MAPK Signal Transduction Pathway Regulation: A Novel Mechanism of Rat HSC-T6 Cell Apoptosis Induced by FUZHENGHUAYU Tablet

Qi Wang,Ping Gao,Hongbo Du,Yue Li,Jun Cheng,Xiaoli Zhang,Min Li,Shunai Liu

doi:10.1155/2013/368103

Abstract

FUZHENGHUAYU Tablets have been widely used in the treatment of liver fibrosis in China. Here, we investigate the apoptotic effect of FUZHENGHUAYU Tablet in rat liver stellate cell line HSC-T6. HSC-T6 cells were incubated with control serum or drug serum from rats fed with 0.9% NaCl or FUZHENGHUAYU Tablet, respectively. Cells exposed to drug serum showed higher proportions of early and late apoptotic cells than controls. The mRNA levels of collagens I and III, TGF-β1 and α-SMA were reduced by drug serum compared to control serum. Differentially expressed mRNAs and miRNAs were analyzed by microarray and sequencing, respectively. We identified 334 differentially expressed mRNAs and also 60 GOs and two pathways related to the mRNAs. Seventy-five differentially expressed miRNAs were down-regulated by drug serum and 1963 target genes were predicted. 134 GOs up-regulated in drug serum group were linked to miRNA targets, and drug serum also regulated 43 miRNA signal transduction pathways. Protein levels were evaluated by Western blot. Drug serum down-regulated (phospho-SAPK/JNK)/(SAPK/JNK) and up-regulated phospho-p38/p38 ratios. The study showed that FUZHENGHUAYU Tablet induced apoptosis in rat HSC-T6 cells possibly in part by activating p38 and inhibiting SAPK/JNK.

Highlights

Liver fibrosis is a consequence of chronic liver disease characterized by replacement of liver tissue by fibrosis, scar tissue, and regenerative nodules, leading to loss of liver function
In order to analyze whether FUZHENGHUAYU Tablet could induce apoptosis in rat Hepatic stellate cells (HSCs)-T6 cells, we treated rat HSC-T6 cells with control or drug serum, respectively
Cells were stained with Annexin V-Fluorescein isothiocyanate (FITC)/7-AAD and gated into lower right (LR) and upper right (UR) quadrants

Summary

Introduction

Liver fibrosis is a consequence of chronic liver disease characterized by replacement of liver tissue by fibrosis, scar tissue, and regenerative nodules, leading to loss of liver function. There are no reports of the effects of FUZHENGHUAYU on HSC apoptosis, and little is known about the role played by miRNA and mRNA related mechanisms with respect to the effects of FUZHENGHUAYU Tablet. Drug and control serums were supplied by Dr Chenghai Liu, Institute of Liver Disease, Shanghai University of Traditional Chinese Medicine, China. The cells were cultured for up to 24 h for RNA and protein extraction and for 72 h for apoptosis analysis. Actinomycin D (2 μL/mL; Sigma) was added into rat HSC-T6 cells exposed to 10% FBS after 12 h as a positive control for the analysis of apoptosis. Rat HSC-T6 cells treated with drug serum or control serum were washed twice in cold PBS and harvested by exposure to trypsin-EDTA solution.

Materials and Methods

Results

Discussion

Conclusions