1、The Improvement Of Compounds WYD1-8 And WYD7-6 On β-amyloid-induced Learning And Memory Impairment In Mice 2、The Role And Mechanism Of Scavenger Receptor Marco In α-synuclein-induced Neuroinflammation

Section 1. The improvement of compounds WYDl-8 and WYD7-6 on β-amyloid-induced learning and memory impairment in miceAlzheimer’s disease (AD) is kind of degenerative disease of the central nervous system with aging. In recent years, studies have shown that inflammation is a key player in the pathophysiology of AD. The notable feature in AD brain is a large number of activated glial cells, accompanied by the productions of inflammatory cytokines and chemokins in the vicinity of β-amyloid protein (Aβ), and these inflammatory mediators may cause neuronal death. Compounds WYD1-8 and WYD7-6 are phloroglucinol phenol derivatives. Previous studies demonstrated that WYD1-8 and WYD7-6 had strong anti-inflammatory property in primary hippocampal mixed cultures stimulated by Aβ or lipopolysaccharide (LPS), and also had neuroprotective effects. So we speculated that compounds WYD1-8 and WYD7-6 might have therapeutic effects on AD. In this study, we aimed to investigate the ability of compounds WYD1-8 and WYD7-6 in improving learning and memory disorders and the underling mechanisms using Aβ1-42-induced AD mouse model. The results showed that compounds WYD1-8 and WYD7-6 significantly decreased the learning and memory impairment induced by Aβ1-42, inhibited acetylcholinesterase (AChE) activity and increased choline acetyltransferase (ChAT) activity and acetylcholine (ACh) level. Further studies found that compounds WYD1-8 and WYD7-6 could inhibit microglia and astrocytes activation in hippocampus of mice and reduce the production of neurotoxic inflammatory mediators such as tumor necrosis factor-a (TNF-a) and interleukin-1β (IL-1β). WYD1-8 and WYD7-6 also decreased the level of cyclooxygenase-2 (Cox-2) and the activity of inducible nitric oxide synthase (iNOS). Meanwhile, WYD1-8 and WYD7-6 could increase superoxide dismutase (SOD) activity. In addition, compounds WYD1-8 and WYD7-6 inhibited apoptosis of hippocampus in mice by reducing Bax expression and increasing Bcl-2 expression, and Cysteine-aspartic protease3 (Caspase-3) activity was also inhibited by WYD1-8 and WYD7-6. We then further investigate the signal transduction pathways related to inflammation, and found that compounds WYD1-8 and WYD7-6 significantly inhibited IκB kinase (IKK), inhibitory κB (IκB) phosphorylation and nuclear transcription factor κB (NF-κB) p65 translocation induced by Aβ, indicating that the NF-κB pathway was inhibited by WYD1-8 and WYD7-6. Meanwhile, c-Jun N-terminal kinase (JNK) and p38 phosphorylation were also significantly inhibited by WYD1-8 and WYD7-6. However, extracellular signal-regulated kinase (ERK) phosphorylation did not change, suggesting that WYD1-8 and WYD7-6 could inhibit inflammation by inhibiting NF-κB and JNK/p38 MAPK signal transduction pathways. Taken together, these results suggested that compounds WYD1-8 and WYD7-6 had therapeutic effects on AD, which might be related to their inhibition of neuroinflammation and apoptosis. Our results also indicated that compounds WYD1-8 and WYD7-6 might be a potent drug candidate for the treatment of AD. Section 2. Therole and mechanism of Scavenger receptor Marco in a-synuclein-induced neuroinflammationParkinson’s disease (PD) is a progressive, neurodegenerative disorder which closely related with aging. Studies have shown that neuroinflammation is important in the aetiology and pathogenesis of PD. Astrocytes are key glial cells that can be activated by a-synuclein or other stimulating factor, and then transduces the signal to neurons and microglia, playing an important role in PD. However, the mechanisms of a-synuclein-induced inflammation in astrocytes are not very clear. Scavenger receptor Marco (SR-Marco) is abundantly expressed on cell membranes of activated astrocytes. It was reported that Aβ or LPS induced neuroinflammation in astrocytes is SR-Marco mediated. So we speculated that the activation of astrocytes by a-synuclein might be SR-Marco mediated. To confirm this speculation, in this study, primary cultured rat astrocytes were stimulated with a-synuclein (A53T), and the role and mechanism of SR-Marco in mediating inflammation were observed. Our results showed that after astrocyts were stimulated with a-synuclein for 48h, the expression of glial fibrillary acidic protein (GFAP) and SR-Marco was significantly increased. Moreover, the inflammatory response was also observed as indicated by increased iNOS activity, TNF-α and IL-1β productions and Cox-2 level. When SR-Marco was silenced by SR-Marco small interfering RNA (siRNA), GFAP expression, iNOS activity, TNF-a and IL-1β release, as well as Cox-2 level were significantly reduced. We further investigated the signal transduction pathways associated with inflammation, and found that a-synuclein stimulating led to IKK and IκB phosphorylation, NF-κB p65 translocation, indicating NF-κB pathway was activated. In addition, Janus kinase 2 (JAK2) expression and signal transducer and transcription activator (STAT3) phosphorylation were also increased by a-synuclein stimulating. However, NF-κB and JAK2/STAT3 pathways were suppressed upon silencing of SR-Marco. In summary, these data suggested that SR-Marco played a critical role in neuroinflammation in astrocytes stimulated with a-synuclein. The results also provided evidence that SR-Marco might be an importamt drug target for the treatment of disorders induced by neuroinflammation, such as PD.Section 3. FLZ attenuates learning and memory deficits via suppressing neuroinflammation induced by LPS in miceLPS, a component of Gram-negative bacteria’s cell wall, is widely used as an inflammatory stimulus. The studies of graduated student Hong-Yan Pang in our group demonstrated that FLZ could significantly inhibit the activation of glial cells induced by intracerebroventricular injection of LPS, reduced the production of neurotoxic inflammatory mediators, and significantly increased learning and memory function and neuronal damage induced by inflammation. Learning and memory function in animals could be detected by channel type water maze test and Morris water maze test, and channel type water maze test was used in the experiment of Hong-Yan Pang. The classic Morris water maze test is relatively simple in terms of the learning process in animals and can be more sensitive to reflect the animal’s learning and memory function, therefore it should be better to combine the two water maze tests in order to get more objective results. In this experiment, we further tested learning and memory function of animals by Morris water maze test to compare the similarities and differences between the two methods, in order to get more objective results. The results showed that FLZ significantly improved the learning and memory impairment in mice intracerebroventricular injected with LPS and significantly decreased TNF-α concentration and inhibited AChE activity.