The Ameliorative Effects of the Ethyl Acetate Extract of Salicornia europaea L. and Its Bioactive Candidate, Irilin B, on LPS-Induced Microglial Inflammation and MPTP-Intoxicated PD-Like Mouse Model

Abstract

Hyperactivation of microglia, the resident innate immune cells of the central nervous system, exacerbates various neurodegenerative disorders, including Parkinson's disease (PD). Parkinson's disease is generally characterized by a severe loss of dopaminergic neurons in the nigrostriatal pathway, with substantial neuroinflammation and motor deficits. This was experimentally replicated in animal models, using neurotoxins, i.e., LPS (lipopolysaccharides) and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Salicornia europaea L. (SE) has been used as a dietary supplement in Korea and Europe for several years, due to its nutritional and therapeutic value. In this study, we intend to investigate the antineuroinflammatory and anti-PD-like effects of the bioactive fraction/candidate of the SE extract. Initially, we screened various fractions of SE extract using an in vitro antioxidant assay. The optimal fraction was investigated for its in vitro antineuroinflammatory potential in LPS-stimulated BV-2 microglial cells and in vivo anti-PD-like potential in MPTP-intoxicated mice. Subsequently, to identify the potential candidate responsible for the elite therapeutic potential of the optimal fraction, we conducted antioxidant activity-guided isolation and purification; the bioactive candidate was structurally characterized using nuclear magnetic resonance spectroscopy and chromatographic techniques and further investigated for its in vitro antioxidative and antineuroinflammatory potential. The results of our study indicate that SE-EA and its bioactive candidate, Irilin B, effectively alleviate the deleterious effect of microglia-mediated neuroinflammation and promote antioxidative effects. Thus, they exhibit potential as therapeutic candidates against neuroinflammatory and oxidative stress-mediated PD-like neurodegenerative complications.

Figure 1

Phytochemical profiling and antioxidant screening of varied SE subfractions. Fractionation of Salicornia europaea L. hot water (SE-HW) extract using organic solvent partitioning. Chemical compositions and antioxidative activities of the SE subfractions obtained from organic solvent partitioning of SE-HW. We yield SE-HW's fraction of n-hexane (SE-H), chloroform (SE-C), ethyl acetate (SE-EA), butanol (SE-B), and remained (SE-Q). (a) Organic solvent partitioning of SE-HW, (b) chemical composition of 6 subfractions, and (c) antioxidant activities of SE subfractions obtained from organic solvent partitioning of Salicornia europaea hot water (SE-HW) extract. Antioxidative activity was measured using DPPH radical scavenging potential (c). ^∗∗∗ p < 0.001 vs. lowest concentration.

Figure 2

Bioactivity-guided isolation and characterization of Irilin-B from SE-EA fraction. Antioxidant activity-guided isolation and characterization of compound L13-1 from SE-EA. (a) Purification scheme of compound L13-1 from SE-EA; (b) representative HPLC profiles of SE-EA, SE-EA-L13, and finally purified compound L13-1, Irilin B. HPLC (1260 Infinity, Agilent Technologies, Santa Clara, CA, USA) equipped with a ZORBAX Eclipse XDB C18 prep column (9.4 × 250 mm, 5 μm, Agilent Technologies) was conducted with a gradient eluent of methanol and 0.04% trifluoroacetic acid as the mobile phase. The UV spectrum and chemical structure of the finally purified compound L13-1 are also depicted. (c) NMR spectra of the purified compound L13-1. c1: ¹H-NMR spectrum; c2: ¹³C-NMR spectrum. (d) Determination of stereographic structure by analysis of two-dimensional ¹H-¹H COSY and HMBC-NMR spectra and assignments of carbon and hydrogen in NMR spectra.

Figure 3

The cytotoxicity effects and NO inhibitory potential of SE-EA in LPS-stimulated BV-2 microglial cells. SE-EA (20,100, and 200 μg/mL) were treated onto BV-2 cells with or without LPS and incubated in a CO₂-supplied incubator for 20 hours. A ROS defense protein and HO-1 expression levels were analysed by a western blot and qRT-PCR (a). SE-EA treatment onto BV-2 microglial cells reduced ROS levels and induced HO-1 expressions. Anti-inflammatory effects of SE-EA in LPS-stimulated BV-2 microglial cells. SE-EA (20, 100, and 200 μg/mL) and LPS (200 ng/mL) were cotreated onto BV-2 cells, which were incubated for 20 hours in a CO₂-supplied incubator. Each group's nitric oxide release was measured using a Griess reagent, and cell viability was assayed by an MTT reagent (b). Of the western blot analysis, inflammatory mediators iNOS and COX-2 expression levels were presented. The intensity of each protein band was measured using ImageJ (c). The expressions of iNOS, COX-2, and proinflammatory cytokines TNF-α, IL-1β, and IL-6 were measured by qRT-PCR analysis (d). SE-EA treatments suppressed the expression of inflammatory genes. Values are mean ± standard deviation. # marks vs. the control group, ∗ marks vs. the LPS-stimulated group. ^∗ p < 0.01, ^∗∗ p < 0.05, and ^∗∗∗ p < 0.001. ns: statistically not significant. p values were achieved using a one-way ANOVA analysis (Tukey method).

Figure 4

SE-EA-attenuated motor deficits and tyrosine hydroxylase depletion in MPTP-intoxicated PD-like mouse model. Animal experiment schedule is presented (a). Mouse coordination function was measured using a pole test. Each result is marked by dots and a thick bar is mean ± standard deviation (b). In IHC/DAB, the staining results indicate TH-positive neurons (substantia nigra pars compacta or SNpc) and its axon terminals (striatum or Str.). In the SNpc region, dopaminergic neurons stained by the IHC/DAB staining method and the TH-positive area were measured using ImageJ (c). In the graph, the relative TH-positive area's mean ± standard deviation is presented (d). ^### p < 0.001 vs. the control group and ^∗ p < 0.01, ^∗∗ p < 0.05, and ^∗∗∗ p < 0.001 vs. LPS-stimulated group. p values were achieved using a one-way ANOVA analysis (Tukey method).

Figure 5

Effects of Irilin B in molecular level alterations of proinflammatory cytokines/mediators and antioxidant biomarker. A major flavonoid of SE-EA, Irilin B (20, 10, and 20 μM) was treated on LPS-stimulated BV-2 microglial cells. Cells were incubated for 20 hours in a CO₂-supplied incubator, and the HO-1 expression levels were measured using a western blot and qRT-PCR (a). Nitric oxide (NO) releases were measured by Griess reagent assay, and cell viabilities were assayed by using MTT reagent (b). Including iNOS and COX-2, proinflammatory cytokines, TNF-α, IL-1, and IL-6 expression levels were analysed using the qRT-PCR method (c). # marks vs. the control group, ∗ marks vs. the LPS-stimulated group. ^∗ p < 0.01, ^∗∗ p < 0.05, and ^∗∗∗ p < 0.001. ns: statistically not significant. p values were achieved using a one-way ANOVA analysis (Tukey method).