Mice
C57BL/6N male mice, aged 8 weeks and weighing between 17 and 21 g, have been procured from the Laboratory Animal Middle of China Medical College. These mice have been supplied with unrestricted entry to each water and meals. The ambient indoor situations have been meticulously regulated; a temperature of 21 ± 1 °C and a relative humidity of fifty% ± 10% have been maintained, and a 12-h gentle–darkish cycle (with lights on from 7:00 AM to 7:00 PM) was enforced. Moral concerns have been rigorously adopted, as all animal dealing with procedures adhered to the Pointers for the Care and Use of Laboratory Animals established by China Medical College. Moreover, these experimental protocols have been carried out in strict accordance with the directives stipulated within the Nationwide Institutes of Well being Information for the Care and Use of Laboratory Animals, as outlined in NIH Publications No. 8023 (revised in 1978).
Group setting
For the primary set of animal fashions, the mice have been subjected to randomized allocation into distinct experimental teams, together with a management (Con) group, a probiotic (Pb) group, a bunch uncovered to a 20% (m/V) ethanol (EtOH) resolution, and a bunch uncovered to a 20% (m/V) ethanol + probiotics (EtOH + Pb). Within the EtOH and EtOH + Pb teams, the fluid offered to the mice consisted solely of an ethanol resolution. The mice within the EtOH and EtOH + Pb teams have been administered a routine of 10% ethanol for an preliminary 2-day interval, adopted by 15% ethanol for the next 5 days, culminating within the most ethanol focus on Day 8 and continued till the conclusion of the behavioral observations. Based on the Nationwide Institute on Alcohol Abuse and Alcoholism, dangerous use is outlined as males below the age of 65 who devour greater than 4 customary drinks of ethanol per day. To simulate the day by day alcohol consumption of human alcoholics, the mice within the alcohol group have been drank roughly 3 g of 20% alcohol per day, adjusted for physique floor space. Beginning on Day 1 of the research, the mice have been subjected to both saline or complicated probiotics [6] (1 × 109 cfu/ml; Bifidobacterium (Bifolac™ 12), Lactobacillus acidophilus (Bifolac™ 5), Lactobacillus rhamnosus (Bifolac™ PB01), bifodan A/S; Denmark) by way of oral gavage at a dose of 200 μl as soon as day by day, which was constantly administered over a period of 30 consecutive days. Upkeep of the ethanol options or ingesting water required day by day renewal. Moreover, the cage bedding was changed day by day to keep up a sanitary setting and facilitate the elimination of feces. The 24-h liquid consumption and physique weight of the mice have been systematically assessed on a weekly foundation.
Within the second set of animal fashions, eight-week-old mice have been randomly divided into 4 teams: GMEVs-Con, GMEVs-EtOH, GMEVs-Pb, and GMEVs-EtOH + Pb. GMEVs from the 4 teams of the primary set of animal fashions at a dose of two.5 mg per kilogram (mg/kg) of protein have been administered intravenously by way of the tail vein each different day. Every particular person animal obtained a complete of three intravenous injections throughout the course of the research. The dose and variety of injections have been modified on the subject of Zhu et al. [46].
Isolation of extracellular vesicles from feces
The feces have been preserved at − 80 °C. One gram of thawed feces was weighed and transferred to a 50 ml centrifuge tube, adopted by the addition of fifty ml of 0.9% physiological saline resolution. The contents have been vigorously vortexed on the most pace for a period of ten minutes, after which the ensuing homogenate was subjected to a single filtration step by way of sterile gauze to get rid of bigger particulate matter. The ensuing filtrate was then transferred to a recent 50 ml centrifuge tube and subjected to a sequence of differential centrifugation steps to take away fecal contaminants and small fragments inside the intestinal flora. Based mostly on the centrifugation technique of Shou-Chao Wei et al. [47], the centrifugation course of was as follows: 500 ×g, 4 °C, 10 min, 2 instances; 1000 ×g, 4 °C, 15 min, 3 instances; 3000 ×g, 30 min at 4 °C; 5000 ×g, 1 h at 4 °C; and at last, 10,000 ×g, 4 °C, 30 min. After every centrifugation, solely the supernatant was retained, and the ultimate supernatant was filtered by way of a filter with pore diameters of 0.45 μm and 0.22 μm to acquire the ultimate filtrate. The ultimate filtrate was subjected to ultracentrifugation at 120,000 ×g for two h at 4 °C using an SW32Ti rotor (Optima xpn-100 ultracentrifuge; Beckman Coulter; United States). The ensuing pellet was washed with 0.9% physiological saline and ultracentrifuged below the identical situations. After this course of, the ultimate pellet yielded the GMEVs. Every GMEVs pellet was resuspended in 1 ml of saline and saved at − 80 °C. The imply protein focus of every GMEV pattern was assessed utilizing a BCA protein assay equipment (Beyotime, Shanghai, China).
