Protective effects of hydro-ethanolic extract of Terminalia chebula on primary microglia cells and their polarization (M1/M2 balance)

https://doi.org/10.1016/j.msard.2018.07.015Get rights and content

Highlights

  • TC extract significantly decreased inflammation responsible to LPS.

  • TC extract also increased anti-inflammation ratio of macrophage in inflammatory condition regarding to M1/M2 balance towards M2.

  • TC extract influences gene expression level of inflammatory and anti-inflammatory mediators.

  • TC extract had promising effects on healing of neuro-inflammatory neuro-degenerative disease through microglia roles.

Abstract

Teminalia chebula (TC) has been traditionally used in the Iranian traditional medicine (ITM) and Ayurvedic medicine primarily for neurologic disorders and inflammation. Mainly, its fruits have been applied for CNS disorders. The effects of Terminalia chebula as herbal medicine with anti-inflammatory and anti-oxidant properties were aimed on lipopolysaccharide (LPS)-induced microglial inflammation. Cytotoxicity of TC extract (0–80) µg/ml on microglial cells was evaluated using the MTT assay. Also, the protective effect of TC extract concentrations with specified amount of LPS-induced mice microglial cells was studied. The concentrations of TNF-α (Tumor Necrosis Factor-α), IL-1β (Interleukin-1β), IL-6 and PGE-2 (Prostaglandin-E2) were evaluated using ELISA. Gene expression of TNF-α, IL-1β, IL-6, COX-2 (Cyclooxygenase-2), iNOS and arginase-1 was also evaluated using the Real-Time PCR method. Nitrite oxide and urea were measured using biochemical methods. The studied concentrations of TC extract did not affect the viability of microglial cells but significantly protected the viability after treatment with LPS. The concentrations and expression levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6, PGE-2, COX-2) were significantly decreased after TC extract treatment in LPS-induced microglial cells with dose dependent manner. The extract also significantly decreased the levels of nitric oxide, increased urea and down regulated the expression of nitric oxide synthesis while arginase-1 expression was enhanced. Our results suggest that TC extract reduces inflammation in microglial cells and can be used as a potential anti-inflammatory agent in central nervous system inflammatory diseases.

Introduction

Inflammation is having a protective role and known major response against infection or injury (Rahimi et al., 2017), which in central nervous system (CNS) disorders are caused by ischemia, hypoxia, trauma and other issues, this leads to produce cytokines carrying signals for activation of microglial cells (Graeber and Streit, 2010). In this way, occurrence of neuro-inflammation is accompanied routine properties of neurodegenerative diseases such as dementia, multiple sclerosis and Alzheimer (Pasqualetti et al., 2015).

Microglial cells has been known the resident macrophage cells of brain and as a part of innate immunity system that act an important role in neuro-inflammatory diseases through putting down the cellular produced substances, and regulation of inflammatory or anti-inflammatory conditions. However, chronic activation of microglial cells caused chronic production of pro-inflammatory and cytotoxic mediators, which is known as an important factor in neurodegenerative diseases (Gaire et al., 2013). These cells, similar to other types of macrophage, could polarize to inflammatory and anti-inflammatory producing cells which typically named type 1 macrophage (M1) and type 2 macrophage (M2) cells, respectively (Murray and Wynn, 2011, Perego et al., 2016a). M1 types of macrophage cells are known inflammatory cells which promoted by type 1 T helper cells from resting macrophage (M0) or inflammatory states and cytokines such as presence of LPS and TNF-α, respectively. M1 cells producing pro-inflammatory mediators consisting of TNF-α, IL-1β, PGE2, NO and iNOS that can antagonize the M2 cells producing anti-inflammatory mediators that include IL-10, arginase 1 (Arg1) and urea, differentiate M2 or resting form of macrophage cell to M1 subtype (Gliem et al., 2016, Murray and Wynn, 2011). Regarding to brain injuries especially neuro-degenerative diseases such as multiple sclerosis, Alzheimer, strokes and other conditions, type 1 of microglia cells are formed due to secreted inflammatory cytokines and mediators. Since these cells encourage the degeneration of brain and its neurons, and also microglia cells have potency to polarize at different conditions from M1 to M2 type or vice versa, it could be applied as new immune-therapeutic approaches (Jetten et al., 2014, Perego et al., 2016a).

