Part Two| An Inhibitor Of NF-κB And An Agonist Of AMPK: Network Prediction And Multi-Omics Integration To Derive Signaling Pathways For Acteoside Against Alzheimer’s Disease
Mar 04, 2022
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Part Two | Acteosides: how to treat Alzheimer's disease as one of the effective ingredients of Cistanche?
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DISCUSSION
Alzheimer’s disease is a progressive neuronal and cognitional dysfunction disease with complex dysregulated mechanisms (Fakhri et al., 2020). Accumulating evidence has demonstrated that there is a significant association between microglia-driven inflammation in the brain. Microglia are macrophages in the brain (Afridi et al., 2020) that could be activated into a classical M1 inflammatory phenotype, characterized by enhanced secretion of proinflammatory cytokines (Hanslik and Ulland, 2020). As a consequence, excessive M1 activation could accelerate neuron damage and neurodegeneration, and even exacerbate Alzheimer's Disease (Bagheri-Mohammadi, 2020). Thus, it is imperative to seek new therapeutic approaches aimed at controlling microglia polarization points, so as to provide adaptive benefits.
Our previous work has verified that Acteoside has significant effects on improving the learning and memory ability, and protecting the neurons in rats (Chen Y. et al., 2020). Consistently, the present study also proved that Acteoside could relieve AlCl3-induced dyskinesia and cholinergic system disorder in zebrafish. Excitedly, Acteoside presented remarkable anti-inflammatory activities in LPS-induced BV-2 cells.
Acteoside suppressed M1 polarization by inhibiting the NK-κB pathway. Besides the NF-κB pathway, RNA-seq and HPLC-Q-TOF-MS analysis also discovered that Acteoside treatment could affect arginine biosynthesis as well as pantothenate and CoA biosynthesis. It has been widely reported that iNOS could metabolize Arg to NO and citrulline whereas Arg-1 could hydrolyze Arg to ornithine and urea, which are associated with neuron repair (Rath et al., 2014). LPS stimulation led to the upregulation of iNOS (Figure 2D) and downregulation of Arg-1 (Figure 2E), resulting in increased NO level (Figure 2C). The data uncovered that ACT alleviated the increased NO level through arginine biosynthesis.
Pantothenic acid (PA) is the primary substrate for pantothenate kinase (Kumar et al., 2020) and the rate-limiting metabolite in CoA biosynthesis. PA is the obligate precursor of acetyl-CoA, which is of particular importance for cholinergic neurons (Xu et al., 2020) and participates in the tricarboxylic acid (TCA) cycle (Atamna, 2004). Recent studies showed that elevated concentrations of CoA would lead to altered mitochondrial morphology and lower ATP content (Kumar et al., 2020). LPS-induced BV-2 cells exhibited a decrease in the number of mitochondria and a change of mitochondrial shape. After LPS induction, the production of ROS in BV-2 cells increased. Then, the overladen ROS caused membrane phospholipid to be attacked by free radicals (Magnani et al., 2020), leading to the loss of MMP and, in turn, mitochondrial dysfunction and ATP depletion. It was outstanding that ACT treatment mitigated the decrease of MMP and ATP content. These data suggested that ACT induced mitochondrial dysfunction by regulating pantothenate and CoA biosynthesis.
It has been extensively reported that microglia polarization is closely associated with cell metabolism (Orihuela et al., 2016). In particular, as the metabolic hub, mitochondria play remarkable roles in regulating cell metabolism. Recently, mitochondria have been positioned as a key determinant point in microglia polarization (Harry et al., 2020). To better understand the mechanism of ACT, we judged the functional axis of mitochondria by Western blot. It revealed that ACT induced mitochondrial dysfunction by the activation of the AMPKα/PGC-1/UCP-2 axis.
PGC-1α and UCP-2 are both related to mitochondrial biogenesis (Uittenbogaard and Chiaramello, 2014; de Oliveira Bristot et al., 2019), and they can be thought of as the master regulators of ROS (Jamwal et al., 2020). Reports indicated that PGC-1α-mediated mitochondrial biogenesis and reduction of ROS are dependent on induction of UCP-2 (Uittenbogaard and Chiaramello, 2014; de Oliveira Bristot et al., 2019; Jamwal et al., 2020). Due to overloading ROS, the expression of PGC-1α and UCP-2 was downregulated in LPS-induced BV-2 cells. It suggested that ACT could eliminate excessive ROS through PGC-1α and UCP-2, thus restoring the mitochondrial function. According to the literature, the alteration of PGC-1α in BV-2 cells could contribute to regulating polarization. Interestingly, the previous report has found that increased PGC-1α expression inhibited the NF- κB activity in LPS-induced BV-2 cells (Yang et al., 2017), which qualified the relationship between PGC-1α and NF- κB in our study.
The expression of PGC-1α is affected by upstream pathway proteins, such as AMPK. AMPK is a key protein for the maintenance of cellular homeostasis (Qiu et al., 2020), playing various roles in promoting the M2 polarization of microglia (Chu et al., 2019). It modulates metabolic pathways in cells (Szewczuk et al., 2020). We found that Acteoside promoted the activation of AMPK. At the same time, the application of compound C (AMPK inhibitor) blocked the effect of ACT on attenuating LPS-induced NO excess. Therefore, ACT also suppressed LPS-stimulated M1 polarization via the AMPK signaling pathway.
It is the first time to report the mechanism of ACT on regulating microglia polarization (Figure 6). The data supported that ACT could be developed as a therapeutic agent for neurodegenerative disease associated with neuroinflammation, such as AD. In particular, we linked the microglia polarization with cell metabolism, explaining the effect of ACT through the alteration of mitochondria function. The identification of this metabolic axis, as a target of a unique entity, may lead to much better therapeutic approaches against microglia M1 polarization, particularly in Alzheimer's Disease.
ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Ethics Committee of China Pharmaceutical University
AUTHOR CONTRIBUTIONS
Y-QL, YC, Y-YS, and S-QJ performed conceptualization and methodology. Y-QL performed sample preparation. Y-QL, S-QJ, Y-YS, and S-SW performed data curation and experimental work. Y-QL, YC, and X-LJ performed writing and original draft preparation. FL and PL performed supervision, reviewing, and editing. All authors contributed to the article and approved the submitted version.
FUNDING
This work was supported by grants from the National Key R&D Program of China (2019YFC1711000), the National Natural Science Foundation of China (Nos. 81860773 and 81873185), the Natural Science Foundation of Jiangsu Province (No. BK20181327), and the Xinjiang Science Fund for Distinguished Young Scholar Project (No. 2018Q003).
ACKNOWLEDGMENTS
The authors were grateful to PZ, H-YW, Yu-Meng Shen, and Wei Jiang for their technical assistance, as well as Jia-Lin Yu for her polish.
