Tanshinone (Salviae miltiorrhizae extract) preparations attenuate aminoglycoside-induced free radical formation in vitro and ototoxicity in vivo

Abstract

Antioxidant therapy protects against aminoglycoside-induced ototoxicity in animal models. A clinically suitable antioxidant must not affect the therapeutic efficacy of aminoglycosides or exhibit any side effects of its own. In addition, the treatment should be inexpensive and convenient in order to be implemented in developing countries where the use of aminoglycosides is most common. Standardized Salviae miltiorrhizae extracts (Danshen) are used clinically in China and contain diterpene quinones and phenolic acids with antioxidant properties. We combined in vitro and in vivo approaches to investigate the effect of a clinically approved injectable Danshen solution on aminoglycoside-induced free radical generation and ototoxicity. In vitro, Danshen inhibited gentamicin-dependent lipid peroxidation (formation of conjugated dienes from arachidonic acid), as well as the gentamicin-catalyzed formation of superoxide (in a lucigenin-based chemiluminescence assay) and hydroxyl radicals (oxidation of N,N-dimethyl-p-nitrosoaniline). Danshen extracts were then administered to adult CBA mice receiving concurrent treatment with kanamycin (700 mg/kg of body weight twice daily for 15 days). Auditory threshold shifts induced by kanamycin (approximately 50 dB) were significantly attenuated. Danshen did not reduce the levels in serum or antibacterial efficacy of kanamycin. These results suggest that herbal medications may be a significantly underexplored source of antidotes for aminoglycoside ototoxicity. Such traditional medicines are widely used in many developing countries and could become an easily accepted and inexpensive protective therapy.

FIG. 1.

Effects of Danshen on gentamicin-mediated free radical reactions. (A) Danshen suppresses gentamicin-catalyzed formation of superoxide. Luminosity was assayed in the hypoxanthine-xanthine oxidase system as described in Materials and Methods. Data are means ± standard deviations for four to six experiments per condition. The concentration of Danshen is given as the final concentration of the commercial extract in the assay. Danshen significantly inhibited gentamicin-stimulated lucigenin luminosity (P < 0.05). Differences among individual concentrations of Danshen were also significant (P < 0.05). RLU, relative luminosity units. (B) Danshen inhibits gentamicin-catalyzed formation of hydroxyl radicals. The formation of hydroxyl radicals was catalyzed by gentamicin-Cu(II) complexes as described in Materials and Methods. The concentration of Danshen is given as the final concentration of the commercial extract in the assay. Data are means ± standard deviations for five experiments. Danshen is effective in suppressing gentamicin-catalyzed hydroxyl radical formation at each concentration (P < 0.05). (C) Danshen decreases gentamicin-induced lipid peroxidation. Lipid peroxidation was measured as described in Materials and Methods. The concentration of Danshen is given as the final concentration of the commercial extract in the assay. Data are means ± standard deviations for four to eight experiments per condition. All values in the presence of Danshen are significantly different from gentamicin alone (P < 0.05).

FIG. 2.

Danshen reduces kanamycin-induced threshold shifts. Kanamycin (700 mg/kg twice daily) and Danshen (10 g/kg twice daily) were administered, and ABR thresholds were measured at 24 kHz (A) and 12 kHz (B) as described in Materials and Methods. Data are means ± standard deviations. Filled circles (•), saline controls, n = 11; filled squares (▪), kanamycin alone, n = 14; filled triangles (▴), kanamycin plus Danshen, n = 5; cross (x), Danshen alone, n = 3. Cotreatment with Danshen significantly attenuated kanamycin-induced threshold shifts at all times (P < 0.05).

FIG. 3.

Danshen reduces kanamycin-induced hair cell loss. Cytocochleograms were obtained from the surface preparations of the organ of Corti as described in Materials and Methods. The percentage of missing hair cells is plotted for the entire length of the cochlea. In the saline-treated CBA mice, outer hair cells in the basal turn were almost completely present (A). After treatment with kanamycin (700 mg/kg twice daily), most of the outer hair cells in the basal turn of the cochlea disappeared (B). Animals receiving kanamycin plus Danshen (10 g/kg twice daily) demonstrated less outer hair cell loss (C) than animals receiving kanamycin alone. Heavy solid line, inner hair cells; light solid line, outer hair cells of row 1; dashed line, outer hair cells of row 2; solid line with dots, outer hair cells of row 3.