Regulation of Gene Expression through Food—Curcumin as a Sirtuin Activity Modulator

The sirtuin family comprises NAD+-dependent protein lysine deacylases, mammalian sirtuins being either nuclear (SIRT1, SIRT2, SIRT6, and SIRT7), mitochondrial (SIRT3, SIRT4, and SIRT5) or cytosolic enzymes (SIRT2 and SIRT5). They are able to catalyze direct metabolic reactions, thus regulating several physiological functions, such as energy metabolism, stress response, inflammation, cell survival, DNA repair, tissue regeneration, neuronal signaling, and even circadian rhythms. Based on these data, recent research was focused on finding molecules that could regulate sirtuins’ expression and/or activity, natural compounds being among the most promising in the field. Curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) can induce, through SIRT, modulation of cancer cell senescence, improve endothelial cells protection against atherosclerotic factors, enhance muscle regeneration in atrophy models, and act as a pro-longevity factor counteracting the neurotoxicity of amyloid-beta. Although a plethora of protective effects was reported (antioxidant, anti-inflammatory, anticancer, etc.), its therapeutical use is limited due to its bioavailability issues. However, all the reported effects may be explained via the bioactivation theory, which postulates that curcumin’s observed actions are modulated via its metabolites and/or degradation products. The present article is focused on bringing together the literature data correlating the ability of curcumin and its metabolites to modulate SIRT activity and its consequent beneficial effects.


Introduction
The concept of healthy ageing is defined by WHO as "the process of developing and maintaining the functional ability that enables well-being in older age" [1]. This is becoming more and more important, as the percentage of elders is expected to rise markedly in the next decades and ageing-associated diseases will represent an important financial burden. Both clinicians and health scientists are looking for ways to reduce the risk of age-related metabolic disorders through diet or dietary supplements and, along the way, to build a strong body of data as a basis for the next generation of therapies addressing high incidence age-related diseases [1,2].
It is well-known that our diet greatly influences our health. For many centuries now, empirical observations proved that certain natural products have the ability to positively influence the course of some diseases, to prevent their development or hinder their evolution. Researchers have tried and succeeded to a great extent, to gather the scientific data to support these observations, so we can clearly point to the precise mechanisms of nutraceuticals' beneficial effects in different pathologies. For example, fruits and vegetables, which are rich in polyphenols, have antioxidant, anti-inflammatory effects, and induce a positive response in the evolution of cardiovascular disease, diabetes, obesity, and numerous other diseases; now, we understand, for the most part, and continue to study the ways through which AND "mice," "rats". For clinical trials, the used the keywords and MeSH terms were: "sirtuin", "epigenetic regulation" AND "curcumin", "curcumin metabolites" AND "clinical trial" AND "effects", "disease". The most relevant papers were selected after eligibility analysis and cross-checking. Additionally, literature was reviewed in order to ascertain the key aspects regarding the characteristics of sirtuins and the intake, bioavailability, and metabolism of curcumin; the keywords and MeSH terms used were: "sirtuin", "sirtuin family", "sirtuin enzymes", OR "curcumin" AND "bioavailability", "metabolism", "metabolites" and "derivates".
Curcumin, as well as its analogues, have a very low water solubility and, consequently, a very low bioavailability, characteristic to most polyphenols, with a significant inter-individual variability. Some studies state that about 1% of the administered doses are found in blood, with peak levels of 3.6 μM obtained after a very high consumption (8 g/day for 3 months) [31,32]; others state that 2 g of curcumin generate a blood Cmax value of 6 ng/mL, and doses of 10-12 g lead to a 50 ng/mL blood concentration [33]. In an effort to overcome the shortcomings of curcumin when it comes to pharmaceutical formulations (low water solubility, poor chemical stability, etc.), modern drug delivery systems were employed. Encapsulation of curcumin in lipid-based nanosystems such as micelles, liposomes, nanoemulsions/complexes, hydrogels or chitosan-based nanocarriers, proved useful in improving its stability and bioavailability [34][35][36].
Curcumin metabolism after oral administration in rats, mice and humans follows two stages ( Figure 2)-reduction, as part of phase I metabolism, and conjugation, as a part of phase II metabolism; these processes lead to glucuronide and sulfate conjugates, together with small quantities of octahydrocurcumin, hexahydrocurcumin, tetrahydro- The structure of curcumin allows for keto-enol tautomerism, as shown in Figure 1, both forms are found as a mixture [27].