Staining of the GMEVs with DiD
A complete of 1 ml of GMEVs resolution was gently mixed with 100 μl of 1 μM lipophilic carbocyanine fluorescent membrane labeling dye DiD (BND0073, Abbkine, Wuhan, China) in saline, adopted by incubation in darkness at room temperature (RT) for a interval of 30 min. Subsequently, the ensuing combination was subjected to filtration utilizing a 0.22 μm filter and ultracentrifugation at 120,000 ×g for two h at 4 °C, thereby eliminating any unbound dye from the answer, which was subsequently discarded. For in vivo experiments, mice have been intravenously administered DiD-labeled GMEVs or a saline management by way of the tail vein. After 24 h, the mice have been sacrificed for mind tissue assortment. These mind tissues have been subsequently sectioned into 20 μM slices, after which the sections have been subjected to imaging utilizing a Zeiss AX10 fluorescence microscope, facilitating the detection of DiD indicators inside mind cells.
Transmission electron microscopy
A 20 μl aliquot of the GMEVs resolution was fastidiously dropped onto a clear sealing movie. Subsequently, a mesh membrane was positioned atop the GMEVs droplets, permitting for a 10-min suspension interval earlier than gently desiccating the mesh membrane utilizing filter paper. Following this step, the service mesh was transferred into an answer containing 2.5% glutaraldehyde, the place it was suspended for five min and subsequently subjected to blotting with filter paper. The mesh was then transferred into deionized water droplets and immersed for two min for a complete of 10 repetitions, every of which included blotting utilizing filter paper. Subsequently, the service mesh was uncovered to drops of an answer containing 40 g/L uranium uranyl acetate for 10 min below darkened situations, adopted by thorough drying by way of filter paper. The ultimate step concerned transferring the service mesh into an answer containing 10 g/L methyl cellulose, sustaining the suspension for five min, and finally making certain full desiccation by way of use of the filter paper. The statement and recording of those ready samples have been carried out utilizing a transmission electron microscope (JEOL-1200, JEOL Ltd.) after pure drying.
Nanoflow evaluation
Nanoflow analyses have been carried out to evaluate the sizes and concentrations of the GMEVs samples. In short, GMEVs have been reconstituted in 1 mL of saline for nanoflow evaluation below particular measurement situations, together with a laser energy vary of 20 to 30 mW, a ten% attenuation coefficient, and a detecting strain between 0.1 and 1.5 kPa. The nanoflow analyses have been carried out utilizing the NanoFCM system (NanoFCM Inc., Xiamen, China), and the outcomes have been analyzed with NF Career 2.0 software program.
Open subject take a look at
The evaluation of anxiety-like behaviors in mice was carried out by way of the open subject take a look at (OFT), a technique in step with established protocols. The OFT equipment was comprised of an unadorned sq. enviornment measuring 40 cm × 40 cm × 30 cm, fabricated from plastic materials, that featured a white base and 4 black partitions. Inside this enviornment, a central space measuring 20 cm × 20 cm was demarcated. Particular person mice have been positioned within the nook of the OFT machine, and the SMART™ monitoring program was used to report the motion of the mouse for 10 min. Subsequent to every mouse, thorough decontamination of the world was carried out, involving the appliance of 75% alcohol to get rid of any lingering olfactory cues.
Elevated plus-maze
The analysis of anxiety-like behaviors in mice was carried out using the elevated plus-maze (EPM), following established methodologies. The EPM equipment was a cross-shaped maze comprised of a central area (measuring 10 cm × 10 cm), two open arms (measuring 50 cm × 10 cm) and two enclosed arms (additionally measuring 50 cm × 10 cm). Every mouse was allowed to discover freely within the machine for 5 minutes. Throughout this exploration, the period of time spent in every arm was meticulously recorded using the SMART™ monitoring program. Subsequent to every testing session, the EPM equipment underwent thorough decontamination involving the appliance of 75% alcohol to get rid of any residual olfactory cues.
