Exploiting conjugated linoleic acid for health: a recent update

Abstract

Conjugated linoleic acid (CLA) is widely used as a dietary supplement due to its reported benefits in enhancing immunity, regulating inflammation, treating obesity, and preventing cancer. However, there is a lack of comprehensive studies on its mechanisms and dose-effect relationships. Moreover, there are insufficient in-depth studies on CLA's new functions, safety, side effects, and clinical utility. This review systematically examines the structure and sources of CLA, summarizes its role in improving human health, and critically reviews the potential mechanisms behind these benefits. It also analyzes the side effects of CLA and addresses issues related to dosing and oxidative decomposition in CLA research. Additionally, the potential of using CLA-producing probiotics to manage diseases is explored. This review can guide and promote further research on CLA's functions and support the development of CLA dietary supplements. It will accelerate the development of CLA nutritional and medical foods, contribute to the improvement of human health, and have important social meaning and economic value.

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1. Introduction

The composition of fatty acids has attracted considerable attention from researchers due to its effect on human health.¹ Conjugated linoleic acid (CLA) is a type of polyunsaturated fatty acid found in ruminant animal foods (dairy products, milk, and meat). It is a positional and geometric isomer of linoleic acid and contains conjugated double bond systems, in which 28 CLA isomers have been identified.² The main isomers of CLA in dietary sources are trans10, cis12-CLA (t10, c12-CLA) and cis9, trans11-CLA (c9, t11-CLA). t10, c12-CLA accounts for 1–10% of the total CLA, while c9, t11-CLA accounts for 90% of the total CLA in dietary sources (Fig. 1).³ Furthermore, CLA's status as a class of naturally occurring essential fatty acids indicates that it is involved in a variety of biological regulatory functions in vivo. CLA can inhibit cancer, reduce blood fat, resist atherosclerosis, and improve immunity. Additionally, it can regulate blood sugar, blood pressure, and other important physiological functions.⁴ Thus, CLA has health benefits that merit further examination.

Fig. 1 Source and structure of CLA. CLA is a ruminant animal foods (dairy products, milk, and meat) found in a kind of polyunsaturated fatty acids. The main isomer of CLA in dietary sources is t10, c12-CLA and c9, t11-CLA, which have proven to have health benefits.

The US Food and Drug Administration (FDA) defines trans fats as “unsaturated fatty acids (non-conjugated) containing one or more independent double bonds in trans configuration”.⁵ According to the above definition, CLA with conjugated bonds is not a trans fatty acid. Moreover, t10, c12-CLA, c9, t11-CLA, and a mixture of the two (50 : 50) have been classified as “generally recognized as safe, GRAS” by the FDA.⁶ According to clinical studies, intakes of up to 3.4 g CLA per day (2 years) or 6 g CLA per day (1 year) are currently considered safe.^7,8 The FDA currently states that “the use of CLA-rich oils in certain specific foods in general categories, such as fruit juices, dairy products, beverages, meal replacement drinks, and soy milk, is considered safe at levels of approximately 1.5 g of CLA”.⁶

The limited availability of CLA-fortified foods currently reflects the challenges of using CLA as a food ingredient. Although CLA has been widely studied for regulating health, the analysis of its mechanism is insufficient. In addition, the side effects of long-term use of CLA have not received enough attention. Some of the new features such as prevent alzheimer's disease and depression in CLA studies lack in-depth clinical and safety studies. As a result, this review summarizes the potential health benefits and mechanisms of CLA, lists the potential side effects of CLA, proposes a dose-effect relationship study of CLA, discusses the efficient delivery of CLA, and recommends the use of CLA-producing microorganisms in future CLA research to regulate human health. This review will guide and promote research on CLA function and the development of dietary products.

2. Regulation of CLA on inflammation

2.1. Regulation of inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic recurrent disease, mainly including Crohn's disease and ulcerative colitis (UC).³ CLA can alleviate IBD in animal models. The mucus layer can against harmful substances and microorganisms: it comprises a firm inner layer (composed of polymerized MUC2) and a loose outer layer (formed by hydrolysis of MUC2).⁹ Dietary supplementation of 20 mg per day of CLA significantly increased the number of goblet cells, upregulated MUC2, and repaired the intestinal mucus layer of mice with UC.³ The tight junctions (TJs) and adhesion junctions between epithelial cells can join epithelial cells and prevent microbial invasion through paracellular pathways.¹⁰ CLA can regulate TJ proteins, thereby affecting the intestinal mechanical barrier. For example, when 1% of CLA was added to the diet of mice, it upregulated mRNA levels of Occludin, ZO-1, MUC2, and Claudin-3 in colitis mice.¹¹ However, oral administration of CLA at 20 mg per day upregulated TJ and AJ proteins in colitis mice.³ In addition, dextran sulphate sodium (DSS) can induce apoptosis of epithelial cells, thereby disrupting the intestinal mechanical barrier. 20 mg per day of CLA can significantly downregulate the apoptosis of epithelial cells in mice with colitis,³ which plays an important role in maintaining the intestinal epithelial cell layer. Therefore, CLA can upregulate MUC2 to improve the mucus layer, regulate TJ proteins, inhibit apoptosis, and improve colitis (Fig. 2 and 3).

Fig. 2 Schematic of CLA in improving health. CLA can regulate IBD, colorectal cancer, atopic dermatitis, arthritis, II diabetes mellitus, obesity, atherosclerosis, breast cancer, and psychiatric disease by regulating gut microbiota, intestinal metabolites, cytokines associated with inflammation, oxidative stress, and intestinal mechanical barrier. Docosahexaenoic acid (DHA); brain derived neurotrophic factor (BDNF); synaptic functional proteins (SFP); vascular cell adhesion molecule 1 (VCAM-1); monocyte chemotactic protein 1 (MCP-1); macrophage-1 antigen (Mac-1); very late appearing antigen 4 (VLA-4); total cholesterol (TC); fasting blood glucose (FBG); low density lipoprotein cholesterol (LDL-C); fatty acid (FA); prostaglandin E2 (PGE2); matrix metalloproteinases (MMP); leukotriene B4 (LTB4).

Fig. 3 The potential mechanisms of CLA in relieving colitis. CLA can upregulate MUC2 to improve mucus layer, regulate TJ proteins, and inhibit apoptosis. CLA can also inhibit oxidative stress. CLA regulates NF-κB signaling pathway and intestinal inflammatory cytokines through macrophage-mediated PPAR-γ. CLA inhibits harmful bacteria, promotes the growth of beneficial bacteria, and improves the imbalance of gut microbiota.

