Introduction

Autacoid Local Injury Antagonism (ALIA)-amides are naturally occurring fatty acid amides capable to preserve the normal reactivity of mast cells, as well as microglia and astrocytes, in the peripheral and central nervous system1,2. The principal component of this class of molecules is N-palmitoyl-ethanolamine, known as PEA, endowed with important anti-(neuro)inflammatory and anti-nociceptive properties3,4,5. Although several direct mechanisms of action have been proposed for PEA, including receptor-specific interactions (i.e., the peroxisome proliferator-activated receptor-α, PPAR-α)6,7,8 and the orphan G-protein coupled receptor 55, GPR559, there is growing evidence that PEA also targets several indirect mechanisms of action due to its interaction with some of the molecules (i.e., receptors and/or enzymes) belonging to the expanded endocannabinoid system, recently renamed with the term endocannabinoidome10. In fact, it has primarily been demonstrated that PEA by inhibiting the expression of fatty acid amide hydrolase, FAAH (the enzyme responsible for the degradation of endocannabinoids), enhances the protective effects of the endocannabinoid anandamide (AEA), preventing its degradation and/or increasing its affinity for cannabinoid receptors, CB1 and CB211. Secondly, it has been shown that PEA also increases the endogenous levels of another endocannabinoid, 2-arachidonoyl-glycerol (2-AG), by stimulating the activity of diacylglycerol lipase (DAGL)-α and -β (the enzymes responsible for the biosynthesis of 2-AG), to counteract the inflammation through a CB2-mediated mechanism12. Additionally, PEA may enhance the protective properties of AEA and 2-AG also by allosterically influencing their activity at the transient receptor potential vanilloid 1 (TRPV1) channel, thus potentiating also their desensitization of this receptor13,14,15,16.

N-palmitoyl-D-glucosamine (PGA) differs from PEA for the presence of a glucosamine moiety instead of ethanolamine. Interestingly, it has long ago been shown that: (i) N-fatty-acyl-glucosamine is the common minimum and the smallest structure necessary and sufficient for nitrogen-fixing bacteria (Rhizobium chitolipooligosaccharides, CLOs) to start a symbiotic engagement with legumes; and (ii) the radiolabeled substrate N-palmitoyl[1-14 C]glucosamine is de-N-acylated by the action of fatty-acyl amidase (FAA II), a cell-lysing enzyme secreted by free-living soil amoeba (Dictyostelium discoideum, Amoebidae)17,18. From a pharmacological perspective, PGA is among the least studied ALIA-amides and only few reports have been published to date. In preclinical models of inflammation and osteoarthritic pain, the oral administration of PGA produced anti-inflammatory, pain-relieving, and joint-protective effects19. In addition, particle size reduction of PGA in the micronized formulation (m-PGA) significantly improved the activity of PGA, principally on joint pain and disability, and resulted to be safe in an acute toxicity study19. Indeed, like PEA, PGA is a highly lipophilic compound, with a predicted logP value of 5.619. Likewise, the dietary supplementation with PGA co-micronized with curcumin resulted in joint pain relief and improved mobility both in experimentally-induced20 and naturally-occurring osteoarthritis21. PGA was also demonstrated to be also potentially beneficial at preventing in vitro lipopolysaccharide (LPS)-induced tissue damage, as well as at decreasing allodynia in the peripheral neuropathy induced by oxaliplatin or formalin in mice22. The same authors also showed that PGA stably binds MD-2, the link between Toll-like receptor 4 (TLR4) and LPS signaling23, and acts as a TLR4 antagonist22, suggesting a possible mechanism of action for its potent anti-inflammatory activity22. Likewise, in the third study, m-PGA was found to reduce colitis severity and improve intestinal mucosa integrity by increasing the expression of tight junction proteins, and downregulating the TLR-4/NLRP3/iNOS pathway via PPAR-α receptor signaling in DNBS-treated mice24. In a 3:1 mixture with hesperidin, m-PGA was also able to counteract urothelial damage, relieve chronic visceral pain and decrease bladder mast cell accumulation in a cyclophosphamide-induced chronic cystitis model25. Finally, it has recently been shown that m-PGA limits mucosal damage and VEGF-mediated angiogenesis by PPARα-dependent suppression of pAkt/mTOR/HIF1α pathway in a model of colorectal carcinoma26. According to the latter study, orally administered m-PGA can increase colon PEA levels26. Nonetheless, the mechanism(s) behind the ability of PGA to tune up the endogenous ALIA-amide PEA has not been studied yet.

