Elsevier

Brain Research

Volume 1717, 15 August 2019, Pages 190-203
Brain Research

Research report
Gastrodin attenuates proliferation and inflammatory responses in activated microglia through Wnt/β-catenin signaling pathway

https://doi.org/10.1016/j.brainres.2019.04.025Get rights and content

Highlights

  • LPS promote microglial proliferation and neuroinflammation both in vitro and in vivo.

  • GAS suppresses proliferation and proinflammatory cytokines in LPS activated microglia.

  • GAS plays an neuroprotective role in activated microglia via Wnt/β-catenin signaling pathway.

Abstract

Microglia contribute to the regulation of neuroinflammation and play an important role in the pathogenesis of brain disorders. Thus, regulation of neuroinflammation triggered by activation of microglia has become a promising therapeutic strategy. Here, we investigated the beneficial effects of Gastrodin in activated microglia and analyzed the underlying molecular mechanisms. Microglia activation was regulated by Gastrodin not only in terms of microglia population size but also production of inflammatory mediators. Gastrodin inhibited the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), cyclin-D1 and Ki67 in lipopolysaccharide (LPS)-stimulated BV-2 or primary microglia. Gastrodin also suppressed the expression of iNOS and Ki67 in activated microglia in three-day-old LPS-injected postnatal rats. In addition, the present results have shown that Gastrodin inhibited LPS-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser 9 and β-catenin activity. We further extended our investigation to determine whether Wnt/β-catenin signaling pathway was involved in the anti-inflammatory and anti-proliferation function of Gastrodin. β-Catenin antagonist (XAV939) was used to block LPS-mediated upregulation of iNOS, TNF-α, cyclin-D1, nitric oxide (NO) and the number of cells in the G2/M+S phase of cell cycle. Moreover, treatment with LiCl, a special Wnt/β-catenin pathway agonist significantly blocked Gastrodin-mediated down-regulation of iNOS, TNF-α, cyclin-D1, NO and the number of cells in the G2/M+S phase of cell cycle in LPS-stimulated BV-2 microglia. Taken together, the present results suggested that Gastrodin mediated anti-inflammatory and anti-proliferation effects in activated microglia by modulating the Wnt/β-catenin signaling pathway.

Introduction

It is widely accepted that neuroinflammation which can be triggered by multiple biological mechanisms including oxidative stress and glial reactions plays a key role in the pathogenesis of the different neurological disorders including Parkinson's disease (PD) and Alzheimer's disease (AD) (Agostinho et al., 2010, Alcendor et al., 2012, Allaman et al., 2011, Niranjan, 2014, Aid and Bosetti, 2011, Aktas et al., 2007). Microglia, the resident innate immune cells in the brain, have been implicated as active contributors to neuronal damage in different neurodegenerative diseases. The hyperactive and uncontrolled activation of microglia may result in progressive neurotoxic sequels (Zecca et al., 2006, McGeer et al., 2006, Kim and Joh, 2006). In most circumstances, overactive microglia can induce significantly and highly detrimental neurotoxicity through excessive production of a plethora of cytotoxic factors such as superoxide (Colton et al., 1987), nitric oxide (NO) (Moss and Bates, 2001, Liu et al., 2002) and tumor necrosis factor-α (TNF-α) (Sawada et al., 1989, Lee et al., 1993). The stimuli that cause microglia excessive activation and dysfunction are diverse, ranging from environmental toxins, such as the pesticide, to neuronal death or damage (Zhang et al., 2015). In neurodegenerative disease, activated microglia have been shown to be present in large numbers and indicative of their active proliferation (Graeber and Streit, 2010). Therefore, it is essential to determine the relative contribution of proliferation and activation of microglia for analyzing the pathological outcome of central nervous system (CNS) (Ponomarev et al., 2005). Furthermore, a closer analysis of the extent of microglia proliferation under pathological conditions is desirable for understanding how the innate inflammatory response might contribute to the onset and progression of disease.

Many signaling pathways have been reported to be involved in microglia activation and among them may be mentioned the canonical Wnt/β-catenin pathway that is known to play an important role in many CNS disorders, including cerebral ischemia, psychiatric disorders, PD, AD and epilepsy (Gómez-Nicola et al., 2013, Cao et al., 2017, Zhang et al., 2008, Mastroiacovo et al., 2009, Okerlund and Cheyette, 2011, L’ Episcopo, et al., 2011, Shruster et al., 2011, Halleskog et al., 2011, Busceti et al., 2007). In the Wnt/β-catenin pathway, Wnt ligands (Wnt1, Wnt3a and Wnt5a) bind to Frizzled (Fzd) and LRP5/6. Subsequent binding of Fzd to the scaffold protein Disheveled (Dvl) leads to the inhibition of the destruction complex, a group of proteins which include Dvl, axin, GSK-3, casein kinase 1 (CK1) and adenomatous polyposis coli (APC). In the absence of Wnt, the destruction complex resides in the cytoplasm, where it binds and phosphorylates β-catenin. Then the phosphorylated β-catenin is ubiquitinated and destroyed in the proteasome. Thus, Wnt signaling allows β-catenin accumulation in the cytoplasm. β-catenin subsequently enters the nucleus to interact with T cell/lymphoid enhancer factor (TCF/LEF) transcription factors and regulate the transcription of target genes (Clevers and Nusse, 2012, Kikuchi et al., 2011). Moreover, recent studies have suggested the involvement of Wnt signaling in generating inflammatory responses in the CNS through glial cell modifications (Marchetti and Pluchino, 2013), yet the underlying mechanisms have remained to be fully clarified.