Oxidative stress can facilitate the release of inflammatory mediators, which lead to the destruction of the intestinal mechanical barrier.¹² The antioxidant effects of CLA have been reported. For example, feeding rats with CLA (19.54 mg g⁻¹ fat) during adolescence increased the activity of superoxide dismutase (SOD) and catalase (CAT) in the blood and liver.¹³ Dietary supplementation with 0.83% CLA significantly increased liver total antioxidant capacity (T-AOC) and CAT activity of large yellow croaker.¹⁴ Moreover, CLA (20 or 40 mg per day) significantly increased the activities of CAT, glutathione peroxidase (GSH-Px), and SOD in colon tissue.³ Thus, inhibition of oxidative stress is an important way for CLA to relieve colitis (Fig. 2 and 3).

IBD may be associated with inflammation of gut mucosa. CLA (100 mg per kg per d) prevented colon shortening, significantly reduced NF-κB expression, and increased the expression of PPAR-γ and treleaf factor family 3 (TFF3).¹⁵ In addition, supplementation of 1% CLA in the diet of colitis mice reduced inflammatory mediator infiltration and mucosal damage, indicating a macrophage-mediated PPAR-γ dependent mechanism.¹⁶ Moreover, CLA intervention significantly downregulated IL-6, IL-1β, and TNF-α expression, while it upregulated IL-10 expression.¹¹ CLA (40 or 20 mg per day) decreased IL-6 and TNF-α and increased IL-10 in the colon. This phenomenon may be associated with activation of PPAR-γ and inhibition of NF-κB, decreasing the secretion of proinflammatory factors.³ Therefore, CLA can improve colitis by activating PPAR-γ to inhibit the NF-κB signaling pathway and intestinal inflammatory cytokines (Fig. 2 and 3).

Gut microbiota are closely related to the pathogenesis of IBD.¹⁷ DSS treatment can affect the gut microbiota, while CLA has a regulatory effect on the gut microbiota. For example, CLA treatment increased Verrucomicrobia, which might be attributed to the high expression of MUC2.¹⁷ At genus level, CLA treatment increased the abundance of Odoribacter and Bifidobacterium but significantly decreased Bacteroides. Bifidobacterium, as a probiotic, has a certain relieving effect on colitis.³ In addition, studies have shown that DSS treatment reduced Odoribacter and increased host inflammation by reducing short-chain fatty acid production.¹⁸Odoribacter can produce butyric acid, which improves the intestinal barrier and relieves colitis.¹⁹ Research has shown that Bacteroides has a proinflammatory effect and can degrade the mucosal layer and destroy the intestinal barrier.²⁰ Therefore, the regulatory effect of CLA on intestinal inflammation is partly attributable to the regulation of gut microbiota (Fig. 2 and 3).

2.2. Regulation of arthritis

Arthritis is an inflammatory disease that occurs in the joints. As a dietary supplement, CLA has been reported to have some regulatory effects on arthritis.²¹ The supplementation of 0.5% c9, t11-CLA and t10, c12-CLA can reduce arachidonic acid (ARA) and the incidence of arthritis in mice.²² In collagen-induced arthritis (CA) and rheumatoid arthritis (RA), prostaglandin E2 (PGE2) produced by ARA cascades promotes Th17 cell differentiation, expansion, and disease. In contrast, inhibition of ARA cascades and certain Th cytokines may inhibit the development of arthritis.²³ Moreover, 0.25% t10, c12-CLA increased IgG to aggravate the severity of CA, while 0.5% c9, t11-CLA increased IL-4 and IL-10 to reduce the incidence of CA.²⁴ Therefore, the effect of t10, c12-CLA on arthritis needs further study. Huebner et al. found that 0.5% c9, t11-CLA reduced plasma concentrations of IL-1β, IL-6, and TNF-α.²⁵ In addition, t10, c12-CLA reduced the inflammatory response in the mouse CA model in a dose-dependent manner.²⁶ In another study, 0.5% t10, c12-CLA or 0.5% c9, t11-CLA significantly reduced arthritis scores due to reduced IL-1β, IL-17, and IL-21.²⁷ Therefore, CLA can reduce arthritis by regulating Th1, Th2, and Th17 cells, inhibiting proinflammatory cytokines, and upregulating anti-inflammatory cytokines (Fig. 2 and 4).

Fig. 4 The potential mechanisms of CLA in relieving arthritis. CLA can regulate Th1, Th2 and Th17 cells, as well as inhibit pro-inflammatory cytokines and upregulate anti-inflammatory cytokines. CLA can also inhibit COX-2 and its derivatives, such as prostacyclin and PGE2. Prostaglandin E2 (PGE2).

In RA mouse models, 0.5% c9, t11-CLA reduced the incidence of arthritis. As a result, c9, t11-CLA may be an effective adjuvant of cyclooxygenase-2 (COX-2) inhibition in the treatment of chronic inflammation.²⁸ COX-2 inhibitors relieve pain and inflammation mainly by inhibiting the production of proinflammatory mediators. Additionally, inhibiting COX-2 also leads to the inhibition of PGE2 and prostacyclin, collectively promoting inflammatory diseases, such as RA.²⁸ Inhibition of COX-2 can promote IL-10 secretion and produce anti-inflammatory properties.²⁸ Therefore, inhibiting COX-2 and its derivatives, such as prostacyclin and PGE2, is an important strategy for using CLA to regulate arthritis (Fig. 2 and 4).