Animal feces and tissue assortment
Fecal samples have been procured earlier than the graduation of behavioral assessments. To make sure sanitary situations, an empty cage was used, and its base was lined with sterile filter paper. Subsequently, a person mouse was launched into the cage, permitting it to maneuver freely. Upon the incidence of defecation, fecal samples have been promptly collected utilizing sterile tweezers and deposited into prelabeled tubes. All pertinent devices had been beforehand sterilized to keep up hygienic requirements. Following every pattern assortment, the sterile filter paper was changed with a recent sheet.
Subsequent to the conclusion of the behavioral assessments, the mice have been sacrificed below anesthesia utilizing isoflurane, and their hippocampal tissue was fastidiously remoted and saved in a − 80 °C freezer for subsequent evaluation. Ileums have been collected after which fastened with paraformaldehyde resolution for hematoxylin and eosin (HE) staining.
16S rRNA gene sequencing
The whole DNA of the micro organism from the fecal samples was extracted utilizing the CTAB technique in strict adherence to the producer’s directions, after which it was meticulously preserved at − 80 °C for subsequent analytical procedures. A complete of seven mice have been chosen from every experimental group for evaluation. Whole DNA was used for amplification with primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) that particularly goal the 16S rRNA-V3-V4 area. Subsequent to PCR amplification, the ensuing merchandise have been purified using AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified by way of Qubit (Invitrogen, USA). Amplicon swimming pools have been ready for sequencing, and the scale and amount of the amplicon library have been assessed with an Agilent 2100 Bioanalyzer (Agilent, USA) and a Library Quantification Equipment designed for Illumina (Kapa Biosciences, Woburn, MA, USA), respectively. The libraries have been then sequenced on the NovaSeq PE250 platform. For sequencing procedures, samples have been processed on an Illumina NovaSeq platform in strict accordance with the producer’s directions, as offered by LC-Bio (Hangzhou, China). Subsequently, the libraries have been sequenced on an Illumina HiSeq instrument, with subsequent filtration and elimination of low-quality segments using Vsearch (v2.3.4). Taxonomic data was annotated using the Silva database. The alpha and beta diversities, to facilitate the visualization of bacterial taxa range among the many experimental teams, have been assessed by OmicStudio instruments (https://www.omicstudio.cn).
Protein extraction and Western blotting
Protein was extracted from the mouse hippocampus or GMEVs on ice utilizing radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime) supplemented with 1 mM phenylmethanesulfonyl fluoride (P1006, Beyotime) and phosphatase inhibitors (P1082, Beyotime). Subsequently, ultrasonication was used to facilitate fragmentation. The ensuing lysates have been collected and subjected to centrifugation at 12,000 revolutions per minute (rpm) for 15 min. The whole protein content material was quantified using a BCA protein assay equipment (P0011, Beyotime).
Equal portions of protein have been meticulously separated by way of 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with nonfat milk for 3 h, the membranes have been incubated in a single day at 4 °C with major antibodies towards NLRP3 (27458-1-AP, Proteintech), IL-1β (16806-1-AP, Proteintech), CD63 (sc-5275, Santa Cruz), NF-κB (10745-1-AP, Proteintech), CD9 (sc-13118, Santa Cruz), Alix (sc-53540, Santa Cruz), GM130 (sc-55590, Santa Cruz), and TLR2 (D121749, Sangon Biotech, Shanghai, China). Following major antibody incubation, the membranes have been incubated with secondary antibodies (Thermo Fisher Scientific, 1:10,000) for a interval of two h. Detection of protein bands was achieved utilizing an enhanced chemiluminescence (ECL) reagent (Merck Millipore, Billerica, MA, USA), with the ensuing chemiluminescent indicators captured and analyzed using the Tanon-5500 chemiluminescent imaging system (Science and Expertise Co., Ltd., Shanghai, China). The grey values of the bands have been quantitatively assessed using ImageJ 2.1.0 software program (Nationwide Institutes of Well being, Bethesda, MD, USA).
