The Hepatoprotective Effect of Leonurine Hydrochloride Against Alcoholic Liver Disease Based on Transcriptomic and Metabolomic Analysis

Abstract

Excessive alcohol consumption can eventually progress to alcoholic liver disease (ALD). The underlying mechanism of ALD toxicity is primarily associated with oxidative damage. Many alkaloids have been reported to possess potential antioxidative efficacy, while the mechanism of their hepatoprotective activity against ALD is still not clear. In this study, eight alkaloids were selected from a monomer library of Traditional Chinese Medicine and evaluated for their antioxidant activity against ALD by the evaluation of Glutathione (GSH) and Malondialdehyde (MDA). The result suggested that Leonurine hydrochloride (LH) was a potent antioxidant that could reduce alcoholic liver damage. To further investigate the underlying mechanism of LH against ALD, the molecular pathway induced by LH was identified by RNA-seq analyses. Transcriptome data revealed the principal mechanism for the protective effect of LH against ALD might be attributed to the differentially expressed genes (DEGs) of PI3K-AKT, AMPK, and HIF-1 signaling pathways involved in the lipid metabolism. Given the hepatoprotective mechanism of LH is involved in lipid metabolism, the lipid metabolism induced by LH was further analyzed by UHPLC-MS/MS. Metabolome analysis indicated that LH significantly regulated glycerophospholipid metabolism including phosphatidylcholine, 1-acyl-sn-glycero-3-phosphocholine, phosphatidylethanolamine and 1-acyl-sn-glycero-3-phosphoethanolamine in the liver. Overall, this study revealed that the hepatoprotective mechanism of LH against alcoholic liver damage might be associated with the genes involved in glycerophospholipid metabolism.

Keywords: alcoholic liver disease; alkaloids; antioxidant; hepatoprotective; leonurine hydrochloride.

FIGURE 1

Chemical structures of different alkaloids.

FIGURE 2

The antioxidative activity of different alkaloids (10 μM) against ethanol-treated LO2 cells. (A) The levels of Glutathione (GSH), (B) the levels of Malondialdehyde (MDA). (C) The levels of GSH in a dose-dependent manner (0–50 μM). (D) The levels of MDA in a dose-dependent manner (0–50 μM). The final result was normalized by protein concentration (mg prot/mL) and the unit of the final result was μmol/g prot. Values are expressed as mean ± SEM (n = 3). * and ** indicated a significant difference at p < 0.05 level, a highly significant difference at p < 0.01 level, respectively, between ET group vs. different alkaloid groups using the independent t-test. (E) Cell viability of LO2 cells after exposure to Leonurine hydrochloride (LH) (0–500 μM) for 24 h. Values are expressed as mean ± SEM (n = 6).

FIGURE 3

Leonurine hydrochloride (LH) affects the genes expression of LO2 cells. (A) Transcriptomic changes of ethanol-treated LO2 cells exposed to LH (10 μM) for 24 h. Cluster heatmap representing differentially expressed genes (DEGs) between the ET group and ET + LH group (n = 2). The scaled expression value of each feature is plotted in red–blue color scale. Positive z-score (red) indicates a predicted up-regulated function and negative z-score (blue) is a down-regulated function. (B) Gene Ontology (GO) enrichment analysis. Different colors represent different GO: Biological processes (BP), Cellular component (CC) and Molecular functions (MF).

FIGURE 4

Bubble plot of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of differentially expressed gene (DEGs) (TOP 15).

FIGURE 5

The genes expression changes of Leonurine hydrochloride (LH) against alcoholic liver disease (ALD). (A) The expression changes of genes determined by Real-time qPCR (RT-qPCR). Ethanol-treated LO2 cells were exposed to LH (10 μM) for 24 h. Then mRNA was extracted, the mRNA expressions were measured by RT-qPCR. * and ** indicated a significant difference at p < 0.05 level, a highly significant difference at p < 0.01 level, respectively, between ET group vs. ET + LH group using the independent t-test. (B) Protein-protein interaction network of LH acting on ALD. Small circles: protein of unknown 3D structure; large circles: some 3D structure is known or predicted.

FIGURE 6

Metabolic analysis of the hepatoprotective effect of Leonurine hydrochloride (LH). (A) Heatmaps of identified metabolites between ET and ET + LH with the description of treatments (ethanol-treated cells and those treated with alcohol and LH). The row Z-score or scaled expression value of each feature is plotted in red–blue color scale. Positive z-score (red) indicates a predicted up-regulated function and negative z-score (blue) is a down-regulated function. (B) Metabolic pathway analysis by MetaboAnalyst 5.0. Pathway impact value based on the pathway topology analysis. The x-axis represents the pathway impact value computed from pathway topological analysis, and the y-axis is the-log of the p-value obtained from pathway enrichment analysis.