Animals and cells
The C57BL/6J male mice (6–8 weeks outdated, 18–22 g) had been bought from Hunan Lake Jingda Laboratory Animal Co., Ltd. (high quality certificates No.430727231102973863), and had been stored within the Animal Experiment Heart of Hunan College of Chinese language Medication. The wild-type AB Zebrafish had been obtained from the Nationwide Zebrafish Useful resource Heart and maintained within the Zebrafish breeding system of Hunan Key Laboratory of Hunan College of Chinese language Medication (Beijing Aiseng Know-how Growth Co., Ltd.). The zebrafish had been positioned in an atmosphere of 28 ± 1℃ with a 14-hour mild / 10-hour darkish cycle (photocycle from 7 am to 10 PM), and grownup fish aged 90–290 days had been chosen for the experiments. Pure mating was employed to breed zebrafish larvae, and fish embryos 1 day after fertilization had been used for formal analyses. All animal experiments had been accredited by the Ethics Committee of the Heart for Animal Experiments of Hunan College of Chinese language Medication (Approval quantity: SLBH-202307190001). The mouse mononuclear macrophages cell line (RAW264.7) was obtained from the Chinese language Academy of Sciences Cell Financial institution.
Reagents
The DMEM fundamental tradition resolution (lot quantity: WH0021D031), phosphate buffer (PBS) (lot quantity: WH0021D081), serum-free non-programmed frozen resolution (lot quantity: WH0021A081), and double antibody (penicillin, streptolycin, lot quantity: WH1021A161) had been all bought from Wuhan Penocai Life Know-how Co., Ltd. The fetal bovine serum (lot quantity: SA210518) was bought from Thermo Fisher Know-how Co., Ltd. The cell-specific lipopolysaccharides (LPS-L2630, lot quantity: 0000155608) and animal-specific lipopolysaccharides (LPS-L2800, lot quantity: 0000189843) had been bought from Sigma-Aldrich. The CCK8 cell proliferation-toxicity detection equipment (lot quantity: BS350A) was bought from Biosharp. The BCA protein quantitative equipment (batch quantity: 81910335A) was bought from Lianke Biotechnology Co., Ltd. The mRNA reverse transcription primers and real-time quantitative polymerase chain response (RT-qPCR) primers had been synthesized by Shenggong BioEngineering (Shanghai) Co., Ltd. The CD86 antibodies (lot quantity: 13395-1-AP) and CD206 antibodies (lot quantity: 18704-1-AP) had been bought from Proteintech. The 4’,6-diamidino-2-phenylindole (4’, 6-Diamidino-2-phenylindole, DAPI) resolution, rhodamine phalloidin, and tetramethyl isothiocyanate (TRITC) had been bought from Beijing Soleibaol Know-how Co., Ltd. The fluorescein isothiocyanate isomer (FITC) was bought from Proteintech. Anti-CD11b-FITC(lot quantity: 4012900) and anti-CD86-PE-Cy7(lot quantity: 4221282) was bought from BD Pharmingen™, USA, Anti-F4/80-BV421(lot quantity: 2920767) and anti-CD206-PE(lot quantity: 2535977) was bought fromInvitrogen, USA.
Devices
The experimental devices used on this research embody: desk prime excessive pace refrigerated centrifuge (Thermo, USA), ELX800-enzyme spectrometer (Bio-tek, ELX800, USA), optima MAX-XP desk prime ultra-centrifuge, ultrafiltration tube (Merck millipore, Amicon Extremely-15, Ultracel-30 Okay), concentrated tube (Beckman, Centrifuge Tubes), Nanoparticle Monitoring analyzer (Malvern Panalytical, Zetasizer Professional), Nanoparticle dimension potentiometric analyzer (Malvern Panalytical, ZetaView), CO2 cell incubator (Thermo Fisher Scientific), inverted microscope (Motic), ultra-high decision laser confocal microscope (Zeiss), real-time quantitative PCR instrument (BioTek), chemiluminescence imager and synergy2 multifunctional enzyme label (BioTek), transmission electron microscopy (Hitachi (China) Co., Ltd.), micropipette (Poland Excessive Tech Lab), electrical thermostatic water bathtub (Shanghai Jinghong Experimental Tools Firm), ultra-microspectrophotometer (Bole Life Medical Merchandise (Shanghai) Co., Ltd.), and SY-5000 rotary evaporation instrument (Shanghai Yarong Biochemical Instrument Manufacturing facility), the BD FACSCanto™ movement cytometer(BD biosciences, USA).