2.3. Regulation of atopic dermatitis

Atopic dermatitis is one of the most common recurrent inflammatory skin diseases in the world.²⁹ It is characterized by clinical signs typical of Th2/Th1 imbalance, IgE hypersensitivity, chronic pruritus, and eczema. Th2 cytokines, including IL-13 and IL-4, predominate in the acute phase of atopic dermatitis. IL-4 can stimulate the synthesis of IgE by B lymphocytes. Th1 cytokines (IL-12 and IFN-γ) progress to chronic atopic dermatitis through the proliferation and differentiation of infiltrating macrophages.²⁹ CLA (100 mg kg⁻¹) reduced the number of CD4+ T cells in the skin and inhibited the production of IFN-γ and IL-4 in serum and atopic dermatitis-like lesion skin.³⁰ In addition, elevated serum total IgE levels are one of the key features of atopic dermatitis.²⁹ Oral administration of 100 mg c9, t11-CLA significantly inhibited allergic dermatitis in mice mainly by reducing the infiltration of inflammatory cells (neutrophils, eosinophils, monocytes, mast cells, and leukocytes) and IgE levels.³¹ Oral CLA mixture (200 μL per day) reduced the clinical score of allergic dermatitis, and this effect was attributed to decreased ear IL-4 and plasma IgE levels.³² Antigen production and IgE conversion stimulate mast cell activation, prompting mast cell release of itch-related substances, such as histamine, leukotrienes, prostaglandins, inflammatory cytokines, and other inflammatory mediators.³³ PGE2 and leukotriene B4 (LTB4) can promote inflammation and pruritus. Therefore, reducing the number of mast cells and the production of pruritus mediators is beneficial to relieving pruritus.³⁴ In addition, supplementation with CLA can reduce levels of PGE2 and LTB4 in the skin, which is consistent with previous studies.³⁵ Thus, CLA can regulate atopic dermatitis by regulating IgE and inhibiting mast cell activity and mast cell release of itch-related substances, such as PGE2 and LTB4 (Fig. 2 and 5).

Fig. 5 The potential mechanisms of CLA in relieving atopic dermatitis. CLA can regulate IgE and inhibit mast cell activity and mast cell release of itch related substances. CLA can also downregulate COX-2 and 5-LOX and inhibit the production of ARA derivatives PGE2 and LTB4. Oral CLA supplementation significantly inhibit atopic dermatitis-like skin lesions and inflammation by attenuating the NF-κB inflammatory signaling pathway and reducing the inflammatory factor. Arachidonic acid (ARA); prostaglandin E2 (PGE2); leukotriene B4 (LTB4).

In addition, CLA can downregulate the expression of NF-κB, myeloid differentiation factor88 (MyD88), 5-lipoxygenase (5-LOX), COX-2, toll-like receptor 4 (TLR4), and TNF-α.³⁰ For example, CLA dose-dependently inhibited proinflammatory cytokines, including inducible nitric oxide synthase (iNOS), COX-2, IL-1β, IL-6, and TNF-α, in the skin of inflamed mice.³⁶ Increased expression of 5-LOX and COX-2 has been proposed as a mechanism leading to increased production of LTB4 and PGE2. 5-LOX and COX-2 are two important enzymes that catalyze the conversion of ARA to bioactive lipid derivatives LTB4 and PGE2.³⁷ COX-2 and 5-LOX can also induce atopic dermatitis, implying that the downregulation of COX-2 or 5-LOX reduces the development of inflammatory processes. CLA can significantly inhibit the expression of 5-LOX/LTB4 or COX-2/PGE2 in mouse skin tissues,³⁰ which is an important mechanism for CLA to regulate atopic dermatitis. Therefore, CLA can improve atopic dermatitis by downregulating COX-2 and 5-LOX, inhibiting the production of ARA derivatives PGE2 and LTB4 (Fig. 2 and 5).

The production of a large number of proinflammatory cytokines (IL-6, TNF-α, and IL-1β), leads to severe and persistent skin inflammation and itching sensation.³⁰ TNF-α produced by macrophages plays a crucial role in the acute phase of atopic dermatitis, mediating its onset.³⁸ In addition, the NF-κB pathway regulates the production of proinflammatory cytokines in skin allergic reactions. Consistent with these findings, the DNFB-induced atopic dermatitis model increased NF-κB production in mouse skin, whereas CLA inhibited NF-κB production.³⁰ Thus, oral CLA supplementation significantly inhibited atopic dermatitis-like skin lesions and inflammation by attenuating the NF-κB inflammatory signaling pathway and reducing the inflammatory factor (Fig. 2 and 5).

3. Regulation of metabolic syndrome

3.1. Regulation of obesity

Metabolic syndrome refers to a pathological state in which carbohydrates, fats, proteins, and other substances in the human body undergo metabolic disorders, including dyslipidemia, hypertension, type 2 diabetes, and obesity, which impose a significant economic burden on patients. Browning of white adipose tissue is a mechanism of fat mobilization that promotes weight and fat loss. For example, CLA (1.5% mixed) promoted uncoupling protein 1 (UCP1) expression in white adipose tissue and browning of epididymal (visceral) white fat in OB/OB spontaneously obese mice.³⁹ Moreover, low doses of t10, c12-CLA (0.03, 0.1, and 0.3%) upregulated the expression of UCP1 in epididymal (visceral) white adipose tissue and inguinal (subcutaneous),⁴⁰ thus promoting browning of white adipose tissue. Supplementation of compound CLA can reduce adipocyte size, increase lipolysis, and reduce body weight and mesenteric fat.⁴¹ In addition, the weight loss achieved by gavage of t10, c12-CLA (0.4%) mainly involved the inhibition of adipogenesis and the prevention of adipocyte differentiation.⁴² Dietary supplementation of t10, c12-CLA (0.1%) decreased the mass of brown adipose tissue and white adipose tissue and increased thermogenesis and adipose oxidative decomposition.⁴³ Thus, the main modes of action of CLA against obesity include (1) suppressing appetite and reducing energy intake, (2) inhibiting fatty acid metabolism and adipogenesis, (3) increasing lipolysis or defatting, (4) decreasing adipocyte size, and (5) increasing fat oxidation and energy expenditure in white liver, muscle, and adipose tissue (Fig. 2).^44,45

Table 1 presents human trials of CLA using obesity as an endpoint. A significant reduction was observed in body fat, while no significant difference was observed in body mass index in the oral CLA group.⁴⁶ When CLA supplementation was extended to a year, subjects lost both fat mass and body weight.^47,48 Many studies have reported beneficial effects of CLA on body weight and obesity-related parameters, including reduced body mass index, fat mass, subcutaneous fat, and appetite and increased fat oxidation rate, satiety, and serum leptin. However, no significant side effects were observed (Table 1).^49–57 Some studies have shown that mixed CLA supplements did not show any affect physical metrics.^58–64 In addition to causing weight and fat loss, CLA may also cause harmful side effects. A daily intake of 4.2 g of CLA was effective in reducing fat mass in obese men,⁶⁵ but promoted insulin resistance. In addition, a daily intake of 3.4 g of t10, c12-CLA significantly reduced body fat and body weight but increased glucose levels and plasma insulin and decreased insulin sensitivity.^66–68 These studies showed that t10, c12-CLA-mediated weight loss may occur at the expense of metabolic health.

