A potential link between senescence and hepatic lipid metabolism has been hypothesized since impaired lipid metabolism is a driving force in NAFLD development and progression and since impaired lipid metabolism is also linked to OxS. Furthermore, steatosis may contribute to DNA damage via OxS and the activation of signaling pathways that lead to cellular senescence
[48]. Evidence from in vitro and in vivo findings has shown that the senescent phenotype in hepatocytes correlates with increased fat accumulation
[49][50][51]. Likewise, experimental evidence suggests that impaired lipid metabolism can drive cellular senescence, most likely via OxS-dependent mechanisms
[52]. Senescent cells also have an altered metabolism during NAFLD development, and because of mitochondrial dysfunction they are less able to metabolize fatty acids, which may contribute to fat accumulation and OxS in the liver
[51][53]. Senescent cells show changes in lipid metabolism, but the importance of these changes to cellular senescence is still unclear. Therefore, bidirectional interaction between cellular senescence and lipid metabolism is plausible; however, its causality needs to be further explored.
Transcriptomic and lipidomic analysis has revealed differential expression patterns of lipid-related genes and different lipid compositions in senescent cells, suggesting an important role of lipid metabolism in senescence. Specific polyunsaturated triacylglycerols species accumulate during the development of cellular senescence, probably via increased CD36-mediated fatty acid uptake
[54]. Interestingly, a significant increase in CD36 expression was able to induce a senescent-like phenotype, with a concomitant accumulation of phosphatidylcholine. This could be linked to membrane remodeling during the induction of senescence
[55]. These findings correlate with the observation that senescent cells accumulate lipid droplets and show increased expression of several lipid regulatory proteins. In addition, in vitro treatment of proliferating cells with specific lipids, such as cholesterol, ceramides or triglycerides, significantly increased cellular senescence
[56]. This evidence suggests that the dysregulation of lipid metabolism can induce cellular senescence. Moreover, increased accumulation of lipid droplets in senescent cells during OxS might be a cellular mechanism of protection against lipotoxicity, but this point remains still unclear.
Lipid molecules and their metabolites can modulate different cellular responses. Lipids have several biological functions: structural, as part of cellular membranes, in the storage of fatty acids, as neutral lipids in lipid droplets and some lipids, e.g., diacylglycerol, sphingolipids and ceramides, can act as signaling molecules
[59]. Sphingolipids and related metabolites such as ceramides, have been proposed as key factors in NAFLD progression because they strongly influence lipid metabolism and lipotoxicity in the liver
[60]. In addition, ceramides have been shown to induce cellular senescence in vitro and it has been proposed that ceramides promote senescence during the development of NAFLD
[61][62]. The modulation of signaling pathways related to cellular senescence can influence lipid accumulation
[12]. However, this modulation is reciprocal, as lipid species are also modulators of cellular senescence and can either be protective or contribute to the progression of NAFLD via ER stress and mitochondrial dysfunction
[63].
4. Modulating OxS-Induced Senescence as a Potential Therapy in NAFLD
4.1. Targeting Oxidative Stress to Modulate Cellular Senescence
OxS might be an interesting therapeutic target to modulate senescence. Results from various experimental models have demonstrated that the inhibition of OxS can effectively inhibit cellular senescence
[65][66][67][68]. The same has been shown in the aging liver: a number of in vivo studies have demonstrated the attenuation of hepatic senescence by preventing ROS production or by enhancing the antioxidant response
[69][70][71]. Nrf2 activation might also attenuate cellular senescence
[72], and Nrf2 activators are able to antagonize cellular senescence in human mesenchymal cells
[73][74]. In fact, it has been found that activation of Nrf2 with specific compounds suppress cellular senescence in mouse livers, with inhibition of the SASP and DDR pathway and with concomitant reduction of ROS production via upregulation of antioxidant enzymes. In the study, the crucial role of ROS in the induction and maintenance of senescence in the liver as well as the potential as a therapeutic target were convincingly demonstrated
[75]. Likewise, the induction of Nrf2 activation in rodents inhibits senescence and the SASP and protects against oxidative damage in different tissues. Interestingly, in the study Nrf2 activation was found to be AMPK-dependent
[68].
