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Triptolide up-regulates metabotropic glutamate receptor 5 to inhibit microglia activation in the lipopolysaccharide-induced model of Parkinson’s disease

https://doi.org/10.1016/j.bbi.2018.04.006Get rights and content

Highlights

  • Triptolide activates mGlu5 to inhibit microglia activation in LPS-induced PD model.

  • Triptolide regulates mGlu5 expression in transcription and post-translation levels.

  • Triptolide inhibits LPS-induced microglia activation via mGlu5-mediated signaling.

Abstract

Metabotropic glutamate receptor (mGlu)5 regulates microglia activation, which contributes to inflammation. However, the role of mGlu5 in neuroinflammation associated with Parkinson’s disease (PD) remains unclear. Triptolide (T10) exerts potent immunosuppressive and anti-inflammatory effects and protects neurons by inhibiting microglia activation. In this study, we investigated the role of mGlu5 in the anti-inflammatory effect of T10 in a lipopolysaccharide (LPS)-induced PD model. In cultured BV2 cells and primary microglia, blocking mGlu5 activity or knocking down its expression abolished T10-inhibited release of proinflammatory cytokines induced by LPS. Moreover, T10 up-regulated mGlu5 expression decreased by LPS through enhancing mRNA expression and protein stability. T10 also reversed the reduction in mGlu5 membrane localization and modulated receptor-mediated mitogen-activated protein kinase activity induced by LPS. Pharmacological inhibition of signaling molecules increased nitric oxide level and inducible nitric oxide synthase (iNOS), tumor necrosis factor-α, and interleukin (IL)-1β and -6 transcript levels that were downregulated by treatment with T10. Consistent with these in vitro findings, blocking mGlu5 attenuated the anti-inflammatory effects of T10 in an LPS-induced PD model and blocked the decreases in the number and morphology of ionized calcium binding adaptor molecule 1-positive microglia and LPS-induced iNOS protein expression caused by T10 treatment. Besides, mGlu5 mediated the effect of T10 on microglia-induced astrocyte activation in vitro and in vivo. The findings provide evidence for a novel mechanism by which mGlu5 regulates T10-inhibited microglia activation via modulating protein expression of the receptor and its intracellular signaling. The study might contribute to the biological effects of Chinese herbs as an approach for protecting against neurotoxicity in PD.

Introduction

Parkinson’s disease (PD) is a common and debilitating neurodegenerative disease resulting from the massive loss of dopaminergic neurons, particularly in the substantia nigra (SN), leading to motor and non-motor symptoms such as rigidity, bradykinesia, and postural instability. In the normal brain, microglia contribute to the maintenance of homeostasis, disrupting this function induces their activation, which results in changes to their immunological and morphological phenotype (Garden and Möller, 2006) including increased expression of specific inflammatory surface markers, chemokines and their receptors, and of receptors for classical neurotransmitters such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and nitric oxide (NO) (Chao et al., 1992, Ghoshal et al., 2007). Evidence shows the influence of microglia on the pathogenesis of PD (Long-Smith et al., 2009). Thus, inflammation underlies the pathology of PD, with hyperactivation of microglia being a main contributor and a potential therapeutic target (Lull and Block, 2010, Pessoa et al., 2014, Richardson and Hossain, 2013).

The standard treatment for PD is levodopa (Schapira, 2007), but its efficacy declines with disease progression (Lang and Lozano, 1998, Schapira, 2007). As an alternative therapy, Chinese herbs and herbal extracts have shown potential clinical benefits in attenuating PD progression in humans (Li et al., 2006, Zhou et al., 2005). Triptolide (T10) is the active extract of the traditional Chinese herb Tripterygium wilfordii Hook F. Pretreatment with T10 dose-dependently reduced lipopolysaccharide (LPS)-induced NO accumulation and TNF-α and IL-1β release from microglia (Zhou et al., 2003), which in turn protected neurons. T10 also inhibited cyclooxygenase (COX)-2 expression and prostaglandin E2 release (Gong et al., 2008). The neuroprotective effects of T10 were confirmed in an inflammatory PD model generated by injecting LPS into SN of rats; movement disorder and dopaminergic neuron death were decreased while dopamine absorption was increased by T10 in this model (Li et al., 2006, Zhou et al., 2005). T10 also blocked the activation of c-Jun N-terminal kinase (JNK), protein kinase B (AKT), and nuclear factor (NF)-κB in LPS-treated microglia (Gong et al., 2008, Liu et al., 2007). Thus, T10 exerts an anti-inflammatory effect via multiple intracellular downstream signaling targets and it is considerable to investigate whether upstream regulation of T10 via a receptor leads to its inhibitory effect on microglia activation in PD.

