Fruits of S. chinensis used in this study were collected from Moonkyeng City, Korea in September, 2005. A voucher specimen (accession no. SC-PNUNPRL-1) was deposited in the Herbarium of Pusan National University. To purify gomisin A, dried fruits of S. chinensis (2.5 kg) were ground into a fine powder and successively extracted at room temperature with n-hexane, EtOAc and MeOH. The hexane extract (308 g) was evaporated under vacuum and chromatographed on a silica gel (40 μm; J.T. Baker, Phillipsburg, NJ, USA) column (70×8.0 cm) with a step gradient of 0, 5, 10, 20 and 30% EtOAc in hexane (each 1 liter) (18). Of these extracts, fraction 29 (1,992 mg) was separated on a silica gel column (100×3.0 cm) with 15% CHCl₃ in acetone to provide gomisin A (973 mg). Pure gomisin A was identified by HPLC on a Phenomenex Luna C18 column (150×4.6 mm ID; 5 μm particle size; Phenomenex) (19). In addition, the chemical structure of gomisin A used in this study was verified by LC-MS (Bruker BioApex FT mass spectrometer) and NMR analysis (Varian Inova 500 spectrometer) (20) (Fig. 1A).

Sprague-Dawley (SD) rats used in this study were purchased from Samtaco BioKorea (Osan, Korea). All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Pusan National University (approval no. PNU-2009-0007). Animals were handled at the Pusan National University-Laboratory Animal Resources Center accredited by the Korea FDA in accordance with USA NIH guidelines (accredited unit no. 000996). All rats were housed under specific pathogen-free (SPF) conditions with a strict light cycle (lights on at 06:00 h and off at 18:00 h) and were fed a standard irradiated chow diet (Purina Mills, Inc.) ad libitum.

Eight-week-old SD rats were randomly divided into three subgroups with six rats/group. The first group of SD rats was not treated with any compounds (non-treated group). The second group received a comparable volume of olive oil via oral gavage (vehicle/CCl₄-treated group) daily, whereas the third group received 100 mg/kg body weight per day of gomisin A via oral injection for four days (gomisin A/CCl₄-treated group). On the fifth day, the second and third groups received 0.1 ml of CCl₄ solution via intraperitoneal injection. At 24 h after CCl₄ injection, the animals were immediately euthanized using CO₂ gas. Body weights as well as weights of internal organs, including the liver, kidney, heart, lung, spleen and thymus, were measured using a chemical balance. Subsequently, liver and kidney tissues as target organs were collected and stored in Eppendorf tubes at -70°C until being assayed. For histological analysis, these tissues were fixed in 10% formalin solution for 24 h.

After the final administration of CCl₄, all rats were fasted for 24 h, and blood was collected from abdominal veins. Serum was obtained by centrifugation of blood incubated for 30 min at room temperature. Serum biochemical components were assayed using an automatic serum analyzer (Hitachi 747; Hitachi, Japan). All assays were assessed using fresh serum and conducted in duplicate.

Liver and kidney tissues collected from rats were fixed with 10% formalin for 24 h, embedded in paraffin wax, and then sectioned into 5-μm slices. The liver and kidney sections were then stained with hematoxylin and eosin (H&E; Sigma-Aldrich, St. Louis, MO, USA). The stained liver and kidney tissue sections were observed by light microscopy, and morphological features of hepatocytes and kidney cells were assessed with Leica Application Suite (Leica Microsystems, Switzerland).

Proteins prepared from tissues of the vehicle/CCl₄- and gomisin A/CCl₄-treated SD rats were separated by electrophoresis on a 4–20% SDS-PAGE gel for 3 h and then transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was incubated separately with the primary antibody: anti-caspase-3 (#9662; Cell Signaling Technology, Boston, MA, USA), anti-Bax (ab7977), anti-Bcl-2 (ab7973; both were from Abcam, Cambridge, UK), anti-ERK (sc-94), anti-p-ERK (sc-7383; both were from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-JNK (#9252), anti-p-JNK (#9251), anti-p38 (#9212), anti-p-p38 (#9211; all were from Cell Signaling Technology), or anti-actin (A5316; Sigma-Aldrich, St. Louis, MO, USA) overnight at 4°C. The membranes were then washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄ and 0.05% Tween-20) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories, Inc., South San Francisco, CA, USA) diluted 1:1,000 at room temperature for 2 h. The membrane blots were developed using a Chemiluminescence Reagent Plus kit (ECL; Pfizer and Pharmacia, New York, NY, USA).

