Antioxidants, Vol. 11, Pages 2396: Mangosteen Pericarp Extract Supplementation Boosts Antioxidant Status via Rebuilding Gut Microbiota to Attenuate Motor Deficit in 6-OHDA-Induced Parkinson’s Disease

1. IntroductionParkinson’s disease (PD) is a progressive neurodegenerative disease that affected over 8 million individuals globally in 2019 [1]. The development of PD damages dopaminergic neurons in the basal ganglia circuit, particularly in the substantia nigra pars compacta (SNc), which consequently results in deprivation of the neurotransmitter dopamine and affects normal motor function, leading to motor function impairments, for instance bradykinesia, rigidity, tremors, and gait dysfunction [2]. In addition to motor dysfunctions, various non-motor dysfunctions (e.g., gut dysbiosis, constipation, sleep disorders, depression, and dysphagia) have been reported to follow the loss of dopaminergic neurons in PD [2,3]. The exact pathogenesis of PD remains obscure. However, several lines of evidence suggest that the progression of oxidative stress [4,5], inflammation [6,7], and mitochondrial dysfunction [8,9,10] in the brain—along with gut microbiota dysbiosis [3,11]—may contribute to the development of PD.The correlation between the progression PD and oxidative stress has been discussed extensively. Oxidative stress occurs when there is an imbalance between the levels of free radicals, such as reactive oxygen species (ROS), and antioxidants. Mitochondrial oxidative phosphorylation is one of the major contributors to the generation of ROS products. ROS products such as the superoxide anion and hydrogen peroxide are generated by the electron transport chain processes in cellular energy production involving oxidative phosphorylation [12]. The generation of these ROS products is exacerbated in PD [13,14] due to depletion of mitochondrial proteins (i.e., complexes I, II, and IV) [8,9,10] and the presence of mitochondrial dysfunction [15]. Antioxidants such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) are innately produced to neutralize the ROS. However, in the pathological state of PD, the host fails to produce sufficient levels of antioxidants to counterbalance ROS [16,17]. As a result, accumulation of ROS further damages mitochondria and aggravates dopaminergic neuron loss [12,18]. Moreover, oxidative stress can induce proinflammatory responses in the brain by activating microglia, which promote neuronal degeneration [7]. Additionally, impaired mitochondrial function has not only been reported in the brain of patients with PD, but also in the peripheral tissues, including the skeletal muscles [19,20].In the past few years, gut dysbiosis has been suggested as a factor involved in the development of PD [3]. Increased abundances of pathogenic bacteria and decreased abundances of commensal bacteria were documented in several clinical studies of patients with PD [11,21,22,23,24,25]. An imbalance in the gut microbiota composition towards proinflammatory bacteria leads to increased inflammatory responses within the host [26]. The antioxidant capacity within the host may also be reduced by decreases in the abundances of short-chain fatty acid-producing bacteria [27,28]. Additionally, the bidirectional axis between the gut and the brain may allow pathogenic bacteria in the gut to promote neuronal degeneration in the brain in PD [29]. Thus, it is imperative to explore PD-related gut dysbiosis since the gastrointestinal disorders in patients with PD occur years before the development of motor deficits [30]. Moreover, this evidence further suggests that the progression of PD is induced by various factors and that the related molecular impairments are not limited to the brain. Thus, targeting the PD-related pathologies that occur in several organs may offer better neuroprotective effects and may even avert the progression of this disease.Mangosteen (Garcinia mangostana Linn.) is a fruit rich in polyphenols [31]. Its pericarp contains various bioactive compounds including xanthones (e.g., α-mangostin, β-mangostin, and γ-mangostin), anthocyanins, and proanthocyanidins [32,33]. Furthermore, xanthones, benzophenones, and anthocyanins within mangosteen pericarp (MP) extract have been reported to possess antioxidant [34], anti-inflammatory [35], antiobesity, antiulcer, and anticancer properties [32] and exert antimicrobial action against pathogenic bacteria [36]. One study [34] reported that the neuroprotective effects of α-mangostin, a phytochemical found in MP, improved motor function and memory function in rats with rotenone-induced PD by decreasing oxidative stress (i.e., reduced nitrite and malondialdehyde (MDA) and increased glutathione (GSH)). In addition, α-mangostin can reduce neuroinflammation [35] and protect neuronal cells from hydrogen peroxide-induced injury [37].Polyphenols are abundant in MP and can affect the health of the host through modulating the gut microbiota. Polyphenols and the gut microbiota can exert synergistic health benefits to the host as polyphenols can modify the gut microbiota composition and metabolite by selectively affecting bacterial cell membranes and the microbiota can improve the bioavailability of polyphenols by transforming them into bioavailable metabolites [38]. Moreover, MP extract has been reported to inhibit the growth of a pathogenic bacterial species [39], suggesting MP has the capacity to alter the gut microbiota.

