Daphnetin Attenuates Inflammatory and Apoptotic Responses Induced by High Glucose/High Free Fatty Acids and Lipopolysaccharide in an HK-2 Cell–Neutrophil Co-Culture System

Abstract

Objectives

The present study aimed to investigate the protective effect of daphnetin (dap) against high glucose and high free fatty acids (HGHF) and lipopolysaccharide(LPS)-induced injury in the aforementioned co-culture system under normal control conditions (normal glucose and no exogenous FFA supplementation), and to explore its potential molecular mechanisms.

Methods

Neutrophils were isolated from the peripheral blood of healthy individuals. Subsequently, HK-2 cells and a neutrophil suspension were co-cultured at 1x10⁵ cells/mL to establish the experimental model. The non-cytotoxic concentration of dap for subsequent experiments was determined by Cell Counting Kit-8 (CCK-8) assay. Heparin-binding protein (HBP) concentration was measured using dry quantitative immunofluorescence assay. Inflammatory cytokines were quantified via cytometric bead array, and cell apoptotic rate was assessed by Annexin V-FITC-based flow cytometry.

Results

Both HGHF treatment and LPS exposure significantly elevated the levels of the inflammatory mediator HBP, cytokines (TNF-α, IFN-γ, IL-1β, IL-2, IL-6 and IL-10) and cellular apoptosis, whereas dap administration markedly attenuated these inflammatory and apoptotic alterations induced by HGHF or LPS (all P<0.05).

Conclusion

In this in vitro model, dap attenuated HGHF- and LPS-induced inflammatory and apoptotic responses. Further studies are needed to assess its relevance to diabetic kidney disease.

Graphical Abstract.

Introduction

Diabetic kidney disease (DKD) is one of the most common microvascular complications of diabetes, which has increasingly become a major cause of end-stage renal disease.^1,2 At present, there remains a lack of effective specific treatment options for DKD. Studies have shown that the inflammatory response is a risk factor for the progression of DKD at all stages.^2,3 Treatment targeting the inhibition of specific inflammatory molecules or inflammatory signaling pathways has been proposed as a potential strategy for delaying the progression of DKD. However, the therapeutic translation of such approaches remains to be established.

Daphnetin (Dap) is a natural coumarin derivative extracted from Chinese herbal medicines, with multiple pharmacological effects including anti-inflammatory, bacteriostatic, immune regulatory and anti-lipid peroxidative properties.^4,5 Studies have shown that dap can inhibit high glucose-induced oxidative stress, inflammatory responses and extracellular matrix accumulation in human glomerular mesangial cells^5,6; however, its specific mechanism has not been fully elucidated.

Our previous in vitro and in vivo experiments have shown that inflammatory pathological damage can occur in patients with DKD, as well as in a co-culture system of human renal cortical proximal tubular epithelial cells (HK-2) and neutrophils induced by high glucose and high free fatty acids (FFA) (HGHF).^7,8 However, it remains to be elucidated as to whether dap can improve HGHF- and LPS-induced pathological damage in the aforementioned co-culture system via its pharmacological effects.

We hypothesized that HGHF and LPS may exert synergistic effects in inducing inflammatory damage in the HK-2/neutrophil co-culture system, and that dap may exert protective effects against these stimuli. Based on previous reports suggesting that dap modulates signaling pathways such as NF-κB, MAPK, and NLRP3, we speculated that its protective actions might be associated with the regulation of inflammatory mediators; however, the present study was not designed to directly test these pathway-specific mechanisms.

Therefore, by observing the effects of dap on the levels of inflammatory factors and cell apoptosis in a HGHF and lipopolysaccharide (LPS)-treated co-culture system, the current study aimed to explore the potential impact of dap on inflammatory responses in this in vitro model, which may offer preliminary insights for future investigations into DKD-related inflammation.

Materials and Methods

The experiments were conducted between March 2025 and August 2025.

Cell line and Culture Conditions

The HK-2 cell line was acquired from the China Center for Type Culture Collection, Wuhan University (Wuhan, China). The cell line has been authenticated by the supplier using short tandem repeat (STR) profiling. In our laboratory, cells were used at passages 5–15 after thawing from master stocks. Routine testing for mycoplasma contamination was performed monthly using the MycoProbe Mycoplasma Detection Kit (R&D Systems, cat. no. CUL001B), and only mycoplasma-negative cells were used for experiments. For cell culture, HK-2 cells were seeded in Minimum Essential Medium (cat. no. C41500034; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (cat. no. SH30396.03; HyClone; Cytiva). The cells were maintained in a humidified incubator at 37°C with 5% CO₂.

In accordance with the proliferative characteristics of HK-2 cells, the culture medium was refreshed every 3 days to facilitate subculture. Only cells in the logarithmic growth phase were selected for subsequent experimental procedures.