Curcumin, as well as its analogues, have a very low water solubility and, consequently, a very low bioavailability, characteristic to most polyphenols, with a significant inter-individual variability. Some studies state that about 1% of the administered doses are found in blood, with peak levels of 3.6 µM obtained after a very high consumption (8 g/day for 3 months) [31,32]; others state that 2 g of curcumin generate a blood Cmax value of 6 ng/mL, and doses of 10-12 g lead to a 50 ng/mL blood concentration [33]. In an effort to overcome the shortcomings of curcumin when it comes to pharmaceutical formulations (low water solubility, poor chemical stability, etc.), modern drug delivery systems were employed. Encapsulation of curcumin in lipid-based nanosystems such as micelles, liposomes, nanoemulsions/complexes, hydrogels or chitosan-based nanocarriers, proved useful in improving its stability and bioavailability [34][35][36].
Curcumin metabolism after oral administration in rats, mice and humans follows two stages ( Figure 2)-reduction, as part of phase I metabolism, and conjugation, as a part of phase II metabolism; these processes lead to glucuronide and sulfate conjugates, together with small quantities of octahydrocurcumin, hexahydrocurcumin, tetrahydrocurcumin and dihydrocurcumin [37][38][39][40][41]. The reduced forms of curcumin, generated by alcohol dehydrogenases are either found free or conjugated in the blood [42]. Glucurono-conjugation takes place both in the liver and in the intestinal cells, both in humans and rodents [38]; also, the reductive metabolism can take place under the action of NADPH reductases of the intestinal microbiota, which generates mostly di-and tetrahydrocurcumin [43,44]. Animal studies using radio-labeled curcumin revealed that most of the compound is eliminated through feces and only a small amount by the kidney [45,46]. alcohol dehydrogenases are either found free or conjugated in the blood [42]. Glucurono-conjugation takes place both in the liver and in the intestinal cells, both in humans and rodents [38]; also, the reductive metabolism can take place under the action of NADPH reductases of the intestinal microbiota, which generates mostly di-and tetrahydrocurcumin [43,44]. Animal studies using radio-labeled curcumin revealed that most of the compound is eliminated through feces and only a small amount by the kidney [45,46]. Taking into consideration the high quantities of curcumin found in the gastrointestinal tract after administration, researchers postulated that this compound might exert direct modulatory effects of the microbiota and interfere with the gut-brain axis. Mutual two-way interactions between curcumin and the microbiota were reported. Curcumin was found to directly regulate the gut microbiota and its capacity to metabolize the parent compound and generate metabolites-key aspects for curcumin's pharmacological activity [47][48][49].
Therefore, most of the curcumin is found in the bloodstream either in the form of metabolites, conjugates or as degradation products, which seem to be responsible for some of the biological effects of this natural compound. Patients with colorectal cancer who received daily oral doses of 3.6 g of curcumin for 4 months presented blood levels of 16 nM glucuronide and 9 nM curcumin sulfate, respectively; for these patients, the urinary concentrations were 1 nM for curcumin, 500 nM for the glucuronide and 40 nM for the sulfate derivative [54].
Studies show that ferulic acid and vanillin yield antioxidant and anti-inflammatory effects but have lower antiproliferative actions compared to the parent compound [55][56][57][58][59]. The curcumin glucuronide is shown to stimulate the expression of antioxidant genes Nrf2, HO-1, and NQO1, but also those involved in epigenetic changes (histone deacetylases-HDAC1, HDAC2, HDAC3, and HDAC4) [60]. The reductive forms have either anti-oxidant properties, through Nrf2 stimulation, or anti-inflammatory, through modula- Taking into consideration the high quantities of curcumin found in the gastro-intestinal tract after administration, researchers postulated that this compound might exert direct modulatory effects of the microbiota and interfere with the gut-brain axis. Mutual two-way interactions between curcumin and the microbiota were reported. Curcumin was found to directly regulate the gut microbiota and its capacity to metabolize the parent compound and generate metabolites-key aspects for curcumin's pharmacological activity [47][48][49].
Therefore, most of the curcumin is found in the bloodstream either in the form of metabolites, conjugates or as degradation products, which seem to be responsible for some of the biological effects of this natural compound. Patients with colorectal cancer who received daily oral doses of 3.6 g of curcumin for 4 months presented blood levels of 16 nM glucuronide and 9 nM curcumin sulfate, respectively; for these patients, the urinary concentrations were 1 nM for curcumin, 500 nM for the glucuronide and 40 nM for the sulfate derivative [54].
