Male C57BL/6J mice (8–10 weeks old, weighing 20–25 g) were obtained from Vital River Laboratory Animal Technology Co Ltd, Beijing, China. Mice were housed and maintained on a 12:12 light–dark cycle with ad libitum access to food and water under specific pathogen free conditions. All procedures were performed in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals (publications no.80-23) revised 1996. All animal protocols were approved by the committee of experimental animals of Tongji Medical College (Wuhan, China).
Experimental grouping and treatment
The experiment was performed in three parts. In part A, sleep-deprived or non-sleep-deprived groups with or without SDV 14 days prior to LPS administration. Mice were intraperitoneally administrated with LPS (5 mg/kg; L-4130, serotype 0111:B4; Sigma-Aldrich, St Louis, MO) or 0.9% saline (5 ml/kg). SDV or sham operation was performed 14 days prior to LPS administration. SD was performed immediately after LPS administration for 3 consecutive days. Mice were euthanized to collect plasma, spleen, lung, liver, kidney and fecal samples on day 4 after LPS administration.
In part B, control pseudo germ-free mice or pseudo germ-free mice with SDV or sham-operation were received fecal microbiota transplantation (FMT) from LPS-treated mice with or without SD. Pseudo germ-free mice underwent SDV or sham operation 14 days prior to antibiotics administration. Mice were euthanized and plasma, lung, liver and kidney were collected after the commencement of FMT.
In part C, pseudo germ-free mice with splenectomy or sham-operation were received FMT from LPS-treated mice with or without SD. Mice were euthanized and plasma was collected after the commencement of FMT. Splenectomy was performed 14 days prior to antibiotics administration.
Sleep deprivation model
Sleep deprivation was performed using a modified multiple platform method [24, 25]. Mice were placed in a ventilated transparent cage (50 × 30 × 17cm; 3 mice/cage) containing 9 circular platform (3.5cm in diameter) surrounded by water up to 1cm beneath the upper surface of the platforms enabling the mice to move between the platforms. When the mice had reached the rapid eye movement stage of sleep, muscle atonia could cause the mouse to fall into the water. The water in the cage was replaced with clean water every day. Food and water were available ad libitum throughout the sleep-deprivation period. The non-sleep-deprived mice were maintained under normal feeding conditions after administering LPS.
Bilateral SDV was performed under anesthesia with 5% isoflurane..Upper abdominal wall was opened through a 1 cm right transverse abdominal incision. Esophagus was exposed by carefully keeping costal arc, liver, and stomach out of sight. The ventral and dorsal branch of the vagus nerve was exposed along the subdiaphragmatic esophagus under a surgical microscope (BX51, Olympus, Japan). Each vagal branch was ligated twice with a surgical thread at an interval of 1 cm, and then dissected between the ligatures. After fluid resuscitation with 0.5 mL sterile saline solution, the abdominal wall was closed. For sham surgery, the vagus nerve were exposed but not cut.
Total splenectomy was performed under the isoflurane anesthesia. Splenectomy was performed by making a 2-cm subcostal incision on the left dorsolateral side of the abdomen, and the spleen was carefully exteriorized through the incision. The afferent and efferent vessels near to the spleen were ligated using 6-0 silk suture and the spleen was removed. The abdominal wall was closed with 4-0 silk suture and the skin was sutured using 3-0 silk suture. Sham-splenectomized mice underwent laparotomy without removal of the spleen.
Pseudo germ-free mice modeling
Based on a previous study , broad-spectrum antibiotics (ampicillin 1 g/L, neomycin sulfate 1 g/L, and metronidazole 1 g/L; Sigma-Aldrich Co. Ltd, USA) dissolved in drinking water were given ad libitum to mice for 14 consecutive days. The drinking solution was renewed every 2 days.
The fecal donor mice received continuous 3 days of SD or non-sleep-deprivation after LPS administration. On day 4, mice were placed in a clean cage with sterilized filter paper on the bottom. Stool samples from the experimental mice were collected immediately after defecation in a sterile micro-tube and immediately stored in a −80 °C freezer until analysis. 1 g of fecal sample from donor mice was diluted in 10 mL of phosphate-buffered saline (PBS). The feces were then suspended and 200 μL of the suspension was given to each mouse recipient by gavage.
Enzyme-linked immunosorbent assay (ELISA)
Blood was collected via cardiac puncture, placed into tubes containing ethylenediaminetetraacetic acid (EDTA), and immediately centrifuged at 3,000 ´ g for 5 min at 4℃, to obtain plasma, and then stored at –80℃ until bioanalysis. The commercial kits were used for the quantitative assay of IL-6 (Cat Number: 88-7064, Invitrogen, Camarillo, CA, USA), IL-10 (Cat Number: EMC005.96, NeoBioscience, China) and TNF-α (Cat Number: 88-7324, Invitrogen, Camarillo, CA, USA) according to the manufacturer’s instructions.
