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NRF2 Exerts Anti-Inflammatory Effects in LPS-Induced gEECs by Inhibiting the Activation of the NF-κB

ABSTRACT: Nuclear factor E2-related factor 2 (NRF2) plays an anti-inflammatory role in several pathological processes, but its function in lipopolysaccharide- (LPS-) induced goat endometrial epithelial cells (gEECs) is still unknown. We designed a study to investigate the function of NRF2 in LPS-induced gEECs. LPS was found to increase the NRF2 expression and the nuclear abundance of NRF2 in gEECs in a dose-dependent manner. NRF2 knockout (KO) not only increased the expression of LPS-induced proinflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) but also increased the expression of TLR4, p-IκBα/IκBα, and p-p65/p65 proteins. Immunoprecipitation experiments showed that NRF2 directly binds to p65 in the nucleus and inhibits the binding of p65 to downstream target genes (TNF-α, IL-1β, IL-6, and IL-8). Even though a NF-κB/p65 inhibitor (PDTC) reduced the LPS-induced NRF2 expression and nuclear abundance of NRF2, overexpressing TNF-α reversed the inhibitory effects of PDTC on the NRF2 expression and on its abundance in the nucleus. Similarly, knockdown of the proinflammatory cytokines (TNF-α, IL-1β, IL-6, or IL-8) significantly decreased the LPS-induced NRF2 expression and NRF2 in the nucleus. In conclusion, our data suggest that proinflammatory cytokines induced by LPS through the TLR4/NF-κB pathway promote the NRF2 expression and its translocation into the nucleus. Our work also suggests that NRF2 inhibits the expression of proinflammatory cytokines by directly binding to p65.

SUBMITTER: Bao H

PROVIDER: S-EPMC8577927 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeTo explore the anti-inflammatory and neuroprotective effects of lithium chloride (LiCl) in LPS-induced retinal injury.MethodsIn vitro, primary retinal microglia were pretreated with LiCl and stimulated with lipopolysaccharide (LPS). Pro-inflammatory cytokine production, microglial morphological changes, and inflammation-associated signaling pathways were measured by real-time PCR (RT-PCR), western blotting, and immunofluorescence. Primary retinal neurons were cultured with microglial-derived conditioned medium in the absence or presence of LiCl. Neurotoxicity was evaluated by Cell Counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and γ-H2AX detection. In vivo, an endotoxin-induced uveitis mice model was established, and each animal was given intraperitoneal injection of LiCl or vehicle. The retinal inflammatory response was measured by hematoxylin and eosin and fluorescent staining, RT-PCR, western blotting, and TUNEL assay. Retinal thickness and function were evaluated by spectral-domain optical coherence tomography and electroretinography.ResultsIn vitro, LiCl exerted no obvious toxic effects on microglia and significantly decreased proinflammatory factor (inducible nitric oxide synthase, tumor necrosis factor α, interleukin 6) production, inhibited microglial activation in morphology, and suppressed nuclear factor kappa B (NF-κB), Akt, and phosphatidylinositol 3-kinase (PI3K) phosphorylation. Moreover, LiCl promoted retinal neuron survival and reduced cell apoptosis and the expression of γ-H2AX. In vivo, LiCl reduced inflammatory infiltrating cells in the vitreous cavity and decreased proinflammatory cytokine expression in retinas. LiCl suppressed LPS-induced microglial activation, proliferation, and migration. Additionally, LiCl reduced LPS-induced apoptosis of ganglion cells and retinal edema and rescued retinal functional damage.ConclusionsThis study demonstrates that LiCl exerts anti-inflammatory and neuroprotective effects by inhibiting microglial activation via the PI3K/Akt/NF-κB pathway in LPS-induced retinal injury. LiCl provides a novel and promising option to treat retinal inflammatory diseases.

Project description:BackgroundEndometriosis is well known as a chronic inflammatory disease. The development of endometriosis is heavily influenced by the estrogen receptor β (ERβ), while NOD-like receptors (NLRs) family CARD domain-containing 5 (NLRC5) exhibits anti-inflammatory properties during endometriosis. However, whether NLRC5-mediated anti-inflammation is involved in the ERβ-mediated endometriosis is still uncertain. This study aimed to assess that relation.MethodsNine cases of eutopic endometrial tissue and ten cases of ectopic endometrial tissue were collected from patients with endometriosis, and endometrial samples from ten healthy fertile women were analyzed, and the expression levels of ERβ were quantified using immunohistochemistry (IHC). Subsequently, we constructed mouse model of endometriosis by intraperitoneal injection. We detected the expression of ERβ, NLRC5, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-10 and measured the volume of ectopic lesions in mice with endometriosis. In vitro, human endometrial stromal cells (hESCs) were transfected respectively with ERβ-overexpressing and NLRC5-overexpressing plasmids. We then assessed the expression of ERβ and NLRC5 using quantitative real-time PCR (qRT-PCR) and western blot analysis. Furthermore, we measured the concentrations of TNF-α, IL-6, and IL-10 in the cell culture supernatant through enzyme-linked immunosorbent assay (ELISA). Additionally, we evaluated the migration and invasion ability of hESCs using transwell and wound healing assays.ResultsInhibition of NLRC5 expression promotes the development of ectopic lesions in mice with endometriosis, upregulates the expression of pro-inflammatory factors TNF-α and IL-6, and downregulates the expression of anti-inflammatory factor IL-10. The high expression of NLRC5 in endometriosis depended on the ERβ overexpression. And ERβ promoted the migration of hESCs partially depend on inflammatory microenvironment. Lastly, NLRC5 overexpression inhibited ERβ-mediated development and inflammatory response of endometriosis.ConclusionsOur results suggest that the innate immune molecule NLRC5-mediated anti-inflammation participates in ERβ-mediated endometriosis development, and partly clarifies the pathological mechanism of endometriosis, expanding our knowledge of the specific molecules related to the inflammatory response involved in endometriosis and potentially providing a new therapeutic target for endometriosis.

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NRF2 Exerts Anti-Inflammatory Effects in LPS-Induced gEECs by Inhibiting the Activation of the NF-κB

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