2.1. Reagents

The experimental reagents, pharmaceuticals, and other materials necessary for this study are described in detail in the supporting information.

2.2. Animals

We used 6–8-week-old healthy male C57BL/6 mice, weighing 20–22 g, for the experiments. These mice were purchased from Beijing Specific Biotechnology Co. Ltd (License No. SCXK [Beijing] 2024-0001). The mice were housed in groups of five per cage under controlled environmental conditions (24 ± 2^°C, 55 ± 5% relative humidity, and a 12-h light–dark cycle) and had ad libitum access to food and water. All experimental procedures followed the Guidelines for Animal Ethics and received approval from Zhejiang Chinese Medical University (IACUC-20250217-07).

2.3. Animal Model Establishment and Grouping

After 1 week of acclimatization, 60 mice were randomly divided into two groups. The control (CON) group, consisting of 10 mice, was fed a normal diet, while the remaining 50 mice were given a high-sugar, high-fat diet. After 8 weeks on this diet, the modeling process began. The procedure, as previously described [17], involved a 12-h fasting period with no water. We then injected 30 mg/kg of STZ intraperitoneally into the experimental group, while the CON group received an equal volume of 1% sodium citrate buffer. Three days postinjection, we collected blood from the tail vein to measure random blood glucose levels. A glucose concentration ≥ 16.7 mmol/L was used as the criterion for the induction of Type 2 diabetes mellitus (T2DM). Following this, all mice continued their respective diets, and 24-h urine protein quantity (24 h-UTP) was measured weekly. DN modeling was confirmed by random blood glucose ≥ 16.7 mmol/L and 24 h-UTP ≥ 20 mg.

The 50 mice that successfully developed the DN model were randomly assigned into five groups, with 10 mice per group: the DN model group (DN), the irbesartan (IRB) positive CON group (DN + IRB), the low-dose COS group (DN + COSL), the medium-dose COS group (DN + COSM), and the high-dose COS group (DN + COSH). The CON and DN groups each received daily saline gavage (0.2 mL/kg), while the DN + IRB group was gavaged with 40 mg/kg of IRB daily [18]. The DN + COSL, DN + COSM, and DN + COSH groups were administered 10 mg/kg, 20 mg/kg, and 40 mg/kg of COS daily, respectively [19]. Each group received the corresponding treatment for 8 weeks. The CON group was fed a normal diet, while the other groups continued with a high-sugar, high-fat diet throughout the experiment. Blood glucose levels and body weight were monitored and recorded weekly. After treatment for 8 weeks, we collected 24-h urine samples from each group using metabolic cages. Blood was obtained via intraocular canthus sampling. Following euthanasia, we performed a necropsy, opening the abdominal cavity to collect the left kidney, which was fixed in 4% paraformaldehyde. The right kidney was snap-frozen in liquid nitrogen and stored at −80°C for further analysis.

2.4. Renal Function Index Test

We centrifuged the collected 24-h urine samples at 4000 rpm for 10 min and separated the supernatant. The 24 h-UTP in each group was measured following the kit instructions. Blood samples were centrifuged at 3500 rpm for 15 min, and the serum was collected. The serum levels of creatinine (Cr) and blood urea nitrogen (BUN) were then quantified according to the kit instructions.

2.5. Renal Histopathology and Morphology Detection

Renal tissues were extracted from the 4% paraformaldehyde solution, dehydrated, paraffin-embedded, and sectioned at 4 μM thickness. The sections were stained with H&E, Masson, and PAS. After sealing the slides, we examined them under a microscope and captured images for analysis. H&E staining was used to assess the pathological morphology of renal tissues, and the scores were quantified. Masson staining was employed to evaluate renal fibrosis, while PAS staining was used to examine glycogen deposition. We quantified them using ImageJ software.

2.6. Transcriptomic Assay

Renal tissues were removed from −80°C, and total RNA was isolated. RNA integrity was assessed using the Bioanalyzer 2100 system (Agilent Technologies, California, United States). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using dUTP instead of dTTP. The directional library was prepared through end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. Library quality was assessed using Qubit and real-time PCR for quantification and the Bioanalyzer for size distribution detection. Libraries were pooled based on effective concentration and targeted data amount, then sequenced using the Illumina platform. The sequencing principle is “Sequencing by Synthesis,” where fluorescently labeled dNTPs, DNA polymerases, and adapter primers are added to the sequencing flow cell. As each sequencing cluster extends its complementary strand, the addition of each fluorescently labeled dNTP releases a corresponding fluorescence signal, captured and converted into sequencing peaks by the sequencer to obtain the target fragment sequence information.

