Centipeda minima extract exerts antineuroinflammatory effects via the inhibition of NF-κB signaling pathway

Abstract

Background

Centipeda minima (L.) A.Br. (C. minima) has been used in traditional Chinese herbal medicine to treat nasal allergy, diarrhea, asthma and malaria for centuries. Recent pharmacological studies have demonstrated that the ethanol extract of C. minima (ECM) and several active components possess anti-bacterial, anti-arthritis and anti-inflammatory properties. However, the effects of ECM on neuroinflammation and the underlying mechanisms have never been reported.

Purpose

The study aimed to examine the potential inhibitory effects of ECM on neuroinflammation and illustrate the underlying mechanisms.

Methods

High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was performed to qualify the major components of ECM; BV2 and primary microglial cells were used to examine the anti-inflammatory activity of ECM in vitro. To evaluate the anti-inflammatory effects of ECM in vivo, the mice were orally administrated with ECM (100, 200 mg•kg⁻¹•d⁻¹) for 2 days before cotreatment with LPS (2 mg•kg⁻¹•d⁻¹, ip) for an additional 3 days. The mice were sacrificed the day after the last treatment and the hippocampus was dissected for further experiments. The expression of inflammatory proteins and the activation of microglia were respectively detected by real-time PCR, ELISA, Western blotting and immunofluorescence.

Results

HPLC-MS/MS analysis confirmed and quantified seven chemicals in ECM. In BV2 and primary microglial cells, ECM inhibited the LPS-induced production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), thus protecting HT22 neuronal cells from inflammatory damage. Furthermore, ECM inhibited the LPS-induced activation of NF-κB signaling pathway and subsequently attenuated the induction of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), NADPH oxidase 2 (NOX2) and NADPH oxidase 4 (NOX4), leading to the decreased production of nitrite oxide, prostaglandin E₂ (PGE₂) and reactive oxygen species (ROS). In an LPS-induced neuroinflammatory mouse model, ECM was found to exert anti-inflammatory activity by decreasing the production of proinflammatory mediators, inhibiting the phosphorylation of NF-κB, and reducing the expression of COX2, iNOS, NOX2 and NOX4 in the hippocampal tissue. Moreover, LPS-induced microglial activation was markedly attenuated in the hippocampus, while ECM at a high dose possesses a stronger anti-inflammatory activity than the positive drug dexamethansone (DEX).

Conclusion

These findings demonstrate that ECM exerts antineuroinflammatory effects via attenuating the activation of NF-κB signaling pathway and inhibiting the production of proinflammatory mediators both in vitro and in vivo. C. minima might become a novel phytomedicine to treat neuroinflammatory diseases.

Introduction

Neuroinflammation is implicated in the pathogenesis of many brain diseases, especially neurodegenerative diseases (Ransohoff, 2016), in which the activation of microglia is one of the main characteristics. In response to inflammatory stimuli, microglia is quickly activated and recruited to the neurinflammatory site, then contributing to the development of neuroinflammation via the production of inflammatory mediators (Simon et al., 2019) .

A variety of inflammatory stimuli such as LPS can directly activate microglia through a cascade of molecular changes. As an inflammatory transcriptional factor, NF-κB is critical in mediating the activation of microglia. Normally, NF-κB is sequestered in the cytoplasm by the inhibitor IκB-α, whereas the inflammatory signal downregulates IκB-α and induces the phosphorylation and nuclear translocation of NF-κB (Li et al., 2013). Once activated, NF-κB stimulates the transcription of genes encoding inflammatory cytokines and inflammatory enzymes, leading to the expression of iNOS and COX2 and the generation of proinflammatory mediators, such as IL-1β, TNF-α, nitric oxide and PGE₂, which extensively contribute to microglia-mediated neurotoxicity (Cunningham et al., 2009). Moreover, oxidative stress that induced by chronic inflammation can further contribute to the development of neuroinflammation (Block et al., 2007). For example, LPS can directly activate the NADPH oxidase, including NOX2 and NOX4, which significantly increase microglia-derived ROS, activate inflammatory-related signaling, and severely accelerate the neuronal damage (Haslund-Vinding et al., 2017; Qin et al., 2004). Therefore, targeting microglia has been regarded as a prospective therapeutic strategy for the treatment of neuroinflammatory diseases.

Medicinal formula and plant extracts have been used for centuries to treat inflammatory diseases. C. minima is widely distributed in East and Southeast Asia and used in the treatment of a number of diseases, such as asthma, rhinitis, sinusitis and diarrhea (Liu et al., 2005; Wu et al., 1991). Recently, the major chemical components of C. minima extract, including terpenoids, polyphenol, phenolics, organic acid and flavonoids, have been determined based on the HPLC-MS/MS system (Ding et al., 2009; Wu et al., 2012). For example, isochlorogenic acid A and 6-O-angeloylplenolin, have been found to be most abundantly distributed in the herb and proposed to be used for the quality control of C. minima extract (Chan et al., 2016, 2019). Pharmacological studies have demonstrated that C. minima and its active components harbors antibacterial, anticancer, antiallergy, antioxidant and anti-inflammatory activities, with good pharmacological profiles and less toxicity (Liu et al., 2011, 2015; Taylor and Towers, 1998; Wu et al., 2012). For example, both the aqueous and hydroalcoholic extract of C. minima have been demonstrated to exert anti-inflammatory and antioxidant activities (Huang et al., 2013). Our recent study also find that ECM significantly alleviates the oxidative stress in the hippocampus and exerts neuroprotective effects (Wang et al., 2019); moreover, 6-O-angeloylplenolin, the major chemical constituents of ECM, has been shown to inhibit the inflammatory response in the central nervous system (CNS) (Li et al., 2019; Zhou et al., 2019), suggesting that ECM has potential anti-inflammatory activities in the CNS.

In this study, ECM with a good quality was prepared to examine the antineuroinflammatory activities and the underlying mechanisms. We found that ECM exhibits neuroprotective effect via the inhibition of LPS-induced neuroinflammation in microglial cells. ECM inhibits the activation of NF-κB signaling and the induction of inflammatory enzymes, leading to the decreased production of proinflammatory mediators. Taken together, this study demonstrates that ECM has a therapeutic potential for the treatment of neuroinflammatory diseases.