HomeNanotechnologyMesoporous silica nanoparticle-encapsulated Bifidobacterium attenuates mind Aβ burden and improves olfactory dysfunction...

Mesoporous silica nanoparticle-encapsulated Bifidobacterium attenuates mind Aβ burden and improves olfactory dysfunction of APP/PS1 mice by nasal supply | Journal of Nanobiotechnology

Antibodies and reagents

An anti-Aβ (6E10) antibody was bought from Invitrogen (Carlsbad, CA, USA). Anti-Iba1 (019-19,741) was acquired from Wako laboratory chemical substances (Osaka, Japan). Pierce streptavidin-coupled Poly-Horseradish Peroxidase (HRP) and protein markers had been ordered from Thermo Fisher Scientific (Ann Arbor, MI, US). Thioflavin S (ThioS), dimethyl sulfoxide (DMSO), and bovine serum albumin (BSA) had been obtained from Sigma-Aldrich (Shanghai, China). Casein tryptone, yeast extract, and agar had been ordered from Sinopharm (Shanghai, China). Bifidobacterium selective (BS) tradition medium and trypticase-phytone-yeast (TPY) Broth had been ordered from Hopebio (Qingdao, China). Cy3-N-hydroxysuccinimide (NHS) ester was bought from Lumiprobe (Hannover, Germany). The 4′,6-diamidino-2-phenylindole (DAPI) resolution was ordered from Beyotime Biotechnology (Shanghai, China), tetrahydrofuran (THF), N-cetyltrimethylammonium bromide (CTAB, 99%) and methanol had been bought from Aladdin (Shanghai, China). Tetraethyl orthosilicate (TEOS, 98%) and paraformaldehyde (PFA) had been obtained from Macklin (Shanghai, China). FITC-d-Lys was obtained from Shengguang Biotechnology Co., Ltd. (Xiamen, China). All further chemical substances had been bought from business suppliers and used as acquired.


4 month-old transgenic APPSwe/PS1dE9 (APP/PS1) mice had been bought from the Beijing Huafukang Biotechnology Co., Ltd (Beijing, China). Age-matched C57BL/6J mice had been bought from Shulaibao Biotech Co. (Wuhan, China). This examine used APP/PS1 and wild-type C57BL/6J mice aged roughly 4–8 months. All mice had been maintained beneath commonplace particular pathogen-free (SPF) circumstances with an ambient temperature of 23 ± 2 °C, air humidity of 40–70%, and a man-made 12 h mild/darkish cycle. All experiments had been permitted by the Institutional Animal Care and Use Committee of Huazhong College of Science and Expertise.

Synthesis of MSNs

MSNs had been ready utilizing the coprecipitation methodology [32]. Briefly, 40 mL of methanol was combined with 110 mL of ultrapure water, and the pH was adjusted to 11 with ammonia. CTAB was added and heated. When the temperature reached 80 °C, a specific amount of TEOS was added dropwise and stirred vigorously for 3 h. After cooling to room temperature, the answer was centrifuged at 9500 rpm for 10 min, washed 3 instances with deionized water, after which vacuum dried in a single day to acquire a white strong powder. Lastly, the dry white powder was dissolved in a combined resolution of 100 mL of absolute ethanol and 10 mL of concentrated hydrochloric acid. The answer was refluxed for twenty-four h to take away CTAB. The mass ratio of TEOS/CTAB was managed at 2:1 to synthesize MSNs. Subsequent, MSNs had been combined with Poly-L-Lysine in a shaker (55 rpm) at 37 °C for 30 min after which vacuum dried to acquire a discolored powder.

MSNs characterization

Right here, 20 μL of 1 mg/mL MSNs was dropped on a copper grid, and extra liquid was eliminated utilizing filter papers. After drying at room temperature, the morphology of the nanomaterials was noticed by transmission electron microscopy (TEM) on a HITACHI HT7700 transmission electron microscope (TEM; Hitachi Excessive-Tech, Japan) at a voltage of 120 kV. Subsequent, dynamic mild scattering (DLS; photon correlation spectroscopy) and the Zetasizer Nano-ZS90 system (Malvern Devices, Worcestershire, UK) had been used to measure the dimensions distribution and floor potential of MSNs.

