2.1.1 AA metabolites are involved in the regulation of molecular signaling in bone metabolism

Many studies have shown that AA metabolites are involved in bone development and bone diseases. Bone development is the result of a series of synchronous events, including osteogenesis and bone resorption. Osteoblasts, osteoclasts and chondrocytes are critical for bone homeostasis.²⁴ AA signaling is important for the differentiation and function of osteoblasts, osteoclasts and chondrocytes. Ca²⁺ is required for osteoblastogenesis, and AA causes a concentration-dependent increase in the intracellular Ca²⁺ concentration due to the action of the COX metabolites—PGE-1 and PGE-2.²⁵ Osteoblasts are differentiated from bone marrow-derived mesenchymal stem cells (BMSCs), which are subject to subtle gene expression regulation. Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator that governs the differentiation of BMSCs into adipocytes through cyclic adenosine monophosphate-protein kinase A system (cAMP–PKA) signaling via the regulation of PGI-2.^26-28 In the inflammatory state, osteoblasts are stimulated by inflammatory factors, such as interleukin-6 (IL-6) and LPS, resulting in the activation of the AA metabolic pathway, causing an imbalance in osteoprotegerin/receptor activator of nuclear factor kappa-B (OPG/RANK) and affecting bone homeostasis due to the proliferation and differentiation of osteoclasts (Figure 2).^{29, 30}

Moreover, treatment of BMSCs with a COX-2 inhibitor suppressed the osteogenic genes encoding runx2, alkaline phosphatase, and osteocalcin.^{31, 32} The effect of AA on bone formation is also reflected in the regulation of cytokine and growth factors expression. Insulin-like growth factor-1 (IGF-1) is a crucial growth factor that regulates bone mass, enhances osteoblast differentiation, and promotes bone formation. PGE-2 has been shown to stimulate IGF-1 and its binding proteins. Studies have suggested that AA might influence bone formation and resorption by regulating the synthesis and action of IGF-1 and IGF binding proteins.^{33, 34} In vitro studies have shown that the bone resorption activity of PGE-2 mediated by receptor activator of nuclear factor-κb ligand (RANKL) is critical for the induction of osteoclast formation. Moreover, bone resorption stimulated by inflammation involves PGE-2 production.^{35, 36} Interestingly, PGE-2 also promotes osteogenesis by stimulating osteoblast proliferation and differentiation.³⁷ These findings indicate that the nature of PGE-2 in bone remodeling is multifaceted. LTB-4 promotes peripheral blood mononuclear cell differentiation into osteoclasts in a RANKL-dependent manner,³⁸ and it reduces mineralized nodule formation and alkaline phophatase (ALP) activity in primary osteoblasts.³⁹ LTD-4, another leukotriene metabolite, significantly increases the expression of p53, p21, and plasminogen activator inhibitor-1 (PAI-1), as well as the activity of senescence-associated β-galactosidase (SA-β-Gal), with commensurate reduction in SIRT1 expression, resulting in the senescence of osteoblasts.⁴⁰ Relatively few studies have been focused on the AA metabolite-mediated differentiation and development of chondrocytes by AA metabolites. One study showed that PGE-2 inhibited the expression of the differentiation-related genes in chondrocytes, such as collagen-X (col-X), vascular endothelial growth factor (VEGF), matrix metalloproteinase-13 (MMP-13), and ALP, in a dose-dependent manner, resulting in inhibited chondrocyte maturation.⁴¹

2.1.2 AA metabolites play essential roles in bone metabolic diseases

The effect of AA metabolites on osteoblasts and osteoclasts is of great importance to bone mineral density and bone mass.⁴² A condition closely related to bone disease is osteoporosis. In vivo studies showed that administration of AA into ovariectomized mice decreased the bone mineral density and weakened biomechanical functions by reducing the mineral apposition rate and impairing the microstructure of trabecular bone. The underlying mechanism was described as AA inducing an increase in the content of serum PGE-2 and causing a marked increase in the expression of tumor necrosis factor associated factor-6 (TRAF6), nuclear factor-kappa b (NF-κB), c-fos proto-oncogene (c-fos), and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), which leads to a decrease in the OPG/RANKL ratio.⁴³ Endocannabinoids produced during AA metabolism via the fatty acid amide hydrolase (FAAH) pathway play important roles in the regulation of bone homeostasis. Administration of FAAH ameliorated ovariectomy-induced bone loss in mice by suppressing RANKL through the regulation of IL-17 signaling.⁴⁴ Lipoxin A4 (LXA-4) is a metabolic product of AA produced by lipoxidase. A study showed that LXA-4 reduced ovariectomy-induced bone loss by inhibiting NF-κB, activator protein-1 (AP-1), and phosphoinositide 3 kinase-threonine kinase (PI3K-AKT) activity, as well as p38, extracellular regulated protein kinase (ERK), and c-Jun N-terminal kinase (JNK) in the mitogen activated protein kinases (MAPKs) activation. Furthermore, LXA-4 prevented the production of ROS and the expression of osteoclast-specific genes, resulting in inhibited osteoclastogenesis.⁴⁵

AA metabolite signaling not only contributes to bone metabolism but also exerts regulatory effects on the occurrence of inflammatory bone diseases such as osteoarthritis, rheumatoid arthritis and synovitis. Ye et al.⁴⁶ discovered that AA-regulated calcium signaling in T cells from rheumatoid arthritis patients led to increased activity of the AA-regulated calcium-selective (ARC) channel and the phosphorylation of components in the T-cell receptor signaling cascade, which resulted in the promotion of synovial inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs) targeting COX-2 have been widely used in the clinic to treat arthritis.^{47, 48} Considering the side effects of COX-2 inhibitors as treatments for arthritis,⁴⁹ many efforts have been made to develop safer drugs. Jiang et al.⁵⁰ found that PGE-2 mediated cell migration and osteoclastogenesis via its prostaglandin E receptor 2 (EP2) and prostaglandin E receptor 4 (EP4) receptors; therefore, drugs aimed at EP4 showed better selectivity in treating osteoarthritis.

