Esculentoside A exerts anti-inflammatory activity in microglial cells

doi:10.1016/j.intimp.2017.08.014

International Immunopharmacology

Volume 51, October 2017, Pages 148-157

https://doi.org/10.1016/j.intimp.2017.08.014 Get rights and content

Highlights

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EsA attenuated LPS-induced inflammatory mediators' production in activated microglia.
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The anti-inflammatory effects of EsA was associated with the inactivation of NF-κB, MAPKs and NLRP3 pathways.
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EsA suppressed mRNA levels of cytokines in Aβ_1–42-induced primary microglia cells.

Abstract

Esculentoside A (EsA) is a saponin isolated from the roots of Phytolacca esculenta. This study was designed to evaluate the pharmacological effects of EsA on lipopolysaccharide (LPS)-stimulated BV2 microglia and primary microglia cells. Our results indicated that EsA pretreatment significantly decreased LPS-induced production of Nitric Oxide (NO) and Prostaglandin E2 (PGE2) and impeded LPS-mediated upregulation of pro-inflammatory mediators' expression such as nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-12 (IL-12) and tumor necrosis factor-a (TNF-α) in both BV2 microglia and primary microglia cells. Moreover, EsA markedly suppressed nuclear factor-κB p65 (NF-κB p65) translocation by blocking IκB-α phosphorylation and degradation in LPS-treated BV2 cells. EsA also decreased phosphorylation level of mitogen-activated protein kinases (MAPKs) and inhibited NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome mediated caspase-1 activation in LPS-stimulated BV2 cells. Additionally, EsA decreased β-amyloid_1–42 (Aβ_1–42)-induced production of TNF-α, IL-1β and IL-6 in primary microglia. Thus, EsA might be a promising therapeutic agent for alleviating neuroinflammatory diseases.

Introduction

Microglia, the immune surveillance cells in the brain and spinal, has been reported to play critical roles in immune defense and tissue repair in the central nervous system (CNS) [1]. In the steady state, microglia maintains a resting phenotype which performs a variety of physical functions, such as secrete neurotrophic factors, support neurogenesis and regulate normal brain development. In the context of neurodegenerative disease, cerebrovascular disease or cancer, microglia can become activated or dysregulated [2]. Microglia are exquisitely sensitive to stimuli of their microenvironment and they can be activated, and then release a variety of pro-inflammatory or cytotoxic factors, such as nitric oxide (NO), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and reactive oxygen species (ROS) [3]. The over activation of inflammatory response by microglia could contribute to neighboring neuronal damage and degeneration [4]. It is now apparent that hyperactivated microglia is associated with multiple chronic neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD) [3], [5], [6]. These observations suggest that regulating microglia activation might be a promising therapeutic approach for various neurodegenerative diseases.

Lipopolysaccharides (LPS), a natural constituent of the cell wall of Gram (−) bacteria, is a robust activator of CNS glia [4]. Interaction of Toll-like receptor-4 (TLR4) with LPS results in the induction of array of immune responses, such as activation of the transcription factors nuclear factor-κB (NF-κB) [7] and mitogen-activated protein kinases (MAPKs) [8], resulting in production of numerous pro-inflammatory mediators, including NO, TNF-α and IL-6. Besides, LPS can also induce activation of NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome and influence secretion of IL-1β. Thus, LPS-stimulated microglia has been widely used as an in vitro model to study characters of activated microglia [9].

Esculentoside A (EsA) is a triterpene saponin isolated from the root of Chinese herb Phytolacca esculenta, which is identified as 3-O-[b-d-glucopyranosyl-(1, 4)-b-d-xylopyranosyl] phytolaccagenin. Chinese herb containing EsA has been used for treatment of breast hyperplasia and ovarian cyst for > 20 years in China [10]. EsA has been reported that it has strong anti-inflammatory effects in several in vivo and in vitro experiment models [10], [11], [12]. Xiao ZY et al. demonstrated that EsA inhibited the pro-inflammatory cytokine production such as IL-1, PGE₂, TNF-α, IL-6 and IL-2 in LPS-activated macrophages and ConA-induced lymphocytes respectively [12]. EsA is also referred to inhibit immune responses through suppressing the proliferative response of T lymphocytes and inducing apoptosis of activated thymocytes [11]. Moreover, EsA treatment can also suppress inflammatory responses in LPS-induced acute lung injury and attenuated CCl₄ and GalN/LPS-induced acute liver injury in mice [13], [14]. Based on this, the anti-inflammatory and neuroprotective effects of EsA on LPS-induced inflammatory response in microglia cells and the underlying possible mechanism were clarified in the present study.