However, PGA may behave like other ALIA-amides, and particularly PEA, by potentiating the endogenous mechanisms of protection. Therefore, it is surprising that the effects of PGA on autocrine signaling effected by the endocannabinoidome have not yet been investigated.

Thus, the aim of the present study was to investigate the potential interaction between PGA and the players, such as lipids and enzymes, of the endocannabinoidome and its involvement in the mechanism of action of PGA, in in vitro cell systems. To achieve this objective, here the human keratinocytes (HaCaT) cell line was preferred as cell model, since under the same conditions PEA was previously shown to produce in these cells an “entourage effect” consisting of increasing the endogenous levels of AEA and 2-AG16.

Results

PGA increases AEA, 2-AG and PEA levels in HaCaT cells

To evaluate if PGA treatment is able to produce the characteristic effect of PEA, i.e., the “entourage” effect on the endogenous levels of the endocannabinoids (AEA and 2-AG), the levels of these lipid mediators were measured in HaCaT cells and supernatants after 40 min and 6 h of PGA treatment (10 µM). In addition, the ability of PGA to produce an entourage effect on the endogenous levels of PEA was also evaluated in the same experimental conditions. The results obtained demonstrate that AEA (t = 4.444; dF = 24; P = 0.0002), 2-AG (t = 5.410; dF = 22; P < 0.0001) and PEA (t = 4.780; dF = 22; P < 0.0001) levels were significantly increased after 40 min of PGA treatment compared to vehicle-treated HaCaT cells (Fig. 1A, B, C). Interestingly, PGA treatment significantly increased AEA (t = 2.896; dF = 24; P = 0.0079), 2-AG (t = 2.198; dF = 24; P = 0.0379) and PEA (t = 2.156; dF = 21; P = 0.0428) levels also after 6 h compared to vehicle-treated HaCaT cells (Fig. 1D, E, F). Moreover, the “entourage” effect of PGA on the endogenous levels of AEA, 2-AG and PEA was also investigated in other cell lines, such as immune cells (RBL-2H3) or neuronal cells (SH-SY5Y). The results obtained show that in SH-SY5Y cells treated with PGA for 40 min the endogenous levels of AEA (t = 3.012; dF = 19; P = 0.0072) and PEA (t = 2.177; dF = 20; P = 0.0416) (but not 2-AG levels) were significantly increased compared to vehicle-treated SH-SY5Y cells (Fig. 2A, B, C). Instead, in RBL-2H3 cells treated with PGA for 40 min, only PEA levels (t = 2.567; dF = 22; P = 0.0176) were significantly increased compared to vehicle-treated RBL-2H3 cells (Fig. 2D, E, F).

Fig. 1
figure 1

Effect of PGA on the endogenous levels of AEA, 2-AG and PEA in HaCaT cells. A-C Concentrations of AEA, 2-AG and PEA in HaCaT cells treated with PGA (10 µM), or Vehicle for 40 min. Student’s t test was used for analysis. *** P < 0.001 and **** P < 0.0001: Vehicle vs. PGA. D-F Concentrations of AEA, 2-AG and PEA in HaCaT cells treated with PGA (10 µM), or Vehicle for 6 h. Student’s t test was used for analysis. * P < 0.05 and ** P < 0.01: Vehicle vs. PGA.

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Fig. 2
figure 2

Effect of PGA on the endogenous levels of AEA, 2-AG and PEA in SH-SY5Y and RBL-2H3 cells. A-C Concentrations of AEA, 2-AG and PEA in SH-SY5Y cells treated with PGA (10 µM), or Vehicle for 40 min. Student’s t test was used for analysis. * P < 0.05 and ** P < 0.01: Vehicle vs. PGA. D-F Concentrations of AEA, 2-AG and PEA in RBL-2H3 cells treated with PGA (10 µM), or Vehicle for 40 min. Student’s t test was used for analysis. * P < 0.05: Vehicle vs. PGA.

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Glucosamine does not increase AEA, 2-AG and PEA levels in HaCaT cells

To investigate if the PGA-induced increase in the cellular concentrations of AEA, 2-AG and PEA could be attributed to glucosamine, released after the potential hydrolysis of PGA, the levels of these lipid mediators were measured in HaCaT cells and supernatants after 40 min and 6 h of glucosamine treatment (10 µM). The results obtained indicate that glucosamine does not mimic the entourage effect of PGA, neither after 40 min (Fig. 3A, B, C) nor after 6 h (Fig. 3D, E, F) of treatment, as compared to vehicle-treated HaCaT cells (Fig. 3).

