Analgesic and Anti-Inflammatory Effects of the Synthetic Neurosteroid Analogue BNN27 during CFA-Induced Hyperalgesia

Dehydroepiandrosterone (DHEA), an adrenal and neurosteroid hormone with strong neuroprotective and immunomodulatory properties, and ligand for all high-affinity neurotrophin tyrosine kinase receptors (Trk), also exerts important effects on hyperalgesia. Its synthetic, 17-spiro-epoxy analogue, BNN27, cannot be converted to estrogen or androgen as DHEA; it is a specific agonist of TrkA, the receptor of pain regulator Nerve Growth Factor (NGF), and it conserves the immunomodulatory properties of DHEA. Our study aimed to evaluate the anti-nociceptive and anti-inflammatory properties of BNN27 during Complete Freund’s Adjuvant (CFA)-induced inflammatory hyperalgesia in mice. Hyperalgesia was evaluated using the Hargreaves test. Inflammatory markers such as cytokines, NGF and opioids were measured, additionally to corticosterone and the protein kinase B (AKT) signaling pathway. We showed for the first time that treatment with BNN27 reversed hyperalgesia produced by CFA. The effect of BNN27 involved the inhibition of NGF in the dorsal root ganglia (DRG) and the increased synthesis of opioid peptides and their receptors in the inflamed paw. We also found alterations in the cytokine levels as well as in the phosphorylation of AKT2. Our findings strongly support that BNN27 represents a lead molecule for the development of analgesic and anti-inflammatory compounds with potential therapeutic applications in inflammatory hyperalgesia.

Encoding of inflammatory hyperalgesia in mice spinal cord

Inflammation modifies the input-output properties of peripheral nociceptive neurons, thus leading to hyperalgesia, i.e., changes in the perception of noxious heat stimuli such that the same stimulus produces enhanced pain. The increased nociceptive output enters the superficial dorsal spinal cord (SDH), which comprises the first CNS network integrating the noxious information. Here we used in vivo calcium imaging and a computational approach to investigate how the SDH network in mice encodes the injury-mediated abnormal input from peripheral nociceptive neurons. We show the application of noxious heat stimuli to the mice hind paw in naive conditions before induction of injury affects the activity of 70% of recorded neurons by either increasing or suppressing it. Application of the same noxious heat stimuli to hyperalgesic skin leads to activation of previously non-responded cells and de-suppression of the "suppressed" neurons. We demonstrate that reduction in synaptic inhibition mimics the response to the noxious stimuli in the hyperalgesic conditions. Using a computational model of SDH network, we predict that inflammation-induced "disinhibition"-like changes result in increased activity of excitatory projection neurons. Our model also predicts that the observed "disinhibitory" effect of hyperalgesic stimuli does not require changes in the inhibitory transmission and may result from the SDH networks' intrinsic cytoarchitecture.

TRPV1 Responses in the Cerebellum Lobules V, VIa and VII Using Electroacupuncture Treatment for Inflammatory Hyperalgesia in Murine Model

Inflammatory pain sensation is an important symptom which protects the body against additional tissue damage and promotes healing. Discovering long-term and effective treatments for pain remains crucial in providing efficient healthcare. Electroacupuncture (EA) is a successful therapy used for pain relief. We aimed to investigate effects and mechanisms of Complete Freund’s Adjuvant (CFA)-inducing inflammatory pain in the cerebellum, and the inhibition of this inflammatory hyperalgesia using EA at Zusanli acupoint (ST36). The results display a significant increase in mechanical and thermal sensitivities in the CFA and CFA + SHAM groups, which was significantly reduced in the CFA+EA and CFA + KO groups. This evidence was substantiated in the protein levels observed using immunoblotting, and presented with significant escalations after CFA inducing inflammatory hyperalgesia in CFA and CFA + SHAM groups. Then, they were significantly attenuated by EA in the CFA + EA group. Furthermore, the CFA + transient receptor vanilloid member 1 (TRPV1)−/− group indicated similar significant decreases of protein expression. Additionally, a concomitant overexpression in lobule VIa was also observed in immunofluorescence. These consequences suggest that CFA-induced inflammatory pain provokes modifications in cerebellum lobules V, VIa and VII, which can subsequently be regulated by EA treatment at the ST36 through its action on TRPV1 and related molecular pathways.

Mas-related G protein-coupled receptor D exacerbates inflammatory hyperalgesia by promoting NF-κB activation in dorsal root ganglia

Background Mas-related G protein–coupled receptor D (MrgprD) is mainly expressed in small-diameter sensory neurons of the dorsal root ganglion (DRG). Results from previous studies suggest that MrgprD participates in mechanical allodynia and nerve injury-induced neuropathic pain. However, it remains elusive whether and how MrgprD is involved in inflammatory pain. Methods we used a mouse model of inflammatory hyperalgesia established by intraperitoneal administration of lipopolysaccharide (LPS). The generation of neuroinflammation and inflammatory hyperalgesia were determined using animal behavioral tests, Western blotting, quantitative real-time PCR and ELISA assay. The expression levels of MrgprD were assessed by Western blotting. The involvements of MrgprD in the LPS-triggered neuroinflammation and inflammation-related signaling pathways were assessed by animal behavioral tests, Western blotting, immunohistochemistry, ELISA, and co-immunoprecipitation assays. Results The LPS injection induced an evident peripheral neuroinflammation and mechanical allodynia in the mice and increased MrgprD expression in the DRG. The LPS administration also augmented the proportion of MrgprD-expressing neurons in the lumbar 4 DRG. The LPS-induced hypersensitivities to mechanical and cold stimuli, but not to a heat stimulus, were substantially attenuated in Mrgprd- knockout mice compared with wildtype littermates. Mrgprd deletion suppressed the LPS-triggered activation of the NF-κB signaling pathway and attenuated LPS-induced up-regulation of proinflammatory cytokines. Moreover, ectopic overexpression of MrgprD in HEK293 cells stably expressing mouse toll-like receptor 4 (TLR4) markedly promoted the LPS-induced NF-κB activation and enhanced NF-κB’s DNA-binding activity. Furthermore, MrgprD physically interacted with TGF-β-activated kinase 1 (TAK1) and I-kappa-B-kinase (IKK) complexes in DRGs and macrophage-like RAW 264.7 cells, and MrgprD agonist treatment was sufficient to elicit the activation of NF-κB signaling, but not of mitogen-activated protein kinases (MAPKs) in RAW 264.7 cells. Conclusions Our findings demonstrate that MrgprD exacerbates LPS-induced inflammatory hyperalgesia by promoting canonical NF-κB activation, indicating that MrgprD could be a potential therapeutic target for managing NF-κB-mediated inflammation and inflammatory pain.