Chinese Herbal Medicine Formula Shenling Baizhu San Ameliorates High-Fat Diet-Induced NAFLD in Rats by Modulating Hepatic MicroRNA Expression Profiles

Abstract

Objective: The purpose of present study was to investigate the potential mechanism underlying the protective effect of Shenling Baizhu San (SLBZS) on nonalcoholic fatty liver disease (NAFLD) by microRNA (miRNA) sequencing.

Methods: Thirty male Wistar rats were randomly divided into a normal control (NC) group, a high-fat diet (HFD) group, and an SLBZS group. After 12 weeks, the biochemical parameters and liver histologies of the rats were assessed. The Illumina HiSeq 2500 sequencing platform was used to analyse the hepatic miRNA expression profiles. Representative differentially expressed miRNAs were further validated by qRT-PCR. The functions of the differentially expressed miRNAs were analysed by bioinformatics.

Results: Our results identified 102 miRNAs that were differentially expressed in the HFD group compared with the NC group. Among those differentially expressed miRNAs, the expression levels of 28 miRNAs were reversed by SLBZS administration, suggesting the modulation effect of SLBZS on hepatic miRNA expression profiles. The qRT-PCR results confirmed that the expression levels of miR-155-5p, miR-146b-5p, miR-132-3p, and miR-34a-5p were consistent with those detected by sequencing. Bioinformatics analyses indicated that the target genes of the differentially expressed miRNAs reversed by SLBZS were mainly related to metabolic pathways.

Conclusion: This study provides novel insights into the mechanism of SLBZS in protecting against NAFLD; this mechanism may be partly related to the modulation of hepatic miRNA expression and their target pathways.

Figure 1

Effects of SLBZS on (a) body weight, (b) nasoanal length, (c) Lee index, (d) liver weight, (e) liver index, and (f) energy intake in HFD-fed rats. The values are presented as the mean ± standard deviations. Differences were assessed by ANOVA. ^∗∗P < 0.01 vs the HFD group.

Figure 2

SLBZS improved the biochemical parameters: (a) ALT levels; (b) AST levels; (c) TC levels; (d) TG levels (e) HDL-C levels; (f) LDL-C levels; (g) FFA levels. Values are presented as the mean ± standard deviations. Differences were assessed by ANOVA. ^∗P < 0.05 and ^∗∗P < 0.01 vs the HFD group.

Figure 3

SLBZS attenuated hepatic steatosis: (a) representative H&E and oil red O images (magnification, 200×); (b) liver TC levels; (c) liver TG levels. Values are presented as the mean ± standard deviations. Differences were assessed by ANOVA. ^∗∗P < 0.01 vs the HFD group.

Figure 4

Cluster analysis of miRNAs expressed in HFD vs NC (a) and SLBZS vs HFD (b) and Venn diagram (c) of the differentially expressed miRNAs. In differentially expressed miRNA scatter plots, red indicates upregulated miRNAs, green indicates downregulated miRNAs, and grey indicates no significant changes in miRNA expression. The threshold set for significantly differential genes was |log 2(fold change)| ≥ 1 and P < 0.05.

Figure 5

Heatmap and hierarchical clustering of 28 differentially expressed miRNAs in HFD-fed rats with SLBZS administration. Each row represents a miRNA, and each column represents a sample. The colour scale shown at the top illustrates the relative expression level of miRNAs; red represents a high relative expression level, and blue represents a low relative expression level.

Figure 6

Relative expression of four selected miRNAs was quantified by qRT-PCR and normalized by U6 expression. Significance was assessed by one-way ANOVA followed by Tukey's multiple comparison tests. Data are presented as the mean ± SDs. ^∗P < 0.05 and ^∗∗P < 0.01 vs the HFD group.

Figure 7

Venn diagram of the target gene counts from three different databases, taking the intersection of them finally resulted in 2802 target genes for further study.

Figure 8

GO analysis for predicted targets of differentially expressed miRNAs (top 10 of fold enrichment).

Figure 9

Pathway enrichment analysis of the target genes of differentially expressed miRNAs. The x-axis indicates the proportion of the enriched differential gene in the background gene of the pathway; the y-axis indicates the pathway name; point sizes indicate the number of differentially enriched genes; dot colours indicate the size of the P value (genes number ≥18 and P value <0.01).

Figure 10

A network of the predicted target genes of differentially expressed miRNAs.