Part 1: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
Abstract:
Background: Alzheimer's disease (AD) is the most frequent type of dementia. While acteoside from cistanche herb (ACT), a compound isolated from Cistanche tubulosa, possesses neuroprotective properties. However, the underlying mechanism in regulating microglia polarization remains ill-de¦ned. Methods: Herein, the AlCl3-induced AD model in zebra¦sh larvae was applied to uncover the therapeutic e¨cacy of ACT. BV-2 cells were used to demonstrate the role of ACT on microglia polarization. RNA- Sequence, HPLC-Q-TOF-MS, western blot, and molecular docking were combined to con¦rm its mechanism. Results: ACT signi¦cantly ameliorated the experimental dyskinesia and nervous system disorders in zebra¦sh. Subsequently, it suppressed M1 polarization and promoted to the M2 phenotype in LPS- induced BV-2 cells. We ¦rst demonstrated that ACT exerted profound transcriptomic impact, which involved regulation of key signaling pathways in in§ammation, arginine biosynthesis, as well as pantothenate and CoA biosynthesis, correlating with mitochondria function. ACT(acteoside from cistanche herb) treatment reduced microglia M1 polarization by inhibiting the NF-κB signaling pathway. And the metabolic pathways were further con¦rmed by HPLC-Q-TOF-MS. In addition, ACT recti¦ed excessive ROS to restore mitochondria function through AMPK-mediated PGC-1α and UCP-2 upregulation, consistent with metabolic changes. Intriguingly, ACT may directly bind to both NF-κB and AMPKα, as evidenced by molecular docking. Conclusions: The research provided an infusive mechanism of ACT and illustrated a new perspective based on mitochondrial dysfunction to reveal the connection between metabolism and microglia polarization.
Background:
Alzheimer's disease(AD) is a common neurodegenerative disease accompanied by cognitive impairment and dyskinesia[1]. It’s characterized by severe neuronal loss, senile plaques, and neuro¦brillary tangles[2]. The pathogenesis of AD is multidimensional and linked to neuroin§ammation. Neuroin§ammation is driven by the activation of glial cells, closely related to the development of AD[3, 4]. During the progression and exacerbation of neuroin§ammation, microglia is considered the key factor. Microglia are the primary immune cells in the central nervous system. It’s closely associated with a cascade of processes, containing brain development, maintaining a neutral environment, as well as responding to injury and repair[1]. Moreover, microglia can be stimulated to an M1 phenotype and the expression of pro-in§ammatory cytokines is increased when neuroin§ammation-related diseases such as AD occurred[5]. Studies also demonstrate that the polarization to the M1 phenotype is often accompanied by metabolic disorders[6], causing energy metabolism imbalance and mitochondrial dysfunction[7]. These adverse changes are derived from neurodegeneration, even AD.
acteoside from cistanche herb(ACT), a phenylethanoid glycoside, is primarily derived from Cistanche tubulosa. Increasing evidence has suggested that ACT possessed numerous pharmacological activities, including neuroprotective[8], anti-in§ammatory[9], and antioxidant[10] effects. Particularly, ACT has been reported to improve learning and memory impairment as well as upregulate energy metabolism in streptozotocin-induced rats[11]. It also has been suggested to inhibit neuronal apoptotic cell death and mitochondrial damage in the experimental autoimmune encephalomyelitis mice[12]. However, fewer studies have been focused on the effect of ACT on microglia M1/M2 polarization. Especially, various mechanisms, such as repair of mitochondria function and the regulation of cell metabolism, have not been performed. In addition, the mechanism of ACT contributed to microglia M1/M2 polarization has remained unexplored.
The present report was aimed to investigate the therapeutic e¨cacy of ACT as well as the underly molecular mechanism of ACT in AD. Herein, ACT showed a signi¦cant neuroprotection effect in AlCl3-induced AD zebra¦sh larvae. In addition, ACT effectively inhibited M1 polarization and promoted the M2 phenotype in LPS-induced BV-2 cells. RNA-Sequencing (RNA-Seq) integrated with metabolomics method to better understand the underlying mechanism of ACT in regulating microglia polarization. The crosstalk between metabolism and microglia polarization in terms of mitochondrial function was investigated. This study will provide new respect for the further investigation of ACT as a potential therapeutic agent for treating AD.
