Epigallocatechin Gallate Protects against Hypoxia-Induced Inflammation in Microglia via NF-κB Suppression and Nrf-2/HO-1 Activation

Abstract

Hypoxia-induced neuroinflammation in stroke, neonatal hypoxic encephalopathy, and other diseases subsequently contributes to neurological damage and neuronal diseases. Microglia are the primary neuroimmune cells that play a crucial role in cerebral inflammation. Epigallocatechin gallate (EGCG) has a protective antioxidant and anti-inflammatory effects against neuroinflammation. However, the effects of EGCG on hypoxia-induced inflammation in microglia and the underlying mechanism remain unclear. In this study, we investigated whether EGCG might have a protective effect against hypoxia injury in microglia by treatment with CoCl₂ to establish a hypoxic model of BV2 microglia cells following EGCG pre-treatment. An exposure of cells to CoCl₂ caused an increase in inflammatory mediator interleukin (IL)-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 expression, which were significantly ameliorated by EGCG via inhibition of NF-κB pathway. In addition, EGCG attenuated the expression of hypoxia-inducible factor (HIF)-1α and the generation of ROS in hypoxic BV2 cells. Furthermore, the suppression of hypoxia-induced IL-6 production by EGCG was mediated via the inhibition of HIF-1α expression and the suppression of ROS generation in BV2 cells. Notably, EGCG increased the Nrf-2 levels and HO-1 levels in the presence of CoCl₂. Additionally, EGCG suppressed hypoxia-induced apoptosis of BV2 microglia with cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3. In summary, EGCG protects microglia from hypoxia-induced inflammation and oxidative stress via abrogating the NF-κB pathway as well as activating the Nrf-2/HO-1 pathway.

Keywords: NF-κB; epigallocatechin gallate; hypoxia; inflammation; microglia.

Figure 1

Effect of EGCG on CoCl₂-induced cytotoxicity in BV2 cells. (a) Cell viability was determined by WST assay in BV2 cells in the presence of different concentrations of EGCG (50, 100, 150, 200, and 250 μM) for 8 h. (b) Cell viability was analysed in various concentration (200, 250, 300, and 350 μM) of CoCl₂-treated BV2 cells by WST assay. (c) The mRNA expression of IL-6, iNOS, and COX-2 was analysed by real time PCR in various concentrations (200, 250, 300, and 350 μM) of CoCl₂-treated BV2 cells. (d) Cells was pre-treated with or without various concentrations of EGCG (50–250 μM) for 1 h before CoCl₂ (350 μM) exposure for 8 h and WST assay was performed. The data represent mean ± SEM based on triplicate independent experiments. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group; ^### p < 0.001 vs. CoCl₂-treated group.

Figure 2

Effect of EGCG on the expression of pro-inflammatory mediators in CoCl₂-treated BV2 cells. Cells were treated with EGCG (200 μM) for 1 h prior to CoCl₂ (350 μM) exposure for 8 h. The mRNA expression of IL-6 (a), iNOS (b) and COX-2 (c) was determined by real-time PCR. (d) The protein expression of IL-6, iNOS, COX-2, and β-actin was detected by Western blot analysis. The data represent mean ± SEM. ns, not significant, *** p < 0.001 vs. control group. ^# p < 0.05, ^### p < 0.001 vs. CoCl₂-treated group.

Figure 3

Effect of EGCG on NF-κB activation in CoCl₂-treated BV2 cells. (a) Cells pre-treated with EGCG (200 μM) for 1 h were treated with CoCl₂ (350 μM) for 8 h. The levels of p-IκB, IκB, p-NF-κB p65, and NF-κB p65 proteins were detected by Western blot analysis and the histograms normalised to β-actin are presented. (b) Cells pre-treated with EGCG (200 μM) or JSH-23 (30 μM) for 1 h were treated with CoCl₂ (350 μM) for 8 h. DNA binding activity of NF-κB p65 was measured from nuclear extracts and quantified by a microplate reader. The data represent mean ± SEM. ns, not significant, * p < 0.05, *** p < 0.001 vs. control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. CoCl₂-treated group.

Figure 4

Effect of EGCG on HIF-1α expression and ROS production in CoCl₂-treated BV2 cells. (a,b) Cells were treated with or without EGCG (200 μM) for 1 h prior to CoCl₂ (350 μM) exposure for 8 h and the expression of HIF-1α was determined using real-time PCR and Western blot analysis, respectively. (c) ROS generation was determined by H₂DCFDA and fluorescent images were analysed microscopically in CoCl₂ (350 μM)-treated cells with EGCG (200 μM), NAC (5 mM), or YC-1 (10 μM). (d) Quantitative analysis of DCF fluorescence by microplate reader. (e) Cells were treated in the presence of CoCl₂ (350 μM) with or without NAC (5 mM) for 8 h and the expression of HIF-1α protein levels was detected by Western blot. (f) Cells were treated with CoCl₂ (350 μM) in the presence or absence of NAC or YC-1 for 8 h and the levels of IL-6 protein released in the supernatant were analysed with ELISA. The data represent mean ± SEM. ns not significant, *** p < 0.001 vs. control group; ^# p < 0.05, ^### p < 0.001 vs. CoCl₂-treated group.

Figure 5

Effect of EGCG on HO-1 expression and Nrf-2 translocation in CoCl₂-treated BV2 cells. (a) Cells were treated with EGCG (200 μM) prior to CoCl₂ (350 μM) treatment for 8 h, and the protein expression of HO-1 was detected by Western blot analysis. (b) The protein levels of Nrf-2 in cytosolic or nuclear fraction were measured by Western blot analysis in CoCl₂ (350 μM)-treated BV2 cells with EGCG (200 μM) or NAC (5 mM) for 8 h. The results represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. CoCl₂-treated group.

Figure 6

Effect of EGCG on CoCl₂-induced apoptosis in BV2 cells. (a) Cells were treated with EGCG (200 μM) or NAC (5 mM) for 1 h prior to CoCl₂ (350 μM) for 8 h in BV2 cells. The proportions of apoptotic cells were determined by flow cytometry. (b) Cells were treated with EGCG (200 μM) for 1 h prior to CoCl₂ (350 μM) for 8 h. The levels of cleaved PARP and cleaved caspase-3 were detected by Western blot analysis using anti-PARP and anti-cleaved caspase-3 antibodies. The results are expressed as mean ± SEM. * p < 0.05 *** p < 0.001 vs. control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. CoCl₂-treated group.