Table 1 CLA studies in humans with the endpoint of body weight/composition

Study participants	Patient group	CLA dose	Treatment period	Effects due to CLA	Year	Ref.
Body mass index (BMI); type 2 diabetes mellitus (T2DM); conjugated linoleic acid triglyceride (CLA-TG); conjugated linoleic acid free fatty acid (CLA-FFA); high-density lipoproteins (HDL); very low-density lipoproteins (VLDL); C-reactive protein (CRP); interleukin (IL); low-density lipoproteins (LDL).
Healthy overweight men and women	Placebo (26) vs. mixed CLA (26)	Mixed CLA (1.7, 3.4, 5.1, or 6.8 g per day) or placebo	3 months	↑ Lean mass	2000	46
				↓ Fat mass
Healthy overweight men and women	Placebo (59) vs. mixed CLA (61)	Mixed CLA (3.4 g per day) or placebo	12 months	↓ Fat, BMI and body weight	2004	47
Healthy, overweight men and women	Placebo (20) vs. mixed CLA (20)	Mixed CLA (4 g per day) or placebo (safflower oil).	12 months	↓ Fat mass, BMI, body weight	2007	48
Overweight but weight stable men and women	Placebo (9) vs. mixed CLA (10)	Mixed CLA (4 g per day) or placebo (safflower oil)	6 months	↓ Body weight	2007	49
				↑ Fat oxidation
Overweight men and women	Placebo (18) vs. mixed CLA (18) vs. 9,11 CLA (18)	Mixed CLA (either 1.8 or 3.6 g per day) or placebo (oleic acid)	3.5 months	↓ Appetite	2003	50
				↑ Satiety
Postmenopausal women with T2DM	Placebo (17) vs. mixed CLA (18)	Mixed CLA (1.7 g per day) or placebo	4 months with a 1 month washout	↓ Trunk adiposity, BMI and body weight	2009	51
Women with metabolic syndrome	Placebo (7) vs. mixed CLA (7)	Mixed CLA (3 g per day) or placebo	3 months, in conjunction with a low-calorie diet.	↓ Body fat mass	2012	52
				↓ Plasma insulin levels
Healthy men and women of stable weight	Placebo (40) vs. mixed CLA (40)	Mixed CLA (1.7 g per day) or placebo	3 months	↓ Body fat, BMI, body weight and subcutaneous fat	2012	53
Healthy men and women	Placebo (17) vs. mixed CLA (20) vs. mixed CLA (18)	Mixed CLA (3.2 g per day or 6.4 g per day) or placebo	3 months	↑ Lean body mass	2007	54
				↑ serum IL-6
Healthy overweight men and women	Placebo (37) vs. mixed CLA (44)	Mixed CLA (3.4 g per day) or placebo	12–24 months	↓ Serum cholesterol	2005	55
				↑ Serum leptin
				↓ Fat, BMI and body weight
Healthy men and women	Placebo (38) vs. mixed CLA (38)	Mixed CLA (5 g per day) or placebo	3.5 months	↑ Muscle mass	2006	56
				↓ Fat mass
6–10 years-old children with a BMI >85th percentile	Placebo (38) vs. mixed CLA (38)	Mixed CLA (5 g per day) or placebo	6 months	↓ Body fat, BMI	2010	57
				↑ Lean body mass
				↓ HDL cholesterol
Healthy weight-stable men and women	Placebo (32) vs. mixed CLA (32)	Mixed CLA (3.9 g per day) or placebo	3 months	No effects on body weight	2007	58
Obese women	Placebo (12) vs. mixed CLA (12)	Mixed CLA (3.2 g per day) or placebo (olive oil).	2 months with concurrent exercise	None	2016	59
Moderately overweight men and women	Placebo (5) vs. mixed CLA (5) vs. 9,11 CLA (5)	Mixed CLA (1.3 g per day), 9,11 CLA (1.3 g per day) or placebo	2 months, treatment period with a 1 months washout.	No effects on body weight or plasma lipids	2010	60
Moderately obese weight-stable men and women	Placebo (61) vs. mixed CLA (61)	Mixed CLA (3.4 g per day) or placebo (olive oil)	13 months	No effects on body weight	2006	61
Healthy men and women	Placebo (20) vs. mixed CLA (20)	Mixed CLA (4.5 g per day), or placebo (olive oil).	3 months	No effect on body weight, BMI, or body fat	2006	62
Overweight and obese women	Placebo (37) vs. mixed CLA (37)	Mixed CLA (3 g per day) or placebo (sunflower oil).	3 months	No effect on body weight, BMI	2016	63
Sedentary males	Placebo (9) vs. CLA (9)	Mixed CLA (3 g per day) or placebo	1 month, concurrently with exercise	No difference in body weight, fat or BMI	2013	64
Abdominally obese older men	Placebo (12) vs. mixed CLA (13)	Mixed CLA (4.2 g per day) or placebo	1 month	↑ Insulin resistance	2001	65
				↓ Sagittal abdominal diameter
Men with signs of metabolic syndrome	Placebo (12) vs. CLA (13)	10,12 CLA (3.4 g per day) or placebo	3 months	↑ Insulin resistance and glycemia	2002	66
				↓ HDL cholesterol

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3.2. Regulation of type II diabetes mellitus

Adipocytes can produce a variety of proinflammatory molecules, or “adipocytokines”, including IL-6, adiponectin, resistin, leptin, monocyte chemotactic protein 1 (MCP-1), IL-1β, and TNF-α. Abnormal expression of these adipokines occurs during obesity and leads to insulin resistance.⁶⁹ Inflammatory mediators released by macrophages and T lymphocytes disrupt important insulin pathways in adipocytes and lead to subsequent accumulation of macrophages into the stromal vascular portion, further enhancing insulin resistance.⁷⁰ Diabetic mice fed with fermented milk containing 0.01% CLA had significantly lower low density lipoprotein cholesterol (LDL-C), triglyceride, body weight, serum total cholesterol (TC), leptin levels, serum insulin level, and fasting blood glucose (FBG).⁷¹ Dietary administration of c9, t11 CLA reduced triglyceride levels, insulin, and fasting glucose and upregulated the production of insulin signaling pathway molecules.⁷² In addition, dietary supplementation with CLA (0.5 or 1%) improved plasma creatinine levels and overall inflammatory response, iNOS, and C-reactive protein in diabetic patients (Fig. 2).⁷³ These results showed that regulation of fatty acid composition through administration of CLA may reduce obesity-induced insulin resistance. However, the effects of CLA observed in Type 2 diabetes mellitus animal models have not been comprehensively validated in human studies.