Studies show that ferulic acid and vanillin yield antioxidant and anti-inflammatory effects but have lower antiproliferative actions compared to the parent compound [55][56][57][58][59]. The curcumin glucuronide is shown to stimulate the expression of antioxidant genes Nrf2, HO-1, and NQO1, but also those involved in epigenetic changes (histone deacetylases-HDAC1, HDAC2, HDAC3, and HDAC4) [60]. The reductive forms have either anti-oxidant properties, through Nrf2 stimulation, or anti-inflammatory, through modulation of COX2, decrease of interleukin release or of Ikβ-α, NF-κβ, and PI3k/Akt/mTOR pathways. They also act on SIRT1 and 2 as well as on HDAC1, thus influencing the epigenetic phenomena associated with curcumin intake [41].
Still, literature shows inferior results when comparing the effects induced by curcumin metabolites, compared to the parent compound on most of the cellular pathways in experimental models. For example, curcumin inhibited TNF-induced NF-κB activation, while its glucuronides had no effects on NF-κB suppression; curcumin, but not the glucuronides, showed anti-proliferative effects on several cancer cell lines [61,62]. Another aspect on curcumin and its metabolites was reported by Jamil et al. [63], who proved in cancer cell lines that curcumin is mainly metabolized through sulfation, and the sulfate exits the cell, thus reducing the intracellular concentration of curcumin and consequently its growth inhibitory effects [41].

The Sirtuin Family
Sirtuins perform lysine deacetylation of several substrates, together with NAD + hydrolysis (NAD + acting as a co-substrate), thus releasing nicotinamide, which in turn modulates SIRT activity; it is clear that NAD + , NADH (generated via mitochondrial processes) and nicotinamide influence the activity of sirtuins [8,64]; for example, upregulation of NAD + biosynthesis significantly prolongs yeast lifespan [65]. The interdependence of sirtuin activity with NAD + /NADH ratio suggests the fact that sirtuins can be considered energy sensors [66].
Mammalian sirtuins are located either in the nucleus (SIRT1, SIRT6, and SIRT7), mitochondria (SIRT3, SIRT4, and SIRT5) or cytoplasm (SIRT2), and are catalysts for different metabolic reactions (different "deacylations" of long-chain fatty acids-demalonylation, desuccinylation, decrotonylation, depropyonylation, delipoamidation, or mono-ADPribosylation), thus regulating several physiological functions, such as energy metabolism, stress response, cell survival, DNA repair, tissue regeneration, inflammation, neuronal signaling, and even circadian rhythms [14,19,67,68]. Interestingly, some of the sirtuins are able to shuttle from one cell compartment to another; for example, SIRT1 can be found not only in the nucleus, but also in the cytoplasm, while mitochondrial sirtuins can migrate to the nucleus; SIRT3 can migrate from mitochondria to the nucleus as a response to stress (DNA damage) [14,69,70].
The protective effect against oxidative stress exerted by SIRT1 is also based on its ability to activate through deacetylation gene transcription of PGC-1α (peroxisome proliferatoractivated receptor-coactivator 1α), PPAR (peroxisome proliferator-activated receptor), nuclear respiratory factor (NRF), and mitochondrial transcription factor A (TFAM), all of these being involved in the mitochondrial function, as well as in glucose and lipid metabolism [77,80]. Through the modulation of PGC-1α deacetylation, SIRT1 prevents mitochondrial dysfunction and, subsequently, apoptosis [81].
The cytoplasmic and mitochondrial sirtuins perform the same type of deacetylation for enzymatic systems, such as eNOS and AMPK, thus contributing to the regulation of metabolic processes and adaptive cellular responses (cellular anti-stress response, energy metabolism and tumorigenesis) [67,68]. Sirtuins contribute to a reduction of fat accumulation and favor insulin secretion and release through the activation of PPARγ coactivator-1α [10,82]; SIRT4 also regulates mitochondrial glutamine metabolism [83].
The general effect of the processes influenced by sirtuins ( Figure 3) is a delayed onset of age-related diseases with the subsequent extension of healthy lifespan, due to their protective effects against age-related pathologies (cancer, liver steatosis, neurodegeneration, cardiovascular disease, diabetes, etc.) [67,68,84]. Therefore, one of the most discussed functions of the sirtuin family is their ability to regulate lifespan; for example, absence of SIRT1 and SIRT6 is correlated with the expression of senescence associated genes and premature ageing [78,85], while SIRT1 and 6 overexpression postponed the senescence for stem cells exposed to stress factors (oxidative stress), but also for human umbilical cord fibroblasts, angiotensin II-treated human coronary artery endothelial cells, primary porcine aortic endothelial cells, stress-exposed lung cells, or bone marrow-derived mesenchymal stem cells and hindered the ageing phenomena for mouse embryonic fibroblasts, human diploid fibroblasts or human endothelial cells [21,72,[86][87][88][89][90][91][92][93]. SIRT1 has a higher activity in embryonic stem cells, which decreases in differentiated cells [20]; SIRT3 is also highly expressed in hematopoietic stem cells [94]. Sirtuins 1, 6 and 7 are up-regulated in correlation with calorie restriction; also, their deletion prevents the health benefits of physical exercise and calorie restriction [84,[95][96][97][98][99][100].