Isolation of Neutrophils and Establishment of a Co-Culture System

Neutrophil Isolation Solution Kit (cat. no. LZS11131) was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd. Following local ethics committee approval(All procedures were approved by the Committee of the First Affiliated Hospital,Medical College of Nanchang University (approval no. 2018-016; approved on June 15, 2018).), written informed consent was obtained from each individual. Blood samples were obtained from six different healthy individuals (n=6; 3 males and 3 females; median age 34 years; age range 26–45 years).Normal human peripheral anticoagulated blood (5 mL) was mixed with an equal volume of neutrophil isolation solution. Following centrifugation at 600 x g for 30 min, the milky white cellular layer (enriched in neutrophils) was carefully collected. Isolated neutrophils were washed 2-3 times with pre-warmed washing solution and subsequently resuspended in HK-2 cell culture medium to prepare a single-cell suspension. The purity of the isolated neutrophils was evaluated by Wright–Giemsa staining, as previously described. ⁷ Only preparations with neutrophil purity >95% were used for subsequent co-culture experiments.

For establishment of the co-culture system, HK-2 cells and the neutrophil suspension were adjusted to the same final concentration of 1x10⁵ cells/mL, and were mixed thoroughly. The co-culture mixture was incubated at 37˚C in a humidified atmosphere containing 5% CO₂. Detailed procedures were performed as described in a previous report. ⁷

The experimental design focused on evaluating the cumulative effects of neutrophil-derived mediators on HK-2 cell injury, rather than on maintaining neutrophil viability throughout the 4-day culture period. This model has been previously validated for investigating inflammatory responses in the context of DKD. ⁷ A detailed discussion of this limitation is provided in the Discussion section.

Experimental Design and Biological Replication

To ensure the reproducibility and statistical validity of the results, neutrophils were isolated individually from six healthy donors (n = 6; 3 males and 3 females; median age 34 years; age range 26–45 years). Each donor’s neutrophils were processed separately and were never pooled with cells from other donors. For each independent biological replicate (one per donor), the isolated neutrophils were co-cultured with HK-2 cells and allocated across all eight treatment conditions in parallel. Experiments for the six donors were conducted on separate days to avoid inter-run contamination and to minimize technical confounding. Within each biological replicate, three technical replicate wells were included for every treatment condition. For CCK-8 assays and supernatant collection, the readings or supernatants from the three technical replicates were averaged or pooled per donor per condition before further analysis. The final value reported for each condition represents the mean of the six biological replicate means (i.e., n = 6 donors), ensuring that each donor contributes equally to the final statistical analysis. This design formally treats the donor as the independent experimental unit, and technical replicates serve only to improve measurement precision without artificially inflating the sample size.

An osmotic control group was included to distinguish the metabolic effects of high glucose from hyperosmolar stress. Specifically, cells were treated with normal glucose (5.5 mM) plus 38.9 mM mannitol (Sigma-Aldrich, cat. no. M4125), which adjusts the osmolarity to match that of the 44.4 mM glucose condition. The mannitol concentration was calculated as the difference between the high-glucose and normal-glucose concentrations (44.4 mM − 5.5 mM = 38.9 mM).

Preparation of Free Fatty Acid (FFA) Stock Solution and FFA-BSA Complex

The FFA mixture consisted of oleic acid (OA; cat. no. O1008, Sigma-Aldrich, St. Louis, MO, USA) and palmitic acid (PA; cat. no. P0500, Sigma-Aldrich) at a 2:1 molar ratio (OA:PA). Each fatty acid was individually dissolved in 100% ethanol to prepare 150 mM stock solutions. The stock solutions were stored at −20°C under nitrogen protection to prevent oxidation.

For conjugation with albumin, fatty acid-free bovine serum albumin (BSA; cat. no. A8806, Sigma-Aldrich) was dissolved in pre-warmed culture medium to a final concentration of 1% (w/v). The required volumes of 150 mM OA and PA stock solutions were added to the BSA-containing medium to achieve a final FFA concentration of 0.5 mM (with OA and PA at a 2:1 molar ratio) and an FFA:BSA molar ratio of 5:1. The mixture was incubated at 37°C for 1 hour with gentle agitation to allow complete conjugation. A vehicle control was prepared identically, containing 1% fatty acid-free BSA and the equivalent concentration of ethanol (0.316% v/v), without the addition of fatty acids.

Based on previously published literature and preliminary experimental results, ⁷ the treatment concentrations of HGHF and LPS were determined as follows: 44.4 mM glucose + 0.5 mM FFA (OA:PA = 2:1, complexed with 1% fatty acid-free BSA) and 10 μg/mL LPS.

