Anti-inflammatory activity of palmitoylethanolamide ameliorates osteoarthritis induced by monosodium iodoacetate in Sprague-Dawley rats

Abstract

Novel treatment strategies are urgently required for osteoarthritis (OA). Palmitoylethanolamide (PEA) is a naturally occurring fatty acid amide with analgesic and anti-inflammatory effects. We aimed to examine its effect on OA and elucidate the molecular mechanism of actions in monosodium iodoacetate (MIA)-induced OA Sprague-Dawley rats. The experimental animals were divided into normal control group (injected with saline + treated with phosphate-buffered saline (PBS), NOR), control group (injected with MIA + treated with PBS, CON), 50 or 100 mg/kg body weight (BW)/day PEA-treated group (injected with MIA + treated with 50 or 100 mg of PEA/kg BW/day, PEA50 or PEA100), and positive control group (injected with MIA + treated with 6 mg of diclofenac/kg BW/day, DiC). The changes in blood parameters, body parameters, gene expression of inflammatory mediators and cytokines, knee thickness, and joint tissue were observed. Oral administration of PEA had no adverse effects on the BW, liver, or kidneys. PEA reduced knee joint swelling and cartilage degradation in MIA-induced OA rats. The serum levels of leukotriene B4, nitric oxide, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and prostaglandin E2 considerably reduced in the PEA100 group compared with those in the CON group. In the synovia of knee joints, the mRNA expression of iNOS, 5-Lox, Cox-2, Il-1β, Tnf-α, and Mmp-2, -3, -9, and -13 apparently increased with MIA administration. Meanwhile, Timp-1 mRNA expression apparently decreased in the CON group but increased to the normal level with PEA treatment. Thus, PEA can be an effective therapeutic agent for OA.

Keywords: Cytokine; Inflammation; Monosodium iodoacetate (MIA); Osteoarthritis; Palmitoylethanolamide (PEA).

Fig. 1

Effect of PEA on OA symptoms in MIA-induced OA rats. MIA-induced OA rats were administered either PEA (50 or 100 mg/kg BW/day) or diclofenac (6 mg/kg BW/day) for 4 weeks. The femorotibial joint was scanned, integrated into three-dimensional micro-CT images, and analyzed with a micro-CT scanner and built-in software. a Three-dimensional micro-CT images. b Two-dimensional micro-CT images. c Gray scale reconstructed image for quantitative analysis and BS/BV (%) of the subchondral bone in the femorotibial joint. d Gray scale reconstructed image for quantitative analysis, BV/TV (%), Tb.N (1/mm), and Tb.Th (mm) of the metaphysis of the tibia. Each bar represents mean ± SEM (n = 5). ^*P < 0.01, ^**P < 0.05, and ^***P < 0.001 significantly different from the NOR group. ^#P < 0.01, ^##P < 0.05, and ^###P < 0.001 significantly different from the CON group. PEA palmitoylethanolamide, OA osteoarthritis, MIA monosodium iodoacetate, BW body weight, NOR normal control group (injected with saline + treated with phosphate-buffered saline (PBS)), CON control group (injected with MIA + treated with PBS), PEA50 or PEA100 50 or 100 mg/kg body weight (BW)/day PEA-treated group (injected with MIA + treated with 50 or 100 mg of PEA/kg BW/day), DiC positive control group (injected with MIA + treated with 6 mg of diclofenac/kg BW/day), micro-CT micro-computed tomography, BS/BV bone surface/bone volume, BV/TV bone volume/total volume, Tb.N trabecular number, Tb.Th trabecular thickness

Fig. 2

Effect of PEA on histological changes in the articular cartilage in MIA-induced OA rats. MIA-induced OA rats were administered either PEA (50 or 100 mg/kg BW/day) or diclofenac (6 mg/kg BW/day) for 4 weeks. Articular cartilage was stained with H&E (a) and safranin O (b), and subjected to Osteoarthritis Research Society International (OARSI) scoring (c). Representative staining images are shown. Scale bar, 50 μm. Each bar represents mean ± SEM (n = 5). ^*P < 0.01, ^**P < 0.05, and ^***P < 0.001 significantly different from the NOR group. ^#P < 0.01, ^##P < 0.05, and ^###P < 0.001 significantly different from the CON group. PEA palmitoylethanolamide, OA osteoarthritis, MIA monosodium iodoacetate, BW body weight, H&E hematoxylin and eosin, NOR normal control group (injected with saline + treated with phosphate-buffered saline (PBS)), CON control group (injected with MIA + treated with PBS), PEA50 or PEA100 50 or 100 mg/kg body weight (BW)/day PEA-treated group (injected with MIA + treated with 50 or 100 mg/kg BW/day), DiC positive control group (injected with MIA + treated with 6 mg of diclofenc/kg BW/day)

