Profound wound healing efficiency and relatively high availability of the source tissue, the placenta, make the amniotic membrane (AM) and amniochorionic membrane (ACM) ones of the most widely used grafts worldwide. Their wound healing effect is given by the interplay of anti-inflammatory, anti-microbial, and anti-fibrotic components [1,2]. The presence of numerous growth factors, cytokines and other bioactive proteins in AM/ACM has been experimentally proven [1–5] and their therapeutic efficacy has been repeatedly confirmed at the clinical level in ophthalmology, surgery, and wound healing [6–9].
The application of an AM/ACM graft on the wound leads to the significant pain relief [10–12]. To date, no specific substances directly responsible for the analgesic effect of these grafts have been reported [13,14]. The pain-relieving effect of AM/ACM is usually explained by the mechanical protection of exposed nerve endings after a tight adherence of the graft to the wound surface, hydration of the wound bed and the presence of anti-inflammatory and anti-scarring components which may alleviate nociception indirectly [13–15]. However, it seems unlikely that such a strong and generalized analgesic effect is invoked without major contribution from a specific compound [13,14]. As specific proteins have been determined to be responsible for the anti-inflammatory and anti-microbial properties of AM, we hypothesized that a similar mechanism could be involved in the analgesic effect of AM/ACM. Therefore, we were interested in establishing whether some known substances that have analgesic properties are present in placental tissue, and thus explain the substantial pain relief generally reported in relation to application of AM/ACM. After an extensive search for potential candidates we shortlisted a group of endogenous bioactive lipid-related signalling molecules N-acylethanolamines (NAEs), in which particularly palmitoylethanolamide (PEA) has been shown to have profound analgesic and anti-nociceptive effect [16,17].
PEA along with other endogenous fatty acid amides, oleoylethanolamide (OEA), and anandamide (arachidonoylethanolamid, AEA), are ubiquitous in organisms from plants to mammalian tissues [18–22]. In the human body, NAEs have been detected in most organs, tissues (e.g. brain, nerves, muscles, gastrointestinal tract, adipose tissue, skin, eye), and fluids (e.g. blood, breast milk, amniotic fluid, saliva) [18–25].
All three lipid mediators (PEA, AEA, OEA) are synthesized constitutively or on demand, i.e., after exposing cells to specific, predominantly harmful/non-physiological stimuli. They are released from cell membrane phospholipid precursors by phospholipase D, and then act locally in the cells to which they are transported by carrier-mediated transport [22,26,27]. PEA and OEA exert their action primarily by activating the nuclear peroxisome proliferator-activated receptor-α (PPAR-α), the transient receptor potential cation channel subfamily V member 1 (TRPV1) and the G protein-coupled receptors GPR55 and GPR119 [28–32]. PEA can indirectly activate cannabinoid receptors CB2R [33]. AEA ligates cannabinoid receptors CB1R, CB2R, TRPV1, PPAR-γ and some evidence points to AEA binding PPAR-α also [34–36]. NAEs are implicated in multiple physiological (immunity, fertilisation, feeding and sleeping behaviours) and pathological conditions (pain, inflammation, allergy) [23,24,27,37].
PEA was first isolated from egg yolk, soybeans, and peanuts where its anti-anaphylactic activity was also reported [18,19]. Later on, anti-inflammatory, neuroprotective, analgesic and anti-nociceptive effects of PEA were shown in experimental animal studies [16,20,21,27,38,39], and confirmed in human clinical trials [40,41]. It has been reported in rodent models that PEA also inhibits lung, liver and retinal fibrosis [42–44]. PEA has also been shown to have a positive effect on viral respiratory infections [38,45], and was recently proposed as a promising nutraceutical in COVID-19 infection [46]. The anti-hyperalgesic effect of PEA has been utilized in the treatment of peripheral neuropathy and chronic pain, e.g. sciatic pain or pain from carpal tunnel syndrome [40,47,48], and its efficiency is independent of the etiopathogenesis of pain [47].
OEA has mostly anorexigenic properties [49], but its ability to reduce nociceptive responses and inflammation has also been shown [50,51]. AEA has been implicated in possessing anti-nociceptive, vasodilation, and anti-inflammatory effects [22,35,52–54]. Recently its role in physiological wound healing has been also suggested [22,53,54].
Binding of PEA to the PPAR-α receptor leads to attenuation of nociceptive and inflammatory responses, as confirmed in PPAR-α null mice [55,56]. The activation of PPAR-α can aid in the regeneration of mice peripheral nerves at the level of axon repair [57], mediated via satellite glial cells [57,58]. PPARα activation downregulates nuclear factor kB (NF-kB) followed by the decrease of proinflammatory proteins, such as inducible NO synthase (iNOS), cyclo-oxygenase-2 (COX2), tumour necrosis factor-α (TNFα) or interleukin 1 and 6 or prostaglandin E2 (PGE2) [41,59], which all can contribute to the anti-inflammatory properties of AM/ACM grafts. PEA also stimulates macrophages to remove invading bacteria and apoptotic neutrophils [60,61].
To date, of the NAEs lipid mediators, only AEA has been reported in frozen placental tissues [25,62]. However, its concentrations were not measured in isolated AM or ACM [63], which are the most important placenta related tissues used for grafting. We therefore designed our study to determine whether NAEs are present in placental tissues and whether their concentrations can explain the analgesic effect of the AM dressing.