Preparation of dap and Lipopolysaccharide

Dap (cat. no. HY-N0370, purity > 99.5% by HPLC) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Stock solutions were prepared in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and stored at −20°C. Working concentrations were freshly diluted in culture medium immediately before use; the final DMSO concentration in all treatment groups was kept below 0.1% (v/v). Lipopolysaccharide (LPS; from Escherichia coli O55:B5, cat. no. L2880) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions were prepared in sterile phosphate-buffered saline (PBS) at 1 mg/mL and stored at −20°C. The LPS concentration of 10 μg/mL was selected based on established literature demonstrating that this concentration reliably induces inflammatory injury in HK-2 cells, including reduced cell viability and upregulated pro-inflammatory cytokine expression. While this concentration exceeds typical circulating endotoxin levels, it is widely used in in vitro HK-2 models to achieve robust and reproducible inflammatory responses within a practical experimental timeframe and serves as a controlled inflammatory challenge for evaluating daphnetin’s anti-inflammatory effects.

Cell Counting Kit-8 (CCK-8) Assay

The co-culture system was seeded into 96-well plates at a density of 1x10⁴ cells/well and treated with different concentrations of dap (0, 5, 10, 20, 30 and 40 μg/mL) dissolved in medium containing 1% fatty acid-free bovine serum albumin (BSA; cat. no. A8806, Sigma-Aldrich). The plates were incubated for 4 days at 37˚C in a humidified incubator containing 5% CO₂. For the CCK-8 assay, three technical replicate wells were used for each dap concentration per biological replicate (n = 6 donors). The absorbance value for each donor at each concentration was calculated as the mean of the three technical replicates, and the final viability data represent the mean ± SD of the six donor-derived values.

At the end of the treatment period, 10 μl CCK-8 solution (cat. no. GK10001; GLPBIO Technology LLC) was added to each well. After further incubation for 4 h at 37˚C, the absorbance was measured at 450 nm using a microplate reader (Model RT-6000; Rayto Life and Analytical Sciences Co., Ltd.). Cell viability was calculated as: Viability (%) = [(OD_experimental − OD_blank)/(OD_control − OD_blank)] × 100%.All CCK-8 experiments were performed in six independent replicates (n = 6). To account for potential absorbance interference from dap, drug-only blank controls (culture medium containing the corresponding concentration of dap and CCK-8 reagent, without cells) were included for all tested concentrations and all experimental conditions. The absorbance values from these blanks were subtracted from the corresponding experimental wells prior to calculating cell viability. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test, with each dap concentration group compared against the respective 0 μg/mL control group within the same condition (Con or HGHF).

Cell Treatment and Experimental Grouping

According to the results of the CCK-8 cytotoxicity screening assay, a non-cytotoxic concentration of dap (10μg/mL) was selected for subsequent experiments. The experiment was divided into eight groups, as follows: i) Normal glucose + no exogenous FFA supplementation (basal medium) group (Con); ii) Normal glucose + no exogenous FFA supplementation + dap group (C-dap); iii) Normal glucose + no exogenous FFA supplementation + LPS group (C-LPS); iv) Normal glucose + no exogenous FFA supplementation + LPS + dap group (C-LPS-dap); v) high glucose and high free fatty acids group (HGHF); vi) HGHF + dap group (H-dap); vii) HGHF + LPS group (H-LPS); viii) HGHF + LPS + dap group (H-LPS-dap).

For each biological replicate (i.e., each donor-derived neutrophil preparation), the co-culture system was seeded into 12-well plates at a density of 1×10⁵ cells/mL (2 mL/well) and treated under the aforementioned eight conditions in parallel for 4 days. Each condition was set up in three technical replicate wells. After treatment, the culture supernatants from the three technical replicates were pooled for each condition within the same donor prior to centrifugation and storage at −80°C, to ensure sufficient volume for downstream multiplex assays while maintaining the biological replicate as the independent unit of analysis.

Experimental Design and Replication Structure

The eight treatment groups were arranged according to a 2 × 2 × 2 full factorial design, with the three factors being metabolic condition (normal glucose + no exogenous FFA supplementation vs. HGHF), LPS (0 vs. 10μg/mL), and dap (0 vs. 10μg/mL). Each of the six biological replicates (one per donor) included all eight treatment conditions in parallel. Technical triplicates were performed for each condition within each experiment. This design enabled formal evaluation of main effects and all interaction terms among the three factors.

Detection of HBP

HBP Reagent (cat. no. 2211001) was procured from Zhonghan Shengtai Biotechnology Co., Ltd. The concentration of HBP in the collected supernatant was measured using an immunoanalyzer (Zhonghan Shengtai Biotechnology Co., Ltd.) based on the dry quantitative immunofluorescence method. According to the manufacturer’s specifications, the assay has a limit of detection of 0.5 ng/mL, a standard curve range of 0.5–200 ng/mL, intra-assay CV < 8%, and inter-assay CV < 12%. Although this assay is commercially available for clinical applications, its detection principle—based on specific antibody-antigen recognition—is equally applicable to cell culture supernatants. To validate the assay for our culture supernatant matrix, we performed spike-and-recovery tests using recombinant HBP spiked into conditioned culture medium (recovery rate: 92–106%) and serial dilution linearity tests (R² > 0.98), confirming the absence of significant matrix interference.

