Unveiling the molecular mechanisms: dietary phytosterols as guardians against cardiovascular diseases

Phytosterols are plant-derived compounds that are structurally similar to cholesterol and are known to have a cholesterol-lowering effect in humans. Phytosterols, which are available as supplements or functional foods, are recognized by the European Union as foods, can be purchased without a prescription, and are frequently taken without the guidance of a healthcare provider. Several studies have also suggested that phytosterols may have protective roles against CVDs, which are a leading cause of mortality worldwide. The following are some of the mechanisms through which phytosterols may exert their protective effects (Fig. 1).

Fig. 1figure 1

Schematic representation of mechanistic pathways involved in the protection against cardiovascular diseases by phytosterols. CAT: catalase; SOD: superoxide dismutase; GSH: glutathione; GPx: glutathione peroxidase; GR: glutathione reductase; APX: ascorbate peroxidase; PPO: polyphenol oxidase; POD: peroxidase; NO: nitric oxide; Cyt c: Cytochrome C; LP: lipid peroxidation; TBARS: thiobarbituric acid reactive substances; HPO: hydroperoxides; LOX: lipoxygenase; Nrf2: nuclear factor-erythroid 2-related factor 2; MCP-1: monocyte Chemoattractant Protein-1; ROS: reactive oxygen species; CVD: cardiovascular diseases; LDL-C: low-density lipoprotein cholesterol; AKT: protein kinase B; JAK 3: janus kinase 3; STAT3: signal transducer and activator of transcription 3; ICAM-1: intracellular adhesion molecule-1; VCAM-1: vascular cell adhesion molecule-1; NF-κB: nuclear factor-κB; IL: Interleukin; TNF-α: tumor necrosis factor-α; ACAT-1: acyl-CoA: cholesterol acyltransferase-1; VEGF: vascular endothelial growth factor; MAPK: mitogen-activated protein kinase; ERK: extracellular signal-regulated kinase; TLR 4: toll-like receptor 4; ACC: acetyl-CoA carboxylase; FAS: FA synthase; FAT: FA translocase; PPARγ: peroxisome proliferator-activated receptor γ; iNOS: inducible nitric oxide; PGE 2: prostaglandin E 2; COX-2: cyclooxygenase-2

2.1 Antioxidant mechanisms

As demonstrated above, it is possible to state that certain phytosterols have a double anti-inflammatory and antioxidant effect, and therefore, additional antioxidant properties. Specifically, several phytosterols have shown promising antioxidant effects via various mechanisms of action classified according to the cellular, sub-cellular, and molecular levels at which they act (Table 1).

Table 1 Antioxidant mechanisms of phytosterols

Regarding cellular antioxidant mechanisms, numerous investigations have shown that phytosterols are able to activate antioxidant enzymes to reduce the production of ROS and prevent oxidative damage. Indeed, β-sitosterol, fucosterol, stigmasterol, and ergosterol all induced the activation of multiple antioxidant enzymes, namely CAT, SOD, GSH, glutathione peroxidase (GPx), glutathione reductase (GR), ascorbate peroxidase (APX), and ceruloplasmin [19,20,21, 28,29,30,31,32,33,34,35,36,37,38,39,40,41,42]. Additionally, stigmasterol restored the levels of two types of SOD present in living cells; Mn-SOD (mitochondrial SOD) and T-SOD (extracellular SOD) [21]. Moreover, levels of certain non-enzymatic antioxidants such as tocopherol, ascorbic acid, carotene, and vitamins C and E were also increased by β-sitosterol [20, 33]. On the other hand, the activity of polyphenol oxidase (PPO) and peroxidase (POD), two types of enzymes that catalyze oxidation reactions and are commonly present in plants, was up-regulated by β-sitosterol [20] and stigmasterol [35].

Importantly, stigmasterol exhibited antioxidant activity by decreasing the adverse effects of salt stress on plants (bean) [36]. This stress, of environmental origin, can accumulate free radicals in plants, which can have negative effects on crop yield and growth. Antioxidants, by reducing oxidative damage, can increase cell survival in response to elevated free radical levels. This was achieved by ergosterol, which improved H2O2-induced damaged cells’ survival rates [43]. Paradoxically, a balanced production of nitric oxide (NO) can also have antioxidant effects under certain conditions, as recently demonstrated by stigmasterol [34].