Considering the role of AA metabolic pathways in the regulation of bone homeostasis and diseases, such as osteoarthritis, targeting related metabolites could be effective in modulating skeletal development and osteoarthritic diseases, thus overcoming the adverse effects of currently used clinical drugs such as glucocorticoids.⁵¹

2.2.1 AA metabolism in liver development and diseases

Studies focused on the expression of CYP-450 EPO family members (CYP2C8, CYP2C9, CYP2C19, and CYP2J2) in human embryonic/fetal tissues have shown that CYP2C8, CYP2C9, and CYP2C19 is initially expressed in the liver in prenatal week 5 and remains steadily expressed until the 20th week. The expression of CYP2J2 is also initiated in the liver in the 5th prenatal week, peaks in the 12th week and then gradually decreased to the normal level.⁵² AA is an essential component of the cell membrane. Jakobsson et al. utilized radioactive phospholipids to identify the types of fatty acids in the hepatocellular membrane. AA constitutes approximately 10-30% of the cell membrane composition.^{53, 54} AA is not only a main component of the hepatocyte membrane but also participates in the proliferation and differentiation of hepatocytes. Transforming growth factor-beta (TGF-β) is a multifunctional cytokine that is pivotal in the regulation of hepatic cell proliferation, differentiation, and migration.^55-57 Han et al.⁵⁸ discovered that TGF-β regulated the growth of primary and transformed hepatocytes through the concurrent activation of Smad and phosphorylation of cPLA₂, which indicated that AA metabolic signaling may be indirectly involved in the regulation of hepatocyte differentiation and development. Additionally, intracellular Ca²⁺ signaling is essential for cell development.⁵⁹ Notably AA inhibited the store-operated Ca²⁺ flow but did not activate Ca²⁺-permeable channels in rat liver cells.⁶⁰

AA is not the only important factor in the liver, liver function and health also depend on feedback mechanisms regulated by AA metabolites. Experiments have shown that the FAAH level is decreased in murine models of liver fibrosis, which leads to increased ROS levels and hepatocellular injury.⁶¹ In addition, 2-arachidonoyl glycol (2-AG) has been reported to selectively induce hepatic cell apoptosis and cause liver dysfunction.⁶² In liver cirrhosis, the level of COX-2 but not that of COX-1 was significantly increased, and the use of selective COX-2 inhibitors in patients with liver cirrhosis was beneficial in reducing inflammation and preventing malignant transformation.⁶³ However, in transgenic animal models, COX-2 did not appear to be the cause of liver fibrosis or cirrhosis because hepatocytes overexpressing human COX-2 and COX-2 knockout mice showed the same level of hepatic injury as wild type mice in response to CCl₄.⁶⁴ In addition, the role of 5-LOX, 15-LOX-1, and 15-LOX-2 in alcohol-induced liver cirrhosis has also been studied. The results showed that increased plasma HETE concentrations were in line with upregulated 5- and 15-LOX-1 and 15-LOX-2 mRNA in liver samples from cirrhosis patients.⁶⁵ AA metabolic signaling plays important roles in liver cancer. COX-2/PGE-2 signaling promotes the survival of liver cancer cells. Studies have shown that when hepatocellular carcinoma cell (HCC) lines were treated with selective COX-2 inhibitors, the proliferation of liver cancer cells was suppressed.^{66, 67} Targeted treatment of HCC revealed that the combined use of COX-2 inhibitors with sorafenib showed a synergistic inhibitory effect on tumor growth and angiogenesis in mice bearing HCC xenografts.⁶⁸ 5-LOX is another AA metabolic signaling pathway that has been studied in depth in liver cancer. Expression of the 5-LOX protein was upregulated both in HCC cell lines and in patient tumors. 5-LOX and its metabolite LTB-4 have been shown to participate in the metastasis of HCC cells. A 5-LOX inhibitor but not a COX inhibitor reduced the number of metastatic foci. Moreover, the administration of an antagonist of the LTB-4 receptor reduced the number of lung metastasis foci.⁶⁹

2.2.2 AA metabolism in kidney development and diseases

In contrast to that in the liver, the expression pattern of the PGE-2 synthetic system in the kidney has been associated with postnatal nephrogenesis. The transient induction of the mRNA and protein expression of microsomal PGE synthase-1 has been observed between postnatal days 4 (P4) and 8 (P8) during the first 10 days after birth, and the protein levels of both COX-1 and COX-2 reached their highest levels on P8.⁷⁰ CYP2J5 expression was the most abundant in the kidney and was lower in the liver. CYP2J5 was expressed before birth and reached maximal levels in the kidney 2−4 weeks of age. However, CYP2J5 is not expressed in the fetus and is first expressed in 1 week of the early postnatal period and remains relatively constant in the liver.⁷¹ Although these studies suggest that the temporal and spatial expression of these AA metabolites is precisely regulated, the underlying mechanisms remain largely unknown. Recently, AA and its metabolites have been shown to be baroreceptors that transduce changes in mechanical pressures via nuclear mechanotransduction mechanisms to change gene expression and renin cell function.⁷²