4. Regulation of atherosclerosis

Atherosclerosis is a progressive dyslipidemia and inflammatory disease in which monocyte-derived macrophages play a key role.⁷⁴ Atherosclerotic plaque is characterized by an accumulation of lipid and inflammatory cells in the artery wall that can gradually become unstable, leading to rupture and subsequent ischemic events.⁷⁵ Endothelial dysfunction and monocyte aggregation are the key markers of atherosclerosis.⁷⁴ The t10, c12-CLA and c9, t11-CLA isomers can reduce the expression of macrophage-1 antigen (Mac-1) and late-appearing antigen 4 (VLA-4) on monocytes,⁷⁶ indicating that CLA can regulate the adhesion of monocytes to endothelial cells. The aggregation and migration of monocytes and macrophages are caused by increased MCP-1 levels. MCP-1-deficient mice have been reported to significantly reduce the incidence of atherosclerosis.⁷⁷ CLA inhibited the migration of monocytes to MCP-1 by PPAR-γ-dependent mechanism in vitro.⁷⁸ This result shows that CLA is an effective inhibitor of monocyte migration in vitro and may be promising in combating migrating pathogenic monocytes in atherosclerosis. In addition to monocytes, CLA affects macrophages in atherosclerotic animals. Mixed CLA supplements can reduce the size of atherosclerotic lesions and macrophage accumulation.^75,79 Feeding the animals with 0.1% CLA also reduced plaque macrophage content.⁸⁰ In addition, CLA inhibited atherosclerosis by inhibiting foam cell formation and regulating the expression of the RCT gene, thereby facilitating the removal of cholesterol from the circulation.^81,82 For example, CLA inhibited foam cell formation in vitro through PPAR-γ and LXRα dependent mechanisms.⁸³ Thus, CLA can improve atherosclerosis by inhibiting monocyte migration, improving endothelial cell dysfunction, inhibiting macrophage secretion, and inhibiting the formation of foam cells (Fig. 2 and 6).

Fig. 6 The potential mechanisms of CLA in relieving atherosclerosis. CLA can improve endothelial cell dysfunction. CLA can also upregulate PPAR-γ to inhibit macrophage secretion and monocyte migration. Moreover, CLA may inhibit inflammatory factor and the formation of foam cells.

In Ldlr and ApoE mice, the improvement effect of t10, c12-CLA was significantly greater than that of c9, t11-CLA,⁸⁴ indicating that t10, c12-CLA were isomers responsible for the protection of atherosclerosis.⁸⁰ These studies collectively showed that CLA (particularly likely t10, c12-CLA) had an antiatherosclerotic effect.⁷⁵ However, other studies showed that CLA supplementation had no significant effect on atherosclerosis. In a study of apoE mice, feeding the mice with a diet mixed with CLA for 12 weeks did not improve atherosclerosis.⁸⁵ Similarly, administration of 0.5% t10, c12-CLA did not affect atherosclerosis in apoE/Ldlr/double knockout mice.⁸⁶ Therefore, future studies are needed to confirm the role of CLA in atherosclerosis.

5. Regulation of cancer

CLA is a good anticancer agent. Different animal models have been used to demonstrate the role of CLA in the development of colorectal and breast cancers (Table 2). A diet containing 1% CLA inhibited the incidence of colon tumors, decreased PGE2 and ARA, and increased the apoptosis-regulating protein Bcl-2/bax ratio and caspase-3 in colon cancer (CRC) rats.^87,88 In Apc^min/+ mice, c9, t11-CLA reduced the number of polyps, while supplementation of t10, c12-CLA increased the diameter of polyps in intestinal tumors through the PPAR-γ signaling pathway, indicating that different CLA isomers had different effects on colon tumors.⁸⁹ However, a mixture of t10, c12-CLA (0.5%) and c9, t11-CLA (0.5%) did not show anticancer effects in Apc^min/+ mice.⁹⁰ Mice injected with cancer cells were fed with a diet containing t10, c12-CLA and c9, t11-CLA for 4 weeks. The results showed that the two CLA isomers were found to be effective against CRC cell metastasis in vivo.⁹¹ However, more research is needed to explain the mechanisms by which various CLA isomers inhibit lung metastasis and CRC. Kim et al. found that CLA supplementation decreased the levels of TXB2 and PGE2 in the colonic mucosa, increased apoptosis, and reduced tumor incidence.⁹² In addition, CLA supplementation inhibited TNF-α and COX-2, increased PPAR-γ, induced apoptosis, and inhibited cell proliferation.^93,94 Therefore, CLA can inhibit CRC cell proliferation and metastasis, promote apoptosis of CRC cells, activate PPAR-γ, inhibit proinflammatory cytokines, and reduce harmful substances, thereby reducing CRC (Fig. 2 and 7).

Fig. 7 The potential mechanisms of CLA in relieving cancer. CLA can inhibit cancer cell proliferation and cell cycle growth. CLA up-regulates bax and caspase-3 and promotes apoptosis of cancer cells. CLA can also inhibit MMP2 and MMP4 and cancer cell metastasis. CLA may activate PPAR-γ to inhibit pro-inflammatory cytokines. CLA inhibits oxidative stress and inflammatory substance receptors. Matrix metalloproteinases (MMP).

