Green tea, cultivated from Cheju island, Korea, was extracted with 80% methanol and freeze-dried.

Cell culture: HSC-T6 cells, an immortalized rat HSC line, were cultured in Dulbecco’s minimal essential medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Gibco) and 0.5% antibiotics. Cultures were placed in a humidified atmosphere of 5% CO₂ at 37°C, and the medium was changed twice a week. Acetaldehyde (175 μmol/L) was added to induce collagen type 1 and morphological features of activated stellate cell.

Cell viability: HSC-T6 cells were seeded into 96-well plates at a density of 1.5 × 10⁴ cells/well until 50% confluence. Cells treated with GT (10, 50, 100 μg/mL) for 48 h were incubated with MTT (1 mg/mL) in a medium for 3 h at 37°C. The supernatant was removed and 100 μL of DMSO was added to each well to dissolve the formazan product. Absorbance at 570 nm was measured using a microplate reader.

Hydroxyproline content: Collagen was determined by estimating the hydroxyproline content, an amino acid characteristic of collagen. HSC-T6 cells were lysed after treatment with GT (100 μg/mL) for 24 h. The lysates were hydrolyzed in 6 mol/L HCl for 16 h at 110°C and evaporated to dryness to remove the acid. The residue, dissolved in distilled water, was mixed with 50% isopropanol and chloramine-T solution and left for 10 min at room temperature. Finally, p-dimethylaminobenzaldehyde in 60% perchloric acid was added and heated to 60°C for 25 min. The absorbance was measured at 558 nm.

Expression of collagen type 1: The expression of collagen type 1 was observed by ELISA. HSC-T6 cells, seeded on 24-well plates at a density of 1.5 × 10⁵ and cultured until 90% confluency, were treated with serum-free DMEM with or without 175 μmol/L acetaldehyde. Ascorbic acid (50 μg/L), and 3-aminopropionitrile fumarate (100 μg/L) were also added to increase the collagen proline hydroxylation and to prevent collagen cross-linking. After 24 h of treatment with GT (100 μg/L), aliquots of medium were transferred into immunowell plates, and glutaraldehyde (0.01%) was added and incubated at room temperature for 1 h. Collagen type 1 antibody (1:4000, Abcam Co., Cambridge, UK) was added and further incubated for 2 h at 37°C. The antigen-coated plates were blocked with casein and incubated with the secondary antibody (1:8000) linked to peroxidase, and subsequently re-incubated with substrate (TMB 10 mg/mL, 3% H₂O₂, 50 mmol/L sodium acetate buffer, pH 5.1) for 15 min. The enzymatic reaction was stopped by adding 1 mol/L H₂SO₄, and the absorbance at 450 nm was measured with a microplate reader.

Animals and treatments: Male albino rats (235-250 g) were purchased from Samtako (Kyunggi-do, Korea) and housed in controlled temperature and relative humidity, and a 12 h light/dark cycle. All experiments were performed according to National guidelines for the use of animals in biomedical research. The rats were randomly assigned to four groups of eight rats each: the normal control group without any treatment (NC), the hepatic fibrosis control group (FC), and hepatic fibrosis with 100 mg/kg GT treated group (FG). Hepatic fibrosis was induced by intraperitoneal injections of 10 mg/kg dimethylnitrosamine (DMN, Sigma, St. Louis, USA) for 3 consecutive days each week over a period of 4 wk. Normal saline was given to NC rats. GT was administered in drinking water which was calculated according to the amount of water consumed the previous day. At the end of the 4 wk experimental period, all rats were killed under ether anesthesia. Blood was obtained from the inferior vena cava, and the liver was excised. The liver was immediately frozen for biochemical measurements or fixed in formalin for histochemical examination.

Hepatotoxicity and lipid peroxidation: Hepatotoxicity was assessed by quantifying the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) using a spectrophotometric diagnostic kit (Youngdong Pharmaceutical Co., Korea). Lipid peroxidation in the liver and serum were determined by measuring the levels of MDA, an end product of lipid metabolism. For the serum sample, 3 mol/L sulfuric acid and 100 g/L phosphotungstic acid were added and incubated at room temperature for 10 min, and then centrifuged. For the liver sample, homogenates of liver in potassium phosphate buffer were prepared. MDA contents in the serum and liver samples were determined using a colorimetric reaction with thiobarbituric acid.

Hepatic hydroxyproline content: A portion of liver tissue (200 mg) was homogenized in 10 volumes of 0.5 mol/L potassium phosphate (KP) buffer and hydroxyproline content was measured as described above.

Histology of liver: Liver tissues were fixed in 10% neutral buffered formalin, dehydrated with 50%-100% ethanol, and embedded in paraffin. Five micrometer sections were cut and stained with hematoxylin-eosin.

All data were analyzed and expressed as mean ± SD. Comparisons were performed by Student’s t-test to detect differences between the groups. A level of P < 0.05 was considered statistically significant.

