Knockout of Purinergic P2Y6 Receptor Fails to Improve Liver Injury and Inflammation in Non-Alcoholic Steatohepatitis

Abstract

Nonalcoholic steatohepatitis (NASH) is a disease that progresses from nonalcoholic fatty liver (NAFL) and which is characterized by inflammation and fibrosis. The purinergic P2Y₆ receptor (P2Y₆R) is a pro-inflammatory G_q/G₁₂ family protein-coupled receptor and reportedly contributes to intestinal inflammation and cardiovascular fibrosis, but its role in liver pathogenesis is unknown. Human genomics data analysis revealed that the liver P2Y₆R mRNA expression level is increased during the progression from NAFL to NASH, which positively correlates with inductions of C-C motif chemokine 2 (CCL2) and collagen type I α1 chain (Col1a1) mRNAs. Therefore, we examined the impact of P2Y₆R functional deficiency in mice crossed with a NASH model using a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Feeding CDAHFD for 6 weeks markedly increased P2Y₆R expression level in mouse liver, which was positively correlated with CCL2 mRNA induction. Unexpectedly, the CDAHFD treatment for 6 weeks increased liver weights with severe steatosis in both wild-type (WT) and P2Y₆R knockout (KO) mice, while the disease marker levels such as serum AST and liver CCL2 mRNA in CDAHFD-treated P2Y₆R KO mice were rather aggravated compared with those of CDAHFD-treated WT mice. Thus, P2Y₆R may not contribute to the progression of liver injury, despite increased expression in NASH liver.

Keywords: inflammation; nonalcoholic steatohepatitis; purinergic P2Y6 receptor.

Figure 1

Increase in mRNA expression levels of P2Y₆R, CCL2 and Col1a1 in NASH patients. (A) The expression of CCL2, Col1a1, and P2Y receptors was analyzed in a Gene Expression Omnibus (GEO) dataset (GSE167523) containing the expression profile of the liver from NASH patients. (B) Correlation diagram between the expression levels of P2Y₆R and the expression levels of CCL2 and col1a1. Data are shown as the mean ± SEM. (NAFL; n = 51, NASH; n = 47) Student’s t test (A). Pearson’s product moment correlation coefficient (B).

Figure 2

Effects of P2Y₆R knockout on body status changes induced by CDAHFD. (A) The mRNA expression of P2Y₆R and CCL2 in the livers of C57BL/6J mice fed with standard diet (control) or CDAHFD for 6 weeks (n = 5 in each group). (B) Correlation diagram between the expression levels of P2Y₆R and the expression levels of CCL2 in mouse liver. (C) Immunohistological staining of P2Y₆R in the liver of mice fed with standard diet or CDAHFD for 6 weeks. Scale bar: 40 μm. CDAHFD were fed to WT and P2Y₆R KO mice for 6 weeks. (D) Body weight. (E) Water intake. (F) Urine volume. (G) Food intake. (H) Stool number. Data are shown as the mean ± SEM (n = 5 in each group). Student’s t test (A,F). Pearson’s product moment correlation coefficient (B).

Figure 3

Liver weights and serum levels of AST and ALT in WT and P2Y₆R KO mice fed with CDAHFD. (A) Liver weights. (B,C) Comparison of serum levels of AST (B) and ALT (C) among P2Y₆R KO and WT mice. Data are shown as the mean ± SEM (control WT: n = 3, control P2Y₆R KO: n = 3, CDAHFD WT: n = 5, CDAHFD P2Y₆R KO: n = 5). Two-way ANOVA followed Sidak’s multiple comparison test.

Figure 4

Effects of P2Y₆R knockout on liver steatosis induced by CDAHFD. (A) H&E-stained images of liver sections. Scale bar: 100 μm. (B) Liver steatosis. Data are shown as the mean ± SEM (control WT: n = 3, control P2Y₆R KO: n = 3, CDAHFD WT: n = 5, CDAHFD P2Y₆R KO: n = 5). Two-way ANOVA followed Sidak’s multiple comparison test.

Figure 5

Knockout of P2Y₆R accelerated expression of liver inflammation markers induced by CDAHFD feeding. Expression of CCL2 (A), IL-6 (B), Col1a1 (C), and TGFβ (D) in liver were quantified by real-time qPCR. Data are shown as the mean ± SEM (control WT: n = 3, control P2Y₆R KO: n = 3, CDAHFD WT: n = 5, CDAHFD P2Y₆R KO: n = 5). Two-way ANOVA followed Sidak’s multiple comparison test.