The formalin test in mice is a reliable and valid model of nociception. The noxious stimulus was injected with 1% formalin in saline under the right hide paw. In this study, we first found that the treatment of CBD cream effectively decreases nociceptive behaviors in mice. Moreover, we found that the treatment of 1% CBD combination with 4% levomenthol cream effectively decreases nociceptive behaviors and higher treatment with 1% CBD cream alone in the early phase. In addition, we found that 4% levomenthol was a synergistic antinociception response of 1% CBD in the early phase (neurogenic pain) while the late phase (inflammatory pain) was antagonistic 1% CBD. Levomenthol is a cooling effect and vasoactive agent 21, acting as a sodium channel blocker. 22 Low concentration of levomenthol depresses cutaneous nociception and may even desensitize nociceptive C-afferent fibers 23, but it did not have anti-inflammatory activity as shown in the late phase.
The nociceptive mechanism of the formalin test produces the inflammatory mediator’s distinct biphasic phases. The early phase of the formalin test represents acute pain and occurs immediately after formalin injection. The nociceptor mediators, including bradykinin, glutamate, and substant P activate the nociceptor through C fiber 18, 24. The late phase of the formalin test activates inflammatory mediators, including prostaglandins, histamine, bradykinin, serotonin, and substance P 25. Moreover, inflammatory mediators of the late phase were activated transient receptor potential subfamily A (TRPA) and transient receptor potential vanilloid (TRPV) indirectly or directly via the activation of downstream signaling pathways 26, 27. The meta-analysis and a systematic review of modulators of the endocannabinoid system found verity in CBD treatment outcomes based on the inflammatory and pain model. The studies CBD has significantly decreased nociceptive behaviors with mixed results in neuropathic and inflammatory pain models 28, 29. Other pre-clinical and clinical studies have found CBD to be a promising antinociceptive agent for decreasing neuropathic and inflammatory pain 22, 30–34.
The acetic acid-induced writhing test, which model represents a nociceptive chemical test. This test is widely employed to measure peripheral analgesic activity or visceral inflammatory pain 35. Nociception is intraperitoneal injection with acetic acid and leads to the stimulation of nociceptive neurons by increasing inflammatory mediators such as histamine, prostaglandin, and bradykinin in peritoneal fluid. The inflammatory mediators stimulate peripheral nociceptive receptors and nociceptive behavior such as abdominal constrictions, pelvic rotation, and subsequent stretching of at least one hind limb 36 37. However, the nociceptive behavior was probably similar to the result of peritonitis 38. The study found that CBD cream had an anti-inflammatory effect and decreased nociceptive behavior in the mice model. However, the effect of combination with 4% levomenthol cream is not synergistic CBD to reduce nociceptive behavior.
Carrageenan-induced inflammation is a well-established inflammation model for evaluating the acute anti-inflammatory 32, 39. This method is susceptible to Non-steroidal anti-inflammatory drugs (NSAIDs) and has been accepted as helpful in investigating new anti-inflammatory drugs 40. The effect of carrageenan has been a descript biphasic event that involves various inflammatory agents. The first phase (0–2 h after carrageenan injection) is rapid-release pro-inflammatory mediators such as serotonin, histamine, and bradykinin 41. The second phase (3–6 h after carrageenan injection) is associated with the release of prostaglandins, inflammatory cytokines, nitric oxide, and other COX products respectively 42. The effect of paw edema occurs immediately and reaches a maximum in the third hour 43, 44.
The study found that 1% CBD cream had rapid onset to decrease paw edema at 1 h and remained the anti-inflammatory effect until 4 h. In contrast, 1% diclofenac cream decreased paw edema at 3 h. Moreover, the literature review of CBD has been shown to be a potential anti-inflammatory and analgesic in animal models 45, 46. CBD provides various anti-inflammatory mechanisms and regulates immune cell functions and cell cycle 47. CBD could suppress products of pro-inflammatory cytokines, such as interleukin-1 beta (IL-1β), tumor necrosis factor (TNF)-α, growth factors, chemokines, as well as inhibition of immune cell, migration, activation, maturation, proliferation, and antigen presentation 48, 49. CBD also showed potential action of oxidative inhibition stress, modulating the expression of inducible nitrotyrosine and nitric oxide synthase (iNOS) and decreasing the production of reactive oxygen species (ROS) 50. However, the mechanism of topical CBD did not been well-identify yet, but it might be an alternative drug for relieving pain and anti-inflammatory conditions 51, 52.