Effects of arachidonic acid, docosahexaenoic acid and prostaglandin E(2) on cell proliferation and morphology of MG-63 and MC3T3-E1 osteoblast-like cells

During bone remodelling bone is resorbed by osteoclasts and replaced again by osteoblasts through the process of bone formation. Clinical trials and in vivo animal studies suggest that specific polyunsaturated fatty acids (PUFAs) might benefit bone health. As the number of functional osteoblasts is important for bone formation the effects of specific PUFAs on in vitro osteoblastic cell proliferation were investigated. Morphological studies were conducted to determine whether exposure of the cells to these agents caused structural damage to the cells thereby yielding invalid results. Results from this study showed that arachidonic acid (AA) and docosahexaenoic acid (DHA) both inhibit cell growth significantly at high concentrations. The anti-mitotic effect of AA is possibly independent of PGE(2) production, as PGE(2) per se had little effect on proliferation. Further study is required to determine whether reduced proliferation due to fatty acids could be due to increased differentiation of osteoblasts to the mature mineralising osteoblastic phenotype.     

Eicosapentaenoic acid decreases expression of anandamide synthesis enzyme and cannabinoid receptor 2 in osteoblast-like cells

Anandamide (AEA) is an endogenous agonist for the cannabinoid receptor 2 (CB2) which is expressed in osteoblasts. Arachidonic acid (AA) is the precursor for AEA and dietary n-3 polyunsaturated fatty acids (PUFA) are known to reduce the concentrations of AA in tissues and cells. Therefore, we hypothesized that n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which reduce AA in cells, could lower AEA in osteoblasts by altering enzyme expression of the endocannabinoid (EC) system. MC3T3-E1 osteoblast-like cells were grown for 6, 10, 15, 20, 25 or 30 days in osteogenic medium. Osteoblasts were treated with 10 μM of AA, EPA, DHA, oleic acid (OA) or EPA+DHA (5 μM each) for 72 h prior to their collection for measurement of mRNA and alkaline phosphatase (ALP) activity. Compared to vehicle control, osteoblasts treated with AA had higher levels of AA and n-6 PUFA while those treated with EPA and DHA had lower n-6 but higher n-3 PUFA. Independent of the fatty acid treatments, osteoblasts matured normally as evidenced by ALP activity. N-acyl phosphatidylethanolamine-selective phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH) and CB2 mRNA expression were higher at 20 days compared to 10 days. NAPE-PLD and CB2 mRNA was lower in osteoblasts treated with EPA compared to all other groups. Thus, mRNA expression for NAPE-PLD, FAAH, and CB2 increased during osteoblast maturation and EPA reduced mRNA for NAPE-PLD and CB2 receptor. In conclusion, EPA lowered mRNA levels for proteins of the EC system and mRNA for AEA synthesis/degradation is reported in osteoblasts.     

Effects of arachidonic acid, docosahexaenoic acid, prostaglandin E(2) and parathyroid hormone on osteoprotegerin and RANKL secretion by MC3T3-E1 osteoblast-like cells

Bone is continuously remodeled through resorption by osteoclasts and the subsequent synthesis of the bone matrix by osteoblasts. Cell-to-cell contact between osteoblasts and osteoclast precursors is required for osteoclast formation. RANKL (receptor activator of nuclear factor-kappaB ligand) expressed on osteoblastic cell membranes stimulates osteoclastogenesis, while osteoprotegerin (OPG) secreted by osteoblasts inhibits osteoclastogenesis. Although polyunsaturated fatty acids (PUFAs) have been implicated in bone homeostasis, the effects thereof on OPG and RANKL secretion have not been investigated. MC3T3-E1 osteoblasts were exposed to the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA); furthermore, the bone-active hormone parathyroid hormone (PTH) and the effects thereof were tested on OPG and RANKL secretion. Prostaglandin E(2) (PGE(2)), a product of AA metabolism that was previously implicated in bone homeostasis, was included in the study. AA (5.0-20 microg/ml) inhibited OPG secretion by 25-30%, which was attenuated by pretreatment with the cyclooxygenase blocker indomethacin, suggesting that the inhibitory effect of AA on OPG could possibly be PGE(2)-mediated. MC3T3-E1 cells secreted very low basal levels of RANKL, but AA stimulated RANKL secretion, thereby decreasing the OPG/RANKL ratio. DHA suppressed OPG secretion to a smaller extent than AA. This could, however, be due to endogenous PGE(2) production. No RANKL could be detected after exposing the MC3T3-E1 cells to DHA. PTH did not affect OPG secretion, but stimulated RANKL secretion. This study demonstrates that AA and PTH reduce the OPG/RANKL ratio and may increase osteoclastogenesis. DHA, however, had no significant effect on OPG or RANKL in this model.     

