Isolation and characterization of Cc-ELNs
Cc-ELNs were isolated from Coptis chinensis juice using a multi-step differential centrifugation protocol. Fresh Coptis chinensis was homogenized to obtain juice, which was subjected to sequential centrifugation at 1,000 g (10 min), 3,000 g (20 min), and 10,000 g (30 min) to remove large fibers. The resulting supernatant was filtered through a 0.22-µm membrane and ultracentrifuged at 120,000 g for 60 min (Optima L-100xp, Beckman Coulter, USA). The pellet was resuspended in PBS, and Cc-ELNs were quantified by protein content using a bicinchoninic acid protein assay kit (Beyotime, China). Samples were stored at − 80 °C until further analysis.
The morphology of Cc-ELNs was examined using transmission electron microscopy (TEM). Briefly, Cc-ELNs were deposited onto a copper grid, stained with 3% (w/v) aqueous phosphotungstic acid for 1 min, and examined using a JEM-1400PLUS (Jeol, Japan). Cc-ELNs were also characterized using high-sensitivity flow cytometry for nanoparticle analysis (N30, NanoFCM, China).
Animals and ethics
Six-week-old male C57BL/6J mice were purchased from the Guangdong Medical Laboratory Animal Center, China. All animal procedures followed the guidelines set by the Animal Care and Use Committee of Guangzhou Medical University (approval number: G2023-726) and complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals in China.
In vivo biodistribution of Cc-ELNs
To assess the in vivo biodistribution of Cc-ELNs, the particles were labeled with DiR fluorescent dye (UElandy, China) by incubating at 37 °C for 30 min, following the manufacturer’s instructions. The solution was ultracentrifuged at 120,000 g for 90 min at 4 °C, and the pellet was resuspended in 200 µL of PBS to obtain DiR-Cc-ELNs. Mice were intraperitoneally injected with 150 µg of DiR-Cc-ELNs per mouse and euthanized 24 h later. Tissues from the intestine, femur, heart, liver, spleen, lungs, kidneys, and brain were collected for ex vivo imaging analysis to monitor Cc-ELN biodistribution. DiR fluorescent signals were detected using the Odyssey CLx (LI-COR Biosciences, USA).
Induction of DSS colitis and treatment
Acute colitis was induced by administering 2.5% (wt/vol) dextran sulfate sodium (DSS, 36–50 kDa, MP Biomedicals) in drinking water for 8 days consecutively (days 0–7). Mice were randomly assigned to the following groups: Normal, DSS, DSS + Cc-ELNs, DSS + BBR, DSS + DNase I, DSS + miR-NC (RIBOBIO, China), and DSS + miR-5106 (RIBOBIO, China). The intervention groups received intraperitoneal injections of Cc-ELNs (50 µg per mouse), BBR (50 µg per mouse), DNase I (20 mg/kg), miR-NC (5 nmol per mouse), or miR-5106 (5 nmol per mouse), respectively. Control groups (normal and DSS) received equivalent volumes of PBS through intraperitoneal injection.
Clinical and histological evaluation of colitis
Disease progression was monitored daily by evaluating body weight changes, stool consistency, and the presence of fecal blood using a hemoccult assay kit (Nanjing Jiancheng Bio-engineering Institute, China). DAI was calculated based on these parameters, following established protocols [56].
On day 7, mice were euthanized, and colon lengths were measured. A blinded observer performed macroscopic evaluation of the colons, considering factors such as hyperemia, wall thickening, ulceration, extent of inflammation, and overall damage (Table 1) [56]. Colonic tissues were fixed in 4% paraformaldehyde, processed, and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) for histopathological analysis, including the assessment of inflammation severity, immune cell infiltration, crypt damage, submucosal edema, goblet cell loss, and epithelial hyperplasia [56].
Goblet cell quantification
Alcian blue-periodic acid Schiff (AB-PAS) staining was used to visualize and quantify goblet cells in colon sections. The number of goblet cells per crypt was quantified.
Flow cytometric analysis of neutrophil subsets
Neutrophil subsets were analyzed in cells isolated from Peyer’s patches, spleens, and femoral BM. Single-cell suspensions were stained with fluorochrome-conjugated monoclonal antibodies targeting CD11b and Ly6G (BioLegend, USA), following the manufacturer’s instructions. Data acquisition was performed using a CytoFLEX S flow cytometer (Beckman Coulter, USA), and neutrophil populations were quantified using CytExpert software (Beckman Coulter, USA).
Neutrophil isolation and in vitro treatments
Neutrophils were isolated from the femur, tibia, and spleens of mice using Percoll density gradient centrifugation. Isolated neutrophils were cultured at 37 °C in a 5% CO2 humidified atmosphere. For in vitro experiments, neutrophils were seeded on coverslips and treated for 24 h with Cc-ELNs (20 µg/mL), BBR (20 µg/mL), miR-NC (20 µM), miR-5106 (20 µM), or ZnCl2 (200 µM). PMA (150 nM) was added 4 h before cell collection to stimulate NET formation.
