Part Ⅱ Molecular Mechanisms And Therapeutic Potential Of α- And β-Asarone in The Treatment Of Neurological Disorders

Neuroprotective Effects of α- and β-Asarone

In preclinical studies, α- and β-asarone show strong neuroprotective activities. At the molecular level, this neuroprotection has been attributed to antioxidant, anti-neuroinflammatory, and antiapoptotic effects of α- and β-asarone, along with their ability to modulate various neuroprotective signaling pathways, such as the phosphatidylinositol-3-kinase (PI3K/Akt), camp response element-binding protein (CREB), mitogen-activated protein kinase (MAPK), neurotrophic factors (NTFs), and Kelch-like ECH-associating protein 1 (Keap1)/nuclear factor erythroid factor 2-related factor 2 (Nrf2)/antioxidant responsive element (ARE) axes. The neuroprotective effects of α- and β-asarone are summarized in Table 1 and the different pathways by which α- and β-asarone exert these effects are examined below.

1. Effects of α- and β-Asarone on Oxidative Stress

Oxidative stress results from an imbalance between the production of ROS and reactive nitrogen species (RNS) and the activity of antioxidant defense systems. Oxidative stress has been associated with the pathogenesis and progression of several neurodegenerative diseases, including AD and PD, and contributes to the damage associated with other neurological conditions (e.g., ischaemic stroke and schizophrenia) [65,66]. The antioxidant defense system neutralizes the formation of excess free radicals in response to increased oxidative stress, preventing cellular damage. Various plant secondary metabolites with antioxidant properties exhibit demonstrable beneficial effects on brain function and overall health in humans [22,23]. Both in vitro and in vivo experiments have shown that α- and β-asarone exhibit antioxidant properties. The free-radical scavenging activities of α- and β-asarone have been demonstrated using various in vitro antioxidant assays, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals scavenging assay and the potassium ferricyanide reduction method, and by monitoring the levels of hydroxyl radicals, superoxide, and lipid peroxidation [67–69]. Interestingly, α- and β-asarone treatment significantly enhanced catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH) activity levels and inhibited the excessive accumulation of malondialdehyde (MDA), lactate dehydrogenase (LDH), and ROS, suggesting that α- and β-asarone could improve enzymatic antioxidant defense systems [67,70,71]. Moreover, β-asarone pre-treatment was also found to activate the Nrf2 signaling pathway and its downstream target heme oxygenase-1 (HO-1), which is involved in the quenching of ROS to mitigate oxidative stress [71]. When small interfering RNA (siRNA) was used to silence Nrf2, the protective effect of β-asarone was reduced, and H2O2-induced oxidative stress was enhanced in PC12 cells [71]. In addition, using an Aβ-stimulated PC12 cell model, Meng et al. observed that β-asarone pretreatment could improve cell viability and mitigate cell damage and apoptosis. β-asarone could also decrease the level of ROS and MDA, increase the level of SOD, CAT, and GSH-PX, and promote the expression of Nrf2 and HO-1 [72] (Figure 2).

Recently, Pages et al. [70] and Saki et al. [91] reported an increase in the expression levels of the endogenous antioxidant enzymes SOD and glutathione peroxidase (GPx) in the brains of mice and rats treated with α- and β-asarone compared with untreated animals. Furthermore, α-asarone treatment attenuated the oxidative stress response in the brains of a rat model of noise-induced stress, an effect mediated by the increased expression of SOD, GPx, and CAT, in addition to the upregulation of other endogenous non-enzymatic antioxidant molecules, such as vitamin C, vitamin E, and GSH [92]. Similarly, α-asarone treatment increased acetylcholinesterase (AChE) activity and normalized MDA and SOD levels in the hippocampus and cerebral cortex of AD-like scopolamine-induced amnesic mice [76]. Another study reported that α-asarone administration attenuated brain and kidney damage induced by γ-radiation exposure by restoring antioxidant levels, such as SOD, GPx, CAT, and GSH, and decreasing the lipid peroxidation levels [93]. Yang et al. [83] observed that β-asarone supplementation significantly restored GSH, glutathione reductase (GR), CAT, and glutathione S transferase (GST) levels and decreased lipid peroxidation levels in the hippocampus of a rat model of ischemia induced by middle cerebral artery occlusion (MCAO). Wang et al. [94] showed that β-asarone treatment could effectively attenuate MDA damage and significantly increase CAT and SOD activities in a rat model of AD generated by the intracerebroventricular injection of Aβ1–42 combined with ischemia. In a rat model of MCAO-induced ischemic stroke, β-asarone treatment activated Nrf2/ARE pathway-related proteins, an effect that was inhibited by an Nrf2 inhibitor [31]. These findings suggest that the antioxidant effects of α- and β-asarone could contribute to their therapeutic benefits in the treatment of neurological disorders.