For subcellular mechanisms, β-sitosterol stimulated mitochondrial ATP-producing capacity in H9c2 cells [44] (Fig. 2). Low ATP production can impair mitochondrial functions, leading to oxidative stress. Maintaining the balance between mitochondrial ATP production and mitochondria protection against oxidative damage is therefore very important.

Fig. 2figure 2

Potential mechanistic pathways of the antioxidant effect of β-sitosterol leading to cardioprotective benefits using in vitro and ex vivo experiments. Mitochondrial ROS generation was enhanced by β-sitosterol. This phytosterol also increased mitochondrial respiration in states 3 and 4, decreasing coupling efficiency. In H9c2 cells, β-sitosterol increased glutathione redox cycling (GR, GSH, and GSSG) and protected against hypoxia/reoxygenation-induced apoptosis. Ex vivo, β-sitosterol protected the myocardium against I/R injury in female rats. The cardioprotective effect of β-sitosterol in this category was most likely mediated by an increase in mitochondrial glutathione redox cycling (GR, GSH, and GSSG). GR: glutathione reductase; GSH: reduced glutathione; GSSG: oxidized glutathione; ROS: reactive oxygen species; I/R: ischemia/reperfusion

Moreover, inhibition of lipid peroxidation (LP), a lipid oxidation process in cell membranes, has emerged as a crucial subcellular defense mechanism against oxidative stress. β-sitosterol, ergosterol, and stigmasterol either prevented LP alterations or reduced levels of lipid peroxidative products [28, 31, 40, 43, 45, 46]. In this sense, β-sitosterol decreased the levels of thiobarbituric acid reactive substances (TBARS) [33, 47], often used as markers of LP. By inhibiting LP, β-sitosterol thus has the ability to reduce TBARS levels, which may ultimately protect tissues and cells from oxidative damage. The antioxidant effectiveness of β-sitosterol is illustrated by the reduction of TBARS levels. The content of another LP by-product used as a marker of cell damage and oxidative stress called malondialdehyde (MDA) has been strongly inhibited by stigmasterol and ergosterol in several studies [32, 35, 36, 38, 42]. Modulation of cell signaling may also constitute another subcellular antioxidant mechanism, namely the modulation of signaling pathways involved in programmed cell death, such as the apoptosis pathway. β-Sitosterol is an interesting molecule in this area, as it has been shown to protect H9c2 cells against hypoxia/reoxygenation-induced apoptosis [44] and to prevent GOX-induced oxidative stress and LP via estrogen receptor (ER)-mediated PI3K/GSK3β signaling [48].

Antioxidants can protect lipids from oxidation in liposomes, structures used for transporting drugs or other substances. However, the lipids that form liposomes are very vulnerable to oxidation, which can affect their effectiveness as drug carriers. This is why it is imperative to protect lipids against oxidation to maintain liposome stability. This mechanism was observed in the study carried out by Dupont et al. [49] using ergosterol.

At the molecular level, several antioxidant mechanisms have been demonstrated. Free radical protection in cell membranes has been a promising therapeutic approach in neutralizing free radicals before they can cause damage. Indeed, stigmasterol, ergosterol, and β-sitosterol reduced the generation and levels of several radicals such as DPPH, ABTS, H2O2, O2⋅−, OH−, NO, hydroperoxides (HPO), and •CH(OH)CH3, as well as that of an enzyme catalyzing free radical formation, lipoxygenase (LOX) [19, 20, 33, 35, 41, 46, 47, 50]. In addition, β-sitosterol [20] and ergosterol [43] showed a significant reduction in intracellular ROS levels. This reduction in ROS is essential to protect cells against oxidative damage. In fact, overproduction of ROS can lead to cellular damage, whereas these phytosterols can neutralize ROS and avoid their harmful effect, as suggested by previous results. By regulating the redox balance, they can also maintain cellular health and prevent possible diseases. This was achieved by β-sitosterol which up-regulated the redox cycle of cellular glutathione [44].