Immunofluorescence analysis
For immunofluorescence analysis of colon tissue sections, specimens were fixed, embedded in paraffin, and sectioned. Sections were deparaffinized, rehydrated, and blocked with 1% bovine serum albumin. The samples were incubated overnight at 4 °C with the following primary antibodies: anti-MPO (1:100; Abcam, UK), anti-H3cit (1:200; CST, USA), anti-Ki67 (1:500; Abcam, UK), anti-GSDMD (1:100; Proteintech, China), and anti-Lgr5 (1:200; Abcam, UK). For cultured cell analysis, cells were fixed with 4% paraformaldehyde for 25 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 4 min, and blocked with PBS containing 2% BSA for 30 min. The cells were incubated overnight at 4 °C with anti-MPO (1:100; Abcam, UK), anti-H3Cit (1:200; CST, USA), anti-Ki67 (1:500; Abcam, UK), anti-GSDMD (1:100; Proteintech, China), Mito-Tracker Red CMXRos (50 nM; Beyotime, China), 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probes (1 µM; Solarbio, China), and Hoechst (1:2000; Beyotime, China). Both tissue sections and cells were incubated with appropriate Alexa Fluor-conjugated secondary antibodies. Nuclear staining was achieved with DAPI. Samples were washed three times with PBS, each wash lasting 1 min, and visualized using a TCS SP8 DLS laser scanning confocal microscope (Leica, Germany).
Scanning electron microscopy
Neutrophils cultured on coverslips were fixed with 2.5% glutaraldehyde overnight. After fixation, samples were washed with PBS and dehydrated using an ethanol gradient, followed by exchange with acetone and isoamyl acetate. Specimens were subjected to critical point drying and gold coating using an ion coater. Imaging was performed using a TM4000Plus II scanning electron microscope (HITACHI, Japan).
Analysis of Cc-ELNs uptake
Cc-ELNs were labeled with PKH26 fluorescent dye (MCE, China) for 5 min, after which an equal volume of 1% BSA was added to terminate the staining. The labeled Cc-ELNs were transferred to a Quick-Seal centrifuge tube (Beckman Coulter, USA) and ultracentrifuged at 120,000 g for 90 min at 4 °C using an Optima L-100xp ultracentrifuge with a swinging-bucket rotor (SW60 Ti, Beckman Coulter). The resulting pellet containing PKH26-labeled Cc-ELNs was resuspended in PBS and underwent a second round of ultracentrifugation under identical conditions to ensure purity.
To assess internalization, neutrophils were incubated with the purified PKH26-labeled Cc-ELNs for 1 h, and prepared for confocal microscopy analysis. Alexa Fluor phalloidin-FITC (CST, USA) was used to label actin, and DAPI was used to detect nuclei.
Purification of NETs and NETs-DNA and mouse IECs treatment
NETs were collected from freshly isolated mouse neutrophils as described previously with minor modifications [35]. Neutrophils were stimulated with 500 nM PMA for 4 h, after which the supernatant was discarded. NETs adhering to the container bottom were resuspended by gently pipetting 2 mL of chilled PBS. NETs were centrifuged at 1,000 g for 10 min at 4 °C. The cell-free supernatant containing the NETs (a DNA-protein complex) was carefully collected. NET-DNA was purified using a MicroElute DNA Clean Up Kit (OMEGA, China), and its concentration was determined by spectrophotometry. Mouse IECs were stimulated with 2.5 µg/mL of NETs or NET-DNA, and the expression of Ki67 and GSDMD was assessed by immunofluorescence.
Small RNA sequencing and analysis
Small RNA sequencing was conducted as previously described [57]. Total RNA was extracted from Cc-ELNs and used for small RNA library preparation. Following cluster generation, libraries were sequenced on an Illumina NovaSeq 6000 platform (Illumina, USA). Data analysis included quality control, comparative analysis, target gene functional annotation, and quantification of miRNA expression levels.
Prediction of potential miR-5106 target genes and dual-luciferase reporter assay
The miRanda algorithm was used to predict potential targets of miR-5106. Target genes were selected based on scores and conserved binding sites identified by miRanda. HEK293T cells were transfected with reporter plasmids encoding either the un-mutated or mutated 3′-UTR of Slc39a2, along with miR-5106 mimic or control mimic, for 48 h using RNAiMAX (Invitrogen). Luciferase activity was measured with a luciferase assay reagent (Beyotime, China) using an Infinite Synergy H1 plate reader (Agilent, USA), and normalized to Renilla luciferase activity.
RNA extraction and qRT-PCR
mRNA expression was quantified using qRT-PCR. Briefly, RNA was extracted from cells by homogenizing 100 mg of colon tissues in TRIzol reagent (Invitrogen, USA) following the manufacturer’s guidelines. The extracted RNA was quantified using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, USA). Complementary DNA (cDNA) was synthesized from 1.0 mg of total RNA using oligo (dT) primers and a Thermo Scientific RevertAid First Strand cDNA synthesis kit (Thermo Scientific), adhering to the manufacturer’s protocol. Expression of GSDMD, Caspase-1, Usp49, Trim66, Slc39a2, Krt84, Epb42, and Col28a1, Ccdc130, P2rx2, Ksr2, Zbtb34 and Selp was analysed using a SYBR Green Master Mix kit (Takara, Japan) with the primers listed in Table 2. GAPDH served as the internal control, and the fold-change in expression was calculated using the 2−ΔΔCT method.
Serum analysis
Blood samples from mice were allowed to clot at room temperature for 1 h and then centrifuged at 3000 rpm for 15 min to obtain serum. Liver function parameters (ALT, AST, and TP) as well as renal function-related indices (CREA and UREA) were measured using commercial assay kits (Kehua Bio-engineering, Shanghai, China) according to the manufacturer’s protocols.
Statistical analysis
Results are presented as mean ± SD. Comparisons between two groups were performed using unpaired two-sample t-tests. For multiple comparisons involving more than two groups, one-way ANOVA followed by Tukey’s multiple comparison tests was employed. P values < 0.05 were deemed statistically significant. Quantification of immunofluorescence and scanning electron microscopy data was carried out using ImageJ software. All statistical analyses were conducted using GraphPad Prism 8.0.