Fig. 3
figure 3

Effect of Glucosamine on the endogenous levels of AEA, 2-AG and PEA in HaCaT cells. A-C Concentrations of AEA, 2-AG and PEA in HaCaT cells treated with Glucosamine (10 µM), or Vehicle for 40 min. D-F Concentrations of AEA, 2-AG and PEA in HaCaT cells treated with Glucosamine (10 µM), or Vehicle for 6 h. Student’s t test was used for analysis.

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The PGA-induced increase of PEA levels in HaCaT cells is mostly due to mechanisms other than PGA hydrolysis

To investigate if [13C]4-PGA is hydrolyzed to glucosamine and [13C]4-palmitic acid and the latter reused by cells in order to biosynthesize [13C]4-PEA, the levels of [13C]4-PEA were measured in HaCaT cells and supernatants after 40 min of [13C]4-PGA treatment (10 µM). The results obtained show a significant increase of [13C]4-PEA levels (t = 3.099; dF = 10; P = 0.0113) in [13C]4-PGA-treated HaCaT cells (Fig. 4A), as compared to vehicle-treated HaCaT cells, where [13C]4-PEA was not detectable (Fig. 4A), indicating that some [13C]4-palmitic acid is produced from [13C]4-PGA hydrolysis and recycled into [13C]4-PEA. However, a significant increase of non-isotopically labelled PEA levels (t = 2.367; dF = 10; P = 0.0395) was also observed in [13C]4-PGA-treated HaCaT cells (Fig. 4B) compared to vehicle-treated HaCaT cells (Fig. 4B). Importantly, after subtraction of basal levels found in vehicle-treated control cells, we estimated that [13C]4-PGA increases the levels of non-isotopically labelled PEA 3-fold more than those of [13C]4-PEA (Fig. 3), suggesting that PGA entourage effect on PEA is mostly due to mechanisms other than PGA hydrolysis and palmitic acid recycling into this mediator.

Fig. 4
figure 4

Effect of [13C]4-PGA on the levels of [13C]4-PEA and endogenous non-isotopically labelled PEA. A Concentrations of [13C]4-PEA in HaCaT cells treated with [13C]4-PGA (10 µM), or Vehicle for 40 min. B Concentrations of endogenous non-isotopically labelled PEA in HaCaT cells treated with [13C]4-PGA (10 µM), or Vehicle for 40 min. Student’s t test was used for analysis. * P < 0.05: Vehicle vs. [13C]4-PGA.

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MAFP inhibits the stimulatory effect of PGA on AEA, 2-AG and PEA levels in HaCaT cells

To investigate if the treatment with an unselective serine hydrolase inhibitor, MAFP, is able to counteract the increases of endocannabinoid and PEA levels induced by PGA treatment, HaCaT cells were treated with MAFP (10 µM) in the presence or absence of PGA (10 µM) for 40 min, and lipid mediators were measured in cell pellets and supernatants. The results obtained indicate that when HaCaT cells were treated with MAFP alone, no significative alteration of AEA, 2-AG and PEA levels was observed (data not shown). On the contrary, when HaCaT cells were treated with MAFP in the presence of PGA, the levels of AEA (F(3,20) = 110.6; P < 0.0001) were decreased by 2.6-fold compared to those measured in HaCaT cells treated with PGA only, although they were still significantly higher by 2.7-fold as compared to vehicle-treated HaCaT cells (Fig. 5A). On the other hand, MAFP fully prevented the increases of 2-AG (F(3,18) = 14.01; P < 0.0001) and PEA (F(3,16) = 32.25; P < 0.0001) levels induced by PGA, as compared to PGA-treated HaCaT cells, and the levels of these two mediators were thus comparable to those observed in HaCaT cells treated with only vehicle (Fig. 5B, C).

Fig. 5
figure 5

Effect of MAFP (10 µM) on the endogenous levels of AEA, 2-AG and PEA in PGA-treated HaCaT cells. A-C Concentrations of AEA, 2-AG and PEA in HaCaT cells co-treated with PGA (10 µM) and MAFP (10 µM), or Vehicle for 40 min. One-way ANOVA followed by Tukey’s multiple comparison test was used for analysis. ** P < 0.01 and **** P < 0.0001: Vehicle vs. PGA; ** P < 0.01 and **** P < 0.0001: Vehicle mix vs. PGA + MAFP.

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PGA does not inhibit FAAH

Enzymatic assays performed with rat brain membranes showed no effect of PGA on FAAH activity. PGA was not able to inhibit the amidase activity of the enzyme at concentrations as high as 10 µM (IC50 > 10 µM).