The effects of polyunsaturated fatty acids and their metabolites on osteoclastogenesis in vitro

Bone homeostasis is maintained by active remodeling through the balance between resorption (by osteoclasts) and synthesis (by osteoblasts). In this study, we examined the effects of polyunsaturated fatty acids (PUFAs) and their metabolites on sRANKL-induced differentiation of bone marrow-derived macrophages (BMMs) into osteoclasts in vitro. Docosahexaenoic acid (DHA) strongly inhibited osteoclastogenesis; however, dihomo-γ-linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid (EPA) enhanced it. The enhancement effect of PUFAs on osteoclastogenesis was mediated predominantly by cyclooxygenase (COX) products, because the effect was inhibited by a COX inhibitor. It was also found that COX products of PUFAs, prostaglandin E₁, E₂, and E₃, clearly increased in osteoclastogenesis. The inhibitory effect of DHA on osteoclastogenesis was reversed by treatment with a lipoxygenase (LOX) inhibitor. Furthermore, resolvin D1, a LOX product of DHA, significantly inhibited osteoclastogenesis. Quantitative analysis of specific mRNA levels revealed that DHA-mediated attenuation of osteoclastogenesis might be due to a decrease in DC-STAMP expression. These results suggested that the effect of DHA on osteoclastogenesis is, at least in part, mediated by lipoxygenase products. This study showed a distinct mechanism of the effect of PUFAs on osteoclastogenesis and will provide evidence for therapeutic treatment with DHA in osteoporotic patients.     

Contrasting effects of arachidonic acid and docosahexaenoic acid membrane incorporation into cardiomyocytes on free cholesterol turnover

The preservation of a constant pool of free cholesterol (FC) is critical to ensure several functions of cardiomyocytes. We investigated the impact of the membrane incorporation of arachidonic acid (C20:4 ω6, AA) or docosahexaenoic acid (C22:6 ω3, DHA) as ω6 or ω3 polyunsaturated fatty acids (PUFAs) on cholesterol homeostasis in primary cultures of neonatal rat cardiac myocytes. We measured significant alterations to the phospholipid FA profiles, which had markedly different ω6/ω3 ratios between the AA and DHA cells (13 vs. 1). The AA cells showed a 2.7-fold lower cholesterol biosynthesis than the DHA cells. Overall, the AA cells showed 2-fold lower FC masses and 2-fold higher cholesteryl ester masses than the DHA cells. The AA cells had a lower FC to phospholipid ratio and higher triglyceride levels than the DHA cells. Moreover, the AA cells showed a 40% decrease in ATP binding cassette transporter A1 (ABCA1)-mediated and a 19% decrease in ABCG1-mediated cholesterol efflux than the DHA cells. The differences in cholesterol efflux pathways induced by AA or DHA incorporation were not caused by variations in ABCs transporter expression and were reduced when ABC transporters were overexpressed by exposure to LXR/RXR agonists. These results show that AA incorporation into cardiomyocyte membranes decreased the FC turnover by markedly decreasing the endogenous cholesterol synthesis and by decreasing the ABCA1- and ABCG1-cholesterol efflux pathways, whereas DHA had the opposite effects. We propose that these observations may partially contribute to the beneficial effects on the heart of a diet containing a high ω3/ω6 PUFA ratio.     

Long chain‐polyunsaturated fatty acids modulate membrane phospholipid composition and protein localization in lipid rafts of neural stem cell cultures

Rat neural stem cells/neural progenitors (NSC/NP) are generally grown in serum‐free medium. In this study, NSC/NP were supplemented with the main long‐chain polyunsaturated fatty acids (PUFAs) present in the brain, arachidonic acid (AA), or docosahexaenoic acid (DHA), and were monitored for their growth. Lipid and fatty acid contents of the cells were also determined. Under standard conditions, the cells were characterized by phospholipids displaying a highly saturated profile, and very low levels of PUFAs. When cultured in the presence of PUFAs, the cells easily incorporated them into the phospholipid fraction. We also compared the presence of three membrane proteins in the lipid raft fractions: GFR and connexin 43 contents in the rafts were increased by DHA supplementation, whereas Gβ subunit content was not significantly modified. The restoration of DHA levels in the phospholipids could profoundly affect protein localization and, consequently, their functionalities. J. Cell. Biochem. 110: 1356–1364, 2010. © 2010 Wiley‐Liss, Inc.     