Preparation of recent liquid extract and distilled liquid from PG
Recent PG roots (sourced from Chifeng, Interior Mongolia, China) had been washed with deionized water, reduce into small items, and absolutely floor at low temperature with acceptable quantity of pre-cooling PBS in a gradual juicer to acquire recent PG juice. The juice was centrifuged at 4 ℃, 2000×g for 20 min. After discarding residues, the supernatant was collected, positioned in a spherical backside flask, and concentrated utilizing a rotary evaporator. The concentrated recent liquid extract (PF) and distilled liquid (PFW) of PG had been collected respectively and saved at -40 ℃.
Preparation of dried PG, dried PG extract, and distilled liquid from dried PG
The recent PG slices had been pre-frozen in a -80 ℃ fridge, then positioned in a vacuum freeze dryer in a single day, and freeze-dried at -40 ℃, 110KPa for 48 h. The dried merchandise had been measured to calculate the drying charge. Subsequently, the dried product was pulverized into powder utilizing a grinder, extracted twice with ten instances distilled water, and concentrated via a rotary evaporator. After focus, the dried PG extract (PD) and distill liquid from dried PG (PDW) had been collected respectively and saved at -40 ℃.
Preparation of PGLNs
The recent PG juice was centrifuged at 4 °C, 500×g for 10 min, 2000×g for 20 min, and at last 10,000×g for 30 min to take away massive particles and fibers. The ultimate supernatant was filtered with a 0.45 μm microporous filter membrane and centrifuged at 4 ℃, 120,000×g ultra-fast for 60 min. The obtained crude extraction precipitate was re-suspended in PBS and centrifuged with the sucrose density gradient (8%, 30%, 45% and 60%) at 4 ℃, 120,000×g for 90 min. The bands of 8-30% sucrose layer and 30-45% sucrose layer had been collected and washed correctly. Lastly, purified PGLNs had been obtained and re-suspended in sterile PBS (for use as recent or retailer at -80 ℃).
Characterization and identification of PGLNs
Electron microscope commentary
The obtained PGLNs had been absorbed by capillary tubes and dropped onto a copper mesh with a supporting movie. After resting nonetheless for 3–5 min, the copper mesh was eliminated, and the remaining water droplets had been absorbed. After the surplus water content material was fully evaporated, a small drop of three% phosphotungstic acid resolution was added on the wax dish. The copper mesh containing the pattern was gently put onto the floor of the dye resolution, permitting the pattern to tightly contact with the dye resolution and stand nonetheless for 3–5 min. Then, the copper mesh was eliminated, and the surplus liquid droplets had been absorbed once more. After pure drying, electron microscope photos had been taken and noticed.
Nanoparticle monitoring evaluation and zeta potential evaluation
The obtained PGLNs had been added to 1mL PBS for re-suspension and mixing. Then, 0.2mL suspension was added to the colorimetric dish. The experimental information had been analyzed by Origin2021 software program utilizing the BT-Zeta100 nanometer particle dimension and Zeta potential analyzer.
Coomassie sensible blue staining
The protein of PGLNs was extracted with RIPA lysate, and was subjected to electrophoresis with 4×SDS polyacrylamide gel after denaturation. Subsequently, 10% SDS-PAGE was reduce off and subjected to Coomassie blue staining for two h, after which rinsed with bleaching resolution for two–3 instances, 1–2 h every (bleaching resolution: 95% ethanol 2,250 mL + glacial acetic acid 250 mL + distilled water 2,500 mL). The protein within the supernatant was analyzed utilizing a gel imaging evaluation system.
Detection of PGLNs focus by BCA
The obtained PGLNs had been added to 1mL PBS for re-suspension. In response to the directions of the BCA equipment, 20 µL suspension was added into every effectively of a 96-well plate, with 3 wells for every group. Then, BCA working fluid was added, and the plate was incubated in an oven at 37℃ for 30 min. The absorbance worth (OD worth) was detected at 562 nm wavelength, and the focus of PGLNs was calculated.
Gel electrophoresis of agarose
The ready agarose gel was poured into the electrophoresis tank and allowed to solidify at room temperature for half-hour. A mix of 5 μL RNA pattern, 1 μL loading buffer, and 1 μL nucleic acid dye was ready and completely combined, then loaded into the pattern wells. The gel was positioned within the electrophoresis chamber, submerged in 1× TAE buffer, and electrophoresed at 120 V for 60 minutes. Following electrophoresis, RNA bands had been visualized and documented utilizing a UV gel imaging system.