PGA modulates the mRNA expression of anabolic and catabolic endocannabinoidome enzymes

Again, with the purpose of deciphering the mechanism underlying the effects of PGA on AEA, 2-AG and PEA levels, the mRNA expression of endocannabinoid and PEA anabolic (NAPEPLD, ABHD4, GDE1, DAGLA and DAGLB) and catabolic (FAAH, MAGL and NAAA) enzymes was measured in untreated and in PGA-treated HaCaT cells for 40 min and 6 h. Untreated HaCaT cells expressed the investigated anabolic and catabolic genes of endocannabinoids and PEA, that was unchanged between 40 min and 6 h of exposure to the vehicle (DMSO 0.05%) (Fig. 6A, B). Importantly, following PGA treatment (10 µM) for 6 h (Fig. 6B), but not after 40 min (Fig. 6A), the mRNA expression of DAGLB (t = 7.408; dF = 4; P = 0.0018), FAAH (t = 2.725; dF = 4; P = 0.0527), GDE1 (t = 3.979; dF = 4; P = 0.0164), MAGL (t = 2.990; dF = 4; P = 0.0403) and NAAA (t = 3.155; dF = 4; P = 0.0343), was significantly reduced, as compared to vehicle-treated HaCaT cells (Fig. 6B). A trend toward the decrease was also observed with NAPEPLD (Fig. 6B).

Fig. 6
figure 6

mRNA expression of genes encoding for metabolic enzymes regulating the endocannabinoidome activity in HaCaT cells treated with PGA. A Bar chart with individual points showing the mRNA expression levels of the indicated genes measured in HaCaT cells treated with PGA (10 µM), or Vehicle for 40 min. B Bar chart with individual points showing the mRNA expression levels of the indicated genes measured in HaCaT cells treated with PGA (10 µM), or Vehicle for 6 h. Student’s t test was used for analysis. * P < 0.05 and ** P < 0.01: Vehicle vs. PGA.

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ABHD4-siRNA does not significantly alter the PGA-induced increase of AEA, 2-AG and PEA levels

The above results suggest that the downregulation of metabolic enzymes may play a role in the effect of PGA, which is in agreement with the fact that these enzymes are all serine hydrolases and with the occlusive effect of MAFP on PGA effect, described above. To support this possibility using a more specific and molecular approach, we could have knocked down several catabolic enzymes at once and see if this treatment would occlude the effect of PGA, as seen with MAFP. However, since these enzymes were multiple and already down-regulated by PGA, we decided instead to investigate the role of anabolic enzymes by knocking down the only such enzyme whose mRNA expression was entirely unaffected by the treatment with PGA, i.e., ABHD4, thus introducing conditions that would leave unaltered the regulatory action of the catabolic enzymes, or, if anything, emphasize them. Indeed, siRNA-mediated silencing of ABHD4 (t = 9.970; dF = 6; P < 0.0001) (Fig. 7A) and then stimulation with PGA (10 µM) for 40 min (Fig. 7B, C, D) resulted in effects very similar to those observed in untreated HaCaT cells. In fact, PEA levels were increased by 1.8-fold compared to those measured in ABHD4-silenced HaCaT cells treated with only vehicle (t = 4.534; dF = 14; P = 0.0005) (Fig. 7D). Likewise, the endogenous levels of both AEA (t = 2.252; dF = 14; P = 0.0409) and 2-AG (t = 2.177; dF = 14; P = 0.0471) were also increased in ABHD4-silenced HaCaT cells after PGA treatment (Fig. 7B, C). In fact, their levels were increased by 1.8-fold and 1.3-fold, respectively, compared to those measured in ABHD4-silenced HaCaT cells treated with vehicle (Fig. 7B, C).

Fig. 7
figure 7

Effect of PGA on the endogenous levels of AEA, 2-AG and PEA in ABHD4-silenced HaCaT cells. A Bar chart with individual points showing the mRNA expression levels of ABHD4 in control HaCaT cells (scramble) and siRNA ABHD4 transfected HaCaT cells. Student’s t test was used for analysis. **** P < 0.0001: scramble vs. siRNA. B-D Concentrations of AEA, 2-AG and PEA in ABHD4-silenced HaCaT cells treated with PGA (10 µM), or Vehicle for 40 min. Student’s t test was used for analysis. * P < 0.05 and *** P < 0.001: Vehicle vs. PGA.

