Effects of Plant and Animal Natural Products on Mitophagy

Mitophagy is a protected cellular process that is essential for autophagic removal of damaged mitochondria and for preservation of a healthy mitochondrial population. In the last years, a particular interest has been devoted in studying the effects of natural compounds on mitophagy. Different natural compounds may modulate mitochondrial oxidative phosphorylation, the production of mitochondrial reactive oxygen species, the expression of mitophagy- and autophagy-related genes, and the activities of transcription factors which regulate the expression of mitochondrial proteins, thereby controlling mitochondrial damage and mitophagy. Remarkably, since mitochondrial function has a crucial role in the pathogenesis of various diseases (e.g., cancer, atherosclerosis, Duchenne muscular dystrophy, diabetes complications, Alzheimer's disease, and hepatic steatosis), these effects might have important therapeutic implications. In this review, preclinical studies investigating the role of different natural compounds in the modulation of mitophagy will be discussed.


Introduction
Mitochondria, double membrane-enclosed organelles of eukaryotic cells, have a crucial role in the regulation of cellular energy homeostasis and cell death [1]. Mitophagy, which was first described over one hundred years ago [2], is the mechanism by which impaired or superfluous mitochondria are engulfed in autophagosomes to be then degraded into lysosomes [3,4]. Mitophagy is critical for the maintenance of proper cellular functions. Removal of damaged mitochondria through mitophagy requires two steps: induction of general autophagy and priming of damaged mitochondria for selective autophagic recognition [5,6]. Several protein receptors, including autophagyrelated protein (Atg)32 in yeast, Nix/BCL2 interacting protein 3 like (Bnip3l), BCL2 interacting protein 3 (Bnip3), and FUN14 domain containing 1 (Fundc1) in mammalian systems, directly act in mitophagy. Atg32 interacts with Atg8 on the surface of mitochondria, promoting core Atg protein assembly for mitophagy. Nix/Bnip3l, Bnip3, and Fundc1 also have a classic motif to directly bind to microtubule-associated protein 1A/1B light chain 3 (LC3) (Atg8 homolog in mammals) for activation of mitophagy ( Figure 1) [3].
In the last decades, a particular interest has been devoted in studying the effects of different natural compounds of vegetable and animal origin, often found in dietary products at biologically active concentrations, on the modulation of pathophysiological processes underlying different diseases [7]. More recently, some of these natural compounds have attracted attention for their ability to modulate mitochondrial homeostasis. In this review, we will discuss recent findings from experimental studies concerning the effects of different natural compounds, including resveratrol, curcumin, E. uniflora, G. formosana, berberine, P. americana, P. suffruticosa, quercetin, quercetogetin, Shanxi aged vinegar (SAV), sulforaphene, tomatidine, and toxicarioside H, on mitophagy (Table 1).

Methods
We searched the literature available in ISI Web of Knowledge, Medline, PubMed, Scopus, and Google Scholar databases for English articles published until January 2019. For this purpose, we used appropriate keywords such as "mitophagy," "natural compound," "plants," "phytochemical," and "nutraceutical." Twenty-four studies were considered eligible for inclusion in this review (Table 1). Abstracts, unpublished articles, and non-English language articles were excluded.