Proteomics of PGLNs
GLNs proteins had been lysed with 2% sodium deoxycholate, adopted by ultrasonic therapy, denaturation at 95 °C, centrifugation, and assortment of the supernatant for quantification and aliquoting. Protein focus was decided utilizing the Bradford methodology, and SDS-PAGE was used to confirm the integrity of the proteins. A complete of fifty μg of protein was digested with Trypsin (37 °C,16 h), desalted utilizing a Waters solid-phase column, and saved frozen. Mass spectrometry was carried out utilizing the Thermo EASY-nLC1200 system outfitted with a home made C18 column (100 μm × 35 cm,1.8 μm) for gradient elution (4%–90% acetonitrile/0.1% formic acid). Information had been acquired in DDA mode on an Orbitrap Exploris480 mass spectrometer with a decision of 60,000 for MS1 and 15,000 for MS2. Database looking was performed utilizing MaxQuant software program (v2.1.4.0) in opposition to the UniProt database, with settings for trypsin digestion, cysteine alkylation as fastened modifications, and methionine oxidation as variable modifications, with a false discovery charge (FDR) <1%. Differentially expressed proteins had been recognized with standards of |FC| >1.2 and P <0.05. GO/KEGG pathway enrichment evaluation was carried out utilizing the metaX bundle in R, and subcellular localization was predicted utilizing WoLF PSORT.
Analysis of biocompatibility of PGLNs
Zebrafish experiment
Two male and one feminine zebrafish had been positioned in a breeding tank in a single day in a darkish atmosphere. Lights had been turned on the following morning and the dividers had been eliminated, leaving the fish free to put eggs and fertilize. To reduce genetic variation, the fish eggs had been collected and randomly assigned to at the least three teams. The fertilized eggs had been then positioned in 6-well plates and divided into 6 teams, with PGLNs concentrations of 1, 5, 10, 50, and 100 µg/mL, respectively, alongside a standard management group. The eggs had been stored at 28 ± 2 °C for 96 h after fertilization, and the teratogenic and deadly results on juvenile fish had been noticed.
Hemolysis check
The recent entire blood of hen was collected in a BD tube containing anticoagulant. The tube was completely shaken to effectively combine the blood with the anticoagulant, and was then centrifuged at 3000 rpm for 15 min. After eradicating the supernatant, the crimson blood cell precipitation was suspended with PBS/ regular saline, centrifuged beneath the identical situations, and washed for 3–4 instances till the supernatant was clarified. Subsequently, the erythrocyte suspension was ready with PBS/ regular saline and 2-4% erythrocyte suspension, and was briefly saved within the fridge at 4 ℃ for later use. A optimistic management group (pure water) and a unfavorable management group (PBS), in addition to 6 PGLNs teams with concentrations of 0, 10, 50, 100, 500 and 1000 µg/mL had been arrange. Additional, 500 uL crimson blood cell suspension was added into EP tubes, which had been supplemented with the identical quantity of pure water, PBS, and completely different concentrations of PGLNs, respectively. After incubation at 37℃ for 1 h, the tubes had been subjected to centrifugation at 2400 rpm for five min, and the supernatant was taken to measure the OD worth at 540 nm. Lastly, the hemolysis charge was decided based mostly on the next equation: hemolysis charge = (OD pattern check group – OD unfavorable management group) ÷ (OD optimistic management group – OD unfavorable management group) × 100%. For the focus of 1000 µg/mL, the hemolysis charge of PGLNs was lower than 20%, exhibiting a low hemolysis exercise.
CCK8
RAW264.7 cells had been added in a 96-well plate (4 × 103 cells/effectively). After 24 h of tradition, the cells had been combined with PGLNs on the concentrations of 0, 0.5, 1, 5, 10, 25, 50, 100, 250 µg/mL, respectively, and had been incubated for twenty-four h. Then, the absorbance worth was decided at 450 nm wavelength in line with the working directions of the cell proliferation and virus detection equipment: cell relative viability charge (%) = (experimental group A imply ÷ management group A imply) × 100%.
Poisonous results of PGLNs on mouse organs noticed by H&E staining
After one week of adaptive feeding, 6 SPF male C57BL/6J mice, weighing 18–22 g, had been randomly divided into the traditional management group (Con) and PGLNs group (PGLNs). After resting for six h, the liver, coronary heart, spleen, lung and kidney tissues had been sampled and glued with 4% paraformaldehyde, dehydrated with ethanol gradient, handled for transparency with xylene, impregnated with wax, and embedded with paraffin. Then, discontinuous 5 μm-thick sections had been sliced, stained with HE, dehydrated, handled for transparency, and sealed correctly. The poisonous results had been noticed beneath a microscope.