AA metabolic signaling also plays important roles in maintaining kidney function. Reportedly, LOX and CYP-450 alter renal blood flow and the glomerular filtration rate.⁷³ Studies based on renal blood flow have shown that although COX-2 is expressed in the macula densa and plays an important role in renin secretion, COX metabolites are not critical components for the renal blood flow regulatory pathway. Similarly, no glomerular LOX metabolites have been shown to contribute to renal blood flow regulation. On the other hand, the critical role of CYP metabolites in renal blood flow has been demonstrated, and 20-HETE is a key component of the afferent arteriolar autoregulatory response.⁷⁴ The roles of COX metabolites, LOX metabolites, and CYP metabolites involved in regulating renal blood flow can be distinguished by selective AA metabolic signaling inhibitors or agonists.⁷⁵ Increased afferent arteriolar constriction to increasing perfusion pressure were greatly attenuated by the selective CYP hydroxylase inhibitor.⁷⁶ The 20-HETE synthesis inhibitor HET0016 and the antagonist 6,15-20-HEDE completely blocked the myogenic response in afferent arterioles.^{77, 78} The mechanisms underlying the roles of CYP and LOX in glomerular filtration mainly involve K⁺ and Ca²⁺ channels, cyclic guanosine monophosphate (cGMP) and cAMP.⁷³ For example, the LOX metabolite 12(S)-HETE has been shown to influence renal microvessels and glomerular mesangial cells through L-type Ca²⁺ channels.⁷⁹ 20-HETE inhibits Na⁺-K⁺-ATPase activity by enhancing the protein kinase C (PKC) pathway activation.⁸⁰ Furthermore, 20-HETE downregulates Na⁺-K⁺-ATPase α1 expression via the ubiquitin‒proteasome pathway, and phosphorylated Na⁺-K⁺-ATPase α1 is a prerequisite for ubiquitination.⁸¹ CYP and LOX metabolites also interact with the nitric oxide (NO) synthase and ROS pathways, which are involved in the glomerular filtration barrier.⁸² 12-/15-LOX catalyze the depletion of NO and prevent NO activation of cGMP, and knocking out of 12-/15-LOX results in increased eNOS expression.⁸³ Because of the important role of AA metabolic signaling in renal blood flow and glomerular permeability, the occurrence and development of kidney diseases such as nephritis, renal fibrosis, and renal cancer are closely related to AA metabolite levels.⁸⁴ Thus, intervening in AA metabolism, such as inhibiting the 5-LOX pathway,⁸⁵ COX-2 pathway,⁸⁶ or CYP-450 pathway,⁸⁷ is a promising approach to regulate renal function. All of the abovementioned studies indicate that targeting the regulation of AA metabolites and related signaling pathways is a potential approach to the clinical treatment of liver and kidney diseases.

Although AA metabolic pathways have their own expression patterns in the liver and kidney, metabolites play essential roles in the development of the liver and kidney. AA metabolic pathways have similar effects on liver and kidney disease, mainly in terms of oxidative stress, fibrosis, and modulation of the vascular microenvironment. The understanding of these common factors will assist in the development of drugs for the treatment of liver and kidney disease.

2.3.1 Effects of AA on fetal neurodevelopment

Neurodevelopment is a complex physiological event that requires an adequate nutrient supply. Among key nutrients, the long-chain polyunsaturated fatty acids (PUFAs) docosahexaenoic acid 22:6n-3 (DHA) and AA 20:4n-6 (ARA) have been proven to be required for fetal neurodevelopment.^{88, 89} Compared with those of DHA, the roles of AA in neurodevelopment are not well characterized. The accumulation of AA in the fetal brain, particularly within the cerebral cortex mainly occurs in the middle and terminal stages of pregnancy.^{90, 91} Data showed that AA accounted for 12% of the fatty acids in the whole brain,⁹² and supplementation with AA in early life benefits to cognition in early and middle childhood.^{93, 94} Studies have shown that AA metabolites affect the transmission of synaptic signals mainly through the G protein-coupled receptors (GPCRs) cannabiniod receptors (CB1 and CB2), which are highly expressed in the hippocampus, basal ganglia, cerebellum, cortex, thalamus, amygdala, and olfactory bulb. Furthermore, non-GPCR molecular targets of AA metabolites have been discovered, such as the membrane cation channels known as TWIK-related acid-sensitive K (TASK-1 K⁺) channels, T-type Ca²⁺ channels, and vanilloid type 1 receptor (VR1).^95-97 In long-term depression state, postsynaptic IP3 receptor-mediated Ca²⁺ release from internal stores, postsynaptic eicosanoids synthesis, and activation of CB1 receptors possibly via release of the gliotransmitter d-serine.⁹⁸ Notably, the underlying mechanism of AA metabolites regulation of Ca²⁺ influx between neuron synapses may rely on reduced N-methyl-D-aspartic acid receptor (NMDAR)-induced calcium influx via CB1-mediated closure of voltage-sensitive calcium channels in the brain (Figure 3).⁹⁹

However, the content of AA in the brain is unaffected by postnatal dietary supplementation, which is different from that of DHA.¹⁰⁰ The reason for this difference between AA and DHA is likely caused by the distinctive gene expression pattern of the fatty acid desaturase (FADS-gene) cluster and differences in sensitivity to dietary supplementation.^{101, 102} The roles of AA metabolic signaling in neurodevelopment have been studied via genetically altered mouse models.¹⁰³ COX-2 knockout mice exhibited decreased susceptibility to ischemia/reperfusion brain injury and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced brain injury.¹⁰⁴ COX-2 plays an important role in cholinergic signaling in the brain, as indicated by the abrogation of AA incorporation in response to arecoline in COX-2 knockout mice, suggesting that COX-2 is required for muscarinic receptor activation.¹⁰⁵ Data from PGE-2 receptor-EP-1 knockout mice showed that the EP1 receptor mediated neurotoxicity after NMDA treatment, oxygen/glucose deprivation, or focal ischemia.¹⁰⁶ However, another PGE-2 receptor, EP-2, exerted a protective effect against NMDA-induced neurotoxicity in the hippocampus.¹⁰⁷ 12-/15-LOX knockout mice were protected against neuronal cell death and oxidative stress caused by transient focal ischemia.¹⁰⁸ These studies indicate that the AA metabolic signaling pathway is involved mainly in mediating oxidative stress and neurotoxicity.