Gastrointestinal Disease
The intestinal epithelium is one of the most rapidly selfrenewing tissues and needs a great amount of energy. In intestinal epithelial cells, a hyperstimulation of mitochondriamediated production of cellular energy may occur, leading to the overproduction of ROS. An excessive ROS production, in turn, may promote mitochondrial damage, disruption of the oxidation respiratory chain, and activation of cell death pathways. Therefore, mitophagy, which can mediate the clearance of damaged mitochondria before they cause activation of cell death, is crucial for the homeostasis of the intestinal epithelium [8].
Cao et al. investigated the effects of dietary administration of resveratrol (100 mg/kg) on mitochondrial dysfunction and mitophagy in intestinal mucosa epithelial cells of diquat-challenged piglets [8]. They showed that resveratrol reduced diquat-induced mitochondrial ultrastructure abnormalities (e.g., swelling and vacuolation) and ameliorated mitochondrial function of intestinal mucosa epithelial cells by inducing mitophagy. Accordingly, resveratrol increased phosphatase and tensin homo-log (PTEN), induced putative kinase 1 (PINK1) and Parkin expression levels, decreased the generation of reactive oxygen species (ROS), and increased the mitochondrial membrane potential, the content of mitochondrial DNA (mtDNA), and the activity of mitochondrial complexes I-IV.
In a study by Martins et al., treatment with resveratrol (1, 10, and 50 μM, for 24 h) increased activated hepatic stellate cell (i.e., GRX cell) death signals by altering mitochondrial dynamics and function. In fact, all resveratrol concentrations stimulated autophagosome formation by increasing the expression levels of autophagy-related proteins [14].
Wu et al. investigated the effects of resveratrol on mitochondrial ROS production and NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in high glucose containing peritoneal dialysis (PD) solution-treated human peritoneal mesothelial cells (HPMCs). In their study, treatment with resveratrol (0, 25, and 50 μmol/l, for 48 h) induced mitophagy through the adenosine monophosphate-activated protein kinase (AMPK) activation and protected HPMCs from oxidative stress and NLRP3-mediated inflammatory injury [15].
Eugenia uniflora (E. uniflora), also called pitanga or Brazilian cherry [16] or Cerisier Carré [17], is a plant of the Myrtaceae family, native of the east coast of tropical South America. Different pharmacological properties of this plant, such as gastroprotective [18], anti-inflammatory, antioxidant [19], antinociceptive [20], and antibacterial [21] effects, have been reported. The effect of ethanolic E. uniflora extract (5, 50, and 100 μg/ml, for 72 h) on autophagy was evaluated in GRX cells (a wellestablished line) by Denardin et al. [22]. In their study, E. uniflora extract increased the expression activated hepatic stellate cell levels of autophagy mediators such as Atg7. In addition, results of flow cytometry and ultrastructural analyses of treated cells indicated that the number of mature autophagosomes and autolysosomes significantly increased after treatment with E. uniflora extract [22]. Quercetin is a natural flavonoid found abundantly in apples, honey, raspberries, onions, red grapes, cherries, citrus fruits, and green leafy vegetables [23]. There is growing evidence suggesting that quercetin has therapeutic potential for the treatment of different diseases, including cardiovascular diseases [24], cancer [25], and neurodegenerative diseases [26]. The effects of quercetin on chronic ethanol-induced hepatic mitochondrial damage in mice were investigated by Yu et al. [27]. In their study, oral quercetin administration (100 mg/kg, for 15 weeks) reduced hepatocyte damage and mitochondrial morphological abnormalities (e.g., fractured endoplasmic reticulum, lipid droplets near the mitochondria, swelling, and restructuring of mitochondrial inner membranes) and dysfunction. In addition, quercetin inhibited ethanol-induced mitophagy suppression by increasing the expression levels of mitophagy mediators, such as LC3-II, p62, the mitochondrial outer membrane protein required for Parkin-dependent mitophagy voltage-dependent anionselective channel 1 (VDAC1), Parkin, and FoxO3a, and by decreasing the expression levels of ubiquitin-specific protease 30 (Usp30), an inhibitor of Parkin-mediated mitophagy. In addition, quercetin induced mitophagy through the upregulation of the AMPK and extracellular signal-regulated kinase 2 (ERK2) signaling pathways [27].
In a study by Liu et al., the effects of oral quercetin administration (100 mg/kg, for 10 weeks) on high-fat diet-(HFD-) induced hepatic steatosis in mice were evaluated. Treatment with quercetin reduced HFD-induced body weight gain and disorders of lipoprotein metabolism by reducing the expression of lipogenic genes, such as fatty acid synthase (FAS), and by increasing the expression of carnitine palmitoyltransferase I (CPT1), a key enzyme in fatty acid βoxidation. These quercetin-mediated beneficial effects against hepatic steatosis were in part dependent on a reduced mitochondrial damage and increased mitophagy activation. In fact, quercetin upregulated the expression of mitophagy mediators (e.g., Parkin, PINK1, Bnip3, Fundc1, LC3-II, p62, Beclin-1, and frataxin) and improved Parkin translocation to mitochondria. In addition, quercetin promoted mitophagy through the stimulation of frataxin-mediated activation of PINK1/Parkin-dependent mitophagy [28]. Shanxi aged vinegar is a traditional Chinese rice vinegar produced by spontaneous solid-state fermentation [29]. Several SAV-mediated pharmacological effects have been described such as antioxidant [30], hypotensive, hypoglycemic, and cholesterol-lowering effects [31]. The protective effects of SAV extract against bromobenzene-(BB-) induced hepatotoxicity were investigated by Yang et al. In their study, SAV extract inhibited hepatocyte ROS production and induced hepatocyte mitophagy by increasing the expression levels of autophagy mediators such as LC3-II, Beclin-1, and p62 through the activation of the protein phosphatase 2A-(PP2A-) Akt signaling pathway [32].

