β-Caryophyllene Inhibits Oxaliplatin-Induced Peripheral Neuropathy in Mice: Role of Cannabinoid Type 2 Receptors, Oxidative Stress and Neuroinflammation

Abstract

Peripheral neuropathy is an important adverse effect caused by some chemotherapeutic agents, including oxaliplatin (OXA). OXA-induced peripheral neuropathy (OIPN) is a challenging condition due to diagnostic complexities and a lack of effective treatment. In this study, we investigated the antiallodynic effect of β-caryophyllene (BCP), a cannabinoid type 2 (CB2) receptor agonist, in a mouse model of OIPN. BCP treatment inhibited OXA-induced mechanical and cold allodynia in both preventive and therapeutic drug treatment regimens. Experiments with the CB2 receptor agonist GW405833 confirmed the role of CB2 receptors in OIPN. The CB2 antagonist SR144528 abrogated the anti-nociceptive effect of BCP on mechanical allodynia, without impacting OXA-induced sensitivity to cold. BCP decreased neuroinflammation, as inferred from TNF, IL-1β, IL-6, and IL-10 profiling, and also reduced ROS production, lipid peroxidation, and 4-hydroxynonenal protein adduct formation in the spinal cords of OXA-treated mice. BCP did not affect the antitumor response to OXA or its impact on blood cell counts, implying that the cytotoxicity of OXA was preserved. These results underscore BCP as a candidate drug for OIPN treatment via CB2 receptor-dependent mechanisms, and anti-inflammatory and antioxidant responses in the spinal cord.

Keywords: neuroinflammation; neuropathic pain; oxaliplatin; oxidative stress; phytocannabinoids.

Figure 1

BCP inhibits mechanical and cold allodynia induced by OXA in the preventive treatment scheme. (A) Schematic representation of the experimental protocols performed in this study (described in the Section 2). (B) von Frey, (C) cold plate, and (D) Hargreaves tests; (E) tumor growth kinetics; and (F) white blood cell count (WBC) in Ehrlich tumor-implanted mice treated with OXA combined with different doses of BCP (10, 50, 100, and 250 mg/kg, 1×/day, gavage) in the preventive treatment scheme. Baseline thresholds were taken on protocol day −1, and OXA and BCP treatments started on day 0. Legend: Control (vehicle-treated); OXA (oxaliplatin); BCP “X” (β-caryophyllene; “X” represents the dose in mg/kg). * Different from the vehicle/control group in (B–D,F) and different from all other groups in (E), considering the same experimental time point; ^# different from the OXA group at the same experimental time point (ANOVA; p < 0.05, n = 8/group).

Figure 2

BCP inhibits OXA-induced mechanical and cold allodynia in the therapeutic protocol. (A) von Frey and (B) cold plate tests in Ehrlich tumor-implanted mice treated with OXA combined with different doses of BCP (25, 50, and 100 mg/kg, 1×/day, gavage) in the therapeutic scheme. Baseline thresholds were taken on protocol day −1, OXA treatment started on day 0, and BCP started on day 6 (detailed in Figure 1A and Section 2). (C) Short-term effect (1, 3, 6, and 12 h) of a single dose of BCP (gavage) on mechanical allodynia in tumor-bearing mice with established OIPN. Using the therapeutic protocol, on day 6, mechanical nociception was monitored using the von Frey test after the first administration of different doses of BCP. (D) Tumor volume/growth kinetics, (E) tumor weight in grams at the end of the experiment, and (F) body weight change in tumor-implanted mice treated with OXA + BCP combination in the therapeutic scheme. Body weight per mouse was expressed as a percentage compared to respective weight on protocol day 0. Legend: Control (vehicle-treated); OXA (oxaliplatin); BCP “X” (β-caryophyllene; “X” represents the dose in mg/kg). * Different from the vehicle/control group in (A–C,E,F), and different from all other groups in (D), considering the same experimental time point; ^# different from the OXA group at the same experimental time point (ANOVA; p < 0.05; n = 8/group).

Figure 3

Role of CB2 receptor and PPARγ in BCP anti-nociceptive effect. von Frey tests showing the effect of the (A) CB2 agonist GW405833 (1mg/kg; i.p. daily), (B) BCP combined with the CB2 receptor antagonist SR144528 (1 mg/kg, i.p. daily), and (C) BCP combined with the PPARγ antagonist GW9662 (1 mg/kg, i.p. daily) in the therapeutic protocol. OXA treatment started on day 0, and BCP (100 mg/kg, gavage, daily) with or without CB2 and PPAR antagonists started on protocol day 6 (see Figure 1A and Section 2 for details). Data shown in panels (A–C) were collected from the same experiment, and split to ease visualization of the tested agonist/antagonists. Note that the “control” and “OXA” groups’ data are the same in the (A–C) panels. (D) Short-term effect (1, 3, 6, and 12 h) of a single dose of BCP (100 mg/kg, gavage) combined with SR144528 (1 mg/kg, i.p. daily) or GW9662 (1 mg/kg, i.p. daily) in animals with established neuropathy caused by OXA. The effect of the CB2 agonist GW405833 (1 mg/kg; i.p. daily) in OXA-treated mice is also shown. Using the therapeutic scheme, on protocol day 6, mechanical nociception was monitored using the von Frey test after the first administration of BCP with/without antagonists. (E) Cold plate test showing the effect of CB2 and PPARγ antagonists combined with BCP, and of the CB2 agonist GW405833, upon cold sensitivity at baseline (protocol day −1) and different time-points in the therapeutic model. (F) Body weight changes in mice treated with OXA/BCP in the presence or absence of CB2 and PPARγ antagonists. Body weight per mouse was expressed as a percentage compared to respective weight on protocol day 0. * Different from the vehicle/control group, ^# different from the OXA group, and ^& different from the OXA + BCP group at the same experimental time point (ANOVA; p < 0.05; n = 8/group).

Figure 4

BCP suppresses OXA-induced inflammatory cytokine production and spinal cord oxidative damage. (A) ELISA assay quantification of TNF, IL-1β, IL-10, and IL-6 levels, (B) ex vivo DCF assay, (C) TBARS, and (D) 4-HNE content in the spinal cords of tumor-bearing animals treated with OXA with/without BCP (100 mg/kg, gavage, daily) (n = 6–7/group). OXA treatment started on day 0, BCP (100 mg/kg, gavage, daily) started on day 6, and spinal cord tissues were isolated on protocol day 15 for cytokine and oxidative stress evaluation. In (A), the dashed line denotes the assay sensitivity cut-off. * Different from vehicle/control group, ^# different from OXA group, and ^& different from both OXA and control groups, considering the same experimental time point (ANOVA; p < 0.05).