Elsevier

Phytomedicine

Volume 23, Issue 8, 15 July 2016, Pages 872-881
Phytomedicine

Anti-neuroinflammatory and neuroprotective effects of the Lindera neesiana fruit in vitro

https://doi.org/10.1016/j.phymed.2016.05.002Get rights and content

Abstract

Background

Lindera neesiana Kurz (Lauraceae), popularly known as Siltimur in Nepal, is an aromatic and spicy plant with edible fruits. It is a traditional herbal medicine widely used for the treatment of diarrhea, tooth pain, headache, and gastric disorders and is also used as a stimulant.

Purpose

The aim of the present study was to examine in vitro cytoprotective, anti-neuroinflammatory and neuroprotective potential of an aqueous extract of L. neesiana (LNE) fruit using different central nervous system (CNS) cell lines.

Methods

In order to study the neuroprotective potential of LNE, we used three different types of CNS cell lines: murine microglia (BV2), rat glioma (C6), and mouse neuroblastoma (N2a). Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent, and prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and nerve growth factor (NGF) release in the culture media was determined using enzyme linked immunosorbent assay (ELISA) kits. Western blot analysis was performed to determine the protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), mitogen activated protein kinase (MAPK) family proteins, Bax, B cell lymphoma (BCL)-2, and cleaved caspase 3. Neurite outgrowth was determined using the IncuCyte imaging system.

Results

LNE treatment not only reduced nitric oxide (NO) production in a dose-dependent manner, but also significantly reduced proinflammatory cytokines, iNOS and COX-2 production by lipopolysaccharide (LPS) stimulated BV-2 cells. LNE increased the expression of phosphorylated (p)-extracellular signal-regulated kinase (ERK), whereas p-p38 and p- janus kinase (JNK) expression was significantly decreased in activated microglia. Furthermore, LNE increased cell viability of N2a cells, which was accompanied by decreased caspase-3 expression and the ratio of Bax/Bcl2 protein expression as well as increased NGF and neurite outgrowth, suggesting its neuroprotective potential against LPS-induced effects. Additionally, LNE substantially increased nuclear factor erythroid 2-related factor 2 (Nrf2) secretion in N2a cells and inhibited lipid dehydrogenase (LDH) release in H2O2-stimulated BV2 cells demonstrating the strong anti-inflammatory and antioxidant effects of LNE in CNS cell lines.

Conclusion

Here we found that water the soluble extract of LNE has promising anti-neuroinflammation and anti-apoptotic properties and identify LNE as a potential natural candidate for neuroprotection.

Introduction

Inflammation and apoptosis are characteristic features of many neurodegenerative diseases. Although inflammation is a necessary and important component for host defense, controlling inflammation is necessary for the treatment of illnesses associated with cellular inflammation (McGeer and McGeer, 2001). Neuroinflammation is involved in the pathogenesis of several central nervous system (CNS) disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, cerebral ischemia/stroke, amyotrophic lateral sclerosis, multiple sclerosis and psychiatric disorders (Erickson et al., 2012, Pizza et al., 2011). In the adult CNS, apoptosis of neuronal populations contributes to the manifestation of clinical symptoms in several neurodegenerative disorders. CNS dysfunction may induce several human pathological conditions associated with both cognitive impairment and a variable degree of neuroinflammation (Pizza et al., 2011).

Over the last few decades, several studies have reported different potential mechanisms for the neuroprotective, anti-inflammatory and anti-apoptotic effects of herbal medicines (Chrubasik et al., 2007). Natural products and phytochemicals may lower the risk of, or even prevent, certain chronic neuro-inflammatory disorders (Saviranta NM, 2011). Based on our preliminary screening of herbal medicines, we hypothesized that the aqueous extract of Lindera neesiana (LNE) fruit may possess anti-neuroinflammatory and anti-apoptotic effects in CNS cell lines in vitro.

L. neesiana (Lauraceae), popularly known as Siltimur in Nepal, is a small tree that grows in the temperate Himalayan regions (Comai et al., 2010). It is commonly found in different province of China, Nepal, Bhutan, India and Myanmar. It is an aromatic and spicy plant with edible fruits. Powdered fruit is used in various food preparations as a spice or stimulant, as well as in several herbal medicine preparations (Gaire BP, 2011). Many people use the powder of L. neesiana fruit to prepare pickles, and it is used daily basis as a food ingredient. In Nepalese traditional medicine, fruit of the L. neesiana is chewed to promote recovery from diarrhea, tooth pain, headache, and gastric disorders (Comai et al., 2010, Gaire and Subedi, 2011). Essential oils from L. neesiana fruit, such as Z-citral, eucalyptol, citronellal, α-pinene and β-pinene, are reported to possess potential antimicrobial activity against Staphylococcus aureus and Candida albicans at non-cytotoxic doses in human keratinocytes (Comai et al., 2010). Previous studies reported that, these essential oils possess significant antioxidant and anti-inflammatory activities (Horvathova et al., 2007, Juergens et al., 2003, Melo et al., 2011, Rabbani et al., 2006, Rufino et al., 2014). However, neuroprotective effect has not been reported for this plant yet. In the current study, we investigated the potential neuroprotective effects of LNE via regulation of inflammation, oxidation and apoptosis using three different types of CNS cell lines.

Section snippets

Reagents

Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin (PS) were purchased from Invitrogen (Carlsbad, CA, USA). Lipopolysaccharide (LPS), N-monomethyl-L-arginine (NMMA), and 6- shogaol were purchased from Wako Pure Chemical (Osaka, Japan). ELISA development kit for tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, prostaglandin E2 (PGE2), and nerve growth factor (NGF) were bought from (R&D Systems, Minneapolis, MN, USA). Primary and secondary

LNE shows cytoprotective effect on different cell line

The cytotoxic effect of different concentrations of LNE (1, 5, 10, 50, 100, and 200 μg/ml), was evaluated 24 h after the treatment. In all cell types, LNE was not cytotoxic at any concentrations examined (Fig. 1A-C). Moreover, LNE protected BV2 cells from LPS-induced cell death (Fig. 1D). The cell viability of LPS-stimulated BV2 cells was increased by LNE in a concentration dependent manner, with a maximum effects at 200 μg/ml (148 ± 3%) of control. These results show that LNE may be cytoprotective

Discussion

In the present study, we report for the first time the cytoprotective, anti-neuroinflammatory, and anti-apoptotic effects of aqueous extracts of Lindera neesiana fruit using three different types of CNS cell lines. We demonstrated that LNE possesses strong inhibitory effects on neuroinflammation, oxidative stress and apoptosis, and thus, LNE could be used as an alternative therapeutic agent for CNS disorders.

The proper development of the CNS environment is highly dependent upon the interaction

Conflicts of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome

Acknowledgments

This research was supported by High Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (114006041HD020).

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