Oral ellagic acid attenuated LPS-induced neuroinflammation in rat brain: MEK1 interaction and M2 microglial polarization

Abstract

Ellagic acid, the marker component of peels of Punica granatum L., is known traditionally to treat traumatic hemorrhage. In this study, the cellular mechanism underlying ellagic acid-induced anti-inflammation was investigated using lipopolysaccharides (LPSs) as a neuroinflammation inducer. Our in vitro data showed that LPS (1 μg/mL) consistently phosphorylated ERK and induced neuroinflammation, such as elevation in tumor necrosis factor-α (TNF-α) and nitric oxide production in treated BV-2 cells. Incubation of ellagic acid significantly inhibited LPS-induced ERK phosphorylation and subsequent neuroinflammation in treated BV-2 cells. Furthermore, our in vivo study of neuroinflammation employed an intranigral infusion of LPS that resulted in a time-dependent elevation in phosphorylated ERK levels in the infused substantia nigra (SN). Oral administration of ellagic acid (100 mg/kg) significantly attenuated LPS-induced ERK phosphorylation. A four-day treatment of ellagic acid did not alter LPS-induced ED-1 elevation but ameliorated LPS-induced reduction in CD206 and arginase-1 (two biomarkers of M2 microglia). A seven-day treatment of ellagic acid abolished LPS-induced increases in heme-oxygenase-1, cyclo-oxygenase 2, and α-synuclein trimer levels (a pathological hallmark) in the infused SN. At the same time, ellagic acid attenuated LPS-induced increases in active caspase 3 and receptor-interacting protein kinase-3 levels (respective biomarkers of apoptosis and necroptosis) as well as reduction in tyrosine hydroxylase-positive cells in the infused SN. In silico analysis showed that ellagic acid binds to the catalytic site of MEK1. Our data suggest that ellagic acid is capable of inhibiting MEK1-ERK signaling and then attenuated LPS-induced neuroinflammation, protein aggregation, and programmed cell deaths. Moreover, M2 microglial polarization is suggested as a novel antineuroinflammatory mechanism in the ellagic acid-induced neuroprotection.

Keywords: Ellagic acid; M2 microglial polarization; MEK-1; in silico assay; neuroinflammation; selumetinib.

Figure 1.

Ellagic acid inhibited LPS-induced ERK phosphorylation and proinflammatory cytokine in BV-2 cells. (A) and (B) BV-2 cells were treated with LPS (1 μg/mL) and ellagic acid (EA, 50, 100 µM) for 20 and 40 min, respectively. p-ERK in (A) and TNF-α in (B) were measured using the western blot assay. Graphs show statistical results from relative optical density of bands on the blots estimated by Image J software. (C) BV-2 cells were treated with LPS (1 μg/mL) and EA (25, 50, 100 µM) for 24 h. NO content was measured using the Griess reagents. Values are the mean ± SEM (n = 3/group). *P < 0.05 in the LPS group compared with the control group; #P < 0.05 in LPS plus EA groups compared with LPS group by one-way ANOVA and t-test.

Figure 2.

Binding models of ellagic acid and selumetinib with MEK1 protein (PDB code: 7JUS). (A) and (D): chemical structures of selumetinib and ellagic acid (EA). (B) and (E): spatial orientation of selumetinib and ellagic acid in MEK1 pocket. (C) and (F): hydrogen bonding formed between MEK1 and selumetinib as well as MEK1 and ellagic acid.

Figure 3.

Ellagic acid inhibited LPS-induced ERK phosphorylation and modulated LPS-induced M1/M2 microglial polarization in rat SN. LPS (4 μg/μL) was locally infused in the SN of anesthetized rats. (A) A time-dependent effect of LPS on ERK phosphorylation was investigated in the SN. Phosphorylated ERK protein levels in SN were measured using the western blot assay. Values are the mean ± SEM (n = 4/group). (B) Oral administration of ellagic acid (EA) was pretreated 1 h prior to the intranigral infusion of LPS. Three hours after LPS infusion, p-ERK protein levels in SN were measured using the western blot assay. Values are the mean ± SEM (n = 4/group). (C) to (E) Oral administration of EA was performed 1 h prior to intranigral infusion of LPS and daily for four days. Protein levels of (C) ED-1, (D) CD206, and (E) arginase 1 (ARG-1) in SN were measured using the western blot assay. Graphs show statistical results from relative optical density of bands on the blots estimated by the Image J software. Values are the mean ± SEM (n = 3–4/group). *P < 0.05 in the LPS group compared with the control group; #P < 0.05 in LPS plus EA group compared with LPS group by one-way ANOVA and t-test. n.s.: no significance.

Figure 4.

Ellagic acid attenuated LPS-induced oxidative stress and protein aggregation in rat SN. LPS (4 μg/μL) was locally infused in the SN of anesthetized rats. Oral administration of ellagic acid (EA) was performed 1 h prior to intranigral infusion of LPS and daily for seven days. (A) The effect of oral administration of EA for seven days on the body weight of rats. Values are the mean ± SEM (n = 10/group). Protein levels of (B) HO-1, (C) COX-2, and (D) α-synuclein aggregation in SN were measured using the western blot assay. Graphs show statistical results from relative optical density of bands on the blots estimated by the Image J software. Values are the mean ± SEM (n = 3–4/group). *P < 0.05 in the LPS group compared with the control group; #P < 0.05 in LPS plus EA group compared with LPS group by one-way ANOVA and t-test.

Figure 5.

Ellagic acid inhibited LPS-induced programmed cell death in rat SN. LPS (4 μg/μL) was locally infused in the SN of anesthetized rats. Oral administration of ellagic acid (EA) was performed for seven days. Protein levels of (A) procaspase 3 and cleaved-caspase 3 as well as (B) RIPK3 in SN were measured by the western blot assay. Graphs show statistical results from relative optical density of bands on the blots estimated by the Image J software. Values are the mean ± SEM (n = 3/group). *P < 0.05 in the LPS group compared with the control group; #P < 0.05 in LPS plus EA group compared with LPS group by t-test. Similar results were observed in duplicates. (C) Representative confocal microscopic data showed TH-positive neurons in the SN of rat. (D) Statistical data showed TH-positive cells in SN receiving intranigral infusion (i.n.) of LPS were counted and expressed as % of that in the contralateral intact SN of the same rat. Values are the mean ± SEM (n = 3/group). *P < 0.05 in the LPS group compared with the control group; #P < 0.05 in LPS plus EA group compared with LPS group by one-way ANOVA and t-test.