2.3.2 AA metabolism in neurodegenerative diseases

In nervous system diseases, the relationship between AA and its metabolites and degenerative brain diseases, such as Alzheimer's disease and Parkinson's disease, has been widely reported.¹⁰⁹ The level of intracellular free AA and the balance between the release and incorporation of membrane phospholipid enzymes played critical roles in neuroinflammation and synaptic dysfunction in mice with Alzheimer's disease before amyloid plaques and neurofibrillary tangles developed.¹¹⁰ COX-2 is overexpressed in the cortex and hippocampus of Alzheimer's disease patients.^{111, 112} 5-LOX protein expression is also upregulated in Alzheimer's disease patients, and immunoreactivity induced using 5-LOX amino terminus-directed antibodies was lacking in neurons but abundant in neurofibrillary tangles, neuritic plaques, and glia.¹¹³ Increasing evidence has proven that 5-LOX is involved in Aβ peptide formation and deposition. Chu and Pratico reported that 5-LOX modulated Aβ peptide generation by activating cAMP-response element-binding protein (CREB) and promoting γ-secretase complex expression at the transcriptional level in Tg2576 and 3xTg mice and N2A-APPswe cells.^{114, 115} In addition, 5-LOX overexpression resulted in the activation of cyclin-dependent kinase 5 (cdk5), which was followed by the significant elevation in the tau phosphorylation rate, which suggests another important mechanism underlying Alzheimer's disease.¹¹⁶ Genetic deletion and inhibition of 5-LOX inhibited cdk5-dependent tau phosphorylation and glial reactions and relieved synaptic dysfunction and memory impairment.^{117, 118} Additionally, abnormal activation of 5-LOX has been found in the pathological progression of cerebral ischemia. A direct connection between 5-LOX products (LTC-4 and LTD-4) and ischemic brain damage was reported in bilateral common carotid occlusion-induced transient forebrain ischemia in gerbils.¹¹⁹ In summary, although further studies are still required to understand the roles of AA metabolites in regulating neurological function, the inhibition of AA metabolic signaling pathways is a promising therapeutic strategy for brain dysfunction and neurodegenerative diseases.

As an important component of fatty acids in the brain, the role of AA in brain development and cognitive function remains a mystery. In some psychiatric diseases, such as bipolar disorder, a decreased turnover of AA and its metabolites was found, instead of docosahexaenoic acid.¹²⁰ In addition, as more evidence accumulates, interventions in AA metabolic pathways may be of great importance for the alleviation of neurodegenerative diseases.

2.4.1 AA metabolism in vascular tone and blood pressure

AA and its metabolites play pivotal roles in cardiovascular function (Figure 4). They function as vasodilators or vasoconstrictors and modulate vasodilation under pathological and physiological conditions.^{121, 122} Some blood vessels carry EPO, and AA can be metabolized to epoxides through EPO. Notably, that 5,6-epoxides exhibit vasodilation-inducing effects. In addition, AA and its metabolites are considered endothelium-derived relaxing factors, which causes endothelium-dependent vasodilation. COX is another important enzyme in AA metabolism and is highly active in platelets. When activated by collagen or thromboxane, platelets release ADP and 5-HT, increase the synthesis of thromboxanes (TXs), and thus cause platelet aggregation.^{123, 124} EPO metabolites inhibit COX activity in platelets, reduce the TX production rate, and thus inhibit platelet aggregation. In this way, EPO metabolites show synergistic effects with other antiplatelet aggregation factors, such as PGD-2 and PGI-2, and their levels are balanced with the levels of platelet aggregation-promoting factors like adenosine diphosphate (ADP), 5-hydroxtryptamine (5-HT), and TXs.

Prostacyclin (PGI-2) is the main product of AA metabolism in the blood vessel wall and is the most effective endogenous inhibitor of platelet aggregation. Investigations with animals and humans have clearly shown the endothelial thromboresistance and atheroprotection conferred by vascular COX-2-derived PGI-2.⁵ Honda et al.¹²⁵ found that oral administration of selexipag, a recently approved, orally available and selective PGI-2 receptor agonist, significantly reduced right ventricular systolic pressure in Sprague‒Dawley SuHx rats.¹²⁶

TXA-2 is a major metabolite of AA in platelets and vascular endothelial cells and promotes platelet aggregation and induces thrombosis. TXA-2 promotes the dissociation of calcium ions in the dense tubule system, causes the contraction of dense bodies, induces the release of ADP and 5-HT, and cause the aggregation of nearby platelets.¹²⁷ TXA-2 in activated platelets contributes to primary hemostasis and atherothrombosis in animal and human models. Additionally, TP receptors are activated by TXA-2 and induce vasoconstriction. Under normal physiological conditions, the levels of TXA-2 and PGI-2 in circulating blood are relatively balanced, which is important maintaining smooth blood circulation. An imbalance in TXA-2 and PGI-2 levels may lead to thrombosis and tissue ischemia. When thrombosis occurs, the TXA-2 level is usually increased or the PGI-2 level is decreased.¹²⁸

LTs constitute a group of inflammatory substances produced via AA metabolism through the 5-LOX pathway, increasing the incidence of myocardial infarction (MI), stroke, atherosclerosis, and aortic aneurysm.¹²⁹ LTB-4 reportedly stimulated the release of contraction factors and NO by activating the LT receptor of aortic endothelial cells and induce endothelium-dependent vasoconstriction, thus promoting atherosclerosis, which is also closely related to the formation of atherothrombosis through NF-κB signaling.^{130, 131} LTC-4, LTD-4, LTE-4, and LTF-4 belong to the CysTL family and are involved in the proliferation and migration of vascular smooth muscle cells.^132-134

EETs and HETEs participate in the regulation of vascular endothelial cells and smooth muscle cells proliferation, migration and apoptosis and function as endogenous vasodilators to mediate the contraction of blood vessels. The normally functioning EETs and HETEs play important roles in maintaining the stability of vascular system functions and angiogenesis. However, abnormal EET and HETE metabolism may readily lead to the occurrence of vascular disorders, such as hypertension.¹³⁵

