microscope was used to capture images.

HK-2 cells were seeded in 6-well plates (1×10⁵/well). Following overnight incubation at 37°C, they were treated at 37°C for 24 h with CP (10 µM), CP (10 µM) + Rg3 (80 µg/ml), CP (10 µM) + Rg3 (80 µg/ml) + Ex527 (30 µM) or Ex527 (30 µM). Cells were washed four times with cold PBS, fixed at room temperature with 4% paraformaldehyde for 10 min and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Following three 5 min PBS washes, cells were blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) at 37°C for 1 h, then incubated overnight at 4°C with rabbit anti-NLRP3 antibody (cat. no. 27458-1-AP, 1:100). Following three PBS washes, cells were incubated with the Alexa Fluor 488-goat anti-rabbit secondary antibody (cat. no. abs20025, 1:100, Absin) at room temperature for 1 h in the dark. Following three PBS washes, cells were stained with DAPI at room temperature for 5-10 min. Fluorescence images were captured using a BX83 fluorescence microscope (Olympus Corporation). Images were analyzed using ImageJ software (version 1.52a, National Institutes of Health).

SOD (A001-302), MDA (A003-401), CAT (A007-1-1) and LDH (A020-2-2) Assay kits (Nanjing Jiancheng Bioengineering Institute) were used to assess intracellular SOD, MDA, CAT and LDH levels according to the manufacturer's instructions.

The structure of Rg3 was retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), and imported into Chem3D software (Chem3D Pro 21.0, revvitysignals.flexnetoperations.com/), where it underwent optimization and energy minimization using the MM2 module. The structures of NLRP3 (ID: 7ALV) and SIRT1 (ID: 4ZZI) were acquired from Research Collaboratory for Structural Bioinformatics Protein Data Bank (rcsb.org/). These structures were processed within the Maestro11.9 (Schrödinger, Inc.) platform employing Schrodinger's Protein Preparation Wizard. This processing involved removal of crystalline water molecules, addition of missing hydrogen atoms, repair of missing bond information and patching of incomplete peptide segments. Finally, the proteins were energy-minimized and geometrically optimized. The binding affinity between NLRP3, SIRT1 and Rg3 was evaluated based on binding energy and the virtual docking model was visualized using Pymol2.1 software (Schrödinger, LLC).

All animal care and experimental procedures were approved by the Center for Experimental Animal Research of the Institute of Basic Medical Sciences, Jilin University (Changchun, China; approval no. 2023226). The experiments with animal followed an Animal Research: Reporting of In Vivo Experiments protocol (15). All animal procedures were performed in strict accordance with the guidelines (16) for tumor-bearing mice established by the Center for Experimental Animal Research, Institute of Basic Medical Sciences, Jilin University. Male Kunming mice (n=32; age, 4-6 weeks; weight, 22±2 g) were purchased from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). Mice were allowed free access to food and water and were maintained on a 12/12-h light/dark cycle at a temperature of 20-25°C and humidity of 50±5%. Tumors were induced by subcutaneous injection of 4×10⁶ LLC cells in 200 µl PBS into the right axilla. Tumor-bearing mice were randomized using a computer-generated random number sequence (Excel 2019, Microsoft Corporation) with block randomization and allocation concealment using sequentially numbered, sealed envelopes prepared by an independent technician; investigators conducting tumor measurements and tissue analysis were blinded to group allocation throughout the experiment. Mice were grouped (n=8/group) as follows: i) Control, receiving intraperitoneal (IP) injection of 0.9% NaCl and daily oral water gavage; ii) CP, receiving IP injection of CP at 4 mg/kg every 2 days, and daily oral water gavage; iii) CP + Rg3, receiving IP injection of CP at 4 mg/kg every 2 days, and daily oral Rg3 (5 mg/kg); and iv) Rg3, receiving daily oral Rg3 (5 mg/kg), as previously described (20) (Fig. S1). Administration was initiated when tumor volumes reached 50 mm³. The tumor volume and body weight were measured every 2 days post-inoculation, with tumor volume calculated as 1/2x tumor length x tumor width². Humane endpoints were tumor diameter ≥15 mm and >20% body weight loss. Following the 10 day drug administration period, no animals were excluded from the final analysis. The blood was collected via abdominal aortic puncture. Mice were euthanized using CO₂ inhalation (chamber volume displaced by the CO₂ flow rate, 30%). Death was confirmed by absence of response to external stimuli, such as a gentle prod with a blunt object and the cessation of heartbeat and breathing. Tumors and kidneys were immediately excised in an aseptic manner for analysis.