Differentially expressed genes (DEGs) between the DN and CON groups, as well as between the DN + COSH and DN groups, were analyzed using DESeq2 and edgeR software. For DESeq2 with biological replicates, differential expression analysis was performed using the DESeq2 R package (1.20.0), which adjusts the resulting p_adj using the Benjamini and Hochberg method to control the false discovery rate. The threshold for significant differential expression was set at p_adj ≤ 0.05 and |log₂(foldchange)| ≥ 1. For edgeR without biological replicates, read counts were adjusted using the edgeR R package (3.22.5) by scaling normalization factors to eliminate sequencing depth differences between samples, followed by differential expression analysis. The resulting p_adj was adjusted using the Benjamini and Hochberg method, with the same significance threshold. Finally, KEGG pathway enrichment analyses were conducted on the identified DEGs.

2.7. Enzyme-Linked Immunosorbent Assay (ELISA)

Frozen renal tissues were homogenized, and the expression levels of IL-1β, IL-6, TNF-α, CCL2, and fibrinogen in the tissue homogenates were measured using ELISA. The assay was performed according to the instructions provided with the corresponding kits.

2.8. Biochemical Assay

Frozen renal tissues were homogenized, and C3 levels were measured using a commercial kit. Additionally, cell samples from each group were collected and homogenized to detect ROS levels. For ROS measurements, ROS levels were normalized to the PMA group (cells permeabilized with 100 nM PMA for 3 h). ROS production was expressed as a fold change from baseline. All operations were carried out according to the operational instructions of the kits.

2.9. Isolation and Extraction of Mice Peripheral Blood Neutrophils

Neutrophils were isolated from the peripheral blood of SPF-grade healthy male C57BL/6 mice. Blood was collected in anticoagulated vacuum blood collection tubes, and neutrophils were separated using the Animal Peripheral Blood Neutrophil Separation Kit (LZS1091), following the manufacturer’s instructions. Specifically, to isolate and purify neutrophils from a blood sample, first, add 5 mL of separation medium to a 15-mL sterile centrifuge tube, and then slowly add 2 mL of an 80% concentration separation medium solution (mixed with sample diluent in a 4:1 ratio) to form a stable gradient interface. Next, mix anticoagulated blood with red blood cell sedimentation liquid in a 2:1 ratio, and carefully layer it on top of the gradient interface, ensuring that the volume of separation medium is not less than that of the blood sample. After that, centrifuge at 800 g for 20 min. Following centrifugation, two ring-shaped, milky white cell layers will appear below the plasma layer: The upper layer contains mononuclear cells, and the lower layer contains neutrophils. Carefully transfer the neutrophil layer to a new centrifuge tube, add three to five times the volume of washing liquid, gently mix the cells, and centrifuge at 250 g for 10 min, discarding the supernatant. Repeat this step twice to ensure thorough washing of the cells. Finally, more than 80% of neutrophils were extracted, resuspended in 0.5 mL of washing solution, and adjusted to a concentration of 1 × 10⁷ cells using a cell counter. The isolated neutrophils were washed with phosphate-buffered saline (PBS), resuspended in RPMI 1640 medium containing 10% fetal bovine serum, and cultured in a humidified incubator at 37°C with 5% CO₂.

2.10. MTT

Freshly isolated neutrophils were seeded at a density of 5 × 10⁴ cells per well in 96-well plates and allowed to adhere for 24 h. After 24 h, the cells were treated with varying concentrations of COS (0.5, 1, 2.5, 5, and 10 μM) for 24 h [14]. After incubation, 10 μL of MTT solution (5 mg/mL) was added to each well. After an additional 4 h of incubation, the culture medium was gently removed, and 100 μL of dimethyl sulfoxide was added. The absorbance of the samples was measured at 570 nm. The concentrations of COS that exhibited low, medium, and high efficacy (1, 2.5, and 5 μM) were selected for further experiments.

2.11. Induction and Intervention of NETs

Freshly isolated neutrophils were resuspended in RPMI 1640 complete medium, seeded in cell culture plates, and incubated for 1 h. Following incubation, the cells were divided into the following treatment groups:

The PMA group was treated with 100 nM PMA for 3 h to induce the formation of NETs [20].

The PMA + COSL group received both 100 nM PMA and 1 μM COS for 3 h.

The PMA + COSM group was treated with 100 nM PMA and 2.5 μM COS for 3 h.

The PMA + COSH group was treated with 100 nM PMA and 5 μM COS for 3 h.

After the treatment period, cells from each group were collected for subsequent analysis.