Current therapies for PD, such as L-DOPA drugs, only alleviate the motor symptoms and do not target the pathological mechanisms, such as oxidative stress and gut dysbiosis. Therefore, novel therapies that target disease-specific pathologies urgently need to be identified. The bioactive compounds in MP exhibited antioxidant and anti-inflammatory activities and altered the gut microbiota composition in various studies, indicating MP has potential as an auxiliary therapy for the treatment of PD. Therefore, in this study, we investigated the therapeutic effects of an MP extract on striatal and SNc dopamine transporter (DAT) binding activity and motor function in a rat model of 6-OHDA-induced PD-like motor deficits. Furthermore, we explored the potential effects of MP supplementation on regulatory pathways in a PD-like condition, particularly antioxidant defense mechanisms and the function of mitochondria in both the brain and muscles, along with inflammatory markers in blood plasma and microbiota profiles in fecal samples. To the best of our knowledge, this is the first study to link the effects of MP on antioxidant defense mechanisms in the brain and muscles with fecal microbiota profiles in a PD-like condition. Moreover, this work provides new insight on the potential of improving the antioxidant capacities and mitochondrial function of muscles to delay the progressive motor deficits in rats with 6-OHDA-induced PD.

4. Discussion

Oxidative stress and gut dysbiosis have been suggested to play critical roles in the deterioration of dopaminergic neurons and motor function in PD. Most of the available therapies, such as L-DOPA drugs, only address the motor symptoms caused by the lack of dopamine. Therefore, new therapies that not only alleviate the motor symptoms, but also attenuate the pathogenic mechanisms, such as oxidative stress and gut dysbiosis, are urgently needed. This study shows that MP extract directly acts as an antioxidant to counterbalance excessive production of free radicals and may also indirectly increase antioxidant capacity by rebalancing the gut microbiota, and the resulting enhanced anti-inflammatory capacity and restoration of mitochondrial function attenuate the motor deficits and slow down the progression of PD.

Supplementation with polyphenol-rich MP extract for 8 weeks at both a low dose (LMP) or a high dose (HMP) attenuated the progression of PD in our 6-OHDA-induced PD-like rat model, as indicated by significant improvements in motor function (Figure 2A,B) and DAT binding activity in the SNc (Figure 2F). These effects may be mainly derived from a cascade of cellular mechanisms involving the antioxidant defense system and gut microbiota. The antioxidant compounds in MP might act directly on the cellular antioxidant defense system to reduce oxidative stress in the brain and increase the endogenous expression of antioxidant genes in the brain, including Sod1, Sod2, Cat, Gpx, and Nrf2. As a result, MP reduced the levels of ROS in the brain (Figure 3A,B). Moreover, MP supplementation also attenuated 6-OHDA-induced decrease in total SOD in the serum (Figure 4) and suppressed production of inflammatory cytokines (i.e., IL-1β, IL-6, TNF-α) in plasma (Table 3), which implies MP may have similar effects in the brain. Furthermore, the MP extract elevated the antioxidant capacity (Figure 3B) and prevented the 6-OHDA-induced impairments to mitochondrial biogenesis in muscle, which consequently improved muscle mitochondrial function and energy metabolism (Figure 5) and may further prevent motor dysfunction.Moreover, MP extract may also indirectly improve the cellular antioxidant defense system through fecal microbiota alterations (Figure 6, Figure 7, Figure 8 and Figure 9). Some of the alterations observed in the abundance of fecal bacteria positively correlated with enhanced antioxidant capacity in the brain and muscles. The correlation between the fecal microbiota and antioxidant capacities in the brain and muscles indicate a potentially important role for the gut microbiota profile in the progression of PD. Additionally, the abundance of several bacterial genera that positively correlated with the levels of pro-inflammatory cytokines were suppressed by MP. This strongly suggests that the mechanisms of action MP extract might be initiated by reshaping the gut microbiota profile, which in turn regulates antioxidative and inflammatory responses at the systemic level and, as a consequence, MP affected the antioxidant capacity and energy metabolism in the brain and muscles. Ultimately, the MP extract protected active dopaminergic neurons and attenuated motor deficits. The proposed mechanisms of action by which MP extract delayed the motor deficits in 6-OHDA-induced PD are presented in Figure 10.Oxidative stress has long been associated with the advancement of neurodegeneration in PD. Aging, neurotoxins, and neuroinflammation are known to induce oxidative stress during the progression of PD [49,50]. Elevated oxidative stress has been reported to result in mitochondrial damage in patients with PD [8,10,51] and the subsequent impairments to mitochondrial function lead to excessive ROS production [12]. Under normal conditions, ROS