EETs are metabolites of CYP-450 epoxidases, and they include 5,6-, 8,9-, 11,12-, and 14,15-EETs, which are endothelium cell-derived hyperpolarizing factors and play important protective roles in the cardiovascular system.¹³⁶ EETs are released from the vascular endothelium and exert effects on smooth muscle cells under endothelial cells. Generally, they activate potassium calcium channels in the cell membrane, thereby hyperpolarizing the cell membrane and leading to the relaxation of smooth muscle cells.¹³⁷ Moreover, EETs contribute to vasomotor tone control of endothelial cells by activating calcium-activated potassium channels, endothelial NO synthase transcription and MAPK, PI3K/AKT, and PKC signaling.^{138, 139}

HETEs are important endogenous factors that facilitate the depolarization of smooth muscle cells and maintain the contraction of smooth muscle cells by inhibiting the activation of many calcium channels on the surface of vascular smooth muscle. Thus, HETEs increase the intracellular calcium concentration.

In 1989, Escalante et al.¹⁴⁰ first reported the vasoconstrictive effects of 20-HETE in the rat aorta. 20-HETE activated PKC, MAPK, and src-type tyrosine kinase, which elevated the cytosolic Ca²⁺ level and increased the Ca²⁺ entry rate through specific channels.¹⁴¹ 20-HETE also maintained the phosphorylation rate of myosin light chains through Rho kinase and increased the Ca²⁺-related contraction rate.^{142, 143}

Studies in animals and humans demonstrated that deficient 20-HETE biosynthesis in tubules and increased 20-HETE levels in the vasculature contribute to hypertension by sensitizing the vasculature to constriction-inducing stimuli, potentiating vascular inflammation, and causing endothelial dysfunction.^{142, 144, 145} Another study of vascular calcification in mice demonstrated that the levels of multiple metabolites of AA were significantly increased in calcified aortas; the abundant metabolites included 12-HETE, 11-HETE, and 15-HETE, of which the most abundant metabolite was 12-HETE. A specific inhibitor of the metabolic enzyme arachidonic 15-LOX (ALOX15) significantly reduced the plasma 12-HETE level, promoted calcium deposition in the aortic arch and increased the calcium level in blood vessels.¹⁴⁶

Blockade of 20-HETE by the nonspecific and specific inhibitors 1-ABT and HET-0016 could lead to reduced mean arterial pressure by 20–30 mmHg in old female rats with spontaneous hypertension.^{147, 148} Probable G-protein coupled receptor 75 (GPR75), a receptor of 20-HETE, is a novel target for hypertension treatment.¹⁴⁹

2.4.2 AA metabolism in heart failure

Ischemic myocardial injury is the most common cause of heart failure. In recent years, many studies have suggested that EETs and 20-HETE play an important role in preventing ischemic myocardial injury.^150-152 In rat and mouse models of ischemia‒reperfusion injury, low concentrations of 11,12-EET perfusion treatment before myocardial ischemia reduced the reperfusion-induced MI area and increased cardiac function.^153-155 In canine and rabbit models of myocardial ischemia/reperfusion, the level of 20-HETE in the coronary artery was significantly increased. When the generation of 20-HETE was inhibited, cardiac function after myocardial ischemia/reperfusion injury was improved.^{156, 157} These above results show that 20-HETE aggravates cardiac function inhibition after myocardial ischemia/reperfusion injury, while EETs alleviate cardiac dysfunction caused by ischemia/reperfusion injury.

Heart failure involves a persistent systemic inflammatory reaction. The cascade of inflammatory cytokines reactions plays an important role in the occurrence and development of heart failure.¹⁵⁸ 20-HETE gradually activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to produce more ROS through the MAPK/ERK pathway, promote inflammatory reactions and aggravate myocardial cell damage.¹⁵⁹ EETs and their metabolites can activate PPARγ and endogenous growth factor receptor (EGFR), inhibit inflammation, reduce myocardial cell damage, and prevent the occurrence and development of heart failure.^160-162

Heart failure can also be caused by cardiomyocyte apoptosis. Myocardial cell apoptosis leads to weakened cardiac contractile function, which causes a decline in cardiac function. EETs promote the expression of antiapoptotic proteins and reduce the activity of apoptotic effector proteins.^{163, 164} 20-HETE causes the release of a large amount of ROS from vascular endothelial cells and directly acts on DNA to induce cell apoptosis.¹⁶⁵ Moreover, it enhances cell membrane permeability, promotes Ca²⁺ influx, and causes intracellular calcium overload to induce cell apoptosis. In addition, the accumulation of ROS exerts a regulatory on the expression of apoptosis-related genes and changes the levels of the proteins they encode, inducing apoptosis. Bao et al.¹⁶⁶ found that after 20-HETE administration, the survival rate of neonatal rat cardiomyocytes decreased, and the number of cells in the early and late stages of apoptosis increased markedly.

Another risk factor for heart failure is cardiac hypertrophy. 20-HETE activates the calcineurin/nuclear factor of activated T-cell signaling pathway, a key pathway in the cardiac hypertrophy signaling pathway, thereby promoting cardiac hypertrophy.¹⁶⁷ In contrast, EETs effectively inhibit cardiac hypertrophy, promote ventricular remodeling, and delay heart failure by regulating the expression of MMPs or activating the EGFR/PI3K/Akt/CREB signaling pathway to promote atrial natriuretic peptide secretion.^{168, 169}

In general, AA metabolites show high biological activity, and balance in the effects of AA metabolites is very important. These metabolites are especially important to research on vascular movement and blood coagulation, atherosclerosis, hypertension, and antithrombotic drugs and the treatment of heart failure.