Fluorescence modification of MSNs

MSNs (10 mg) had been dissolved in 1 mL of PBS resolution. 5 μL of Cy3-NHS (100 μg) in DMSO resolution was slowly added to the MSN suspension, stirred in a single day in the dead of night, adopted by centrifugation (5000 rpm, 10 min) to take away unloaded Cy3 from the outer floor of the MSNs.

FITC-d-Lys staining of Bifidobacterium in vitro

Bifidobacterium was bought from the Tradition Assortment Heart of Yunnan Institute of Microbiology. Bifidobacterium was grown within the BS tradition medium at 37 °C till OD600 reached 0.6. The medium was diluted to an OD600 of 0.3 with recent medium containing FITC-d-Lys (0.1 mM). The diluted micro organism had been additional incubated at 37 °C till the OD600 was 1.0–1.5. The micro organism had been centrifuged and washed 3 instances with the BS tradition medium.

Preparation and characterization of MSNs-Bi

The classy FITC-labeled Bifidobacterium was centrifuged at 8000 ×g for 10 min at 4 °C, then the pellet was washed and resuspended in 5 mL of physiological saline. Then, 1 mL of 40 µg/mL Cy3-MSNs was combined with 1 mL of FITC labeled-Bifidobacterium tradition (2.31 ×1011 CFU/mL) for 30 min incubation at 37 °C in a shaker (255 rpm). After washing 3 instances with PBS, FITC labeled-Bifidobacterium and MSNs-Bi had been suspended with 2.5% glutaraldehyde resolution at 4 °C for 10 h, after which dehydrated in rising sequence of water–ethanol options (35%, 50%, 70%, 85%, 95%, and 100%), and eventually vacuum dried. The morphology of MSNs-Bi and Bifidobacterium was examined by scanning electron microscope (SEM; Sirion 200, the Netherlands).

In vitro stability of MSNs-Bi

To guage the steadiness of MSNs-Bi, on the one hand, the change of fluorescence depth of Cy3-MSNs-Bi with time was detected. Briefly, 20 μg Cy3-MSNs-Bi had been incubated in 200 μL Krebs–Henseleit resolution (D-glucose 2.0 g/L, magnesium sulfate 0.141 g/L, potassium phosphate monobasic 0.16 g/L, potassium chloride 0.35 g/L, sodium chloride 6.9 g/L, calcium chloride dihydrate 0.373 g/L, and sodium bicarbonate 2.1 g/L) at 37 °C for 0, 0.5, 1, 2, 3, 4, 6, 9, and 12 h. The Cy3 fluorescence indicators of samples had been detected utilizing a multiscan FC microplate photometer (Thermos, USA) at an emission wavelength of 490 nm (excitation at 440 nm). The discharge curve of Cy3 in MSNs is proven in Extra file 1: Fig. S10, and the Cy3 fluorescence depth of MSNs-Bi will superimpose the free launch quantity of Cy3 from MSNs.

Alternatively, the exercise of Bifidobacterium at completely different time factors was detected. 1 μL of MSNs-Bi at completely different time factors was combined with 100 μL of tradition medium, and their exercise was evaluated by counting the variety of colonies shaped by Bifidobacterium on the tradition medium.

Results of MSNs on bacterial proliferation

Antimicrobial kinetics had been evaluated utilizing a co-culture assay [15]. Briefly completely different concentrations (5, 10, 40, 60, 80, 100, and 160 μg/mL) of MSNs had been added to 10 mL of Escherichia coli (E. coli) and Bifidobacterium cultures (1 ×105 CFU/mL). Subsequently, the E. coli and Bifidobacterium cultures with MSNs had been shaken at 37 °C for 12 h. At every time interval, 100 μL of the medium was transferred to a 96-well plate, and the optical density at 600 nm was measured with a NanoDrop 2000 (Thermo Scientific, USA) and the minimal inhibitory focus was calculated.