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2. Effects of α- and β-Asarone on Neuroprotective Signaling Pathways

A variety of pro-survival signaling pathways, such as PI3K/Akt and extracellular signal-regulated kinase (ERK)1/2, are activated by α- and β-asarone (Figure 2) [72,73]. These pathways play an important role in cellular function, synaptic plasticity, and memory by altering the phosphorylation condition of molecules and modulating gene expression [95–97]. Oxidative stress triggers MAPK cascades that result in the activation of pro-survival p38 MAPKs, c-Jun N-terminal kinase (JNK), and ERK signaling pathways. The activation of ERK signaling suppresses the death-inducing complex formation and boosts cell survival by upregulating the anti-apoptotic proteins Bcl-xL and Bcl-2 and inhibiting Fas-mediated apoptosis [98].

In vitro, results collected over the years have shown that α- and β-asarone activate the PI3K/Akt/Nrf2 and protein kinase A (PKA) signaling pathways, which play crucial roles in protecting the cells against abnormal ROS levels and neuronal damage, as well as improving cell viability and neuroprotective function [43,72,73]. In Aβ-treated PC12 cells, pre-treatment with β-asarone increased cell viability and decreased cell apoptosis [88]. The protective effect of β-asarone against β-amyloid-induced neurotoxicity was partly mediated through the inhibition of Aβ-induced JNK activation. Additionally, β-asarone significantly attenuated the Aβ-induced downregulation of Bcl-w and Bcl-xL and inhibited mitochondrial cytochrome C release and the activation of caspase-3 [88]. In primary cultured rat astrocytes and SH-SY5Y cells, α- and β-asarone upregulated Akt signaling and protecting cells from oxidative stress, an effect that was shown to be mediated by the scavenging of ROS formation and the stimulation of the Nrf2-ARE self-defense mechanism, and the subsequent triggering of the expression of antioxidant enzymes, and increased the levels of the anti-apoptotic protein Bcl-2 [73,82]. Another recent study showed that β-asarone protects cells from Aβ1–42-induced cytotoxicity and attenuates autophagy via activation of the Akt-mTOR signaling pathway, which may be involved in the neuroprotection of β-asarone against Aβ toxicity in PC12 cells [99]. α- and β-asarone activate not only PI3K/Akt but also ERK/CREB/BDNF pathways in vivo and in vitro, which help to enhance memory function, protect neurons, and recover behavioral changes including immobility time, locomotor activity, and escape latency [77,90,100,101].

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3. Effects of α- and β-Asarone on Proteostasis, ER Stress, and Autophagy

The accumulation of misfolded intracellular and extracellular protein aggregates is a major hallmark of neurodegenerative disease pathogenesis [102–104]. In the brain, several native proteins (Aβ, tau, and α-synuclein) undergo conformational changes due to genetic and environmental factors [104]. Neurodegeneration has been associated with mutated and misfolded protein monomers, oligomers, and insoluble aggregates, formed through altered β-sheet interactions, which cause dysfunction in cellular proteolytic pathways [105,106]. Pathogenic misfolded protein aggregation exerts neurotoxic effects on the CNS, resulting in eventual neuronal cell death and the development of neurodegenerative diseases [3,107]. Many therapeutic approaches to neurodegenerative diseases have aimed to diminish the accumulation of toxic oligomers, fibril deposits, and aggregation intermediates [108,109]. Several studies have suggested that some phytochemicals can inhibit amyloidogenic monomer synthesis and fibrillar aggregate formation, enhancing the formation of nontoxic aggregates and stimulating the proteolytic system to target the neurotoxic pathogenic factors associated with neuronal loss in neurodegenerative disease [110,111].