The general effect of the processes influenced by sirtuins ( Figure 3) is a delayed onset of age-related diseases with the subsequent extension of healthy lifespan, due to their protective effects against age-related pathologies (cancer, liver steatosis, neurodegeneration, cardiovascular disease, diabetes, etc.) [67,68,84]. Therefore, one of the most discussed functions of the sirtuin family is their ability to regulate lifespan; for example, absence of SIRT1 and SIRT6 is correlated with the expression of senescence associated genes and premature ageing [78,85], while SIRT1 and 6 overexpression postponed the senescence for stem cells exposed to stress factors (oxidative stress), but also for human umbilical cord fibroblasts, angiotensin II-treated human coronary artery endothelial cells, primary porcine aortic endothelial cells, stress-exposed lung cells, or bone marrowderived mesenchymal stem cells and hindered the ageing phenomena for mouse embryonic fibroblasts, human diploid fibroblasts or human endothelial cells [21,72,[86][87][88][89][90][91][92][93]. SIRT1 has a higher activity in embryonic stem cells, which decreases in differentiated cells [20]; SIRT3 is also highly expressed in hematopoietic stem cells [94]. Sirtuins 1, 6 and 7 are up-regulated in correlation with calorie restriction; also, their deletion prevents the health benefits of physical exercise and calorie restriction [84,[95][96][97][98][99][100]. The impairment of sirtuin activity is correlated with age-related pathological processes, such as cardio-vascular disease, neurodegeneration or cancer [69,101,102]. SIRT1 is the most studied member of this enzymatic family, its dysregulation being involved in inflammation, redox stress, neurodegeneration, fat accumulation, diabetes, tumorigenesis, etc. [10,69]. The SIRT1 level is reduced in liver cells during the ageing process (which is associated with an increase in DNA damage), probably as a result of decreased NAD + The impairment of sirtuin activity is correlated with age-related pathological processes, such as cardio-vascular disease, neurodegeneration or cancer [69,101,102]. SIRT1 is the most studied member of this enzymatic family, its dysregulation being involved in inflammation, redox stress, neurodegeneration, fat accumulation, diabetes, tumorigenesis, etc. [10,69]. The SIRT1 level is reduced in liver cells during the ageing process (which is associated with an increase in DNA damage), probably as a result of decreased NAD + availability [103]; also, SIRT1 decreases in ageing arteries and endothelial cells suggesting its involvement in age-related cardiovascular disease [14,104].
If, in most cases, the up-regulation of sirtuins has beneficial effects on cell metabolism and general physiological processes, there are some pathologies where the inhibition of these enzymes rather than their activation might provide beneficial effects; for example, there are studies arguing that SIRT2 inhibition (as provided by AGK2 or MIND4) results in protective actions concerning neurodegenerative diseases, such as Parkinson's or Huntington's disease [105][106][107].

Cellular Effect of Curcumin
According to experimental studies, curcumin is a modulator for the action of different signaling molecules, such as protein kinase C, JNK and advanced glycation end-product (AGE) pathways; it can also act against oxidative stress induced by NADPH oxidase, in- hibiting apoptosis via JNK, PI3K-Akt, etc., including in experimental settings associated with high glucose levels [68,108,109]. In a similar manner, curcumin is a direct protector of cardiomyocytes, preventing the oxidative stress-related effects developed under hyperglycemic conditions, thus preventing complications associated with metabolic imbalance in diabetes of metabolic syndrome [24].
Curcumin exerts its antioxidant effects through activation of Nrf2, kelch-like ECHassociated protein 1 (Keap1), a cysteine rich metalloprotein, and antioxidant response element (ARE) [110,111]. Under physiological conditions, Keap1 keeps Nrf2 in the cytoplasm, where its ubiquitination and proteasomal hydrolysis is favored; under the effect of different stressors, Keap1 is blocked through a phosphorylation stage and, consequently, Nrf2 is translocated to the nucleus where it promotes the transcription of genes coding antioxidant molecules (SOD, GSH and GPx and glutathione transferases, HO-1, NADPH, and NAD(P)H dehydrogenase (quinone) 1). Curcumin activates the Keap1/Nrf2 system, through binding of thiol groups from the Keap1 structure, which permits Nrf2 to move to the nucleus and act as a transcription factor for the above-mentioned antioxidant and detoxifying systems [112,113].
