β-caryophyllene and docosahexaenoic acid, isolated or associated, have potential antinociceptive and anti-inflammatory effects in vitro and in vivo

Abstract

Inflammation is a complex biological response involving the immune, autonomic, vascular, and somatosensory systems that occurs through the synthesis of inflammatory mediators and pain induction by the activation of nociceptors. Staphylococcus aureus, the main cause of bacteremia, is one of the most common and potent causes of inflammation in public health, with worse clinical outcomes in hospitals. Antioxidant substances have been evaluated as alternative therapeutic analgesics, antioxidants, anti-inflammatory agents, antitumor agents, and bactericides. Among these, we highlight the essential oils of aromatic plants, such as β-caryophyllene (BCP), and polyunsaturated fatty acids, such as docosahexaenoic acid (DHA). The objective of this study was to evaluate the biological activities of BCP-DHA association in in vitro and in vivo experimental models of antinociception and inflammation. To determine the anti-inflammatory effects, monocytes isolated from the peripheral blood of adult male volunteers were infected with methicillin-resistant S. aureus and incubated with treatment for cytokine dosage and gene expression analysis. Antinociceptive effects were observed in the three models when comparing the control (saline) and the BCP-DHA treatment groups. For this purpose, the antinociceptive effects were evaluated in animal models using the following tests: acetic acid-induced abdominal writhing, paw edema induced by formalin intraplantar injection, and von Frey hypernociception. There was a significant reduction in the GM-CSF, TNFα, IL-1, IL-6, and IL-12 levels and an increase in IL-10 levels in the BCP-DHA treatment groups, in addition to negative regulation of the expression of the genes involved in the intracellular inflammatory signaling cascade (IL-2, IL-6, IRF7, NLRP3, and TYK2) in all groups receiving treatment, regardless of the presence of infection. Statistically significant results (p < 0.05) were obtained in the acetic acid-induced abdominal writhing test, evaluation of paw edema, evaluation of paw flinching and licking in the formalin intraplantar injection model, and the von Frey hypernociception test. Therefore, BCP and DHA, either administered individually or combined, demonstrate potent anti-inflammatory and antinociceptive effects.

Figure 1

Concentration of GM-CSF (A), TNFα (B), IL-1β (C), IL-6 (D), IL-10 (E), and IL-12 (F) cytokines in the peripheral blood of male volunteers. C = control (sterile saline); BCP = beta-caryophyllene; DHA = docosahexaenoic acid; B/D = BCP and DHA association; LPS = lipopolysaccharides; C80 = strain C80; IT B = infected and treated with BCP; IT D = infected and treated with DHA; IT B/D = infected and treated with BCP and DHA association; TI B = treated with BCP and infected; TI D = treated with DHA and infected; TI B/D = treated with BCP and DHA association and infected. Data are expressed as mean ± DPM. a p < 0.05 vs. C; b p < 0.05 vs. LPS; c p < 0.05 vs. C80.

Figure 2

Comparison of the gene expression of the treated group with the association of β-caryophyllene and docosahexaenoic acid (50 + 50 mg/dL) with the infected-only group with methicillin-resistant Staphyloccocus aureus (A), the group infected with methicillin-resistant Staphyloccocus aureus and the group treated with BCP-DHA (B) and the group treated with BCP-DHA and infected with methicillin-resistant Staphyloccocus aureus (C). Data are expressed as mean ± DPM. *p < 0.05.

Figure 3

Number of abdominal writhing induced by acetic acid in Balb-C mice. (A) Female mice. (B) Male mice. (C) Comparison between sexes. S = saline; BCP = beta-caryophyllene; DHA = docosahexaenoic acid; B/D = BCP and DHA combination; I = indomethacin. Data are expressed as mean ± DPM. a p < 0.05 vs. S; b p < 0.05 vs. I; c p < 0.05 vs. BCP; *p < 0.05.

Figure 4

Number of flinches exhibited by Balb-C mice after intraplantar formalin administration (1.5%). (A) Females in 0–5 min period. (B) Females in 530 min period. (C) Males in 0–5 min period. (D) Males in 5–30 min period. (E) Comparison between sexes in the period 0–5 min. (F) Comparison between sexes in the period 0–5 min. S = saline; BCP = beta-caryophyllene; DHA = docosaehexanoic acid; BD = BCP and DHA combination; M = morphine. Data are expressed as mean ± DPM. a p < 0.05 vs. S; b p < 0.05 vs. M; c p < 0.05 vs. BCP; d p < 0.05 vs. BD; *p < 0.05.

Figure 5

Paw weights of Balb-C mice that received formalin intraplantar injection (1.5%). (A) Female mice. (B) Male mice. (C) Comparison between sexes. S = saline; BCP = beta-caryophyllene; DHA = docosaehexanoic acid; B/D = BCP and DHA association; M = morphine. Data are expressed as mean ± DPM. a p < 0.05 vs. S.

Figure 6

Nociception intensity by ∆ limit of the paw removal after Carrageenan injection (100 µg) in Balb-C mice. (A) Female mice. (B) Male mice. (C) Comparison between sexes. S = saline; BCP = beta-caryophyllene; DHA = docosahexaenoic acid; BD = BCP and DHA association. Data are expressed as mean ± DPM. a p < 0.05 vs. S; b p < 0.05 vs. BCP; * p < 0.05.

Figure 7

Anti-inflammatory characterization of the BCP-DHA treatment in the face of MRSA infection. The recognition of S. aureus by toll-like receptors present in the cell membrane initiates an intracellular signaling cascade that results in the dissociation of the NF-κB/Iκb complex, NF-κB translocation to the cell nucleus, and stimulation of the synthesis of pro-inflammatory cytokines. BCP-DHA treatment acts through inhibiting different pathways that result in the activation of NF-κB, negatively regulating the expression of the MD2, IRF7, NLRP3, and TYK2 genes, which culminates in lower synthesis and acting of pro-inflammatory cytokines.