Palmitoylethanolamide and luteolin ameliorate development of arthritis caused by injection of collagen type II in mice

Abstract

Introduction: N-palmitoylethanolamine (PEA) is an endogenous fatty acid amide belonging to the family of the N-acylethanolamines (NAEs). Recently, several studies demonstrated that PEA is an important analgesic, antiinflammatory, and neuroprotective mediator. The aim of this study was to investigate the effect of co-ultramicronized PEA + luteolin formulation on the modulation of the inflammatory response in mice subjected to collagen-induced arthritis (CIA).

Methods: CIA was induced by an intradermally injection of 100 μl of the emulsion (containing 100 μg of bovine type II collagen (CII)) and complete Freund adjuvant (CFA) at the base of the tail. On day 21, a second injection of CII in CFA was administered. Mice subjected to CIA were administered PEA (10 mg/kg 10% ethanol, intraperitoneally (i.p.)) or co-ultramicronized PEA + luteolin (1 mg/kg, i.p.) every 24 hours, starting from day 25 to 35.

Results: Mice developed erosive hind-paw arthritis when immunized with CII in CFA. Macroscopic clinical evidence of CIA first appeared as periarticular erythema and edema in the hindpaws. The incidence of CIA was 100% by day 28 in the CII-challenged mice, and the severity of CIA progressed over a 35-day period with a resorption of bone. The histopathology of CIA included erosion of the cartilage at the joint. Treatment with PEA or PEA + luteolin ameliorated the clinical signs at days 26 to 35 and improved histologic status in the joint and paw. The degree of oxidative and nitrosative damage was significantly reduced in PEA + luteolin-treated mice, as indicated by nitrotyrosine and malondialdehyde (MDA) levels. Plasma levels of the proinflammatory cytokines and chemokines were significantly reduced by PEA + luteolin treatment.

Conclusions: We demonstrated that PEA co-ultramicronized with luteolin exerts an antiinflammatory effect during chronic inflammation and ameliorates CIA.

Figure 1

Effect of PEA-LUT combination therapy on the clinical expression of CIA and on radiographic analysis. No clinical signs were observed in sham mice (A). CIA developed rapidly in mice immunized with CII and clinical signs like periarticular erythema and edema (B). Hindpaw erythema and swelling increased in frequency and severity in a time-dependent mode (L). CIA-PEA-treated mice demonstrated a significant reduction in the clinical signs of CIA (C). Co-ultramicronized PEA + LUT formulation showed an enhanced reduction of clinical signs of CIA (D). In addition, radiographic analysis was evaluated. No evidence of pathology in the femoral growth plate or in the tibiotarsal joints of normal mice (E, I). Hindpaws from CII-immunized (35 days) vehicle-treated mice showed bone resorption in the femoral growth plate as well as in the tibiotarsal joints (F, I). PEA-treated mice showed less bone erosion in the femoral growth plate, as well as in the tibiotarsal joints of CIA mice (G, I). A significant difference was showed between PEA and PEA-LUT combination therapy as well as between PEA-LUT combination therapy and LUT-alone treatment (H, I). Figure is representative of at least three experiments performed on different days. Values are expressed as mean ± SEM of 20 animals for each group. *P < 0.01 versus sham-control. °P < 0.01 versus CIA. ^#P < 0.01 versus CIA-PEA. ^§P < 0.01 versus CIA-LUT.

Figure 2

Effect of PEA- LUT combination therapy on paw edema and body weight. CIA developed rapidly in mice immunized with CII, leading to a 100% incidence of CIA at day 28 (B). Swelling of hindpaws (A) over time was measured at 2-day intervals. Beginning on day 25, the CII-challenged mice gained significantly less weight, and this trend continued through day 35 (C). CIA-PEA mice demonstrated a significant reduced incidence of weight loss (C), as well as less paw edema (A). CIA-LUT mice did not demonstrate a reduced incidence of weight loss (C) as well as less paw edema (A) compared with the PEA group. Furthermore, the combination therapy with PEA and LUT enhanced the reduction of incidence of body-weight loss and paw edema (A, C). Figure is representative of all the animals in each group. Values are expressed as mean ± SEM of 20 animals for each group. *P < 0.01 versus Sham-control. °P < 0.01 versus CIA. ^#P < 0.01 versus CIA-PEA. ^§P < 0.01 versus CIA-LUT.