can be neutralized by endogenous antioxidants (e.g., SOD, catalase, and GPx). However, these endogenous antioxidants are depleted in PD [52,53]. Specifically, several lines of evidence indicate SOD1 expression and activity is reduced in both in vitro and clinical studies of PD [54,55,56,57], and copper deficiency has been suggested as one possible explanations for these changes [58]. Herein, we revealed that supplementation of MP extract elevated the endogenous expression levels of antioxidant-related genes (Sod1, Sod2, Gpx, and Cat; Figure 3) and reduced the levels of ROS in the brain (Figure 3A), and also preserved dopaminergic neurons in the SNc (Figure 2F). Importantly, supplementation with the higher dose of MP (HMP) stimulated the expression of Nrf2, a key regulator of antioxidant and redox status.Polyphenol-rich MP extract has been reported to possess antioxidant [34] and anti-inflammatory properties [35] as it contains various bioactive compounds, including xanthones (e.g., α-mangostin, β-mangostin, and γ-mangostin), anthocyanins, and proanthocyanidins [32,33]. The xanthones contained in MP were demonstrated to exert potent antioxidant capacity to counterbalance the excessive ROS production during the PD-like pathogenesis [34,59,60,61]. MP was also shown to enhance protein expression of NRF2 [62], which upregulates secretion of endogenous antioxidant enzymes including SOD, CAT, and GPx [63,64]. Furthermore, the reduction in oxidative stress conferred by the antioxidant activity of MP was shown to inhibit mitochondrial-dependent apoptosis of dopaminergic neurons [59,60]. The antioxidant properties of α-mangostin were found to alleviate neuroinflammation [35,65], which may prevent dopaminergic neuronal loss in PD. In addition, the xanthones contained in MP were reported to suppress monoamine oxidase (MAO) activity [66,67,68], which produces ROS by oxidation of neurotransmitters such as dopamine [69,70].The oxidative stress observed in PD is commonly followed by inflammation. Studies have reported increased numbers of activated microglia cells and astrocytes, along with higher levels of proinflammatory cytokines, in the brain of patients with PD [71]. Importantly, some studies suggest that the inflammation is not only present in the brain, but also in the peripheral blood [71,72]. In this study, we showed that LMP and HMP reduced the levels of pro-inflammatory cytokines (i.e., IL-1β, IL-6, and TNF-α) in plasma; these reductions were associated with increased total SOD activity in serum. This suggests that antioxidant activity of MP enhanced the endogenous antioxidant activity, which in turn reduced the levels of pro-inflammatory cytokines in the blood. These results also indirectly suggest that the antioxidant activity of MP might potentially reduce neuroinflammation in the brain.Prolonged exposure to free radicals can also exacerbate the mitochondrial dysfunction in PD. Mitochondrial dysfunction has been documented in the SNc region of the brain in patients with PD [2,15] and is associated with depletion of mitochondrial proteins (i.e., complexes I, II, and IV) [8,9,10]. The depletion of these protein complexes impairs the electron transport reaction during oxidative phosphorylation, which consequently leads to decreased energy production and excessive ROS production [51] and creates a vicious cycle that continually damages mitochondrial function [18]. In this study, we observed that supplementation with HMP for 8 weeks increase the expression of antioxidant-related genes in the brain and slightly increased the oxygen consumption rate (OCR) in the brain, indicating HMP may potentially improve mitochondrial function (Figure S2). Our results are in line with Hao et al. [61], who reported α-mangostin treatment improved mitochondrial function, increased ATP production, and also prevented dopaminergic neuronal death in rotenone-induced neuroblastoma cells.Previous studies reported low mitochondrial respiratory function in the skeletal muscle of patients with PD, which might be associated with motor impairment [20,73,74,75]. Clinical studies also observed a loss of efficiency in oxidative metabolism in the muscles of patients with PD [76,77]. Animal studies of PD-like conditions have also reported mitochondrial dysfunction in the skeletal muscles of rats treated with 6-OHDA [44] and Parkin-knockout mice [78]. Similarly, we observed unilateral 6-OHDA injection induced mitochondrial dysfunction in the skeletal muscles. However, HMP supplementation for 8 weeks restored the muscle mitochondrial function. The antioxidant effects of HMP supplementation were associated with higher expression of Sod1 and the antioxidant transcriptional regulator Nrf2 in muscles (Figure 3C) and higher total SOD activity in serum (Figure 4), and these changes may further reduce oxidative stress in muscles. The reduction in oxidative stress may lead to upregulation of mtDNA biogenesis (Pgc1) and transcription (Tfam), and result in the increased mtDNA copy numbers [complex 1 (Nd1) and complex V (Atp6)] (Figure 5C,D). Preservation of the mtDNA copy number may have improved mitochondrial function in skeletal muscles (Figure 5A,B) and thus prevented severe