The preparation of EUP – SeNP
SeNP was ready by reacting sodium selenite (Aladdin Industrial Company, Shanghai, China) with ascorbic acid (Solarbio, Beijing, China) in a sure proportion. Briefly, 10 mL sodium selenite (20 mM) was added to 10 mL ascorbic acid (80 mM) for mixing and stirring for 1 h underneath darkish setting, after which Eucommia ulmoides polysaccharide (Tianrui Biology, Xian, China) (0.32, 0.64, 1.28, 1.71, 2.13 mg/mL) was added to stir for 12 h to acquire EUP-SeNP answer. Extra sodium selite and ascorbate have been subsequently eliminated by dialysis with Mili-Q water in regenerated cellulose tubes (Mw cutoff worth 5000) till the Se regenerated cellulose tubes have been undetectable by inductively coupled plasma emission spectrometry (ICP-OES) in exterior answer. After dialysis, a few of the response merchandise have been lyophilized. The remaining liquid is saved at 4 ℃.
Characterization of EUP-SeNP
The Se content material in EUP-SeNP was quantitated by ICP-OES on an ICP spectrometer at a particular wavelength of 196.08 nm. The Fourier rework infrared (FTIR) spectra of EUP and SeNP and EUP-SeNP have been decided. Briefly, 5 mg of every pattern was uniformly blended with dry KBr stable powder. The powder was sufficiently floor with a mortar and pressed by a vacuum pill machine. The infrared absorption spectra of those samples have been decided on a Nicolet 6700 FTIR (Thermoelectric (Shanghai) Expertise Instrument Firm, China). The EUP-SeNP have been characterised by transmission electron microscope and the vitality dispersive X ray spectrometer (TEM-EDX) (TECNAI G220; FET firm, USA). A small quantity of the pattern was positioned on the membrane floor of the copper mesh used for electron microscopy. Then, the copper mesh was dried naturally within the oven to keep away from the contamination of mud and different impurities. The dried copper mesh was noticed with a TEM, and the basic composition of the composite particles within the goal space was noticed utilizing a spot sweep methodology along with an EDX. Particle measurement was decided by Zetasizer Nano ZS (ZS90; Malvern Instrument Co., Ltd., UK). Vitro antioxidant capability was evaluated utilizing 1,1-diphenyl-2-picrylhydrazyl(DDPH), hydroxyl radical(·OH), and a couple of, 2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) free radical scavenging equipment. (Nanjing Jiancheng Bioengineering Institute, China)
Animals and experimental design
A complete of 60 6–8 week previous male C57BL/6JNifdc mice (20-21 g) have been bought from Important River Laboratory Animal Expertise Firm (Beijing, China). All animals have been raised in a managed setting: relative humidity was 40 ± 10% and temperature was 20 ± 5 ℃, with a 12/12 h gentle–darkish cycle. After 1 week of adaptation, the mice have been randomly divided into 6 teams, specifically management group, 3percentDSS group, 3percentDSS + EUP group, 3percentDSS + SeNP group, 3percentDSS + EUP-SeNP group and threepercentDSS + selenite group, every with 10 mice. Besides the management group, the opposite teams got 3% w/v DSS ingesting water for 7 consecutive days, and the management group was given ingesting water with out DSS. From the eighth day, within the remedy group, 200 µL of the drug was intragastric for five days, and the selenium content material within the EUP-SeNP remedy group was 0.5 ppm. The management group and DSS group got the identical quantity of PBS. Physique weight and illness exercise index (DAI) scores of mice in every group have been noticed and recorded every day. The size of the colon was measured when the mice have been sacrificed. All animal procedures have been authorized by the China Agricultural College Institutional Animal Care and Use Committee (AW80212202-2-1).