Table 2 Effects of CLA in carcinogenesis (animal models)

Cancer type	Inducer	Animal	CLA dose	Treatment period	Effects	Year	Ref.
1,2-Dimethylhydrazin (DMD); azoxymethane (AOM); N-nitroso-N-methylurea (NMU); 1,2-dimethylhydrazine (DMH); 7,12-dimethylbenz[a]anthracene (DMBA); N-methyl-nitrosourea (MNU); 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP); N-nitrosobis-2-oxopropylamine (BOP).
Colon	DMD	SD rats	CLA mixture (1%)	14 weeks	↑ Apoptotic index	2001	87
					↓ Number of tumors
					↓ Prostaglandin E2, thromboxane B2 and arachidonic acid
Colon	AOM	SD rats	CLA mixture (1%)	44 weeks	↑ Apoptosis and Caspase-3	2010	88
					↓ Wnt signaling and arachidonic acid
Colon	—	Apc(min/+) mice	c9, t11-CLA (1%)	8 weeks	↓ Polyp size (c9, t11-CLA, CLA mixture)	2008	89
			CLA mixture (1%)		↑ Polyp size (t10, c12-CLA)
			t10, c12-CLA (1%)
Colon	—	Apc(min/+) mice	CLA mixture (1%)	7 weeks	No effect	2000	90
Colon	NMU	BALB/c mice	c9, t11-CLA (0.1%)	4 weeks	↓ Cancer cell migration, pulmonary nodules	2007	91
			t10, c12-CLA (0.1%)
Colon	DMH	SD rats	CLA mixture (1%)	30 weeks	↓ Tumor incidence, arachidonic acid, thromboxane B2 and DAG	2003	92
					↑ Apoptosis
Colon	AOM + DSS	C57BL/6 mice	CLA mixture (1%)	12 weeks	↓ Macrophage	2010	93
					↑ PPAR g
					↓TNF-α
Colon	AOM, colonic aberrant crypt foci	Male F344 rats	c9, t11-CLA (0.1%, or 1%)	4 weeks	Induced apoptosis and suppressed cell proliferation, ↑ PPAR g	2007	94
					↓ COX-2
Mammary	DMBA	SD rats	CLA mixture (80 mg per kg BW)	14 weeks	Inhibit PUFA oxidation, MDA	2019	95
Mammary	DMBA	BALB/c mice	CLA mixture (0.1%, 0.5% or 1%)	4 weeks	Decresed tumor burden	2000	96
Mammary	MNU	SD rats	c9, t11-CLA (1%) or CLA mixture (1%)	20 weeks	Decressed tumor mass	2003	97
Mammary	DMBA	SD rats	c9, t11-CLA (80 mg per kg BW)	20 weeks	↓ Decreased tumor weight and Volume	2020	98
					↓ Ki-67
Mammary	DMBA	SD rats	CLA mixture (0.5%, 1%, 1.5% or 2%)	24 weeks	No effect	1997	99

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CLA can downregulate the proliferation of breast cancer cells and also has certain effects on breast cancer animals. For example, mixture CLA intervention in rats reduced the incidence of breast cancer by inhibiting PUFA oxidation and regulating the expression of COX-2 and PGE2 receptors.⁹⁵ Another study showed that CLA (0.1–1%) reduced lung tumor load in mice.⁹⁶ Oral administration of c9, t11-CLA reduced tumor weight and volume, hormone receptor PR, and Ki-67 expression, demonstrating a protective effect on breast cancer.^97,98 Therefore, CLA can inhibit oxidative stress, inhibit inflammatory substance receptors, and thus regulate breast cancer (Fig. 2 and 7). However, another study showed that CLA did not significantly affect breast cancer. For example, rats with breast cancer were fed with 0.5, 1, 1.5, and 2% CLA. The results showed that CLA did not affect breast cancer.⁹⁹

6. Regulation of psychiatric disease

Alzheimer's disease (AD) is a progressive neurodegenerative disease. c9, t11-CLA diet significantly reduced amyloid beta and regulated the expression of anti-inflammatory cytokines IL-19 and IL-10 in AD model mice.¹⁰⁰ Neuroinflammation caused by Aβ accumulation is thought to be the key to memory loss in AD.¹⁰¹ Thus, dietary supplementation of c9, t11-CLA can upregulate anti-inflammatory cytokines in the brain and may inhibit neuroinflammation by activating PPAR-γ. In addition, CLA supplementation can enhance cognitive function and reduce cerebral oxidative stress, inflammation, apoptosis, and acetylcholinesterase activity in AD mice.¹⁰² Therefore, CLA can inhibit amyloid beta and cell apoptosis and activate PPAR-γ to regulate inflammatory cytokines, thereby improving AD (Fig. 2 and 8).

Fig. 8 The potential mechanisms of CLA in relieving psychiatric disease. CLA can inhibit amyloid beta and cell apoptosis, and activate PPAR-γ to regulate inflammatory cytokines, leading to improvement of AD. Supplementation of CLA can significantly improve redox homeostasis, inhibit Nrf2 overactivation, and increase DHA, BDNF and SFP in depressed mice. Alzheimer's disease (AD); docosahexaenoic acid (DHA); brain derived neurotrophic factor (BDNF); synaptic functional proteins (SFP).

Depression is a global mental illness characterized by anhedonia, insomnia, loss of appetite, sadness, and even suicide.¹⁰³ Supplementation of 30 mg mixed CLA significantly improved redox homeostasis and inhibited Nrf2 overactivation. Additionally, it increased synaptic functional proteins (SFP), brain-derived neurotrophic factor (BDNF), and docosahexaenoic acid (DHA) in depressed mice.¹⁰³ Oxidative stress, one of the etiology of depression/neurodegenerative disease, is often accompanied by activation of the Nrf2 pathway. This pathway is a key antioxidant/detoxification mechanism and is considered a promising target for the treatment of psychiatric disorders and depression-like behaviors.¹⁰⁴ The beneficial effects of CLA supplementation are clearly multifactorial. Nrf2 is involved in regulating multiple physiological pathways (lipid metabolism, inflammation, and redox homeostasis), this mechanism may at least partially explain the antidepressant effects of CLA (Fig. 2 and 8). Currently, there are a limited amount of studies on the effects of CLA on depression and AD. Therefore, more animal and even clinical studies are needed to expand the current results in the future.

7. Limitations, challenges, and prospects in functional studies of CLA

7.1. Effects of isomer, dose, animal model, and test time on results of CLA functional studies

Commercial CLAs usually contain equal proportions of t10, c12-CLA and c9, t11-CLA. Both isomers are currently used in animal and clinical studies. Most of the beneficial properties of CLA are attributed to the two main isomers. In addition, different CLA isomers may have different effects, and the effects of different concentrations of isomers in CLA mixtures are difficult to distinguish. For example, t10, c12-CLA is involved in lipid oxidative catabolism, while c9, t11-CLA can act as active anabolic agents with anti-inflammatory effects.¹⁰⁵ Both isomers have anticancer properties, although they have different effects on apoptosis regulation, oncogene regulation, and lipid metabolism.¹⁰⁶ Therefore, the function of each CLA isomers should be studied to make the research results more explicit and clearer.

