A novel role of RNase L in the development of nonalcoholic steatohepatitis

Abstract

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and affects about 25% of the population globally. NAFLD has the potential to cause significant liver damage in many patients because it can progress to nonalcoholic steatohepatitis (NASH) and cirrhosis, which substantially increases disease morbidity and mortality. Despite the key role of innate immunity in the disease progression, the underlying molecular and pathogenic mechanisms remain to be elucidated. RNase L is a key enzyme in interferon action against viral infection and displays pleiotropic biological functions such as control of cell proliferation, apoptosis, and autophagy. Recent studies have demonstrated that RNase L is involved in innate immunity. In this study, we revealed that RNase L contributed to the development of NAFLD, which further progressed to NASH in a time-dependent fashion after RNase L wild-type (WT) and knockout mice were fed with a high-fat and high-cholesterol diet. RNase L WT mice showed significantly more severe NASH, evidenced by widespread macro-vesicular steatosis, hepatocyte ballooning degeneration, inflammation, and fibrosis, although physiological and biochemical data indicated that both types of mice developed obesity, hyperglycemia, hypercholesterolemia, dysfunction of the liver, and systemic inflammation at different extents. Further investigation demonstrated that RNase L was responsible for the expression of some key genes in lipid metabolism, inflammation, and fibrosis signaling. Taken together, our results suggest that a novel therapeutic intervention for NAFLD may be developed based on regulating the expression and activity of RNase L.

Keywords: HFHCD; NAFLD; NASH; RNase L; mouse.

Figure 1.. H&E staining of liver tissues from HFHCD-fed mice

Age-matched adult male RNase L WT and KO mice (n=8/group) were fed with HFHCD containing 2% cholesterol for either 12 or 22 weeks. Histological changes in the liver structures after fed with HFHCD are shown. Representative images of H&E stained sections of liver tissues are present at 10× and 20× magnifications. Black arrows indicate the infiltrated immune cells (blue).

Figure 2.. Infiltration of immune cells in the livers of mice fed with HFHCD

Immune cells were infiltrated into the livers of RNase L WT and KO mice fed with HFHCD containing 2% cholesterol for either 12 or 22 weeks. The cell types were identified by immunohistostaining with (A) F4/80, (B) CD4, and (C) CD8. Representative images are present at 10× and 20× magnifications. Black arrows indicate the infiltrated immune cells. (D) Quantification of positive cells per field (10X objective). Values are present as means ± SEM (****p < 0.0001).

Figure 2.. Infiltration of immune cells in the livers of mice fed with HFHCD

Immune cells were infiltrated into the livers of RNase L WT and KO mice fed with HFHCD containing 2% cholesterol for either 12 or 22 weeks. The cell types were identified by immunohistostaining with (A) F4/80, (B) CD4, and (C) CD8. Representative images are present at 10× and 20× magnifications. Black arrows indicate the infiltrated immune cells. (D) Quantification of positive cells per field (10X objective). Values are present as means ± SEM (****p < 0.0001).

Figure 2.. Infiltration of immune cells in the livers of mice fed with HFHCD

Immune cells were infiltrated into the livers of RNase L WT and KO mice fed with HFHCD containing 2% cholesterol for either 12 or 22 weeks. The cell types were identified by immunohistostaining with (A) F4/80, (B) CD4, and (C) CD8. Representative images are present at 10× and 20× magnifications. Black arrows indicate the infiltrated immune cells. (D) Quantification of positive cells per field (10X objective). Values are present as means ± SEM (****p < 0.0001).

Figure 2.. Infiltration of immune cells in the livers of mice fed with HFHCD

Immune cells were infiltrated into the livers of RNase L WT and KO mice fed with HFHCD containing 2% cholesterol for either 12 or 22 weeks. The cell types were identified by immunohistostaining with (A) F4/80, (B) CD4, and (C) CD8. Representative images are present at 10× and 20× magnifications. Black arrows indicate the infiltrated immune cells. (D) Quantification of positive cells per field (10X objective). Values are present as means ± SEM (****p < 0.0001).

Figure 3.. Trichrome staining of liver tissues from mice fed with HFHCD

Age-matched adult male RNase L WT and KO mice (n=8/group) were fed with HFHCD containing 2% cholesterol for either 12 or 22 weeks. The fibrotic status in the liver tissues after fed with HFHCD is shown (blue). Representative images of Trichrome staining processed sections of liver tissues are present at 10× and 20× magnifications.

Figure 4.. Bodyweight and liver/body weight ratio of mice fed with HFHCD

RNase L WT and KO mice (n= 8/group) were fed with HFHCD for either 12 (A-C) or 22 (D-F) weeks, and the mice were weighed every week; values are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5.. Liver biochemistry parameters of mice fed with HFHCD

RNase L WT and KO mice (n=6/group) were fed with HFHCD for either 12 or 22 weeks. Plasma biochemistry parameters for the liver function were analyzed by a veterinary chemistry analyzer; values are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 6.. Levels of certain cytokines and chemokines in the plasma of mice fed with HFHCD

RNase L WT and KO mice (n=7-8/group) were fed with HFHCD for either 12 or 22 weeks. Cytokines in plasma were analyzed by ELISA; values are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 7.. Expression of key genes involved in cholesterol metabolism, inflammation, and fibrosis pathways in the livers of mice fed with HFHCD.

Tissue extracts (100 mg protein/ each sample) prepared from the livers of RNase L WT and KO mice fed with HFHCD for either 12 or 22 weeks were analyzed by Western blot with antibodies against HMGCR, NLRP3, Caspase-1, Notch 2, α-SMA and COL1A1 (Cell Signaling); MMP9 (Abcam). GAPDH (Santa Cruz) was used for protein normalization. The same control was used for MMP9/α-SMA or NLRP3/Caspase-1in each of the experiments.