Hypertonic saline regulates microglial M2 polarization via miR-200b/KLF4 in cerebral edema treatment

https://doi.org/10.1016/j.bbrc.2018.03.161Get rights and content

Highlights

  • Hypertonic saline (HS) alleviates cerebral edema via regulating neuroinflammation.

  • miR-200b/KLF4 regulates microglial M2 polarization during HS treatment.

  • KLF4 directly binds to Arg1 promoter and induces its transcription.

  • Modulation of KLF4 in vivo affects HS treatment of cerebral edema.

Abstract

Background

Hypertonic saline (HS) has been used clinically for treatment of cerebral edema for decades. Previously we have demonstrated that HS alleviates cerebral edema via regulating water/ion channel protein and attenuating neuroinflammation. However, whether HS treatment triggers microglia polarization and its regulatory mechanism during this process is unclear.

Methods and results

The Sprague-Dawley (SD) rats that underwent right-sided middle cerebral artery occlusion (MCAO) were used for assessment of neuroinflammation and microglia functions. Treatment of 10% HS not only significantly reduced infarct size and ipsilateral ischemic hemispheric brain water content (BWC) via attenuating ischemia-induction of TNF-α, IL-1β, microglia M1 markers (iNOS, CD86) and miR-200b, but also increased neurotrophic factors such as IL-10 and IL-4, microglia M2 markers (Arg1, CD206) and Krüppel-like factor 4 (KLF4). Similar changes were confirmed in primary microglial cells subjected to hypoxia with/without HS in vitro. Importantly, overexpression of miR-200b was able to induce microglia M1 polarization via directly targeting KLF4. Restoring KLF4 expression abolished this effect. On the contrary, miR-200b inhibitor or KLF4 overexpression led to microglia M2 polarization. Mechanistically, KLF4 directly binds to promoter region of Agr1, thus inducing its transcription. Similar to treatment of HS, experimental overexpression of KLF4 in vivo exerted significant beneficial effects on ischemia-induced cerebral edema. However, knockdown of KLF4 abrogated the benefits of HS.

Conclusions

Hypertonic saline regulates microglial M2 polarization via miR-200b/KLF4 during its treatment of cerebral edema. This study may provide new insights of HS-related therapy for cerebral edema.

Introduction

Cerebral edema, represented as excess accumulation of fluid in the brain which causes intracranial hypertension (ICH), is a life-threatening condition that leads to severe outcome of patients [1]. Most frequently, this is the consequence of brain trauma, ischemic stroke, cancer, or brain inflammation [2]. The clinical management includes osmolar therapy and anti-inflammatory treatment. The former utilizes, most commonly, mannitol and hypertonic saline (HS) to remove free water from the intracellular into the extra-cellular space through osmotic force and reduction of peripheral vascular resistance [3]. Osmolar therapy has been well-established in the past decades [4], while anti-inflammatory treatment was introduced to target reperfusion injury (R/I) in recent years [5]. R/I, occurred when revascularization is delayed after critical injury of brain and vascular, not only increases lesion size but also aggravates the breakdown of blood barrier, leading to brain edema and hemorrhage [5]. Post-stroke inflammation was considered as a major underlying mechanism of R/I [6]. Interestingly, our previous studies have demonstrated that in addition to its osmotic force, 10% HS alleviates cerebral edema not only through regulating water channel protein or related ion channel transporters [7,8], but also via attenuating proinflammatory signaling and mediators in the brain [9]. Nevertheless, detailed mechanisms of HS-mediated anti-inflammation await elucidation.

Microglia are resident macrophage-like immune cells in the central nervous system (CNS) that are important for maintaining homeostasis and normal function of the CNS. Microglia regulate multiple aspects of inflammation, such as cytotoxicity, regeneration, repair and immunosuppression depending on their activation states or phenotypes [10]. Numerous studies have suggested that polarization of microglia may largely determine the outcome of CNS inflammation [[11], [12], [13]]. In inflammatory process of the CNS, polarized M1 microglia produce pro-inflammatory cytokines and neurotoxic molecules, which contribute to dysfunction of neural network and promote inflammation reaction, whereas polarized M2 microglia secrete anti-inflammatory mediators and neurotrophic factors that are involved in restoring homeostasis [14]. However, whether HS treatment triggers microglia polarization and its underlying molecular mechanisms during cerebral edema is not fully understood [15].

MiRNAs, consist of approximately 22 nt that repress target genes usually by binding to the 3′ untranslated region (UTR) of messenger RNA (mRNA), have emerged as key post-transcriptional regulators of gene expression in the past decade [16]. MiRNAs play important roles in various biological processes and in disease development and progression [[16], [17], [18]]. Accumulating evidences have revealed that miRNAs are acting as a fine tuner for brain endothelial barrier function [19], neuronal survival [20], microglia activation and neuroinflammation [21] after ischemic stroke or brain injury. Of note, dual effects on microglia polarization of miRNAs have been observed, either pro-inflammatory M1 [22] or anti-inflammatory M2 [23] phenotype. It's currently unclear whether miRNA is involved in HS-modulated microglia function switch related to polarization during cerebral edema treatment.

In this study, we continued our previous work on molecular mechanisms of HS alleviating cerebral edema. In a rat model of middle cerebral artery occlusion (MCAO), pro-inflammatory cytokines such as TNF-α, IL-1β were markedly upregulated and microglia underwent M1 polarization. 10% HS treatment could block these changes by inducing microglia M2 polarization. Importantly, we were able to prove that these processes were mediated by aberrant expression pattern of transcriptional factor KLF4 and its regulator miR-200b.

Section snippets

10% HS decreases the infarct size and ipsilateral ischemic hemispheric brain water content (BWC) via attenuating inflammation

To confirm the therapeutic effects of 10% HS on cerebral edema, infarct size and volume, ipsilateral ischemic hemispheric BWC and neuroinflammation were assessed. Results showed that the infarct size in the 10% HS group was much smaller than that of ischemic group (Fig. 1A) and the volume of cerebral infarct was significantly decreased with treatment of HS (Fig. 1B). Compared to sham group, BWC in the ipsilateral ischemic hemispheres was significantly increased, which could be attenuated by 10%

Discussion

Osmotherapy agents, such as hypertonic saline and mannitol, are currently being used for treatment of cerebral edema resulting from ischemic stroke [4]. Studies have showed that HS exerts 20% more effective than an equal volume of mannitol [33]. The present study confirmed that the infarct size and BWC in the 10% HS group was significantly decreased compared to the ischemic group, consistent with our previous data [8]. We have demonstrated that HS mediated downregulation of AQP4 and NKCC1 in

Ischemia-induced cerebral edema animals model

All animal protocols were approved by the Administrative Panel on Laboratory Animal Care at Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, and followed the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). A total of 125 male Sprague-Dawley (SD) rats (220–250 g) were randomly divided into a sham- operated group (sham group, n = 25), cerebral ischemic + normal saline group

Acknowledgement

This study is supported by Natural Science Foundation of Guangdong Province, China (2016A030313763), Science and Technology Program of Guangzhou, China (201707010322, 155700027) and Pilot Project of Guangzhou Clinical Medical Research and Translational Medicine Center (201508020005).

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