Isolation and characterization of Cc-ELNs
Cc-ELNs had been remoted from Coptis chinensis juice utilizing a multi-step differential centrifugation protocol. Contemporary Coptis chinensis was homogenized to acquire 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 take away giant fibers. The ensuing supernatant was filtered by 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 had been quantified by protein content material utilizing a bicinchoninic acid protein assay package (Beyotime, China). Samples had been saved at − 80 °C till additional evaluation.
The morphology of Cc-ELNs was examined utilizing transmission electron microscopy (TEM). Briefly, Cc-ELNs had been deposited onto a copper grid, stained with 3% (w/v) aqueous phosphotungstic acid for 1 min, and examined utilizing a JEM-1400PLUS (Jeol, Japan). Cc-ELNs had been additionally characterised utilizing high-sensitivity stream cytometry for nanoparticle evaluation (N30, NanoFCM, China).
Animals and ethics
Six-week-old male C57BL/6J mice had been bought from the Guangdong Medical Laboratory Animal Middle, China. All animal procedures adopted the rules set by the Animal Care and Use Committee of Guangzhou Medical College (approval quantity: G2023-726) and complied with the Nationwide Institutes of Well being Pointers for the Care and Use of Laboratory Animals in China.
In vivo biodistribution of Cc-ELNs
To evaluate the in vivo biodistribution of Cc-ELNs, the particles had been labeled with DiR fluorescent dye (UElandy, China) by incubating at 37 °C for 30 min, following the producer’s directions. The answer was ultracentrifuged at 120,000 g for 90 min at 4 °C, and the pellet was resuspended in 200 µL of PBS to acquire DiR-Cc-ELNs. Mice had been intraperitoneally injected with 150 µg of DiR-Cc-ELNs per mouse and euthanized 24 h later. Tissues from the gut, femur, coronary heart, liver, spleen, lungs, kidneys, and mind had been collected for ex vivo imaging evaluation to observe Cc-ELN biodistribution. DiR fluorescent alerts had been detected utilizing the Odyssey CLx (LI-COR Biosciences, USA).
Induction of DSS colitis and therapy
Acute colitis was induced by administering 2.5% (wt/vol) dextran sulfate sodium (DSS, 36–50 kDa, MP Biomedicals) in consuming water for 8 days consecutively (days 0–7). Mice had been randomly assigned to the next teams: Regular, DSS, DSS + Cc-ELNs, DSS + BBR, DSS + DNase I, DSS + miR-NC (RIBOBIO, China), and DSS + miR-5106 (RIBOBIO, China). The intervention teams obtained 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. Management teams (regular and DSS) obtained equal volumes of PBS by intraperitoneal injection.
Medical and histological analysis of colitis
Illness development was monitored each day by evaluating physique weight adjustments, stool consistency, and the presence of fecal blood utilizing a hemoccult assay package (Nanjing Jiancheng Bio-engineering Institute, China). DAI was calculated primarily based on these parameters, following established protocols [56].
On day 7, mice had been euthanized, and colon lengths had been measured. A blinded observer carried out macroscopic analysis of the colons, contemplating elements comparable to hyperemia, wall thickening, ulceration, extent of irritation, and total injury (Desk 1) [56]. Colonic tissues had been mounted in 4% paraformaldehyde, processed, and embedded in paraffin. Sections had been stained with hematoxylin and eosin (H&E) for histopathological evaluation, together with the evaluation of irritation severity, immune cell infiltration, crypt injury, submucosal edema, goblet cell loss, and epithelial hyperplasia [56].
Goblet cell quantification
Alcian blue-periodic acid Schiff (AB-PAS) staining was used to visualise and quantify goblet cells in colon sections. The variety of goblet cells per crypt was quantified.
Stream cytometric evaluation of neutrophil subsets
Neutrophil subsets had been analyzed in cells remoted from Peyer’s patches, spleens, and femoral BM. Single-cell suspensions had been stained with fluorochrome-conjugated monoclonal antibodies focusing on CD11b and Ly6G (BioLegend, USA), following the producer’s directions. Information acquisition was carried out utilizing a CytoFLEX S stream cytometer (Beckman Coulter, USA), and neutrophil populations had been quantified utilizing CytExpert software program (Beckman Coulter, USA).
Neutrophil isolation and in vitro therapies
Neutrophils had been remoted from the femur, tibia, and spleens of mice utilizing Percoll density gradient centrifugation. Remoted neutrophils had been cultured at 37 °C in a 5% CO2 humidified ambiance. For in vitro experiments, neutrophils had been seeded on coverslips and handled for twenty-four 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 earlier than cell assortment to stimulate NET formation.
Immunofluorescence evaluation
For immunofluorescence evaluation of colon tissue sections, specimens had been mounted, embedded in paraffin, and sectioned. Sections had been deparaffinized, rehydrated, and blocked with 1% bovine serum albumin. The samples had been incubated in a single day at 4 °C with the next major 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 aesthetic cell evaluation, cells had been mounted 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 had been incubated in a single day 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 Crimson CMXRos (50 nM; Beyotime, China), 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probes (1 µM; Solarbio, China), and Hoechst (1:2000; Beyotime, China). Each tissue sections and cells had been incubated with applicable Alexa Fluor-conjugated secondary antibodies. Nuclear staining was achieved with DAPI. Samples had been washed thrice with PBS, every wash lasting 1 min, and visualized utilizing a TCS SP8 DLS laser scanning confocal microscope (Leica, Germany).