Curcumin is also able to reduce the differentiation of preadipocytes, thus acting as an anti-obesity molecule; curcumin decreases the expression of FABP4 and C/EBPα (biomarkers for cell differentiation) in adipocytes [114][115][116], fat accumulation through a decrease of mRNA of the membrane fatty acid transporter [117,118], the expression of fatty acid synthase and glycerol-3-phosphate acyltransferase-1, important enzymes in the synthesis of fatty acids and triglycerides [114,118], these effects being observed for 5, 10 and 20 µM of curcumin. Curcumin, in the same concentrations mentioned before, not only decreases the biosynthesis, but also increases the catabolism of lipids in adipocytes, by increasing the carnitine palmitoyltransferase 1 (CPT-1) expression involved in β-oxidation [115]. Moreover, 0.1 to 10 µM curcumin reduces the NF-κB activity and its nuclear translocation, and so inhibits the recruitment of macrophages into the enlarged adipose tissue and reduces the release of IL-6, TNF-α, MCP-1 from adipocytes [117,119]; the results of in vitro studies are confirmed in animal models of obesity [30].

Anti-Proliferative and Antiaging Effects
The ability of curcumin to modulate the activity of sirtuins and to facilitate adaptation to certain cellular settings, including stress factors, allows it to be included into the hormetin category [32,120,121]. There are studies focused on the adaptative dosedependent biphasic effects of curcumin on skin fibroblasts [122]. Additionally, experimental research using a replicative senescence model, as well as a model for premature senescence using doxorubicin, showed that curcumin at low levels of exposure (0.1-1 µM) did not delay senescence and up-regulated sirtuins in vascular smooth muscle cells (VSMCs) and endothelial cells [32]; on the other hand, higher curcumin concentrations (2.5-10 µM) induced senescence in cancer and endothelial cells [123]. Therefore, it can be concluded that, at lower concentrations, curcumin has beneficial, protective, anti-senescence actions, while at higher concentrations it can be pro-senescent and even cytotoxic. Studies showed that endothelial cells are more susceptible to these effects compared to VSMCs, probably due to a cell-specific ability to up-regulate sirtuin levels [32,123,124]. There are also studies demonstrating that the anti-proliferative effects induced by curcumin in VSMCs, due to SIRT7 downregulation, could be a useful approach for mitigating cardiovascular pathologies [125].
Sirtuin up-regulation contributes to the anti-senescence effect reported for curcumin in different experimental studies; for example, curcumin improved keratinocytes differentiation ability in a senescence model [126][127][128]. The lifespan of C. elegans was prolonged by curcumin, this effect being abolished by mutations of SIRT2 gene [129].

Anti-Inflammatory and Antioxidant Effects
SIRT1 controls the transcription and consequent expression of inflammatory markers (IL-1, TNF-α, IL-8, IL-6, etc.) by influencing the acetylation of NF-κB and p65 [24,77,80,130]; it also interacts with JAK/STAT pathway, suppressor of cytokine signaling (SOCS), and TLR4/MyD88/NF-κB axis thus modulating the inflammatory networks [112]. Curcumin is one of the molecules able to inhibit STAT3 phosphorylation and thus inhibits the inflammatory pathway; the natural compound acts both in vitro and in vivo to support SOCS1 and 3 expression (as antagonists of JAK/STAT pathway) and through this mechanism, it blocks the release of TNF-α, IL-6, and PGE2 [69,81,112,131,132].
The anti-inflammatory action of curcumin is associated with beneficial effects in different inflammatory pathologies. Administering this natural compound in a mouse model of acute lung injury had a beneficial outcome associated with a decreased expression of NF-κB together with an increased level of SIRT1 [133]; in rat models of hemorrhage shock after lung injury or COPD, curcumin increased pulmonary barrier function, reduced pulmonary oxidative stress and lung inflammation, alleviated COPD by promoting autophagy and inhibiting ER stress and induced the activity of SIRT1 [134,135]. Additionally, in another rat model of COPD (cigarette smoke exposure combined with intratracheal administration of lipopolysaccharide), curcumin induced a reduction of oxidative stress and inflammation markers and increased the expression of SIRT3 [136]. Moreover, curcumin proved protective effects in a rat model of aluminum phosphide induced lung injury, modulated by a decrease in oxidative stress and an increased expression of SIRT1, FOXO1 and FOXO3 [137]. The protective effects were also confirmed in lymphocytes isolated from COPD patients, exposed to 1 µM curcumin, which increased SIRT1 expression and decreased steroid resistance, IFNγ and TNF-α production [138].