Figure 3

Effect of PEA- LUT combination therapy on locomotor activity and pain evaluation. CIA-subjected mice shortened the time to stay on a rotating rod compared with sham mice (A). Locomotor abilities on the rotarod are better maintained in CIA + PEA than in CIA + Vehicle mice (A). Locomotor abilities on the rotarod are not better maintained in CIA + LUT than on CIA + PEA mice (A). A significant difference in locomotor activity was found between PEA and PEA-LUT combination therapy, as well as between PEA LUT combination therapy and LUT-alone treatment (A). In addition, pain evaluation in CIA + vehicle, CIA-LUT, CIA + PEA, CIA + PEA-LUT, and sham mice was measured by a hotplate test and a plantar test. Measurements were recorded in mice able to ambulate. CIA + vehicle mice exhibit increased pain sensitivity and thermal hyperalgesia compared with normal controls. (B, C). PEA treatment reduced significantly pain sensitivity and thermal hyperalgesia in CIA-PEA-treated mice (B, C). LUT treatment did not significantly reduce pain sensitivity and thermal hyperalgesia compared with the PEA group (B, C). The combination therapy with PEA-LUT enhanced the reduction of pain sensitivity and thermal hyperalgesia compared with a higher dose of PEA (B, C). Figure is representative of all the animals in each group. Values are given as mean ± SEM of 20 animals for each group. *P < 0.01 versus Sham-control. °P < 0.01 versus CIA. ^#P < 0.01 versus CIA-PEA. ^§P < 0.01 versus CIA-LUT.

Figure 4

Morphologic changes of CIA. Representative hematoxylin/eosin-stained section of the joint was examined with light microscopy. The histologic evaluation of a joint from CIA-control mice (A, A1, and I) revealed inflammatory cell infiltration and bone erosion. The histologic alterations were significantly reduced in the tissues from CIA-PEA-LUT treated mice (B, B1, and I). Toluidine blue staining was also performed. A significant mast cell infiltration was observed in joint tissues of CIA-subjected mice (C, C1) compared with sham animals (data not shown). PEA-LUT enhanced the reduction of mast cell infiltration (D, D1). In addition, a significant increase in chymase and tryptase expression was found mainly in the joint tissues collected after CIA induction (E, E1, G, G1, and L). Chymase and tryptase expression was significantly attenuated in the joint from CIA-PEA-LUT-treated mice (F, F1, H, H1, and see L). Densitometry analysis of immunocytochemistry photographs (n = 5) for chymase and tryptase from paw sections was assessed (L). Data are expressed as percentage of total tissue area. *P < 0.01 versus Sham-control. °P < 0.01 versus CIA. ^#P < 0.01 versus CIA-PEA. ^§P < 0.01 versus CIA-LUT.

Figure 5

Effect of PEA-LUT combination therapy on cytokines, chemokine expression, and neutrophil infiltration. A substantial increase in the expression of MIP-1α (A), MIP-2 (B), IL-1β (C), IL-6 (D), TNF-α (E), and MPO activity (F) was found in CIA-control mice 35 days after CII immunization. CIA-PEA-treated mice demonstrated a significant reduction in the expression of MIP-1α (A), MIP-2 (B), IL-1β (C), IL-6 (D), TNF-α (E), and MPO activity (F). CIA-LUT-treated mice did not significantly reduce the expression of MIP-1α (A), MIP-2 (B), IL-1β (C), IL-6 (D), TNF-α (E), and MPO activity (F). The combination therapy with PEA-LUT significantly reduced the expression of MIP-1α (A), MIP-2 (B), IL-1β (C), IL-6 (D), TNF-α (E), and MPO activity (F). Values are shown as mean ± SEM of 20 animals for each group. *P < 0.01 versus Sham-control. °P < 0.01 versus CIA-control. ^#P < 0.01 versus CIA-PEA. ^§P < 0.01 versus CIA-LUT.

Figure 6

Effect of PEA-LUT combination therapy on nitrotyrosine immunostaining and MDA levels. A marked increase in nitrotyrosine (A, see particularly A1 and C), staining was evident in the paw 35 days after initiation of CIA. A marked reduction was seen in the immunostaining for nitrotyrosine (B, see particularly B1 and C) in the paws of CIA-PEA-LUT mice. Densitometry analysis of immunocytochemistry photographs (n = 5) for nitrotyrosine from paw sections was assessed (C). The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). Data are expressed as percentage of total tissue area. *P < 0.01 versus Sham-control. °P < 0.01 versus CIA. In addition, MDA levels, a marker of lipid peroxidation, were evaluated. A substantial increase in MDA levels (D) was found in CIA-control mice 35 days after CII immunization. CIA-PEA-treated mice demonstrated a significant reduction in MDA levels (D). The combination therapy with PEA-LUT enhanced the reduction in MDA levels (D). Values are given as mean ± SEM of 20 animals for each group. *P < 0.01 versus Sham-control. °P < 0.01 versus CIA-control. ^#P < 0.01 versus CIA-PEA. ^§P < 0.01 versus CIA-LUT.