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Discussion

N-palmitoyl-D-glucosamine (PGA) is the amide of palmitic acid with glucosamine, structurally similar to N-palmitoyl-ethanolamine (PEA) that is the amide of palmitic acid with ethanolamine. In addition to this structural similarity, these two molecules also show a functional similarity because they share, at least in part, a similar mechanism of action. In fact, both compounds are able to downmodulate mast cell activity through the ALIA mechanism27,28. These features of PGA and PEA make the two compounds belong to the same class of molecules known as ALIA-amides. PEA, the principal component of this family, is able to exert anti-(neuro)inflammatory and anti-nociceptive effects also through an endocannabinoid-mediated mechanism known as the “entourage effect”. In particular, PEA acts by potentiating the activity of the endocannabinoids, AEA and 2-AG, at CB1 and CB2 receptors, by increasing their endogenous levels through: (i) the inhibition of the expression of the AEA hydrolytic enzyme FAAH11; or (ii) the stimulation of the activity of the 2-AG biosynthetic enzyme DAGL12. Although being much less studied, PGA, like PEA also exerts anti-inflammatory and anti-nociceptive activities19,22,24, but nothing is known about its ability to interact with endocannabinoids and the endocannabinoidome. Therefore, the present study aimed at investigating the capability of PGA to modulate the endocannabinoidome and to decipher the possible involvement of the latter in the mechanism of action of this molecule in in vitro cell systems, such as human HaCaT keratinocyte cells, where we have previously reported several anti-inflammatory actions of PEA29.

First, we investigated if PGA was able to produce the characteristic effect of PEA, i.e., the “entourage effect” on the endogenous levels of AEA and 2-AG. Our results show that PGA treatment was able to increase the endogenous levels of AEA and 2-AG, as well as those of PEA, in HaCaT cells. Additionally, our results identified other cell types, such as neuronal and immune cells, wherein PGA exerted a similar “entourage” effect, especially on PEA levels. However, it is plausible that these increased PEA levels are not sufficient to determine an entourage effect on 2-AG levels, which we have previously demonstrated in a study published in the same cell type (i.e., RBL-2H3) wherein PEA was exogenously added12. These observations led us to hypothesize that the possible enzymatic hydrolysis of PGA into a palmitic acid and glucosamine could occur, followed by PEA formation and, eventually PEA or glucosamine stimulatory action on AEA and 2-AG levels. This hypothesis was supported by previous reports showing how the radiolabeled substrate N-palmitoyl[1-14 C]-glucosamine is de-N-acylated by the action of fatty-acyl amidases (FAA II), a cell-lysing enzyme secreted by a soil ameba, Dictyostelium discoideum (Amoebidae)17, and was investigated here in HaCaT cells. Thus, we measured the endogenous levels of [13C]4-PEA after [13C]4-PGA treatment and the endogenous levels of PEA, AEA and 2-AG after glucosamine treatment. [13C]4-PGA treatment was performed in order to evaluate if [13C]4-PEA could be produced using [13C]4-palmitic acid derived from the hydrolysis of [13C]4-PGA. Our results show that although [13C]4-PEA was measurable after [13C]4-PGA treatment of HaCaT cells, its levels were 3-fold lower compared to those of endogenous, non-isotopically labelled PEA levels following the same treatment. Additionally, glucosamine had no effect on AEA, 2-AG and PEA levels. These findings indicate that its possible hydrolysis to palmitic acid cannot account for most of the effect of PGA on PEA levels in HaCaT cells, and that glucosamine formed from this putative hydrolysis is not responsible for the “entourage” effect of PGA.

