The antioxidant effects of the flavonoids rutin and quercetin inhibit oxaliplatin-induced chronic painful peripheral neuropathy

Abstract

Background: Oxaliplatin, the third-generation platinum compound, has evolved as one of the most important therapeutic agents in colorectal cancer chemotherapy. The main limiting factor in oxaliplatin treatment is painful neuropathy that is difficult to treat. This side effect has been studied for several years, but its full mechanism is still inconclusive, and effective treatment does not exist. Data suggest that oxaliplatin's initial neurotoxic effect is peripheral and oxidative stress-dependent. A spinal target is also suggested in its mechanism of action. The flavonoids rutin and quercetin have been described as cell-protecting agents because of their antioxidant, antinociceptive, and anti-inflammatory actions. We proposed a preventive effect of these agents on oxaliplatin-induced painful peripheral neuropathy based on their antioxidant properties.

Methods: Oxaliplatin (1 mg/kg, i.v.) was injected in male Swiss mice, twice a week (total of nine injections). The development of sensory alterations, such as thermal and mechanical allodynia, was evaluated using the tail immersion test in cold water (10°C) and the von Frey test. Rutin and quercetin (25-100 mg/kg, i.p.) were injected 30 min before each oxaliplatin injection. The animals' spinal cords were removed for histopathological and immunohistochemical evaluation and malondialdehyde assay.

Results: Oxaliplatin significantly increased thermal and mechanical nociceptive response, effects prevented by quercetin and rutin at all doses. Fos immunostaining in the dorsal horn of the spinal cord confirmed these results. The oxidative stress assays mainly showed that oxaliplatin induced peroxidation in the spinal cord and that rutin and quercetin decreased this effect. The flavonoids also decreased inducible nitric oxide synthase and nitrotyrosine immunostaining in the dorsal horn of the spinal cord. These results suggest that nitric oxide and peroxynitrite are also involved in the neurotoxic effect of oxaliplatin and that rutin and quercetin can inhibit their effect in the spinal cord. We also observed the preservation of dorsal horn structure using histopathological analyses.

Conclusions: Oxaliplatin induced painful peripheral neuropathy in mice, an effect that was prevented by rutin and quercetin. The mechanism of action of oxaliplatin appears to be, at least, partially oxidative stress-induced damage in dorsal horn neurons, with the involvement of lipid peroxidation and protein nitrosylation.

Figure 1

Decrease of mechanical and cold nociceptive threshold induced by oxaliplatin. Each mouse received two intravenous injections per week for 4,5 weeks, totalizing nine injections. Withdrawal responses were determined once a week until the 49th day. Panel A: paw withdrawal threshold of control (C, vehicle, n = 12) and treated (oxaliplatin 1 mg/kg, 2 mg/kg and 4 mg/kg, n = 12) mice to the electronic pressure meter (electronic von Frey) applied to the plantar surface of the hind paw. Panel B: Tail withdrawal threshold of Control (C, vehicle, n = 12) and treated (oxaliplatin 1 mg/kg, 2 mg/kg and 4 mg/kg, n = 12) mice to tail immersion in cold non-noxious (10°C) water. The results are reported as the means ± SEM paw withdrawal threshold (g) for mechanical threshold (panel A) and tail withdrawal response (s) for the cold threshold (panel B). A significant reduction in mechanical and thermal threshold (***p < 0.001, ANOVA followed by Student Newman-Keuls post hoc test), was observed when compared with the vehicle group.

Figure 2

Antinociceptive effects of rutin and quercetin on oxaliplatin-induced mechanical nociceptive threshold decrease (von Frey). The mice received two intravenous injections of oxaliplatin (OXL; 1 mg/kg) per week, for 4.5 weeks for a total of nine injections. Rutin (A) and quercetin (B) were injected intraperitoneally 30 min before every OXL administration. The control group received saline instead of rutin or quercetin. Mechanical threshold was assessed before and every 7 days after each treatment. The data points represent the mean ± SEM the paw withdrawal response in grams (g) in six animals.*p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA followed by Student Newman-Keuls post hoc test).

Figure 3

Antinociceptive effects of rutin and quercetin on cold nociceptive threshold in oxaliplatin (OXL)-treated mice (tail immersion). The mice received two intravenous injections of OXL (1 mg/kg) per week for 4.5 weeks for a total of nine OXL injections. Rutin (A) and quercetin (B) were injected intraperitoneally 30 min before every OXL administration. The control group received saline instead of rutin or quercetin. The thermal test (tail immersion test in cold water, 10°C) was conducted before and every 7 days after each treatment. The data points represent the mean ± SEM of the reaction time in seconds after tail immersion in cold water in six animals.*p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA followed by Student Newman-Keulspost hoc test).