2.5.1 AA metabolism in obesity

Obesity is an independent risk factor for systemic diseases. A study assessing the fatty acids in adipose tissues of children in Crete and Cyprus demonstrated that the subcutaneous AA levels in all of the obese children were in the highest quartile.¹⁷⁰ A clear correlation between body weight and the regulation of anandamide level was found. The level of circulating 2-AG in obese patients was significantly increased and positively correlated with body mass index and abdominal adiposity.¹⁷¹ Ailhaud et al.²⁷ found that AA stimulated adipogenesis and weight gain through its effect on PGI-2 pathway by upregulating CCAAT/enhancer-binding protein B (C/EBPB) or activating PPARγ and PPARβ/PPARδ.

Lipoxin is a biologically active substance synthesized from AA in a reaction catalyzed by LOXs, mainly LXA-4 and LXB-4. LXA-4 inhibits IκBα degradation and NF-κB translocation to attenuate the inflammatory the M1 macrophage phenotype acquired in the context of obesity.¹⁷² Börgeson et al.¹⁷³ found that LXA-4 and LX analogs reduced obesity-induced inflammatory reaction in adipose tissue, changed the ratio of M1/M2 macrophages in adipose tissues, and decreased obesity-induced autophagic flux. In addition, lipoxins also reduce obesity-induced liver disease and chronic kidney disease. Börgeson and Sharma¹⁷⁴ reported that lipoxins restored insulin sensitivity and inhibited renal fibrosis by regulating leukocyte infiltration and promoting the elimination of inflammation in visceral adipose tissue. These results indicate that lipoxins exhibit therapeutic potential in obesity-induced pathological damage.

Obesity is one of the causes of diabetes, especially type 2 diabetes. Obese patients usually have more visceral fat. This accumulation of visceral fat can lead to insulin resistance and reduce the action of insulin effect, leading to failed glucose utilization in the body and a gradual increase the plasma glucose level, which leads to diabetes. The specific role of AA metabolism in diabetes mellitus (DM) is described in the following subsection.

2.5.2 AA metabolism in DM

DM is a metabolic disease characterized by hyperglycemia caused by defective insulin secretion or insulin dysfunction. As a PUFA, AA in diabetes patients plays a contradictory role. On the one hand, the AA level in the circulatory system of diabetes patients is low, and exogenous intake of AA effectively attenuates the abnormal lipid metabolism and insulin resistance characteristic of DM.¹⁷⁵ On the other hand, a significant increase in the AA level in the circulatory system of diabetes patients inhibits insulin secretion, promoting the occurrence and development of DM.¹⁷⁶

Reportedly, the AA metabolic pathway in type 2 diabetes model mice is abnormal. The expression levels of fatty acid synthase, phospholipase A2, COX-2, and LOX-5 and the levels of fasting blood glucose, insulin, AA, and related metabolites in the liver of diabetic mice are significantly increased, while the expression of carnitine palmitoyltransferase 1A and CYP-450 family 4A are significantly reduced.

Studies on AA metabolites showed that different metabolites contribute differentially to insulin resistance, depending on the cell and tissue type. In white adipose tissues, PGE-2 promotes adipogenesis and induces glycogen decomposition and gluconeogenesis, thereby reducing the insulin resistance of adipocytes.^{177, 178} PGE-2 also enhances the dysfunction and destruction of pancreatic islet β-cells and hinder insulin secretion. In contrast, PGI-2 increases the insulin sensitivity of pancreatic cells. Recently, scholars found that a higher level of PGF-2α in diabetic mice was related to gluconeogenesis in the liver, and PGF-2α was also the main factor associated with fasting hyperglycemia in type 2 diabetes patients.¹⁷⁹ In addition, the 12-/15-LOX enzyme induces the production of various HPETEs. These HPETEs interact with PPARα and PPARβ and are involved in cytokine-mediated damage to β-cells in pancreatic islets. 12-/15-LOX-knockout mice showed resistance to the development of diabetes to some extent.¹⁸⁰ Similarly, LTs produced by HETEs also showed an inhibitory effect on insulin secretion.^{181, 182} LTB-4 is vitally important for the recruitment and activation of B2 lymphocytes in adipose tissue, which may contribute to insulin resistance after a high-fat diet.¹⁸³ In contrast, 20-HETE and EET could protect pancreatic β-cells from apoptosis and promote the secretion of insulin.¹⁸⁴

Reportedly, human and rat pancreatic islet cells can produce EETs and stimulate the release of insulin and glucagon. 5,6-EETs play important roles in the excitation–coupling process of pancreatic islet β-cells, which increase insulin secretion by activating the concentration of Ca²⁺.¹⁸⁵ 8,9-EETs, 11,12-EETs, and 14,15-EETs stimulate the secretion of glucagon.¹⁸⁶ Increasing the level of EETs tighten the control of blood glucose levels by increasing insulin sensitivity. In addition, Xu et al.¹⁸⁷ found that CYP2J3/EETs may reduce insulin resistance via the PI3K/AKT and MAPK pathways or membrane translocation of GLUT-4 through the HO-1/adiponectin pathway. Diabetes has also been associated with the increased expression of sEH, which is the enzyme critical for the degradation of EETs. In a diabetic mouse model, the deletion of the sEH gene led to an increase in insulin sensitivity and antiapoptotic effects on pancreatic islet cells.¹⁸⁸ The therapeutic potential of sEH inhibitors is currently being evaluated in clinical trials.

In conclusion, AA metabolites play different roles in the pathogenesis of both obesity and diabetes. Studies into AA metabolism and related enzyme pathways may lead to the identification of new targets for clinical treatment.