The above data suggest the existence of a mechanism, independent of PGA hydrolysis, responsible for the modulation of PEA, AEA and 2-AG levels in HaCaT cells. Since, we previously demonstrated that glycerophosphodiester phosphodiesterase 1 (GDE-1), an N-acylethanolamine biosynthetic enzyme, is more strongly expressed in these cells than another N-acylethanolamine producing enzyme, NAPE-PLD30, we hypothesized that PGA could act by modulating the activity of GDE1, first to produce PEA and thus subsequently, explain the “entourage effect” on both PEA and AEA levels. Alternatively, this effect of PGA could be due, as previously shown for PEA (see above), to effects on other endocannabinoidome enzymes, responsible for the biosynthesis (in this case PGA would enhance their expression or activity) or degradation (in this case PGA would reduce their expression or activity) of the mediators found here to be enhanced by this compound. Interestingly, these enzymes belong, in most cases, to the wide family of serine hydrolases (the only exception being NAAA). For these reasons, we next conducted three series of experiments. First, we evaluated whether PGA was able of modulating the catabolic enzyme relevant to all three lipids, i.e., FAAH. Secondly, we evaluated whether MAFP, a non-selective serine hydrolase inhibitor that strongly affects the activity of most of the endocannabinoidome metabolic enzymes, would occlude the effects of PGA on PEA, AEA and 2-AG levels. Thirdly, we investigated the effect of PGA on the expression of these enzymes. Our results show that PGA was not able to inhibit FAAH enzymatic activity, while co-treatment with MAFP was able to attenuate (in the case of AEA levels) or completely block (in the case of 2-AG and PEA levels) the effect of PGA. On the other hand, PGA reduced the expression of all catabolic enzymes (i.e., NAAA and FAAH, for PEA and AEA, and MAGL, for 2-AG). Intriguingly, PGA also reduced the expression of some anabolic enzymes (i.e., GDE1 and NAPEPLD, for AEA and PEA, and DAGLB, for 2-AG), although some of these down-regulations did not reach statistical significance (as in the case of NAPEPLD). These findings, taken together, allow us to exclude that PGA acts as a direct FAAH-inhibitor, and lead us to hypothesize that instead: (1) PGA may act by reducing the expression of degradation enzymes for AEA, PEA and 2-AG, rather than enhancing the expression of their biosynthetic enzymes, and (2) the expression of anabolic enzymes does not play a key role in determining the levels of these mediators in HaCaT cells. The former hypothesis was supported by the aforementioned effect of MAFP, which, although used here at a concentration inactive per se on the levels of these mediators, may still have occluded the effect of the down-regulation of the catabolic enzymes by PGA, and hence the increase by the latter compound of AEA, PEA and 2-AG levels. We thus sought molecular evidence for hypothesis 2) by knocking-down the only enzyme whose mRNA expression had been totally unaltered by the molecule, i.e., ABHD4. Indeed, the other possibility, i.e., to knock-down all catabolic enzymes, was not taken into consideration due to the multiplicity and redundancy of these enzymes and to the fact that they were already down-regulated by PGA. We reasoned that, if ABHD4 plays a secondary role in determining the levels of AEA and PEA, and hence in the effect of PGA thereupon, by knocking it down using a specific siRNA (which would also impede the capability of GDE1 to produce AEA and PEA, since GDE1 action is down-stream of ABHD4), we should have observed either a potentiation or no change of the “entourage” effect of PGA. Indeed, we found that the siRNA against ABHD4, whilst being very effective at reducing ABHD4 mRNA levels in HaCaT cells, did not alter the effect of PGA on PEA or AEA (nor 2-AG) levels after a 40 min incubation.

In conclusion, we provided evidence for the first time that PGA strengthens the autocrine signaling of three key mediators of the endocannabinoidome by increasing the levels of the endocannabinoid-like molecule (and PGA naturally occurring analogue) PEA, as well as those of the two endocannabinoids, AEA and 2-AG. We have also provided preliminary evidence that this effect of PGA might be due, at least in part, to down-regulation of catabolic enzymes, such as FAAH, MAGL and NAAA. Although, further studies are needed to explain the exact mechanism of action of PGA and identify other potential targets beyond those belonging to the endocannabinoidome documented in this study, our findings point to this molecule as a modulator of the endocannabinoidome with potential anti-inflammatory and neuroprotective effects.

Materials and methods

Reagents

All reagents were purchased from Sigma-Aldrich (Milano, Italy) unless otherwise stated. N-palmitoyl[13C]4-D-glucosamine ([13C]4-PGA) and unlabeled PGA were obtained from Epitech Group SpA (Saccolongo, Padova, Italy). Deuterated standards—[2H]8-AEA, [2H]5−2-AG and [2H]4-PEA—were purchased from Cayman Chemical (Cabru, Arcore, Italy). The human keratinocyte (HaCaT) cell line was purchased from CLS Cell Lines Service (Eppelheim, Germany). The rat basophilic leukemia (RBL-2H3) cell line and the human neuroblastoma (SH-SY5Y) cell line were purchased from LGC Standards (Milano, Italy). Total mRNA was isolated from HaCaT cells using Trizol Reagent (Thermo Fisher, Milano, Italy) following the manufacturer instructions. cDNA preparation from RNA was performed using iScript Reverse Transcription enzyme (Biorad, Milano, Italy). Specific primer sequences were designed using Primer3 Software and synthetized by Eurofin (Milano, Italy). MISSION® siRNA targeting human ABHD4 was purchased from Levanchimica (Bari, Italy).