Interestingly, besides its anti-inflammatory role, curcumin also acts by stimulating SIRT1 in tert-butyl hydroperoxide-treated rat chondrocytes and consequently inhibits ER stress, thus contributing to an improvement of osteoarthritis development in animal models [139].
By stimulating the level of phosphorylated mTOR and stimulating SIRT1 expression in the colon tissue, curcumin had protective effects in a dextran sulfate sodium-induced ulcerative colitis in mice, resulting in a reduction of body weight loss and of disease severity [140]. Additionally, in a model of necrotizing microscopic colitis in newborn rats, curcumin activated the SIRT1/Nrf2 pathway and reduced the TLR4 expression, improving disease evolution [141].
The antioxidant effect of curcumin is also correlated with its ability to modulate sirtuin expression. In a rat model of cisplatin-induced renal impairment, curcumin stimulated SIRT1, SIRT3 and SIRT4 activities [142] reduced oxidative stress and protected the kidney from pathological changes [143]. Additionally, curcumin improved the testis oxidative stress and inflammation induced by cyclosporine use in a rat model of male infertility, by stimulating SIRT1 expression [144]. In a mouse model of sepsis-induced acute kidney injury, tetrahydrocurcumin increased the survival rate, improved the kidney function and ameliorated the renal histological damage, reduced the inflammatory response (IL-1β, IL-6, and TNF-α), alleviated the oxidative stress (measured by MDA level, SOD, GSH, CAT, and GPx activities), and prevented cell apoptosis in renal tissues of septic mice through an increased expression of SIRT1, associated with reduced downstream molecules Ac-p65 and Ac-foxo1 [145]. Moreover, curcumin exerted protective kidney effects in a rat model of gentamicin-induced acute kidney injury, reduced the apoptosis of tubular cells, reduced oxidative stress and induced SIRT1 and Nrf2/HO-1 expression [146].
Curcumin use in mouse and cell models of iron-overload, resulted in an increased SIRT3 expression, associated with a decreased SOD activity, thus protecting against the oxidative stress, and suppressed iron loading-induced autophagy [147].
Studies performed in a diabetes mouse model showed that curcumin treatment modulates SIRT1 through AMPK, thus contributing to an improved glucose absorption and metabolism [148]. Curcumin acts to protect VSMC by activating AMPK and subsequently stimulating ATP and superoxide synthesis, as well as SIRT1 activity; in models of myocardial infarction/reperfusion injury, curcumin reduced oxidative stress damaging effects on mitochondria and lessened the infarction size through a SIRT1-mediated pathway [149,150]. Experimental studies on cardiomyocytes exposed to hypoxia/reoxygenation cycles pointed out the ability of curcumin to reduce apoptosis and oxidative stress in association with an increased SIRT1 expression [151,152]. Additionally, tetrahydrocurcumin, administered in a streptozotocin diabetes mouse model, improved cardiac function and ameliorated myocardial fibrosis and cardiac hypertrophy, accompanied by reduced reactive oxygen species (ROS) generation and increased SIRT1 expression [153]. Curcumin, administered in a model of coronary artery ligation in adult mice, induced the expression of SIRT1 and exerted an anti-fibrotic general effect [154]; the age-related impairment of endothelial cells and VSMC were also abridged through the SIRT1 pathway and the NF-κB inhibitor, and this type of effect was also revealed in muscle cells [32,[154][155][156].
All these effects of curcumin could be extrapolated to different pathologies such as diabetes, ischemia/reperfusion injury or cardiac fibrosis. The antiatherosclerotic effects of curcumin were supported by experimental studies showing that this natural compound decreased foam cell formation and intracellular lipid accumulation, and favored cholesterol efflux in macrophages, through an activation of SIRT6 [157] and through activating AMPK-SIRT1-LXRα signaling [158]. Additionally, in a study using a mouse model of atherosclerosis induced by high-fat diet, long term administration of curcumin (80 weeks) protects against the decrease of SIRT1, and also against the increase in senescent cells and inflammation at vascular level [159].