motor dysfunction (Figure 2A). MP treatment is known to increase the OCR [63] and the activities of oxidative respiratory enzymes, including nicotinamide adenine dinucleotide-cytochrome c reductase (NCCR), succinate-cytochrome c reductase (SCCR), and cytochrome c oxidase (CCO) [79]. Furthermore, MP supplementation could upregulate antioxidant enzymes and decrease mitoROS, which could prevent further mitochondrial damage [63].Gut dysbiosis has been associated with the development of PD. Several studies have reported increased abundance of inflammation-related genera, such as Streptococcus [21], Ralstonia [22], Akkermansia [23], and Sutterella [24], and decreased abundance of anti-inflammation-related genera, such as Prevotella [25], Blautia [21], and Roseburia [22], in fecal samples from clinical and preclinical models of PD. Our previous study also demonstrated changes in the microbiota profile in a 6-OHDA-induced model of PD [40]. Herein, we observed similar alterations in the fecal microbiota profile. In the untreated PD group, the abundance of Proteobacteria at the phylum level and abundance of Streptococcus and Sutterella at the genus level were enriched, while Lactobacillus and Roseburia were reduced (Figure 8). HMP supplementation attenuated the alterations in these taxa induced by the 6-OHDA PD injection. MP contains beneficial polyphenols that may promote a gut environment that supports commensal bacteria and limits colonization of pathogenic microbiota in the gut [80]. Due to its capacity to modulate the gut microbiota profile, polyphenol-rich HMP may increase the antioxidant status (Figure 3 and Figure 4) and could prevent prolonged inflammation under PD-like conditions, as indicated by the observed decreases in the plasma levels of IL-1β, IL-6, and TNF-α (Table 3).In most clinical studies, the abundance of Prevotella is reported to be lower in PD compared to healthy controls. However, Heintz-Buschart et al. found Prevotella sp. was more abundant in the gut of patients with PD than in healthy subjects [81]. Prevotella is associated with immunoregulatory function in the gut. Overgrowth of Prevotella in the gut is linked to mucosal inflammation through enhanced production of proinflammatory cytokines and chemokines, activation of T helper type 17 cells (Th17), and increased activation of neutrophils [26,82]. The genus Rothia, which is commonly found in the human mouth and upper respiratory tract, was reported to be highly enriched in the oral cavity of patients with PD [83]. A high abundance of Rothia is known to cause peritoneal infection [84] and cerebrospinal fluid infection [85] in immunocompromised patients. In an in vitro study, the species Aggregatibacter actinomycetemcomitans was shown to produce a leukotoxin that promoted the secretion of IL-1β from human macrophages [86]. In this study, HMP supplementation for 8 weeks suppressed the enrichment of Rothia, Prevotella, and Aggregatibacter in the PD rats (Figure 8). In addition, the increase in Aggregatibacter was accompanied by an increase in the level of IL-1β in plasma in the untreated PD group. Moreover, our correlation analysis suggested that the abundance of Prevotella, Rothia, and Aggregatibacter correlated negatively with antioxidant-related gene expression (Table 4). This suggests that HMP not only directly exerted antioxidant effects, but also indirectly increased antioxidant status through gut microbiota alterations. This finding further supports the suggestion that rebuilding of the gut microbiota composition may boost the antioxidant status of the host.We also observed a trend towards increased abundance of the genus Turicibacter in groups supplemented with HMP, while the untreated PD group showed a slight decrease in this genus. This finding may be positively correlated with the observed improvements in antioxidant capacity at the systemic level and in the muscles and brain. Turicibacter is known to promote production of butyric acid in the gut [87] and upregulates antioxidant enzymes, such as SOD and GPx [27]. Furthermore, increased levels of butyric acid induced by Turicibacter may also possibly mediate the optimization of mitochondrial function observed in the muscles [88]. We also observed a positive correlation between Turicibacter and Gpx expression in the brain. These results further strengthen the evidence of a role for the gut microbiota in antioxidant defense mechanisms in PD-like conditions.Here, we evaluated the effects of MP on DAT binding activity in our PD animal model using [18F]FE-PE21 PET scans. Although it may not represent the precise number of dopaminergic neurons in the brain, the [18F]FE-PE21 PET scanning technique used in this study is a reliable tool to estimate active dopaminergic neurons. Additionally, DAT expression can propose a potential surrogate marker for dopaminergic neurons in nigrostriatal pathway and can be used to predict motor function in PD [48]. Moreover, we propose that in addition to the indirect evidence of neuroinflammation, neuroinflammation in the brain could be directly assessed in future studies to characterize the evidence of neuronal damage of PD.

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