Tests for significance between the vehicle- and gomisin A-treated SD rats were performed using a one-way ANOVA test of variance (SPSS for Windows, release 10.10, standard version; SPSS, Chicago, IL, USA). Tests for significance between the non-treated and CCl₄-treated groups (vehicle/CCl₄ and gomisin A/CCl₄) were performed using a post-hoc test (SPSS for Windows, release 10.10, standard version) of variance, and significance levels are provided in the text. All values are reported as the means ± standard deviation. A P-value <0.05 was considered to indicate a statistically significant result.

Generally, toxic effects on animal and human bodies are confirmed by alterations in body and organ weights (21). In order to investigate the protective effects of gomisin A against CCl₄-induced toxicity, we first measured the body weights of non-treated vehicle- as well as gomisin A-pretreated rats for five days, including one day following CCl₄ exposure. No significant differences in body weight were detected among the three groups (Fig. 1B). However, results of the organ weight analysis were different from those of the body weight analysis. Of the six organs, five organs, including the kidney, heart, lung, spleen and thymus, showed slightly lower weights in the gomisin A/CCl₄-treated group compared to these values in the vehicle/CCl₄-treated group, although these results were not statistically significant. In contrast, the liver weight was significantly higher in the gomisin A/CCl₄-treated group compared to the vehicle/CCl₄-treated group. In addition, the weights of the liver, lung and spleen in the CCl₄-treated group were significantly lower than those in the non-treated group (Fig. 2). Therefore, the present results suggest that gomisin A did not affect body and organ weights, apart from that of the liver.

Increased concentrations of alkaline phosphatase (ALP), alanine transaminase (ALT) and aspartate transaminase (AST) in serum are well-known factors indicating liver toxicity, whereas blood urea nitrogen (BUN) and creatinine (CRE) are evidence of kidney toxicity (21,22). To investigate the protective effects of gomisin A against CCl₄-induced toxicity in terms of serum biochemical indicators, the levels of five indicators, including ALP, AST, ALT, BUN and CRE, were measured in the vehicle/CCl₄- and gomisin A/CCl₄-treated rats. In regards to the liver toxicity factors, the levels of ALP, AST and ALT were higher in the vehicle/CCl₄-treated rats than these levels in the non-treated rats. However, these levels were significantly decreased in the gomisin A/CCl₄-treated rats when compared with the vehicle/CCl₄-treated rats, although the rate of decrease varied for each factor (Fig. 3A). Furthermore, the factors representing kidney toxicity showed similar patterns as those representing liver toxicity. Specifically, serum levels of BUN and CRE were significantly increased upon CCl₄ exposure. However, the concentrations of these two factors were restored by gomisin A pretreatment to similar levels as those in the non-treated group (Fig. 3B). Therefore, these results demonstrated that pretreatment with gomisin A conferred protective effects against liver and kidney damage, although the protective effects varied according to each factor.

Generally, histological staining is performed with H&E to visualize the differences between tissue components under normal and pathological conditions. Histological alterations during hepatocellular damage along with the protective effects of gomisin A were first identified by histological analysis of the liver section. In the non-treated group, the histopathology of the liver displayed a normal distribution of hepatocytes with clear visible nuclei, a portal triad and central vein (Fig. 4). However, following CCl₄ treatment, extensive centrolobular necrosis was observed in and around the terminal hepatic venule (THV) of the liver. Furthermore, the central vein was significantly dilated in the vehicle/CCl₄-treated group compared with the non-treated group (Fig. 4). However, the liver section of the group pretreated with gomisin A for four days displayed low hepatocellular necrosis, a poorly dilated central vein, and regular arrangement of hepatocytes.