Stimulation of prostaglandin E2 (PGE2) production by arachidonic acid, oestrogen and parathyroid hormone in MG-63 and MC3T3-E1 osteoblast-like cells

Polyunsaturated fatty acids (PUFAs) as well as oestrogen (E2) and parathyroid hormone (PTH) affect bone cells. The aim of the study was to determine whether arachidonic acid (AA), E2, and PTH increase prostaglandin E₂ (PGE₂) synthesis in MG-63 and MC3T3-E1 osteoblastic cells and the level of mediation by COX-1 and COX-2. PGE₂ levels were determined in the conditioned culture media of MG-63 and MC3T3-E1 osteoblasts after exposure to AA, PTH and E2. Cells were pre-incubated in some experiments with the unselective COX inhibitor indomethacin or the COX-2 specific blocker NS-398. Indirect immunofluorescence was performed on MG-63 cells to detect the presence and location of the two enzymes involved. AA increased PGE₂ secretion in both cell lines; production by MC3T3-E1 cells, however, was significantly higher than that of MG-63 cells. This could be due to autoamplification via the EP₁ subtype of PGE receptors in mouse MC3T3-E1 osteoblasts. Both COX-1 and COX-2 affected the regulation of PGE₂ synthesis in MG-63 cells. E2 had no effect on PGE₂ secretion in both cell lines, while PTH caused a slight increase in PGE₂ synthesis in the MG-63 cell line.     

Sodium hydrogen sulfide inhibits nicotine and lipopolysaccharide‐induced osteoclastic differentiation and reversed osteoblastic differentiation in human periodontal ligament cells

Although previous studies have demonstrated that hydrogen sulfide (H₂S) stimulated or inhibited osteoclastic differentiation, little is known about the effects of H₂S on the differentiation of osteoblasts and osteoclasts. To determine the possible bioactivities of H₂S on bone metabolism, we investigated the in vitro effects of H₂S on cytotoxicity, osteoblastic, and osteoclastic differentiation as well as the underlying mechanism in lipopolysaccharide (LPS) and nicotine‐stimulated human periodontal ligament cells (hPDLCs). The H₂S donor, NaHS, protected hPDLCs from nicotine and LPS‐induced cytotoxicity and recovered nicotine‐ and LPS‐downregulated osteoblastic differentiation, such as alkaline phosphatase (ALP) activity, mRNA expression of osteoblasts, including ALP, osteopontin (OPN), and osteocalcin (OCN), and mineralized nodule formation. Concomitantly, NaHS inhibited the differentiation of tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts in mouse bone marrow cells and blocked nicotine‐ and LPS‐induced osteoclastogenesis regulatory molecules, such as RANKL, OPG, M‐CSF, MMP‐9, TRAP, and cathepsin K mRNA. NaHS blocked nicotine and LPS‐induced activation of p38, ERK, MKP‐1, PI3K, PKC, and PKC isoenzymes, and NF‐κB. The effects of H₂S on nicotine‐ and LPS‐induced osteoblastic and osteoclastic differentiation were remarkably reversed by MKP‐1 enzyme inhibitor (vanadate) and expression inhibitor (triptolide). Taken together, we report for the first time that H₂S inhibited cytotoxicity and osteoclastic differentiation and recovered osteoblastic differentiation in a nicotine‐ and periodontopathogen‐stimulated hPDLCs model, which has potential therapeutic value for treatment of periodontal and inflammatory bone diseases. J. Cell. Biochem. 114: 1183–1193, 2013. © 2012 Wiley Periodicals, Inc.     