Prior to euthanasia, mouse body weight was measured. Tumors were surgically removed following euthanasia, dried with filter paper and weighed. Tumor-to-body weight ratio was calculated as (tumor weight/body weight) ×100%.

Tumor tissue was fixed in 10% formalin at room temperature for 24 h, embedded in paraffin and sectioned at 4 µM. These sections underwent deparaffinization, rehydration and hematoxylin-eosin staining at room temperature (hematoxylin for 5 min and eosin for 2 min), with histopathological changes examined at 400X magnification via a light microscope (Nikon Corporation; Eclipse TS200). Kidney tissue was fixed in 10% neutral buffered formalin at room temperature for 24 h, then embedded in paraffin and sectioned at 4 µM. Sections were mounted on glass slides and dried overnight at 37°C. Paraffin sections were deparaffinized in xylene twice for 10 min each, then rehydrated through a graded ethanol series, followed by rinsing in PBS. Antigen retrieval was performed by heating in 10 mM sodium citrate buffer (pH 6.0) in a microwave oven at 95-100°C for 15-20 min. After cooling to room temperature, sections were washed three times with PBS for 5 min each. Deparaffinized sections were treated with 3% hydrogen peroxide (v/v) in methanol for 15 min at room temperature, followed by washing twice with PBS. Non-specific binding was blocked with 10% BSA at room temperature for 30 min. Sections were incubated with primary antibody against OCT2 (1:100, ab179808, Abcam) overnight at 4°C in a humidified chamber. After washing three times with PBS for 5 min each, sections were incubated with the Alexa Fluor 488-goat anti-rabbit secondary antibody (cat. no. abs20025, 1:100 dilution, Absin) at room temperature for 30 min. Immunoreaction products were observed under a light microscope (Olympus BX53). Images were captured and analyzed using ImageJ software (version 1.52a, National Institutes of Health).

Apoptosis was identified using the TUNEL kit. Kidney tissue was fixed in 10% neutral buffered formalin at room temperature for 24 h, embedded in paraffin and sectioned at 4 µM. Sections were mounted on glass slides and dried overnight at 37°C. Paraffin sections were deparaffinized in xylene twice for 10 min each, then rehydrated through a graded ethanol series (100, 95, 85, 75, and 50% ethanol, 2 min each), followed by rinsing in phosphate-buffered saline (PBS). Sections were permeabilized with Proteinase K (20 µg/ml) in PBS at 37°C for 30 min, followed by washing twice with PBS. TUNEL staining was performed using the TUNEL BrightGreen Apoptosis Detection kit (Beyotime Biotechnology, cat. no. C1088) according to the manufacturer's instructions. Briefly, sections were incubated with TUNEL reaction mixture at 37°C for 60 min in a humidified chamber protected from light. Sections were stained with DAPI (1 µg/ml) at room temperature for 5-10 min, then rinsed three times with PBS. Sections were mounted with antifade mounting medium (Vector Laboratories, H-1700 or Fluoromount-G) and covered with glass coverslips. TUNEL-positive cells were visualized using a fluorescence microscope (Nikon Eclipse TS200). For each tissue section, three randomly selected fields of view were captured. TUNEL-positive (green fluorescent) cells and total cells (DAPI-stained blue nuclei) were counted, and the apoptotic index (%) was calculated as follows: (number of TUNEL-positive cells/total number of DAPI-stained nuclei) ×100%.