Musculoskeletal Disease
Duchenne muscular dystrophy (DMD) is a severe type of muscular dystrophy in humans characterized by progressive weakness of skeletal muscles and cardiomyopathy. DMD is caused by genetic mutations of the dystrophin gene. The absence of dystrophin causes intracellular Ca 2+ dysregulation, resulting in mitochondrial dysfunction and increased production of ROS [33].
The effects of resveratrol on cardiomyocyte mitochondrial damage and autophagy of damaged mitochondria in the mdx mouse (a Duchenne muscular dystrophy model) heart were evaluated by Kuno et al. [33]. In their study, oral administration of resveratrol (0, 5, 50, and 500 mg/kg, for 56 weeks) ameliorated the cardiomyopathy by reducing the amount of mtDNA deletion, ROS production by damaged mitochondria, and the number of mitochondria-containing autophagosomes. Such effects were mediated by resveratrolinduced accumulation of the nuclear transcription factor Forkhead box O3 (FoxO3a) and FoxO-induced expression of autophagy-related genes [33].
In a similar study, Sebori et al. investigated the protective effects of resveratrol in skeletal muscle cells of the mdx mice [34]. In their study, oral administration of resveratrol (0.04, 0.4, and 4 g/kg, for 56 weeks) reduced myofiber wasting and promoted skeletal muscle cells maturation, leading to a significant reduction of the number of immature myofibers and to an increased number of thicker myofibers in the quadriceps. Accordingly, resveratrol reduced the creatine kinase levels and improved the animals' physical performance in rotarod and four-limb hanging tests. An increased clearance of damaged mitochondria probably contributed to these beneficial effects as resveratrol significantly increased the expression levels of mitophagy-and autophagy-related genes such as PINK1, Parkin, sequestosome-1 (SQSTM1), Bnip3, Fundc1, Atg5, Beclin-1 (Becn1), microtubule-associated proteins 1A/1B light chain 3B (Map1lc3b), transcription factor EB (Tfeb), and lysosomal-associated membrane protein 1 (Lamp1) [34].

