*3.3. Genes Associated with HSC Activation are Significantly Upregulated in TGR5 KO Mice as Compared to WT Littermates*

Gene expression analysis by semi-quantitative real-time PCR revealed a similar pattern of gene regulation in both genotypes after LCA feeding. As expected, induction of cholestasis by LCA feeding resulted in a significant downregulation of Cyp7a1 and Cyp8b1, the rate limiting enzymes of bile acid synthesis. In line with the increase in liver damage, a significant upregulation of the genes encoding receptor-interacting serine/threonine-protein kinases 1 and 3 (RIPK1 and RIPK3), as well as caspase-3, was observed (Table 1). Furthermore, we found a significant induction of pro-inflammatory genes such as tumor necrosis factor-α (TNF-α), CCL2, interleukin-1β (IL-1β) and pro-fibrogenic chemokine receptors CCR1, CCR5 (Table 1) [21,22,39]. Interestingly, genes related to sinusoidal endothelial dysfunction, activation of HSCs, and fibrosis development, such as platelet-derived growth factor receptor-α (PDGFRα), PDGFRβ, transforming growth factor-β1 (TGF-β1), and endothelin-1 (ET-1), were upregulated in both WT and TGR5 KO mice following LCA feeding. However, the observed increase was significantly higher in TGR5 KO mice (PDGFRα: WT LCA (*n* = 7) 3.52 ± 0.8 vs. KO LCA (*n* = 10) 7.30 ± 1.4, 2.1-fold increase in TGR5 KO, *p* < 0.05; PDGFRβ: WT LCA (*n* = 12) 4.0 ± 0.6 vs. KO LCA (*n* = 12) 6.23 ± 0.75, 1.6-fold increase, *p* < 0.05; TGF-β1: WT LCA (*n* = 7) 7.81 ± 0.8 vs. KO LCA (*n* = 9) 11.8 ± 1.4, 1.5-fold increase, *p* < 0.05; ET-1: WT LCA (*n* = 14) 8.58 ± 1.1 vs. KO LCA (*n* = 11) 13.9 ± 2.4, 1.6-fold increase, *p* < 0.05) (Figure 3A–D). Sirius red staining of liver tissue after 3.5 days of LCA feeding revealed a slight increase in periportal collagen deposition, but no overt hepatic fibrosis in both genotypes, which was in line with measurements of hydroxyproline content in liver tissue (Figure 4A,B). Expression of genes encoding collagen-1α1, collagen-1α2 were upregulated in both genotypes following LCA feeding for 3.5 days, indicating initiation of extracellular matrix synthesis, as described earlier (Figure 4B) [21,22].

**Table 1.** Selection of genes significantly regulated in livers of TGR5 wildtype (WT) and TGR5 knockout (KO) after LCA feeding for 84 h as compared to chow-fed animals of the same genotypes. Data were generated by real-time PCR, as described in Section 2—Materials and Methods. Gene expression is presented in relation to the house-keeping gene SDHA. Values for WT mice on chow diet were set to 1.0. \* Significantly different as compared to chow-fed animals of the same genotype (*p* < 0.05); \*\* significantly different as compared to chow-fed animals of the same genotype (*p* < 0.01); # significantly different as compared to WT animals of the same treatment group (*p* < 0.05). All data are expressed as mean ± SEM. Green and red boxes indicate a statistically significant downregulation or upregulation, respectively, of a gene in TGR5 KO as compared to WT littermates receiving the same treatment.



**Table 1.** *Cont.*

**Figure 3.** Expression of genes related to portal hypertension and fibrosis development are significantly higher in liver tissue of TGR5 KO mice as compared to WT littermates in response to LCA feeding. (**A**–**D**) Semi-quantitative real-time PCR analysis of liver tissue revealed an upregulation of mRNA expression PDGFRα, PDGRFβ, endothelin-1, and TGF-β1 following LCA feeding. The increase was more pronounced in mice deficient for TGR5. Data are presented as mean ± SEM (*n* = 4–12). \* Statistically significant difference between the LCA- and chow-fed mice of the same genotype (*p* < 0.05); # statistically significant difference between WT and TGR5 KO mice fed the same diet (*p* < 0.05; using the two-sided Student's *t*-test).

**Figure 4.** LCA-feeding triggers an increase in extracellular matrix production in both genotypes. (**A**) Representative Sirius red staining from one chow-fed and two LCA-fed animals of each genotype demonstrates a slight increase in fibrous tissue in both genotypes. Bars = 200 μm. (**B**) Measurement of hydroxyproline content in liver tissue showed a slight increase following LCA feeding, which did not reach significance (*n* = 8–10 animals per genotype LCA group, *n* = 3 animals per genotype for chow group). However, expression of collagen-1α1 and collagen-1α2 increased significantly after LCA feeding in both genotypes (qPCR*: n* = 12–14 LCA-fed animals per genotype, *n* = 4 chow-fed animals per genotype). Liver tissue from WT animals is shown in grey, while tissue from TGR5 KO mice is represented by black bars. All data are shown as mean ± SEM. \* Statistically significant difference between the LCA- and chow-fed mice of the same genotype (*p* < 0.05) (*n* = 8–10).