Terminalia chebula (TC) is identified as medicinal plant from Combretaceae, with complementary application in Ayurveda for urinary disorders, colic pain, CNS disorders, asthma and chronic cough as well as sore throat, inflammation and hiccups for many years in Iran, tropical India, Burma and Ceylon (Gaire and Kim, 2014, Sadeghnia et al., 2017). Terminalia chebula is also defined as “Halileh” by Iranian traditional medicine (ITM) reference text books in Persian such as the canon of medicine by Avicenna, that have explained the medicinal part of the plant is the fruits (Jokar et al., 2016). Recently, phyto-pharmacological studies have been demonstrated anti-inflammatory properties of the plant acting through inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in Lipopolysaccharides (LPS)-stimulated macrophages (Yang et al., 2014). Moreover, anti- arthritis and analgesic effects of this plant have been also observed (Seo et al., 2012). Terminalia chebula contains medicinal constituents such as tannin, chebulinic acid, galic acid, ellagic acid, chebulanin, rutin, casuarinin, quercetin and ascorbic acids (Jokar et al., 2016). It has also been indicated that this plant is able to inhibit nuclear translocation of NF-κB because of diminishing IκBα separation, that is known factor in inflammation and immune system responses in human lymphoblastic T cells (Das et al., 2011, Jacobs and Harrison, 1998, Vancurova and Vancura, 2012). Based on these, we were encouraged to investigate the possible anti-inflammatory effects of TC extract on isolated primary mice microglia and identify the ratio of type 1 and 2 macrophages in presence of LPS.

Section snippets

Materials

DMEM/F12 culture media, penicillin and streptomycin (pen/strep), amphotericin B, FBS, DMSO, Ficoll, DNAse I, Dispase II, LPS and other cell culture materials were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). RBC Lysis Buffer (10x, Cat No:420301) was from Biolegend company (San Diego, CA, USA). Proliferation assay kit (MTT), and ELISA kits (PGE2, IL-6, IL-1β, TNF-α and IFN-γ) were provided from Roche Diagnostic (Mannheim, Germany) and eBioscience (San Diego, CA, USA),

Yield, TPC and TFC of the extract

The yield, TPC and TFC of TC extract were estimated about 28% w/w, 128.362 mg of GAE/g of dried extract and 6.37 mg of QE/g of dried extract, respectively.

Effects of LPS and TC extract on cell proliferation

We observed that TC extract at the range of concentrations 5–80 μg/ml had no cytotoxicity on microglial cells proliferation and survives were approximately %100 compared to control group (Fig. 1A). Incubation with LPS led to a significant reduction in cell proliferation in comparison to normal control group (p < 0.001, Fig. 2B). In contrast,

Discussion

Based on our knowledge, this is the first reporting of anti-inflammatory effects of TC extract on microglia cells and their balances. In this study, LPS as innate immune system activator increased inflammatory cytokines and imbalance the M1/M2 ratio towards M1 inflammatory state, while TC extract could significantly reversed this outcome, and also prepare the M2 condition regarding to decrease the inflammatory cytokines, iNOS, NO and increase the Arg1 and urea.

Clearly, the inflammation is a

Conclusion

In summary, our results displayed anti-inflammatory and immune-modulatory properties of TC extract based on reduction in inflammatory biomarkers including TNF-α, IL-1β, IL-6, PGE2 and iNOS as well as NO and up-regulation of Arg1 and urea level. These results indicated that TC could be effective as complementary therapy to modulate disease with M1 predominance state such as MS, brain ischemia, Alzheimer and etc., to prepare healing and regeneration.

Declaration of competing interests

The authors declare that have no conflict of interest to disclose.

Author's contribution

Study concept and design: Vahid Reza Askari, Vafa Baradaran Rahimi; Performing the experiments: Vahid Reza Askari, Vafa Baradaran Rahimi, Pouria Rahmanian-Devin, Nafiseh Askari and Zahra Sane-Far. Analysis and interpretation of data: Vahid Reza Askari, Razieh Ghodsi and Sina Soheili-Far; Helping in extraction: Reza Shirazinia; Cytokines measurement: Vahid Reza Askari and Vafa Baradaran Rahimi; Drafting of the manuscript: Vahid Reza Askari; and Critical revision of the manuscript for important

Acknowledgements

This study financially supported by self-foundation of corresponding authors.

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