3.2 PIC Ⅱ may attenuate hepatic fibrosis via improving hepatic
immune microenvironment in Mdr2−/− mice
In order to investigate whether the anti-fibrotic effects of PIC Ⅱ were
relied on the regulation of different immune cells, we systematically
analyzed the RNA-sequencing data and
try to figure out the primary target genes that PIC Ⅱ might focus on.
Based on the different modules of gene expression of all DEGs, WGCNA was
used to construct a gene co-expression network with the Pearson’scorrelation coefficient applying to cluster the sample, and
simultaneously, eight modules were then obtained through the
hierarchical clustering of the predetermined dissimilarities inFig. 2A . To pick out the liver fibrosis-related modules, the
relationship between the modules and the result of hepatic pathology and
function was studied. Among the 8 modules, the turquoise and blue module
was highly associated with pathological manifestation, thus they were
selected for further analysis. Additionally, three co-expression
modules, including inflammatory infiltration, collagen deposition and
oxidative stress were mainly enriched, especially for inflammatory
infiltration governing the liver fibrosis (Fig. 2B ). Therefore,
we speculate that PIC Ⅱ might exert a potential effect through immune
cells and thus we plotted DEGs implicated in immune-related targets as a
heatmap and prepared relative clusters (Fig. 2C ). Compared with
the WT group, it was found that most differential genes were enriched in
cluster 3, which were upregulated in model and PIC Ⅱ mice, including the
macrophage makers Cd86 ,
colony stimulating factor
2 receptor subunit alpha (Csf2ra ) and Csf3r , and NK cell
activation cytokines like
C-C motif chemokine ligand
3 (Ccl3 ) and
interleukin 4 receptor,
alpha (Il4ra ). After noticing the significant changes in immune
reactions, different subtypes of immune cells in three different groups
were analyzed, we then investigated that PIC Ⅱ could remarkably affect
the proportion of macrophages (Fig. 2D ). Generally, M1
macrophages were known as a stimulator of inflammatory reaction while M2
macrophage more likely contributes to the remission of liver fibrosis
(Cheng et al., 2021). Notably, a further grouping of quantitative
differences exhibited that PIC Ⅱ increased the numbers of M1 macrophages
and monocytes but didn’t decrease the number of M2-type macrophages and
NK cells, compared with Mdr2−/− mice (Fig. 2Eand 2F ). These results indicated that PIC Ⅱ might alleviated
fibrosis through establishing intricate links among diversified immune
cells, which was far more complex than we traditionally imaged.