Forsythiaside prevents β-amyloid-induced hippocampal slice injury by upregulating 2-arachidonoylglycerol via cannabinoid receptor 1-dependent NF-κB pathway

https://doi.org/10.1016/j.neuint.2019.02.008Get rights and content

Highlights

  • Forsythiaside inhibits COX-2 and MAGL over-expression induced by Aβ to up-regulate 2-AG.

  • Forsythiaside prevents inflammation and apoptosis from Aβ via CB1R-dependent NF-κB pathway.

  • Forsythiaside functionally improves Aβ-caused LTP deficits.

Abstract

In the study, the neuroprotectivities of forsythiaside, a main constituent of Forsythia suspensa (Thunb.) Vahl (F. suspensa, Lianqiao in Chinese), were investigated in the hippocampal slices. Forsythiaside suppressed the overexpression of cyclooxygenase-2 (COX-2) and monoacylglycerol lipase (MAGL) proteins induced by β-amyloid (Aβ25-35) to upregulate the levels of 2-arachidonoylglycerol (2-AG), an endogenous endocannabinoids. Then the inhibition of forsythiaside on COX-2 was deeply studied by the molecular docking. Forsythiaside prevented neuroinflammation and apoptosis from Aβ25-35 insults, and this action appeared to be mediated via cannabinoid receptor 1 (CB1R)-dependent nuclear factor-κB (NF-κB) signaling pathways. More importantly, forsythiaside functionally improved Aβ25-35-induced learning and memory deficits, which was indicated by long term potentiation (LTP). Taken together, forsythiaside may have therapeutic potential for Alzheimer's diseases (AD) by increasing the levels of 2-AG.

Introduction

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive deterioration of cognitive function and loss of memory in association with widespread neuronal death and neuroinflammation. The neuropathological hallmarks of AD are extracellular senile plaques composed of β-amyloid (Aβ) deposits (Selkoe, 1989), consequent intracellular neurofibrillary tangles and eventual cerebral atrophy, etc (Centonze et al., 2007). In recent years the amyloid cascade-neuroinflammation hypothesis was raised: Aβ deposits also trigger glial/microglia cells that start proinflammatory cytokines, e.g., interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), etc. and chemokines, thereby causing pronounced neuroinflammation, which even further enhances the production of Aβ and brain-damaging effects and ultimately leads to neurodegeneration (Akiyama et al., 2000; Karl et al., 2012; Wyss-Coray, 2006). Indeed, production of proinflammatory cytokines and chemokines and the activation of the complement cascade have been observed in AD patients (Koppel and Davies, 2008). The inflammation within the brain plays a pivotal role in AD progress, so the antiinflammation provides novel insights into potential AD therapeutics.

The endocannabinoid system consists of G-protein-coupled cannabinoid receptors (CBRs) that can be activated by endocannabinoids (eCBs) plus associated biochemical machinery (including precursors, synthetic and degradative enzymes, transporters) (Alger and Kim, 2011). CBRs comprise Ⅰtype (CB1R), expressed in largely pre-synaptic neurons in the brain (i.e., the highest levels in cerebral cortex, hippocampus, basal ganglia and cerebellum) and Ⅱ type (CB2R), abundant in immune cells (e.g., macrophages and T cells) and also highly expressed by the activated microglia in the central nervous system (Freund et al., 2003). As one of eCBs, 2-arachidonoylglycerol (2-AG) is a full agonist for CBRs, is mainly produced from diacylglycerol by diacylglycerol lipase (DAGL) to be involved in a variety of physiological and pathological processes and is hydrolyzed to arachidonic acid (AA) by monoacylglycerol lipase (MAGL) (Nomura et al., 2011; Sugiura et al., 2006). Moreover, 2-AG is a substrate for cyclooxygenase-2 (COX-2), mainly expressed in the hippocampal regions (Ho et al., 1999) and is oxygenated by COX-2 to form new types of prostaglandins: prostaglandin glycerol esters (PGEs) and prostaglandin ethanolamides, etc (Kozak et al., 2000). And thus COX-2 and/or MAGL inhibition can elevate endogenous 2-AG level. 2-AG elevation robustly suppressed production and accumulation of Aβ, protected neurons from inflammatory stimuli and neurotoxicity, reduced cellular apoptosis, inhibited neurodegeneration, maintained integrity of hippocampal synaptic structure and function and improved long-term synaptic plasticity, spatial learning and memory in AD animals, although the mechanisms involved were not fully elucidated (Bedse et al., 2015; Chen et al., 2011, 2012; Diego et al., 2007; Fernandez-Ruiz et al., 2010; Janefjord et al., 2014; Karl et al., 2012; Koppel and Davies, 2008; Nomura et al., 2011; Turcotte et al., 2015).

