Uric acid inhibits HMGB1-TLR4-NF-κB signaling to alleviate oxygen-glucose deprivation/reoxygenation injury of microglia

https://doi.org/10.1016/j.bbrc.2020.12.097Get rights and content

Highlights

  • Uric acid has promise for the treatment of acute ischemic stroke.

  • Uric acid inhibits the HMGB1-TLR4-NF-κB signaling to alleviate ODG/R injury in BV2 cells.

  • Uric acid exerts an anti-inflammatory effect intracellularly.

Abstract

Mounting evidence has implicated inflammation in ischemia–reperfusion injury following acute ischemic stroke (AIS). Microglia remain the primary initiator and participant of brain inflammation. Emerging evidence has indicated that uric acid has promise for the treatment of AIS, but its explicit mechanisms remain elusive. Here, we observed that uric acid reduced the severity of cerebral infarction and attenuated the activation of microglia in the cerebral cortex in a mouse middle cerebral-artery occlusion/reperfusion model. Thus, we speculated that uric acid may play a role by directly interfering with the inflammatory response of microglia. First, we investigated whether the HMGB1-TLR4-NF-κB signaling plays a role in oxygen glucose deprivation and reperfusion (OGD/R) injury of BV2 cells. Inhibition of the signaling significantly reduced the release of the proinflammatory cytokines tumor necrosis factor α (TNF-α), interleukin 1β (IL1β), and IL6 caused by OGD/R in BV2 cells. Second, uric acid weakened the decreased cell viability and lactate dehydrogenase release induced by OGD/R in BV2 cells. Finally, uric acid reduced the release of the proinflammatory cytokines TNF-α, IL1β, and IL6 caused by OGD/R in BV2 cells by dampening HMGB1-TLR4-NF-κB signaling, which was reversed by probenecid treatment, an inhibitor of the uric acid channel. Hence, uric acid halted the release of inflammatory factors and the decreased cell viability induced by ODG/R via inhibiting the microglia HMGB1-TLR4-NF-κB signaling, thereby alleviating the damage to microglia. This may be part of the molecular mechanisms by which uric acid protects mice against the brain damage of middle cerebral-artery occlusion/reperfusion.

Introduction

Stroke is the second leading cause of death in the world and the first cause of death among Chinese people [1,2]. Acute ischemic stroke (AIS), characterized by arterial occlusion, is the most dominant type and can lead to neurological deficits involving consciousness, language, and movement, even life-threatening events.

Vascular recanalization is currently the only effective method for treating AIS with improved prognosis, but the treatment time window is limited, and the reperfusion easily leads to ischemia–reperfusion injury (IRI). IRI is an essential pathophysiological process of AIS, and inhibiting cerebral IRI is key element of AIS therapy. The pathogenesis of IRI is not completely clear and may involve oxidative/nitrative stress [3], excitatory glutamate toxicity [4], inflammatory response [5,6], apoptosis and necrosis as well as intestinal flora disorder [7]. The inflammatory response is a key molecular mechanism, and the innate immune response it causes is related to the release of various damage-associated molecular patterns (DAMPs), which further activates pattern recognition receptors, including Toll-like receptors (TLRs) that are abundantly expressed on the membrane of microglia, TLR4 being the most important.

There is evidence that TLR4 is activated by endogenous proteins released by damaged brain cells and plays a pivotal role in IRI, with HMGB1 protein widely studied [8]. Studies have shown that plasma HMGB1 concentration in patients with cerebral ischemia is significantly increased [9], and HMGB1 concentration is positively correlated with cerebral infarction area. Anti-HMGB1 neutralizing antibodies reduced infarct size in rats after MCAO [10]. In activated microglia, HMGB1 undergoes acetylation and phosphorylation, which reduces the binding force with DNA, and is actively released outside the cell.

Originated from yolk-sac hematopoietic progenitor cells [11], microglia are resident immune cells of the central nervous system responsible for local immune surveillance and homeostasis regulation [12]. They express TLRs and Nod-like receptors to sense pathogens or damage signals and phagocytic cell debris and also induce inflammation. After AIS, microglia are activated to phagocytose damaged or necrotic nerve cells before peripheral macrophages infiltrate the brain tissue.

Uric acid (UA) is the oxidative end-product of human purine nucleotide, contributing to two-thirds of the total antioxidant capacity of plasma [13], and its neuroprotective effect is generally concerned. A multicenter randomized double-blind phase 2b/3 clinical trial (URICO-ICTUS) reported that uric acid administration reduced the incidence of early ischemic worsening, improved nerve function (modified Rankin Scale score), and decreased disability rate without any safety concerns [14,15]. In vivo experiments suggested that uric acid reduces the volume of cerebral infarction and improves the neurological function score in the rat MCAO model [[16], [17], [18], [19]].

In summary, uric acid shows good prospects in the treatment of AIS but with elusive molecular mechanisms. Whether uric acid has neuroprotective effects by inhibiting the HMGB1-TLR4-NF-κB signaling of microglia is unknown. Here, we showed that uric acid reduced the volume of cerebral infarction with attenuated activation of microglia in vivo. Then, we found that uric acid reduced the release of proinflammatory cytokines TNF-α, IL1β, and IL6 caused by OGD/R in BV2 cells by dampening HMGB1-TLR4-NF-κB signaling, which was reversed by probenecid, an inhibitor of the uric acid channel.

Section snippets

Reagents

The BV2 cell line was purchased from Kunming Cell Bank, Chinese Academy of Sciences. TAK-242 and glycyrrhizic acid were from MCE (USA); BAY 11-7085 and probenecid were from Santa Cruz Biotechnology (USA); uric acid and methyl thiazolyl tetrazolium (MTT) were from Sigma (USA); fetal bovine serum (FBS) was from Gibco (USA); BCA Protein and LDH release Assay Kits were from Beyotime (China); Nuclear and Cytoplasmic Protein Extraction Kit was from Sangon Biotech (China); radioimmunoprecipitation

Results

Uric acid reduces the cerebral infarction area and abolishes the activation of microglia in a mouse model of MCAO.

We constructed the MCAO model to uncover the role of uric acid in AIS. TTC staining and MRI revealed that uric acid significantly reduced the area of cerebral infarction in MCAO mice (Fig. 1A–B, Figs. S1A–C). Meanwhile, immunohistochemistry (IHC) was performed to observe the morphology of microglia labeled by anti-Iba1 antibody. Cerebral ischemia-reperfusion insult conferred a

Discussion

Inflammation has been considered a pivotal pathophysiological process of AIS accompanied by hyperactive microglia in ischemic penumbra, and suppressing the excessive activation of microglia is expected to alleviate the IRI of AIS. As the most important endogenous antioxidant, uric acid has promise for AIS therapy according to epidemiological and clinical studies. Within 6 h after the onset of AIS, serum uric acid concentration gradually decreases, which suggests that uric acid is a consumable

Ethics approval and consent to participate

All animal research procedures were performed as approved by the Institutional Animal Care and Use Committee (IACUC) of Xiamen University. This article does not contain any study with human participants performed by any author.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (81570772, 81471081 and 81703742), the Natural Science Foundation of Guangdong Province (2015A030313434), the Natural Science Foundation of Fujian Province (2020J01010) and the Gout Research Foundation (Japan, 2019). We thank Core Facility of Biomedical Sciences, Xiamen University.

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