RESUMEN
By observing the cytotoxic effects of anthraquinones on HepG2 cell and using the precision-cut liver slices technique to authenticate the cytotoxic constituents, the paper aims to explore the material basis of Polygonum multiflorum root to cause liver toxicity. Firstly, MTT method was used to detect the effect of 11 anthraquinone derivatives on HepG2 cell. Then, the clear cytotoxic ingredients were co-cultured with rat liver slices for 6h respectively, and the liver tissue homogenate was prepared. BCA method was used to determine the content of protein in the homogenate and continuous monitoring method was used to monitor the leakage of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamine amino transpeptidase (GGT) and lactate dehydrogenase (LDH). The toxic effect of these ingredients on liver tissue was tested by calculating the leakage rate of the monitored enzymes. As a result, rhein, emodin, physcion-8-O-β-D-glucopyranoside and physcion-8-O-(6'-O-acetyl)-β-D-glucopyranoside showed cytotoxic effects on HepG2 cell and their IC₅₀ values were 71.07, 125.62, 242.27, 402.32 μmol•L⁻¹ respectively, but the other 7 compounds are less toxic and their IC₅₀ values can not be calculated. The precision-cut liver slices tests showed that rhein group of 400 μmol•L⁻¹ concentration significantly increased the leakage rate of ALT, AST and LDH (P<0.01), and the rhein group of 100 μmol•L⁻¹ concentration only increased the leakage rate of LDH (P<0.05). With the increase of rhein concentration, the protein content in liver slices decreased significantly (P<0.05) with a certain range of does. Emodin group of 400 μmol•L⁻¹ concentration significantly increased the leakage rate of ALT, GGT and LDH (P<0.01). Physcion-8-O-β-D-glucopyranoside group of 800 μmol•L⁻¹ concentration also significantly increased the leakage rate of ALT, AST and LDH (P<0.01 or P<0.05), but the group of 200 μmol•L⁻¹ concentration only significantly increased the LDH leakage (P<0.05). Along with the increase of the concentration of physcion-8-O-β-D-glucopyranoside, the leakage rate of ALT, AST and LDH showed a trend of increase, but the protein content in liver slices was in decline. Furthermore, MTT reduction ability of liver slices significantly decreased (P<0.01) in the physcion-8-O-β-D-glucopyranoside group of 800 μmol•L⁻¹ concentration. The results suggested that rhein, emodin and physcion-8-O-β-D-glucopyranoside at high concentrations (≥400 μmol•L⁻¹) can produce some damage to the liver tissue. However, the exposure levels of these constituents are very low, so to reach the toxic concentration (400 μmol•L⁻¹ or 800 μmol•L⁻¹) an adult of 65 kg body weight will need at least a single oral 4 898 g, 339 g and 5 581 g of P.multiflorum root respectively, which is far from the statutory dose of crude P. multiflorum root (3-6 g) or its processed product (6-12 g). Therefore, the conclusion that anthraquinones are the prime constituents of the hepatotoxicity of P. multiflorum root are still not be proved.
RESUMEN
Aim To investigate effects of indole-3-carbinol (I3C) on hepatic stellate cells (HSCs) activated by acetaldehyde in precision-cut liver slices (PCLS). Methods PCLS were incubated with 700 ?mol?L-1 acetaldehyde and 200 ~ 800 ?mol?L-1 I3C for 6 h. The expression of ?-smooth muscle actin(?-SMA) in liver slices was analyzed by immunohistochemistry. The leakages of glutathione S-transferase (GST), lactate dehydrogenase (LDH) and content of transforming growth factor-?1 (TGF-?1) in media were assayed. Contents of malondialdehyde (MDA) and hydroxyproline (Hyp) in tissue were also determined. Expressions of matrix metalloproteinases-1 (MMP-1) and tissue inhibitor of metalloproteinase (TIMP-1) in media were analyzed by the western blot. Results The increase of activated HSCs due to acetaldehyde was inhibited by I3C (200~800 ?mol?L-1). Meanwhile, I3C treatment (200~800 ?mol?L-1) showed significant and concentration-dependent antagonistic actions on the increment of GST, LDH leakages into the media and MDA, Hyp contents in tissues induced by acetaldehyde. The increase of TGF-?1 was also remarkable inversed by I3C (200~800 ?mol?L-1). As compared with acetaldehyde group, the ratio of MMP-1/TIMP-1 was increased significantly by I3C treatment (800 ?mol?L-1)(P