Section snippets

Reagents

Esculentoside A (purity, > 98%, #A0351) was purchased from Chengdu Must Bio-Technology Co., Ltd. (Sichuan, China). EsA was dissolved in culture medium before experiments. Minimum essential medium (MEM, Gibco™ #11095080), fetal bovine serum (FBS,Gibco™ #10099–141), penicillin and streptomycin (Gibco™ #15140122), trypsin/EDTA (Gibco™ #25200072) and antibody against NLRP3 (# PA5-20838)were purchased from Thermo Fisher scientific (USA). LPS from Escherichia coli serotype O55:B5 was purchased from

Effects of EsA on BV2 microglia cell viability

To test the cytotoxicity of EsA, various concentrations of EsA were applied with or without LPS (500 ng/ml) to BV2 microglia for 24 h. The viability of cell was determined by MTT assay. As shown in Fig. 1 either treatment of EsA alone or EsA with LPS had no toxic effect on cultured BV-2 cells in the performed concentrations. Based on these results, EsA at concentrations of 6, 12 and 24 μM was used in the subsequent experiments.

EsA attenuated NO production and iNOS expression in LPS-stimulated microglia

To investigate the effects of EsA on LPS-induced inflammatory

Discussion

Microglia, the macrophages of the central nervous system, are widely distributed in the brain parenchyma. With the change of surrounding microenvironment in the brain, microglia can change their phenotype rapidly and induce various gene expression. Under normal conditions, microglia are in a state of surveying the surrounding environment and neurons neighboring microglia deliver signals which keep microglia in surveillance mode [17]. However, tiny vascular or tissue damage can rapidly prompt

Acknowledgements

This study was supported by the National Natural Science Foundation of China (81230026, 81630028), the Natural Science Foundation (BE2016610) and Jiangsu Province Commission of Health and Family Planning (LJ201101) of China.

References (38)

M.T. Heneka et al.
Neuroinflammation in Alzheimer's disease
Lancet Neurol.
(2015)
H. Qin et al.
LPS induces CD40 gene expression through the activation of NF-kappaB and STAT-1alpha in macrophages and microglia
Blood
(2005)
C. Guo et al.
Anti-neuroinflammatory effect of Sophoraflavanone G from Sophora Alopecuroides in LPS-activated BV2 microglia by MAPK, JAK/STAT and Nrf2/HO-1 signaling pathways
Phytomedicine: International Journal of Phytotherapy and Phytopharmacology
(2016)
Z. Hu et al.
Effects of esculentoside A on autoimmune syndrome induced by Campylobacter jejuni in mice and its modulation on T-lymphocyte proliferation and apoptosis
Int. Immunopharmacol.
(2010)
W.T. Zhong et al.
Protective effect of esculentoside A on lipopolysaccharide-induced acute lung injury in mice
J. Surg. Res.
(2013)
L. Minghetti et al.
Microglia as effector cells in brain damage and repair: focus on prostanoids and nitric oxide
Prog. Neurobiol.
(1998)
M. Mecha et al.
Microglia activation states and cannabinoid system: therapeutic implications
Pharmacol. Ther.
(2016)
L. Han et al.
Dalesconols B inhibits lipopolysaccharide induced inflammation and suppresses NF-kappaB and p38/JNK activation in microglial cells
Neurochem. Int.
(2013)
H. Liu et al.
Paeoniflorin attenuates Abeta1-42-induced inflammation and chemotaxis of microglia in vitro and inhibits NF-kappaB- and VEGF/Flt-1 signaling pathways
Brain Res.
(2015)
V. Selvaraj et al.
Inhibition of MAP kinase/NF-kB mediated signaling and attenuation of lipopolysaccharide induced severe sepsis by cerium oxide nanoparticles
Biomaterials
(2015)

K. Biber et al.

Central nervous system myeloid cells as drug targets: current status and translational challenges

Nat. Rev. Drug Discov.