Cells were rinsed with cold PBS and lysed in RIPA buffer (Beyotime Biotechnology) supplemented with 1% (w/v) PMSF. Tumor and kidney tissue were minced and homogenized in RIPA buffer containing 1% PMSF on ice. Cell suspensions and tissue homogenates were centrifuged at 15,000 g for 15 min at 4°C. Protein concentrations were determined using the bicinchoninic acid method. Aliquots containing 30 µg protein per lane were mixed with 5X loading buffer and heated at 100°C for 5 min to denature. Protein samples were separated by 10-12% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST (0.1% Tween-20) at room temperature for 1 h, followed by overnight incubated with primary antibodies at 4°C. The primary antibodies were as follows: SIRT1 (1:1,000 dilution, 13161-1-AP, Proteintech), NLRP3 (1:1,000, 27458-1-AP, Proteintech), p65 (1:1,000 dilution, 80979-10RR, Proteintech), p-p65 (1:1,000 dilution, 82335-1-RR, Proteintech), MIF (1:1,000 dilution, ab7207, Abcam), p53 (1:2,000 dilution, ab32049, Abcam), p-p53 (1:2,000 dilution, ab338899, Abcam), P-gp (1:1,000 dilution, ab170904, Abcam), OCT2 (1:5,000 dilution, ab179808, Abcam), VCAM1 (1:2,000 dilution, ab134047, Abcam), ICAM1 (1:1,000 dilution, ab282575, Abcam), Caspase-3 (1:1,000 dilution, YM8058, Immunoway), Caspase-9 (1:1,000 dilution, AF1264, Beyotime), and GAPDH (1:1,000 dilution, AF0006, Beyotime) at 4°C. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (cat. nos. A0208, 1:1,000, Anti-mouse IgG, A0216, 1:1,000, Beyotime) at room temperature for 1 h. Protein bands were detected using an ECL kit (New Cell & Molecular Biotech Co., Ltd.), and visualized on X-ray film (Kodak). Band images were quantified using ImageJ 1.37c software (National Institutes of Health).

GraphPad Prism 9.0 software (Dotmatics) was employed for statistical analysis. Data are presented as the mean ± standard error of the mean from ≥3 independent experiments. One-way ANOVA followed by Tukey's post hoc test was applied. P<0.05 was considered to indicate a statistically significant difference.

MTT assay was employed to measure cell viability (17). The findings revealed that both CP and Rg3 reduced LLC cells viability in dose- and time-dependent manner (Fig. 1B-D). The combination of Rg3 and CP resulted in a time-dependent decrease in cell viability, with significant reductions at 12, 24 and 48 h compared with CP treatment alone. CP + Rg3 (40 and 80 µg/ml) groups exhibited slower migration than the CP group (Figs. 1E and S2). These results indicated that Rg3 enhanced therapeutic efficacy of CP.

To evaluate whether Rg3 enhances CP anti-tumor activity in vivo, tumor volume and weight were measured in a xenograft model. There was no significant difference in tumor volume between any groups. Only the CP + Rg3 group exhibited significantly decreased tumor weight compared with CP group (Fig. 2A-C). This suggests that Rg3 increased CP antitumor effect and slowed tumor growth.

HE and TUNEL staining were used for histopathological analysis. Tumor cells were closely packed with clear nucleoli and a diffuse pattern. The CP + Rg3 group demonstrated a larger area of unclear borders with cell nucleus disappearance compared with CP alone, supporting a role of Rg3 in enhancement of chemosensitivity (Fig. 2D). The TUNEL assay demonstrated increased apoptosis in the co-treatment group, as indicated by higher brown staining intensity (Fig. 2E). This suggested that the combination of CP and Rg3 enhanced tumor cell apoptosis.

Western blotting was performed to quantify the alterations in apoptotic protein expression following treatment with CP and Rg3. Rg3 significantly elevated the levels of cleaved caspase-3 and -9 and p-p53, which are pivotal for apoptosis (18,19), both in LLC cells and tumor tissue (Fig. 3A-D). The enhancement in these apoptotic markers following CP + Rg3 treatment suggested a potential therapeutic strategy to induce programmed cell death.

