GA and α-smooth muscle actin (SMA) antibody were purchased from Sigma-Aldrich (St Louis, MO, United States). MMP2 and MMP9 antibodies were bought from Abcam (Cambridge, MA, United States), CTGF antibody was bought from Santa Cruz Biotechnology (Santa Cruz, CA, United States), caspase-3 and Bax antibodies were purchased from Cell Signaling Technology (Beverly, MA, United States). GAPDH and tubulin antibodies were bought from Beyotime Biotechnology (Haimen, Jiangsu, China), horseradish peroxidase (HRP)-conjugated anti-mouse, anti-goat and anti-rabbit IgG antibodies were purchased from Cell Signaling Technology. The chemiluminescence reaction kit (ECL Plus) was purchased from Millipore (Billerica, MA, United States).

Male Sprague-Dawley rats weighing 150-200 g were supplied by the Experimental Animal Center of Zhongshan Hospital, Fudan University. The animals were acclimatized to laboratory conditions (23 °C, 12 h/12 h light/dark, 50% humidity, ad libitum access to food and water) for 2 wk prior to experimentation. Forty-five rats were randomly and equally divided into the control group, CCl₄ group and GA treatment group. In order to induce the liver fibrosis model, rats were given a 40% solution of CCl₄ in olive oil by hypodermic injection at a dose of 3 mL/kg biweekly for 8 wk, while the rats in the control group were given the same dose of olive oil by subcutaneous injection, with an initial double-dose injection. In the GA group, rats were also treated with a 40% solution of CCl₄ by hypodermic injection at a dose of 3 mL/kg plus 0.2% GA solution in water (3 mL) by intraperitoneal injection three times weekly, beginning at the first week, following a previously published method[17]. Rats in the control group were treated with the same isovolumetric dose of olive oil and water. Animals were sacrificed 24 h after the last injection. All animals were euthanized by barbiturate overdose (intravenous injection, 150 mg/kg pentobarbital sodium) for tissue collection. Liver tissues were removed and rinsed with 0.9% saline, some were fixed in 10% buffered formaldehyde and embedded in paraffin for hematoxylin-eosin (HE) and Sirius-red staining, and the remaining sections were stored at -70 °C for analysis.

Liver tissues were embedded in paraffin and 5-μm-thick slices were cut and placed on glass slides, stained with HE, and examined under a light microscope (Olympus, Tokyo, Japan). HE staining was performed to assess pathological changes in the liver. Sirius-red staining was performed to detect collagen deposition and observed under a light microscope.

The deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (Roche, Germany) was performed following the manufacturer’s protocol. Nuclei were respectively counterstained with 4, 6-diamidino-2-phenylindole (DAPI) in the same sections. Cells stained by TUNEL were evaluated using fluorescence microscopy (Olympus).

Total proteins in liver tissue were extracted and quantified using the bicinchoninic acid protein concentration assay kit (Beyotime Biotechnology). Sample proteins were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% bovine serum albumin for 2 h, and then incubated overnight at 4 °C with primary antibodies rabbit caspase-3, Bcl-2, Bax, MMP2 and MMP9 antibodies, mouse α-SMA, GAPDH and tubulin antibodies, and goat CTGF antibody. On the next day, the membranes were incubated with the secondary antibodies conjugated with horseradish-peroxidase goat anti-rabbit IgG, goat anti-mouse IgG and rabbit anti-goat IgG (1:5000 dilution) at room temperature for 2 h, and then washed three times with Tris-buffered saline with 0.1% Tween-20 (TBST) , and detected by enhanced chemiluminescence. The intensities of the bands were analyzed by Image J software.

Total RNA in liver tissue was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, United States), subsequently converted to cDNA, which was carried out using PrimeScript reagent kit with gDNA Eraser (Takara Bio,. Dalian, China), and subjected to real-time PCR using SYBR Premix Ex TaqII (Takara Bio). The results were calculated in line with the dissociation curves and normalized against a housekeeping gene (GAPDH). This was performed using a 7300HT Fast Real-time polymerase chain reaction (PCR) System. Primer sequences used for real-time PCR were as follows: α-SMA: sense 5’-AGGGAGTGATGGTTGGAATG-3’, antisense 5’-GATGATGCCGTGTTCTATCG’; collagen I: sense 5’-TCAAGATGGTGGCCGTTACT-3’, antisense 5’-GCGGATGTTCTCAATCTGCT-3’; collagen III: sense 5’-ACCTCCTGGTGCTATTGGTC-3’, antisense 5’-TCTCTCCATTGCGTCCATC-3’, GAPDH: sense 5’-GACATGCCGCCTGGAGAAAC-3’, antisense 5’-AGCCCAGGATGCCCTTTAGT-3’. The mRNA expression levels of samples relative to the control groups were analyzed by the comparative CT (2^-∆∆CT) method[18].

