Suaeda glauca Attenuates Liver Fibrosis in Mice by Inhibiting TGFβ1-Smad2/3 Signaling in Hepatic Stellate Cells

Abstract

Chronic liver injury due to various hepatotoxic stimuli commonly leads to fibrosis, which is a crucial factor contributing to liver disease-related mortality. Despite the potential benefits of Suaeda glauca (S. glauca) as a natural product, its biological and therapeutic effects are barely known. This study investigated the effects of S. glauca extract (SGE), obtained from a smart farming system utilizing LED lamps, on the activation of hepatic stellate cells (HSCs) and the development of liver fibrosis. C57BL/6 mice received oral administration of either vehicle or SGE (30 or 100 mg/kg) during CCl₄ treatment for 6 weeks. The supplementation of SGE significantly reduced liver fibrosis induced by CCl₄ in mice as evidenced by histological changes and a decrease in collagen accumulation. SGE treatment also led to a reduction in markers of HSC activation and inflammation as well as an improvement in blood biochemical parameters. Furthermore, SGE administration diminished fibrotic responses following acute liver injury. Mechanistically, SGE treatment prevented HSC activation and inhibited the phosphorylation and nuclear translocation of Smad2/3, which are induced by transforming growth factor (TGF)-β1 in HSCs. Our findings indicate that SGE exhibits anti-fibrotic effects by inhibiting TGFβ1-Smad2/3 signaling in HSCs.

Keywords: Suaeda glauca extract; TGFβ1; hepatic stellate cells; liver fibrosis; smart farming system.

Figure 1

The effect of SGE on CCl₄-induced liver fibrosis in mice. (A) A schematic diagram depicting the experimental setup for investigating the anti-fibrotic effect of SGE. (B) Sirius Red staining and H&E staining in the liver of mice (left) (scale bar: 20 μm). Mice were subjected to CCl₄ treatment in the presence or absence of SGE, as shown in panel A. Arrows and arrowheads mark hepatocyte damage and macrophage infiltration, respectively. The area of Sirius Red positive staining was quantified (right) (n = 3 for each group). (C) ALT and AST activities were measured in the serum of mice (n = 6–8 for each group). (D) Immunoblotting for α-SMA in the liver of mice (left). The relative protein levels of α-SMA were quantified and normalized to those of GAPDH (right) (n = 3 for each group). The data are presented as mean ± SEM. The statistical significance of the differences between the groups was determined as follows: * p < 0.05 or ** p < 0.01 compared with the vehicle group and # p < 0.05 or ## p < 0.01 compared with the CCl₄ alone group.

Figure 2

The effect of SGE on fibrogenic and inflammatory responses upon acute liver injury. (A) A schematic diagram depicting the experimental setup to evaluate the impact of SGE on CCl₄-induced acute liver damage. (B) ALT and AST activities were measured in the serum of mice (n = 4–6 for each group). (C) H&E staining in the liver of mice (scale bar: 20 μm). Arrows and arrowheads mark hepatocyte damage and macrophage infiltration, respectively. (D) qRT-PCR assay for hepatic fibrogenic and inflammatory genes. Mice were treated according to the experimental protocol illustrated in panel A (n = 4–6 for each group). The data are presented as mean ± SEM. The statistical significance of the differences between the groups was determined as follows: ** p < 0.01 compared with the vehicle group and # p < 0.05 or ## p < 0.01 compared with the CCl₄ alone group. N.S., not significant.

Figure 3

The effect of SGE on the fibrogenesis of HSCs. (A) Schematic diagram illustrating the experimental design for monitoring the effect of SGE on HSC activation. (B) Immunoblotting for α-SMA in mouse primary HSCs (left). Activated HSCs (5 days) were treated with SGE during the culture activation period, as depicted in panel A. The relative protein levels of α-SMA were quantified and normalized to those of β-Actin (right) (n = 3). (C) Immunofluorescence staining for α-SMA in activated HSCs (5 days) (scale bar: 20 μm). (D) qRT-PCR assay for fibrogenic genes in activated HSCs (5 days). Like panel A, the cells were treated with SGE (300 μg/mL). (E) MTT assay for cell viability in HSCs. Both LX-2 cells and mouse primary HSCs were treated with various concentrations of SGE as indicated for 24 h. The data are presented as mean ± SEM. The statistical significance of the differences between the groups was determined as follows: * p < 0.05 or ** p < 0.01 compared with the control group. N.S., not significant.

Figure 4

The effects of SGE on the migration and proliferation of HSCs. (A) Migration assay in mouse primary HSCs. The cells were treated with SGE (100 or 300 μg/mL) for 72 h. The relative wound closure was quantified (n = 3). (B) Immunoblotting for PCNA in LX-2 cells (left). The cells were treated with SGE for 24 h. The relative protein levels of PCNA were quantified and normalized to those of α-Tubulin (right) (n = 3). The data are presented as mean ± SEM. The statistical significance of the differences between the groups was determined as follows: ** p < 0.01 compared with the control group. N.S., not significant.

Figure 5

The effect of SGE on TGFβ1-Smad2/3 signaling in HSCs. (A) Immunoblotting for p-Smad2/3. LX-2 cells were pre-treated with 300 μg/mL of SGE for 30 min and then exposed to TGFβ1 (5 ng/mL) for 15 min. The relative protein levels of p-Smad2 and p-Smad3 were quantified and normalized to those of β-Actin (n = 3). (B) Immunofluorescence staining for p-Smad2 and p-Smad3 in LX-2 cells (scale bar: 20 μm). (C) Fluorescence intensities of p-Smad2 and p-Smad3 in panel B were quantified (n = 3). (D) SBE luciferase assay in LX-2 cells. The cells were transfected with the reporter vector and then treated with SGE (300 μg/mL) and TGFβ1 (5 ng/mL, 24 h). (E) qRT-PCR assay for fibrogenic genes in LX-2 cells. Like panel D, the cells were similarly treated with SGE (300 μg/mL, 30 min) and TGFβ1 (5 ng/mL, 12 h). The data are presented as mean ± SEM. The statistical significance of the differences between the groups was determined as follows: ** p < 0.01 compared with the control group and ## p < 0.01 compared with the TGFβ1 alone group.