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.