Isolation and characterization of Cc-ELNs
Cc-ELNs have been remoted from Coptis chinensis juice utilizing a multi-step differential centrifugation protocol. Recent 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 way of 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 have been quantified by protein content material utilizing a bicinchoninic acid protein assay package (Beyotime, China). Samples have been saved at − 80 °C till additional evaluation.
The morphology of Cc-ELNs was examined utilizing transmission electron microscopy (TEM). Briefly, Cc-ELNs have 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 have been additionally characterised utilizing high-sensitivity move cytometry for nanoparticle evaluation (N30, NanoFCM, China).
Animals and ethics
Six-week-old male C57BL/6J mice have 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 Tips 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 have 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 have 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 have been collected for ex vivo imaging evaluation to observe Cc-ELN biodistribution. DiR fluorescent alerts have 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 have 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 way of intraperitoneal injection.
Scientific 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 based mostly on these parameters, following established protocols [56].
On day 7, mice have been euthanized, and colon lengths have been measured. A blinded observer carried out macroscopic analysis of the colons, contemplating elements comparable to hyperemia, wall thickening, ulceration, extent of irritation, and general injury (Desk 1) [56]. Colonic tissues have been mounted in 4% paraformaldehyde, processed, and embedded in paraffin. Sections have 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.
Movement cytometric evaluation of neutrophil subsets
Neutrophil subsets have been analyzed in cells remoted from Peyer’s patches, spleens, and femoral BM. Single-cell suspensions have been stained with fluorochrome-conjugated monoclonal antibodies concentrating on CD11b and Ly6G (BioLegend, USA), following the producer’s directions. Knowledge acquisition was carried out utilizing a CytoFLEX S move cytometer (Beckman Coulter, USA), and neutrophil populations have been quantified utilizing CytExpert software program (Beckman Coulter, USA).
Neutrophil isolation and in vitro remedies
Neutrophils have been remoted from the femur, tibia, and spleens of mice utilizing Percoll density gradient centrifugation. Remoted neutrophils have been cultured at 37 °C in a 5% CO2 humidified ambiance. For in vitro experiments, neutrophils have 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 have been mounted, embedded in paraffin, and sectioned. Sections have been deparaffinized, rehydrated, and blocked with 1% bovine serum albumin. The samples have 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 classy cell evaluation, cells have 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 have 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 have been incubated with applicable Alexa Fluor-conjugated secondary antibodies. Nuclear staining was achieved with DAPI. Samples have been washed 3 times 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 have been mounted with 2.5% glutaraldehyde in a single day. After fixation, samples have been washed with PBS and dehydrated utilizing an ethanol gradient, adopted by alternate with acetone and isoamyl acetate. Specimens have been subjected to important 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 have 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 have 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 below an identical circumstances to make sure purity.
To evaluate internalization, neutrophils have 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 have been collected from freshly remoted mouse neutrophils as described beforehand with minor modifications [35]. Neutrophils have been stimulated with 500 nM PMA for 4 h, after which the supernatant was discarded. NETs adhering to the container backside have been resuspended by gently pipetting 2 mL of chilled PBS. NETs have been centrifuged at 1,000 g for 10 min at 4 °C. The cell-free supernatant containing the NETs (a DNA-protein complicated) 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 have 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 era, libraries have been sequenced on an Illumina NovaSeq 6000 platform (Illumina, USA). Knowledge 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 have been chosen based mostly on scores and conserved binding websites recognized by miRanda. HEK293T cells have 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 tips. The extracted RNA was quantified utilizing a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, USA). Complementary DNA (cDNA) was synthesized from 1.0 mg of whole 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 methodology.
Serum evaluation
Blood samples from mice have 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) have been measured utilizing industrial assay kits (Kehua Bio-engineering, Shanghai, China) based on the producer’s protocols.
Statistical evaluation
Outcomes are introduced as imply ± SD. Comparisons between two teams have 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 assessments was employed. P values < 0.05 have been deemed statistically important. Quantification of immunofluorescence and scanning electron microscopy knowledge was carried out utilizing ImageJ software program. All statistical analyses have been performed utilizing GraphPad Prism 8.0.
