Intraplantar β-Caryophyllene Alleviates Pain and Inflammation in STZ-Induced Diabetic Peripheral Neuropathy via CB2 Receptor Activation

Abstract

Diabetic peripheral neuropathy (DPN) is a debilitating complication of diabetes, characterized by mechanical allodynia, neuroinflammation, and oxidative stress. Current treatments offer limited efficacy and are often associated with systemic side effects. Emerging evidence suggests that activation of cannabinoid receptor type 2 (CB₂) may represent a promising target for managing neuropathic pain and inflammation. This study investigates the therapeutic potential of intraplantar β-Caryophyllene (BCP), a selective CB₂ receptor agonist, administered as a topical intervention in a streptozotocin (STZ)-induced DPN mouse model. Hyperglycemia was induced by STZ injections, and diabetic mice received intraplantar BCP (9, 18, or 27 µg) daily for 21 days. Mechanical allodynia was assessed using von Frey filaments, and levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and oxidative stress markers (MDA, SOD, CAT) were quantified in hind paw tissues. BCP dose-dependently alleviated STZ-induced mechanical allodynia, with the 27 µg dose producing the most pronounced effect (p < 0.001). The anti-allodynic effects of BCP were mediated through CB₂ receptor activation, confirmed by reversal with the CB₂ antagonist AM630 (p < 0.001), while the CB₁ antagonist AM251 had no significant impact. In addition, BCP significantly reduced pro-inflammatory cytokines (p < 0.01) and oxidative stress markers (p < 0.001) while restoring antioxidant enzyme activities (p < 0.05). A control group treated with a clinically available topical analgesic cream containing capsaicin 0.075% exhibited limited efficacy. These findings position topical BCP administration as a novel therapeutic strategy for DPN, offering sustained pain relief and modulation of neuroinflammatory and oxidative pathways with minimal systemic exposure. Further clinical studies are warranted to validate its potential for translation into therapeutic practice.

Keywords: STZ-induced neuropathy; cannabinoid receptors 2; diabetic peripheral neuropathy; β-Caryophyllene.

Figure 1

Schematic representation of the experimental timeline. This diagram illustrates the experimental timeline, outlining the procedures for diabetes induction and BCP treatment. Mice were induced with diabetes via STZ injections (90 mg/kg, i.p.). Starting on day 14, daily intraplantar (i.pl.) injections of β-Caryophyllene (BCP, 27 µg) were administered for 21 days to six groups: non-diabetic, diabetic + vehicle, diabetic + BCP, diabetic + BCP + AM251 (CB₁ antagonist), diabetic + BCP + AM630 (CB₂ antagonist), and diabetic + topical analgesic cream with capsaicin 0.075% (Zostrix^® HP). Behavioral and functional parameters, including mechanical allodynia (assessed by the Von Frey test), fasting blood glucose levels, and body weight, were evaluated at baseline (before STZ injection) and then on days 7, 14, 21, 28, and 35. On day 35, mice were sacrificed, and hind paw skin was collected to analyze oxidative stress biomarkers and pro-inflammatory cytokines. The figure was created with BioRender.com.

Figure 2

Effects of STZ-induced diabetes and BCP treatment on blood glucose and body weight. (A) STZ-induced diabetes resulted in glucose levels exceeding 250 mg/dL. BCP treatment, either alone or with AM251 (CB₁ antagonist) or AM630 (CB₂ antagonist), did not significantly alter glucose levels in diabetic mice. Non-diabetic controls maintained normal glucose levels. (B) Diabetic mice showed significant weight loss starting on day 7 post-STZ injection, with this reduction persisting throughout the study. BCP and antagonist treatments had no significant impact on body weight. Data are presented as mean ± SEM (n = 8 per group). Statistical analysis: Two-way ANOVA with Tukey’s post-hoc test. ns, not significant, ** p < 0.01, *** p < 0.001 vs. non-diabetic controls.

Figure 3

Time course of mechanical withdrawal thresholds in STZ-induced diabetic mice. Mechanical allodynia was assessed using von Frey filaments in STZ-induced diabetic and non-diabetic control mice. Diabetic mice exhibited a significant reduction in paw withdrawal thresholds starting from day 7 post-STZ injection, with the reduction becoming more pronounced by day 14 and persisting through day 35. Non-diabetic controls maintained stable thresholds throughout the study. Thresholds are expressed in grams (g) and represent mean ± SEM (n = 8 per group). Statistical analysis: Two-way ANOVA with Bonferroni correction. ns, not significant, ** p < 0.01, *** p < 0.001 vs. non-diabetic controls.