Detection of Cell Apoptosis

After 4 days of treatment, the culture supernatant was carefully collected and centrifuged at 500 × g for 5 min at 4°C to pellet any floating (detached) cells. The adherent cells were washed 2–3 times with pre-cooled PBS and subsequently digested with 0.25% trypsin-EDTA at 37°C for 2–3 min. The digestion was terminated by adding an equal volume of complete culture medium. The detached cell pellet obtained from the supernatant was then combined with the trypsinized adherent cells and centrifuged at 500 × g for 5 min to collect the total HK-2 cell population for apoptosis analysis.

For flow cytometric analysis, cell doublets were excluded using FSC-A/FSC-H gating. The HK-2 cell population was specifically identified and gated based on its characteristic FSC (high) and SSC (moderate) parameters, which clearly distinguish HK-2 cells from smaller and more granular neutrophils. Fluorescence compensation was established using single-stained controls (Annexin V-FITC only and PI only). Apoptosis was detected using an Annexin V-FITC/PI Apoptosis Detection Kit… (rest of original text).

The apoptosis rate was calculated as the percentage of Annexin V-positive cells, which included both early apoptotic (Annexin V⁺/PI^-, Q3) and late apoptotic/necrotic (Annexin V⁺/PI⁺, Q2) populations. The gating strategy and quadrant definitions are presented in representative dot plots (Figure 7).

Figure 7.

Representative flow cytometry dot plots of HK-2 cell apoptosis. HK-2 cells were gated based on FSC/SSC parameters to exclude neutrophils and debris. Quadrant definitions: Q1 (UL), necrotic cells (PI⁺/Annexin V^-); Q2 (UR), late apoptotic cells (PI⁺/Annexin V⁺); Q3 (LR), early apoptotic cells (PI^-/Annexin V⁺); Q4 (LL), live cells (PI^-/Annexin V^-). The total apoptosis rate was calculated as the sum of Q2 and Q3 (Annexin V-positive cells). Fluorescence compensation was performed using single-stained controls. Data shown are representative of six independent biological replicates (n = 6)

Osmotic Effects of Mannitol in the Co-Culture System

The osmotic control (mannitol) treatment did not significantly alter cell apoptosis, HBP secretion compared with the normal control group (P > 0.05; Supplementary Figure S1), indicating that the effects observed in the HGHF group were primarily mediated by glucose-specific metabolic pathways rather than by hyperosmolar stress.

Cytokine Detection

The concentrations of six cytokines [tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-6 and IL-10] in the supernatant were detected using a commercial human cytokine assay kit (Immunofluorescence Assay; cat. no. 20230801; Jiangxi Saiji Biotechnology Co., Ltd.). Cytokine detection was based on cytometric bead array (CBA) technology on a flow cytometer (Model Cytomics FC 500; Beckman Coulter, Inc.).According to the manufacturer’s specifications, the limits of detection for the six cytokines were: TNF-α: 1.2 pg/mL; IFN-γ: 1.5 pg/mL; IL-1β: 0.8 pg/mL; IL-2: 1.0 pg/mL; IL-6: 1.5 pg/mL; and IL-10: 1.2 pg/mL. The standard curve range for all cytokines was 0–5,000 pg/mL. Intra-assay coefficients of variation were < 10%, and inter-assay coefficients of variation were < 15% for all cytokines. All sample measurements fell within the respective standard curve ranges.

The principle of the CBA assay is analogous to that of a sandwich enzyme-linked immunosorbent assay: Target cytokines were captured by specific antibodies conjugated to microspheres (4 or 5 μm in diameter) with distinct allophycocyanin fluorescence intensities. Each cytokine binds to a unique microsphere population, which was further recognized by a biotinylated secondary antibody. After adding 20 μl streptavidin-phycoerythrin to each well, the mixture was incubated for 30 min at 37˚C in the dark. Detailed experimental procedures were performed as described in a previous report. ⁹

Statistical Analysis

All experiments were performed with six independent biological replicates, each using neutrophils isolated from a distinct healthy donor (n = 6 donors). To minimize the confounding effect of this inter-donor variability and to enable meaningful comparisons across independent experiments, all outcome measures (HBP, cytokines, and apoptosis rate) were normalized to the respective control group (Con) within each donor prior to statistical analysis. Specifically, for each donor, the mean value of the control group was set to 100%, and the values of all other treatment groups were expressed as a percentage of that donor’s control mean. Within each experiment, technical triplicates were included for all measurements. To account for inter-donor variability and to avoid treating observations from different donors as fully independent, donor identity was included as a random intercept in a linear mixed-effects model (LMM). The fixed factors were metabolic condition (normal vs. HGHF), LPS stimulation (absent vs. present), and dap treatment (absent vs. present), as well as their two-way and three-way interaction terms. Normality of residuals was assessed using the Shapiro–Wilk test, and homogeneity of variances was verified using Levene’s test. When the LMM revealed significant fixed effects or interactions, post-hoc pairwise comparisons were performed using Tukey’s adjustment for multiple testing. All statistical analyses were conducted using SPSS version 26.0 (IBM Corp., Armonk, NY, USA) and R version 4.2.2 (lme4 package). A two-tailed P < 0.05 was considered statistically significant.

