5.1. Therapeutic agents targeting the SIRT3-FOXO3-PRKN pathway
As mentioned above (Ma et al., 2018; Reiter, Ma & Sharma, 2020; Reiter, Tan, Rosales-Corral, Galano, Jou & Acuna-Castroviejo, 2018), melatonin activates the SIRT3-FOXO3-PRKN pathway to trigger mitophagy and block inflammation. In this regard, applying pharmaceutical or natural therapeutic agents to modulate SIRT3 and FOXO3 may aid melatonin to reinstate mitophagy in macrophages.
A natural biphenolic compound, honokiol, was reported to exert anti-oxidative and anti-inflammatory properties and reverse cardiac hypertrophy due to its capacity to mediate SIRT3 upregulation bothin vivo and in vitro (Pillai et al., 2015). Honokiol also binds with SIRT3 in mitochondria to enhance SIRT3 activity (Pillai et al., 2015).
Furthermore, resveratrol is a natural phenol abundant in peanuts and grape skins, which significantly attenuate mitochondrial ROS by enhancing SIRT3 levels in the mitochondria and evoke FOXO3 upregulation in human vascular endothelial cells (Zhou et al., 2014). In addition, resveratrol activates the SIRT1-FOXO3 pathway, upregulates PINK1, BNIP3, RAB7A (RAB7A, member RAS oncogene family), and BECN1, and therefore, resulting mitophagy induction (Kuno et al., 2018; Ren & Zhang, 2018). Resveratrol is also capable of inhibiting AKT1 (AKT serine/threonine kinase 1), resulting in FOXO3 activation and consequently activation of antioxidant enzymes (Franco et al., 2014).
Troxerutin is also a natural flavonol extracted from flavonoid rutin, and its administration remarkably upregulates SIRT3 and SIRT1, leading to suppression of oxidative stress, apoptosis, and acute neuroinflammation in Wistar rats following lipopolysaccharide challenge (Jamali-Raeufy, Kardgar, Baluchnejadmojarad, Roghani & Goudarzi, 2019).
Metformin is perhaps one of the most widely used medication clinically that can enhance FOXO3 activation through AMPK activation and may enhance mitophagy due to the activation of the AMPK-FOXO3 axis (Sato et al., 2012). Auranofin is an approved therapeutic agent with diverse biological affects, one of which is the activation of FOXO3 and promotion of its nuclear localization (Park, Lee, Berek & Hu, 2014).
6,8-diprenylorobol, extracted from Glycyrrhiza uralensis  Fisch roots, is a phytochemical compound with anti-cancer properties, which are attributed to FOXO3 upregulation (Lee et al., 2020).
Taken together, a combinational therapeutic system comprising melatonin and SIRT3 or FOXO3 modulators might be a potential package for provoking mitophagy in macrophages. However, some of these modulators have not yet been clinically approved and much effort should be engaged for their optimization to meet clinical expectations.
Besides, a growing trend shows that miRNAs play a crucial role in the regulation of SIRT3 and FOXO3, and thus mitophagy induction. For instance, Mir214 blocks SIRT3 expression as its target molecule, and its knockdown restores SIRT3 expression, as well as mitochondrial activity and morphology, in a murine model of angiotensin II-induced cardiomyopathy (Ding et al., 2020). Furthermore, MIR708-5ptargets and blocks SIRT3 expression in cancer cells (Huang, Guo, Cao & Xiong, 2019), suggesting that MIR708-5p knockdown might induce SIRT3 upregulation. Similarly, MIR494 suppresses SIRT3 expression in hepatoma cell lines and its inhibition might induce SIRT3 upregulation (Zhang, Zhu, Hu, Yan & Chen, 2019).
In the case of FOXO3, Mir182 transfection into rat muscle cells targets Foxo3 mRNA and suppresses its expression (Hudson, Rahnert, Zheng, Woodworth-Hobbs, Franch & Russ Price, 2014). Further,MIR96 binds to the seed region in FOXO3 mRNA, and remarkably reduces its expression, whereas MIR96 downregulation induces FOXO3 upregulation (Li et al., 2015). Moreover, MIR629negatively regulates FOXO3 at the posttranscriptional stage and suppresses its expression (Yan et al., 2017), indicating that inhibitory targeting of MIR629 may reverse its effect on FOXO3 expression. Overall, miRNAs regulate the expression of FOXO3 and SIRT3, and, thereby, their modulation could be a part of melatonin-based combinational therapies.
Apart from microRNAs, two major lifestyle medication factors, exercise and diet, mediate SIRT3 and FOXO3 upregulation. Exercise training, caloric restriction, and fasting upregulate SIRT3, whereas a high-fat intake downregulates SIRT3 in skeletal muscles (Palacios et al., 2009). Mechanistically, caloric restriction activates SIRT3, which in turn deacetylates lysine residues on SOD2 (superoxide dismutase 2; an antioxidant enzyme residing in mitochondria), leading to removal of ROS (Qiu, Brown, Hirschey, Verdin & Chen, 2010). In addition, caloric restriction activates mammalian SIRT2, which in turn deacetylates FOXO3 and enhances its function (Wang, Nguyen, Qin & Tong, 2007). Calorie restriction in aged rats also preserves the melatonin rhythm which probably helps to maintains SIRT3 activity (Stokkan, Reiter, Nonaka, Lerchl, Yu & Vaughan, 1991). Thus, caloric restriction can also boost mitophagy through upregulation of FOXO3 and SIRT3.