To find out the minimal bactericidal focus (MBC) of MSNs, 100 μL of E. coli and Bifidobacterium cultures (1 ×105 CFU/mL) had been inoculated on Luria Broth (LB) agar or BS tradition medium pretreated with completely different concentrations of MSNs (5, 10, 40, 60, 80, 100, and 160 μg/mL) in a single day at 37 °C. After 24 h, colonies had been noticed and digital photos of every plate had been captured to guage the impact of MSNs.

Bacterial exercise take a look at of MSNs-E. coli and MSN-Bi in gastrointestinal stimulated fluid

To find out the discharge traits of E. coli and Bifidobacterium loaded with MSNs beneath physiological circumstances of the gastrointestinal tract, simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) had been ready in keeping with the strategy laid out in United States Pharmacopoeia. Briefly, SGF was ready by dissolving 2 g/L NaCl and three.2 g pepsin (350 exercise models per mg) to make a ultimate quantity of 1 L ddH2O with a pH of two.0. The SIF at pH 7.2 was ready utilizing 0.05 mol/L KH2PO4 and 1.2% (w/v) bile salts. Then, 100 μL of MSN, MSNs-E. coli, and MSNs-Bi (1 × 105 CFU/mL) had been cultured in 1 mL of SGF and SIF at 37 °C for five, 30, 60, and 120 min. The variety of colonies shaped by E. coli and Bifidobacterium on LB agar and BS tradition medium was used to guage the impact of MSNs on Bifidobacterium viability.

Fluorescence imaging and biodistribution research of MSNs-Bi

To guage the biodistribution of MSNs-Bi, 4 month-old male C57BL/6J mice (n = 3) had been intranasally injected with MSNs-Bi (1 ×109 CFU/mL). An equal quantity of PBS was used as a management. Main organs (e.g. stomach, mind, lung, spinal wire, gut, and intestinal contents) had been collected, washed with PBS, and imaged utilizing a home made whole-body imaging system at 5 h post-injection. As well as, tissues of the mind, stomach, backbone, and gut had been mounted in 4% (w/v) PFA in a single day and sliced in 50 μm sections utilizing a CM1950 Leica cryostat (Leica Biosystems, Germany). Fluorescence indicators of Cy3-MSNs and FITC-labeled Bifidobacterium in tissues had been noticed by confocal imaging.

In vivo monitoring of Bifidobacterium

To guage the prevalence of nasally administered MSNs-Bi over typical oral administration of Bifidobacterium, 4 month-old male C57BL/6J mice (n = 3) had been intranasally injected with Cy3-MSNs-Bi (1 ×109 CFU/mL; Bifidobacteria had been labeled with FITC). An equal quantity of FITC-labeled Bifidobacterium was administered orally as a management. Then, recent intestinal contents (abdomen, small gut, and enormous gut) had been collected and imaged utilizing a home made whole-body imaging system at 0, 0.25, 0.5, 3, 6, and 9 h post-injection [33, 34].

Assessing the impact of MSNs-Bi on intestinal irritation

To guage whether or not intranasal instillation of MSNs-Bi was more practical in assuaging intestinal irritation in APP/PS1 mice than gavage with Bifidobacterium, 4 month-old APP/PS1 mice had been handled with PBS, MSNs, MSNs-Bi, and Bifidobacterium, respectively. The Bifidobacterium had been dissolved in PBS earlier than therapy and administered at a dose of 1 ×109 CFU/kg each 4 days for a complete of seven instances. After therapy, mice had been allowed to relaxation for two months. Then, the mice had been sacrificed. Therapy results had been evaluated in keeping with neuroinflammation, mind and intestinal Aβ plaques, intestinal flora, and behavioral standing.

