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. 2024 Aug 19;25(16):8995.
doi: 10.3390/ijms25168995.

In Vitro Investigation of the Anti-Fibrotic Effects of 1-Phenyl-2-Pentanol, Identified from Moringa oleifera Lam., on Hepatic Stellate Cells

Watunyoo Buakaew  1   2 , Sucheewin Krobthong  3   4 , Yodying Yingchutrakul  4 , Nopawit Khamto  5 , Pornsuda Sutana  2 , Pachuen Potup  2 , Yordhathai Thongsri  2 , Krai Daowtak  2 , Antonio Ferrante  6   7 , Catherine Léon  8 , Kanchana Usuwanthim  2
Affiliations

In Vitro Investigation of the Anti-Fibrotic Effects of 1-Phenyl-2-Pentanol, Identified from Moringa oleifera Lam., on Hepatic Stellate Cells

Watunyoo Buakaew et al. Int J Mol Sci. .

Abstract

Liver fibrosis, characterized by excessive extracellular matrix deposition, is driven by activated hepatic stellate cells (HSCs). Due to the limited availability of anti-fibrotic drugs, the research on therapeutic agents continues. Here we have investigated Moringa oleifera Lam. (MO), known for its various bioactive properties, for anti-fibrotic effects. This study has focused on 1-phenyl-2-pentanol (1-PHE), a compound derived from MO leaves, and its effects on LX-2 human hepatic stellate cell activation. TGF-β1-stimulated LX-2 cells were treated with MO extract or 1-PHE, and the changes in liver fibrosis markers were assessed at both gene and protein levels. Proteomic analysis and molecular docking were employed to identify potential protein _targets and signaling pathways affected by 1-PHE. Treatment with 1-PHE downregulated fibrosis markers, including collagen type I alpha 1 chain (COL1A1), collagen type IV alpha 1 chain (COL4A1), mothers against decapentaplegic homologs 2 and 3 (SMAD2/3), and matrix metalloproteinase-2 (MMP2), and reduced the secretion of matrix metalloproteinase-9 (MMP-9). Proteomic analysis data showed that 1-PHE modulates the Wnt/β-catenin pathway, providing a possible mechanism for its effects. Our results suggest that 1-PHE inhibits the TGF-β1 and Wnt/β-catenin signaling pathways and HSC activation, indicating its potential as an anti-liver-fibrosis agent.

Keywords: 1-phenyl-2-pentanol; Moringa oleifera Lam.; hepatic stellate cell; liver fibrosis; molecular docking analysis; proteomics.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
The dose–response curves of LX-2 cell viability after treatment with varying concentrations of crude Moringa extract or 1-phenyl-2-pentanol.
Figure 2
Figure 2
The expression of liver fibrotic-associated genes and the MMP-9 level in crude MO extract and 1-PHE treated cells. The LX-2 cells at 2 × 105 cells/well were incubated with different concentrations of crude MO extract or 1-PHE in the presence of 10 ng/mL TGF-β1 for 48 h. The cells were harvested and the mRNA expression measured using real-time qRT-PCR. (A) The candidate liver fibrotic-associated genes, as shown above, include COL1A1, TIMP1, MMP2, SMAD2, and SMAD3. The relative gene expression was normalized to GAPDH. (B) The cell culture supernatant was collected and evaluated for the MMP-9 level. The data are presented as mean ± SD. p-value < 0.0332 (*), p-value < 0.0021 (**), p-value < 0.0002 (***), p-value < 0.0001 (****).
Figure 3
Figure 3
The proteomic profiling of DEPs and GO annotation analysis. In total, 1570 DEPs were identified following treatment of LX-2 cells with 1-PHE and TGF-β1 (A). Statistical thresholds (log2 fold change ≤1.5 or ≥1.5; p-value < 0.05) were applied to distinguish significantly upregulated (n = 68; red dots) and downregulated (n = 30; blue dots) proteins (B). Protein–protein interaction (PPI) network analysis demonstrated interconnectedness within the sets of upregulated and downregulated DEPs (C,D). To illuminate the potential functions of these DEPs, Gene Ontology (GO) annotation was employed, categorizing proteins by biological process, cellular component, and molecular function (E,F).
Figure 4
Figure 4
KEGG signaling pathway enrichment analysis of DEPs. KEGG signaling pathway enrichment analysis was performed on DEPs to elucidate the potential impact of 1-PHE on signaling pathways within LX-2 cells. Notably, Parkinson’s disease emerged as the most significantly enriched pathway associated with upregulated DEPs (A). In contrast, the renin secretion signaling pathway demonstrated the strongest enrichment among downregulated proteins (B). Intriguingly, the Wnt signaling pathway, a pathway strongly implicated in liver fibrosis and HSC activation, was also found to be downregulated (C). Downregulated genes were indicated by the color blue, while other genes within the pathway were represented by green. LRP5, LDL-receptor-related protein 5; PRKACA, protein kinase cAMP-activated catalytic subunit alpha.
Figure 5
Figure 5
The 2D and 3D structural characteristics of ligand-protein complexes. Specifically, the interactions between PRKACA and two ligands, 1-PHE and 3SB, were visualized (A,B). Additionally, the interaction between 1-PHE and LRP5 was investigated (C).
Figure 6
Figure 6
Illustration of the results of 250-nanosecond molecular dynamics simulations comparing the interactions of 1-PHE with PRKACA (A–D) and LRP5 (E–H). (A,D) present root mean square deviation (RMSD) plots, (B,F) display root mean square fluctuation (RMSF) plots, (C,G) depict numbers of hydrogen bonds, and (D,H) showcase radius of gyration (Rg) plots.
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