Experimental animal models
Male SD rats (120–150g) (Shanghai laboratory animal center, Shanghai, China) were randomly divided into the control group (CT) and the diabetes mellitus group (DM). 24 h after fasting, the streptozotocin (65 mg/kg body weight, dissolved in 0.01 M citrate buffer, pH 4.5; Catalog: S8050, Solarbio, Beijing, China) were intraperitoneal injected to the rats of DM group while CT group were injected with equal volume of the vehicle. 72 h after diabetes conduction, the rats with blood glucose above 16.7 mmol/L were considered the successful diabetes models.
After six weeks, all rats were anesthetized by sodium pentobarbital (45 mg/kg body weight). Blood samples were obtained from hepatic portal vein to evaluate the level of IL-1β. The liver and the body weight were also recorded. The liver of half rats in each group were removed and fixed in 4% paraformaldehyde solution overnight for hematoxylin-eosin (H&E) and immunohistochemical staining, while another half liver in each group were preserved in liquid nitrogen for Western blot and ELISA assay.
Gut microbiota analysis
Fresh fecal samples were collected at the time of blood sampling, snap frozen in liquid nitrogen, and stored at -80°C until analysis. Sequencing and data processing were conducted by Illumina MiSeq (Illumina, San Diego, USA). Microbial DNA was extracted from fecal samples using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s protocols. Quantitative polymerase chain reaction was conducted using the following program: 3 min of denaturation at 95 °C, 27 cycles of 30 s at 95 °C, 30 s for annealing at 55 °C, and 45 s for elongation at 72 °C, and a final extension at 72 °C for 10 min by thermocycler PCR system (GeneAmp 9700, ABI, USA). PCR reactions were performed in triplicate 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase and 10 ng of template DNA. The resulted PCR products were extracted from a 2 % agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor ™-ST (Promega, USA) according to the manufacturer’s protocol. Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Raw fastq files were demultiplexed, quality-filtered by Trimmomatic and merged by FLASH with the following criteria: (i) The reads were truncated at any site receiving an average quality score <20 over a 50 bp sliding window. (ii) Primers were exactly matched allowing 2 nucleotide mismatching, and reads containing ambiguous bases were removed. (iii) Sequences whose overlap longer than 10 bp were merged according to their overlap sequence. Operational taxonomic units (OTUs) were clustered with 97 % similarity cutoff using UPARSE（version 7.1 http://drive5.com/uparse/) and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier algorithm (http://rdp.cme.msu.edu/) against the Silva (SSU123) 16S rRNA database using confidence threshold of 70%.
Hematoxylin-eosin staining (H&E) and immunohistochemical staining
The livers (in 4% paraformaldehyde solution) were embedded in paraffin, then cut into 6-μm section. The sections were respectively stained with H&E to determine pathological changes. Images were observed by the microscope (BX-53, Olympus Corp, Tokyo, Japan).
The rest sections from each group were stained with immunohistochemical assay and incubated with the primary antibodies against TLR9 (TLR9, Catalog: ab134368, 1:200, Abcam, USA). After overnight incubation at 4 ℃, sections were washed with PBS (3 × 5 min) and incubated with biotinylated and affinity-purified IgG secondary antibodies for 1 h at room temperature. Images were observed by the microscope (BX-53, Olympus Corp, Tokyo, Japan) and analyzed by Image-Pro Plus 6.0 (Media Cybernetics, USA).
KCs were cultured with TLR9 agonist and inhibitor
We used 1640 medium (Catalog: gnm31800, China) with 10% (V/V) fetal bovine serum (Catalog: 10099-141, Gibco, Invitrogen, USA) to culture KCs (Catalog: CP-R132, Procell Life Secience&Technology, Wuhan, China) in the medium inside a tri-gas incubator (Thermo Fisher Scientific, Marietta, OH, USA) composed of 5 % CO2 and 95% air at 37 °C.
5 × 105/well KCs were seeded in 6-well (3 μM) system and divided into six groups: the control group (CT), the control group treated with unmethylated CpG-DNA (5.8 μM, dissolved in 9.91 mM DMSO; Catalog: HY-101929, MedChemExpress, USA) (CT-OV), the control group treated with TLR9 inhibitor E6446 dihydrochloride (10 μM, dissolved in 14.55 mM DMSO; Catalog: HY-12756A, MedChemExpress, USA) (CT-SI), the diabetes group (DM), the diabetes group treated with unmethylated CpG-DNA (DM-OV) and the diabetes group treated with E6446 dihydrochloride (DM-SI). CT, CT-OV and CT-DM group were supplied with 5.5 mM glucose, while DM, DM-OV and DM-SI group were supplied with 30 mM high glucose to simulate the diabetic environment. After 24 h treatment, the cell culture supernatant from all groups were collected in the flow tube and centrifuged for 20 min at 1000 × g at 2~ 8 ℃ for ELISA assay. Cells were washed in PBS (3 × 5 min), lysed in Western & IP buffer (Catalog: P0013, Beyotime Institute of Biotechnology, Shanghai, China) then used for Western blot assay.
Western blot analysis
The expression of proteins in liver and cultured cells were detected by Western blot assay. Equal amounts of protein were separated by 10 % SDS-PAGE. It was transferred from the gel to nitrocellulose membrane, then blocked by 5 % skimmed milk (Tris-buffered saline of 0.1 % Tween 20) for 1 h at room temperature. The membranes were incubated with the primary antibodies (TLR9, Catalog: ab134368, 1:1000, Abcam, USA; IL-1β, Catalog: ab9722, 0.8 μl/ml, Abcam, USA) overnight at 4 °C. The next day, the membranes were washed with TBST (4 × 5 min) and incubated with infrared labeled secondary antibodies at room temperature for 1 h. The immunoblotted bands were captured by a CLX Odyssey infrared imaging system (Li-COR biosciences, USA).
ELISA analysis of IL-1β levels in tissue homogenates, serum and cell culture supernatant of KCs were detected by Rat IL-1β ELISA Kit (Catalog: E-EL-R0012c, Elabscience, Wuhan, China). Sample collection, reagent preparation and assay procedure were strictly according to the kit instruction. The microplate reader (Thermo, USA) was used to assay the optical density (OD) values at 450 nm.
The data are presented as mean ± standard deviation (SD) and analyzed by SPSS 18.0 software (SPSS Inc., USA). The differences among both groups were performed with one-way analysis of variance (ANOVA). P values <0.05 were considered statistically significant.