Scanning electron microscopy
Neutrophils cultured on coverslips had been mounted with 2.5% glutaraldehyde in a single day. After fixation, samples had been washed with PBS and dehydrated utilizing an ethanol gradient, adopted by alternate with acetone and isoamyl acetate. Specimens had been subjected to crucial level drying and gold coating utilizing an ion coater. Imaging was carried out utilizing a TM4000Plus II scanning electron microscope (HITACHI, Japan).
Evaluation of Cc-ELNs uptake
Cc-ELNs had been labeled with PKH26 fluorescent dye (MCE, China) for five min, after which an equal quantity of 1% BSA was added to terminate the staining. The labeled Cc-ELNs had been transferred to a Fast-Seal centrifuge tube (Beckman Coulter, USA) and ultracentrifuged at 120,000 g for 90 min at 4 °C utilizing an Optima L-100xp ultracentrifuge with a swinging-bucket rotor (SW60 Ti, Beckman Coulter). The ensuing pellet containing PKH26-labeled Cc-ELNs was resuspended in PBS and underwent a second spherical of ultracentrifugation underneath an identical circumstances to make sure purity.
To evaluate internalization, neutrophils had been incubated with the purified PKH26-labeled Cc-ELNs for 1 h, and ready for confocal microscopy evaluation. 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 therapy
NETs had been collected from freshly remoted mouse neutrophils as described beforehand with minor modifications [35]. Neutrophils had been stimulated with 500 nM PMA for 4 h, after which the supernatant was discarded. NETs adhering to the container backside had been resuspended by gently pipetting 2 mL of chilled PBS. NETs had been centrifuged at 1,000 g for 10 min at 4 °C. The cell-free supernatant containing the NETs (a DNA-protein advanced) was rigorously collected. NET-DNA was purified utilizing a MicroElute DNA Clear Up Package (OMEGA, China), and its focus was decided by spectrophotometry. Mouse IECs had been 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 evaluation
Small RNA sequencing was performed as beforehand described [57]. Complete RNA was extracted from Cc-ELNs and used for small RNA library preparation. Following cluster technology, libraries had been sequenced on an Illumina NovaSeq 6000 platform (Illumina, USA). Information evaluation included high quality management, comparative evaluation, goal gene purposeful annotation, and quantification of miRNA expression ranges.
Prediction of potential miR-5106 goal genes and dual-luciferase reporter assay
The miRanda algorithm was used to foretell potential targets of miR-5106. Goal genes had been chosen primarily based on scores and conserved binding websites recognized by miRanda. HEK293T cells had been transfected with reporter plasmids encoding both the un-mutated or mutated 3′-UTR of Slc39a2, together with miR-5106 mimic or management mimic, for 48 h utilizing RNAiMAX (Invitrogen). Luciferase exercise was measured with a luciferase assay reagent (Beyotime, China) utilizing an Infinite Synergy H1 plate reader (Agilent, USA), and normalized to Renilla luciferase exercise.
RNA extraction and qRT-PCR
mRNA expression was quantified utilizing qRT-PCR. Briefly, RNA was extracted from cells by homogenizing 100 mg of colon tissues in TRIzol reagent (Invitrogen, USA) following the producer’s pointers. The extracted RNA was quantified utilizing a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, USA). Complementary DNA (cDNA) was synthesized from 1.0 mg of complete RNA utilizing oligo (dT) primers and a Thermo Scientific RevertAid First Strand cDNA synthesis package (Thermo Scientific), adhering to the producer’s protocol. Expression of GSDMD, Caspase-1, Usp49, Trim66, Slc39a2, Krt84, Epb42, and Col28a1, Ccdc130, P2rx2, Ksr2, Zbtb34 and Selp was analysed utilizing a SYBR Inexperienced Grasp Combine package (Takara, Japan) with the primers listed in Desk 2. GAPDH served as the interior management, and the fold-change in expression was calculated utilizing the two−ΔΔCT technique.
Serum evaluation
Blood samples from mice had been allowed to clot at room temperature for 1 h after which centrifuged at 3000 rpm for 15 min to acquire serum. Liver perform parameters (ALT, AST, and TP) in addition to renal function-related indices (CREA and UREA) had been measured utilizing industrial assay kits (Kehua Bio-engineering, Shanghai, China) in accordance with the producer’s protocols.
Statistical evaluation
Outcomes are offered as imply ± SD. Comparisons between two teams had been carried out utilizing unpaired two-sample t-tests. For a number of comparisons involving greater than two teams, one-way ANOVA adopted by Tukey’s a number of comparability exams was employed. P values < 0.05 had been deemed statistically vital. Quantification of immunofluorescence and scanning electron microscopy information was carried out utilizing ImageJ software program. All statistical analyses had been performed utilizing GraphPad Prism 8.0.