Neuroprotective Actions
Curcumin is one of the natural compounds that has not only hormetic properties, but also, specifically, neuro-hormetic properties, improving in a dose-dependent manner the adaptive stress response through an increased expression of: antioxidant enzymes, sirtuins, FOXO transcription factors, chaperones, neurotrophic factors and anti-apoptotic proteins and, thus, suppressing the development of pathological processes in animal/cell models of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases [160]. Sirtuins, PGC-1α, oxidative stress, inflammation and mitochondrial dysfunction are potential targets and, through their modulation, curcumin could exert neuroprotective effects against the pathological pathways associated with neuronal lesions/neurodegeneration (Alzheimer's, Parkinson, multiple sclerosis, etc.) [161,162].
Curcumin, together with other natural compounds, may possess the ability to reduce the hallmarks of neuronal damage and slow down the characteristic cognitive decline and positively modulate the pathological pathways in Alzheimer's (amyloid deposits, neurofibrillary tangles, synaptic loss, inflammation, regulating autophagy and extensive oxidative stress) [4,163], some of these effects are being associated with increased SIRT expression [164]. The antioxidant and other beneficial effects associated with increased SIRT2 level were pointed out in the hippocampus of adult rats treated with curcumin [165].
Some other neuronal protective effects were also observed; for example, curcumin attenuated memory deficits in amyloid-induced neuronal metabolic dysfunction and improved cognition in transgenic mice through increasing SIRT3 activity [166], improved mitochondrial membrane potential and inhibited apoptotic cell death in Aβ25-35 treated neurons, activated the expression of SIRT1 and subsequently decreased the expression of Bax (apoptosis regulator) in the presence of Aβ25-35 [167]. Additionally, curcumin reduced glutamate excitotoxicity in cultured neurons, through an increased expression of SIRT1 and reduced the level of ac-PGC1α due to its deacetylation under the effect of SIRT1 [168]. Curcumin reduced the levels of inflammatory markers and induced SIRT1 and Bax expressions in a cerebral ischemia/reperfusion injury rat model, thus demonstrating clearly neuroprotective effects [169]. Bisdemethoxycurcumin, a curcumin derivative, manifested also neuroprotective effects in experimental models of Alzheimer's disease, the effects being associated with increased SIRT1 activity and modulation of GSH and SOD [170].
Curcumin's effects on sirtuins were also pointed out in clinical settings; in a randomized, double-blind clinical study including 72 patients with PCOS, results proved that administering 1500 mg curcumin, 3 times/day significantly reduced oxidative stress markers, increased gene expression for PGC1-α and also a non-significantly increased gene expression of SIRT1 and the activity of the SOD enzyme [171]. Similar results were obtained for animal models; female mice treated i.p. with curcumin showed increased expression for SIRT1 and 3, associated with an increase in number of follicles and oocyte maturation, fertilization and embryo development as well as decreased oxidative stress [172].

Anti-Cancer Potential
Related to the anti-cancer potential of curcumin, there are some studies proving that the natural compound induces apoptosis in cancer cells [156,173]; experimental studies show that SIRT1 is overexpressed in colon cancer cells, compared to normal cells; knockdown of SIRT1 led to the decreased viability and migration of cancer cells as well as a decrease in tumor volume and growth rate. Curcumin treatment reduced the expression of SIRT1 protein in colon cancer cells and facilitated the proteasomal degradation of oncogenic SIRT1 [174].

Other Effects
Curcumin activates the SIRT1 pathways in all cellular compartments; experimental and preclinical studies pointed out that SIRT1, as a cardioprotective factor, is inhibited by stress factors associated with increased risk of myocardial infarction; reduced SIRT1 activity is observed in inflammatory settings, as well as during the ageing process, both phenomena being associated with oxidative stress. Hyperglycemia and hypercholesterolemia inhibit SIRT1 and curcumin is able to restore its activity, thus acting as a protective agent against ischemia-reperfusion injury. On the other hand, SIRT1 contributes to an enhancement of mitochondrial processes and reduces oxidative stress and inflammation in cardiomyocytes; also, curcumin treatment of hyperglycemia/osmotic stress exposed cardiomyocytes exerted a protective effect, improving cell viability and also enhancing SIRT3 expression and activity [175,176].
