Peroxisome proliferator-activated receptor-alpha (PPAR-α). Oleoylethanolamide (OEA) acts as a high-affinity endogenous ligand of PPAR-α. All anti-fibrotic effects of OEA in vivo and in vitro were mediated by PPAR-α activation. [1]
PPAR-α (peroxisome proliferator-activated receptor-alpha)
TRPV1 (vanilloid receptor)
GPR119 [1]

In CFSC hepatic stellate cells (HSCs) stimulated with TGF-β1 (5 ng/mL), OEA (3, 10, 30 μM) dose-dependently suppressed the mRNA expression of α-SMA and Col1a. OEA also dose-dependently inhibited the protein expression of α-SMA as shown by immunofluorescence staining and Western blot. [1]
OEA (10 μM) reduced TGF-β1-induced phosphorylation of Smad2/3 in CFSC cells, as demonstrated by Western blot and immunofluorescence staining. This inhibitory effect on Smad2/3 phosphorylation was blocked by the PPAR-α antagonist GW6471 (10 μM). [1]
The inhibitory effects of OEA on HSC activation (α-SMA mRNA and protein expression) were completely blocked by the PPAR-α antagonist MK886 (10 μM). [1]
OEA treatment increased the mRNA expression of PPM1A (a phosphatase that dephosphorylates activated Smad2/3) but did not affect the mRNA expression of TGFBR1, TGFBR2, Smad4, or Smad7. OEA did not directly repress TGF-β1 promoter activity. [1]

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Hepatic stellate cells are the target of oléoylethanolamide (OEA), an endogenous PPAR-α ligand that attenuates liver fibrosis. Through PPAR-α, oleoylethanolamide inhibits the activation of hepatic stellate cells (HSCs) elicited by TGF-β1 in vitro. qPCR is used to measure the expression levels of Col1a and α-SMA in TGF-β1-stimulated HSCs in order to evaluate the effect of oleoylethanolamide on HSC activation. When TGF-β1 (5 ng/mL) is stimulated for 48 hours in the group of CFSC cells, the mRNA levels of α-SMA and Col1a are significantly increased; however, the mRNA levels are inhibited in a dose-dependent way when Oleoylethanolamide is applied. The results of immunofluorescence and western blot demonstrate that treatment with oleoylethanolamide dose-dependently suppresses the production of the HSC activation marker α-SMA in protein form. Oleoylethanolamide's inhibitory effects on HSC activation are totally prevented by the PPAR-α antagonist MK886 (10 μM). Furthermore, PPAR-α's mRNA and protein expression levels are down-regulated in response to TGF-β1 stimulation; however, these changes are dose-dependently restored by oleoylethanolamide therapy. Furthermore, it has been discovered that TGF-β1 stimulation causes an upregulation of Smad 2/3 phosphorylation, which is in line with the effects on HSC activation. On the other hand, phosphorylation of Smad 2/3 in CFSC mimicked with TGF-β1 is decreased by oleoylethanolamide (10 μM).

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In CFSC cells (a rat hepatic stellate cell line), treatment with OEA (30 μM, 10 μM, 3 μM) dose-dependently suppressed the mRNA expression levels of α-SMA and Col1a induced by TGF-β1 (5 ng/mL) stimulation for 48 hours. [1]
Immunofluorescence staining and western blot analysis showed that OEA treatment dose-dependently inhibited the protein expression of α-SMA, a marker of hepatic stellate cell activation, in TGF-β1-stimulated CFSC cells. [1]
The inhibitory effects of OEA (10 μM) on TGF-β1-induced HSC activation (α-SMA mRNA and protein expression) were completely blocked by the PPAR-α antagonist MK886 (10 μM). [1]
OEA (10 μM) reduced the phosphorylation of Smad2/3 in CFSC cells stimulated with TGF-β1 (5 ng/mL) for 30 minutes. This effect was blocked by the PPAR-α antagonist GW6471 (10 μM). [1]
OEA treatment (30, 10, 3 μM) had no effect on TGF-β1 promoter activity. [1]
OEA treatment increased the mRNA expression of TGFBR1, Smad4, and PPM1A, while it did not affect the mRNA expression of TGFBR2 and Smad7. [1]
TGF-β1 repressed the mRNA expression of PPAR-α in CFSC cells, which could be reversed by SB-431542, a TGF-β pathway inhibitor. [1]