Histopathological evaluation

After therapy with PBS, MSNs, Bifidobacterium, and MSNs-Bi, colons had been collected from mice after which mounted in 4% (w/v) PFA resolution in a single day. Colon tissues had been embedded in paraffin sections and stained with hematoxylin and eosin (H&E). H&E stained sections had been imaged on a Nikon Ni-E microscope (Nikon, Minato, Tokyo, Japan). Colonic crypt size in mice handled with PBS, MSNs, Bifidobacterium, and MSNs-Bi was evaluated utilizing ImageJ software program.

16 s DNA library preparation and metagenomic evaluation

After accumulating feces from APP/PS1 and C57BL/6 mice handled with MSNs, MSNs-Bi, and Bifidobacterium, the full DNA of fecal samples was extracted utilizing a high-throughput DNA isolation equipment (Boao Basic, Beijing). To arrange the 16 s rDNA library, primers (ahead: CCTAYGGGRBGCASCAG; reverse: GGACTACNNGGGTATCTAAT) had been utilized to amplify the V3–V4 area of the 16 s rRNA. In keeping with the producer’s protocol, sequencing was carried out on the Illumina HiSeq 2500 platform. Moreover, high quality management was carried out utilizing QIIME pipelines. Uncooked reads had been filtered and trimmed utilizing default settings. Subsequently, the pair-end reads had been mixed and the primers had been eliminated. Subsequent, the taxonomy was assigned by search with a similarity threshold of 97%. Downstream evaluation was carried out utilizing the R packages ‘MicrobiotaProcess’ and ‘ggplot2’. Alpha variety is measured based mostly on 4 completely different metrics, together with Chao1, Abundance-based Protection Estimator (ACE), Shannon, and Simpson. The differential abundance of the microbiome was calculated utilizing LEfSe of the R package deal ‘lefser’.

Immunoprecipitation and western blotting

After therapy with PBS, MSNs, Bifidobacterium, and MSNs-Bi, APP/PS1 mice had been anesthetized with 0.4 mL Avertin (25 mg/mL) and perfused transcardially with 1 M PBS for 30 min; the tissues (mind, abdomen, duodenum, jejunum, ileum, caecum, and colon) had been saved at − 80 °C earlier than evaluation. Frozen tissues had been homogenized in liquid nitrogen and dissolved in tris-buffered saline resolution (TBS, 20 mmol/L Tris and 137 mmol/L NaCl, pH 7.6) as beforehand described [1, 35]. Briefly, the homogenized supernatant was centrifuged (10,000 g, 30 min) at 4 °C to acquire TBS-soluble proteins. The supernatant was aliquoted and saved at –80 °C earlier than evaluation. Moreover, 100 μLof mind homogenate supernatant or blood was incubated with 40 μg/mL of 6E10-conjugated protein A/G magnetic beads for 30 min at room temperature. Magnetic beads had been washed 5 instances with PBS on a magnetic stand. Then, 20 μL of PBS and 4 μL of loading buffer (Boster Biotech, USA) had been added and the combination was boiled at 95 °C for 10 min. Lastly, beads had been loaded and subjected to 12% tris-tricine sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Proteins separated by SDS-PAGE had been transferred onto polyvinylidene fluoride (PVDF) membranes, which had been blocked with PBS-T containing 5% skimmed milk for 30 min. Subsequently, membranes had been incubated with main antibody 6E10 (1:1000, 5% skimmed milk) in a single day at 4 °C and washed with PBS-T. Subsequent, the membranes had been incubated with a second antibody HRP-conjugated goat anti-mouse IgG (H +L) (1:4000, PBS-T) for 1 h. Lastly, immunoreactive proteins had been recognized utilizing ECL substrates (Vazyme, China) on a Tanon 5200 Multi (Shanghai, China).