Redox‐modulated colorimetric detection of ascorbic acid and alkaline phosphatase activity with gold nanoparticles

Gold nanoparticles (AuNPs) exhibit characteristic absorption peaks in the ultraviolet visible region due to their special surface plasmon resonance effect. This characteristic absorption peak would change with the relative colour varying from wine red to orange‐yellow upon sequential addition of ascorbic acid (AA) into the mixture of AuNPs and Ag(I). Similar observations also could be found when the hydrolysis product of sodium l ‐ascorbyl‐2‐phosphate with alkaline phosphatase (ALP) was used as an alternative to AA. Results of structure characterization confirmed that the phenomena were due to the reduction of Ag(I) to Ag(0) on the surface of AuNPs and the formation of core‐shell AuNPs@Ag. Therefore, a colorimetric assay for rapid visual detection of AA and ALP based on redox‐modulated silver deposition on AuNPs has been proposed. Under the optimal experimental conditions, the absorbance variation ΔA_{522 nm}/A_{370 nm} of AuNPs was proportional to the concentration of AA (5–60 μmol/L) and ALP (3–18 U/L) with the corresponding detection limit of 2.44 μmol/L for AA and 0.52 U/L for ALP. The assay showed excellent selectivity towards AA and ALP. Moreover, the assay has been applied to detect AA and ALP activity in real samples with satisfying results.     

Successful high‐level accumulation of fish oil omega‐3 long‐chain polyunsaturated fatty acids in a transgenic oilseed crop

Omega‐3 (also called n‐3) long‐chain polyunsaturated fatty acids (≥C20; LC‐PUFAs) are of considerable interest, based on clear evidence of dietary health benefits and the concurrent decline of global sources (fish oils). Generating alternative transgenic plant sources of omega‐3 LC‐PUFAs, i.e. eicosapentaenoic acid (20:5 n‐3, EPA) and docosahexaenoic acid (22:6 n‐3, DHA) has previously proved problematic. Here we describe a set of heterologous genes capable of efficiently directing synthesis of these fatty acids in the seed oil of the crop Camelina sativa, while simultaneously avoiding accumulation of undesirable intermediate fatty acids. We describe two iterations: RRes_EPA in which seeds contain EPA levels of up to 31% (mean 24%), and RRes_DHA, in which seeds accumulate up to 12% EPA and 14% DHA (mean 11% EPA and 8% DHA). These omega‐3 LC‐PUFA levels are equivalent to those in fish oils, and represent a sustainable, terrestrial source of these fatty acids. We also describe the distribution of these non‐native fatty acids within C. sativa seed lipids, and consider these data in the context of our current understanding of acyl exchange during seed oil synthesis.     

Growth Inhibitory Effect of Polyunsaturated Fatty Acids (PUFAs) on Colon Cancer Cells via Their Growth Inhibitory Metabolites and Fatty Acid Composition Changes

Background

Colorectal cancer is common. Polyunsaturated fatty acids (PUFAs) exert growth-inhibitory and pro-apoptotic effects on colon cancer cells. Metabolites of PUFAs such as prostaglandins (PGs), leukotrienes (LTs) and lipoxins (LXs) play a significant role in colon cancer.

Methods

Human colon cancer LoVo and RKO cells were cultured with different concentration of PUFAs and 5-fluorouracil (5-FU) in vitro. Cell morphological changes, fatty acid composition, formation of PGE2, LTB4 and LXA4 and expression of COX-2, ALOX5, PGD synthase (PGDS), microsomal prostaglandin E synthase (mPGES) were assessed in LoVo and RKO cells when supplemented with PUFAs and 5-FU.

Results

PUFAs and 5-FU inhibited growth of LoVo and RKO cells to the same extent at the doses used and produced significant alterations in their shape. As expected, higher concentrations of supplemented PUFAs were noted in the cells compared to control. LA, GLA, AA, ALA and EPA supplementation to LoVo cells suppressed production of PGE₂, LTB₄,and ALOX5, mPGES expression, but enhanced that of LXA₄; whereas DHA enhanced PGE₂ and LXA₄ synthesis but decreased LTB₄ formation and COX-2, ALOX5, mPGES expression. In contrast, 5-FU enhanced formation of PGE₂, LTB₄ and mPGES expression, but suppressed LXA₄ synthesis and COX-2 expression. PGE₂, LTB₄ synthesis and ALOX5 expression was suppressed by LA, GLA, ALA and DHA; whereas AA, EPA and 5-FU enhanced PGE₂ but paradoxically AA decreased and EPA and 5-FU enhanced LTB4 synthesis in RKO cells. All the PUFAs tested enhanced, while 5-FU decreased LXA₄ formation in RKO cells; whereas GLA, AA, and 5-FU augmented while LA, ALA, EPA and DHA enhanced COX-2 expression in RKO cells.