Cancer
Recent studies have shown that the expression of a number of proteins involved in the mitophagy processes, including MFN1, Parkin, BNIP3, and BNIP3L/NIX, is dysregulated in different cancer types [35]. However, the role of mitophagy in tumor development and progression remains largely unclear.
The anticancer effects of resveratrol (0.001-1000 μM, for 48 h) on HeLa cells were investigated by Rodríguez-Enríquez et al. [35]. In their study, biochemical mechanisms underlying resveratrol-induced inhibition of HeLa cell growth and promotion of HeLa cell death were an excessive cellular ROS production corresponding with a significant decrement in the superoxide dismutase (SOD) activity and glutathione (GSH) levels, a decreased oxidative phosphorylation, and a strong mitophagy activation [35].
Effects of curcumin (25 μM, for 24 h) on apoptosis of human hepatoma-derived Huh-7 cells were investigated by Moustapha et al. In these cells, curcumin promoted the formation of autophagic vacuoles containing degraded mitochondria and induced autophagy by increasing the expression levels of LC3-II [49].
In a study by Maiti et al., the effects of curcumin or solid lipid curcumin particles (SLCP) (25 μM for 24 h) on autophagic responses were evaluated in cultured U-87MG, GL261, F98, C6-glioma, and N2a cells. Treatment with curcumin or SLCP increased the expression levels of autophagy markers such as Atg5, Atg7, Beclin-1, LC3, and p62 and decreased the expression levels of mitophagy markers such as Fundc1, Bnip3, PINK1, and hypoxia-inducible factor 1-alpha (HIF-1α) by inhibiting the phosphoinositide 3-kinase (PI3K)/protein kinase b (Akt)/mammalian target of rapamycin (mTOR) pathway. In addition, cell survival markers were downregulated and cell death markers were upregulated by curcumin. All these effects were amplified in SLCP-treated cells in comparison to curcumin-treated cells [50].
Accordingly, the clearance of p62, a common autophagic substrate, and the conversion of LC3-I to LC3-II were enhanced in a time-and dose-dependent manner [54].
Paeonia suffruticosa (P. suffruticosa), which belongs to the Paeoniaceae family, has been widely used in traditional Chinese medicine for the treatment of various pathological conditions, including macula, epilepsy, and menstrual disorders [55]. Several pharmacological effects can be mediated by P. suffruticosa, including antispasmodic, antidiabetic [56], anti-inflammatory [57], antioxidant, anticancer [58], and antimelanogenic effects [59]. The effects of P. suffruticosa aqueous extracts on the survival, proliferation, and migration of pancreatic cancer (PC) cells (i.e., PANC1, AsPC1, and BxPC3) were evaluated by Liu et al. [60]. Treatment with P. suffruticosa (25-2500 μg/ml, for 24 h) decreased cell survival in a dose-dependent manner through an increased expression of LC3-II, a reduced expression of MFN2 and MFN1, and the promotion of mitophagy through an increased autophagosome and autolysosome formation. Additionally, P. suffruticosa inhibited cell cycle progression and cell migration through the downregulation of cyclin and cyclindependent kinase (CDK) and the stabilization of F-actin cytoskeleton [60].
Sulforaphene is a natural isothiocyanate extracted from Raphanus sativus, a medicinal herb used for over a thousand years in traditional Chinese medicine. Several sulforaphenemediated pharmacological effects have been described such as anti-inflammatory [61], antioxidant [62], antiasthmatic [63], and anticonvulsant effects [64]. In a study by Wang et al., treatment with sulforaphene (0-20 μM, for 72 h) induced apoptosis and cell cycle arrest in human lymphoma cell lines, such as U937, HUT78, Raji, JeKo-1, and U2932, by triggering simultaneous mitophagic cell death. Sulforaphene-induced mitophagy occurred through the chromosomal maintenance 1-(CRM1-) mediated p62 overexpression and AMPK activation. In fact, different autophagy-related genes such as SQSTM1, valosin-containing protein (VCP), and apoptosis regulator Bcl-2 (BCL2) were expressed after sulforaphene treatment and a timedependent elevation of different autophagy mediators was seen in the total cell lysate [65].
Toxicarioside H is a cardenolide extracted from the seeds of the tropical medicinal plant Antiaris toxicaria [66] and is commonly used in the treatment of congestive heart failure and arrhythmias [67]. Recently, toxicarioside H has been shown to block tumor cell proliferation and induce tumor cell apoptosis through the regulation of different signaling pathways [68]. In a study by Huang et al., treatment with toxicarioside H (0-0.8 μM, for 24 h) was reported to inhibit the proliferation of human lung cancer cell line (i.e., A549 and H460) by promoting mitochondrion-mediated apoptosis. However, in the same study, toxicarioside H was also reported to exert a cytoprotective effect by inducing mitophagy through the upregulation of SIRT3 and the increased interaction of Parkin with VDAC1 [69]. Therefore, toxicarioside H induced lung cancer cell damage and mitochondrionmediated apoptosis, but it simultaneously allowed lung cancer cells to counteract mitochondrion-mediated apo-ptosis. Accordingly, the inhibition of toxicarioside Hinduced mitophagy by siSIRT3 interference resulted in a more significant toxicarioside H-mediated proapoptotic effect.