In Sv/129 wild-type mice fed a methionine-choline-deficient (MCD) diet for 8 weeks, intraperitoneal administration of OEA (5 mg/kg/day) significantly attenuated liver fibrosis development. OEA treatment ameliorated steatosis, reduced hepatocyte ballooning, and decreased collagen deposition (Sirius red staining). OEA also partially prevented the MCD diet-induced increases in serum ALT (P < 0.01), AST (P < 0.05), and hepatic triglyceride (TG) levels (P < 0.05). OEA treatment reduced leukocyte infiltration and suppressed mRNA expression of ICAM and VCAM. OEA also downregulated the hepatic mRNA expression of TGF-β1, α-SMA, Col1a, Col3a, TIMP1, MMP-2, and MMP-9. These protective effects were not observed in PPAR-α knockout mice. [1]
In wild-type mice treated with thioacetamide (TAA, 160 mg/kg, i.p., three times per week for 6 weeks), OEA (5 mg/kg/day, i.p.) significantly prevented the progression of hepatic fibrosis. OEA reduced collagen deposition (Sirius red staining), decreased ICAM and VCAM mRNA expression, reduced TGF-β and α-SMA mRNA expression, and decreased Col1a, Col3a, TIMP1, MMP-2, and MMP-9 mRNA expression. These effects were absent in PPAR-α knockout mice. [1]

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In mice models of hepatic fibrosis, oleoylethanolamide (OEA) can highly reduce the pro-fibrotic cytokine TGF-β1 and adversely regulate genes in the TGF-β1 signaling pathway (α-SMA, collagen 1a, and collagen 3a). By preventing the activation of hepatic stellate cells (HSCs), treatment with oleoylethanolamide (5 mg/kg/day, intraperitoneal injection, ip) considerably slows the progression of liver fibrosis in both experimental animal models[1].

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In MCD diet-fed wild-type Sv/129 mice, OEA administration (5 mg/kg/day, i.p., 8 weeks) ameliorated steatosis, reduced hepatocyte ballooning, and decreased fibrillary collagen deposition (as shown by H&E, Oil red O, and Sirius red staining). OEA treatment also reduced leukocyte infiltration in the liver. These improvements were not observed in PPAR-α knockout mice. [1]
In MCD diet-fed wild-type mice, OEA treatment partially prevented the increases in serum ALT (P<0.01), AST (P<0.05), and hepatic TG levels (P<0.05), but did not attenuate these increases in PPAR-α knockout groups. [1]
In MCD diet-fed wild-type mice, OEA treatment reduced the mRNA expression of adhesion molecules ICAM and VCAM, as well as hepatic mRNA levels of TGF-β1, α-SMA, Col1a, Col3a, TIMP1, MMP-2, and MMP-9. These suppressive effects were not observed in PPAR-α knockout mice. [1]
In TAA-treated wild-type mice (TAA 160 mg/kg, i.p., three times per week for 6 weeks), OEA administration (5 mg/kg/day, i.p.) remarkably prevented the progression of hepatic fibrosis as identified by H&E and Sirius red staining, decreased ICAM and VCAM mRNA expression, decreased TGF-β and α-SMA mRNA expression, reduced Col1a and Col3a mRNA expression, and reduced TIMP1, MMP-2, and MMP-9 mRNA expression. PPAR-α knockout mice were completely insensitive to OEA treatment. [1]
The mRNA and protein expression levels of PPAR-α were reduced with MCD treatment in wild-type mice, but these changes were reversed by OEA treatment. [1]

For western blot analysis, cells were lysed and proteins were separated by SDS-polyacrylamide gel electrophoresis, then transferred to membranes. The target proteins were detected using primary antibodies against α-SMA (1:500), p-Smad2/3 (1:1000), Smad2/3 (1:1000), PPAR-α (1:1000), and β-actin (1:5000). [1]
For immunofluorescence analysis, cells seeded in 24-well plates were fixed with 4% paraformaldehyde for 15 minutes at room temperature. After blocking with 2% bovine serum albumin (BSA) in PBS for 1 hour at room temperature, cells were incubated with primary antibodies against α-SMA (1:100) or p-Smad2/3 (1:100) diluted in 1% BSA overnight at 4°C. After washing, cells were incubated with secondary antibodies (donkey anti-mouse or anti-rabbit IgG conjugated with Alexa Fluor 594) diluted in 1% BSA for 1 hour at room temperature. Cell nuclei were stained with DAPI, and images were captured using a laser scanning confocal microscope. [1]

