Exosomal miRNAs as Potential Biomarkers to Monitor Phosphodiesterase 5 Inhibitor Induced Anti-Fibrotic Effects on CCl4 Treated Rats

Abstract

MicroRNAs (miRNAs) are short, non-coding RNA species that are important post-transcriptional regulators of gene expression and play an important role in the pathogenesis of non-alcoholic fatty liver disease. Here, we investigated the phosphodiesterase 5 (PDE5) inhibitor induced effects on hepatic and plasma exosomal miRNA expression in CCl₄-treated rats. In the present study, hepatic miRNA profiling was conducted using the Nanostring nCounter technology and mRNA profiling using RNA sequencing from PDE5 treated rats in the model of CCl₄-induced liver fibrosis. To evaluate if the PDE5 inhibitor affected differentially expressed miRNAs in the liver can be detected in plasma exosomes, qRT-PCR specific assays were used. In livers from CCl₄-treated rats, the expression of 22 miRNAs was significantly increased (> 1.5-fold, adj. p < 0.05), whereas the expression of 16 miRNAs was significantly decreased (> 1.5-fold, adj. p < 0.05). The majority of the deregulated miRNA species are implicated in fibrotic and inflammatory processes. The PDE5 inhibitor suppressed the induction of pro-fibrotic miRNAs, such as miR-99b miR-100 and miR-199a-5p, and restored levels of anti-fibrotic miR-122 and miR-192 in the liver. In plasma exosomes, we observed elevated levels of miR-99b, miR-100 and miR-142-3p after treatment with the PDE5-inhibitor compared to CCl₄/Vehicle-treated. Our study demonstrated for the first time that during the development of hepatic fibrosis in the preclinical model of CCl₄-induced liver fibrosis, defined aspects of miRNA regulated liver pathogenesis are influenced by PDE5 treatment. In conclusion, miRNA profiling of plasma exosomes might be used as a biomarker for NASH progression and monitoring of treatment effects.

Keywords: CCl4; PDE 5 inhibitor; exosomes; gene expression; liver fibrosis; microRNAs.

Figure 1

Histology assessment of liver sections from control rats, from rats with CCl₄-induced liver injury and from CCl₄/PDE5 inhibitor treated rats. (A) Representative images of Masson’s trichrome stain (Masson). (B) NAFLD activity score (NAS). (C) Fibrosis scores. Data in (B) and (C) represent scores of individual animals (n = 9–10). (D) Image-based quantification of collagen-positive area in Masson’s trichrome stained liver sections. (E) Hepatic αSMA content. (F) Hepatic HYP content. Data in (D–F) represent mean (n = 5) ± SEM, * p < 0.05; scale bar: 100µm.

Figure 2

Up-regulated (A) and down-regulated (B) hepatic miRNA expression. MiRNA levels of control, CCl₄/Vehicle and CCl₄/PDE5 inhibitor treated rats revealed by Nanostring analysis. Absolute values are displayed for each animal by normalized reporter cell counts (RCC). MiRNAs whose expression is significantly changed in one of the treatment groups are highlighted with boxes. * p < 0.05.

Figure 3

Correlation of fibrosis scores with up-regulated hepatic (A) and down-regulated (B) miRNA expression. MiRNA levels of control, CCl₄/Vehicle and CCl₄/PDE5 inhibitor treated rats were correlated with fibrosis scores. Absolute values are displayed for each animal by normalized reporter cell counts (RCC). Spearman’s correlation coefficient and corresponding p-values are indicated.

Figure 4

Gene set enrichment analysis of miRNA-mRNA pairs according to 37 hallmark pathways. Effects on distinct pathways are represented by colors indicating enrichment scores.

Figure 5

Plasma exosomal miRNA expression. Plasma exosomal levels of miR-99b, miR-100, miR-142-3p, miR-122, and miR-192 in control, CCl₄/Vehicle and CCl₄/PDE5 inhibitor treated rats. Relative plasma exosomal miRNA expression analyzed by qRT-PCR was normalized to the mean expression of control rats. The median is depicted and significant differences between experimental groups are indicated by * (p < 0.05).