Figure 4

Effects of localized β-Caryophyllene (BCP) injections on mechanical allodynia in diabetic mice. (A) Dose-dependent effects of intraplantar (i.pl.) administration of BCP (8, 18, and 27 µg) on paw withdrawal thresholds in diabetic mice. Paw withdrawal thresholds were measured over 90 min post-BCP injection on day 14 after STZ injection. *** p < 0.001 compared to non-diabetic controls; ~~~ p < 0.001 compared to diabetic controls; ns indicates no significant difference compared to diabetic controls. (B) Effects of CB receptor antagonists on BCP (27 µg)-induced alleviation of mechanical allodynia. Diabetic mice were pretreated with AM630 (CB₂ antagonist) or AM251 (CB₁ antagonist) 30 min before BCP injection. As a positive control, mice were treated with a topical analgesic cream containing capsaicin 0.075%. Data are presented as mean ± SEM (n = 8 per group). Statistical analysis: two-way ANOVA with Tukey’s post-hoc test. ns, not significant, *** p < 0.001, compared to diabetic controls; ns indicates no significant difference compared to diabetic controls; ~~~ p < 0.001 compared to BCP (27 µg)-treated diabetic mice. Statistical analysis: two-way ANOVA with Tukey’s post-hoc test.

Figure 5

Sustained anti-allodynic effects of chronic topical BCP injections in STZ-induced DPN mouse model. Daily intraplantar (i.pl.) injections of β-caryophyllene (BCP) (27 µg) significantly alleviated mechanical allodynia in diabetic mice compared to vehicle-treated diabetic controls on days 7, 14, and 21. In contrast, topical analgesic cream containing capsaicin 0.075% showed a significant effect only on day 21. CB₂ receptor antagonist AM630 pretreatment abolished this effect, while CB₁ receptor antagonist AM251 had no significant impact. Non-diabetic mice maintained higher thresholds throughout the study. Data are presented as mean ± SEM (n = 8 per group). Statistical analysis: two-way ANOVA with Tukey’s post-hoc test. ns, not significant, * p < 0.05, *** p < 0.001 compared to diabetic controls; ns indicates no significant difference compared to diabetic controls; ~~~ p < 0.001 compared to BCP (27 µg)-treated diabetic mice.

Figure 6

Topical intraplantar (i.pl.) BCP administration modulates pro-inflammatory cytokines in hind paw skin tissues of STZ-induced DPN mouse model. (A) Tumor necrosis factor-alpha (TNF-α), (B) interleukin-1β (IL-1β), and (C) interleukin-6 (IL-6) levels were assessed in hind paw tissues of non-diabetic and diabetic mice using ELISA. Diabetic mice exhibited significantly elevated cytokine levels compared to non-diabetic controls. Topical i.pl. treatment with BCP (27 µg) significantly reduced these cytokine levels. Pretreatment with the CB₂ antagonist AM630 abolished the anti-inflammatory effects of BCP, while the CB1 antagonist AM251 had no significant impact. Topical capsaicin 0.075% significantly reduced TNF-α and IL-6 levels but did not affect IL-1β. Data are expressed as mean ± SEM (n = 8 per group). Statistical analysis: one-way ANOVA with Tukey’s post-hoc test. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to diabetic controls; ns indicates no significant difference compared to diabetic controls; ~~ p < 0.01, ~ p < 0.05 compared to BCP (27 µg)-treated diabetic mice.

Figure 7

Effect of topical β-Caryophyllene (BCP) administration on oxidative stress biomarkers in hind paw skin tissues of STZ-induced DPN mouse model. (A) malondialdehyde (MDA) levels, (B) superoxide dismutase (SOD) activity, and (C) catalase (CAT) activity were measured in skin homogenates from hind paw tissues. BCP treatment significantly restored SOD and CAT activities while reducing MDA levels in diabetic mice. These effects were reversed by pretreatment with AM630 (CB₂ receptor antagonist), confirming CB₂-mediated mechanisms. AM251 (CB₁ receptor antagonist) had no significant impact. Topical capsaicin 0.075% did not alter oxidative stress markers. Statistical analysis: one-way ANOVA with Tukey’s post-hoc test. *** p < 0.001, ** p < 0.01, * p < 0.05 compared to diabetic controls; ns indicates no significant difference compared to diabetic controls; ~~ p < 0.01, ~ p < 0.05 compared to BCP (27 µg)-treated diabetic mice. Data are presented as mean ± SEM (n = 8 per group).