Glutamate receptors have been implicated in brain injury and neurodegenerative diseases (Ambrosi et al., 2014, Mehta et al., 2013). There are three groups of metabotropic glutamate (mGlu) receptors classified according to pharmacological profiles and downstream signaling mechanisms: group I (mGlu1 and mGlu5), group II (mGlu2 and mGlu3), and group III (mGlu4, mGlu6, mGlu7, and mGlu8) (Pin and Duvoisin, 1995). Agonists of group I mGlu receptors activate phospholipase C (PLC), which leads to Ca2+ release from the endoplasmic reticulum and protein kinase C (PKC) activation. Group I mGlu receptors are implicated in several neurological disorders, including PD, amyotrophic lateral sclerosis, and Alzheimer’s disease (Ma et al., 2006, Ribeiro et al., 2010). mGlu5 is expressed in microglia (Biber et al., 1999) and activation of mGlu5 with CHPG was reported to reduce microglia activation in response to LPS via inhibition of TNF-α and production of reactive oxygen species and NO (Byrnes et al., 2009b). T10 treatment reduced LPS-induced microglia activation (Zhou et al., 2003). However, it remains unclear if mGlu5 plays a role in the suppression of microglia activation by T10 in a PD model.

Based on the above evidence, the aim of this study was to investigate the mechanism underlying T10 targeting on mGlu5 for the protection against microglia activation in PD. We found that T10 up-regulate the expression of mGlu5 by increasing mRNA level and protein stability and thereby suppressing inflammation via mGlu5-mediated signaling. These results provide evidence that T10 activates mGlu5 to protect against neurotoxicity associated with increased inflammation in neurological disorders.

Section snippets

Cell cultures

The murine BV2 microglia cell line was provided by Professor Xiao-Min Wang (Capital Medicine University, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 (Corning, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C and 5% CO2. Rat primary microglia were isolated from whole brains of 0 or 1-day-old Sprague-Dawley rats (from Beijing Weitong Lihua Laboratory Animal Center, SCXK 2012-0001, Beijing, China) as previously

T10 suppresses LPS-induced toxicity in microglia

LPS induces microglia activation and increases pro-inflammatory cytokine production in vitro (Awada et al., 2014, Wang et al., 2015, Wu et al., 2015). Meanwhile, T10 dose-dependently inhibits NO accumulation and TNF-α and IL-1β release from LPS-activated microglia (Zhou et al., 2003). To confirm that T10 inhibits LPS-induced microglia activation, BV2 cells were incubated with different concentrations of LPS for 24 h. NO and IL-1β were upregulated in a dose-dependent manner by LPS treatment (

Discussion

T10 has anti-inflammatory and immunosuppressive effects and the excessive production of TNFα, IL-1β, and NO induced by LPS is abolished by T10 administration (Zhou et al., 2005, Zhou et al., 2003). Meanwhile, mGlu5 exerts neuroprotective effects in microglia by reducing oxidative stress and inhibiting inflammatory cytokine release in vitro (Byrnes et al., 2009b, Qiu et al., 2015). However, the role of mGlu5 in the inhibitory effect of T10 on microglia activation and the underlying mechanism are

Acknowledgments

This work was supported by the National Natural Science Foundation of the People’s Republic of China (No. 81372587; 81171886), Beijing Municipal Natural Science Foundation (No. 7132018), Beijing City Board of Education Development Project (KZ201310025021), and the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality (No. IDHT20140514). In addition, we thank Dr. Ling Zhang (Capital Medical University, Beijing, China)

Financial disclosures

The authors declare that they have no conflict of interest.

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