Fig. 3

Effect of PEA on the expression of aggrecan and COL2A1 in the articular cartilage of MIA-induced OA rats. MIA-induced OA rats were administered either PEA (50 or 100 mg/kg BW/day) or diclofenac (6 mg/kg BW/day) for 4 weeks. Articular cartilage was stained with aggrecan (a) and COL2A1 (b) antibodies. Representative IF staining images are shown. Scale bar, 50 μm. (c), (d) Staining intensity of the indicated proteins was quantified. Each bar represents mean ± SEM (n = 5). ^*P < 0.01, ^**P < 0.05, and ^***P < 0.001 significantly different from the NOR group. ^#P < 0.01, ^##P < 0.05, and ^###P < 0.001 significantly different from the CON group. COL2A1 collagen type II alpha 1, PEA palmitoylethanolamide, IF immunofluorescence, OA osteoarthritis, MIA monosodium iodoacetate, BW body weight, NOR normal control group (injected with saline + treated with phosphate-buffered saline (PBS)), CON control group (injected with MIA + treated with PBS), PEA50 or PEA100 50 or 100 mg/kg body weight (BW)/day PEA-treated group (injected with MIA + treated with 50 or 100 mg of PEA/kg BW/day), DiC positive control group (injected with MIA + treated with 6 mg of diclofenac/kg BW/day)

Fig. 4

Effect of PEA on the expression of inflammatory mediators and cytokines in the synovia of MIA-induced OA rats. MIA-induced OA rats were administered either PEA (50 or 100 mg/kg BW/day) or diclofenac (6 mg/kg BW/day) for 4 weeks. The total RNA was extracted from the synovia and reverse transcribed, followed by amplification using real-time PCR. Expression of target mRNA was normalized to that of GAPDH and represented relative to that of the NOR group. Each bar represents mean ± SEM (n = 10). ^*P < 0.01, ^**P < 0.05, and ^***P < 0.001 significantly different from the NOR group. ^#P < 0.01, ^##P < 0.05, and ^###P < 0.001 significantly different from the CON group. PEA palmitoylethanolamide, OA osteoarthritis, MIA monosodium iodoacetate, iNos inducible nitric oxide synthase, Cox-2 cyclooxygenese-2, 5-Lox 5-lipoxygenase, Tnf-α tumor necrosis factor-α, Il-1β interleukin-1β,Gapdh glyceraldehyde 3-phosphate dehydrogenase, NOR normal control group (injected with saline + treated with phosphate-buffered saline (PBS)), CON control group (injected with MIA + treated with PBS), PEA50 or PEA100 50 or 100 mg/kg body weight (BW)/day PEA-treated group (injected with MIA + treated with 50 or 100 mg of PEA/kg BW/day), DiC positive control group (injected with MIA + treated with 6 mg of diclofenac/kg BW/day)

Fig. 5

Effect of PEA on the expression of MMPs and TIMP-1 in the synovia of MIA-induced OA rats. MIA-induced OA rats were administered either PEA (50 or 100 mg/kg BW/day) or diclofenac (6 mg/kg BW/day) for 4 weeks. The total RNA was extracted from the synovia and reverse transcribed, followed by amplification using real-time PCR. Expression of target mRNA was normalized to that of Gapdh and represented relative to that of the NOR group. Each bar represents mean ± SEM (n = 10). ^*P < 0.01, ^**P < 0.05, and ^***P < 0.001 significantly different from the NOR group. ^#P < 0.01, ^##P < 0.05, and ^###P < 0.001 significantly different from the CON group. PEA palmitoylethanolamide, OA osteoarthritis, MIA monosodium iodoacetate, MMP matrix metalloproteinase, TIMP-1 tissue inhibitor of metalloproteinase-1, NOR normal control group (injected with saline + treated with phosphate-buffered saline (PBS)), CON control group (injected with MIA + treated with PBS), PEA50 or PEA100 50 or 100 mg/kg body weight (BW)/day PEA-treated group (injected with MIA + treated with 50 or 100 mg of PEA/kg BW/day), DiC positive control group (injected with MIA + treated with 6 mg of diclofenac/kg BW/day)