Immunofluorescence staining
Mouse mind tissues have been immobilized with 4% paraformaldehyde resolution for at least 24 h. Following fixation, they have been transitioned to a sucrose resolution at 4 °C for dehydration. Subsequently, the mind tissue was frozen in liquid nitrogen after which embedded in O.C.T. compound (Sakura). After the mind tissue sections have been rinsed with phosphate-buffered saline (PBS), a 0.1% Triton X-100 resolution (Solarbio) was used to permeabilize the membrane for 15 min. Subsequent, blocking buffer containing 5% donkey serum was utilized for two h, after which the tissue sections have been incubated in a single day at 4 °C with major antibodies towards GFAP (16825-1-AP, Proteintech) and Iba1 (019-19741, Wako). The subsequent day, the tissue part was incubated with secondary antibody (Thermo Fisher Scientific) at room temperature for two h, adopted by washing with PBS. After the nuclei have been stained with DAPI (Thermo Fisher Scientific), the slides have been sealed with antifade mounting medium (ZSGB-BIO). Imaging was carried out utilizing a laser confocal microscope (Leica TC).
Paraffin sectioning and hematoxylin and eosin (HE) staining
Ileal tissues have been initially fastened in a 4% paraformaldehyde resolution for at least 24 h. Following fixation, the tissues underwent a sequence of preparatory steps, together with flushing, dehydration, transparency therapy, dipping in mushy paraffin wax and embedding in arduous paraffin wax. Subsequently, typical paraffin sections have been generated using a microtome. The dried sections have been then subjected to dewaxing, hydration, and marking with hematoxylin for 1 min. Subsequent, they have been differentiated utilizing hydrochloric acid alcohol. After thorough rinsing with faucet water to revive blue coloration, eosin staining was carried out for 20 s, adopted by dehydration, transparency therapy, and finally sealing with impartial gum.
Cell viability take a look at
C8-D1A astrocytes have been cultured in DMEM (Gibco, USA, GB) containing 10% fetal bovine serum (Biosharp, China) with the vesicles eliminated after ultracentrifugation and 1% antibiotics and incubated at 37 °C with a mix of 5% CO2 and 95% air. After a time period, the cells have been handed by way of 96-well plates. When the cells reached the logarithmic development part, the GMEVs (20 μg/ml) from the 4 teams or the corresponding solvent (saline) have been added to the cells for twenty-four h. Then, 100 μl of diluted MTS resolution (CellTiter96® AQueous One Resolution Cell Proliferation Assay, PROMEGA, USA) was added to every effectively, and the absorbance was measured at a wavelength of 490 nm after tradition at 37 °C for two h.
Liquid chromatograph mass spectrometer (LC/MS)
After weighing the mouse hippocampus tissue, carry out pattern pretreatment utilizing a mix of containing 0.3% trifluoroacetic acid (TFA) methanol and acetonitrile. Centrifuge the pattern at 12,000 ×g for 10 min and acquire the supernatant for subsequent experiments. For the evaluation, make the most of the Agilent 1260–6420 QQQ LC/MS system with the next specs: the chromatographic column is an Agilent Zorbax SB C18 (2.1 mm × 50 mm, 1.8 μm); set the column temperature to 30 °C; the cell part consists of 20 mmol/L ammonium acetate and 0.1% formic acid buffer, with acetonitrile at a ratio of 30:70 (v/v); keep a circulation fee of 0.20 mL/min and an injection quantity of 5 µL. Make use of the ESI ion supply in optimistic ion mode, with a drying fuel temperature of 350 °C, a drying fuel circulation fee of 10 L/min, and a nebulizer strain of fifty psi; function in a number of response monitoring (MRM) mode.
Statistical evaluation
All experimental information are introduced because the imply values accompanied by their respective customary deviations (SD), except particularly indicated in any other case. The statistical evaluation of the information was carried out with the Kruskal–Wallis take a look at with Dunn’s a number of comparisons take a look at and one-way ANOVA with Tukey’s a number of comparisons take a look at. Significance ranges have been established with a threshold of p < 0.05. Each the statistical evaluation and graphical illustration have been carried out utilizing GraphPad Prism 9 software program (GraphPad Software program, San Diego, CA, USA).