Findings in a hamster model of vitamin D deficiency demonstrated that vitamin D has anti-senescence effects, which are mediated by Nrf2 activation and subsequent reduction of ROS production
[66]. Other experimental results from animal models of NAFLD induced by HFD, demonstrated that vitamin D deficiency was associated with increased cellular senescence and vitamin D supplementation normalized biochemical parameters. Vitamin D supplementation correlated with increased level of SMP-30, a senescence biomarker that is usually downregulated in NAFLD
[76]. These results imply that the beneficial antioxidant effects of vitamin D are associated with its potential as a senescence modulator and might be useful in the treatment of NAFLD.
NAD
+ metabolism, AMPK and SIRT proteins have been identified as potential molecular targets of the beneficial effects of OxS reduction on cellular senescence
[77]. Restoring cellular NAD
+ levels protects against senescence and eliminating senescent cells or antagonizing the SASP improves NAD
+ homeostasis
[78]. Similar findings have been demonstrated with paricalcitol, an agonist of vitamin D, which is able to reduce OxS-induced senescence in bile duct ligated mice. A decreased number of SA-β-gal-positive cells and reduced expression of senescence markers such as p53, p21 and p16 were also observed. The effects of paricalcitol on cellular senescence were due to the inhibition of OxS. Paricalcitol effectively prevented downregulation of SIRT1 expression in bile duct ligated mice and in biliary epithelial cells treated with t-BHP (tert-butyl hydroperoxide), confirming the protective role of the SIRT1 pathway against ROS-induced damage
[79]. The protective role of SIRT1 against OxS has also been demonstrated in relation to the beneficial effects of resveratrol
[80][81], a well-known antioxidant compound. Resveratrol improved mitochondrial function and biogenesis, increased NAD
+ levels and induced AMPK activation in a SIRT1-dependent manner, demonstrating that SIRT1 plays an important role in AMPK activation
[80]. Several studies reported that resveratrol protects against OxS and prevents hepatic steatosis in experimental models
[82][83], and its antioxidant effects are linked to therapeutic targets such as SIRTs, AMPK and Nrf2, which might be also be involved in the regulation of senescence
[84][85].
SIRTs have been proposed as a molecular target for the treatment of NAFLD due to their regulatory role in hepatic metabolism, inflammation and cellular senescence
[86]. Studies in SIRT1 knockout mice have confirmed that SIRT1 limits hepatic lipid accumulation and hepatic oxidative stress, which especially prevents mitochondrial ROS production. Thus, SIRT1 seems to play a protective role during the progression of NAFLD
[87][88]. Naringenin, a citrus flavonoid, has been found to decrease ROS production and improve the liver antioxidant response via the activation of SIRT1, and to attenuate NAFLD progression in a mouse model
[89]. Similar findings have been reported with the alkaloid Berberine. This compound was found to reduce lipid accumulation and steatosis via activation of SIRT3 in a dietary mouse model of NAFLD
[90]. Various other antioxidants have been reported to reduce OxS via SIRTs in NAFLD models, including resveratrol, dietary polyphenols and melatonin
[91][92][93][94][95].