Forsythia suspensa (Thunb.) Vahl (F. suspensa, Lianqiao in Chinese) fruits are well-known traditional Chinese herbal medicines that have been widely used in China, Korea and Japan to treat inflammation, based on its antibacterial, antiinflammatory and antioxidant activities for long time. F. suspensa is one of authentic Chinese medicinal materials in Shanxi Province. Forsythiaside (Fig. 1, Fig. 3, Fig. 4- dihydroxy-β-phenethyl-O-α-L-rhamnopyranosyl- (1 → 6)-4-O-caffeoyl-β-D- glucopyranoside), a phenylethanoid glycoside, is a major component to play the bioactivities in F. suspensa (Chinese Pharmacopoeia Commission, 2015; Qu et al., 2008). Recent studies proved that forsythiaside showed neuroprotective effects on Aβ-induced cells by downregulating acetylcholinesterase (Yan et al., 2017) and had the ability of improving learning and memory in AD model mice (Kim et al., 2009, 2011; Wang et al., 2013). Based on previous statement, we hypothesized that forsythiaside had the ability to prevent AD by upregulating the signal of 2-AG. Among the Aβ fragments studied so far, as the shortest fragment of Aβ processed in vivo by brain proteases, Aβ25-35 peptide represents significant levels of molecular aggregation and exhibits the same bioactivities with the full-length peptide (Frozza et al., 2009; Jo et al., 2011; Suh et al., 2008). Thus, this fragment is widely used in both in vitro and in vivo by neuroscience researches to establish the AD models. In the present work, we established Aβ25-35-induced AD models in hippocampal slices and investigated the protection of forsythiaside against brain neuroinflammation and thus cognitive dysfunction by upregulating the signal of 2-AG.

Section snippets

Preparation of organotypic hippoc ampal slices

ICR mice with 14- to 15-day-old were purchased from Experimental Animal Center of Shanxi Medical University (Grade II and Certificate No. of SCXK (JIN) 2015-0001). After mice were decapitated, the brains were dissected and kept in cold artificial cerebrospinal fluid (ACSF) containing 2.5 mM KCl, 108 mM NaCl, 45 mM NaHCO3, 1 mM NaH2PO4, 5 mM MgSO4, 0.5 mM CaCl2, 12 mM glucose, and 0.5 mM ascorbic acid (Du et al., 2013). 400 μm thick slices were immediately cut in cold ACSF on a vibrating slicer

Forsythiaside increased the contents of endogenous 2-AG

2-AG is an endogenous lipid mediator involved in a variety of physiological, pharmacological and pathological processes. Firstly our aims were to track down the effects of forsythiaside on the 2-AG signal. The results showed that 5 μM Aβ25-35 markedly decreased endogenous 2-AG levels to 35.21 ± 6.43% compared to control group (Ctrl, P < 0.001, n = 4) and forsythiaside at 80 μM moderately rescued this process (51.23 ± 7.04%, P < 0.01 vs. Aβ group, n = 4), as shown in Fig. 2. In a word,

Discussion

AD is known to be associated with various inflammatory reactions and resulting neuronal degeneration that leads to dysfunction and loss of the synapses that are involved in learning and memory deficits (Tuppo and Arias, 2005; Wyss-Coray, 2006). In the development process of AD, Aβ promotes inflammation that in turn fastens and worsens AD. Recently, as it has the ability to modulate a range of aspects of AD pathology, the endocannabinoid system emerged as a novel potential therapeutic target to

Conflicts of interest

There is no conflict of interest.

Acknowledgments

The work was supported by National Natural Science Foundation of China (No. 81403130), Shanxi Scholarship Council of China (No. 2017-021) and Natural Science Foundation of Shanxi Province (No. 201801D121290). We thank Dr. Jinping Jia for the technical help of LC-MS/MS analysis in Scientific Instrument Center, Shanxi University.

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