Amyloidogenic Aβ1–42 peptides are primarily generated by the cleavage of amyloid precursor protein (APP) by β- and γ-secretase [112]. Recent studies have found that α- and β-asarone suppress the expression of the β-secretase BACE1, improving cognitive and behavioral function in animal models of AD [113,114]. A very recent study found that α-asarone potentially targets the Aβ and tau pathology pathways by inhibiting Aβ42 aggregation, in addition to inhibiting tau phosphorylation, resulting in improved spatial learning memory in APP/presenilin-1 (PS1) transgenic mice [28] (Figure 3). Another study demonstrated that treatment with β-asarone reduced the number of senile plaques and decreased Aβ40, Aβ42, and APP expression levels in the hippocampus of APP/PS1 transgenic mice [85]. Moreover, β-asarone displayed a significant therapeutic effect against toxic protein deposition and increased the expression of the presynaptic protein synapsin 1 (SYN1), which should remove toxic superoxide anion radicals produced in cells [114]. A thioflavin T (ThT) fluorescence assay conducted in PC12 cells treated with α- and β-asarone showed an efficient and dose-dependent reduction in Aβ aggregation, protecting PC12 cells from Aβ aggregate-induced toxicity [115] (Figure 3).

Presynaptic α-synuclein aggregation is considered the primary pathogenic factor in the development of α-synucleinopathies, such as PD [103]. Large α-synuclein aggregates, associated with the presence of missense mutations in the α-synuclein gene, including A30P, A53T, and E46K, may exert toxic effects through increased oxidative stress, mitochondrial dysfunction, altered patterns of phosphorylation, and interactions with the phospholipid membrane [116]. Moloney et al. [117] demonstrated that heat shock protein 70 (HSP70) protects dopaminergic neurons from protein aggregation and inhibits microglial activation, ultimately preventing apoptosis. The overexpression of HSP70 exerts a protective effect against early-onset α-synuclein–induced pathology, as demonstrated in an adeno-associated virus model of α-synuclein aggregation [118]. β-asarone treatment has also been found to alleviate dopaminergic cell death induced by 6-hydroxydopamine (6-OHDA) through the upregulation of HSP70 mRNA and protein expression levels and the downregulation of α-synuclein mRNA and protein expression levels [29] (Figure 4). In addition, in SH-SY5Y cells transfected with α-synuclein, β-asarone treatment protected against cell death induced by MPP+ [119]. In a mouse model, the β-asarone treatment also exerted neuroprotective effects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD, preventing the typical decrease in tyrosine hydroxylase (TH)-positive cells and increasing α-synuclein expression levels, thus protecting dopaminergic neurons in the midbrains [119] (Figure 4).

Pathogenic proteins associated with neurodegenerative diseases cause dysfunction in the cellular proteolytic systems, including the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway [120]. Misfolded and unfolded proteins are identified by ubiquitination and targeted for degradation by the proteasome, and UPS dysfunction has been associated with the aggregation of misfolded proteins in PD, although the specific role played by the UPS in PD pathogenesis remains unclear [121]. Disease-specific protein aggregates that are too large for the UPS, including Aβ, tau, and α-synuclein aggregates, in addition to damaged organelles, such as mitochondria, are typically targeted for degradation by the autophagy-lysosome pathway [105].

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The endoplasmic reticulum (ER) plays a critical role in the proper folding of membrane-bound and non-cytoplasmic proteins [122–124]. The accumulation of misfolded or unfolded proteins in the ER causes cellular stress and triggers the unfolded protein response (UPR). Chronic or excessive UPR activation can eventually lead to cell death. UPR activation is mediated by three ER stress sensors: protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme-1 (IRE1), and activating transcription factor 6 (ATF6) [122–124]. The glucose-regulated protein GRP78 (also, known as BiP) primarily regulates the initiation of the UPR through its direct interactions with each signal-transducing sensor [122,125]. Like ATF6, ATF4 is a basic leucine zipper (bZIP) transcription factor important for maintaining intracellular homeostasis through the upregulation of UPR-target genes involved in efficient protein folding, antioxidant response, and amino acid biosynthesis and transport. In addition to promoting an adaptive response, ATF4 upregulates the bZIP transcription factor C/EBP homologous protein (CHOP), which promotes cell death [122,126]. Collectively, the UPR pathways serve to halt protein biosynthesis, promote protein degradation, and generate chaperones to refold misfolded proteins.