In the kidney, significant changes were detected only in the cortex containing the Bowman’s capsule and convoluted tubules, whereas the medulla region maintained its morphology. Regarding the Bowman’s capsule, the vehicle/CCl₄-treated group showed an increased diameter of the glomerulus as well as a higher number of capillaries in the glomerulus compared with the non-treated group. In the gomisin A pretreatment group, the diameter of the glomerulus, the number of capillaries, and Bowman’s space were significantly decreased. In particular, Bowman’s space completely disappeared in the gomisin A/CCl₄-treated group (Fig. 4). Regarding the convoluted tubules, their diameters were dramatically increased in the CCl₄-exposed group compared with the non-treated group. However, gomisin A pretreatment induced recovery of the diameters of the convoluted tubules back to normal (Fig. 4). The above results suggest that gomisin A pretreatment contributed to the reduction of hepatic necrosis and dilation of the THV in livers of the SD rats after CCl₄ exposure. Furthermore, this lignan reduced the occurrence of renal defects, including increased diameter of the glomerulus, a higher number of capillaries and convoluted tubules.

In order to investigate whether or not gomisin A prevents the activation of apoptosis, alterations in apoptosis-related proteins were examined in the liver and kidney tissues of the rats pretreated with vehicle or gomisin A. First, changes in the levels of these proteins were measured in liver tissue. The pro-caspase-3 level was reduced significantly in the vehicle/CCl₄-treated group, whereas the level of active caspase-3 increased. In the gomisin A/CCl₄-treated group, the levels of pro-caspase-3 and active caspase-3 were recovered to the same levels as those of the non-treated group. Bcl-2 belongs to a family of proteins that includes both pro- and anti-apoptotic members. Among these members, Bcl-2 proteins stimulate anti-apoptosis while the Bax protein significantly inhibits the anti-apoptotic actions of the Bcl-2 protein (23,24). To assess the effects of gomisin A pretreatment on proteins associated with the apoptotic signaling pathway, the expression levels of the Bcl-2 and Bax proteins were determined in the vehicle/CCl₄-treated and gomisin A/CCl₄-treated groups using western blot analysis. The expression of the Bax protein was slightly increased in the vehicle/CCl₄-treated group compared to the non-treated group, whereas it was further increased in the gomisin A/CCl₄-treated group. However, the expression level of the Bcl-2 protein was maintained at a certain level regardless of gomisin A pretreatment (Fig. 5). Therefore, western blot analysis indicated that gomisin A reduced the expression levels of proteins associated with anti-apoptosis in the liver tissue.

Additionally, alterations in the expression levels of these proteins were detected in the kidney tissue. The expression levels of pro-caspase-3 and active caspase-3 were markedly increased in the vehicle/CCl₄-treated group, while their levels were decreased in the gomisin A/CCl₄-treated group (Fig. 6). The expression pattern of the Bax protein very much resembled its pattern in liver tissue, whereas the pattern of Bcl-2 expression differed from that observed in the liver tissue. Following CCl₄ exposure, the expression of the Bcl-2 protein dramatically increased ~3-fold. However, gomisin A pretreatment prevented an increase in the expression of this protein (Fig. 6). Therefore, these results indicate that gomisin A reduced the expression of proteins associated with anti-apoptosis in the kidney tissue.

We investigated the roles of different MAPK signaling proteins on CCl₄-induced liver and kidney damage following gomisin A pretreatment. In regards to the liver, the phosphorylation levels of ERK and JNK were decreased in the vehicle/CCl₄-treated group when compared with levels in the non-treated group, whereas the phosphorylation level of p38 did not significantly change. However, in the gomisin A/CCl₄-treated group, the phosphorylation levels of ERK and p38 were significantly higher compared to those in the vehicle/CCl₄-treated group (Fig. 7).

Furthermore, the MAPK signaling pathway in the kidney showed a different response compared to the liver. In the vehicle/CCl₄-treated group, phosphorylation of JNK increased when compared to its level in the non-treated group. In contrast, phosphorylation of p38 was reduced by 50%, whereas the phosphorylation level of ERK was maintained at a constant level. However, in the gomisin A/CCl₄-treatment group, the phosphorylation level of ERK was significantly decreased when compared to that in the vehicle/CCl₄-treated group, whereas phosphorylation of p38 and JNK was increased (Fig. 8). Therefore, these results showed that gomisin A pretreatment had protective effects against liver and kidney damage induced by CCl₄ exposure through differential regulation of the MAPK signaling pathway.