The exogenous administration of CB2 specific agonist, GW405833, inhibits inflammation by reducing cytokine production and oxidative stress

Abstract

The present study aimed to investigate the role of cannabinoid 2 (CB2) receptors in a rat model of acute inflammation. Therefore, the potential of anti-inflammatory effects of CB2 receptor agonist (GW405833), CB2 receptor antagonist (AM630), and diclofenac, were investigated in carrageenan induced paw oedema in rats: as were assessed by measuring paw oedema; myeloperoxidase (MPO) activity in paw tissue; malondialdehyde (MDA) concentration; glutathione (GSH) level in paw tissue for oxidant/antioxidant balance; cytokine (interleukin-1β, IL-1β; tumour necrosis factor-α, TNF-α) levels in serum; histopathology of paw tissue for inflammatory cell accumulations. The results showed that GW405833 or diclofenac significantly reduced carrageenan-induced paw oedema. GW405833 also inhibited the increase of MPO activity, the recruitment of total leukocytes and neutrophils, and MDA concentration during carrageenan-induced acute inflammation, along with reversed nearly to the normal levels the increased of TNF-α, and IL-1β in serum. AM630 did not affect inflammation alone however clearly reversed the effects of agonist when co-administered. The mechanism of GW405833's suppression of inflammation is supported by these results, which are achieved by the inhibition of neutrophil migration, which regulates the reduction of oxidative stress, TNF-α and IL-1β levels. Finally, the activation of CB2 receptor, by selective agonist, has a major role in peripheral inflammation, and in the near future, targeting the peripheral cannabinoid system as a promising alternative to treat inflammation diseases may be considered a novel pharmacologic approach.

Keywords: CB2 receptor agonist; Carrageenan; GW405833; Paw oedema; oxidative stress.

Figure 1.

Effects of GW405833 (CAR+AGO), AM630 (CAR+ANTA), their combination (CAR+AGO+ANTA) and diclofenac (CAR+DIC) on the carrageenan-induced paw edema formation. Rats were evaluated for paw edema at 0, 1, 2, 3 and 4 h post-carrageenan injection. Results were expressed as percentage increase in paw thickness. Each point represents the mean ± SEM of six rats. *Statistically significant compared with carrageenan group at P<0.05. AGO, agonist; ANTA, antagonist; DIC, diclofenac; CAR, carrageenan.

Figure 2.

Effects of GW405833 (CAR+AGO), AM630 (CAR+ANTA), their combination (CAR+AGO+ANTA) and diclofenac (CAR+DIC) on serum (A)TNF-α and (B) IL-1β activities in carrageenan-induced paw inflammation of rat (4th hour). Each point represents the mean ± SEM of rats (n=6). *Statistically significant compared with saline control group, ^#statistically significant compared with carrageenan group, and ⁺statistically significant compared with agonist-treated group at P<0.05. TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin 1β. AGO, agonist; ANTA, antagonist; DIC, diclofenac; CAR, carrageenan.

Figure 3.

Effects of GW405833 (CAR+AGO), AM630 (CAR+ANTA), their combination (CAR+AGO+ANTA) and diclofenac (CAR+DIC) on (A) MDA and (B) GSH levels in carrageenan-injected paw tissues (4th hour). Each point represents the mean ± SEM of rats (n=6). *Statistically significant compared with saline control group, ^#statistically significant compared with carrageenan group, and ⁺statistically significant compared with agonist-treated group at P<0.05. GSH, glutathione; MDA, malondialdehyde. AGO, agonist; ANTA, antagonist; DIC, diclofenac; CAR, carrageenan.

Figure 4.

Effects of GW405833 (CAR+AGO), AM630 (CAR+ANTA), their combination (CAR+AGO+ANTA) and diclofenac (CAR+DIC) on MPO activity in carrageenan-injected paw tissues (4th h). Each point represents the mean ± SEM of rats (n=6). *Statistically significant compared with saline control group, ^#statistically significant compared with carrageenan group, and ⁺statistically significant compared with agonist-treated group at P<0.05. MPO, myeloperoxidase. AGO, agonist; ANTA, antagonist; DIC, diclofenac; CAR, carrageenan.

Figure 5.

Mean numerical density of inflammatory cells in rat paw tissues (cell/mm²). Each point represents the mean ± SEM of rats (n=6). *Statistically significant compared with saline control group, ^#statistically significant compared with carrageenan group, and ⁺statistically significant compared with agonist-treated group at P<0.05.

Figure 6.

Histological changes in oedema paws 4 h after injection of carrageenan. Light micrograph of rat paw in all studied groups. Paws were harvested 4 h after injection of carrageenan and subjected to histochemical staining of paw tissues. Histopathological pictures of all groups are summarized in Fig. 6. Saline control group was showing no histopathological changes. The amount of inflammatory cells in the carrageenan group was higher than that of the saline control group and agonist or diclofenac groups. There is a slight difference between saline control and treatments group in term of tissue content. The amount of inflammatory cells in the antagonist and agonist+antagonist groups were higher than that of the saline control group and agonist or diclofenac groups. The amount of inflammatory cells in the vehicle group was higher than that of the saline control group and agonist or diclofenac groups. H&E staining, the magnifications of images a, b, and c are at ×4, ×10 and ×40, respectively. Epi, epidermis; Der, dermis; AT, adipose tissue; SG, Sweet gland; Fb, fibroblast; bv, blood vessel; Ef, elastic fiber; Cf, collagen fiber. IA, inflammation area; Rbc, red blood cell; Ic, inflammatory cell; MC, macrophage like cell. Ac, adipose cell; MT, muscle tissue. (A) Saline control group; (B) carrageenan model group; (C) diclofenac group; GW405833, (D) CB2 agonist group; (E) AM630, CB2 antagonist group; (F) CB2 agonist+antagonist group; and (G) Vehicle group. AGO: Agonist, ANTA, antagonist; DIC, diclofenac; CAR, Carrageenan, Vehicle, dimethyl sulfoxide.