Anti-Fibrotic Effects of DL-Glyceraldehyde in Hepatic Stellate Cells via Activation of ERK-JNK-Caspase-3 Signaling Axis

Abstract

During liver injury, hepatic stellate cells can differentiate into myofibroblast-like structures, which are more susceptible to proliferation, migration, and extracellular matrix generation, leading to liver fibrosis. Anaerobic glycolysis is associated with activated stellate cells and glyceraldehyde (GA) is an inhibitor of glucose metabolism. Therefore, this study aimed to investigate the anti-fibrotic effects of GA in human stellate LX-2 cells. In this study, we used cell viability, morphological analysis, fluorescence-activated cell sorting (FACS), western blotting, and qRT-PCR techniques to elucidate the molecular mechanism underlying the anti-fibrotic effects of GA in LX-2 cells. The results showed that GA significantly reduced cell density and inhibited cell proliferation and lactate levels in LX-2 cells but not in Hep-G2 cells. We found that GA prominently increased the activation of caspase-3/9 for apoptosis induction, and a pan-caspase inhibitor, Z-VAD-fmk, attenuated the cell death and apoptosis effects of GA, suggesting caspase-dependent cell death. Moreover, GA strongly elevated reactive oxygen species (ROS) production and notably increased the phosphorylation of ERK and JNK. Interestingly, it dramatically reduced α-SMA and collagen type I protein and mRNA expression levels in LX-2 cells. Thus, inhibition of ERK and JNK activation significantly rescued GA-induced cell growth suppression and apoptosis in LX-2 cells. Collectively, the current study provides important information demonstrating the anti-fibrotic effects of GA, a glycolytic metabolite, and demonstrates the therapeutic potency of metabolic factors in liver fibrosis.

Keywords: Apoptosis; Collagen type-I; DL-glyceraldehyde; Hepatic stellate cells; MAPKs; α-SMA.

Fig. 1

Inhibitory effects of GA on Hep-G2 and LX-2 cell viability. Cells were seeded and treated with different concentrations of GA (0.25, 0.5, and 0.75 mM) for 24 h. Cell viability was measured using an MTT assay. (A, C) Morphological analysis of Hep-G2 and percent of cell viability. (B, D) Morphological analysis of LX-2 and percent of cell viability. (E, F) Cells were treated with different concentrations of GA for 24 h and examined the effects on cell proliferation and L-lactate production of LX-2 cells. **p<0.01, ***p<0.001 vs. control.

Fig. 2

GA induced apoptosis in LX-2 cells. Cells were seeded and treated with different concentrations of GA (0.25, 0.5, and 0.75 mM) for 24 h. (A) Apoptosis was detected using Annexin V-staining kit (PI) by flow cytometry. (a) control group (b) 0.25 mM of GA (c) 0.5 mM of GA (d) 0.75 mM of GA. (B) Quantitative analysis of apoptotic cells. ***p<0.001 vs. control. (C) Cells were seeded and treated with different concentrations of GA (0.25, 0.5, and 0.75 mM) for 24 h, and cells were lysed and analyzed the pro and cleaved protein expression of caspase-3, caspase-9, and PARP by western blotting. (D) Cells were seeded and treated with GA at 0.75 mM in duplicate for 24 h, and cells were lysed and analyzed the pro and cleaved protein expression of caspase-3, caspase-9, and PARP by western blotting. Effects of Z-VAD-fmk on the GA induce grow inhibition and apoptosis in LX-2 cells. Cells were pretreated with Z-VAD-fmk for 1 h and then exposed to GA at 0.75 mM for a further 23 h. (E) Cell viability was measured by MTT assay. (F) Cells harvested from control, 0.75 mM of GA, 20 mM of Z-VAD, and Z-VAD+GA groups were lysed and analyzed the pro and cleaved protein ex-pression PARP by western blotting. (G) Densitometry graph of (F). **p<0.01 vs. control, ^#p<0.05 vs. only GA treatment.

Fig. 3

GA induced ROS production and MAPK activation in LX-2 cells. Cells were seeded and treated with different concentrations of GA (0.25, 0.5, and 0.75 mM) for 24 h. (A) ROS production was analyzed by flow cytometry. (a) M1=control, M2=GA 0.25 mM (b) M1=control, M2=GA 0.5 mM (c) M1=control, M2=GA 0.75 mM. (B) Quantitative analysis of representative cytograms of (A) (DCF-DA staining. (C) Cells were seeded and treated with or without different concentrations of GA (0.25, 0.5, and 0.75 mM) for 1 h. Cell lysates were prepared, and the phosphorylated and total protein expression levels of MAPK (ERK, JNK, and P-38) were analyzed via western blotting. (D) Cells were seeded and treated with or without GA at 0.75 mM in duplicate for 1 h. Cell lysates were prepared, and the phosphorylated and total protein expression levels of MAPK (ERK, JNK, and P-38) were analyzed via western blotting. **p<0.001 vs. control.

Fig. 4

GA at 0.75 mM strongly suppressed fibrotic markers. (A) Cells were seeded and treated with different concentrations of GA (0.25, 0.5, and 0.75 mM) for 24 h, and cells were lysed and analyzed the protein expression of α-SMA and colla-1 by western blotting. (B, C) Densitometry graph of (A). (D, E) Cells were seeded and treated with different concentrations of GA (0.25, 0.5, and 0.75 mM) for 24 h, and the total RNA was extracted from cells and analyzed by qRT-PCR for the measurement of mRNA levels of α-SMA and colla-1. (F, G) Cells were seeded and pretreated with or without GA 0.75 mM for 1 h before Tgf-β1 (10 ng/mL) treatment for an additional 1 h. Cells were lysed and analyzed the protein expression of Smad2/3 and housekeeping protein α-Tubulin by western blotting. *p<0.05 vs. control, **p<0.01 vs. control, ***p<0.001 vs. control, ^#p<0.05 vs. Tgf-β1 treatment.

Fig. 5

Involvement of MAPK in GA induced apoptosis in LX-2 cells. Cells were pretreated with ERK inhibitor (U0126), p-38 inhibitor (SB203580), and JNK inhibitor (SP600125) for 2 h and then exposed to GA at 0.75 mM for a further 22 h. (A) Cell viability was measured by MTT assay. (B) Morphological analysis of control, 0.75 mM of GA, U016+GA, and SP600125+GA groups were done under phase electron microscopy. (C) Whole-cell lysates harvested from control, 0.75 mM of GA, U016+GA, SB203580+GA, and SP600125+GA groups were prepared and analyzed for the pro and cleaved protein expression of caspase-3, and PARP by western blotting. (D) Densitometry graph of (C). **p<0.001 vs. control, ***p<0.0001 vs. control, ^#p<0.01, ^##p<0.001 vs. only GA treatment.