Synthesis of N-palmitoyl-D-glucosamine and N-palmitoyl[13C]4-D-glucosamine

N-palmitoyl-D-glucosamine (PGA) was synthesized according to Dufes and collaborators31. Briefly, D-glucosamine (86.3 mg) was dissolved in dimethylsulphoxide (15 mL) and triethanolamine (93 mL). Palmitic acid N-hydroxysuccinimide ester (283 mg) dissolved in chloroform (4 mL) was then added. After stirring the mixture (room temperature for 48 h), chloroform was evaporated off and the remaining liquid freeze-dried. The resulting powder was purified by consecutive washing with water, and finally crystallized from hot methanol (isolated yield: 85%). N-palmitoyl[13C]4-D-glucosamine was prepared by acylation of glucosamine free base with the corresponding acyl chloride. [13C4]-palmitic acid (1.57 g) was converted into the corresponding chloride by reacting with an excess of thionyl chloride at 60 °C for 30 min. under nitrogen atmosphere. Residual thionyl chloride was then evaporated under vacuum at 15 mbar. The crude residue was dissolved in tetrahydrofuran (15 mL) and cooled down to.

−15 °C under nitrogen atmosphere, then added slowly dropwise to a solution obtained by dissolving D-glucosamine hydrochloride (2.15 g) and sodium carbonate (2.3 g) in 40 mL water cooled to 0 °C and kept under vigorous stirring. The obtained mixture was stirred at 0 °C for further 30 min., then at room temperature overnight. Crude product was separated by filtration, accurately washed with pure cool water, dried under vacuum and, finally, crystallized from hot methanol (isolated yield: 75%).

Cell culture

HaCaT cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) complemented with glutamine (2 mM), penicillin (100 U mL−1), streptomycin (100 µg mL−1), and 10% Fetal Bovine Serum (FBS), in a humidified 5% CO2 atmosphere at 37 °C, plated on 100 mm diameter Petri dishes.

RBL-2H3 cells were grown in Eagles Modified Essential Medium (EMEM) supplemented with glutamine (2 mM), penicillin (50 U mL−1), streptomycin (50 µg mL−1) and 15% FBS, in a humidified 5% CO2 atmosphere at 37 °C, plated on 100 mm diameter Petri dishes.

SH-SY5Y cells were grown in DMEM complemented with penicillin (100 U mL−1), streptomycin (100 µg mL−1), 10% FBS, and non-essential amino acids, in a humidified 5% CO2 atmosphere at 37 °C, plated on 100 mm diameter Petri dishes.

Quantification by LC-APCI-MS of endogenous AEA, 2-AG and PEA levels

HaCaT cells or RBL-2H3 cells or SH-SY5Y cells were plated in 6-well culture dishes at a cell density of 9 × 105 cells per well, and after 1 day, were treated with unlabeled PGA (10 µM), or Glucosamine (10 µM), or vehicle of unlabeled PGA or Glucosamine (DMSO or H2O, 0.05%, respectively), and incubated for 40 min and 6 h at 37 °C in the presence of 5% CO2. HaCaT cells were also treated with unlabeled PGA (10 µM) in the presence of MAFP (10 µM), and incubated for 40 min at the indicated condition. After the treatment, cells and supernatants were collected and homogenized in a solution of chloroform/methanol/Tris-HCl 50 mM pH 7.4 (2:1:1 by vol.) containing 10 pmol of [2H]8-AEA, and 50 pmol of [2H]5−2-AG and [2H]4-PEA as internal standards. The lipid-containing organic phase was dried, weighed, and pre-purified by open-bed chromatography on silica gel. Fractions derived by eluting the column with a solution of chloroform/methanol (90:10 by vol.) were analyzed by LC-APCI-MS by using a Shimadzu (Shimadzu, Kyoto, Japan) HPLC apparatus (LC-10ADVP) coupled to a Shimadzu (LCMS-2020) quadrupole MS via a Shimadzu APCI interface. LC-APCI-MS analyses of AEA, 2-AG and PEA were performed in the selected ion monitoring (SIM) mode32,33, using m/z values of 356 and 348 (molecular ion + 1 for deuterated and undeuterated AEA), 384.35 and 379.35 (molecular ion + 1 for deuterated and undeuterated 2-AG), and 304 and 300 (molecular ion + 1 for deuterated and undeuterated PEA). AEA, 2-AG and PEA levels were determined on the basis of their area ratio with the internal standard signal areas to provide the amounts in pmol/mg of lipid extract.

