MicroRNA-374a-5p inhibits neuroinflammation in neonatal hypoxic-ischemic encephalopathy via regulating NLRP3 inflammasome targeted Smad6

Abstract

Aims

Neonatal hypoxic-ischemic encephalopathy (HIE) is still an important cause of neurological dysfunction. At present, there is no reliable biochemical index in clinical examination. Increasing evidence demonstrates that microRNAs (miRNAs) are involved in the process of HIE, and miR-374a-5p is down-regulated in HIE infants. In this study, the aim is to investigate the role and mechanism of miR-374a-5p in HIE.

Main methods

Sprague-Dawley (SD) rats were used to establish model of neonatal HIE, pathologic changes and inflammatory response of brain tissues were measured. Subsequently, primary microglia were induced by LPS (1 μg/ml) in vitro, the miR-374a-5p mimic, Ad-Smad6 adenovirus vector and NLRP3 siRNA oligo were applied for microglial transfection. Furthermore, the target relationship between miR-374a-5p and Smad6 was analyzed, while microglia activity and inflammatory factor (IL-1β, TNF-α and IL-6) levels were detected.

Key findings

Herein, we found that over-expression of miR-374a-5p significantly attenuated brain injury and strongly inhibited the release of pro-inflammatory cytokines in neonatal rat HIE model. In vitro, miR-374a-5p inhibited LPS-induced microglial pro-inflammatory cytokines production by regulating NLRP3 inflammasome. In addition, Smad6 was identified as a direct target of miR-374a-5p, and miR-374a-5p had a negative regulatory effect on Smad6 expression. By targeting Smad6, miR-374a-5p inhibited the activation of NLRP3 inflammatory signals in microglia and the subsequent release of pro-inflammatory factors.

Significance

Our study recognized that miR-374a-5p as a novel regulator of microglial activation in neonatal HIE highlighted potential therapeutic target for the treatment of neonatal hypoxic-ischemic brain injury.

Introduction

Neonatal hypoxic-ischemic encephalopathy (HIE) is attributed to hypoxia in the brain, causing approximately one million deaths each year, and is one of the leading causes of neonatal morbidity and mortality worldwide [1,2]. Although the continued progress of the treatment in the neonates, the molecular mechanisms of brain damage in neonates with hypoxia-ischemia is still unclear. Brain damage intensifies in the first few hours of birth and may be accompanied by long-term neurological disorders [3]. Consequently, targeted therapeutic interventions should be taken immediately after delivery for infants at risk of HIE. Hypothermia treatment (HT) can reduce oxygen, metabolic demand and inflammation, and inhibit apoptosis and cell death in clinical setting, thus improving the nervous system [4]. However, the range of treatment is very narrow, and nearly half of the infants who received hypothermia still died or had obvious neurological dysfunction [5]. At present, the criteria used to predict neonatal HIE severity are unreliable, so early biochemical markers will greatly contribute to clinical decisions.

Neuroinflammation is a chronic pathological process involving all classes of brain injury and disorder [6]. More and more reports confirm that the neuroinflammation induced by cerebral hypoxia-ischemia is the main factor of perinatal brain injury [7,8]. Cytokines released by neuroinflammation are considered to be the mediators of neonatal brain injury [9]. Dammann et al. expounded from the animal model of perioperative brain injury in infected environment [10]. Typically, the levels of inflammatory cytokines such as interleukin-1β (IL)-1β, IL-6 and IL-8 in cerebrospinal fluid of infants after HIE were increased [11]. Neuroinflammation is characterized by the activation of glial cells, especially microglia. Microglia are innate immune cells of the central nervous system that monitor and deal with invasive pathogens and environmental deterioration, and show resting phenotype under normal circumstances. In the case of stimuli such as brain injury, it may lead to adjacent neurons injury, eliciting more microglia reactivation [12]. In view of the implication of microglia in the neonatal HIE. Consequently, comprehending the regulation mechanism of microglia activation is conducive to the prevention of neonatal HIE.

MicroRNAs (miRNAs) are a highly conserved small RNA molecule (16–25 nucleotides in length) that recognize the 3′ untranslated region (3′UTR) of downstream target genes and induce mRNA degradation and/or inhibit translation. According to our understanding, the research on miRNA in HIE is very limited. Looney et al. published the first description about the miRNA analysis of the neonates, and reported that the miR-374a-5p is down-regulated in the umbilical blood of HIE infants [13]. Since miRNA regulates the expression of target mRNA by inhibition or degradation, it is a reliable bio-marker in molecular biological diagnosis [14] and may provide novel insights for HIE and other diseases. The aim of this study was to elucidate the biological function of miR-374a-5p and further explore its role in neonatal HIE.