Therapy of ALI mannequin mice by PF, PD, PFW and PDW
After one week of adaptive feeding, 24 SPF male C57BL/6J mice, weighing 18–22 g, had been randomly divided into the Con group, LPS group, LPS + PF group, LPS + PD group, LPS + PFW group and LPS + PDW group, with 4 mice in every group. The Con and LPS teams had been handled with pure water, whereas the opposite teams had been handled with PF, PD, PFW and PDW, respectively, for 7 days (Dosage: PD 1 g/kg, PF 10 g/3 kg, 100 µL/day). Then, the mice in every group aside from the Con group got 5 mg/kg LPS for intratracheal instillation modeling, and the Con group was given the identical quantity of PBS [18]. After 24 h of modeling, the alveolar lavage fluid and organ tissues had been separated for additional testing. All through the complete experimental course of, the final indicators of the mice together with exercise, hair coloration, food regimen, and weight had been monitored and recorded day by day.
PGLNs therapy on ALI mannequin mice
After one week of adaptive feeding, 18 SPF grade male C57BL/6J mice, weighing 18–22 g, had been randomly chosen and assigned to the Con group. The remaining mice had been first given 5 mg/kg LPS for endotracheal injection modeling, after which randomly divided into the LPS group and LPS + PGLNs group. After 6 h of modeling, the mice within the LPS + PGLNs group had been injected with 5 mg/kg PGLNs by way of tail vein, whereas the Con group and LPS group had been injected with equal quantities of PBS [19]. After 24 h of administration, the alveolar lavage fluid and organ tissues had been separated for additional testing. All through the complete experimental course of, the final indicators of the mice together with exercise, hair coloration, food regimen, and weight had been monitored and recorded day by day.
Detection of protein content material in alveolar lavage fluid by BCA
The obtained alveolar lavage liquid of every group was added to 1 mLPBS for re-suspension. In response to the directions of the BCA equipment, 20 µL suspension was added to every effectively of a 96-well plate, with 3 wells for every group. Then, BCA working fluid was added, and the plate was incubated in an oven at 37 ℃ for 30 min. Subsequently, the OD worth was detected at 562 nm wavelength, and the useful exosome focus was calculated for every group.
Detection of mRNA expression in mouse lung tissues by RT-qPCR
The overall RNA was extracted from mouse lung tissues by the Trizol methodology, and 1 µg RNA was reversely transcribed into cDNA in line with directions of the reverse transcription equipment, which was amplified by the fluorescence quantitative PCR equipment. The qPCR response situations had been set as follows: predenaturation at 95 ℃ for 30 s, denaturation at 95 ℃ for 10 s, and annealing and extension at 60 ℃ for 30 s, for 40 cycles. The relative expression of associated genes was calculated by the two-ΔΔCt methodology with β-actin as the inner reference. The primer sequences had been proven in Desk 1.
Commentary of histopathological modifications by H&E staining
Mouse lung tissues had been extracted and subjected to HE staining following the earlier process. Then, the histopathological modifications of mice in every group had been noticed.
Cell experiment
Cell tradition
RAW264.7 macrophages had been cultured with 10% fetal bovine serum, 1% double antibody and 90% fundamental tradition medium in a 5% CO2 incubator at 37℃, with the cell tradition medium changed every day. When the cells grew to 90%, 0.25% trypsin was used for digestion and passage.
CCK8
RAW264.7 cells had been added right into a 96-well plate (4 × 103 cells/effectively). After 24 h of tradition, the cells had been combined with LPS on the concentrations of 0, 1, 5, 10, 15, 20, 25, 30 µg/mL, respectively, and had been incubated for twenty-four h. Then, the absorbance worth was decided at 450 nm wavelength in line with the working directions of the cell proliferation and virus detection equipment: cell relative inhibition charge (%) = (1- experimental group A imply ÷ management group A imply) × 100%.
Building of acute cell harm mannequin and intervention with PGLNs
RAW264.7 macrophages had been divided into the Con group, LPS group and LPS + PGLNs group. The macrophages in every group had been inoculated in 6-well plates on the density of 1 × 106 cells/effectively and cultured for twenty-four h. Then, the LPS group and LPS + PGLNs group had been added with 1 µg/mL LPS, whereas the LPS + PGLNs group was added with each 1 µg/mL LPS and 20 µg/mL PGLNs for intervention for twenty-four h.