Figure 4

Photomicrographs of the dorsal horn of the spinal cord of mice subjected to oxaliplatin (OXL)-induced neurotoxicity and treated with rutin (RUT) or quercetin (QT). The mice received two intravenous injections of OXL (1 mg/kg) per week for 4.5 weeks for a total of nine OXL injections. Rutin and quercetin (50 mg/kg) were injected intraperitoneally 30 min before every OXL administration. The control group received saline instead of rutin and quercetin. The figure shows hematoxylin-eosin staining (400× magnification).

Figure 5

Photomicrographs of the skin harvested from the paws of mice subjected to oxaliplatin (OXL)-induced neurotoxicity and treated with rutin (RUT) or quercetin (QT). The mice received two intravenous injections of OXL (1 mg/kg) per week for 4.5 weeks for a total of nine OXL injections. Rutin and quercetin (50 mg/kg) were injected intraperitoneally 30 min before every OXL administration. The control group received saline instead of rutin and quercetin. The figure shows hematoxylin-eosin staining (400× magnification).

Figure 6

Effect of rutin and quercetin on malondialdehyde (MDA) levels in the spinal cord. The mice received two intravenous injections of OXL (1 mg/kg) per week for 4.5 weeks for a total of nine OXL injections. Rutin and quercetin (50 mg/kg) were injected intraperitoneally 30 min before every OXL administration. The control group received saline instead of rutin and quercetin. Fourteen days after the first dose, a portion of the spinal cord was collected and processed to measure MDA levels. The results are expressed as mean ± SEM. *p < 0.05, compared with saline plus oxaliplatin-treated group; ^#p < 0.05, compared with naïve (C) group (ANOVA followed by Student Newman-Keuls post hoc test).

Figure 7

Fos immunostaining in the dorsal horn of the spinal cord in mice subjected to oxaliplatin (OXL)-induced neurotoxicity and treated with rutin or quercetin. The mice received two intravenous injections of OXL (1 mg/kg) per week for 4.5 weeks for a total of nine OXL injections. Rutin or quercetin (50 mg/kg) were injected intraperitoneally 30 min before every OXL administration. The control group received saline instead of rutin and quercetin. (A) Naive animals; (B) Oxaliplatin plus saline; (C) Oxaliplatin plus rutin (50 mg/kg); (D) Oxaliplatin plus quercetin (50 mg/kg). 400× magnification. (E) Bars show the percentage of positive Fos staining area, mean ± SEM (n = 4). ***p < 0.001, compared with saline plus oxaliplatin-treated group (S); ^###p < 0.001, compared with naive (C) group (ANOVA followed by Student Newman-Keuls post hoc test).

Figure 8

Nitrotyrosine immunostaining in the dorsal horn of the spinal cord of mice subjected to oxaliplatin (OXL)-induced neurotoxicity and treated with rutin or quercetin. The mice received two intravenous injections of OXL (1 mg/kg) per week for 4.5 weeks for a total of nine OXL injections. Rutin or quercetin (50 mg/kg) were injected intraperitoneally 30 min before every OXL administration. The control group received saline instead of rutin and quercetin. (A) Naive animals; (B) Oxaliplatin plus saline; (C) Oxaliplatin plus rutin (50 mg/kg); (D) Oxaliplatin plus quercetin (50 mg/kg). 400× magnification. (E) Bars show the percentage of positive nitrotyrosine staining area, mean ± SEM (n = 4). *p < 0.05, compared with saline plus oxaliplatin-treated group (S); ^#p < 0.05, compared with naive (C) group (ANOVA followed by Student Newman-Keuls post hoc test).

Figure 9

iNOS immunostaining in the dorsal horn of the spinal cord of mice subjected to oxaliplatin (OXL)-induced neurotoxicity and treated with rutin or quercetin. The mice received two intravenous injections of OXL (1 mg/kg) per week for 4.5 weeks for a total of nine OXL injections. Rutin or quercetin (50 mg/kg) were injected intraperitoneally 30 min before OXL administration. The control group received saline instead of rutin and quercetin. (A) Naive animals; (B) Oxaliplatin plus saline; (C) Oxaliplatin plus rutin (50 mg/kg); (D) Oxaliplatin plus quercetin (50 mg/kg). 400× magnification. (E) Bars show the percentage of positive iNOS staining area, mean ± SEM (n = 4). **p < 0.01, compared with saline plus oxaliplatin-treated group (S); ^##p < 0.01, compared with naive (C) group (ANOVA followed by Student Newman-Keuls post hoc test).