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:
- Alpha-smooth muscle actin (α SMA), forward:
5’-CCCAGACATCAGGGAGTAATGG-3’; reverse: 5’-TCTATCGGATACTTCAGCGTCA-3’,
- Transforming growth factor-beta1 (TGF β1), 5’-CTCCCGTGGCTTCTAGTGC-3’;
reverse: 5’-GCCTTAGTTTGGACAGGATCTG-3’,
- Collagen Type I (Col 1), 5’- GCTCCTCTTAGGGGCCACT-3’; reverse:
5’-CCACGTCTCACCATTGGGG-3’,
- Vimentin, 5’-CGTCCACACGCACCTACAG-3’; reverse:
5’-GGGGGATGAGGAATAGAGGCT-3’,
- Actin, forward: 5’-GTGACGTTGACATCCGTAAAGA-3’; reverse:
5’-GCCGGACTCATCGTACTCC-3’.
- 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.