Palmitoylethanolamide-Mediated Inhibition of Nogo-a Signaling Attenuates Pain in Sickle Mice

Sickle cell disease (SCD) is characterized by multiple comorbidities including pain. SCD patients often use cannabinoids to alleviate pain, but their psychoactive effects and social stigma impose major challenges. Strategies to elevate endogenous cannabinoids (eCBs) are devoid of such challenges, but pharmacologic approaches showed adverse-effects in clinical trials. Therefore, we examined the potential of non-pharmacologic integrative approaches to elevate eCBs. Enriched high-energy diet has been shown to increase levels of eCBs (Argueta et al., Front Physiol 2019) and when combined with companionship reduced hyperalgesia in sickle mice (Tran et al., Blood 2016). We hypothesized that enriched diet and companionship would enhance eCBs without adverse effects and reduce hyperalgesia by inhibiting peripheral and central pro-nociceptive mechanisms. We fed male homozygous-BERK (sickle) mice, regular Rodent Diet (RD; 2018, Harlan) or customized high calorie enriched Sickle Mouse Diet (SD; 59M3, TestDiet), housed with or without a female companion (C+ or C-, respectively) for 3-weeks. RD/SD contain 18.6/26.4% protein, 6.2/11.1% fat, 24/27.5% carbohydrates and 18/26% kcal/g, respectively; and SD contains higher minerals, vitamins and ω-3 fatty acids compared to RD. Control HbAA-BERK and sickle mice were divided into 4 groups: [i] R/C-, RD, no companion, [ii] S/C-, SD without companion, [iii] R/C+, RD with companion, and [iv] S/C+, SD with companion. After 3-weeks of treatment, spinal cord eCBs were analyzed using targeted lipid quantitation with liquid chromatography mass spectrometry (LCMS). We observed a 20% decrease in palmitoylethanolamide (PEA), in sickle mice compared to control mice, in R/C- group (p<0.05). Further, we observed increased spinal PEA in S/C+ compared to R/C- sickle mice (~40%, p<0.05), which was concomitant with reduced mechanical, heat, and cold hyperalgesia in S/C+ sickle mice group (~80%, p<0.001; ~60%, p<0.01; & ~30%, p<0.001, respectively). Therefore, sickle diet and companionship enhances endogenous spinal PEA which has an inhibitory effect on hyperalgesia in sickle mice. Treatment of control and sickle mice in R/C- group with PEA (i.p. 20 mg/kg/day) led to acute (1 hour) reduction of mechanical- (~40%, p<0.01) and cold-hyperalgesia (~40%, p<0.001) in sickle mice compared to pre-treatment, which was sustained during 3 day treatment, but had no effect on control mice which do not have hyperalgesia. PEA inhibits substance P (SP)-induced mast cell activity, and sickle mice show increased spinal SP, neuronal sensitization, peripheral nerve injury and mast cell activation (Tran et al., Blood 2017). Pain in SCD is both neuropathic and inflammatory. We examined if PEA inhibited the mechanisms that underlie spinal nerve repair by neurite outgrowth inhibitor, NOGO-A/reticulon-4, which regulates nerve regeneration via Rho Kinase (ROCK) signaling. NOGO-A contributes to inflammatory pain and hyperalgesia following spinal cord injury via NOGO receptor 1 in spinal cord. We observed that spinal NOGO-A expression and ROCK activity are upregulated (20% & 100%, respectively) in sickle mice compared to control mice (all R/C-), which were inhibited upon 3-day treatment with PEA. We validated ROCK activity downstream of NOGO-A using SH-SY5Y neuroblastoma cells, simulating a sickle microenvironment with hemin (40 µM) and TNFα (1 ng/ml)(H+T). ROCK activity increased in H+T-treated SH-SY5Y cells compared to vehicle (~30%, p=0.05). In parallel, we analyzed the effect of PEA on extracellular traps (ET) in cutaneous mast cells from sickle mice induced by H+T in vitro. PEA treatment inhibited ET formation and extravasation of nuclear contents in H+T induced mast cells. Thus, PEA has the potential to attenuate neuropathic and inflammatory pain by inhibiting neuronal NOGO-A/ROCK pathway and mast cell activation in a sickle milieu. PEA has analgesic and anti-inflammatory effects on chronic pain in several clinical conditions. Therefore, our data suggest that diet and pleasure have the potential to upregulate pro-analgesic PEA that inhibits NOGO-A signaling and mast cell activation, leading to attenuation of hyperalgesia in sickle mice.