In contrast, activation of Nrf2 and Nrf2/HO-1 signaling pathways can be used against oxidative stress. It has been shown that during cellular stress, these pathways are activated, and as a result the protein called nuclear factor-erythroid 2-related factor 2 (Nrf2) present in the cell cytoplasm moves to the nucleus and activates gene expression involved in toxic substance detoxification, DNA repair, and defense against oxidative stress [51]. Recently, it was discovered that stigmasterol, ergosterol, and fucosterol are able to activate these two pathways, allowing them to be effective natural compounds in oxidative stress management [25, 38, 42, 52,53,54].

It was indicated in the previous section that the protein NF-κB/p65 plays an important role in inflammation and immune response, but an over-activation of this protein can increase the generation of free radicals and increase OS in cells. This indicates that inhibition of its activation is an additional antioxidant mechanism at the molecular level. Indeed, Sun et al. [21] recorded a decrease in CS-induced OS through the inhibition of NF-κB/p65 activation by administering ergosterol.

This phytosterol also inhibited cytochrome c [42, 54], a protein involved in the electron transport chain of cellular respiration. However, excess electron production can lead to the formation of free radicals. In this context, cytochrome c inhibition may be an optional strategy to protect cells from free radical damage and prevent cellular oxidation.

Moreover, several investigations have found that decreased OS markers may constitute a crucial molecular antioxidant mechanism [55, 56]. In our context, β-sitosterol and stigmasterol reduced and stabilized oxidative stress markers in different tissues (in vivo/in vitro) [28, 31, 57], whereas ergosterol reduced the levels of two biomarkers, namely lactate dehydrogenase (LDH) and creatine kinase MB (CK-MB) [42, 54], used to assess cell damage as they may also be overexpressed in various conditions, including CVDs.

As previously demonstrated, stigmasterol has antioxidant potential by stimulating the production of NO that can act as an antioxidant [34]. In the same study, authors highlighted a molecular antioxidant effect related to increased expression levels of the iNOS enzyme, which catalyzes the synthesis of NO. It is therefore clear that increased NO production is closely dependent on iNOS expression. However, it should be noted that the mechanisms regulating the expression of this enzyme can be complex and depend on many factors.

2.2 Anti-inflammatory mechanisms

Several investigations have examined the anti-inflammatory potential of various phytosterols by shedding light on the mechanisms of action. Indeed, the majority of these studies have shown that these natural compounds exert their anti-inflammatory effects at various levels via cellular, subcellular, and molecular mechanisms.

At the cellular level, several phytosterols inhibited experimentally induced edema in animals, in particular paw and ear edema, such as β-sitosterol [61,62,63,64], stigmasterol [65,66,67], and ergosterol [68]. In addition to the reduction in paw edema recorded by Zhang et al. [64] with β-sitosterol, a decrease in the polyarthritic index used to assess the severity of joint damage in subjects with polyarticular forms of arthritis. It has been shown that in rheumatoid arthritis, the immune system attacks various body tissues, particularly joints, subsequently inducing chronic inflammation.

Additionally, β-sitosterol and stigmasterol showed notable anti-inflammatory effects (in vivo) against colonic inflammation (colitis) by alleviating its severity [69,70,71] and score [69] or inhibiting colon shortening [72] that is a major consequence of inflammation.

On the other hand, a powerful anti-inflammatory activity was obtained by β-sitosterol, which stimulated the secretion of certain molecules involved in inflammatory processes, namely histamine, bradykinin, serotonin, and prostaglandins [62].

Another mechanism associated with an inflammatory immune response has been observed with this phytosterol, which is the increase in calcium absorption in activated neutrophils [73]. Activation of these white blood cells (neutrophils) in response to tissue damage or infection, triggers biochemical reactions to produce free radicals and inflammatory enzymes that target damaged cells and pathogens [74]. In activated neutrophils, the production of these enzymes is closely linked to the absorption of calcium having the role of a cellular messenger [

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