*2.1. PGE2 Is Responsible for the Long-Lasting Antinociceptive Effect Induced by Crotalphine*

The data show that the magnitude of the crotalphine antinociceptive effect depends on tissue inflammation [8]. Therefore, we first characterized the effect of one single systemic administration of crotalphine on naïve rats and rats sensitized with an intraplantar injection of Prostaglandin E2 (PGE2) at day one. The mechanical threshold was assessed 1 h after crotalphine (3 h after PGE2) and every day for 8 days (192 h) (Figure 1A). The intraplantar injection of PGE2 (100 ng/paw) induced a significant decrease in the mechanical threshold with a peak response occurring 3 h after the administration, compared with the basal values obtained before any treatment, which characterize hypersensitivity. The systemic administration of crotalphine (20 ng/kg or 1 μg/kg, p.o.) blocked the PGE2-induced hypersensitivity and increased the nociceptive threshold of the animals when compared with the basal values (analgesia). This effect is detected at 3 h and lasts until 120 h (5 days) after one single administration of crotalphine (Figure 1B). Next, we tested the crotalphine effect in non-sensitized (naïve) rats. A single administration of crotalphine (20 ng/kg and 1 μg/kg, p.o.) induces a significant increase in the mechanical nociceptive threshold (analgesia), starting at 3 h and lasting until 5 h after treatment, when compared with the baseline. A lower dose of crotalphine (8 pg/kg, p.o.) did not change the mechanical threshold (Figure 1C). As expected, no difference in the nociceptive threshold was detected in rats treated with saline (control). Together, these results demonstrate that a single administration of PGE2 enhances and prolongs crotalphine-induced analgesia.

#### *2.2. Crotalphine Increases the Spinal ERK and JNK and Decreases p38 Activation*

In order to investigate the potential mechanisms involved in the antinociception induced by systemic crotalphine, we sought to determine whether crotalphine interferes with the expression and phosphorylation of ERK, JNK and p38 (Figure 2A). Importantly, since the inhibition of MEK, an up-stream MAPK kinase, blocks PGE2 induced hyperalgesia, the following experiments were conducted in naïve rats, i.e., without sensitization to focus on the mechanism involved in crotalphine-induced analgesia [7]. Moreover, we selected the 1 μg/kg dose of crotalphine for the next experiments.

**Figure 1.** Effect of crotalphine in the mechanical nociceptive threshold of sensitized and non-sensitized rats. (**A**) Schematic representation of treatment and rat paw pressure test. (**B**) PGE2 (100 ng/paw) was injected 2 h before the oral crotalphine administration and nociceptive threshold was assessed before (0) and 3, 24, 48, 72, 96, 120, 144, 168 and 198 h after the PGE2 injection. Data are presented as mean ± SEM. \* significantly different from baseline. # significantly different from the saline + PGE2 group, *n* = 6 per group (*p* < 0.05). (**C**) Nociceptive threshold was obtained before (0) and 1, 3, 5, 24, 48, 72, 96, 120, 144, 168 and 198 h after the oral administration of crotalphine. Data are presented as mean ± SEM. \* significantly different from the saline group, *n* = 5 per group (*p* < 0.05). The observer was blinded to the experimental conditions.

**Figure 2.** Systemic crotalphine administration increases ERK1/2 and JNK and decreases p38 phosphorylation levels in the spinal cord. (**A**) Schematic representation of treatment and immunoblot. (**B**) Representative blots showing the phosphorylated and total ERK1/2, JNK and p38 levels in the total lysate of spinal cord. Changes in protein expression of ERK 1/2 (**C**), JNK (**D**) and p38 (**E**) MAPKs at different time points were determined by Western blot analysis in lumbar spinal cord extracts from crotalphine or saline-treated rats. Graphs represent the ratio between the phosphorylated protein and the total amount of the targeted protein. Data are presented as mean ± SEM and expressed as % of control (saline) animals. \* significantly different from mean values of saline treated animals, *n* = 6 per group (*p* < 0.05). \*\* significantly different from mean values of saline treated animals, *n* = 6 per group (*p* < 0.01). \*\*\* significantly different from mean values of saline treated animals, *n* = 6 per group (*p* < 0.001). The observer was blinded to the experimental conditions.

Crotalphine increases the activation of ERK1/2 in the lumbar spinal cord at 1, 3 and 5 h after administration, when compared with saline-treated rats (Figure 2B,C). Crotalphine also increases the levels of JNK phosphorylation at 1 and 5 h (Figure 2B,D). Conversely, crotalphine decreased the phosphorylation of p38 for up to 96 h after administration (Figure 2B,E). Together, these data indicate that ERK1/2 and JNK are activated in the period in which crotalphine is inducing analgesia, whereas p38 is repressed for a longer period.

#### *2.3. Spinal ERK and JNK Activation Participates in Crotalphine-Induced Analgesia*

To evaluate the functional significance of the increased MAPKs activation in the antinociceptive effect of crotalphine, we used a pharmacological MEK inhibitor (MAPK-I, PD98059) (Figure 3A). First, to investigate whether the peripheral MAPKs participate in crotalphine-induced analgesia, the inhibitor was injected by an intraplantar route in two different doses (15 and 30 μg/paw). MAPK-I did not interfere with crotalphine-induced antinociception (Figure 3B). However, the intrathecal MAPK-I injection (30 μg) completely reversed crotalphine-induced mechanical analgesia, showing that spinal MAPKs, but not intraplantar, are involved in crotalphine's beneficial effects (Figure 3C). We next examined which MAPK would be responsible for the crotalphine effects.

**Figure 3.** Spinal MAPKs are involved in crotalphine-induced analgesia. (**A**) Schematic representation of treatment and behavior assay. Nociceptive threshold was obtained in the rat paw pressure test before (0) and 3 h after systemic administration of crotalphine (CRP, 1 μg/kg). MEK inhibitor (MAPK-I) was administered immediately after crotalphine (**B**) intraplantarly or (**C**) intrathecally. Data represent mean values ± SEM. \* significantly different from baseline (dotted line), *n* = 6 per group (*p* < 0.05). The observer was blinded to the experimental conditions.

The ERK-I and JNK-I intrathecal administration prevented the antinociceptive effect of crotalphine. Nevertheless, the p38 inhibitor did not interfere with crotalphine-induced analgesia (Figure 4B). The control animals injected with the inhibitors alone did not display changes in the nociceptive threshold [7]. Together, these results indicate that crotalphine-induced ERK and JNK activation is responsible, at least in part, for the analgesic effect of this peptide.
