Resveratrol ameliorates oxaliplatin-induced neuropathic pain via anti-inflammatory effects in rats

Abstract

Oxaliplatin (OXA) is a common chemotherapy drug and exhibits clinical activity in several cancer types. Its anticancer clinical effect is frequently accompanied by neurotoxicity. The symptoms include paresthesia and pain, which adversely affect the quality of life of patients. In the present study, five consecutive intraperitoneal injections of 4 mg/kg OXA were used to mimic chemotherapy in rats. OXA administration induced mechanical allodynia, activated spinal astrocytes and triggered the inflammatory response. To explore potential therapeutic options for OXA-induced neuropathic pain, resveratrol (Res) was intrathecally injected into the spinal cord of OXA-treated rats. Paw withdrawal threshold values of OXA-treated rats were increased, indicating an antinociception effect of Res on OXA-induced pain. Additionally, Res treatment reduced the levels of glial fibrillary acidic protein, TNF-α, IL-1β and NF-κB, which were upregulated in OXA-treated rats (compared with control). Furthermore, Auto Dock data showed that Res binds to cyclooxygenase-2 (COX-2) through six hydrogen bonds. Western blot analysis and reactive oxygen species (ROS) assays indicated that Res treatment decreased COX-2 expression and suppressed ROS production. In summary, intrathecal injection of Res reduced the spinal COX-2-mediated ROS generation and inflammatory reaction, suppressed astrocytic activation, and alleviated OXA-induced neuropathic pain.

Keywords: cyclooxygenase-2; oxaliplatin-induced neuropathic pain; resveratrol; spinal inflammation.

Figure 1

Res treatment alleviates OXA-induced neuropathic pain. (A) PWT values of rats in the Control and OXA groups after 5 days of OXA induction. Data are presented as the mean ± SEM (n=6). ^*P<0.05 vs. Control. Effect of Res treatment on PWT values in (B) OXA and OXA + Res and (C) Control and Res groups. Data are presented as the mean ± SEM (n=6). ^*P<0.05 vs. OXA. PWT, paw withdrawal threshold; OXA, oxaliplatin; Res, resveratrol.

Figure 2

Res treatment decreases the OXA-induced spinal inflammatory response. (A) Representative images of H&E staining of spinal cord sections from the Control, OXA and OXA + Res groups (scale bar, 20 µm). (B) Representative IL-1β immunofluorescence staining images of spinal cord sections from Control, OXA and OXA + Res groups (scale bar, 20 µm). (C) Relative inflammation score of Control, OXA and OXA + Res groups. (D) Quantitative data analysis of IL-1β fluorescence intensity shown as the mean ± SD (n=3). ^*P<0.05 vs. Control. ^#P<0.05 vs. OXA. (E) Western blot analysis of the expression levels of TNF-α and IL-1β in spinal cord of the Control, OXA and OXA + Res groups. (F) Semi-quantitative data analysis of TNF-α and IL-1β levels presented as the mean ± SD (n=3). ^*P<0.05 vs. Control. ^#P<0.05 vs. OXA. OXA, oxaliplatin; Res, resveratrol.

Figure 3

Res administration inhibits spinal astrocytic activation. (A) Representative immunofluorescence staining images of spinal GFAP in the Control, OXA and OXA + Res groups (scale bar, 20 µm). (B) Quantitative data analysis of GFAP fluorescence intensity of spinal GFAP presented as the mean ± SD (n=3). ^*P<0.05 vs. Control. ^#P<0.05 vs. OXA. (C) Western blot analysis of GFAP protein levels in the spinal cord of the Control, OXA and OXA + Res groups. (D) Semi-quantitative data analysis of GFAP levels presented as the mean ± SD (n=3). ^*P<0.05 vs. Control. ^#P<0.05 vs. OXA. OXA, oxaliplatin; GFAP, glial fibrillary acidic protein; Res, resveratrol.

Figure 4

Res treatment reduces COX-2 expression. (A) 3D structure model of COX-2 docked with Res generated using Auto Dock. (B) Enlarged view of the binding site in the box. (C) Detail of the COX-2/Res interaction. COX-2 is shown in red and cyan, and Res is shown in yellow. The interaction bonds are shown as yellow dotted lines and the numbers represent the bond lengths. (D) Representative images of immunofluorescence staining of COX-2 in the spinal cord section of the Control, OXA and OXA + Res groups (scale bar, 20 µm). (E) Quantitative data analysis of COX-2 fluorescence intensity presented as the mean ± SD (n=3). ^*P<0.05 vs. Control. ^#P<0.05 vs. OXA. (F) Western blot analysis of expression levels of spinal COX-2 in the Control, OXA and OXA + Res groups. (G) Semi-quantitative data analysis of COX-2 expression presented as the mean ± SD (n=3). ^*P<0.05 vs. Control. ^#P<0.05 vs. OXA. COX-2, cyclooxygenase-2; OXA, oxaliplatin; Res, resveratrol.

Figure 5

Res treatment decreases NF-κB expression. (A) Representative immunofluorescence staining images of spinal NF-κB in the Control, OXA and OXA + Res groups (scale bar, 20 µm). (B) Quantitative data analysis of NF-κB fluorescence intensity presented as the mean ± SD (n=3). ^*P<0.05 vs. Control. ^#P<0.05 vs. OXA. (C) Western blot analysis of NF-κB in the spinal cord of Control, OXA and OXA + Res groups. (D) Semi-quantitative data analysis of NF-κB expression presented as the mean ± SD (n=3). ^*P<0.05 vs. Control. ^#P<0.05 vs. OXA. OXA, oxaliplatin; Res, resveratrol.

Figure 6

Effect of Res on the levels of inflammatory factors and ROS production in C6 cells. (A) Western blot analysis of expression levels of COX-2 and IL-1β in the Control, TNF-α and TNF-α + Res groups. (B) Semi-quantitative data analysis of COX-2 and IL-1β expression presented as the mean ± SD (n=3). ^*P<0.05 vs. Control. ^#P<0.05 vs. TNF-α. (C) Change in ROS detected in the Control, TNF-α and TNF-α + Res groups. (D) Quantitative analysis of the relative ROS generation presented as the mean ± SD (n=3). ^*P<0.05 vs. Control. ^#P<0.05 vs. TNF-α. COX-2, cyclooxygenase-2; DCFH-DA, 2,7-Dichlorodi-hydrofluorescein diacetate; Res, resveratrol; ROS, reactive oxygen species.

Figure 7

Schematic of the underlying mechanism of action of resveratrol in reducing spinal inflammation and reversing OXA-induced neuropathic pain. OXA, oxaliplatin; ROS, reactive oxygen species; COX-2, cyclooxygenase-2.