ER stress has been identified in several experimental cellular models of PD [127], and the increased expression of wild-type α-synuclein is sufficient to cause ER stress [128]. ER, stress is closely related to oxidative stress, which can also trigger UPR activation [129]. In a 6- OHDA-induced PD model, β-asarone treatment downregulated the mRNA levels of GRP78 and CHOP, resulting in the blockade of two of the three UPR activation pathways [130] (Figure 5). GRP78 preferentially binds unfolded or misfolded proteins in the ER, releasing its inhibitory hold on PERK, ATF6, and IRE1 [131]. Furthermore, β-asarone treatment was shown to reduce the expression of GRP78, phosphorylated PERK (p-PERK), and CHOP, regulating ER stress response and autophagy in a 6-OHDA-induced rat model of PD [132] (Figure 5). Interestingly, the α-asarone treatment prevented 7-hydroxycholesterol-triggered macrophage apoptosis by alleviating ER stress-specific signalings, such as caspase induction and CHOP activation [133]. Furthermore, α-asarone treatment significantly reduced chronic constriction injury-induced ER stress in the spinal cord, in addition to decreasing microglial activation and alleviating neuropathic pain [134]. In a recent in vitro study, pre-treatment with α-asarone protected hippocampal cells from oxidative and ER stress by decreasing ROS production and suppressing PERK signaling in glutamate- and tunicamycin-induced hippocampal HT22 cells [135].

Autophagy activation can upregulate the clearance of protein aggregates, prevent mitochondrial damage, control axon homeostasis and neurogenesis, ensure cell survival, and reduce growth factor deficiency and ER stress, with potential therapeutic benefits in slowing the pathological progression of AD, PD, and other neurodegenerative diseases [136,137]. Beclin-1, phosphorylated (p)-Akt, and mammalian target of rapamycin (mTOR) are known autophagy regulators, and some plant-derived secondary metabolites have been shown to exert neuroprotective effects via the stimulation of autophagy through both mTOR-dependent and independent mechanisms [138]. In a 6-OHDA-induced PD mouse model, β-asarone treatment significantly decreased the levels of both mRNA and protein of Beclin-1 and LC3B, and increased p62 expression, indicating autophagy activation [29]. Another study found that treatment with α-asarone enhanced autophagy in macrophages by upregulating autophagolysosomal formation [139]. A recent canonical correlation analysis revealed that β-asarone treatment can inhibit Aβ aggregation through the promotion of autophagy in a PC12 cell model of AD [114]. Another experiment demonstrated that α-asarone reduced the activation of eIF2α-CHOP by 7β-hydroxycholesterol, enhanced autophagy in macrophages through the upregulation of autophagolysosomal formation, increased phosphorylation of Bcl-2, and facilitated the entry of Beclin-1 into the autophagic process [139]. These findings suggest that α- and β-asarone may have the potential to reduce protein aggregation and ER stress through autophagy modulation, Antioxidants which can have regulatory effects on neurodegenerative disease progression.

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How Cistanche extract can help improve brain disorders？

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Cistanche extract is rich in antioxidants and anti-inflammatory compounds that play a crucial role in maintaining optimal brain health. Studies show that the natural compounds in Cistanche extract can help to reduce inflammation in the brain, which is a major contributor to the development of various neurological disorders. By reducing inflammation in the brain, Cistanche extract can help to improve memory, learning, and cognitive functions.

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Rengasamy Balakrishnan 1,2, Duk-Yeon Cho 1 , In-Su Kim 2 , Sang-Ho Seol 3 and Dong-Kug Choi 1,2

1. Department of Applied Life Science, Graduate School, BK21 Program, Konkuk University, Chungju 27478, Korea; balakonkuk@kku.ac.kr (R.B.); whejrdus10@kku.ac.kr (D.-Y.C.)

2. Department of Biotechnology, Research Institute of Inflammatory Disease (RID), College of Biomedical and Health Science, Konkuk University, Chungju 27478, Korea; kis5497@kku.ac.kr

3. Research and Development, Sinil Pharmaceutical Co., Ltd., Seongnam-si 13207, Korea; seol@sinilpharm.com