4.4 Immunohistochemical analysis
Immunohistochemistry was performed as we described previously20. Briefly, paraffin embedded sections were dewaxed, rehydrated, blocked in 3% hydrogen peroxide (20 mins, 37°C), and followed by antigen retrieval treatment using EDTA antigen repair buffer (PH 8.0). After blocking with 10% goat serum, sections were incubated with anti-Collagen Type I (Cell Signaling Technology#72026S, 1:400 dilution), anti-Collagen Type III (Proteintech#22734-1-AP, 1:800 dilution), anti-Vimentin (Cell Signaling Technology#5741, 1:400 dilution) primary antibody at 4 ℃ overnight, followed by incubation with horseradish peroxidase (HRP) labeled goat anti rabbit IgG (H+L) (Beyotime#A0208, 1:50 dilution). Subsequently, the sections were treated with Diaminobenzidine (DAB) chromogen and restained with hematoxylin. Digital images were created and assessed using CaseViewer software (3DHISTEC, Sysmex, Switzerland).
Real-time quantitative PCR (RT-qPCR)
Total RNA was isolated from liver samples using TRIZOL reagent (Invitrogen). The quality and quantity of extracted RNA were assessed by a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Synthesis of the first strand cDNA was performed and then qPCR was conducted using the SYBR Green PCR system according to the manufacturer’s recommendations (Yisheng Biotech). The relative mRNA expression was analyzed by 2-△△Ct method. All primers were purchased from Sangon Biotech (Shanghai, China) and the sequences are shown as follows:
  1. Alpha-smooth muscle actin (α SMA), forward: 5’-CCCAGACATCAGGGAGTAATGG-3’; reverse: 5’-TCTATCGGATACTTCAGCGTCA-3’,
  2. Transforming growth factor-beta1 (TGF β1), 5’-CTCCCGTGGCTTCTAGTGC-3’; reverse: 5’-GCCTTAGTTTGGACAGGATCTG-3’,
  3. Collagen Type I (Col 1), 5’- GCTCCTCTTAGGGGCCACT-3’; reverse: 5’-CCACGTCTCACCATTGGGG-3’,
  4. Vimentin, 5’-CGTCCACACGCACCTACAG-3’; reverse: 5’-GGGGGATGAGGAATAGAGGCT-3’,
  5. Actin, forward: 5’-GTGACGTTGACATCCGTAAAGA-3’; reverse: 5’-GCCGGACTCATCGTACTCC-3’.
  6. Western blotting analysis
Liver samples were lysed using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime) and protease inhibitor (Beyotime), and protein concentration was determined by a BCA assay kit (Beyotime). Western blotting analysis was performed as described previously20. The antibodies used for protein quantification are shown as follows: α SMA (Signalway Antibody#40482, 1:1000 dilution), Col1 (Proteintech#66761-1-Ig, 1:2000 dilution), HSP90 (Proteintech#60318-1-Ig, 1:10000 dilution) along with secondary antibody HRP-conjugated goat anti-rabbit (Beyotime#A0208, 1:1000 dilution) or mouse (Proteintech#SA00001-1, 1:10000 dilution) IgG. Data were normalized to HSP90 and were represented by values relative to the control group.
Fecal sample collection and 16S rRNA amplicon sequencing
After 6 weeks of administration, fecal samples (>0.5 g) of three groups of mice were collected in standard tubes, and then immediately stored in a -80°C freezer. Total genomic DNA (gDNA) was extracted from fecal samples by Fast DNA SPIN extraction kit (MPB Biomedicals, USA) and the quality of extracted DNA was examined by 1% agarose-gel electrophoresis (5 V/cm, 20 min), and DNA concentration and purity were determined by NanoDrop 2000 spectrophotometry (Thermo Fisher Scientific, USA). PCR amplification of the bacterial 16S rRNA genes V3-V4 region was performed using the forward primer 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and the reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Amplicon libraries were run and sequenced on the Illumina system (Illumina, San Diego, CA, USA) for paired-end reads. Paired sequence data were analyzed by QIIME 1.9.1 software. Chimeric sequences were removed using UCHIME software and clustered by operational taxonomic units (OTU) according to the default similarity value of 97% using UPARSE software. Taxon abundance at the genus level was compared by Kruskal test using R software (4.1.1). Alpha and beta diversity were evaluated using QIIME software. In addition, spearman correlation analysis was conducted between liver fibrosis score or indicators related to glucose metabolism and the relative abundance of bacteria at genus level.
Statistical analysis
Data are presented as mean ± standard deviation (SD). The histograms were drawn using GraphPad Prism 8 (San Diego, California). The differences between the two groups were performed by Student’s t-test for normal distribution and the Wilcoxon test for non-normal distribution. Comparisons among multiple groups were evaluated using one-way analysis of variance (ANOVA) for normal distribution and Kruskal test for non-normal distribution. P-value less than 0.05 is considered statistically significant.