Select steroid hormone glucuronide metabolites can cause toll-like receptor 4 activation and enhanced pain

Abstract

We have recently shown that several classes of glucuronide metabolites, including the morphine metabolite morphine-3-glucuronide and the ethanol metabolite ethyl glucuronide, cause toll like receptor 4 (TLR4)-dependent signalling in vitro and enhanced pain in vivo. Steroid hormones, including estrogens and corticosterone, are also metabolized through glucuronidation. Here we demonstrate that in silico docking predicts that corticosterone, corticosterone-21-glucuronide, estradiol, estradiol-3-glucuronide and estradiol-17-glucuronide all dock with the MD-2 component of the TLR4 receptor complex. In addition to each docking with MD-2, the docking of each was altered by pre-docking with (+)-naloxone, a TLR4 signaling inhibitor. As agonist versus antagonist activity cannot be determined from these in silico interactions, an in vitro study was undertaken to clarify which of these compounds can act in an agonist fashion. Studies using a cell line transfected with TLR4, necessary co-signaling molecules, and a reporter gene revealed that only estradiol-3-glucuronide and estradiol-17-glucuronide increased reporter gene product, indicative of TLR4 agonism. Finally, in in vivo studies, each of the 5 drugs was injected intrathecally at equimolar doses. In keeping with the in vitro results, only estradiol-3-glucuronide and estradiol-17-glucuronide caused enhanced pain. For both compounds, pain enhancement was blocked by the TLR4 antagonist lipopolysaccharide from Rhodobacter sphaeroides, evidence for the involvement in TLR4 in the resultant pain enhancement. These findings have implications for several chronic pain conditions, including migraine and tempromandibular joint disorder, in which pain episodes are more likely in cycling females when estradiol is decreasing and estradiol metabolites are at their highest.

Glial contributions to visceral pain: implications for disease etiology and the female predominance of persistent pain

“…For instance, glucuronide metabolites (which typically have a longer half-life than the parent molecule) of estrogen cause potent activation of TLR4 in vitro , correlating with enhanced mechanical allodynia in rats in vivo . 120 The proinflammatory response to LPS is potentiated by estrogen in female but not male neonatal microglia. 121 Moreover, although adult hippocampal microglia from ovariectomized rats in ex vivo preparations show a downregulation in LPS-induced inflammation upon estrogen supplementation, IL-1β mRNA is potentiated when estrogen is administered in vivo .…”

Opioid‐Induced Central Immune Signaling: Implications for Opioid Analgesia

“…Chronic administration of the female sex hormone estradiol in male rats and ovariectomized female rats increased the LPS‐stimulated inflammatory response in hippocampal microglia ex vivo and increased proinflammatory cytokine transcription in vivo . Furthermore, the estradiol metabolites, estradiol‐3‐glucuronide, and estradiol‐17‐glucuronide not only dock to MD2 in silico , but also activate TLR4 signaling in vitro and induce allodynia in vivo . These data predict elevated glial reactivity in females at basal levels and upon stimulation, potentially resulting in exaggerated opioid‐induced central immune signaling, though this has yet to be tested.…”

Therapeutic Developments Targeting Toll‐like Receptor‐4‐Mediated Neuroinflammation

“…Different from the classical opioid receptors, TLR4 is activated by the morphine metabolite morphine-3-glucuronide (M3G) instead of morphine-6-glucuronide (M6G) [24] . The most recent studies from the Watkins group show that both estradiol-3-glucuronide (E 2 -3-G) and estradiol-17-glucuronide (E 2 -17-G) can enhance neuropathic pain [27] . In silico docking was able to predict that E 2 -3-G and E 2 -17-G could bind to MD2 in the TLR4-MD2 complex.…”