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Phillygenin Inhibits TGF-β1-induced Hepatic Stellate Cell Activation and Inflammation: Regulation of the Bax/Bcl-2 and Wnt/β-catenin Pathways

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Abstract

Hepatic fibrosis (HF), a precursor to cirrhosis and hepatocellular carcinoma, is caused by abnormal proliferation of connective tissue and excessive accumulation of extracellular matrix in the liver. Notably, activation of hepatic stellate cells (HSCs) is a key link in the development of HF. Phillygenin (PHI, C21H24O6) is a lignan component extracted from the traditional Chinese medicine Forsythiae Fructus, which has various pharmacological activities such as anti-inflammatory, antioxidant and anti-tumour effects. However, whether PHI can directly inhibit HSC activation and ameliorate the mechanism of action of HF has not been fully elucidated. Therefore, the aim of the present study was to investigate the in vitro anti-HF effects of PHI and the underlying molecular mechanisms. Transforming growth factor-β1 (TGF-β1)-activated mouse HSCs (mHSCs) and human HSCs (LX-2 cells) were used as an in vitro model of HF and treated with different concentrations of PHI for 24 h. Subsequently, cell morphological changes were observed under the microscope, cell viability was analyzed by MTT assay, cell cycle and apoptosis were detected by flow cytometry, and the mechanism of anti-fibrotic effect of PHI was explored by immunofluorescence, ELISA, RT-qPCR and western blot. The results showed that PHI suppressed the proliferation of TGF-β1-activated mHSCs and LX-2 cells, arrested the cell cycle at the G0/G1 phase, decreased the levels of α-SMA, Collagen I, TIMP1 and MMP2 genes and proteins, and promoted apoptosis in activated mHSCs and LX-2 cells. Besides, PHI reduced the expression of inflammatory factors in activated mHSCs and LX-2 cells, suggesting a potential anti-inflammatory effect. Mechanically, PHI inhibited TGF-β1-induced HSC activation and inflammation, at least in part through modulation of the Bax/Bcl-2 and Wnt/β-catenin pathways. Overall, PHI has significant anti-HF effects and may be a promising agent for the treatment of HF.

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Data Availability

The analyzed data sets generated during the present study will be provided by the corresponding author on reasonable request.

Abbreviations

HF:

Hepatic fibrosis

ECM:

Extracellular matrix

HSCs:

Hepatic stellate cells

TGF-β1:

Transforming growth factor-β1

PHI:

Phillygenin

mHSCs:

Mouse hepatic stellate cells

FBS:

Fetal bovine serum

MTT:

3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide

DMSO:

Dimethyl sulfoxide

PI:

Propidium iodide

IL-1β:

Interleukin 1β

IL-6:

Interleukin 6

TNF-α:

Tumor necrosis factor α

BSA:

Bovine serum albumin

GSK-3β:

Glycogen synthase kinase 3β

α-SMA:

α-Smooth muscle actin

TIMP1:

Tissue inhibitors of metalloproteinase 1

Bax:

Bcl-2 associated X

Bcl-2:

B-cell lymphoma 2

ELISA:

Enzyme-linked immunosorbent assay

RT-qPCR:

Real-time quantitative PCR

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Funding

This study was supported by National Natural Science Foundation of China (No: 81891012, 81630101, and U19A2010), Sichuan TCM Science and Technology Industry Innovation Team (No: 2022C001), Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (No: ZYYCXTD-D-202209), Sichuan Province Science and Technology Support Program (No: 2021JDRC0041, 2022ZYD0088).

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Authors and Affiliations

  1. State Key Laboratory of Southwestern Chinese Medicine Resources, Key Laboratory of Standardization for Chinese Herbal Medicine, Ministry of Education, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, Sichuan, China

    Cheng Wang, Shenglin Zhang, Yanzhi Li, Lihong Gong, Chenhao Yao, Ke Fu & Yunxia Li

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  1. Cheng Wang
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  2. Shenglin Zhang
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Contributions

Cheng Wang: conceptualization, methodology, writing-original draft, writing-reviewing and editing. Shenglin Zhang: writing-original draft. Cheng Wang and Yanzhi Li: project administration. Lihong Gong and Chenhao Yao: software, validation. Ke Fu: data curation. Yunxia Li: writing-reviewing and editing, funding acquisition. All authors read and approved the final manuscript.

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Correspondence to Yunxia Li.

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The whole experimental scheme was approved by the Animal Experiment Ethics Committee of Chengdu University of traditional Chinese medicine and carried out under its supervision (Permit number: SYXK 2020-124).

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Wang, C., Zhang, S., Li, Y. et al. Phillygenin Inhibits TGF-β1-induced Hepatic Stellate Cell Activation and Inflammation: Regulation of the Bax/Bcl-2 and Wnt/β-catenin Pathways. Inflammation (2024). https://doi.org/10.1007/s10753-024-01984-w

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