Effects of scoparone on non-alcoholic fatty liver disease revealed by RNA sequencing

Abstract

Scoparone (SCO) is known to have curative effect of alleviating liver injury. The purpose of this study was to observe the therapeutic effect and possible mechanism of SCO against high-fat diet (HFD) induced non-alcoholic liver disease (NAFLD) through in vivo experiments and RNA sequencing. Male Kunming mice were fed with HFD for 8 weeks to establish a mouse model of NAFLD, and SCO was used to treat NAFLD. Histopathology and biochemical indicators were used to evaluate the liver injury and the efficacy of SCO. RNA sequencing analysis was performed to elucidate the hepatoprotective mechanism of SCO. Finally, the differentially expressed genes of cholesterol synthesis and fatty acid (triglyceride) synthesis pathways were verified by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The histopathological results showed that HFD could lead to significant steatosis in mice, while SCO could alleviate liver steatosis remarkably in NAFLD mice. The determination of biochemical indicators showed that SCO could inhibit the increased serum transaminase activity and liver lipid level induced by HFD. RNA sequencing analysis of liver tissues found that 2742 and 3663 genes were significantly changed by HFD and SCO, respectively. SCO reversed the most of genes involved in cholesterol synthesis and fatty acid (triglyceride) metabolism induced by HFD. the results of the validation experiment were mostly consistent with the RNA sequencing. SCO alleviated liver injury and steatosis in NAFLD mice, which may be closely related to the regulation of cholesterol and fatty acid (triglyceride) metabolism.

Keywords: cholesterol metabolism; fatty acid (triglyceride) metabolism; non-alcoholic liver disease (NAFLD); ribonucleic Acid (RNA) sequencing; scoparone.

Figure 1

Overview of the NAFLD model procedure.

Figure 2

Pathological observation of main organs in mice. (A–C) Body weight and liver index of mice. (D) liver morphology. (E, F) Histological photomicrographs of liver sections stained with HE or Oil Red O (Scale bar = 100 μm), fat vacuoles and fatty degeneration (black arrow). (G) Histological photomicrographs of brain, heart lung, liver, spleen, kidney sections stained with HE (Scale bar = 100 μm). The HFD group was compared to the Control group; the HFD + SCO (60, 120 mg/kg) groups and HFD + Silymarin group were compared to the HFD group, ^** P < 0.01.

Figure 3

The results of detection of biochemical indicators. (A) Detection of serum liver function indicators of ALT, AST and γ-GT. (B) Detection of hepatic lipid indicators of TG, TC, HDL-C and LDL-C. All data are presented as the mean ± SD (n = 10). The HFD group was compared to the Control group; the HFD + SCO (60, 120 mg/kg) groups and HFD + Silymarin group were compared to the HFD group, ^* P < 0.05, ^** P < 0.01.

Figure 4

The hierarchical cluster analysis and scatter plot of all differentially expressed genes. (A) The Control group vs the HFD group. (B) The HFD group vs the HFD + SCO group.

Figure 5

The differentially expressed genes of cholesterol biosynthesis in liver tissue. (A) Terpenoid backbone biosynthesis genes. (B) Cholesterol biosynthesis genes. ** Fold change ≥ 1.5 and P < 0.01.

Figure 6

The differentially expressed genes of fatty acid metabolism in liver tissue. (A) Fatty acid synthesis genes. (B) Fatty acid (triglyceride) degradation genes. ^*Fold change ≥ 1.5 and P < 0.05. ** Fold change = 1.5 and P < 0.01.

Figure 7

The results of validation experiments detected by qRT-PCR and western blot. (A) Relative mRNA expressions of Hmgcr, Fasn, Acacα and Cpt1a in mice liver tissue. (B) The gene expressions of Prkaa1, Prkaa2 and Srebf1. (C): Relative mRNA expressions of Prkaa1, Prkaa2, Srebf1. (D, E): The protein expressions of p-AMPK, SREBP-1c, Fasn, Acacα and Cpt1a. The relative expressions of protein were normalized against the control group, with GAPDH as the internal reference. All data are presented as the mean ± SD (n = 3). ^* P < 0.05, ^** P < 0.01.

Figure 8

Proposed model depicting the process of cholesterol metabolism and fatty acid (triglyceride) metabolism in NAFLD mouse, and the underlying mechanisms of SCO in improving NAFLD. The Control group vs the HFD group: up-regulated:; down-regulated:. The HFD group vs the HFD + SCO group: up-regulated:; down-regulated:. Dashed arrows indicate that SCO may inhibit or promote the expression of these genes.