Results

High Concentrations of Daphnetin Reduce Cell Viability

The CCK-8 assay revealed that dap at concentrations ≤ 10 μg/mL exhibited no significant cytotoxicity (cell viability > 90%), while concentrations ≥ 20 μg/mL significantly reduced cell viability compared with the respective controls (P < 0.05 for both Con and HGHF conditions; Figure 1). Based on this safety profile and published evidence that 5–10 μg/mL dap exerts anti-inflammatory effects in various cell types, ¹⁰ we selected 10 μg/mL—the highest concentration that did not significantly impair overall cell viability—as the non-cytotoxic concentration for subsequent experiments. The specific protective effect of dap on HK-2 cells was subsequently confirmed by flow cytometric apoptosis analysis with HK-2 cell-specific gating (see Figures 3 and 7).

Figure 1.

Cytotoxicity screening of dap in the co-culture system. HK-2 cells and neutrophils were co-cultured and treated with increasing concentrations of daphnetin (0–40 μg/mL) for 4 days. Cell viability was assessed by CCK-8 assay. Bars represent mean values; error bars represent SD; individual replicate points (n = 6) are overlaid. Data are from six independent biological replicates (n = 6). The left panel shows viability in the Con group; the right panel shows viability in the HGHF group. * P < 0.05 vs. the respective 0 μg/mL control group within the same condition (one-way ANOVA with Tukey’s post-hoc test). ns, not significant

Figure 3.

Effects of dap treatment on HK-2 cell apoptosis in co-cultured cells. Apoptosis rate was calculated as the sum of early (Annexin V⁺/PI^-, Q3) and late (Annexin V⁺/PI⁺, Q2) apoptotic populations. Bars represent mean values; error bars represent SD; individual replicate points (n = 6) are overlaid. Data are from six independent biological replicates. Statistical symbols and abbreviations are as defined in Figure 2. Representative flow cytometry dot plots are shown in Figure 7

Effects of dap on HBP Secretion: Interaction between HGHF and LPS

Linear mixed-effects modeling revealed significant main effects of HGHF (P < 0.001) and LPS (P < 0.001) on HBP secretion, as well as a significant HGHF × LPS interaction (P < 0.05), indicating that the combination of HGHF and LPS produced a synergistic enhancement of HBP release beyond their additive effects. Importantly, a significant HGHF × LPS × Dap interaction (P < 0.05) was also detected, demonstrating that dap treatment differentially attenuated the synergistic HBP response induced by combined HGHF and LPS stimulation. Post-hoc comparisons confirmed that dap significantly reduced HBP levels in the HGHF + LPS group compared to the untreated HGHF + LPS group (P < 0.05) (Figure 2).

Figure 2.

Effects of dap treatment on HBP secretion in co-cultured cells. Bars represent mean values; error bars represent SD; individual replicate points (n = 6) are overlaid. Data are from six independent biological replicates. * P < 0.05 vs. Con; # P < 0.05 vs. HGHF; & P < 0.05 vs. the respective group without dap treatment within the same condition. Con, normal glucose + no exogenous FFA; C-dap, Con + dap; C-LPS, Con + LPS; C-LPS-dap, Con + LPS + dap; HGHF, high glucose + high FFA; H-dap, HGHF + dap; H-LPS, HGHF + LPS; H-LPS-dap, HGHF + LPS + dap

Effects of dap on Cell Apoptosis: Modulation of HGHF and LPS Synergy

Both HGHF and LPS independently increased the apoptotic rate of co-cultured cells (main effects: P < 0.001 for both). A significant HGHF × LPS interaction (P < 0.01) indicated a synergistic pro-apoptotic effect when the two stimuli were combined. Dap treatment significantly reduced apoptosis across all stimulated groups (P < 0.05), and the significant three-way interaction (HGHF × LPS × dap, P < 0.05) suggested that the anti-apoptotic effect of dap was more pronounced under combined HGHF + LPS stimulation than under either stimulus alone (Figure 3). As shown in Figure 3 and the representative dot plots (Figure 7), HGHF treatment significantly increased the apoptosis rate of HK-2 cells (gated specifically by FSC/SSC parameters in the co-culture system), and LPS treatment further enhanced this apoptotic effect. Conversely, dap treatment significantly reversed the HGHF- and LPS-induced elevation of HK-2 cell apoptosis (all P < 0.05). Notably, because the supernatant was collected and combined with adherent cells prior to analysis, our data reflect the total HK-2 cell population, including both adherent and detached (floating) apoptotic cells.