The reported cellular effects can be correlated with results from animal studies, SIRT1 stimulating insulin secretion, while reducing glucagon secretion, both mechanisms leading to an increased cellular use of glucose and a reduction glycemia [22,24,88,[177][178][179][180]. Protective effects regarding mitochondrial metabolism were also reported by a study employing an acute liver injury model in mice, when curcumin reduced oxidative stress and apoptosis through modulation of hepatic SIRT1 activity [181]. Additionally, in a model of nonalcoholic fatty liver disease, curcumin reduced inflammation and steatosis, through an increase in SOD1 and SIRT1 expression [182]; another experiment showed that dietary curcumin reduced hepatic steatosis and improved mitochondrial function via SIRT3 activity, in a hepatic steatosis model in postnatal overfed rats [183]. Additionally, curcumin is one of the phytochemicals able to induce a calorie restriction-type metabolic state, through an induction of AMPK, yielding a tumor suppressing effect. These types of metabolic effects are determined by a SIRT1 induction, associated with the reduction of IL-6 and NF-κB, as well as an increased PPARα level [184].
Some of curcumin's effects on sirtuin signaling pathways are still under debate, since some of the experimental and preclinical results are not in complete agreement; for example, Yin et al. [185] pointed out that 30-50 µM curcumin did not alter SIRT2 expression in mouse bone marrow-derived macrophages treated with nigericin for NLRP3 inflammasome activation. So, even if other literature data demonstrate that curcumin is a histone deacetylase inhibitor [186], the above mentioned results suggest that inhibition of histone deacetylases did not significantly influence the NLRP3 inflammasome activation [185]. On the other hand, Wang X et al. [187] demonstrated that curcumin promotes cell survival and mitochondrial quality of bone marrow mesenchymal stem cells subjected to hypoxic preconditioning, through the up-regulation of PGC-1α and SIRT3, thus accelerating the wound healing process in mice. Moreover, curcumin was proven to support the metabolic activity and muscle force gain in a mouse model of cancer-induced cachexia; this effect being correlated with an increase in SIRT1 activity [188]; results were similar in a mouse model of disuse muscle atrophy, curcumin stimulating the number of muscle cells and SIRT1 activity [189], and also in a study proving that muscle performance was improved by curcumin through a stimulation of SIRT1 activity [190].
Experimental and preclinical results regarding the effects of curcumin on sirtuin activity, along with experimental models and doses used, are presented in Tables 1 and 2. Mouse model of disuse muscle atrophy 1 mg/kg/day i.p., 14 days ↑ SIRT1 activity ↑ number of muscle progenitor cells [189] Exercise performance and endurance in male Wistar rats 100 mg/kg, 6 weeks followed by intense effort ↑ PGC-1α, thioredoxin-1, SIRT1, Nrf2, ↑ SIRT1 expression ↑ muscle performance [190]

Conclusions
Recent research established the fact that sirtuins are keynote players in the ageing process, acting in order to delay cellular senescence and extending cellular longevity, while their activity is dysregulated in different pathologies associated with the ageing process. The discovery of more and more details regarding the structure and function of sirtuins, their involvement in the beneficial effects of calorie restriction, as well as their modulation through mitochondrial processes generating NAD + /NADH, unraveled the perspective that molecules influencing their action could be used to combat the decline of cellular pathways encountered along the timeline of every living organism. Some of the most abundant and easy to access sirtuin modulators are natural compounds, especially polyphenols, and in this class, curcumin seems to be an important molecule with relevant modulatory action on the sirtuin-mediated cellular processes, with a good perspective to become an important tool to actively protect against age-related diseases. There is still much to be learned about the functions of sirtuins and of the molecules that modulate their activity.
We conclude that curcumin can positively influence numerous physio-pathological processes, contributing to the prevention of cardiovascular, malignant and neurological damage, sirtuins being its key targets in these respects. Future research will be able to tell if curcumin can be used as clinical modulator of sirtuin activity. Furthermore, there is much to be unraveled regarding the potential of curcumin metabolites to modulate the expression and the activity of sirtuins, thus contributing to the overall effects induced in senescent/young cells by the parent compound. Pharmaceutical studies regarding the potential changes in bioavailability through modern drug delivery systems represent an emerging topic that should develop and merge with the ongoing biochemical research regarding curcumin's effects on cellular signaling pathways. Additionally, the assessment of the pharmacokinetic profile of curcumin and other natural compounds in healthy subjects, but also in the elderly, is critical for ascertaining if the observed effects of these molecules are determined by the parent compound, their metabolites or, to a certain degree, of both.
The in vivo effect of every sirtuin modulator found, including curcumin and its derivatives, the extent to which they can influence the activity of sirtuins, should be assessed through clinical trials. These types of studies are paramount in the search for molecules able to contribute to achieving healthy ageing and improving quality of life, an objective sought by clinicians and researchers alike over the last 20 years.

Conflicts of Interest:
The authors declare no conflict of interest.