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Tetramethylpyrazine induces G0/G1 cell cycle arrest and stimulates mitochondrial-mediated and caspase-dependent apoptosis through modulating ERK/p53 signaling in hepatic stellate cells in vitro

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Abstract

Activation of hepatic stellate cells (HSCs) is a pivotal event in the pathogenesis of liver fibrosis. Pharmacological induction of HSC apoptosis could be a promising strategy for fibrosis regression. Natural product tetramethylpyrazine (TMP) exhibits potent antifibrotic activities in vivo. However, the molecular mechanisms remain to be defined. The present study aimed at investigating the anti-proliferative and pro-apoptotic effects of TMP on HSCs and elucidating the underlying mechanisms. Our results demonstrated that TMP had no apparent cytotoxic effects on hepatocytes, but significantly inhibited HSC proliferation and induced cell cycle arrest at the G0/G1 checkpoint. These effects were associated with TMP regulation of cyclin D1, p21, p27 and p53. Furthermore, we found that TMP disrupted mitochondrial functions and led to activation of caspase cascades in HSCs. Mechanistic investigations revealed that TMP selectively blocked the extracellular signal-regulated kinase (ERK) signaling and activated p53, which was required for TMP induction of caspase-dependent mitochondrial apoptosis in HSCs. Autodock simulations predicted that TMP could directly bind to ERK2 with two hydrogen bonds and low energy score, indicating that ERK2 could be a direct target molecule for TMP within HSCs. Moreover, TMP altered expression of some marker proteins relevant to HSC activation. These data collectively revealed that TMP modulation of ERK/p53 signaling led to mitochondrial-mediated and caspase-dependent apoptosis in HSCs in vitro. These studies provided mechanistic insights into the antifibrotic properties of TMP that may be exploited as a potential option for hepatic fibrosis.

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Abbreviations

α-SMA:

Alpha smooth muscle actin

ALT:

Alanine aminotransferase

APAP:

N-acetyl-p-aminophenol

AST:

Aspartate aminotransferase

CDKs:

Cyclin-dependent kinases

CKIs:

CDK inhibitors

CPM:

Counts per minute

Cyt c:

Cytochrome c

DMEM:

Dulbecco’s modified eagle medium

DMSO:

Dimethylsulfoxide

ECM:

Extracellular matrix

EGF:

Epidermal growth factor

ERK:

Extracellular signal-regulated kinase

FBS:

Fetal bovine serum

HSC:

Hepatic stellate cell

LDH:

Lactate dehydrogenase

MAPK:

Mitogen-activated protein kinase

MTP:

Mitochondrial transmembrane potential

MTS:

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium

NF-κB:

Nuclear factor-kappa B

PARP-1:

Poly (ADPribose) polymerase-1

PDGF-βR:

Platelet-derived growth factor-β receptor

PI:

Propidium iodide

PI3K:

Phosphoinositide 3-kinase

PPARγ:

Peroxisome proliferator-activated receptor-γ

TdR:

Thymin deoxyriboside

TGF-β:

Transforming growth factor-β

TMP:

Tetramethylpyrazine

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Acknowledgments

The financial support was from National Natural Science Foundation of China (81270514, 30873424), Doctoral Discipline Foundation of Ministry of Education of China (20103237110010), Jiangsu Natural Science Foundation (BK2008456), Project for Supporting Jiangsu Provincial Talents in Six Fields (2009-B-010), Open Program of Jiangsu Key Laboratory of Integrated Acupuncture and Drugs (KJA200801), Open Project Program of National First-Class Key Discipline for Traditional Chinese Medicine of Nanjing University of Chinese Medicine (2011ZYX4-008), and the ‘‘Eleven-Five’’ National Science and Technology Supporting Program (2008BAI51B02). We are grateful to Dr. Lei Zhang (Anhui Provincial Hospital, China) and Dr. Fangtian Fan (Nanjing University of Chinese Medicine, China) for assistance in experiments, and to Professor Shile Huang (Louisiana State University Health Sciences Center, USA) for insightful discussion and suggestions.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Clinical Pharmacy, College of Pharmacy, Nanjing University of Chinese Medicine, 282 Hanzhong Road, Nanjing, 210029, Jiangsu, China

    Feng Zhang, De-Song Kong, Zi-Li Zhang, Na Lei, Xiao-Jing Zhu, Xiao-Ping Zhang, Li Chen, Yin Lu & Shi-Zhong Zheng

  2. Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, 210046, China

    Yin Lu & Shi-Zhong Zheng

  3. National First-Class Key Discipline for Traditional Chinese Medicine of Nanjing University of Chinese Medicine, Nanjing, 210046, China

    Yin Lu & Shi-Zhong Zheng

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  1. Feng Zhang
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Correspondence to Shi-Zhong Zheng.

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Zhang, F., Kong, DS., Zhang, ZL. et al. Tetramethylpyrazine induces G0/G1 cell cycle arrest and stimulates mitochondrial-mediated and caspase-dependent apoptosis through modulating ERK/p53 signaling in hepatic stellate cells in vitro. Apoptosis 18, 135–149 (2013). https://doi.org/10.1007/s10495-012-0791-5

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