All results were expressed as mean ± SD of three independent experiments. Data were analyzed using one-way analysis of variance. Differences were considered statistically significant if P was < 0.05. All analyses in the study were carried out using SPSS for Windows version 18.0.

Under fluorescence microscopy, no staining was observed and non-apoptotic nuclei were found in normal liver tissue slices using the TUNEL assay. High quantities of TUNEL cells were observed in the liver tissue slices from the CCl₄ group, and numerous condensed and fragmented nuclei were seen. In the GA-treated group, few TUNEL cells were observed in the liver tissue sections, and fewer condensed and fragmented nuclei, in the same slice, were observed. In the same view, co-location of green and blue staining indicated TUNEL-positive cells, and numerous TUNEL-positive cells were found in the CCl₄ group, while a significant reduction in TUNEL-positive cells was observed in the GA-treated group (Figure 1). Overall, these results demonstrated that GA treatment reduced apoptosis during the process of liver injury.

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Figure 1 Effects of glycyrrhizic acid on hepatic apoptosis in carbon tetrachloride-induced rat liver fibrosis were detected by TUNEL assay. Liver tissue sections from different treatment groups were stained with TUNEL (green) and the same sections were respectively counterstained with DAPI to localize the nuclei (blue), and the co-location of green and blue indicated TUNEL-positive cells in the same view (magnification × 200).

Expression of apoptosis-related proteins in liver tissue from the different treatment groups was evaluated by western blot analysis. As shown in Figure 2, the expression level of Bax, the pro-apoptotic protein, was markedly increased in the CCl₄-induced hepatic injury group, whereas GA treatment significantly decreased the expression level of Bax (Figure 2). Caspase activation plays a vital role in apoptosis, and cleaved caspase-3 is a typical feature of apoptosis[19]. In the present study, cleaved caspase-3 (17 kDa) was increased in the CCl₄ group, which suggested severe apoptosis. In addition, the expression level of cleaved caspase-3 significantly decreased in the GA treatment group (Figure 2).

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Figure 2 Effects of glycyrrhizic acid on expression of Bax and caspase-3 were evaluated by western blotting. Total proteins were extracted from the livers of rats in the different treatment groups. GAPDH and tubulin were used to confirm the same sample loading in each lane. Data are shown as mean ± SD. ^aP < 0.05 vs CCl₄ group. GA: Glycyrrhizic acid; CCl_4: Carbon tetrachloride.

These findings indicated that GA suppressed CCl₄-induced hepatocyte apoptosis by inhibiting the activation of caspase-3. Thus, GA treatment ameliorated CCl₄-induced hepatocyte apoptosis.

We examined the effects of GA on α-SMA protein and mRNA expression in CCl₄-induced liver injury using western blotting and real-time PCR. As shown in Figure 3A, the expression level of α-SMA protein was maintained at a low level in the control group, and was up-regulated in the CCl₄-induced hepatic injury group. In contrast, GA significantly down-regulated expression of α-SMA protein. The trend in mRNA expression of α-SMA was in accordance with the results of Western blotting (Figure 3B).

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Figure 3 Effects of glycyrrhizic acid on α-SMA expression were estimated by western blotting and real-time polymerase chain reaction. A: Total proteins were extracted from the livers of rats in the different treatment groups. GAPDH was used to confirm the same sample loading in each lane; B: Total RNA was extracted from the livers of rats in the different treatment groups. GAPDH mRNA was included to normalize the expression of mRNA. Data are shown as mean ± SD. ^aP < 0.05 vs the CCl₄ group.

Using Western blotting, we also examined the effects of GA on CTGF, MMP2 and MMP9 protein expression levels in CCl₄-induced liver injury. As indicated in Figure 4, expression of CTGF, MMP2 and MMP9 proteins was up-regulated in CCl₄-induced hepatic injury. In contrast, following GA treatment, expression of CTGF, MMP2 and MMP9 proteins was down-regulated compared to that in the CCl₄ group.

