Morroniside, a secoiridoid glycoside from Cornus officinalis, attenuates neuropathic pain by activation of spinal glucagon-like peptide-1 receptors

Abstract

Background and purpose: Iridoid glycosides containing the double bond scaffold of cyclopentapyran are reversible and orthosteric agonists of glucagon-like peptide-1 (GLP-1) receptors and exert anti-nociceptive and neuroprotective actions. Morroniside, derived from the medicinal herb Cornus officinalis, is an atypical secoiridoid containing a six-membered cyclic inner ether fragment. Here we investigated whether morroniside was an orthosteric GLP-1 receptor agonist and had anti-hypersensitivity activities in a model of neuropathic pain.

Experimental approach: We used a model of neuropathic pain, induced by tight ligation of L5/L6 spinal nerves in rats. Hydrogen peroxide-induced oxidative damage was also assayed in N9 microglial cells and human HEK293 cells stably expressing GLP-1 receptors.

Key results: Morroniside protected against hydrogen peroxide-induced oxidative damage in N9 microglial and HEK293 cells that expressed mouse or human GLP-1 receptors, but not in HEK293T cells without GLP-1 receptors. The GLP-1 receptor orthosteric antagonist exendin(9-39) also concentration-dependently shifted the concentration-protective response curves of morroniside and exenatide to the right without affecting maximal protection, with similar pA₂ values. Furthermore, morroniside given by oral gavage or intrathecally in neuropathic rats dose-dependently attenuated mechanical allodynia, with comparable E_max values and ED₅₀ s of 335 mg·kg^-1 and 7.1 μg and completely blocked thermal hyperalgesia. Daily intrathecal injections of morroniside over 7 days did not induce anti-allodynic tolerance. Pretreatment with intrathecal exendin(9-39) completely blocked systemic and intrathecal morroniside-induced mechanical anti-allodynia.

Conclusion and implications: Our data demonstrated that morroniside was an orthosteric agonist of GLP-1 receptors and produced antihypersensitivity in a neuropathic pain model by activation of spinal GLP-1 receptors.

Figure 1

Flowers of Cornus officinalis (A) and the chemical structure of morroniside (B).

Figure 2

Expression of GLP‐1 receptors (A) and the protective effects of exenatide and morroniside against hydrogen peroxide‐induced loss of cell viability in N9 microglial cells (B, C) and HEK293 cells (D). Double immunofluorescence staining was conducted using anti‐GLP‐1 receptor and anti‐OX42 antibodies. Hydrogen peroxide‐induced loss of cell viability was assessed using the MTT assay. The concentration–response curves of morroniside and exenatide were constructed in the presence and absence of the GLP‐1 receptor antagonist exendin(9‐39). Schild plots of exendin(9‐39) on morroniside and exenatide are inserted in B and C. Data are presented as means ± SEM ;n = 6 with triplicates at each concentration. *P < 0.05, significantly different from control groups; two‐tailed and unpaired Student's t‐test.

Figure 3

Mechanical antiallodynic and thermal antihyperalgesic effects of oral gavage (A–C) and intrathecal (D–F) morroniside administration in a rat model of neuropathic pain. Peripheral neuropathy was induced by tight ligation of L5/L6 spinal nerves and the paw withdrawal responses to electronic von Frey filaments and radiant heat were measured (B and E). Dose–response analyses of morroniside on mechanical hypersensitivity in the ipsilateral paw were calculated using the thresholds measured 1 h after drug administration, as projected by the non‐linear least‐squares method. Data are presented as means ± SEM; n = 6 per group. *P < 0.05, significantly different from saline control group; repeated measures two‐way ANOVA followed by post hoc Student–Newman–Keuls test.

Figure 4

Mechanical antiallodynic effects of repeated daily intrathecal injections of morroniside (100 μg) in a rat model of neuropathic pain. Peripheral neuropathy was induced by tight ligation of L5/L6 spinal nerves and the paw withdrawal thresholds were measured using an electronic von Frey filaments 1 h after each drug administration. Data are presented as means ± SEM; n = 6 for each group. *P < 0.05, significantly different from saline control group; repeated measures two‐way ANOVA followed by post hoc Student–Newman–Keuls test.

Figure 5

Blockade of intrathecal injection of GLP‐1 receptor antagonist exendin(9‐39) on the mechanical antiallodynic effects of oral (A) and intrathecal (B) morroniside in a rat model of neuropathic pain, induced by tight ligation of L5/L6 spinal nerves. Rats received intrathecal injection of saline (10 μL) or exendin(9‐39), 30 min followed by morroniside administered by oral gavage or intrathecal injection. Hindpaw withdrawal thresholds were measured using an electronic von Frey filaments before and after morroniside administration. Data are presented as means ± SEM; n = 6 of each group. *P < 0.05, significantly different from saline control group; repeated measures two‐way ANOVA followed by post hoc Student–Newman–Keuls test.