2.12. Immunofluorescence

Paraffin-embedded renal tissue sections were subjected to immunofluorescence staining. CD41 staining was performed to assess platelet activation within the renal tissue. Double immunofluorescence staining for MPO and CitH3 was conducted to visualize NET formation. For double immunofluorescence staining to visualize NET formation, primary antibodies against MPO and CitH3 were applied simultaneously and incubated overnight at 4°C. After washing, secondary antibodies were applied and incubated for 1 h at room temperature. Nuclei were counterstained with DAPI. ImageJ software was used to quantify the fluorescence intensity and colocalization of MPO and CitH3.

For neutrophils extracted and treated in vitro, double immunofluorescence staining using SYTOX Green and CitH3 was performed to evaluate NET formation. Specifically, neutrophils were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 for 5 min. Cells were then blocked with 5% BSA in PBS for 1 h. Primary antibodies against CitH3 were applied and incubated overnight at 4°C. The revised statement is “After washing with PBS, secondary antibodies were applied and incubated for 1 h at room temperature. SYTOX Green was used to stain extracellular DNA and was applied for 10 min at room temperature. Nuclei were counterstained with DAPI. The fluorescence intensity of SYTOX Green was quantified using ImageJ software. NET formation was normalized to the PMA group (permeabilized cells treated with 100 nM PMA for 3 h) to account for variations in cell density and staining intensity.

2.13. Western Blot

Total protein and nuclear protein were extracted from renal tissues and neutrophils of each group. The extraction procedures followed the instructions provided by the respective kits. Protein concentrations were determined using the BCA assay. The extracted proteins were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with a 5% skimmed milk solution for 2 h, then incubated overnight at 4°C with the appropriate primary antibody. After three washes with TBST, the membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. The membranes were then washed again and developed using ECL chemiluminescence. Protein bands were quantified using ImageJ software.

2.14. Statistics

Data were analyzed using SPSS 23.0 software and are presented as mean ± standard deviation ( ⁻x ± s). Statistical graphs were generated with GraphPad Prism 8.30. One-way ANOVA was used to compare data across multiple groups, and LSD’s post hoc test was applied for pairwise comparisons. A p value of < 0.05 was considered statistically significant.

3.1. COS Demonstrates Therapeutic Effect on DN Mice

Blood glucose levels showed that mice in the DN group had significantly higher blood glucose compared to the CON group. However, after COS intervention, the DN + COSH group exhibited significantly lower blood glucose (Figure 1a). Regarding body weight, mice in the DN group had significantly reduced body weight compared to the CON group, while COS treatment significantly alleviated the weight loss. The DN + COSH group displayed the most pronounced improvement in body weight (Figure 1b).

Renal function tests revealed that the 24 h-UTP content and serum levels of Cr and BUN were significantly elevated in the DN group compared to the CON group. These markers were notably reduced after COS intervention, particularly in the DN + COSH group, which showed the greatest improvement, approaching the levels seen in the positive CON group (DN + IRB) (Figures 1c, 1d, and 1e). Histological analysis by H&E staining indicated severe renal damage in the DN group compared to the CON group. This damage included glomerular hypertrophy and increased stromal hyperplasia. In contrast, renal tissue from the DN + IRB group and the DN + COS dose groups displayed varying degrees of improvement, with a reduction in glomerular hypertrophy and stromal changes (Figure 1f,g). Collagen fibers, which were more dispersed and intensely stained in the DN group, showed improved distribution and less intense staining in the DN + IRB and all COS treatment groups (Figure 1h,i). PAS staining results demonstrated that, compared to the CON group, the DN group exhibited thickened glomerular basement membranes, pronounced matrix hyperplasia in the mesangial region, darker purplish-red coloration, and increased glycogen deposition in renal tissue. In contrast, the DN + IRB group and the COS treatment groups showed reduced glomerular basement membrane thickening, less matrix hyperplasia, lighter purplish-red coloration, and lower glycogen deposition (Figure 1j,k). These findings suggest that the effect of COS in ameliorating DN is dose-dependent, with the DN + COSH group showing the most significant therapeutic effect, similar to that of the positive CON (DN + IRB). Based on these results, we selected the high-dose COS for further investigation of the underlying mechanisms in subsequent experiments.