Functional studies of CLA involve different animal models, including monkeys, rabbits, rats, mice, and pigs. Differences in animal genetic background and internal structure can lead to varying effects. Using different animals may result in different, and sometimes opposite, outcomes. Therefore, in the functional study of CLA, animal models should be clarified, and in the clinical study of CLA, age, gender, and physical status of subjects should also be clarified to facilitate reference and inspection by other researchers. In addition, CLA supplement time can affect the experimental results. As a result, it is necessary to clarify the CLA supplement time in the study. In conclusion, in the study of CLA function, clarifying the animal model, determining the composition, dose, and duration of CLA, and controlling these factors are necessary to standardize the study of CLA function and obtain valuable results.

7.2. Limitations, challenges, and prospects in dose-effect studies of CLA

At present, there are limited dose-effect studies on the function of CLA. Beef and dairy products are major sources of CLA in foods. Many people supplement these foods due to the difficulty of quantifying dietary CLA intake. Different countries have published recommended intakes for CLA: 52–137 mg per day in the United States, 600–800 mg per day in the United Kingdom, and 1500 mg per day in Australia.¹⁰⁷ The health benefits of CLA can only be achieved if sufficient doses are taken.¹⁰⁸ Studies have shown that a daily intake of 3–6 g of CLA can produce significant health benefits.^109,110 In human studies, a mixture of t10, c12-CLA and c9, t11-CLA has been used. Therefore, in the study of the function of CLA, more attention should be paid to the dosage. Animal models can be used to study the influence of different doses of CLA on a certain disease to determine the dose-effect relationship and find the optimal dose of CLA. Further study on the dose effect of CLA on humans based on the results of animal experiments would be beneficial for the development of CLA products (Fig. 10).

7.3. Limitations, challenges, and prospects in the studies of side effects of CLA

Although many of the effects of CLA are well known, the potential health problems of CLA are rarely reported. Most studies have not reported the relationship between glucose or insulin levels and CLA supplementation.^66,111 Some studies have shown that CLA supplementation has no significant effect on glucose homeostasis,^{52,53,112,113} whereas some showed that CLA supplementation can cause impaired glucose tolerance and disrupt glucose homeostasis.^64,67 Studies have reported that potential adverse effects of CLA on glucose homeostasis were related to the isomer t10, c12-CLA, while c9, t11-CLA showed no relationship with glucose homeostasis.⁶⁷ When the two isomers of CLA are jointly used, c9, t11-CLA may antagonize t10, c12-CLA.^67,68 In addition, the effects of CLA on glucose homeostasis are transient. Moreover, in relatively long-term human studies, the use of CLA has no effect on glucose homeostasis.^55,114 Therefore, the effects of CLA on glucose homeostasis need to be evaluated.

In addition, CLA supplementation can cause fatty liver (hepatic steatosis) (Fig. 9). The c9, t11-CLA can decrease G6PC and PCK1 to reduce hepatic gluconeogenesis, while t10, c12-CLA increases hepatic gluconeogenesis by increasing G6PC expression.¹¹⁵ Regardless of species, CLA induces hepatic steatosis by influencing insulin secretion, regulating hepatic fatty acid (FA) composition, and promoting the secretion of inflammatory response and adipokine.¹¹⁶ CLA can increase liver PKCε, diacylglycerol, and TG to induce insulin resistance, hepatic steatosis, and liver enlargement, which is attributed to the up-regulation of hepatic gluconeogenic gene expression (Fig. 9).^86,117–119 Moreover, high doses of CLA led to fat atrophy, steatosis and inflammation in mice.⁴⁰ Long-term feeding of t10, c12-CLA (0.3%) diets activates lipogenesis, induces FA oxidation and inhibits LDL secretion, which leads to increase liver mass and lipid and TG accumulation.¹²⁰ Therefore, caution should be exercised when using t10, c12-CLA, and attention should be paid to the adverse reactions caused by long-term supplementation.¹²¹

Fig. 9 Possible pathways by which CLA induces hepatic steatosis. CLA in adipose tissue causes a reduction in fatty acid oxidation, lipogenesis and uptake of lipid and glucose, leading to higher circulatory levels of free fatty acids and glucose. Pancreas-induced systemic insulin resistance. Alterations in hepatic lipid metabolism and increased hepatic lipid accumulation and liver mass, leading to hepatic steatosis.

In addition, studies have shown that CLA supplementation inhibits milk fat synthesis in goats, dairy cows, and mice.^122,123 This problem raises questions about the use of CLA in humans, particularly concerning infant health. However, there are significant differences in milk fat sources between humans and ruminants, with milk fat in ruminants being more sensitive to CLA.¹²⁴ Dietary supplementation of t10, c12-CLA can decrease milk fat production.¹²⁵ In addition to animals, several studies reported the effects of CLA on milk fat content in humans. For example, compared with placebo, a daily intake of 1.5 g of CLA for 5 days significantly reduced milk fat, although milk fat levels remained within normal ranges.¹²⁶ Another study showed no significant change in milk fat content after 5 days of CLA supplementation (750 mg CLA isomer per day or 2–4 g per day).¹²⁷ The effect of CLA on the fat content in human milk needs to be confirmed by more clinical trials.

Thus, the effects of CLA on milk fat suppression, hepatic steatosis, and glucose homeostasis have shown different results in animal and clinical studies. More long-term clinical trials are needed to confirm these results in the future (Fig. 10).

Fig. 10 The limitations, challenges, and prospects in functional studies of CLA. In future basic research on CLA in improving health, attention should be paid to the dose-effect relationship (the function and dose of each CLA isomers should be studied), CLA embedding (influence of different embedding methods on CLA function), side effects (the effects of CLA on milk fat suppression, hepatic steatosis, and glucose homeostasis), effects of CLA-producing LAB on health (CLA-producing microorganisms can be used to improve inflammation-related diseases).

7.4. Limitations, challenges, and prospects of CLA embedding and encapsulation

CLA has many functions, but it needs to be taken orally into the intestine. CLA is a polyunsaturated FA that is lipophilic (or hydrophobic) and is easily degraded and oxidized during storage and digestion.¹²⁸ The low solubility of CLA also significantly reduced its bioacceptability and bioavailability. CLA is extremely unstable and even more prone to autooxidation than oleic acid, linolenic acid, or ARA. To overcome these challenges, some carriers have been developed to encapsulate CLA. We summarize the current carrier of CLA packaging and its characteristics and prospects. Chang developed isopass linear dextrin encapsulated CLA, which improved the solubility of the complex and enabled targeted delivery of CLA to the small intestine.¹²⁹ Moreover, the degradation rate of CLA was reduced by encapsulating CLA in whey protein concentrate or a mixture of whey protein concentrate and maltodextrin by spray drying.¹³⁰ However, CLA is rapidly decomposed into furan FAs when heated in air.¹³¹ Therefore, spray drying and other heating methods have limitations and are ineffective for CLA packaging (Fig. 10).