2.7.1 AA metabolism regulates signaling pathways involved in inflammation

AA and its metabolites have strong biological activity and can participate in the inflammatory response by activating various signaling pathways. Abnormal fatty acid metabolism can amplify the inflammatory response of infection by regulating the p38 MAPK signaling pathway.^{220, 221} Calcium signaling pathways in T cells promote synovial inflammation in patients with rheumatoid arthritis.²²² The phenotype of alternatively activated macrophages (M2-type macrophages) differs from that of classically activated macrophages (M1-type macrophages). Both phenotypes are important for the innate and adaptive immune systems. AA and its metabolites were also involved in the macrophage phenotype switching. This effect is mainly dependent on the regulation of PPARγ mediated oxidative phosphorylation.²²³

2.7.2 AA metabolism regulates inflammation at different levels

AA can regulate inflammatory responses independently of its metabolites. In cultured cardiomyocytes and macrophages, AA directly regulates inflammatory responses by regulating toll-like receptor 4 (TLR4) activity. AA inhibits the formation of the TLR4 complex and accessory protein induced by saturated fatty acids. This effect is mainly achieved through direct binding of AA to the TLR4 coreceptor myeloid differentiation factor 2 and the blocking of TLR4 proinflammatory signaling pathway activation by saturated fatty acids.²²⁴

Disorders of the intestinal flora can cause disorders of AA metabolism.²²⁵ This disorder further exacerbates the inflammation associated with atherosclerosis.²²⁶ Peritoneal macrophages (rpMACs) produce inflammatory lipid mediators by secreting PUFAs and AA in response to infection or tissue damage. Chronic Acsl4 deficiency in rpMACs reduces the incorporation of AA into phospholipids, thereby reducing lipid mediator synthesis and inflammation.^{227, 228} Intervention of the AA pathway in mouse macrophages prevents endotoxin-induced inflammation, which is mediated by aldose reductase.^{229, 230} Obesity is associated with low levels of chronic inflammation, in which AA cascades play a key role. AA treatment resulted in significant downregulation of proinflammatory markers and COX pathways. AA treatment can effectively reduce adipocyte inflammation induced by a high-fat diet in obese mice.²³¹ AA may influence obesity through the enteric–hypothalamic–adipose–liver axis.²³² The development of nonalcoholic fatty liver disease is accompanied by inflammation. Early screening indicators are of great significance for the prevention of the disease. Studies have shown that AA can be used as an early detection indicator.²³³ In chronic obstructive pulmonary disease, AA increases inflammation but inhibits extracellular matrix protein expression.²³⁴ Studies on mouse-derived pulp tissue during acute inflammation have shown that AA and LA metabolites regulate the expression of catalase.²³⁵ In addition, air pollution also exacerbates inflammatory progression. Studies have shown that AA- and LA-derived hydroxyl metabolites are associated with air pollution and interact with systemic inflammation in the process of body response to air pollution.²³⁶

2.7.3 Chinese herbal medicine regulates inflammation through AA metabolism

Chinese herbal medicine can also regulate AA metabolism to play an anti-inflammatory role. Magnolol is the main active ingredient in Magnolia officinae. Studies have found that the active ingredient in Magnolia officinae interferes with its function mainly by directly inhibiting the activity of enzymes related to AA metabolism, such as COX-2, or by inhibiting the mRNA and protein expression of COX-2 to affect the AA metabolic pathway and exert a good anti-inflammatory effect.²³⁷ Saikosaponin can improve chronic pelvic inflammatory disease by regulating niacin and niacinamide metabolism and AA metabolism,²³⁸ and Paeoniflorin can improve COX-2 expression in the AA pathway.²³⁹ Inflammation can be controlled by disrupting the body's AA metabolic network in a number of ways.

2.8.1 AA metabolism activates tumor-related cell signaling pathways

AA can regulate tumor proliferation through a variety of signaling pathways. In MDA-MB-231 breast cancer cells, AA and its metabolites are involved in cell migration by activating FAK. In the process of malignant transformation, AA passes through PLA2α. Src, ERK1/2, and LOX activity-dependent pathways promoted GalT I expression in MDA-MB-231 breast cancer cells.²⁴⁹ AA also activated Akt2 through Src, EGFR, and PI3K and promoted the migration and invasion of MDA-MB-231 cells. In addition, AA promoted NF-κB-DNA binding activity in an Akt-dependent manner.²⁵⁰ Triple-negative breast cancer is an aggressive subtype of breast cancer that poses a challenge to treatment because it does not respond to estrogen and progesterone receptor inhibitors. The endogenous AA synthetic pathway, delta 6 desaturase (D6D) activity, and PGE-2 levels are increased in breast tumors, particularly those of the ER- genotype.²⁵¹ Store-operated Ca²⁺ entry has been implicated in the migration of some cancers. Studies have found that AA regulates Ca²⁺-selective channels (ARC channels), activates Ca²⁺ entry, and then inhibits tumor invasion. However, in MDA-MB-231 cells, AA was found to impair the proliferation and migration capacity of MDA-MB-231 cells by activating apoptosis and reducing cell viability. However, these effects are not related to changes in Ca²⁺.²⁵² In neuroendocrine cancer cell lines, AA treatment could promote cell migration, and this migration could be inhibited by selective inhibition of AA-induced Ca²⁺ influx.²⁵³

2.8.2 AA metabolism regulates tumor-related gene expression

The expression of AA and its downstream metabolites is regulated by other abnormally activated genes in tumor cells. In pancreatic cancer, untargeted metabolomics analysis revealed that AA and its downstream metabolites are regulated by HDAC5, which inhibits cPLA2 expression by deacetylating GATA1. Knockdown of HDAC5 results in a significant increase in AA and its downstream metabolites. HDAC5 negatively regulates the expression of the gene encoding calcium-dependent phospholipase A2 (cPLA2), a key enzyme in the formation of AA from phospholipids.²⁵⁴ Increased ATF6α expression in prostate cancer (PCa) leads to a CrPC-like phenotype in PCa cells.²⁵⁵ High ATF6α expression enhances Pla2G4A-mediated AA metabolism and protects tumor cells against iron downregulation to promote the progression of PCa. Therefore, genetic inhibitors targeting ATF6α can promote the apoptosis of iron cells and delay the progression of PCa in tumor cells.²⁵⁶

