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. 2023 Jun 17:17:1819-1829.
doi: 10.2147/DDDT.S402864. eCollection 2023.

Luteolin Alleviates Liver Fibrosis in Rat Hepatic Stellate Cell HSC-T6: A Proteomic Analysis

Batudeligen  1 , Zhiqiang Han  1 , Hongmei Chen  1 , Narisu  1 , Yanhua Xu  1 , Anda  1 , Gegentaoli Han  1
Affiliations

Luteolin Alleviates Liver Fibrosis in Rat Hepatic Stellate Cell HSC-T6: A Proteomic Analysis

Batudeligen et al. Drug Des Devel Ther. .

Abstract

Background: Traditional Chinese medicine (TCM) with single or compound materials is an effective cure for liver fibrosis. Hepatic stellate cells (HSCs) play a key role in liver fibrosis pathology and have become a novel drug target for this condition.

Methods: CCK-8 assay was used to determine the cytotoxicity of four components, SYPA, HSYPA, Apigenin, and Luteolin, from Deduhonghua-7 powder on HSC-T6 cells. Transforming Growth Factor β 1 (TGFβ1)-induced fibrotic cell model and CCI4-induced fibrotic rat model were constructed, the expression of fibrosis-related genes, the pathological changes and serum biochemical markers were evaluated. Proteomic analysis was performed to determine the mechanism by which luteolin attenuated liver fibrosis, which were further confirmed by Western blot.

Results: Luteolin attenuates liver fibrosis in HSC-T6 cells and luteolin decreases the liver fibrosis index level in vivo. A total of 5000 differentially expressed proteins (DEPs) were obtained using proteomic analysis. KEGG analysis found that DEPs were concentrated in various metabolic pathways, including DNA replication and repair and lysosomal signaling. GO analysis showed that molecular functions included the activity and binding of various enzymes, related cellular components included the extracellular space, lysosomal lumen, mitochondrial matrix, and nucleus, and biological processes included collagen organization and biosynthesis and the positive regulation of cell migration. Western blot results showed that CCR1, CD59, and NAGA were downregulated in TGFβ1 treatment, while upregulated both in Lut2 and Lut10 treatment. Meanwhile, eight proteins, ITIH3, MKI67, KIF23, DNMT1, P4HA3, CCDC80, APOB, FBLN2, that were upregulated in TGFβ1 treatment, while downregulated both in Lut2 and Lut10 treatment.

Conclusion: Luteolin was shown to have a strong protective effect on liver fibrosis. CCR1, CD59, and NAGA may promote liver fibrosis while ITIH3, MKI67, KIF23, DNMT1, P4HA3, CCDC80, APOB, and FBLN2 may facilitate protection against fibrosis.

Keywords: TGFβ1; deduhonghua-7 powder; liver fibrosis; luteolin; proteomic analysis.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Effect of Luteolin on HSC-T6 cell viability and fibrosis. (A) HSC-T6 cell viability following treatment with various concentrations of (A) Safflower yellow pigment A (SYPA) (B) Hydroxyl safflower yellow pigment A (HSYPA), (C) Apigenin, (D) Luteolin, or (E) TGFβ1 (5 ng/mL) and drugs. (F) α-SMA mRNA expression in HSC-T6 cells. (G) Collagen I mRNA expression in HSC-T6 cells. (H) α-SMA and collagen I protein expression in HSC-T6 cells. ***P < 0.001, by one-tailed Student’s t-test.
Figure 2
Figure 2
Effect of Luteolin treatment on liver fibrosis. (A) Representative images of HE and Masson-stained liver tissue treated with CCl4 and different concentrations of Luteolin (n-4). Bar=200um, 100X. (B–D) Serum ALT, AST, and ALP levels after treatment with CCl4 and different concentrations of Luteolin. “‑”, no treatment; “+”, treatment; “L”, 2uM; “H”, 10 uM. **P <0.01, ***P <0.001.
Figure 3
Figure 3
DEPs analysis. Vaccaro plot of DEPs in the (A) TGFβ1 vs Vehicle group, (B) Lut 2 vs TGFβ1 group, (C) Lut 10 vs TGFβ1 group, and (D) Lut 10 vs Lut 2 group. Bubble diagrams of DEPs in KEGG pathways between (E) the TGFβ1 vs Vehicle group, (F) The Lut 2 vs TGFβ1 group, (G) The Lut 10 vs TGFβ1 group, and (H) The Lut 10 vs Lut 2 group. Vehicle: blank group; TGFβ1: model group; Lut 2: low dose group; Lut 10: high dose group. The Rich factor is displayed on the x-axis, and the p-value is indicated by the color of each circle, the size of which reflects the number of each pathway.
Figure 4
Figure 4
GO analysis of the DEPs in the different comparison pairs. Bubble diagrams of DEPs in GO pathways between (A) The TGFβ1 vs Vehicle group, (B) The Lut 2 vs TGFβ1 group, (C) The Lut 10 vs TGFβ1 group, and (D) The Lut 10 vs Lut 2 group. Vehicle: blank group; TGFβ1: model group; Lut 2: low dose group; Lut 10: high dose group. The Rich factor is displayed on the x-axis, and the p-value is indicated by the color of each circle, the size of which reflects the number of each pathway.
Figure 5
Figure 5
Analysis of proteins expression patterns. (A) Venn diagram analysis of the DEPs. (B) The protein level of CCR1, CD59 and NAGA in the four experimental groups. (C) The protein level of ITIH3, MKI67, KIF23, DNMT1, P4HA3, CCDC80, APOB and FBLN2 in the four experimental groups. Vehicle: blank group; TGFβ1: model group; Lut 2: low dose group; Lut 10: high dose group. *P <0.05, **P <001, ***P <0.001.
Figure 6
Figure 6
Validation of differentially expressed proteins by Western blot. *P <0.05, **P <001, ***P <0.001.
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References

    1. Dong S, Cai FF, Chen QL, et al. Chinese herbal formula Fuzheng Huayu alleviates CCL4-induced liver fibrosis in rats: a transcriptomic and proteomic analysis. Acta Pharmacol Sin. 2018;39(6):930–941. doi:10.1038/aps.2017.150 - DOI - PMC - PubMed
    1. Thompson AI, Conroy KP, Henderson NC. Hepatic stellate cells: central modulators of hepatic carcinogenesis. BMC Gastroenterol. 2015;15:63. doi:10.1186/s12876-015-0291-5 - DOI - PMC - PubMed
    1. Carloni V, Luong TV, Rombouts K. Hepatic stellate cells and extracellular matrix in hepatocellular carcinoma: more complicated than ever. Liver Int. 2014;34(6):834–843. doi:10.1111/liv.12465 - DOI - PubMed
    1. Lee YA, Wallace MC, Friedman SL. Pathobiology of liver fibrosis: a translational success story. Gut. 2015;64(5):830–841. doi:10.1136/gutjnl-2014-306842 - DOI - PMC - PubMed
    1. Semela D, Das A, Langer D, Kang N, Leof E, Shah V. Platelet-derived growth factor signaling through ephrin-b2 regulates hepatic vascular structure and function. Gastroenterology. 2008;135(2):671–679. doi:10.1053/j.gastro.2008.04.010 - DOI - PMC - PubMed