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Figure 4 Effects of glycyrrhizic acid on CTGF, MMP2 and MMP9 expression were examined by Western blotting. Total proteins were extracted from the livers of rats in the different treatment groups. GAPDH was included to confirm the same sample loading in each lane. Data are shown as mean ± SD. ^aP < 0.05 vs the CCl₄ group. GA: Glycyrrhizic acid; CCl_4: Carbon tetrachloride.

We evaluated the expression levels of type I and III collagen mRNA using real-time PCR. In the CCl₄-treated group, expression of type I and III collagen mRNA was enhanced, however, the pattern and degree of change clearly differed between the two transcripts (Figure 5). Type I collagen mRNA expression level increased in parallel with worsening liver disease. In the CCl₄-induced liver injury group, this enhancement was > 30 times higher than that in the control group, and type III collagen mRNA expression was about 6 times higher than that in the control group. Moreover, GA significantly decreased expression of type I and III collagen mRNA, which was about 20 times and about 4 times that in the control group, respectively.

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Figure 5 Effects of glycyrrhizic acid on expression of collagen type I and III mRNA. Total RNA was extracted from the livers of rats in the different treatment groups. GAPDH mRNA was included to normalize the expression of mRNA. Data are shown as mean ± SD. ^aP < 0.05 vs the CCl₄ group. GA: Glycyrrhizic acid; CCl_4: Carbon tetrachloride.

After 8 wk CCl₄ administration, HE and Sirius red staining showed an integrated lobular structure with central venous and hepatic cord radiation in liver slices from the control group (Figure 6), in which there were few positive areas of Sirius red staining around small central venous walls. These disorders of lobular structure, including wide fibrous tissue hyperplasia and fibrous septa formation, were identified in the CCl₄-treated group, and some pseudolobuli were present (Figure 6). Many positive areas of Sirius red staining were seen in the boundaries of the hepatic lobules in the CCl₄-treated group. Less fibrous tissue hyperplasia and fibrous septa formation were observed in the GA treatment group compared with the CCl₄-treated group, and positive areas of Sirius red staining were significantly reduced in the GA treatment group.

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Figure 6 Effects of glycyrrhizic acid on the pathological histology and collagen of livers in carbon tetrachloride-induced rat liver fibrosis were evaluated by hematoxylin-eosin and Sirius red staining. Liver tissue sections from the different treatment groups were stained with hematoxylin-eosin (magnification × 100) and Sirius red (magnification × 40).

Glycyrrhizic acid (GA), a major active component of Glycyrrhiza glabra roots, is commonly used in Asia to treat patients with chronic hepatitis. Liver fibrosis is a common outcome in many chronic liver diseases. Necrosis and apoptosis are involved in the development of liver fibrosis. In a previous study, the authors found that hepatocyte apoptosis could induce liver fibrosis. In this study, the authors investigated the mechanism how GA exerts its antiapoptotic effect against fibrosis in carbon tetrachloride (CCl₄)-induced liver injury, and its contributing factors in this process.

In the present study, the authors found that GA treatment significantly ameliorated CCl₄-induced liver fibrosis by inhibiting hepatocyte apoptosis and hepatic stellate cell (HSC) activation, which may provide potential therapeutic strategies for fibrosis.

This study investigated the mechanism how GA exerts its anti-apoptotic effect against fibrosis in CCl₄-induced liver injury, and its contributing factors in this process. The results of this study demonstrated that GA treatment significantly ameliorated CCl₄-induced liver fibrosis by inhibiting hepatocyte apoptosis and HSC activation.

The results of this study may provide potential therapeutic strategies for fibrosis.

Hepatocyte apoptosis, a cardinal feature of many liver diseases, is considered to play a part in initiating and maintaining HSC activation. Apoptosis of parenchymal cells, an important inflammatory stimulus, activates HSCs, which display a surprising capacity to phagocytize apoptotic bodies, rather than being a silent consequence of liver injury. Thus, factors which affect apoptosis may be used to modulate liver fibrosis.

In this study, the authors investigated how GA exerted its anti-apoptotic effect against fibrosis in CCl₄-induced liver injury and its contributing factors. The study is well designed and presented. The results shown in the manuscript is interesting and GA is a potential therapeutic agent for liver cirrhosis.