Detection of mRNA expression in cells by RT-qPCR
Identical because the process in animal experiment. The primer sequences had been proven in Desk 1.
Immunofluorescence
RAW264.7 cells had been divided into 3 teams (Con group, LPS group, and LPS + PGLNs group), and had been inoculated in 6-well plates paved with climbing plates, with 2 mL of full tradition resolution in every effectively, in a relentless temperature (37 ℃) incubator for 12 h. The PGLNs had been incubated with PKH67 dye at room temperature for two h for fluorescence labeling. The waste liquid within the plates was discarded. Then, the LPS group was added with LPS and the LPS + PGLNs group was added with each LPS and PGLNs, and had been incubated at 37 ℃ for twenty-four h. Subsequently, the cell crawling tablets had been collected and soaked with PBS twice (3 min every) for the next operations: (1) repair with 4% paraformaldehyde for 15 min, and soak with PBS 3 instances (3 min every); (2) permeate with 0.5% TritonX-100 at room temperature for 20 min, and soak with PBS 3 instances (3 min every); (3) absolutely soak up the PBS, drip with rhodamine, incubate at room temperature for 20 min away from mild, and soak with PBS 5 instances (3 min every); (4) absolutely soak up the PBS, drip with DAPI, incubate at room temperature for five min away from mild, and soak with PBS 8 instances (3 min every). The anti-fluorescence quench agent was added to the tablets, which had been inverted onto the slide to watch beneath a fluorescence microscope.
Stream cytometry
Building of acute cell harm mannequin and intervention with PGLNs is similar as in different cell experiments. To detect CD11b, F4/80 macrophages, anti-CD11b-FITC, anti-F4/80-BV421, anti-CD86-PE-Cy7 and anti-CD206-PE had been used to stain cells. Isotype controls had been run in parallel. Stream cytometry was carried out utilizing the BD FACSCanto™ movement cytometer.
Metabolomics
Macrophages had been collected from every group, and had been added with an acceptable quantity of extraction resolution and magnetic beads for grinding and ultrasonic therapy. The supernatant was taken for vacuum drying after static centrifugation, and was then added with an acceptable quantity of extraction resolution for redissolution and machine testing. Chromatographic separation was carried out on Waters ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) at 45 ℃ utilizing 0.1% formic acid aqueous resolution (A) or methanol (B) because the cell section (movement charge: 0.35 mL/min, injection quantity: 10 µL). The next gradient process was adopted as elution situations: 0–1.5 min, 95percentA; 1.5–3 min, 95%-70percentA; 3–5 min, 70%-40percentA; 5–7 min, 40%-20percentA; 7–12 min, 20%-0 A; 12–16 min, 0 A; 16–18.5 min, 0–95percentA. The mass spectrum situations had been set as follows: vary, 60–900 Da; full scanning decision, 70,000; ddMS2 mass decision, 17,500; movement charge of sheathing fuel, 30 Arb; capillary temperature, 320 ℃. The mass spectrum information had been collected in each optimistic ionization (ESI+) and unfavorable ionization (ESI-) modes (spray voltage: optimistic, 3,500 V; unfavorable, 3,100 V). The metabolites had been recognized utilizing OSI/SMMS software program (Dalian Institute of Chemical Physics, Chinese language Academy of Sciences, Dalian Chemical Information Data Know-how Co., Ltd.) in line with the outcomes of comparability between precursor ions and secondary fragments. Customary databases together with Human Metabolome Database (HMDB, https://hmdb.ca/) and metabolite database (METLIN, http://metlin.scripps.Edu) had been known as the supply database. The optimistic and unfavorable ion information had been merged into a knowledge matrix and imported into the SIMCA platform for partial least sq. discriminant evaluation (PLS-DA) and orthogonal partial least sq. discriminant evaluation (OPLS-DA). Differential metabolites had been screened based mostly on the factors of |log2FC| > 1 and P < 0.05. Then, the biomarkers had been additional screened by combining the usual databases with the HMDB database, and the MetoboAnalysis6.0 (https://www.metaboanalyst.ca) database was used for pathway evaluation.
Statistical evaluation
The measurement information had been examined to evolve to regular distribution and homogeneity of variance, and had been expressed as imply ± SD. One-Method ANOVA was used for comparisons amongst a number of teams. P < 0.05 was thought of statistically important. Statistical significance was outlined as follows: ***P<0.01, **P<0.05; ###P<0.01, ##P<0.05.
