Piper sarmentosum Roxb. confers neuroprotection on beta-amyloid (Aβ)-induced microglia-mediated neuroinflammation and attenuates tau hyperphosphorylation in SH-SY5Y cells
Graphical abstract
Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder which is histopathologically recognised by the presence of extracellular beta amyloid (Aβ) plaques and intracellular neurofibrillary tangles (NFTs). Interestingly, widespread inflammatory events have been observed around Aβ plaques surrounded by activated microglia in the AD brain (Cai et al., 2014, Wang et al., 2015). Previous in vivo study showed that the injection of Aβ into the nucleus basalis of rats induced microglial activation and inflammatory reaction (Wilkaniec et al., 2018). These studies thus suggest that Aβ promotes neuronal degeneration via microglia-mediated neuroinflammation.
Microglia, the resident brain macrophages, serve complex roles in the central nervous system (CNS). Under normal conditions, activated microglia are essential for immune surveillance as well as protection against infections and insults in the brain (Sochocka et al., 2017). However, in AD, persistent microglial activation stimulated by Aβ surpasses the threshold of beneficial function and becomes deleterious following the overproduction of neurotoxic pro-inflammatory mediators including nitric oxide (NO), interleukin-1β (IL-1β), and tumour necrosis factor-α (TNF-α). These mediators subsequently cause uncontrolled inflammatory damage on neuronal cells, which in turn induces reactive microgliosis, therefore resulting in a continuous cycle of microglial activation and neuronal death (Sochocka et al., 2017, Cai et al., 2014).
In AD, what triggers the formation of NFTs is unknown, but neuroinflammation could play a role. Numerous studies have demonstrated that neuroinflammation can stimulate tau phosphorylation (Metcalfe et al., 2010; Lee et al., 2010). Specifically, pro-inflammatory cytokines affect the regulation of tau kinases, leading to abnormal hyperphosphorylation of tau proteins (Domingues et al., 2017). When tau is abnormally hyperphosphorylated, it goes through conformational changes that render it insoluble and lead to its aggregation into paired helical filaments, which are the main components of NFTs (Metcalfe and Figueiredo-Pereira, 2010). In vivo study on mutant human tau transgenic mice showed that immunosuppressive treatment can diminish tau pathology, ameliorate hippocampal atrophy and increase lifespan (Yoshiyama et al., 2007). This evidence suggests that attenuation of neuroinflammation and subsequent tau hyperphosphorylation may be an effective therapeutic strategy against AD. However, the challenge in the development of AD therapeutics is to prevent neurotoxicity linked to inflammation without compromising its neuroprotective role. Hence, there is a keen interest in identifying natural products that can reduce or prevent neuroinflammation, and at the same time confer neuroprotective effects.
Piper sarmentosum Roxb. (PS) (family Piperaceae) is a terrestrial herb widely distributed in the Asian regions. It is an edible plant utilised for various medicinal purposes (Hematpoor et al., 2016). In Malaysia, PS is applied to the forehead to relieve headache (Zakaria et al., 2010) and used by the elderly to improve memory (Khan et al., 2012). Apart from that, the indigene of China utilised PS in the treatment of various CNS disorders such as insomnia, anxiety and depression (Li et al., 2017). The leaves of PS are also employed as Indonesian traditional medicine to relieve chest and rheumatic pain (Rahman et al., 2016), reflecting its anti-inflammatory property. Pharmacological studies have reported that PS exhibits anti-inflammatory (Ridtitid et al., 2007, Zakaria et al., 2010), anti-oxidant (Amran et al., 2011, Hafizah et al., 2010, Lee et al., 2014, Mohd Zainudin et al., 2015), and anti-acetylcholinesterase (Hematpoor et al., 2016, Khan et al., 2012) activities. Interestingly, PS has also been demonstrated to produce anti-depressant-like effects in rodents through regulation of hypothalamic-pituitary-adrenal axis (Li et al., 2017), which suggests that PS is able to modulate brain physiology. This study aimed to evaluate the neuroprotective effects of the extracts from the leaves of PS against Aβ-induced microglia-mediated neurotoxicity associated with the overproduction of pro-inflammatory mediators.
Section snippets
Chemicals and reagents
Dulbecco's modified Eagle's Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S) and 0.25% trypsin-EDTA were obtained from GIBCO/Life Technologies (Grand Island, NY, USA). Human amyloid-β peptide (1−42) and Protein Quantification Kit (Bradford Assay) were purchased from Genscript (New Jersey, USA) and BioRad (California, USA), respectively. Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) reagent were obtained from Calbiochem
Maximum non-toxic concentration (MNTC) of PS leaf extracts on BV-2 and SH-SY5Y
Compared with control, 1.56 μg/mL and 12.50 μg/mL of LHXN were not cytotoxic to BV-2 and SH-SY5Y cells respectively (p > 0.05; Fig. 1A and E); whereas LDCM, LEA and LMEOH at concentrations of 12.50 μg/mL, 0.78 μg/mL and 6.25 μg/mL respectively did not demonstrate significant cytotoxic effects on both BV-2 and SH-SY5Y cells (p > 0.05; Fig. 1B–D and F–H). These concentrations were determined as the MNTCs of the extracts, given that the concentrations below MNTC did not display any significant
Discussion
Microglia-mediated neuroinflammation plays a significant role in the pathogenesis of AD. The overactivation of microglia in response to both neuronal damage and persistent Aβ stimulation initiates additional neuronal loss and causes a perpetuating cycle of neurodegeneration (Sochocka et al., 2017). The present study demonstrated the anti-inflammatory actions of PS and its potential neuroprotective effects against Aβ-induced microglia-mediated neurotoxicity as well as evaluated the underlying
Conclusions
In this study, polar extracts (LEA and LMEOH) from leaves of PS demonstrated neuroprotective effects against Aβ-induced microglia-mediated neurotoxicity in SH-SY5Y cells. These protective effects were shown to be mediated by the inhibition of inflammatory mediator production through de-activation of microglial cells that further attenuate tau hyperphosphorylation in neuronal cells. The extracts exhibited anti-inflammatory actions possibly by inhibiting NO production and downregulating the mRNA
Acknowledgement
This work was supported by International Medical University [Project No.: BP I-01/13(03) 2016].
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