Immunofluorescence evaluation of tissue sections

Mind, abdomen, duodenum, jejunum, ileum, caecum, and colon tissues had been collected from APP/PS1 mice handled with intranasal instillation of PBS, MSNs, MSNs-Bi, and Bifidobacterium. Subsequent, the tissues had been pipetted into 4% (w/v) PFA in a single day after which dehydrated in 30% (w/v) sucrose resolution. Tissues had been then minimize into 25 μm sections utilizing a cryo-Leica CM3050S microtome and mounted on adhesion microscope slides (Jiangsu, China). Sections had been blocked with 3% BSA for two h, handled with 0.2% Triton X-100 (Macklin, China) for 30 min at room temperature, and incubated in a single day at 4 °C with the anti-Aβ main antibody 6E10 (1:1000). Subsequently, sections had been incubated with Cy3-conjugated goat anti-mouse IgG (H +L). Lastly, all tissue sections had been counterstained with 0.002% ThioS resolution for 30 min and washed 3 instances for five min every in 50% ethanol (v/v). Confocal photos had been acquired on a Zeiss LSM710 at 10 ×magnification and analyzed by Picture J software program.

Quantitative evaluation of Aβ and inflammatory components

For quantitative evaluation of Aβ and inflammatory components, frozen tissues of APP/PS1 mice handled with PBS, MSNs, MSNs-Bi, and Bifidobacterium had been homogenized in liquid nitrogen and extracted utilizing TBS resolution. The homogenized supernatant was then centrifuged at 10,000 g for 30 min at 4 °C to acquire TBS-soluble proteins. Following this, Aβ ranges within the mouse mind and gut had been quantified utilizing the Aβ42 human ELISA equipment (Catalog #KHB3441, Invitrogen). The BD Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine equipment (Catalog #560485, BD Biosciences) was used to quantify the degrees of inflammatory components within the blood.

Willpower of SCFAs

Feces from completely different teams of APP/PS1 mice handled with PBS, MSNs, MSNs-Bi, and Bifidobacterium had been collected and saved at − 80 °C earlier than evaluation. Whole SCFAs had been extracted from 50 mg stool samples utilizing the Mouse Brief-Chain Fatty Acid ELISA Package (Catalog #RJ17989, Renjie Biosciences). Knowledge are expressed as imply content material (pg/g) ± SD.

Behavioral evaluation

Odor cross-habituation take a look at

To evaluate the olfactory perform of mice after completely different remedies, an odor cross-habituation take a look at was carried out utilizing a beforehand described methodology to detect olfactory defects in mice [36]. Monomolecular odorants together with heptanone, isoamyl acetate, ( +) enantiomer of limonene, and ethyl valerate (Sigma Aldrich, China) had been diluted to 1 ×10−3 mol/L in mineral oil and coated on a cotton ball. The cotton ball was then wrapped in an odorless plastic bag with small holes and positioned within the bottle cap to forestall the odor liquid from reaching the cage or animals, however enable the risky odor fumes to diffuse. Odor cross-habituation was examined in 4 20 s trials by putting a cotton ball on the facet of the animal’s cage with a 30 s interval between them. Throughout sniffing, the time recording was terminated when the mouse’s nostril was inside 1 cm of the odor of the cotton ball.

Nest building assay

Nesting habits is an easy and versatile habits take a look at that has been broadly used to evaluate motor deficits. Constantly sized paper towels (5 cm ×5 cm) had been positioned in the identical place within the mouse cages within the PBS, MSNs, Bifidobacterium, and MSNsBi therapy teams. After 24 h, the place of the paper towels inside the nest was noticed.

Step down take a look at

Through the coaching part, mice had been initially positioned individually in a shock chamber to freely study the surroundings for 3–5 min. Then, mice acquired unconditioned stimulation (0.4 mA). The time the mice jumped to the security station when it first acquired an electrical shock was thought-about to be its notion capacity. Subsequent, mice had been positioned in a shock chamber for 30 s to measure fast response, and the method was repeated 4 instances. After 24 h, every mouse was returned to the identical security station within the shock chamber, and the full time and variety of shocks inside 5 min had been recorded.

Statistical evaluation

Statistical analyses had been carried out utilizing GraphPad Prism model 8.0 for Home windows. Knowledge are offered as imply ± commonplace error of the imply (SEM). One-way or two-way evaluation of variance (ANOVA) was used for a number of group comparisons. Statistical significance is indicated within the graph with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and n.s. (means no significance).



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