Gastrodin, a natural phenol, is one of the constituents of the Gastrodia elata BI (Zhu et al., 2010). Gastrodin could penetrate through the blood-brain barrier and rapidly decompose to p-hydroxybenzyl alcohol (HBA) in the brain (Lin et al., 2008b). Recent studies revealed that Gastrodin has a neuroprotective action against hypoxia-induced toxicity in the cultured cortical neurons (Xu et al., 2007), ameliorates cerebral damage after transient focal cerebral ischemia (Zeng et al., 2006) and protects primary cultured rat hippocampal neurons against Aβ peptide induced neurotoxicity (Zhao et al., 2012). All these findings suggest that Gastrodin may be effective in attenuating neuroinflammation which is implicated in different neurodegenerative diseases. Previous studies have reported that Gastrodin inhibited expression of inducible NO synthase, cyclooxygenase-2 and proinflammatory cytokines in cultured LPS-stimulated microglia via MAPK pathways (Dai et al., 2011). While the inhibitory effects of Gastrodin on microglia activation may be linked to various signaling routes, the actual mechanistic link between them has remained uncertain.

This study was aimed to determine the neuroprotective role of Gastrodin through mediating activated microglia in vitro and in vivo. We showed here that Gastrodin inhibited LPS-induced microglia activation by suppressing the Wnt/β-catenin signaling pathway. The results also suggested that manipulation of Wnt/β-catenin signal pathway by Gastrodin may offer a promising therapeutic strategy for treatment of brain disorders in which microglia activation is implicated.

Section snippets

Gastrodin suppressed the expression of TNF-α and iNOS in LPS-activated microglia

An increase in expression of inflammatory mediators is considered a hallmark feature of activated microglia (Ponomarev et al., 2005). In order to ascertain whether Gastrodin can mediate the microglia activation, BV-2 cells were stimulated with LPS in the absence or presence of Gastrodin (10, 20, 40 and 80 μM). After LPS treatment, the effect of Gastrodin on mRNA expression of iNOS and TNF-α was assessed by RT-PCR. The mRNA expression of these inflammatory mediators was very low and hardly

Discussion

Neuroinflammation is a subject of intense focus in many basic and clinical studies, as it involves some complex pathophysiological processes. Evidence accumulated during the last three decades has shown the existence of inflammation in AD, including activated microglia within and surrounding senile plaques (McGeer et al., 1987, McGeer et al., 1988). In addition, numerous studies have highlighted a potential role for neuroinflammation in PD (Tansey and Goldberg, 2010). In this connection,

Conclusion

The present results have demonstrated unequivocally that Gastrodin plays an important role in inhibiting proliferation and production of proinflammatory cytokines in LPS activated microglia. In vitro, Gastrodin decreased microglia cell proliferation whose underlying mechanism involves its inhibition of Wnt/β-catenin signal pathway. To this end, decreasing and inhibiting the associated immune response initiated by microglia through use of Gastrodin would be a potential and beneficial therapeutic

BV-2 cells culture and treatment

BV-2 microglia cell line was cultured in DMEM containing 10% FBS (Cat. No. 04-001-1ACS, Biological Industries, BI, USA) and antibiotics (100 IU/ml penicillin and 100 mg/ml streptomycin) at a density of about 5 × 105 cells/ml and maintained at 37 °C in a humidified incubator with 5% CO2. Cells (approximately 2 × 105 cells/well) were seeded in six-well plates before being subjected to treatments. Gastrodin (Purity > 99%, Kunming Pharmaceutical Corporation, Kunming, China) at 10, 20, 40 and 80 μM

Funding

This work was supported in part by National Science Foundation of China (No. 31760292, 81760087, 81460210, 81460350, 81360200) and the Yunnan Applied Basic Research Project [No. 2017FA035, 2018IA048, 2018FE001(-029), 2017FE467(-008), 2018FE001(-017)].

Contributions

Y.Y. and L.B. performed experiments and prepared the manuscript; P.Y. and Y.C. conducted all the experiments; L.S. and Q.A. analyzed the data; D.L. and L.Z. designed experiments, completed the manuscript, and obtained science grant support; Y.S. and R.L. contributed to in vivo imaging studies. All authors read and approved the final version of this manuscript.

Declaration of interest

The authors declare that they have no competing interests.

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    Yueyi Yao and Ligong Bian contributed equally to this work.

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