Nociceptive Effects Of Non-peptidic GLP-1 Receptor Agonists And Their Mechanisms Of Actions

Pain is one of the most common clinical symptoms, which appears in many diseases and disorders, and seriously affects the quality of life of patients, so treatment of pain has become one of the most considered factors in health care and disease treatment. The GLP-1R is a 463-amino acid transmembrane-spanning protein in the family of B/secretin G-protein coupled receptors. It mediates the effects of the endogenous GLP-1 and oxyntomodulin. Preliminary work for the first time find that activation of spinal cord GLP-1R generates strong analgesic actions. It has recently reported that two peptidic GLP-1R agonists, GLP-1(7-36) and exenatide, produced antinociception during chronic phase of formalin pain, neuropathic, bone-cancer and diabetic pains, without affecting the acute phase of formalin pain. GLP-1R has become a research hot spot. But the peptidic GLP-1R agonists which are macromolecules, can not cross the blood-brain barrier, so their application is restricted. However, non-peptidic GLP-1R agonists with small molecular weight, which can be orally administrated, crossing the blood-brain barrier, are more potential to develop into clinical application drugs, and become the new research objectives and new targets, causing widespread interest.The non-peptidic GLP-1R agonists WB4-24 and shanzhiside methyl ester molecular weight are approximately 1000 Da and 300 Da, the previous study is not only find that peptidic GLP-1R agonists GLP-1 and exenatide could dose-dependently inhibit formalin induced II phase pain, bone-cancer pain, neuropathic pain and diabetic pain without affecting acute pain in formalin pain test, also discusses its mechanism of the analgesic pathway, GLP-1 and exenatide may activate GLP-1R of microglial cells, then the neurotransmitter β-endorphin release and produce the analgesic effect. In this manuscript we want to figure out that the non-peptidic GLP-1R agonists WB4-24 and shanzhiside methyl ester can also produce analgesia through activating GLP-1R of microglial cells, releasing analgesic neurotransmitters of β-endorphin or not. This pathway hypothesis, which has been put forward recently, need further verification in various different ways. We chose the non-peptidic agonist WB4-24, which has higher active potency of GLP-1R and study the analgesic effect and mechanism of analgesic pathway. At the same time, we also choose shanzhiside methyl ester(smaller molecular weight, 300 Da), previous research suggests, Lamiophlomis rotata(Benth.) Kudo(L. rotata) extract can inhibit the formalin pain, neuropathic pain and bone-cancer pain, and the activation of GLP-1R can be antagonised by exendin9-39, while shanzhiside methyl ester is one of the quality control ingredients of Lamiophlomis rotata and its preparation. Therefore, we also study analgesic effect and mechanisms of shanzhiside methyl ester deeply, and figure out whether microglia and GLP-R are directly related or not, and microglial cells are excited through p38 MAPK pathway, JNK ERK MAPK pathway or ERK MAPK pathway, and microglia release neurotransmitter β-endorphin or not, access to verify the proposed hypothesis.WB4-24 is derived from Boc5, also a non-peptidic GLP-1R agonist, this study will investigate the analgesic effect of non-peptidic GLP-1R agonist WB4-24 on inflammatory pain, and observe thermal hyperalgesia and mechanical allodynia on animal models after administration of WB4-24, and further study the impact of WB4-24 on the release of β-endorphin of microglia as well as its effect on the inflammatory cytokines. Lamiophlomis rotate consists of iridoid glycosides containing nuclear structure of iridoid, and shanzhiside methyl ester(SM), one of the iridoid glycosides, is the quality control target compound of Lamiophlomis rotata and its preparation included in the 2010 edition Chinese Pharmacopoeia. Shanzhiside methyl ester binds GLP-1R reversibly, its biological effect can be reversed by exendin9-39. This study aims to discuss the non-peptidic GLP-1R agonist analgesic effect of Shanzhiside methyl ester on neuropathic pain, and observe the mechanical