Effects of dap on Inflammatory Cytokines: Comprehensive Interaction Analysis

HGHF and LPS each significantly elevated the levels of TNF-α, IFN-γ, IL-1β, IL-2, IL-6, and IL-10 (main effects: all P < 0.001). For all six cytokines, the HGHF × LPS interaction term was significant (all P < 0.05), confirming a synergistic pro-inflammatory effect of combined metabolic and endotoxin stimulation. Dap treatment significantly reversed the elevation of all measured cytokines (main effect of dap: all P < 0.001). Furthermore, significant HGHF × LPS × dap interactions were observed for TNF-α, IL-1β, and IL-6 (P < 0.05 for each), indicating that dap preferentially attenuated the synergistic inflammatory response induced by combined HGHF and LPS. For IL-10, although dap reduced its levels in stimulated groups, the three-way interaction did not reach significance (P = 0.067), suggesting that the regulation of this anti-inflammatory cytokine may follow a different mechanism (Figures 4-6).

Figure 5.

Effects of dap treatment on IL-1β and IL-2 levels in co-cultured cells. Cytokines were measured by cytometric bead array. Bars represent mean values; error bars represent SD; individual replicate points (n = 6) are overlaid. Data are from six independent biological replicates. Statistical symbols and abbreviations are as defined in Figure 2

Figure 4.

Effects of dap treatment on TNF-α and IFN-γ levels in co-cultured cells. Cytokines were measured by cytometric bead array. Bars represent mean values; error bars represent SD; individual replicate points (n = 6) are overlaid. Data are from six independent biological replicates. Statistical symbols and abbreviations are as defined in Figure 2

Figure 6.

Effects of dap treatment on IL-6 and IL-10 levels in co-cultured cells. Cytokines were measured by cytometric bead array. Bars represent mean values; error bars represent SD; individual replicate points (n = 6) are overlaid. Data are from six independent biological replicates. Note: IL-6 is a pro-inflammatory cytokine; IL-10 is an anti-inflammatory cytokine. Their changes should be interpreted separately and with caution (see Discussion). Statistical symbols and abbreviations are as defined in Figure 2

IL-10, a key anti-inflammatory cytokine, was also significantly upregulated in the HGHF and LPS groups compared with the control group (both P < 0.05). Dap treatment significantly reduced the elevated IL-10 levels induced by HGHF and LPS (P < 0.05; Figure 6). The potential implications of this observation—particularly the fact that IL-10 reduction is not necessarily synonymous with anti-inflammatory benefit—are considered in the Discussion section below.

Discussion

DKD is one of the most prevalent microvascular complications of diabetes, imposing a substantial burden on global healthcare systems.^1,2 At present, there is a lack of effective specific therapeutic strategies for DKD in clinical practice. Dap, a natural coumarin derivative isolated from traditional Chinese medicinal herbs, has been well-documented to possess multiple pharmacological activities, including anti-inflammatory, bacteriostatic, immune regulatory and anti-lipid peroxidative properties.^4,5 In the present study, we investigated whether daphnetin could modulate inflammatory responses in an in vitro model relevant to DKD-associated metabolic and inflammatory stress. However, the following findings should be interpreted within the context of this model’s inherent limitations.

It is important to distinguish between the concepts of cytotoxicity and cytoprotection in interpreting our results. The CCK-8 assay established that 10 μg/mL dap does not impair baseline cell viability (>90%). The subsequent reduction in inflammatory cytokines and apoptosis under HGHF and LPS stimulation therefore represents genuine cytoprotective activity rather than an indirect consequence of cell death. This pattern---safety under basal conditions with efficacy under pathological stress---suggests that daphnetin possesses a favorable safety profile for further investigation.

Our previous study demonstrated that, in the HK-2/neutrophil co-culture system under HGHF conditions, the function of HK-2 cells was impaired through the activation of oxidative stress and inflammatory responses. ⁷ In the present study, the effects of dap were observed on two pathological states induced by HGHF and LPS. HBP is an inflammatory mediator primarily derived from neutrophils, which not only serves as a sensitive biomarker for bacterial infections but also has a critical role in amplifying inflammatory responses and tissue damage.^11,12 The current study revealed that HGHF treatment alone significantly promoted HBP secretion in the co-culture system (to 1.46 ± 0.02 of control), and this effect was further enhanced by additional LPS stimulation (to 7.65 ± 0.07 of control). Notably, dap intervention markedly reversed the upregulation of HBP (to 1.13 ± 0.02 and 5.84 ± 0.06 of control, respectively; Figure 2). This result aligns with the findings of previous literature reports, that inflammatory stimuli could trigger the release of HBP from neutrophils, and anti-inflammatory agents may alleviate tissue injury by inhibiting HBP secretion.^13,14 To the best of our knowledge, there are no existing reports on the association between dap and HBP. The present study confirmed that HBP served a crucial role in mediating HGHF/LPS-induced renal tubular inflammatory damage, whereas dap could regulate HBP expression to interfere with this pathological process.