Quantification by LC-APCI-MS of [13C]4-PEA and endogenous PEA levels

HaCaT cells, plated as above described, were treated with [13C]4-PGA (10 µM), and incubated for 40 min at 37 °C in the presence of 5% CO2. After the treatment, cells and supernatants were collected and homogenized in a solution of chloroform/methanol/Tris-HCl 50 mM pH 7.4 (2:1:1 by vol.) containing 10 pmol of N-heptadecanoyl- ethanolamine as internal standard. The fractions, obtained as above described, were analyzed by LC-APCI-MS. The analyses of [13C]4-PEA and endogenous PEA levels were carried out in the selected ion monitoring mode, using m/z values of 314, 304 and 300 (molecular ions + 1 for the standard, and for [13C]4-PEA and endogenous PEA); retention times were 17 for the standard, and 13–14 min for [13C]4-PEA and endogenous PEA34. [13C]4-PEA and endogenous PEA levels were calculated on the basis of their area ratios with the internal standard signal areas to give the amounts in pmol/ml of volume.

Fatty acid amide hydrolase (FAAH) assay

AEA hydrolysis was measured by incubating the 10,000 g membrane fraction of whole rat brain (70 µg/sample) in Tris-HCl 50 mM, at pH 9.0–10.0 at 37 °C for 30 min, with N-arachidonoyl-[14C]-ethanolamine (110 mCi/mmol, ARC) properly diluted with unlabeled AEA (Tocris Bioscience), containing vehicle or increasing concentrations of PGA.

Quantitative real-time PCR

The mRNA expression of endocannabinoids and PEA anabolic (N-acyl phosphatidylethanolamine-specific phospholipase D, NAPEPLD, Alpha/Beta Hydrolase Domain-Containing Protein 4, ABHD4, Glycerophosphodiester Phosphodiesterase 1, GDE1, DAGLA and DAGLB) and catabolic enzyme genes (FAAH; N-acylethanolamine-hydrolyzing acid amidase, NAAA; and monoacylglycerol lipase, MAGL), was studied by comparison of transcriptional expression in untreated HaCaT cells (plated on 100 mm diameter Petri dishes) vs. the expression of these enzymes in HaCaT cells treated with PGA (10 µM), for 40 min and 6 h at 37 °C in 5% CO2 atmosphere. For each target, all mRNA sequences were aligned and common primers were designed (Table 1). Total RNA was isolated from HaCaT cells by use of TRIzol Reagent (cat# 15596018, Life Technology Milan IT), reacted with DNase-I (cat# AMPD1 Merk Life Technology Milan IT) for 15 min at room temperature, followed by spectrophotometric quantification. Subsequently, the RNA integrity number (RIN) for each RNA sample was analyzed on the Agilent 2100 bioanalyzer. Purified RNA was reverse-transcribed by using the iScript cDNA Synthesis Kit (cat# 1708841 Biorad, Milan IT). Quantitative PCR (qPCR) was carried out in a real-time PCR system CFX384 (BioRad) using the SYBR Green PCR Kit (Cat# 1725274, Bio-Rad; Milan IT) detection technique and specific primer sequences are reported in Table 1. Quantitative PCR was performed on independent biological samples for each experimental group. Each sample was amplified simultaneously in quadruplicate in a one-assay run with a no template control blank for each primer pair to control for contamination or primer-dimer formation, and the cycle threshold (Ct) value for each experimental group was determined. The housekeeping gene ribosomal protein S16 was used to normalize the Ct values, using the 2^−ΔCt formula. Differences in mRNA content between groups were expressed as 2^−ΔΔCt.

Table 1 List of primer sequences.
Full size table

Transfection

HaCaT cells (9 × 105 cells plated in a 6-well plate) were transfected with small interfering RNA (siRNA) for ABHD4 (10 nM) and/or a standard negative control (5 nM), re-suspended in MegaTran 2.0 Transfection Reagent and Opti-MEM® (Thermo Fisher Scientific, Waltham, Massachusetts, USA) in a total volume of 100 µL and added drop by drop to cells for 4 h. The transfection was then stopped by adding DMEM supplemented with 20% FBS overnight and replaced with a complete culture medium incubated at 37 °C, 5% CO2 for 48 h. Silencing efficiency was controlled by performing RT-PCR and the obtained ABHD4-silenced HaCaT were used as due. After transfection, ABHD4-silenced HaCaT cells (9 × 105 cells plated in a 6-well plate) were treated with PGA (10 µM), or vehicle (DMSO 0.05%), and incubated for 40 min at 37 °C in the presence of 5% CO2.

Statistical analysis

Each experiment was performed at least three times with sextuplicate groups. Data were expressed as means ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism software version 10.0 (GraphPad Software Inc., San Diego, CA). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test and Student’s t test were used for analysis. P values < 0.05 were considered statistically significant. Figures were generated in GraphPad Prism software version 10.0.