(2016)

K. Saijo et al.

Microglial cell origin and phenotypes in health and disease

Nat. Rev. Immunol.

(2011)

M.L. Block et al.

Microglia-mediated neurotoxicity: uncovering the molecular mechanisms

Nat. Rev. Neurosci.

(2007)

C. Cunningham

Microglia and neurodegeneration: the role of systemic inflammation

Glia

(2013)

R.M. Ransohoff

How neuroinflammation contributes to neurodegeneration

Science

(2016)

R.M. Ransohoff et al.

Microglial physiology: unique stimuli, specialized responses

Annu. Rev. Immunol.

(2009)

Shuofeng Wang

Molecular Mechanisms of the Anti-Inflammatory Immune Regulation Actions of EsA

(2008)

J. Fang et al.

Inhibitory effect of esculentoside A on platelet activating factor released from calcimycin induced rat peritoneal macrophages

Yao xue xue bao = Acta Pharmaceutica Sinica

(1991)

Z.Y. Xiao et al.

Effects of esculentoside A on production of interleukin-1,2, and prostaglandin E2

Acta Pharmacol. Sin.

(2004)

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Fig. 1A). Previous studies found that EsA could regulate the inflammatory response and oxidative stress injury in various experimental models (Li-Hua et al., 2017; Wang et al., 2016; Wu et al., 2007; Yang et al., 2017; Yang et al., 2015; Zhong et al., 2013). According to both in vivo and in vitro researches, EsA exerts its biological role by regulating PPARγ activity.
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The current research aimed at investigating the protective effects and underlying mechanisms of EsA on the mitigation of cognitive deficits and pathology in triple transgenic AD mice.
Triple transgenic AD mice (3 × Tg-AD) of 8 months old received intraperitoneal treatment of 5 or 10 mg/kg EsA for 8 consecutive weeks. Morris water maze test and open field test were made to evaluate the cognitive function and degree of anxiety of the mice. Liquid chromatography with tandem mass spectrometry analysis was performed to characterize and to quantify EsA in the blood and brain of mice. Immunofluorescence assay and Western blot were adopted to measure the levels of peroxisome proliferator-activated receptor gamma (PPARγ) and key proteins in Aβ pathology, ER stress- and apoptosis-associated pathways. The combination of EsA with PPARγ were theoretically calculated by molecular docking programs and experimentally confirmed by the bio-layer interferometry technology.
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These natural products exhibit marked anti-inflammatory, antifungal, and anticancer effects [44]. The main compounds are ES-A and ES-B, which exert marked anti-inflammatory effects [45] and ES-H, which displays anti-inflammatory and anticancer properties (Figure 2) [46]. These compounds have the capacity to inhibit the release of pro-inflammatory cytokines (such as IL-1α/β, IL-2, and TNF-α), leading to major anti-inflammatory effects that can also contribute to their anticancer activities observed in vitro and in vivo [44] (Figure 3).
(Shanglu) is a well-known traditional herbal medicine that has been used for thousands of years in China. Phytolacca is also used worldwide in different traditional phyto-medicines and homeopathic preparations. The present work reviewed advances in the traditional uses, plant origin, chemical constituents, pharmacology, and medicinal properties of Phytolacca. Phytolacca is usually made from the roots of Phytolacca acinosa and P. esculenta, but the invasive plant P. americana (American pokeweed) is also widely used. Different types of medicinal products made with Phytolacca are available, as well as various types of Phytolacca extracts, showing a range of pharmacological activities including antioxidant, anti-inflammatory, anti-parasitic, antifungal, anticancer, and insecticidal effects. The marked anti-inflammatory activity of Phytolacca extracts supports its use in treating diseases such as arthritis, nephritis, and rheumatism, as well as in combatting cancer. Several bioactive natural products have been identified from Phytolacca, including glycosylated saponins such as esculentosides and phytolaccosides, as well as a few flavones (cochliophilin A) and phytosterols (α-spinasterol), which contribute to Phytolacca’s anti-inflammatory and/or anticancer effects. A quality evaluation of Phytolacca-based extracts and products is highly recommended; nevertheless, the use of Phytolacca can be encouraged to help regulate cytokine production and combat inflammatory diseases.

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