Part 2:Acteoside Repressed Microglia M1 Polarization Through Inhibited NF-κB Signalling Pathway And AMPK-Mediated Mitochondria Function Recovery
Mar 06, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
AD (Alzheimer's disease) is a progressive neuronal and cognitional dysfunction disease, with complex dysregulated mechanisms[17]. Accumulating evidence has demonstrated a signi¦cant association between microglia-driven in§ammation in the brain. It seems to play a critical role in the progression of AD(Alzheimer's disease). Microglia are macrophages in brain[18]. It could be activated to a classically M1 in§ammatory phenotype, characterized by enhanced secretion of proin§ammatory cytokines[4]. Excessive M1 activation could accelerate neuron damage and neurodegeneration, even exacerbating AD(Alzheimer's disease) [19]. Thus, it’s imperative to seek new therapeutic approaches aimed at controlling microglia polarization points that could provide adaptive bene¦ts.
Our previous work has veri¦ed that ACT(acteoside from cistanche) had signi¦cant effects of improving the learning and memory ability and protecting the neurons in rats [20]. Consistently, the present study also proved that ACT could relieve AlCl3-induced dyskinesia and cholinergic system disorder in zebra¦sh. Excitedly, ACT(acteoside from cistanche) presented remarkable anti-in§ammatory activities in LPS-induced BV-2 cells. The transcriptomic pro¦le con¦rmed the signi¦cant changes in LPS-induced cells compared with control cells, as well as ACT-treated cells compared with LPS-induced.
ACT(acteoside from cistanche) suppressed M1 polarization by inhibiting the NK-κB pathway. Except for the NF-κB pathway, RNA-seq also discovered that ACT(acteoside from cistanche) treatment could affect arginine biosynthesis as well as pantothenate and CoA biosynthesis. Interestingly, the two metabolic pathways were further con¦rmed by HPLC-Q-TOF-MS analysis. It’s widely reported that iNOS could metabolize Arg to NO and citrulline whereas Arg-1 could hydrolyze Arg to ornithine and urea, associated with neuron repair[21]. LPS stimulation led to the upregulation of iNOS (Fig. 3a) and downregulation of Arg-1 (Fig. 3b), resulting in increased NO level (Fig. 2f). The data uncovered that ACT(acteoside from cistanche) alleviated the increased NO level through arginine biosynthesis.
Pantothenic acid (PA) is the primary substrate for pantothenate kinase[22], as a rate-limiting metabolite in CoA biosynthesis. PA is the obligate precursor of acetyl-CoA, which is of particular importance for cholinergic neurons[23] and participates in the tricarboxylic acid cycle (TCA cycle)[24]. A recent study showed that elevated concentration of CoA would lead to altered mitochondrial morphology, and lower ATP content[22]. LPS-induced BV-2 cells exhibited a decrease in the number of mitochondria and a change of mitochondrial shape. After being induced by LPS, the production of ROS increased in BV-2 cells. Then the overladen ROS caused membrane phospholipid to be attacked by free radical[25]. It led to the loss of MMP, in turn, mitochondrial dysfunction and ATP depletion. It was outstanding that ACT(acteoside from cistanche) treatment mitigated the decrease of MMP and ATP content. These data suggested that ACT(acteoside from cistanche) induced mitochondrial dysfunction by regulating pantothenate and CoA biosynthesis.
It has been extensively reported that microglia polarization is closely associated with cell metabolism[14]. Particularly, 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[26]. To better understand the mechanism of ACT(acteoside from cistanche), we judged the functional axis of mitochondria by western blot analysis. It revealed that ACT(acteoside from cistanche) 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[27, 28], and they can be thought of as the master regulator of ROS[29]. Reports indicate that PGC-1α-mediated mitochondrial biogenesis and reduction of ROS are dependent on the induction of UCP-2[27–29]. Due to overloading ROS, the expression of PGC-1α and UCP-2 was down-regulated in LPS-induced BV-2 cells. It suggested that ACT(acteoside from cistanche) 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, a previous report has found that increased PGC-1α expression inhibited the NF-κB activity in LPS-induced BV-2 cells[30]. It quali¦ed 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 the key protein for the maintenance of cellular homeostasis[31], playing various roles in promoting the M2 polarization of microglia[32]. It modulates metabolic pathways in cells[33]. We found that ACT(acteoside from cistanche) 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’s the ¦rst time to report the mechanism of ACT(acteoside from cistanche) on regulating microglia polarization (Fig. 10). The data supported that ACT could be developed as a therapeutic agent for neurodegenerative disease associated with neuroin§ammation, such as AD(Alzheimer's disease) . Especially, we linked the microglia polarization to cell metabolism, explaining the effect of ACT(acteoside from cistanche) through the alteration of mitochondria function. The identi¦cation of this metabolic axis, the targeting of this as a unique entity, could allow much better therapeutic approaches against microglia M1 polarization, particularly in AD(Alzheimer's disease) .