Cell Culture: CFSC cells (rat hepatic stellate cell line) were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin. Cells were pretreated with various concentrations of OEA (3, 10, 30 μM) or PPAR-α antagonists (MK886 10 μM, GW6471 10 μM) before stimulation with TGF-β1 (5 ng/mL). [1]
RNA Isolation and qPCR: Total RNA from liver tissues and cells was extracted using TRIzol. cDNA was synthesized using a qPCR RT kit. qPCR was performed using SYBR Green to measure mRNA levels of α-SMA, Col1a, Col3a, TGF-β1, TIMP1, MMP-2, MMP-9, ICAM, VCAM, PPAR-α, TGFBR1, TGFBR2, Smad4, Smad7, and PPM1A. Values were normalized to GAPDH or 18S. [1]
Immunofluorescence: Cells were fixed in 4% paraformaldehyde, blocked with 2% BSA, and incubated with primary antibodies against α-SMA (1:100) or p-Smad2/3 (1:100) overnight at 4°C, followed by Alexa Fluor 594-conjugated secondary antibodies. Nuclei were stained with DAPI. Images were captured using a confocal microscope. [1]
Western Blot: Protein lysates were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against α-SMA (1:500), p-Smad2/3 (1:1000), Smad2/3 (1:1000), PPAR-α (1:1000), and β-actin (1:5000). [1]

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CFSC cells (rat hepatic stellate cell line) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in 5% CO2. The medium was replaced every two days, and cells were harvested and diluted at a ratio of 1:3 twice a week. In experiments, HSCs were pretreated with experimental concentrations of OEA before stimulation with 5 ng/mL TGF-β1. [1]
Total RNA from cells was extracted using TRIzol reagent. cDNA was synthesized from total RNA using a qPCR RT kit. Quantification of mRNA was carried out on a real-time PCR system using SYBR Premix Ex Taq. The quantitative values of mRNA were normalized relative to the levels of GAPDH or 18s. [1]

MCD Diet Model: Sv/129 wild-type and PPAR-α knockout mice were fed a methionine-choline-deficient (MCD) diet for 8 weeks. OEA (5 mg/kg/day) or vehicle (5% Tween-80 + 5% PEG400 + 90% saline, 5 mL/kg/day) was administered via intraperitoneal (i.p.) injection. Liver tissues were collected for histological analysis (H&E, Sirius red, Oil red O), and blood was collected for serum ALT and AST measurements. Hepatic TG levels were also measured. [1]
TAA Model: Wild-type and PPAR-α knockout mice were injected with thioacetamide (TAA, 160 mg/kg, i.p., three times per week for 6 weeks). OEA (5 mg/kg/day, i.p.) or vehicle was co-administered. Liver tissues were collected for histological and gene expression analysis. [1]

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Sv/129 mice and PPAR-α knockout mice were maintained in a room with controlled temperature (21-23°C), humidity (55-60%), and lighting (12h light/dark cycles) with water ad libitum. [1]
In the MCD diet-feeding experiment, wild-type and PPAR-α knockout mice were divided into three groups (n=8/group): (i) control group received normal diet; (ii) fed with MCD diet and injected with vehicle (5% Tween-80 + 5% PEG400 + 90% saline, 5 mL/kg/day, i.p., 8 weeks); (iii) fed with MCD diet along with OEA administration (5 mg/kg/day, i.p., 8 weeks). OEA was dissolved in saline supplemented with 5% PEG400 and 5% Tween-80. [1]
In the TAA experiment, wild-type and PPAR-α knockout mice on standard chow diet were randomly separated into three groups: control group (saline injection), TAA group (TAA 160 mg/kg, i.p., three times per week for 6 weeks, dissolved in saline, plus vehicle), and OEA group (TAA injection plus OEA 5 mg/kg/day, i.p., 6 weeks). [1]
For liver histological studies, fresh liver biopsy specimens were fixed in 10% neutral-buffered formalin for 3 days and then embedded in paraffin. Sections of 5 μm were cut and stained with hematoxylin-eosin (H&E) or Sirius red. For lipid accumulation, frozen sections of formalin-fixed liver were stained with Oil red O. Stained areas were viewed and imaged through standard microscopy. [1]
For plasma and hepatic biochemistry, plasma ALT, AST, and hepatic triglyceride (TG) concentrations were determined using a microplate spectrophotometer with commercial kits. [1]

No specific toxicity data for OEA are described in this article. OEA treatment was well-tolerated at the administered dose (5 mg/kg/day i.p.) in mice. [1]

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[1]. Oleoylethanolamide, an endogenous PPAR-α ligand, attenuates liver fibrosis targeting hepatic stellate cells. Oncotarget. 2015 Dec 15;6(40):42530-40.