Diabetes
Mitochondria not only play an important role in cellular respiration and ROS production but also have a crucial role in the glucose metabolism of some cells, including myocytes. In patients with diabetes, skeletal muscle dysfunction due to mitochondrial deficits may occur, leading to reduced muscle strength. Particularly, mitochondrial homeostasis and mitochondrial quality control are essential for the maintenance of muscle mass since they indirectly control myocyte insulin sensitivity [70].

Alzheimer's Disease
Alzheimer's disease is an irreversible, progressive brain disorder, which is characterized by the development of amyloid plaques and neurofibrillary tangles consisting of aggregated β-amyloid and tau, respectively. Recent studies indicated that β-amyloid could induce mitochondrial abnormalities via disrupting electron transfer chain, increasing ROS production, and impairing mitochondrial function [71].
The effect of resveratrol on mitophagy in amyloid betapeptide 1-42 -(Aβ 1-42 -) treated PC12 cells, an in vitro model of Alzheimer's disease, was evaluated by Wang et al. [71]. In their study, treatment with resveratrol (3 μM, for 24 h) reduced Aβ 1-42 -induced cell death and mitochondrial damage and significantly increased mitophagy, as shown by the increased number of acidic vesicular organelles, by the enhanced expression of LC3-II, Parkin, and Beclin-1, and by the LC3 and translocase of outer mitochondrial membrane 20 (TOMM20) colocalization [71].

Kidney Disease
Cisplatin is a chemotherapy drug that is used widely to treat different cancers including testicular, germ cell, head and neck, bladder, and lung cancer; however, acute kidney injury is an important side effect of this treatment. Different mechanisms have been identified in cisplatin-induced renal injury, such as oxidative stress, apoptosis, and mitochondrial damage of renal cells. Therefore, mitochondrial homeostasis is crucial for maintaining renal function and for counteracting kidney injury [72].
In a study by Ortega-Domínguez et al., the effect of oral administration of curcumin (200 mg/kg, for 3 days) on cisplatin-induced renal damage in rats was investigated. In this study, curcumin significantly reduced tubular epithelial cell damage and necrosis. Such a nephroprotective effect was dependent on curcumin-mediated reduction of cisplatin-induced alterations in mitochondrial dynamics and function. Accordingly, curcumin prevented the increase of FIS1 and the decrease of optic atrophy 1 protein (OPA1) and NAD+-dependent deacetylase sirtuin-3 (SIRT3), crucial regulators of mitochondrial bioenergetics.
In addition, curcumin decreased the expression levels of some mitophagy-associated proteins, such as PINK1 [72].

Cardiovascular Disease
Mitochondrial function is vital to those cells with a high energy expenditure like myocardiocytes. Previous studies showed that hyperglycemia may induce myocardium hypertrophy via meddling mitochondrial normal function [73].
The protective action of berberine on high glucoseinduced cardiomyocyte hypertrophy was evaluated by Hang et al. In their study, berberine (100 nM, for 30 min) reduced H9C2 cell hypertrophy. This effect was dependent on the improvement of mitochondrial function due to the restoration of balance between fusion and fission in mitochondrial  dynamics, the promotion of mitogenesis and mitophagy. The increased clearance of damaged mitochondria was mediated by the AMPK signaling pathway [73].
Periplaneta americana (P. americana), also called cockroach, is one of the largest and oldest insect groups worldwide. It has been employed as a traditional Chinese medicine for over 2,000 years, for its beneficial effects in activating blood circulation, dissipating blood stasis, promoting digestion, and inducing diuresis. Different P. americana extract-mediated pharmacological effects have been reported such as gastric protection [83], wound healing [84], antitumor activity [85], immunomodulation [86], and antifibrotic activity [87]. The effects of P. americana extract on lipopolysaccharide-(LPS-) induced cardiomyocyte injury was investigated by Li et al. [88]. In their study, P. americana extract significantly increased the viability of H9C2 cells. The expression levels of inflammatory mediators (e.g., interleukin-(IL-) 1β, IL-6, and tumor necrosis factor-(TNF-) α) were signifi-cantly reduced in P. americana extract-treated H9C2 cells. In addition, P. americana extract exerted cytoprotective effects through the regulation of mitophagy by the PINK1/-Parkin pathway. In fact, the release of LC3 and the expression of PINK1, Parkin, Bnip3l, and Beclin-1 were significantly decreased in P. americana extract-treated H9C2 cells [88].