The anti-proliferative activity in HSC-T6 cells was determined by cell viability using the MTT assay. As shown in Figure 1, HSC-T6 cell proliferation was dose-dependently inhibited by GT. GT at 10, 50 and 100 μg/mL caused dose-dependent inhibition of HSC-T6 cell proliferation by 29.5% ± 4.2%, 30.6% ± 5.6%, and 44.8% ± 1.2 %, respectively (P < 0.05, Figure 1). The antiproliferative effects were not related to the nonspecific cytotoxic effects of green tea because cells showed normal morphology (data not shown).

To assess the effect of GT on ECM production, hydroxyproline content and type 1 collagen expression, assessed by ELISA, were examined in activated HSC-T6 cells. Serum-starved HSC-T6 cells were cultured with acetaldehyde and GT treatment for 24 h. Treatment with 100 μg/mL GT significantly reduced cell hydroxyproline content by 23.0% ± 2.1% compared to the control group. Furthermore, the expression of type 1 collagen was up-regulated by acetaldehyde stimulation, and GT markedly reduced collagen type 1 expression in a dose-dependent manner. Acetaldehyde at a concentration of 175 μmol/L induced collagen type 1 expression by 17.4% ± 0.1%, and 10, 50 and 100 mg/mL GT reduced collagen type 1 expression by 15.2% ± 2.2%, 15.5% ± 1.3%, and 23.0% ± 1.1%, respectively (Figure 1).

As shown in Table 1, the liver index, which is the percent of liver weight at final body weight, was not significantly different among the experimental groups. In contrast, relative spleen weight was increased 3.5-fold by DMN treatment, and GT administration restored the relative spleen weight (P < 0.05).

AST and ALT concentrations in serum were used as biochemical markers to evaluate hepatic injury. ALT is a cytosolic enzyme, primarily present in the liver. An increase in plasma ALT indicates liver damage more specifically than AST. AST, which is a mitochondrial enzyme present in large quantities in the heart, liver, skeletal muscle, and kidney, in part indicates liver injury. Serum activities of ALT and AST were markedly increased with DMN treatment and GT supplementation attenuated the elevation of AST and ALT activities (Table 1).

Liver fibrosis was evaluated by hematoxylin & eosin staining. The control group showed normal architecture (Figure 2A), whereas the DMN-treated group exhibited necrosis, congestion, hemorrhage, and destruction of the lobular architecture (Figure 2B). Red blood cells from blood vessels were found in liver tissue due to the collapse of the matrix structure. GT administration exhibited notable recovery effects (Figure 2C).

The histological findings were corroborated by biochemical parameters of liver tissue collagen content determined by hydroxyproline, and lipid peroxide determined by MDA.

Hydroxyproline, a product of collagen metabolism, is an amino acid characteristic of collagen. The total collagen present in liver was, therefore, determined by estimating the hydroxyproline content. As shown in Figure 3, hydroxyproline content was significantly increased following DMN treatment (FC), indicating that the liver fibrosis model was successfully established. GT administration (FG, 100 mg/kg) restored the hydroxyproline content in fibrotic liver. Lipid peroxides, measured in terms of the formation of MDA, were significantly increased in DMN-induced rat liver. GT administration significantly reduced the lipid peroxide level.

Hepatic stellate cells (HSC) are recognized as the primary cellular source of matrix components in chronic liver diseases, and therefore play a critical role in the development and maintenance of liver fibrosis. Overproduction of extracellular matrix components, particularly collagen, is a characteristic of activated HSC, and activation and proliferation of HSC have been implicated in the pathogenesis of liver fibrosis.

Hepatoprotective effects of green tea against carbon tetrachloride, cholestasis and alcohol induced liver fibrosis were reported in many studies. However, the hepatoprotective effect of green tea in dimethylnitrosamine (DMN)-induced models has not been studied.

The present study demonstrates that consumption of green tea prevents the development of hepatic fibrosis in a rat model of DMN-induced liver fibrosis. These results were confirmed both by liver histology and by quantitative measurement of hepatic hydroxyproline content, a marker of collagen deposition in the liver. Accordingly, inhibition of proliferation, reduced collagen content, and type 1 collagen expression were observed in activated HSC cells following green tea treatment.

This study demonstrates that green tea may protect liver cells and reduces the deposition of collagen fibers in the liver. Green tea provides a safe and effective strategy for improving hepatic fibrosis.

HSC are the main collagen producing cells in the liver, which proliferate and undergo a process of activation during the development of fibrosis resulting in an increased capacity for collagen synthesis.

This study examined the protective effect of green tea extract on hepatic fibrosis in a HSC line and in a rat model of DMN-induced liver fibrosis. Green tea administration prevents the development of hepatic fibrosis in the rat model of liver fibrosis. Furthermore, inhibition of proliferation, reduced collagen deposition, and type 1 collagen expression were observed in activated HSC cells following green tea treatment. The results imply that green tea may protect liver cells and reduce the deposition of collagen fibers in the liver. Green tea provides a safe and effective strategy for improving hepatic fibrosis.