3.2. Transcriptomic Analysis of the Effect of COS Intervention on Differential Genes in Renal Tissues of DN Mice

We next conducted transcriptomic analysis of renal tissues from the CON, DN, and DN + COSH groups to identify DEGs between DN and CON and between DN + COSH and DN, using the criteria |log₂(foldchange)| ≥ 1 and p_adj ≤ 0.05. The volcano plot revealed that, compared to the CON group, the DN group had 1093 upregulated genes and 594 downregulated genes. In contrast, the DN + COSH group exhibited 228 upregulated genes and 278 downregulated genes compared to the DN group (Figure 2a,b). We then performed KEGG pathway enrichment analysis on these DEGs. From the enriched pathways, we focused on those that intersected between the DN versus CON and DN + COSH versus DN comparisons. These pathways were primarily associated with inflammation and platelet activation (Figure 2c,d). Heatmap analysis of the intersecting genes within these pathways revealed that COS intervention downregulated the expression of genes related to NET formation (Figure 2e). Studies have demonstrated a significant correlation between NET formation and both inflammatory responses and platelet activation [21]. We postulate that COS may attenuate inflammatory processes and suppress platelet activation via the inhibition of NET biogenesis. Consequently, we investigated the modulatory effects of COS on both inflammatory cascades and platelet activation dynamics.

3.3. COS Inhibits Renal Tissue Inflammation and Platelet Activation in DN Mice

ELISA results showed that the expression of IL-1β, IL-6, TNF-α, and CCL2 was significantly elevated in the DN group compared to the CON group. In contrast, the expression of these markers decreased significantly in the DN + COSH group compared to the DN group, indicating that COS exerted a strong anti-inflammatory effect (Figures 3a, 3b, 3c, and 3d). CD41 is a well-characterized surface glycoprotein that serves as a canonical activation marker of platelets, with its expression level being closely associated with platelet aggregation dynamics [22]. Immunofluorescence staining revealed a marked increase in CD41 expression in the glomeruli of mice in the DN group compared to the CON group. Conversely, CD41 expression was significantly reduced in the glomeruli of mice in the DN + COSH group compared to the DN group (Figure 3e,f).

3.4. COS Intervention Reduces Expression of NET-Related Factors in Renal Tissues of DN Mice

Transcriptomic analysis revealed that the DEGs were closely associated with NET formation following COS intervention. Additionally, the literature reports that NET formation is a key contributor to both the inflammatory response and platelet activation–mediated thrombosis in DN [21]. Based on these findings, we next examined the effect of COS on NET formation in DN mice. We then extracted cytosolic proteins from renal tissues, and western blot analysis revealed that CitH3 expression was significantly elevated in the DN model. However, COS treatment notably reduced CitH3 protein levels (Figure 4a,b). Among the NET-related genes identified in the transcriptomic analysis, Fgb, Fga, and C3 are ligands for Itgam, which can activate PAD4 through NADPH-generated ROS. Moreover, the activation of C5ar1, Tlr2, and Fcgr4 also promotes PAD4 activation, ultimately leading to increased CitH3 expression (Figure 4c). To further investigate this pathway, we measured fibrinogen content in mouse renal tissues using an ELISA kit. The results showed that COS significantly reduced elevated fibrinogen levels in the DN model. We also measured C3 levels in renal tissues using a biochemical kit, and the results demonstrated that COS significantly lowered the elevated C3 levels in DN mice (Figure 4d,e). Additionally, western blot analysis revealed that ITGAM and PAD4 expression were significantly increased in the DN model, but COS intervention effectively reversed this increase (Figures 4f, 4g, and 4h).

3.5. COS Inhibits NET Formation In Vitro

The inhibitory effect of COS on NET formation was further validated in vitro. We extracted peripheral blood neutrophils from mice and screened COS doses (1, 2.5, and 5 μM) as low, medium, and high doses, respectively, using MTT for dose selection (Figure 5a). To induce NET formation, we treated the neutrophils with PMA and administered COS at the selected doses. To confirm PMA’s effectiveness in inducing NETs, SYTOX Green and CitH3 expression levels and ROS levels were compared between normal and PMA-treated groups. The results showed that PMA induction increased SYTOX Green and CitH3 expression, along with ROS levels (Figure S1). Immunofluorescence analysis showed that COS treatment, at all doses, reduced the expression of SYTOX Green and CitH3 after PMA induction, with varying degrees of inhibition across the dose groups (Figure 5b,c). Additionally, COS treatment decreased CitH3 protein levels in the nuclear extracts from all groups, indicating that COS effectively inhibited NET activation following PMA induction (Figure 5d,e). ROS assays demonstrated that COS also reduced ROS levels after PMA induction (Figure 5h). Western blot analysis further revealed that COS treatment significantly reduced PAD4 protein expression following PMA stimulation (Figure 5f,g). These in vitro results collectively confirm that COS can inhibit the formation of NETs in neutrophils.