AMPK activation attenuates hepatic OxS and steatosis in NAFLD models
[96][97][98]. AMPK activation also modulates cellular senescence
[99]. For instance, AMPK activation via dietary modulation or exercise ameliorates NAFLD and decreases hepatic senescence markers in mice models of NAFLD. The beneficial effects of AMPK activation have (partially) been attributed to increased autophagy and lipophagy
[100][101]. Recent in vitro and in vivo results have shown that AMPK activators, e.g., licochalcone D, a compound found in a Chinese licorice herb, reduced OxS-induced senescence by triggering AMPK-mediated autophagy
[97]. This demonstrates that AMPK is an interesting signaling hub connecting OxS-induced senescence and NAFLD pathophysiology, implying that AMPK activators with demonstrated inhibitory effects on cellular senescence are potential targets to treat NAFLD
[102]. In this regard, it is interesting to note that metformin is known as an AMPK activator with hepatoprotective effects. Metformin has been proposed as a useful therapy in NAFLD and has also been reported as a potential senostatic, as discussed below. Previous results from researchers' group have demonstrated that metformin protects against oxidative stress
[103], palmitate-induced lipotoxicity
[104] and diclofenac-induced liver toxicity in primary rat hepatocytes
[105]. Most of these effects are related to the inhibition of mitochondrial ROS production and the preservation of mitochondrial function. Interestingly, metformin has been reported to inhibit cellular senescence
[106]. Moreover, results in human adipose stromal cells demonstrated that metformin attenuated oxidative-stress-induced senescence and improved lipid metabolism and adipocyte function. These effects appeared to be mediated by AMPK activation
[107]. Further studies should be conducted to confirm whether the observed hepatoprotective effects of metformin are related to cellular senescence.
4.2. SASP Inhibition: Senostatic/Senomorphic Drugs
Senostatics suppress (part of) the senescence phenotype in cells without causing cell death. Ideally, these drugs should suppress the characteristics of senescent cells that contribute to paracrine tissue damage, i.e., the SASP and ROS production. According to their mechanisms of action, senostatics are classified into two groups: generalized senostatics and precision senostatics. The first group modulates the SASP, while the latter inhibits a specific component of the secretome
[108]. The limitations related to these drugs are due to the existence of multiple SASP targets, some of which have essential functions besides senescence, which prohibits using them as targets. Moreover, the high variability of the SASP between tissues and during disease development might make it difficult to implement this type of therapy
[109][110].
Different senostatic drugs have been proposed for therapeutic application. For instance, rapamycin, a drug targeting mTOR signaling, can reduce the rate of aging. It is known that the mTOR pathway is related to the detrimental effects of aging and its inhibition could improve age-related diseases, lifespan and health span
[111]. The anti-aging effect of rapamycin was reported in a study using old mice (aged 25 months) with a 21-month dietary treatment with rapamycin. Transcriptome and pathway analysis showed that the pathway related to mitochondrial function was the most significantly altered one, among a total of 13 significantly altered pathways in the rapamycin-treated group
[112]. Likewise, rapamycin has been shown to reduce mitochondrial ROS production
[113], and to improve liver function and decrease fibrosis in a rat model of cirrhosis
[114]. Similar beneficial findings were reported in mice treated with rapamycin (14 mg/kg diet for 7 weeks) and middle-aged mice: rapamycin attenuated severe age-induced damage to mitochondria, including ROS production, accumulation of mitochondrial DNA fragments, mitochondrial lipoperoxidation and lipofuscin accumulation
[115]. In this regard, the modulation of autophagy using a mTOR inhibitor has been proposed as an effective way to modulate cellular senescence
[116], and suggests that this is an interesting novel therapeutic target in NAFLD, that might connect OxS and senescence mechanisms.
Other drugs have been shown to have senostatic effects on the liver, most likely via inhibition of the SASP. It has been demonstrated that metformin inhibits the expression of several pro-inflammatory cytokines that are part of the SASP. It also prevents the translocation of NF-κB to the nucleus and inhibits the phosphorylation of IκB and IKKα/β, thus inhibiting the NF-κB pathway
[117][118]. In the context of NAFLD, a number of studies have demonstrated that metformin reverses steatosis in murine models with NAFLD
[119], NASH
[120] and in patients with NAFLD
[121]. Glucocorticoids have also been proposed as potential senostatics, due to their inhibitory effects on the SASP, although their effect in chronic liver disease has not been well established
[122]. Finally, several other molecules have been proposed as senostatics, such as JAK inhibitors, JNK inhibitors, HDAC inhibitors and small molecule MDM2 antagonists. These molecules modulate (part of) the SASP, enhance mitochondrial activity and reduce cytoplasmic chromatin fragments in senescent cells
[123][124][125].