Confirmation that Magnesium Lithospermate B Ameliorates Paraquat-Induced Injury in Cultured Renal Epithelial Cells

This article is also accessible online at: http://BioMedNet.com/karger Dear Sir, Paraquat is a bipyridyl herbicide which is hardly absorbed into the body by inhalation, but causes serious injuries when ingested; death usually results within 2 days after a dose of 50 mg/kg. The cytotoxic action of paraquat is said to be mediated by the production of paraquat radicals through reduction of paraquat in cells and the subsequent induction of lipid peroxidation [1]. Therefore, antioxidants and radical scavengers such as superoxide dismutase, vitamin C, vitamin E, selenium, N-acetylcysteine and clofibrate have been used for the treatment of paraquat poisoning [2–6]. Focusing attention on the clinical finding that decreased urinary excretion of paraquat is associated with aggravation of the pathological condition, we investigated the effect of magnesium lithospermate B, a substance known to interfere with the progression of renal failure [7–13], using a cultured cell line. Dulbecco’s modified Eagle medium/nutrient mixture F-12 (D-MEM/F-12) and fetal calf serum (FCS) were purchased from Life Technologies, Inc. (Grand Island, N.Y., USA) and Cell Culture Laboratories (Cleveland, Ohio, USA), respectively. Paraquat was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Magnesium lithospermate B was isolated and purified from a root extract of Salvia miltiorrhiza Bunge produced in China, as reported previously [14]. Its chemical structure is shown in figure 1. LLC-PK1 cells were cultured in 5% FCSsupplemented D-MEM/F-12 medium. After confluence had been reached, the cells were seeded in culture plates at 104/well. Paraquat and/or magnesium lithospermate B were added to the culture 2 h later, and the plates were incubated for 48 h. Leakage of lactate dehydrogenase (LDH) into the culture medium was assayed as an index of cytotoxicity using a commercial kit from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The extent of lipid peroxidation was estimated by measuring the concentration of malondialdehyde (MDA) as described by Yagi [15]. Results are presented as the mean B SE of 5 determinations. The data were analyzed for statistical significance using Dunnett’s method. Differences at p ! 0.05 were considered statistically significant. Glomerular filtration and active secretion from uriniferous tubules are responsible for excretion of paraquat from the kidney. When tubular necrosis occurs, urinary excretion of paraquat decreases rapidly, probably resulting in aggravation of the disease [16]. Since porcine kidney-derived LLC-PK1 cells, an established cultured cell line of renal epithelial cells, have proximal uriniferous tubule-like features, we examined the effect of magnesium lithospermate B on these cells. The lysosomal enzyme LDH released from injured cells into the culture medium was used as an index for determining the effect of magnesium lithospermate B. As shown in table 1, the amount of LDH released from LLC-PK1 cells into the medium in the presence of 0.01 ÌM paraquat was about 1.4 times higher than that in the absence of paraquat, reflecting the cytotoxicity of paraquat. In contrast, when magnesium lithospermate B was added to the medium at graded concentrations from 2.5 to 50 ÌM, LDH leakage was suppressed in proportion to an increase in the magnesium lithospermate B concentration, the amount of LDH being almost as