Western blot analysis was performed to assess the effects of Rg3 and CP on the expression of VCAM-1, ICAM-1, p-p65 and MIF in LLC cells and mouse tumor tissue samples (Fig. 3E-H). Notably, the co-treatment with Rg3 + CP resulted in a significant downregulation of VCAM-1 and ICAM-1 compared with CP alone. This reduction in adhesion molecules suggested that Rg3 may influence cell interactions and migration, which are key in tumor progression (20). Furthermore, the expression of p-p65, a marker of NF-κB pathway activation, was decreased in the presence of Rg3, indicating its potential to inflammatory responses. Similarly, MIF, a key molecule in immune cell function (21), showed decreased expression, indicating that Rg3 altered the tumor immune microenvironment. These results collectively suggested that Rg3, when administered with CP, modulated proteins involved in cell adhesion, inflammation and immune regulation, underscoring its potential as a therapeutic agent in cancer and immune modulation.

The expression of OCT2, a key organic cation transporter (21), was elevated by CP. However, this increase was counteracted when Rg3 was added with CP (Figs. 4A-C and S3). Similarly, the expression of P-gp, a membrane-bound drug efflux pump (22), was significantly enhanced by Rg3. P-gp expression in the Rg3 group was 1.5-2.0-fold higher than that in the control group, indicating a significant upregulation of P-gp in response to Rg3 (Fig. 4A and B). These results demonstrated that Rg3 had a significant impact on the expression of both OCT2 and P-gp in renal tissue. Consequently, Rg3 may decrease the accumulation of CP in the kidney, potentially mitigating its nephrotoxic effects.

Rg3 exhibited strong binding affinity with both the SIRT1 and NLRP3 target proteins, as evidenced by their high docking scores (Fig. 5A and B), with binding energies of -7.492 and -6.764 kcal/mol, respectively. Visualization of the docked compound-protein complexes demonstrated the binding modes of the compounds within the protein pockets, including interactions with specific amino acid residues. Rg3 demonstrated binding with the active sites of both SIRT1 and NLRP3 target proteins, as well as favorable docking scores, indicating the formation of stable complexes with both proteins. This suggested a potential binding interaction between Rg3 and the SIRT1 and NLRP3 proteins.

Cell viability was not diminished by the SIRT1 inhibitor Ex527 at concentrations of 10, 20 and 30 µM (Fig. 5C). The co-treatment with CP, Rg3 and Ex527 did not significantly alter cell viability compared with the CP + Rg3 group (Fig. 5D). These results indicated that SIRT1 deficiency did not alter the protective capacity of Rg3 on HK-2 cells.

Compared with the control group, the CP group showed a marked increase in SOD activity, along with a significant decrease in MDA levels and CAT activity (Fig. 5E-G). The combination of CP and Rg3 weakened these effects, with SOD activity being significantly decreased and MDA and CAT activity significantly elevated. However, Ex527 reversed these effects of Rg3.

CP significantly increased LDH release compared to the control group, indicating enhanced cell membrane damage (Fig. 5H). Rg3 co-treatment significantly attenuated CP-induced LDH release, demonstrating its protective effect against CP-induced HK-2 cell injury. However, Ex527 did not reverse the protective effect of Rg3. This results were consistent with the results from MTT assay (Fig. 5D). These findings suggested that SIRT1 deficiency may influence the antioxidant capacity of Rg3 but did not affect its ability to protect against cell damage.

Western blot analysis revealed significant upregulation of NLRP3, p-p65, caspase-1 and ASC protein expression in the CP group compared with the control (Fig. 6A and B). Rg3 mitigated the CP-induced increase in these proteins, suggesting an anti-inflammatory effect. However, Ex527 counteracted the suppressive effects of Rg3, indicating a complex interaction between these compounds. Immunofluorescence (Fig. 6C) demonstrated the impact on NLRP3 expression in HK-2 cells. Nuclei were stained blue, while NLRP3 protein expression was indicated by green fluorescence. CP led to an increase in NLRP3 expression, which was attenuated by Rg3. Ex527 reversed the Rg3-mediated decrease in NLRP3 expression. Collectively, these findings indicated that Rg3 served an anti-inflammatory role by modulating the inflammatory response induced by CP, potentially via the SIRT1 pathway.