Respiratory Disease
Chronic obstructive pulmonary disease (COPD), including emphysema and chronic bronchitis, refers to pathological conditions characterized by airway damage leading to progressive airflow blockage and breathing-related problems. The primary cause of COPD is exposure to cigarette smoke. Previous studies showed that cigarette smoke exposure caused mitochondrial dysfunction through the decrease of mitochondrial membrane potential and the increase of mitochondrial ROS production [89].  Figure 3: Specific molecular targets of natural compounds in the mitophagy pathway.
Quercetogetin is a polymethoxyflavone found in citrus peels [90]. Different quercetogetin-mediated pharmacological effects have been reported, such as anticarcinogenic, antiviral, anti-inflammatory, antioxidant, antithrombogenic, and antiatherogenic effects [91]. The effects of quercetogetin on cigarette smoke extract-induced apoptosis of lung epithelial cells were evaluated by Son et al. [89]. In their study, treatment of Beas-2B and NHBE human bronchial epithelial cells with quercetogetin (6.25, 12.5, 25, 50, and 100 μM, for 3-16 h) inhibited apoptosis by suppressing the expression of cleaved caspase-3, caspase-8, and caspase-9 and downregulating caspase activity. In addition, quercetogetin improved mitochondrial function in human bronchial epithelial cells by decreasing mitochondrial ROS production and decreased the expression of mitophagy regulatory proteins such as p-DRP1 and PINK1 [89].

Aging
The growth of the elderly population is considered a global phenomenon, and it is becoming an international challenge for healthcare systems in both developed and developing countries. Mitochondrial dysfunction contributes to aging and age-associated disease phenotypes. With aging, mitochondria undergo progressive changes in morphology, mutations in mtDNA, increase in oxidative stress, epigenetic changes in mitochondrial proteins, and defects in quality control, leading to the progressive accumulation of dysfunctional mitochondria [92].
Tomatidine is a steroidal alkaloid that has been found in the skins and leaves of tomatoes [93]. Different therapeutic effects have been described for tomatidine such as antiasthmatic [94], anti-inflammatory [95], antimicrobial [96,97], and anticancer [98] effects. In a study by Fang et al., the effects of tomatidine (0, 25, and 50 μM) on lifespan and healthspan of N2 (wild type) Caenorhabditis elegans (C. elegans) were examined. In this study, tomatidine extended lifespan and improved many C. elegans behaviors related to healthspan, including pharyngeal pumping and swimming movement. These beneficial tomatidine-mediated effects were due to a reduced percentage of severely damaged muscle cells. In fact, tomatidine was reported to reduce muscle cell stress by maintaining mitochondrial homeostasis and inducing mitophagy through the activation of the skinhead-1 protein (SKN-1)/nuclear factor erythroid 2-(NFE2-) related factor 2 (Nrf2) pathway [92].

Conclusion
In this review, the effects of different natural products on various aspects of mitochondrial biology, such as mitochondrial biogenesis, membrane potential regulation, ROS production, and mitophagy, were discussed. Overall, the regulatory effects of natural products on mitophagy are exerted through multiple mechanisms ( Figure 2) and through multiple molecular targets (Figure 3), which suggest the potential application of these agents as therapeutic agents in several pathological conditions associated with impaired mitophagy. Further experimental studies are required to demonstrate the exact mechanisms of action of these natural compounds and to elucidate their potential clinical applications more clearly.

Conflicts of Interest
The authors declare that there are no conflicts of interest.