The Multitarget Antinociceptive Compound Affinin and Its Effects on Hypothermia, Hypolocomotion, and Sickness Behavior in Lipopolysaccharide-Treated Mice

Abstract

Affinin (spilanthol) is the main bioactive alkylamide present in Heliopsis longipes roots, exerting antinociceptive and anti-inflammatory effects that involve the activation of TRP channels. Previous studies indicated that affinin reduces the LPS-induced increase in pro-inflammatory cytokine production in murine macrophages. However, no studies have evaluated whether affinin produces antinociceptive, anti-inflammatory, and behavioral effects in experimental animals treated with LPS, nor has the mechanism of action involved in these pharmacological effects been established. The present study evaluated whether affinin induces hypothermia, catalepsy, hypolocomotion, and analgesia and, moreover, whether the analgesia involves the activation of the CB1 cannabinoid receptor and TRPV1 and TRPA1 channels. Subsequently, the anti-inflammatory activity and behavioral effects induced by affinin (20 mg/kg) in mice were evaluated via LPS (2.5 mg/kg)-induced hypothermia. The results of the experiments indicate that the analgesic effect of affinin involves the activation of the CB1 cannabinoid receptors and the TRPV1 and TRPA1 channels. Additionally, affinin reduced the severity of LPS-induced hypothermia and attenuated the increase in TNF-α and IL-6 levels in serum. The results obtained demonstrate that affinin induces antinociceptive, anti-hypothermic, and anti-inflammatory activities, which involve the CB1 receptor and the TRPV1 and TRPA1 channels and the suppression of pro-inflammatory cytokines.

Keywords: CB1; N-alkylamide; TRPA1; TRPV1; affinin; analgesia; anti-inflammatory; hypothermia.

Figure 1

(A) Representative images of the trajectories (purple line) followed by the mice, the square indicates the center of the OFT; (B) heat maps of the effects of affinin on the OFT; (C) the total distance traveled for three minutes (cm); (D) total immobile time (s). Data were obtained from videos recorded and analyzed using the ANY-maze software. VEH—vehicle; AF—affinin, administered at 10, 20, and 40 mg/kg. Values are mean ± SD (n = 5); p > 0.05 using a one-way ANOVA followed by Tukey’s post hoc test.

Figure 2

(A) Delta (Δ) of body temperature (°C); (B) catalepsy (s). VEH—vehicle; WIN 55—WIN 55,212-2; AF—affinin (at 10, 20, and 40 mg/kg). Values are mean ± SD (n = 5); ^# p < 0.0001 using a one-way ANOVA followed by Tukey’s post hoc test.

Figure 3

(A) Latency to falling in the rotarod test (s); (B) analgesia (s). VEH—vehicle; WIN 55—WIN 55,212-2; AF—affinin at 10, 20, and 40 mg/kg. Values are mean ± SD (n = 5); ^# p < 0.0001 vs. VEH using a one-way ANOVA followed by Tukey’s post hoc test.

Figure 4

The analgesic effect of affinin. (A) One hour after the administration of affinin, (B) 2 h after the administration of affinin, and (C) the area under the curve (AUC). Values are expressed as the mean ± SD (n = 5); ** p < 0.001 using one-way ANOVA followed by Tukey’s post hoc test.

Figure 5

The effect of the prior administration of rimonabant (RMB), HC-030031 (HC), and capsazepine (CZP) on the analgesic effect of affinin (AF20) in the hot plate test. VEH—vehicle. Values are expressed as the mean ± SD (n = 5); ^# p < 0.0001 vs. AF20, * p < 0.01 using a one-way ANOVA followed by Tukey’s post hoc test.

Figure 6

(A) Representative images of the trajectories (purple line) followed by the mice, the square indicates the center of the OFT; (B) heat maps of the effects of LPS and affinin on the OFT; (C) total distance traveled in the OFT (cm); (D) total immobile time (s). VEH—vehicle; LPS—lipopolysaccharide; AF20—affinin (20 mg/kg). Values are mean ± SD (n = 6); ^# p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05 using a one-way ANOVA followed by Tukey’s post hoc test. Data were obtained via video recordings and analyzed using the ANY-maze software.

Figure 7

(A) Change in body temperature over time (h); (B) area under the curve (AUC) showing the body temperature change over four hours. VEH—vehicle; LPS—lipopolysaccharide; AF20—affinin at 20 mg/kg. Data show mean ± SD (n = 5); ^# p < 0.0001; ** p < 0.001 using a one-way ANOVA followed by Tukey’s post hoc analysis.

Figure 8

Levels of pro-inflammatory cytokines in the serum of BALB/c mice. (A) IL-1β; (B) TNF-α; (C) IL-6. VEH—vehicle; AF20—affinin (20 mg/kg); LPS—lipopolysaccharide. The values are expressed as the ratio of the mean VEH + SD (VEH = 1) (n = 6), ^# p < 0.0001 vs. VEH, *** p < 0.001, ** p < 0.01, * p < 0.05 using a one-way ANOVA followed by Tukey’s post hoc test.

Figure 9

The pathway involved in the anti-inflammatory effect of affinin. Affinin attenuates hypothermia caused by LPS by preventing the exacerbated production of pro-inflammatory cytokines. CB1—cannabinoid type 1 receptor; POA—hypothalamic preoptic area; IKK—IkB-kinase; IkB—inhibitor of kappa B; IL-1β—interleukin-1β; IL-6—interleukin-6; LPS—lipopolysaccharide; NEMO—NF-κB essential modulator; NF-kB—nuclear factor kappa-light-chain-enhancer B cells; PI3K—phosphatidylinositol-3-kinase; ROS—reactive oxygen species; TLR4—toll-like receptor 4; TRPA1—transient receptor potential ankyrin 1 channel; TRPV1—transient receptor potential vanilloid 1 channel; TNF-α—tumor necrosis factor-α.

Figure 10

A diagram of individual experiments: (A) OFT; (B) cannabinoid tetrad test; (C) LPS-induced hypothermia. The groups are labeled as follows: 1. VEH—vehicle; 2. WIN 55—WIN 55,212-2; 3. AF10—affinin (10 mg/kg); 4. AF20—affinin (20 mg/kg); 5. AF40—affinin (40 mg/kg); 6. Antagonist + AF20; 7. LPS—lipopolysaccharide.