allodynia in neuropathic pain animal models after administration of shanzhiside methyl ester, and to further investigate the mechanism of analgesic effect. And we try to find out that whether microglia activation, the p38 pathway and the release of β-endorphin are related. To further investigate the self-tolerance of shanzhiside methyl ester after long term administration and cross tolerance to morphine and β-endorphin.Rat formalin-, carrageenan- and complete Freund’s adjuvant(CFA)-induced inflammatory nociceptive models and neuropathic pain model were employed, and thermal hyperalgesia or mechanical allodynia were tested using a 390 G Plantar Test and a 2391 electrical von Frey hair. β-Endorphin releasing in supernatant of primary microglia, nerons and astrocytes and homogenates of spinal cord were measured using the real-time quantitative PCR and fluorescent immunoassay. Pro-inflammatory cytokines were measured also with the real-time quantitative PCR. Intrathecal catheterization and injection were also adopted. The improved microglia, neurons and astrocytes separation technology was applied during the process of primary cell culture.WB4-24 displaced the specific binding of exendin(9-39)-FITC in microglia, HEK293(stably expressing transfected human GLP-1R) and PC12 cells(endogenously expressing rat GLP-1R), and concentration-dependently shifted the binding curve of GLP-1-FITC to the right, without affecting maximum binding in HEK293 and PC12 cells. Single intrathecal injection of WB4-24(0.3, 1, 3, 10, 30 and 100 μg) exerted, in a dose-dependent manner, specific anti-hypersensitive effects in acute and chronic inflammatory nociception induced by formalin, carrageenan and CFA, with a maximal inhibition of 60-80%. Spinal WB4-24 was not effective in altering nociceptive pain. Subcutaneous injection of WB4-24 also led to anti-hyperalgesic and anti-allodynic effects in CFA-treated rats, with the inhibition of 52.6% and 48.6%. WB4-24 evoked β-endorphin release but did not inhibit the expression of pro-inflammatory cytokines in either cultured microglia stimulated by lipopolysaccharide or the spinal cords of CFA-treated rats. WB4-24 antinociception was prevented by the microglial inhibitor, β-endorphin antiserum and μ-opioid receptor antagonist. Intrathecal injection of Shanzhiside methyl ester(SM)(10, 30, 100 and 300 μg) exerted, in a dose-dependent manner, specific anti-allodynic effects in neuropathic pain, with a maximal inhibition of 48.8% and ED50 value of 40.0 μg. Shanzhiside methyl ester evoked β-endorphin release, which was blocked by minocyline in primary microglial cells, not neurons and astrocytes. β-Endorphin release was inhibited by p38 inhibitor SB203580 in microglial cells. Shanzhiside methyl ester anti-allodynia was prevented by the microglial inhibitor minocycline, β-endorphin antiserum and μ-opioid receptor antagonist CTAP. Shanzhiside methyl ester did not induce self-tolerance and cross tolerance to morphine and β-endorphin.Our results suggest that non-peptidic GLP-1R agonists WB4-24 and Shanzhiside methyl ester inhibit formalin-, carrageenan- and complete Freund’s adjuvant(CFA)-induced inflammatory nociception or neuropathic pain by releasing analgesic neurotransmitter β-endorphin. Our results also indicate that non-peptidic GLP-1R agonists inhibits antinociception by releasing analgesic β-endorphin in spinal microglia, but not induce self tolerance and cross tolerance to morphine and β-endorphin.Furthermore,WB4-24 exerts analgesic effects by exciting β-endorphin release rather than inhibiting the expression of proalgesic pro-inflammatory cytokines in spinal microglia. Non-peptidic GLP-1R agonists WB4-24 and Shanzhiside methyl ester activates microglia, and release β-endorphin through p38 MAPK pathway, but not JNK MAPK pathway and ERK MAPK pathway, microglial released β-endorphin selectivly activates μ-opioid receptor, but not κ-opioid receptor or δ-opioid receptor, and then produce analgesia. In addition, these results further prove the notion that the spinal GLP-1R is a potential target molecule for the treatment of pain hypersensitivity including inflammatory nociception and neuropathic pain.