Increasing the bioavailability of CLA at the nanoscale is a promising approach. This approach delays the degradation of CLA through oxidation and other chemical reactions, thus preventing the development of undesirable odors and loss of metabolic value.¹³² Soybean lecithin is a good emulsifying surfactant owing to its high affinity with cell membranes and its ability to increase the absorption of bioactive compounds. For example, in a study of obese mice, nanoemulsified water-soluble-conjugated linoleic acid increased CLA availability, reduced triglyceride and cholesterol volumes in liver tissue, and significantly increased CLA anti-obesity activity.¹³³ Moreover, results of human intestinal Caco-2 cells showed that nano-emulsification enhanced the absorption rate of CLA.¹³⁴ These results provide important information for the development of nanoemulsion carriers, which may improve the thermal stability and bioavailability of CLA (Fig. 10). Although emulsion technology is particularly suitable for the design and manufacture of delivery systems that encapsulate bioactive lipids such as CLA, these lipids are highly sensitive to oxidation due to their high interfacial area.¹³⁵ Therefore, future research needs to overcome this limitation. Soybean pro-lipoprotein (LP) is characterized by high loading capacity, antioxidant activity, and sustained release in vitro. Therefore, soybean proto-lipoprotein (LPP) nanoparticles were prepared as a new CLA carrier, which encapsulated CLA to prevent oxidation and achieve continuous release.¹³⁶ Further experiments on animals and humans are needed in the future to determine the effect.

7.5. Limitations, challenges, and prospects in the studies of health effect of CLA-producing lactic acid bacteria

Although CLA has many functions, long-term use of CLA has certain side effects. Side effects of CLA have been reported in animal studies, including altered glucose/insulin metabolism and liver dysfunction. CLA supplementation in improving health or preventing disease remains a controversial topic. In addition, CLA supplementation can affect liver transglutaminase and liver fatty acid synthesis and oxidation. A large number of CLA-producing bacteria such as Butyrivibrio, Lachnospiraceae, Bulleidia, and Coriobacteriaceae naturally exist in the rumen of animals that eat food containing high linoleic acid content.¹³⁷ In addition, many non-ruminant bacteria, including strains naturally present in the human gastrointestinal tract, such as Lactococcus, Pediococcus, Propionibacterium, Roseburia, Bifidobacterium and Lactobacillus, can use LA to synthesize CLA.¹³⁸ These bacteria similarly express linoleic isomerase to ruminant bacteria in order to convert linoleic acid to c9, t11-CLA. Only a few studies have reported the production of t10, c12-CLA by gastrointestinal bacteria. Some researchers have considered using CLA-producing strains to directly produce CLA in the colon, thereby eliminating the side effects of taking large doses of CLA directly. For example, for colitis, Yang et al. screened several strains, including Lactobacillus plantarum ZS2058, Bifidobacterium breve CCFM683, B. pseudocatenulatum MY40C, B. pseudocatenulatum CCFM680, and B. longum CCFM681. The effect of these bacteria on UC remission was investigated in a series of studies.^11,139–142 The results showed that these CLA-producing probiotics inhibited NF-κB and inflammatory cytokines. Additionally, they regulated the immune response and played an anti-inflammatory role by activating PPAR-γ. Moreover, they could promote the secretion of MUC2 through goblet cells to protect the intestinal mucus layer and reduce the apoptosis of colon epithelial cells. In addition, they could regulate the diversity and composition of gut microbiota (Fig. 10). These features indicate that colitis can be regulated by the use of CLA-producing microorganisms. Therefore, CLA-producing microorganisms can be used to improve inflammation-related diseases, such as arthritis, dermatitis, and colon cancer, in future studies.

8. Conclusion

CLA has been shown to have some regulatory effects on health in animal experiments, but human studies are lacking. In addition, the side effects of CLA need to be further explored. Based on the summary, analysis, and reflection of numerous studies, the following recommendations are made: (1) identify and control animal models, CLA composition, dose, and duration; (2) control the optimal intake of CLA through dose-effect studies; (3) encapsulate CLA using spray drying, microcapsule, emulsion technology, and embedding to reduce oxidative decomposition and improve bioavailability; and (4) use CLA-producing microbes to improve inflammatory diseases. This review will accelerate the development of nutritional and medical foods containing CLA, ultimately helping to improve human health.

Abbreviation

FDA	The US Food and Drug Administration
IBD	Inflammatory bowel disease
UC	Ulcerative colitis
TJs	Tight junctions
SOD	Superoxide dismutase
CAT	Catalase
T-AOC	Total antioxidant capacity
GSH-Px	Glutathione peroxidase
DSS	Dextran sulphate sodium
COX-2	Cyclooxygenase-2
iNOS	Inducible nitric oxide synthase
TFF3	Treleaf factor family 3
CA	Collagen-induced arthritis
RA	Rheumatoid arthritis
PGE2	Prostaglandin E2
ARA	Arachidonic acid
DHA	Docosahexaenoic acid
BDNF	Brain derived neurotrophic factor
SFP	Synaptic functional proteins
VCAM-1	Vascular cell adhesion molecule 1
MCP-1	Monocyte chemotactic protein 1
Mac-1	Macrophage-1 antigen
VLA-4	Very late appearing antigen 4
TC	Total cholesterol
FBG	Fasting blood glucose
LDL-C	Low density lipoprotein cholesterol
FA	Fatty acid
MMP	Matrix metalloproteinases
LTB4	Leukotriene B4
AD	Alzheimer's disease
5-LOX	5-Lipoxygenase
TLR4	Toll-like receptor 4
MyD88	Myeloid differentiation factor88
UCP1	Uncoupling protein 1
CRC	Colon cancer
TXB2	Thromboxane 2
LPP	Lipoprotein

Consent for publication

All authors have read and agreed with the manuscript.

Data availability

No primary research results, software or code have been included and no new data were generated or analysed as part of this review.

Conflicts of interest

The authors declare that they have no financial conflicts of interest.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (No. 3227161449).

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