Conclusions

Tumoricidal action of PUFAs on colorectal LoVo and RKO cancer cells in vitro was associated with increased formation of LXA₄, decreased synthesis of PGE₂ and LTB₄ and suppressed expression of COX-2, ALOX5, mPGES, whereas 5-FU produced contrasting actions on these indices.     

Arachidonic acid and Docosahexanoic acid enhance platelet formation from human apheresis-derived CD34+ cells

An Aberration in megakaryopoiesis and thrombopoiesis, 2 important processes that maintain hemostasis, leads to thrombocytopenia. Though platelet transfusions are used to treat this condition, blood banks frequently face a shortage of platelets. Therefore, methods to generate platelets on a large scale are strongly desirable. However, to generate megakaryocytes (MKs) and platelets (PLTs) in numbers sufficient for clinical application, it is essential to understand the mechanism of platelet production and explore efficient strategies accordingly. We have earlier reported that the N-6 and N-3 poly-unsaturated fatty acids (PUFAs), Arachidonic acid (AA)/Docosahexanoic acid (DHA) have beneficial effect on the generation of MKs and PLTs from umbilical cord blood derived CD34⁺ cells. Here we tested if a similar effect is observed with peripheral blood derived CD34⁺ cells, which are more commonly used in transplantation settings. We found a significant enhancement in cell numbers, surface marker expression, cellular ploidy and expression of cytoskeletal components during PLT biogenesis in cultures exposed to media containing AA/DHA than control cultures that were not exposed to these PUFAs. The test cells engrafted more efficiently in NOD/SCID mice than control cells. AA/DHA appears to have enhanced MK/PLT generation through upregulation of the NOTCH and AKT pathways. Our data show that PUFAs could be valuable additives in the culture system for large scale production of platelets for clinical applications.     

Selective regulation of UGT1A1 and SREBP‐1c mRNA expression by docosahexaenoic,eicosapentaenoic, and arachidonic acids

We evaluated, in human cell line HepG2, the action of individual dietary polyunsaturated fatty acids (PUFAs) on the expression of several lipid metabolism genes. The effects of docosahexaenoic acid, 22:6, n‐3 (DHA), eicosapentaenoic acid, 20:5, n‐3 (EPA), and arachidonic acid, 20:4, n‐6 (AA) were studied alone and with vitamin E (Vit.E). DHA, EPA, and AA down‐regulated mRNAs and encoded proteins of stearoyl‐CoA desaturase (SCD) and sterol regulatory element binding protein (SREBP‐1c), two major factors involved in unsaturated fatty acids synthesis. DHA affected SREBP‐1c mRNA less markedly than EPA and AA. Vit.E did not affect these products, both when individually added or together with fatty acids. The expression of UDP‐glucuronosyl transferase 1A1 (UGT1A1) mRNA, an enzyme of phase II drug metabolism with relevant actions within lipid metabolism, resulted also differentially regulated. DHA did not essentially reduce UGT1A1 mRNA expression while EPA and AA produced a considerable decrease. Nevertheless, when these PUFAs were combined with Vit.E, which by itself did not produce any effect, the result was a reduction of UGT1A1 mRNA with DHA, an increase reverting to basal level with EPA and no variation with AA. Observed regulations did not result to be mediated by peroxisome proliferator‐activated receptor (PPAR). Our data indicate that major dietary PUFAs and Vit.E are differentially and selectively able to affect the expression of genes involved in lipid metabolism. The different actions of these slightly different molecules could be associated with their physiological role as relevant nutrient molecules. J. Cell. Physiol. 226: 187–193, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of arachidonic acid on the concentration of hydroxyeicosatetraenoic acids in culture media of mesenchymal stromal cells differentiating into adipocytes or osteoblasts