Oleylethanolamine (OEA) is an N-(long-chain acyl)ethanolamine, a glycolamide derivative of oleic acid. It is a monounsaturated analog of the endocannabinoid anandamide. It possesses multiple functions, including as a PPARα agonist, an EC 3.5.1.23 (ceramidinase) inhibitor, and an anti-aging agent. It is an N-(long-chain acyl)ethanolamine, endocannabinoid, and N-acylethanolamine in a 18:1 ratio. Its function is related to oleic acid. OEA has been reported in fruit flies (Drosophila melanogaster), honeybees (Apis cerana), and other organisms with relevant data. Mechanism of Action: Oleylethanolamine (OEA) is a major N-acylethanolamine and endocannabinoid fatty acid. Although it is an endocannabinoid-like compound, it does not bind to cannabinoid receptors. Conversely, this lipid sensor is a peroxisome proliferation-activating receptor-α (PPAR-α) agonist and a neuraminidase inhibitor, thereby inhibiting the sphingolipid signaling pathway.

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Oleoylethanolamide (OEA) is an endogenous PPAR-α ligand that plays a role in modulating lipid metabolism and has anti-inflammatory properties. Unlike synthetic PPAR-α agonists (Wy-14643, fenofibrate), OEA can also act via other receptors such as TRPV1 and GPR119. [1]
This study demonstrates that OEA ameliorates liver fibrosis in MCD diet- and TAA-induced mouse models through a PPAR-α-dependent mechanism by inhibiting hepatic stellate cell (HSC) activation and suppressing the TGF-β1/Smad2/3 signaling pathway. OEA treatment reduced the expression of fibrosis markers (α-SMA, Col1a, Col3a), inflammation-related adhesion molecules (ICAM, VCAM), and matrix remodeling enzymes (TIMP1, MMP-2, MMP-9). [1]

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Liver fibrosis is characterized by excessive production and deposition of extracellular matrix (ECM) proteins. Hepatic stellate cells (HSCs) are the main producers of matrix components and trans-differentiate into myofibroblast-like cells expressing α-SMA and producing type I and type III collagen. The trans-differentiation is mainly regulated by canonical TGF-β1/Smad signaling. Suppression of HSC activation has been proposed as a therapeutic strategy for liver fibrosis. [1]
PPAR-α is predominantly present in the liver and plays a critical role in energy balance, hepatic lipid metabolism, and fibrogenesis. Synthetic PPAR-α ligands such as fenofibrate, Wy-14643 have been shown to prevent hepatic fibrosis. OEA is an endogenous PPAR-α ligand that also acts on TRPV1 and GPR119. [1]
The anti-fibrotic effects of OEA are mediated by PPAR-α activation, as they were absent in PPAR-α knockout mice. OEA inhibits HSC activation and TGF-β1/Smad2/3 signaling in a PPAR-α-dependent manner. OEA may act as a potential therapeutic agent for liver fibrosis. [1]

C20H39NO2

325.5291

325.298

C, 73.79; H, 12.08; N, 4.30; O, 9.83

111-58-0

Oleoylethanolamide-d4;946524-36-3;Oleoylethanolamide-d2;1245477-09-1; 111-58-0; 68511-29-5

5283454

White to off-white solid powder

0.9±0.1 g/cm3

496.4±38.0 °C at 760 mmHg

50-60ºC

254.0±26.8 °C

0.0±2.9 mmHg at 25°C

1.474

6.36

2

2

17

23

277

0

CCCCCCCC/C=C\CCCCCCCC(NCCO)=O

BOWVQLFMWHZBEF-KTKRTIGZSA-N

InChI=1S/C20H39NO2/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-20(23)21-18-19-22/h9-10,22H,2-8,11-19H2,1H3,(H,21,23)/b10-9-

(Z)-N-(2-hydroxyethyl)octadec-9-enamide

N-(2-Hydroxyethyloleamide; AM-1301; AM1301; OEA

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~20.83 mg/mL (~63.99 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.39 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.39 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)