Analysis of colitis
After administration of DSS ingesting water, the illness exercise index rating was recorded daily. The DAI rating is a composite rating of weight change, stool consistency and stool bleeding. Colon tissues have been eliminated and glued with 4% paraformaldehyde. The colons have been lower into 5 μm sections and stained with hematoxylin & eosin (H&E) and Alcian blue-periodic acid-Schiff (AB-PAS) based on customary procedures. (Solarbio, Beijing, China) Histopathological rating is a complete measure of inflammatory cell infiltration and modifications in tissue injury. The modifications in goblet cells in colon tissue have been noticed by AB-PAS staining. Detailed scoring standards for DAI and histology are primarily based on earlier articles [28, 29].
ELISA and measurement of oxidative stress
Proteins have been extracted from colon tissues with physiological saline, then detected with a BCA protein evaluation equipment (CoWin Biotech Co., Inc., Beijing, China). IL-6, IL-10, IL-12, IL-17, IL-1β, TNF-ɑ (Laibotairui Tech Co., Ltd., Beijing, China), have been decided by an ELISA equipment. The outcomes have been normalized to the protein focus of every pattern. A microplate reader (BioTek Co., Ltd., Beijing, China) was used for detection at 450 nm. Indicators of intestinal antioxidant capability: Whole antioxidant capability (T-AOC), Superoxide Dismutase (SOD), Catalase (CAT), glutathione peroxidase(GPX), Malondialdehyde(MDA), Glutathione/oxidized Glutathione(GSH/GSSG), myeloperoxidase(MPO) ranges have been decided by utilizing industrial kits based on the producer’s directions (Nanjing Jiancheng Bioengineering Institute, China).
Western blotting
Proteins extracted from the colon tissue have been separated by 8–12% SDS-PAGE after the focus was detected by the BCA protein assay equipment (CW0014S, CoWin Biotech Co., Inc., Beijing, China). They have been then transferred to 0.2 μm polyvinylidene fluoride membranes (Merck KGaA Co., Ltd., Darmstadt, Germany). Membranes have been blocked with 5% skim milk for 1.5 h. After blocking, the membranes have been incubated with completely different main antibodies at 4 ℃ in a single day. Claudin 1 (1/3000, Abcam Co., Inc., Cambridge, UK), Claudin 3 (1/1000, Abcam Co., Inc., Cambridge, UK), Occludin (1/1000, Abcam Co., Inc., Cambridge, UK), ZO-1 (1/1000, Abcam Co., Inc., Cambridge, UK), TRL-4(1/1000, Proteintech Co., Wuhan, China), IKB(1/1000, Abmart Co., Inc, Shanghai, China), p65(1/5000, Abmart Co., Inc, Shanghai, China), p-IKB(1/1000, Abcam Co., Inc., Cambridge, UK), pp65(1/1000Abcam Co., Inc., Cambridge, UK), β-actin(1/1000, LABLEAD Biotech Co., Ltd., Beijing, China). Then, the membranes have been washed with Trisbuffered saline Tween (TBST) after which incubated with horseradish peroxidase-conjugated goat anti-mouse IgG, or goat anti-rabbit IgG (CoWin Biotech Co., Inc., Beijing, China) for 1.5 h. The membranes have been imaged with a Tanon 5200 imaging system. (Tanon Science & Expertise Co., Ltd., Shanghai, China)
Immunohistochemistry
Colon tissue sections have been dewaxed with xylene and hydrated in gradient ethanol. Then, the antigen was repaired with 0.01 M sodium citrate buffer. Tissue sections have been washed with phosphate buffer (PBS, pH7.0) 3 occasions for five min every time. Endogenous peroxidase exercise was blocked by 3% hydrogen peroxide for 30 min, and non-specific staining was blocked by 5% goat serum for 30 min. Tissue sections have been incubated with Ki67 antibody (1/200) and anti-MUC2 (1/2000, Abcam Co., Inc., Cambridge, UK) at 4 ℃ in a single day. On the two day, after being washed with PBS, they have been incubated with biotin-conjugated goat anti-rabbit IgG (CoWin Biotech Co., Inc., Beijing, China) for two h after which incubated with horseradish peroxidase (HRP)-streptavidin (CoWin Biotech Co., Inc., Beijing, China) for two h. DAB chromogenic reagent equipment (Zhongshan Jinqiao Biotech Co., Ltd., Beijing, China) was used for chromogenic reagent detection. Hematoxylin was used for nuclear re-staining. The first antibody of the unfavorable management group was changed with PBS. Photographs have been captured utilizing a microscope (Nanjing Jiangnan Novel Optics Co., Ltd., Nanking, China).