Synergistic interaction between HGHF and LPS and its modulation by dap. Our factorial analysis formally identified a significant HGHF×LPS interaction for HBP secretion, apoptosis, and all pro-inflammatory cytokines measured, supporting the interpretation that metabolic stress (HGHF) and endotoxin stimulation (LPS) exert synergistic rather than merely additive effects on inflammatory injury in the HK-2/neutrophil co-culture system. Although the present study did not directly investigate these signaling pathways, previous reports have suggested that metabolic stimuli such as high glucose and free fatty acids can activate NF-κB and MAPK cascades.This synergy may be explained by the “priming” phenomenon, wherein metabolic stress activates intracellular signaling pathways (such as NF-κB and MAPK) that enhance cellular responsiveness to subsequent inflammatory triggers.^4,6,15 This finding has important clinical implications, as patients with DKD often experience both metabolic dysregulation and increased susceptibility to endotoxemia. Notably, the significant three-way interactions (HGHF×LPS×dap) observed for HBP, apoptosis, and several key cytokines indicate that dap does not merely reduce the overall inflammatory response but specifically counteracts the synergistic component of HGHF+LPS stimulation. This suggests that dap’s anti-inflammatory mechanisms—potentially involving MAPK/NF-κB/NLRP3, JAK/STAT6, and STING/TBK1/IRF3 pathways—may be particularly effective when inflammatory signaling is amplified through synergistic activation.^4,6,15,16 The enhanced efficacy of dap under combined stimulation underscores its potential therapeutic value in the complex inflammatory microenvironment of DKD, where multiple pathogenic stimuli coexist.

Apoptosis is a vital biological process for maintaining tissue homeostasis, as it efficiently eliminates aged, damaged or dysfunctional cells to preserve normal tissue structure and function.^17,18 In cell experiments, detecting apoptosis is crucial for evaluating drug effects in inflammatory and other pathological conditions. Cell apoptosis is a prominent pathological feature of renal tubular injury in DKD and inflammatory responses.^19,20 In the present study, the results showed that HGHF treatment significantly increased the apoptotic rate of cells in the co-culture system (to 1.15 ± 0.11 of control), and LPS treatment further exacerbated this apoptotic effect (to 2.36 ± 0.21 of control). By contrast, dap intervention significantly reduced the HGHF- and LPS-induced increase in cell apoptosis (to 0.99 ± 0.07 and 1.45 ± 0.13 of control, respectively; Figure 3), demonstrating its anti-apoptotic activity in the context of inflammatory tubular injury.

An imbalance of inflammatory cytokines is a core event in the formation of the inflammatory microenvironment in DKD, and the excessive production of pro-inflammatory cytokines and the dysregulation of anti-inflammatory cytokines can collectively promote renal tissue damage and disease progression. In the present study, the results showed that HGHF treatment significantly elevated the levels of all detected cytokines, and LPS treatment further enhanced this pro-inflammatory effect (Figures 4-6). TNF-α can activate the NF-κB signaling pathway to amplify the inflammatory cascade. ²¹ Furthermore, IL-1β and IL-6 are key mediators of renal interstitial inflammation, which can promote the recruitment of immune cells and the progression of tubular interstitial fibrosis.^22,23 IFN-γ and IL-2 regulate the activation and differentiation of immune cells, thereby further amplifying the inflammatory response in the renal microenvironment.^22-24

IL-10 is a classical anti-inflammatory cytokine that functions as a potent inhibitor of macrophage and dendritic cell activation and suppresses the production of pro-inflammatory mediators such as TNF-α, IL-1β, IL-6, and various chemokines. ²⁵ In the present study, IL-10 levels were significantly elevated under HGHF and LPS stimulation and reduced following dap treatment (Figure 6). However, this observation requires careful interpretation. Unlike pro-inflammatory cytokines, for which a reduction is generally regarded as a favorable anti-inflammatory outcome, a decrease in IL-10 is not automatically interpretable as an anti-inflammatory benefit. Several possible explanations exist, none of which can be definitively distinguished based on our current data:(i) Compensatory response hypothesis: The elevation of IL-10 under HGHF and LPS conditions may represent a compensatory anti-inflammatory mechanism by which the system attempts to counteract excessive inflammation. Under this interpretation, the reduction in IL-10 following dap treatment could reflect diminished demand for compensatory IL-10 production, as dap directly suppresses the upstream inflammatory stimuli. This explanation is conceptually plausible but remains speculative, as we did not perform IL-10 neutralization or functional blocking experiments to test this causal relationship.(ii) Cellular source heterogeneity: IL-10 in our co-culture system may originate from multiple cell types, including HK-2 cells and residual neutrophils. Dap may differentially regulate IL-10 production from these distinct cellular sources, and the net IL-10 level we measured represents the sum of these potentially opposing effects. We did not distinguish IL-10 production by cell type in the present study, and thus cannot determine whether the observed reduction reflects a change in HK-2-derived IL-10, neutrophil-derived IL-10, or both.(iii) Potential immunosuppressive effects: A reduction in IL-10 could, in theory, attenuate regulatory T cell (Treg)-mediated immune suppression or reduce the anti-inflammatory capacity of the local microenvironment. However, this possibility cannot be assessed from our data, as we did not evaluate immune cell function or Treg/Th17 balance in this in vitro co-culture system.Taken together, our IL-10 data should be considered as descriptive observations that require further mechanistic and functional validation. Future studies employing IL-10 neutralization antibodies, cell-type-specific IL-10 reporter systems, or Treg/Th17 profiling will be necessary to definitively interpret the biological significance of IL-10 modulation by dap in this model.

