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. 2021 Sep 17:9:684857.
doi: 10.3389/fcell.2021.684857. eCollection 2021.

Tunicamycin Induces Hepatic Stellate Cell Apoptosis Through Calpain-2/Ca2 +-Dependent Endoplasmic Reticulum Stress Pathway

Haiying Liu  1 , Linyu Dai  1 , Ming Wang  1 , Fumin Feng  1 , Yonghong Xiao  1
Affiliations

Tunicamycin Induces Hepatic Stellate Cell Apoptosis Through Calpain-2/Ca2 +-Dependent Endoplasmic Reticulum Stress Pathway

Haiying Liu et al. Front Cell Dev Biol. .

Abstract

It has been reported that calpain/caspase-mediated apoptosis induced by endoplasmic reticulum stress (ERS) in hepatic stellate cells (HSCs) by previous studies. At present, the activation of HSC is an important cause of liver fibrosis, and the induction of HSC apoptosis plays an irreplaceable role in reversing liver fibrosis. Therefore, it is of great significance to explore mechanisms of action that can induce HSC apoptosis for the reversal of hepatic fibrosis and the clinical prevention and treatment of hepatic-fibrosis-related diseases such as hepatitis, cirrhosis, and liver cancer. In the current study, we demonstrated that tunicamycin (a novel ERS inducer) can induce the apoptosis of HSCs and increase the concentration of intracellular Ca2+ and the expression of ERS protein GRP78, apoptosis protein caspase-12, and Bax, while it can decrease the antiapoptosis protein expression of Bcl-2. Our findings indicate that tunicamycin can induce HSCs apoptosis through calpain-2/Ca2+-dependent ERS pathway.

Keywords: apoptosis; calpain-2; endoplasmic reticulum stress; hepatic stellate cells; tunicamycin.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Tunicamycin concentration and treatment time screening by MTT. After the HSCs were treated by TM with different concentrations and treatment time, the OD value of each treatment was assayed, and the proliferation rate (PR) of HSCs was calculated. (A) HSCs were treated with various concentrations of TM for 24 h. (B) HSCs were incubated in the presence of 2 μg/ml TM for the indicated time periods.
FIGURE 2
FIGURE 2
The ultrastructural changes of HSC in each group. (A) Control group was cultured in complete medium for 48 h. (B) TGF-β1 group was cultured with TGF-β1 blank medium for 48 h. (C) TGF-β1 + ALLN + TM group was cultured with the same concentration of TGF-β1 blank culture medium for 24 h, then pretreated with ALLN for 30 min and added TM for 24 h. (D) TGF-β1 + TM group was incubated with TGF-β1 blank medium for the same time, then added TM for 24 h; yellow arrows marked the villi, red arrows marked the mitochondria, and blue arrows marked the vacuoles. TM, tunicamycin; ALLN, N-acetyl-leu-leu-norleucinal; HSC, hepatic stellate cell; TGF-β1, transforming growth factor-β1.
FIGURE 3
FIGURE 3
The cell cycle changes in HSC in each group. After treated with TGF-β1, ALLN, and TM, HSC cell cycle of each group was determined by flow cytometric analysis. (A) Figures represent the distribution of different cell cycle phase. (B) Columns represent the percentages of the corresponding cell cycle phase. (*p < 0.05 compared with the blank group; #p < 0.05 compared with the TGF-β1 group; $p < 0.05 compared with the ALLN + TM group).
FIGURE 4
FIGURE 4
Cell apoptosis was observed by AO/PI staining. Cell apoptosis was detected by staining with acridine orange/propidium iodide (AO/PI) after treatment with TGF-β1, ALLN, and TM in HSC. (A) Blank group. (B) TGF-β1 group. (C) ALLN+TM group. (D) TM group. Green fluorescence represented survival, and red or orange represented apoptosis.
FIGURE 5
FIGURE 5
The changes in Ca2+ fluorescence intensity in HSC. The changes in Ca2+ concentration in each group were detected by a laser scanning confocal microscope. (A) Green fluorescence represents the Ca2+ fluorescence intensity in HSC. (B) Columns represent Ca2+ fluorescence intensity in HSC (*p < 0.05 compared with the blank group; #p < 0.05 compared with the TGF-β1 group; $p < 0.05 compared with the ALLN + TM group).
FIGURE 6
FIGURE 6
The protein expression level of calpain-2 and caspase-12 in HSC in different groups. Expressions of calpain-2 and caspase-12 protein were detected by immunocytochemistry. (A) Brown part in the figures represent the protein expression in HSC. (B) Columns represent protein expression level of calpain-2 and caspase-12 in HSC (*p < 0.05 compared with the blank group; #p < 0.05 compared with the TGF-β1 group; $p < 0.05 compared with the ALLN + TM group).
FIGURE 7
FIGURE 7
The protein expression level of GRP78, Bax, and Bcl-2 in different groups. The protein expression level of GRP78, Bax, and Bcl-2 were detected by western blot. GAPDH was used as a loading control. (A,C) The gel electrophoresis figure of GRP78, Bax, and Bcl-2 of different groups in HSC. (B,D) Related proteins gray value analysis (*p < 0.05 compared with the blank group; #p < 0.05 compared with the TGF-β1 group; $p < 0.05 compared with the ALLN + TM group).
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