TUNEL
By the one step TUNEL apoptosis assay equipment (LABLEAD Biotech Co., Ltd., Beijing, China), terminal deoxynucleotidyl transferase dUTP nick finish labeling (TUNEL) assay was used to detect the apoptotic degree of colon, IEC-6 and Caco-2. The photographs have been taken with upright DP72 microscope. (Olympus Co., Inc., Tokyo, Japan)
Microbiota evaluation
The contents of the mouse colon have been collected and saved in liquid nitrogen. An MN Nucleo Spin 96 Soi DNA extraction equipment (MACHEREY-NAGEL GmbH & Co. KG, Duren, Germany) was used to extract whole bacterial DNA from the pattern. The primers have been designed based on the conserved area of micro-organism V3 + V4. The primers have been used for PCR amplification, and the merchandise have been purified, quantified and homogenized to kind a sequencing library. The constructed library was first subjected to high quality inspection, and the certified library was sequenced with Novaseq 6000 (Illumina, Co., Inc., San Diego, CA, USA). Sequence similarity better than 97% was categorised as an operational taxonomic unit (OTU). The OTU composition of various samples was analyzed utilizing principal part evaluation (PCA), principal coordinates evaluation (PCoA) and non-metric multidimensional scaling (NMDS) primarily based on Bray Curtis evaluation. Line discriminant evaluation (LDA) impact measurement, often known as LEfSe, was used to investigate the importance of variations between teams from the phylum to genus degree. LEfSe evaluation required an LDA rating > 4.
Consider the safety of EUP-SeNP
We evaluated short-term and long-term toxicity by orally administering EUP-SeNP to mice for 7 days and 30 days, respectively, after which detected Aspartate transaminase (AST) and Alanine transaminase (ALT) of serum. (Nanjing Jiancheng Bioengineering Institute, China) Lastly, H&E staining was carried out on coronary heart, liver, spleen, lung, kidney and colon.
Cell viability evaluation and mobile uptake of EUP-SeNP
IEC-6 and Caco-2 have been cultured in DMEM medium supplemented with 10% FBS, 100 U/mL of penicillin, and 100 µg/mL of streptomycin at 37 °C with 5% CO2 in a humidified ambiance. Cells have been incubated with 50 µg/mL LPS for 36 h, and EUP-SeNP was incubated with 10 µg/mL, 20 µg/mL and 40 µg/mL for pretreatment and post-treatment for 12 h, respectively.
IEC-6 was incubated right into a 96-well plate, after which incubated for twenty-four and 48 h by including completely different concentrations of EUP-SeNP. Subsequent, the medium was discarded and washed thrice with PBS. Lastly, 100 µL PBS and 10 µL CCK8 (Solarbio, Beijing, China) have been added to every properly and incubated for one hour at 37℃, the optical density was measured at a wavelength of 570 nm. IEC-6 was seeded right into a 6-well plate, labeled with fluorescence EUP-SeNP utilizing coumarin-6 as a fluorescence probe, cell nucleus have been stained with DAPI (Solarbio, Beijing, China) and cell membranes have been stained with Dil (Beyotime Institute of Biotechnology, Jiangsu, China). Cell uptake was noticed inside 1 h, 2 h, and 4 h, and pictures have been taken with fluorescence microscope.
Statistical analyses
Information evaluation was carried out with GraphPad Prism (model 8.0.2 for Home windows, GraphPad). Information are expressed because the means ± customary errors of the imply (SEM). All comparisons of variant parameters between teams have been made with one-way evaluation of variance, with statistical significance as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001. “*” represents the importance between the management group and the DSS mannequin group; “#” represents the importance between the DSS mannequin group and the remedy group.
