Isotalatizidine, a C19-diterpenoid alkaloid, attenuates chronic neuropathic pain through stimulating ERK/CREB signaling pathway-mediated microglial dynorphin A expression

Abstract

Background: Isotalatizidine is a representative C₁₉-diterpenoid alkaloid extracted from the lateral roots of Aconitum carmichaelii, which has been widely used to treat various diseases on account of its analgesic, anti-inflammatory, anti-rheumatic, and immunosuppressive properties. The aim of this study was to evaluate the analgesic effect of isotalatizidine and its underlying mechanisms against neuropathic pain.

Methods: A chronic constrictive injury (CCI)-induced model of neuropathic pain was established in mice, and the limb withdrawal was evaluated by the Von Frey filament test following isotalatizidine or placebo administration. The signaling pathways in primary or immortalized microglia cells treated with isotalatizidine were analyzed by Western blotting and immunofluorescence.

Results: Intrathecal injection of isotalatizidine attenuated the CCI-induced mechanical allodynia in a dose-dependent manner. At the molecular level, isotalatizidine selectively increased the phosphorylation of p38 and ERK1/2, in addition to activating the transcription factor CREB and increasing dynorphin A production in cultured primary microglia. However, the downstream effects of isotalatizidine were abrogated by the selective ERK1/2 inhibitor U0126-EtOH or CREB inhibitor of KG-501, but not by the p38 inhibitor SB203580. The results also were confirmed in in vivo experiments.

Conclusion: Taken together, isotalatizidine specifically activates the ERK1/2 pathway and subsequently CREB, which triggers dynorphin A release in the microglia, eventually leading to its anti-nociceptive action.

Keywords: CREB; Dynorphin A; ERK1/2 MAPK; Isotalatizidine; Microglia; Neuropathic pain.

Fig. 1

The effect of isotalatizidine on acetic acid-induced somatic pain in mice. (a) The chemical structure of isotalatizidine. Isotalatizidine significantly decreased the writhing times (b) and increased the rate of analgesia (c) in acetic acid-induced mice. The ED₅₀ of analgesic efficiency of isotalatizidine was 0.43 mg/kg (d). Data are expressed as mean ± SEM (n = 8 mice in each group). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. vehicle group

Fig. 2

Isotalatizidine treatment improved sciatic nerve ligation-induced chronic neuropathic pain. Neuropathic pain mice were induced by tight ligation of sciatic nerve, the withdrawal thresholds within 12 days after surgery (a) and intrathecal injection of vehicle (0.9% normal saline) or isotalatizidine (0.1, 0.3, 1.0 mg/kg) 8 days after surgery (b) were measured by Von Frey Monofilament in both contralateral and ipsilateral paws. Data are expressed as means ± SEM (n = 6 mice in each group). ^*P < 0.05, ^**P < 0.01 vs. saline group, ^***P < 0.001 vs. sham group

Fig. 3

Effects of isotalatizidine on phosphorylation of p38, ERK1/2, and JNK in BV-2 and primary microglial cells. Effects of isotalatizidine on phosphorylation of p38, ERK1/2, and JNK in BV-2 (a) and primary microglial cells (b). Isotalatizidine (25 μM) obviously promoted the phosphorylation of p38 (c) and ERK1/2 (d), but not JNK (e) in cultured microglial cells. Selective p38 inhibitor SB 203580 (f, h) and ERK1/2 inhibitor U0126-EtOH (g, i) significantly blocked the isotalatizidine activated phosphorylation of p38 or ERK1/2, respectively. The data are expressed as mean ± SEM of three independent experiments. ^**P < 0.01, ^***P < 0.001 vs. control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. isotalatizidine group

Fig. 4

Isotalatizidine stimulating the CREB activation and dynorphin A expression via ERK1/2 pathway in the primary microglial cells. In cultured primary microglial cells, isotalatizidine (25 μM) markedly induced the phosphorylation of CREB and expression of dynorphin A, and the phosphorylated levels of CREB and increased dynorphin A level were markedly prevented by ERK1/2 inhibitor of U0126-EtOH (a) but not p38 inhibitor of U0126-EtOH (b). Immunofluorescence analysis showed that isotalatizidine-stimulated IBA-1 expression and dynorphin A production also could be blocked by ERK1/2 inhibitor of SB203580 (c). The data are expressed as mean ± SEM (n = 3). ^*P < 0.05, ^**P < 0.01 vs. control group; ^#P < 0.05 vs. isotalatizidine group

Fig. 5

Isotalatizidine exerts analgesic effect by increasing dynorphin A in the spinal cord tissue via the ERK1/2-CREB pathway. Isotalatizidine (1 mg/kg, n = 6) induced a time-dependent mechanical antiallodynia of ipsilateral paws in CCI-induced mice, which was completely prevented by ERK inhibitor of U0126-EtOH and CREB inhibitor of KG-501, but not p38 inhibitor of SB203580 (a). Phosphorylated level of ERK1/2 and CREB and expression of dynorphin A stimulated by isotalatizidine treatment could be blocked by U0126-EtOH or KG-501 in spinal dorsal tissue, respectively (b). Isotalatizidine treatment resulted in the releasing of dynorphin A in spinal cord tissue, which could be suppressed by U0126-EtOH or KG-501, but not SB230580 (c). Data are expressed as means ± SEM. ^*P < 0.05 vs. CCI mice; ^#P < 0.05 vs. isotalatizidine treated CCI mice. In cultured BV-2 cells, isotalatizidine-induced mRNA expression of prodynorphin also could be inhibited by U0126-EtOH or KG-501, but not SB230580 (d). Data are expressed as means ± SEM of three independent experiments. ^**P < 0.01 vs. control group; ^#P < 0.05 vs. isotalatizidine group

Fig. 6

Proposed analgesic mechanism of isotalatizidine for ERK/CREB signal pathway-induced expression of dynorphin A in spinal microglia