Metabolites derived from the polyunsaturated fatty acids (PUFA) may modulate the mesenchymal stromal cell (MSC) differentiation. Such cells can differentiate into different cellular types, including adipocytes and osteoblasts. Aging favors the bone marrow MSC differentiation toward the former, causing a loss of bone density associated with pathologies like osteoporosis. The omega-6 arachidonic acid (AA) favors MSC adipogenesis to a greater extent than omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In this work, we study the joint action of both PUFA. Thus, not induced and induced to adipocyte or osteoblast MSC were treated with 20 μM of each PUFA (either AA, AA + DHA or AA + EPA). The expression of osteogenic and adipogenic molecular markers, the alox15b lipoxygenase gene expression and the 5-, 8-, 11-, 12- and 15-hydroxyeicosatetraenoic acids (HETE) derived from the AA metabolism in the culture media were determined. The results show that the adipogenesis induction of AA is not suppressed by the joint presence of EPA and DHA. In fact, both increased the adipogenic effect of AA on MSC differentiated into osteoblasts. The different HETE concentrations increased in cultures supplemented with AA, albeit such concentrations were lower in the cultures induced to differentiate, mainly at day 21 after the induction. Furthermore, the reduction in the HETE concentration was correlated with a higher expression of the alox15b gene. These results highlight the PUFA metabolism differences between uninduced and induced MSC to differentiate into adipocytes and osteoblasts, besides the relevant role of the lipoxygenase gene expression in adipogenesis induction.     

Low concentration of lipopolysaccharide acts on MC3T3‐E1 osteoblasts and induces proliferation via the COX‐2‐independent NFκB pathway

The translocations of lipopolysaccharide (LPS) from the gut and its effects on bone healing are usually of clinical interest during bone fracture. As already widely stuided, Cyclooxygenase‐2 (COX‐2) is a key enzyme for prostaglandin E2 (PGE₂) production, which induces the nuclear factor kappa B (NFκB) activation and is beneficial to fracture healing. In order to know their roles in skeletal regeneration, mouse MC3T3‐E1 osteoblasts were treated with NFκB inhibitor BAY 11‐7082 and sc791 (a selective COX‐2 inhibitor), in the presence of LPS. Interestingly, LPS could induce osteoblasts proliferation through increasing NFκB activation and translocation. This induction was not related to COX‐2 expression, suggesting that LPS‐induced NFκB activiation is independent of COX‐2. It is possible that low concentration of LPS can act as a stimulating factor of the NFκB pathway in nonstimulated cells such as osteoblasts. COX‐2 is not necessary for the NFκB pathway during LPS‐induced proliferation of osteoblasts since sc791 had no effects on this induction. These studies provide insight into a potential mechanism by which LPS can affect bone tissue repair in the initial phase of inflammation. Copyright © 2009 John Wiley & Sons, Ltd.     

Arachidonic acid alleviates the detrimental effects of acetylsalicylic acid on human granulosa cells performance in vitro

Here, we investigated the biological effects of arachidonic acid (AA) in human cumulus granulosa cells (CGCs) after exposure to ASA. Cells were isolated from the follicular fluid and incubated with 0.5 mM acetylsalicylic acid (ASA) and 50 µM AA. Cell viability was analyzed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. E₂ and P₄ levels were measured by chemiluminescence assay. Expression of genes including CYP19A1, FACN, and SCD1 was measured by real‐time polymerase chain reaction assay. Oxidative status was analyzed by monitoring glutathione peroxidase activity. The fatty acid profile was analyzed by the gas chromatography technique. Enzyme‐linked immunosorbent assay was used to measure prostaglandin E₂ (PGE₂) in CGCs after exposure to ASA and AA. Protein levels of the estrogen receptor were studied by immunofluorescence staining. Ultrastructural changes were evaluated by transmission electron microscopy imaging. ASA treatment reduced E₂ production, Cyp19a1 expression, glutathione peroxidase (GPx) activity, and estradiol receptor expression in CGCs. The addition of AA prevented the ASA‐induced E₂ reduction (p < .05) and expression of Cyp19a1. Moreover, AA increased the antioxidant capacity of CGCs exposed to ASA by promoting GPx activity (p < .05). AA increased monounsaturated fatty acid/saturated fatty acid ratio compared with the ASA group (p < .05). AA supplementation triggered the synthesis and secretion of PGE₂ in ASA‐treated CGCS (p < .05). Cytoplasmic vacuolation observed in the ASA group and treatment with AA intensified vacuolation rate. The expression of the estrogen receptor was increased after AA supplementation. Data demonstrated that AA decreased the detrimental effects of ASA on human CGCs after 72 hr.