Stool sample collection and microbial sequencing
Fecal samples were collected and placed in 1.5ml tubes, snap-frozen on dry ice, and stored at −80 °C. The 16S rRNA analysis of the fecal samples was performed by GENEWIZ Biotech Co., Ltd. (Suzhou, China). Briefly, total genome DNA from samples was extracted using Soil DNA Kit according to manufacturer’s protocols. DNA samples were PCR-amplified in in 25 μL triplicate reactions with bacterial 16S rRNA gene (V3–V5 region)-specific forward primers containing the sequence “ACTCCTACGGGAGGCAGC” and reverse primers containing the sequence “GGACTACHVGGGTWTCTAAT”. The amplified DNA libraries were validated and quantified for sequencing on an Illumina MiSeq instrument (Illumina, San Diego, CA, USA). Through quality filtering out, the effective sequences were grouped into operational taxonomic units (OTUs). α-diversity and β-diversity were performed by the MOTHUR program.
Periodic acid-schiff (PAS) staining and hematoxylin-eosin (HE) staining
Spleen, lung, liver and kidney tissues were collected and immediately fixed in 4% paraformaldehyde (pH 7.4) at 4℃ for 48 hours and then embedded in paraffin for sectioning at 5 μm. Paraffin-embedded kidney tissue sections were stained with PAS stain according to a standard protocol. Paraffin-embedded Lung, liver and spleen tissue sections were stained with HE. Stained sections were photographed under light microscopy (BX51, Olympus, Japan).
The lung, liver and kidney samples were lysed in RIPA lysis buffer containing protease inhibitors (KeyGen Biotech, Nanjing, China). An equal amount of protein was resolved on a 10% SDS-PAGE gel (Beyotime Institute of Biotechnology, Shanghai, China) and blotted to a PVDF membrane (Millipore; Merck KGaA) using an electrophoresis apparatus (Bio-Rad Laboratories, Inc., Hercules, CA, United States). Membranes were blocked with 5% skim milk diluted in Tris-buffered saline containing 0.1% tween-20 (TBST) at room temperature for 1 hour. Subsequently, the membranes were incubated with primary antibodies against IL-6 (1:500; ABclonal Biotechnology, Co., Ltd., Wuhan, China) and β-actin (1:2,000; Proteintech group, Wuhan, China) overnight at 4℃. After primary antibody incubation, membranes were washed with TBST and incubated with an appropriate peroxidase-conjugated secondary antibody (1:3000; Proteintech group, Wuhan, China) for 2 hours at room temperature. Following three washes with TBST, chemiluminescent signals were visualized using electrochemiluminescence western blotting detection reagents (Millipore; Merck KGaA) and bands were captured using an UVP gel documentation system (UVP, LLC, Phoenix, AZ, USA). Band intensity was quantified using Image J software (version 1.41; National Institutes of Health, Bethesda, MD, USA).
Quantitative Real-Time PCR (RT‑qPCR) measurements
Lung, liver and kidney samples were homogenized before total RNA isolation using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. cDNA was obtained using Taqman reverse transcriptase (Applied Biosystems; Thermo Fisher Scientific, Inc.). β-actin cDNA was amplified using Power SYBR Green (Applied Biosystems; Thermo Fisher Scientific, Inc.). Two step qPCR was performed (95 ℃ for 15 s, 60 ℃ for 60 s for 40 cycles) with specific primers for IL-6 (forward, 5'-GAGGATACCACTCCCAACAGACC-3'; reverse: 5'-GAGGGATATCTATCAGG GTCTTCAT-3') and β-actin (forward, 5'-AAGGCCAACCGTGAAAAGAT-3' and reverse, 5'-GTGGTACGACCAGAGGCATAC-3'). The relative quantitation value is expressed as 2-ΔΔCq, where ΔCq is the difference between the mean ΔCq value of duplicate measurements of the sample and β-actin control.
Data were expressed as the mean ± standard error of mean (SEM). Multiple comparisons were performed by one-way analysis of variance (ANOVA) followed by post-hoc Tukey’s multiple comparison tests. Two groups were compared by unpaired student t test. Correlation was analyzed by Pearson correlation. P < 0.05 was considered to be statistically significant. Statistical analyses were performed using Prism 8 software (GraphPad Software).