Notably, dap intervention significantly reversed the HGHF- and LPS-induced upregulation of the aforementioned cytokines (P<0.05). This broad regulatory effect of dap on the cytokine network suggests that it can restore the balance of the inflammatory microenvironment, rather than simply inhibiting pro-inflammatory cytokines. This finding is consistent with the findings of Pei et al ¹⁵ ; this previous study reported that the anti-inflammatory effect of dap against benzene-induced leukemia was mediated by a reduction in pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-2 and IL-6. Furthermore, Guo et al demonstrated that Dap exhibited a beneficial therapeutic effect on LPS-induced lung injury, ¹⁶ primarily by decreasing the secretion of pro-inflammatory cytokines and suppressing apoptotic processes. The synchronized inhibition of cytokines by daphnetin observed in the present study is phenotypically consistent with the hypothesis, proposed in previous literature, that dap may exert anti-inflammatory effects by suppressing cytokine release. Nevertheless, our data do not establish the causal chain or the upstream molecular events underlying this cytokine suppression; further mechanistic studies are warranted to elucidate the pathway(s) through which dap exerts its observed effects.

Dap treatment significantly reversed the HGHF- and LPS-induced upregulation of IL-10. The mechanistic basis for this regulation may be multifactorial. On one hand, daphnetin has been shown to directly modulate key inflammatory signaling pathways—including NF-κB, MAPK, and NLRP3—which orchestrate the expression of both pro- and anti-inflammatory cytokines.^4,6,15 On the other hand, previous studies have reported that daphnetin can promote regulatory T cell (Treg) differentiation through the NR4A1/BACH2 axis, and Treg cells are a major source of IL-10. ²⁵ Based on these literature findings, one might speculate that the reduction in IL-10 levels following dap treatment in our model could reflect a combination of direct anti-inflammatory effects (reducing the stimulus for compensatory IL-10 production) and active immunomodulation. However, it is important to emphasize that we did not directly assess Treg/Th17 balance or NR4A1/BACH2 expression in the present study; therefore, these mechanistic interpretations remain speculative and require confirmation in future investigations, such as flow cytometric analysis of Treg/Th17 populations or targeted gene expression studies in purified HK-2 cells and/or neutrophils.

The present study has several limitations. First, we did not assess neutrophil viability, proportion, or activation state at the endpoint of the 4-day co-culture. Although HBP is a pre-formed granule protein released rapidly upon neutrophil stimulation, and the measured levels at the 4-day endpoint reflect cumulative mediator release rather than a snapshot of viable cells at that single time point, the culture composition likely changes over time, which may influence the profile and quantity of mediators released. Second, the absence of parallel monoculture controls precludes definitive determination of the relative contribution of each cell type to the observed responses. Third, the molecular mechanisms underlying the protective effects of dap remain incompletely defined, as we did not directly examine signaling pathways such as NF-κB, MAPK, NLRP3, JAK/STAT, or STING/TBK1/IRF3. Our data—limited to measurements of HBP, cytokines, and apoptosis—are purely descriptive at the phenotypic level and do not permit mechanistic conclusions. Future studies incorporating time-course viability analyses, monoculture comparisons, pathway-specific inhibitors, and in vivo animal models of DKD will be required to definitively establish the mechanisms and translational relevance of our findings.

Conclusion

Under the conditions of this in vitro HK-2/neutrophil co-culture model, dap treatment was associated with reduced HBP secretion, altered cytokine concentrations, and lower Annexin V positivity following HGHF and/or LPS exposure. Further mechanistic and in vivo studies are required to determine the relevance of these findings to diabetic kidney disease.

Appendix.

Abbreviations

dap

daphnetin

HGHF

high glucose and high free fatty acids

LPS

lipopolysaccharide

HBP

heparin-binding protein

DKD

diabetic kidney disease

TNF

tumor necrosis factor

IFN

interferon

IL

interleukin

ORCID iD

Hong Wu https://orcid.org/0000-0002-9075-4820

Ethical Considerations

All procedures were approved by the Committee of the First Affiliated Hospital, Medical College of Nanchang University (approval no. 2018-016; approved on June 15, 2018).

Consent to Participate

Following local ethics committee approval, written informed consent was obtained from each individual.

Data Availability Statement

Representative flow cytometry dot plots for apoptosis detection are presented in Figure 7. The raw experimental data, including flow cytometry standard (.fcs) files and primary records, have been archived in the public data repository of the First Affiliated Hospital of Nanchang University in accordance with the institutional data management policy. All data are fully anonymized and contain no personally identifiable information. The datasets are available from the corresponding author upon reasonable request.*