Methods:
Animals and model grouping:
Wild-type zebra¦sh (AB strain, 4 months old) were chosen in this study (Nanjing Qi Wu Biotechnology Co., Ltd.). They were maintained under a 14/10 h light/dark cycle at 28°C, following the previous method[13]. Natural fertilizer and normally developed embryos were generated and cultured to 3 days post-fertilization (dpf) in an illumination incubator. All zebra¦sh experiments were carried out under the supervision of the Animal Ethics Committee of China Pharmaceutical University.
Zebra¦sh larvae were divided into six groups and treated from 3 dpf to 7 dpf: control group, model group, model + donepezil hydrochloride (DPZ) group, model + ACT groups. The control group was maintained in the medium with 0.2% DMSO and the model group was treated with 150 μM AlCl3 (pH 5.8). The model + DPZ group was co-treated with AlCl3 and 8 μM DPZ. The model + ACT groups were co-treated with AlCl3 and different concentrations of ACT (200, 100, 50 μM). ACT(acteoside from cistanche herb) (HPLC purity ≥ 98%) was obtained from Baoji Herbest Bio-Tech Co., Ltd. (Baoji, China). AlCl3·6H2O and DPZ were purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD. (Shanghai, China).
Behavioral analysis
Zebra¦sh larvae movements were recorded with a ViewPoint behavioral analyzer (Zebralab 2018, ViewPoint Life Sciences Co., Ltd.) at 28°C. Brie§y, the behavioral parameters, and result processing were consistent with the method we established earlier[13]. Here, average speed (AS), speed change (ΔS), dyskinesia recovery rate (DRR), and response e¨ciency (RE, %) were selected to evaluate dyskinesia recovery in zebra¦sh.
After treatment from 3 dpf to 7 dpf, zebra¦sh larvae were collected to measure AChE and ChAT activity. Based on the manufacturer's protocol, the activity was detected by the enzyme-linked immunosorbent assay (ELISA) kits (MLBIO biotechnology Co. Ltd., Shanghai, China). And the protein concentrations of different samples were determined by the BCA method.
BV-2 cells were seeded in a 6-well dish separately (n=6/group). After treatment, the medium was removed, and the cells were washed three times with cold PBS. Then immediately exposed to liquid nitrogen to suppress cells metabolism. The cells were harvested with cold 80% methanol (1 mL/well) and the suspension was transferred to a 2 mL Eppendorf tube. To facilitate protein precipitation, vigorously vortexed for 1 min and centrifuged at 13,000 rpm for 15 min at 4°C. The cell suspension was transferred to a new 2 mL Eppendorf tube and dried under a stream of nitrogen and stored at -80°C until analysis. The dried residue was reconstituted in 150 μL of pre-cooled 25% acetonitrile. In order to ensure the stability and accuracy of the sequence analysis, equal volumes (10 μL) of each cell sample were combined as quality control (QC) samples. During metabolite detection, these samples were injected after every six cell samples to con¦rm their stability. A 1 μL aliquot was injected for HPLC-Q-TOF-MS.
HPLC-Q-TOF-MS analysis was performed on Agilent 1290 HPLC system connected with the Agilent 6530 Quadrupole Time-of-Flight (Q-TOF) mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The separation was carried out on an ACQUITY UPLC BEH C18 column (2.1×100 mm, 1.7 μm). The mobile phase was composed of 0.1% formic acid-water (v/v; A) and acetonitrile (B).
The §ow rate was set at 0.4 mL/min with the following optimal gradient elution condition: 0 to 2 min, 5% B; 2 to 20 min, 5% to 95% B (positive ion mode); 0 to 2 min, 5% B; 2 to 20 min, 5% to 95% B (negative ion mode). The operation parameters of the mass spectrometer were set as follows: gas temperature, 320℃; drying gas, 10 L/min; nebulizer, 35 psi; VCap, 4000 V; fragment, 120 V.
The raw data were operated under MassHunter Workstation Software version B.07.00 (Agilent Technologies, Santa Clara, CA, USA). The raw data were pre-processed by the XCMS platform. Principal components analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) of the normalized data were conducted with MetaboAnalyst (https://www.metaboanalyst.ca). Combined with literature, the differential metabolites (VIP > 1, T-test P< 0.05) were identi¦ed on HMDB (https://hmdb.ca). Finally, pathway analysis was conducted with MetaboAnalyst.