Bone metastasis of PCa is a major challenge in clinical treatment. Studies have shown that some SNPs in AA metabolism genes may influence PCa susceptibility. A case‒control study found that SNPs in the COX-2, PTGES2, ALOX5, ALOX5AP, and LTA4H genes were associated with PCa susceptibility.^{257, 258} Noel Clarke found that AA induces PCa cells to enter the bone marrow, a new finding that may help explain why taking statins, commonly used cholesterol-lowering drugs, can slow the progression of the disease in some cases. Using PCa cells, AA has been shown to attract PCa cells to bone marrow. When PCa cells were exposed to AA, the researchers found that the tumor cells changed shape, which helped the cells pass through gaps in the surrounding tissue and establish metastases in the bone marrow, but statin administration disrupted the tumor cells’ ability to make cholesterol, stopping the cells from acquiring these properties. These studies show how fatty acids naturally produced by the bone marrow directly interact with the body's cholesterol-producing system to improve the ability of PCa cells to spread around the body. This information provides important clues as to how PCa patients might benefit from drugs such as statins.²⁵⁹

In colon cancer HT-29 cells, AA inhibits lipogenesis, leading to a lack of sufficient fatty acids to support cell division and subsequently inducing endoplasmic reticulum stress and apoptosis.²⁶⁰ Other studies have shown that DHA and AA exert different anticancer activities in colorectal cancer cells in vitro. DHA inhibits the proliferation of HT-29 cells more than AA, and its effect is mainly through decreasing proteasome granules, while ARA has a significant effect on all six DNA replication helicase granules.^{261, 262} Methyl donor restriction in colon cancer has been shown to cause significant changes in fatty acid metabolism. Regulation of fatty acid metabolism by restricting methyl donors can achieve certain therapeutic effects.²⁶³ In addition to regulating tumor cell survival, AA can attenuate tumor progression by remodeling the tumor microenvironment.

2.8.3 AA metabolism in the tumor microenvironment

The tumor microenvironment is characterized by inflammation and immunosuppression and includes high levels of unsaturated fatty acids. A large number of studies have confirmed that the presence of AA and its metabolites is an important factor promoting tumor metastasis and invasion.²⁶⁴ Researchers have identified phospholipase PLA2G2A as the most clinically relevant extracellular AA-producing enzyme. This finding offers potential treatment options for further diagnosis. AA in the ovarian cancer microenvironment promotes the survival of ovarian cancer cells by disrupting the structure of lipid rafts, destroying janus tyrosine kinase-signal transducer and activator of transcription (JAK–STAT signaling in macrophages and inhibiting the recognition of immune cells by macrophages.²⁶⁵ In addition to the direct effects of AA, its metabolites are also involved in the immunosuppressive effects of the tumor microenvironment.²⁶⁶ PGI-2 released by tumor-associated fibroblasts was found to promote immunosuppression and metastatic macrophage polarization in the ovarian cancer microenvironment.²⁶⁷ AA contributes to an unfavorable clinical outcome of OC by impacting the phenotype of tumor-associated macrophages via the ASK1–p38δ/α (MAPK13/14) regulatory axis.²⁶⁸ Through comprehensive analysis of three databases, the researchers found that the AA metabolism level was positively correlated with the prognosis of breast cancer patients, indicating that AA might promote the immune killing function of the body toward tumor cells, that is, AA metabolism is a cancer suppressor factor for breast cancer.²⁶⁹ The team also found a positive correlation between AA metabolism levels in breast cancer and the levels of CD8+ T cells and activated NK cells.

2.8.4 AA metabolism regulates ferroptosis

Iron is an indispensable trace element that can easily become deficient in the human body and is also essential for the proliferation and development of tumor cells. Ferroptosis is an iron-dependent nonapoptotic cell death mode.²⁷⁰ There is growing evidence that ferroptosis may be associated with a variety of pathological conditions, including acute kidney injury, tissue ischemia‒reperfusion injury, neurodegeneration, and cancer.²⁷¹ Recent studies have shown that AA is also related to the mechanism of ferroptosis induction.²⁷² The polyunsaturated fatty acid biosynthesis pathway determines susceptibility to ferroptosis in gastric cancer.²⁷³ Liao et al.²⁷⁴ found that T-cell-derived interferon (IFN) γ binds to AA to induce ferroptosis in immunogenic tumors. IFNγ alone or single fatty acids failed to induce cell death in the two mouse melanoma lines Yumm5.2 and B16F10 and the human melanoma line A375. However, AA induces effective cell death in all three tumor cell lines by synergistic action with IFN-γ.²⁷⁴

2.8.5 AA metabolism modulates chemotherapy sensitivity

Chemotherapy sensitivity is an important factor that affects the efficacy of tumor treatment. AA can modulate the chemotherapy sensitivity of tumor cells. The protective effect of bleomycin on IMR-32 cells was further enhanced by exogenous apoptotic pathway activation by AA, and reprogramming of arachidonate metabolism conferred temozolomide resistance to glioblastoma by enhancing mitochondrial fatty acid oxidation activity.^{275, 276} Sp1-regulated PGE2 production activates fatty acid oxidation (FAO) and the tricarboxylic acid cycle (TCA cycle) in mitochondria through EP1 and EP3 receptors, resulting in temozolomide (TMZ) resistance in glioblastoma multiforme (GBM). These results will provide us with a new strategy to attenuate drug resistance or to resensitize recurrent GBM. In malignant mesothelioma, AA drives adaptive responses to chemotherapy-induced stress. Pemetrexed promoted the release of excess AA from the malignant mesothelioma cell line cPLA2 and activated the NF-kB signaling pathway. Increased AA mediated the expression regulation of drug resistance-related genes.²⁷⁷