Ca2+ is an important secondary messenger in cells. It is involved in the regulation of various physiological functions, such as cell growth, proliferation, differentiation and apoptosis . In the resting state, the intracellular Ca2+ concentration is maintained at a very low level, and is about 1/10,000 of the extracellular Ca2+ concentration. In response to a suitable stimulus, the intracellular Ca2+ concentration increases.
The calcium ionophore A23187 is the chain ion carrier. External lipophilic complexes of Ca2+ A23187 form at a 1:2 ratio, with two molecules of A23187 end-to-end. The transport of Ca2+ is performed by the carrier complex in the membrane. The effect is different in different cells, which indicates that the signal to the channel is also different. For instance, increased Ca2+ as a secondary messenger can promote protein kinase C and other calcium-dependent protein kinase activation, and catalyze intracellular phosphorylation of various proteins to initiate cell activation and proliferation. A23187 can rapidly increase intracellular Ca2+ concentration in HL-60 cells, disrupting the intracellular Ca2+ steady-state balance to initiate cell apoptosis .
To investigate the effect of A23187 on apoptosis in rat HSCs, the cells were stimulated with TGF-β1. Electron microscopy showed that the cytoplasm had numerous collagen fibers and the nucleus was in the split phase, suggesting that myofibroblasts participated in the development of HF, mainly through their own abnormal proliferation. Furthermore, numerous collagen fibers contain collagen secretion from the extracellular matrix (ECM).
Activated HSCs are the primary source of ECM in HF. They play a key role in its formation and development. TGF-β1 is the most powerful fibrosis promoting factor that can be used to stimulate HSCs to create a model of HF in vitro. TGF-β1 can be used for mechanism research into the regulation of HSC gene expression and the potential of related pathways as therapeutic modalities .
The cells were stimulated with TGF-β1 and then different doses (1, 2 and 4 μM) of A23187 were added. The effect of A23187 on the apoptotic rate was detected using flow cytometry. The apoptosis rate of HSCs was increased with an increase in calcium ionophore concentration. A23187 can form stable complexes with Ca2+ and open the calcium channel on the cell membrane, leading to increase in intracellular calcium concentration. In this study, Fluo-3/AM-loaded cells exposed to low, middle and high doses of A23187 were used to observe the intracellular Ca2+ steady-state imbalance. Ca2+ elevation could activate the ERS pathway, which could be the cause of apoptosis.
Our study shows that in these HSCs, the intracellular Ca2+ homeostasis was mainly maintained through the endoplasmic reticulum (ER). Various factors can cause changes in Ca2+ channels on the ER, resulting in Ca2+ deprivation or Ca2+ overload and activation of the ERS pathway.
GRP78 is an ERS chaperone that plays an important role in maintaining ER protein synthesis, proper protein folding and intracellular calcium homeostasis. It is also an important marker of ERS [11, 12]. GRP78 plays a key regulatory role in ERS and is an important defense mechanism for cells. In this study, the expression of GRP78 protein was detected via immunoblotting. Different doses of A23187 led to high expression of GRP78 protein, suggesting that intracellular ERS was activated.
Ca2+ initiates an early step in apoptosis, which is closely linked to the ER. ERS has a unique caspase-12 pathway. Caspase-12 belongs to the caspase family and is considered to be a specific apoptotic signal in the ERS apoptotic pathway. It is activated by ERS and can mediate mitochondrial-independent apoptosis . Under normal conditions, caspase-12 is located on the ER in the form of inactive zymogens. When ERS occurs, IRE1α activates TRAF2 to activate caspase-12 and caspase-9, causing caspase-mediated apoptosis .
Xie et al.  induced ER stress in cells of the human hepatocarcinoma cell line Huh-7 with toxic carotene (TG). Western blotting was used to detect procaspase-12, which was found to be significantly associated with TG and apoptosis. Bitko et al.  found that apoptosis of A549 human lung epithelial cells caused by respiratory syncytial virus was associated with caspase-12 activation and ERS. Here, we found that low, middle and high doses of A23187 significantly upregulated the expression of intracellular caspase-12 and caspase-9.