Safflor Yellow B Attenuates Ischemic Brain Injury via Downregulation of Long Noncoding AK046177 and Inhibition of MicroRNA-134 Expression in Rats

Sounds interesting. Now to just get our incompetent doctors, stroke hospitals and stroke associations to get human research going. Or is this the responsibility of stroke survivors?  With NO STRATEGY AND NO LEADERSHIP, nothing ever gets done in stroke.

Safflor Yellow B Attenuates Ischemic Brain Injury via Downregulation of Long Noncoding AK046177 and Inhibition of MicroRNA-134 Expression in Rats

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Research Article | Open Access

Volume 2020 |Article ID 4586839 | 20 pages | https://doi.org/10.1155/2020/4586839

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Chaoyun Wang,¹ Hongzhi Wan,² Qiaoyun Wang,² Hongliu Sun,² Yeying Sun,² Kexin Wang,³ and Chunxiang Zhang⁴

¹Hearing and Speech Institute, Binzhou Medical University, Yantai 264003, China
²School of Pharmaceutical Sciences, Binzhou Medical University, Yantai 264003, China
³Third Class of Senior High School, No. 2 Middle School of Yantai Shandong, Yantai, China
⁴Department of Biomedical Engineering, School of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA

Academic Editor: Reggiani Vilela Gonçalves

Received08 Jul 2019

Revised11 Mar 2020

Accepted29 Apr 2020

Published04 Jun 2020

Abstract

Stroke breaks the oxidative balance in the body and causes extra reactive oxygen species (ROS) generation, leading to oxidative stress damage. Long noncoding RNAs (lncRNAs) and microRNAs play pivotal roles in oxidative stress-mediated brain injury. Safflor yellow B (SYB) was able to effectively reduce ischemia-mediated brain damage by increasing antioxidant capacity and inhibiting cell apoptosis. In this study, we investigated the putative involvement of lncRNA AK046177 and microRNA-134 (miR-134) regulation in SYB against ischemia/reperfusion- (I/R-) induced neuronal injury. I/R and oxygen-glucose deprivation/reoxygenation (OGD/R) were established in vivo and in vitro. Cerebral infarct volume, neuronal apoptosis, and protein expression were detected. The effects of SYB on cell activity, cell respiration, nuclear factor erythroid 2-related factor 2 (Nrf2), antioxidant enzymes, and ROS were evaluated. I/R or OGD/R upregulated the expression of AK046177 and miR-134 and subsequently inhibited the activation and expression of CREB, which caused ROS generation and brain/cell injury. SYB attenuated the effects of AK046177, inhibited miR-134 expression, and promoted CREB activation, which in turn promoted Nrf2 expression, and then increased antioxidant capacities, improved cell respiration, and reduced apoptosis. We suggested that the antioxidant effects of SYB were driven by an AK046177/miR-134/CREB-dependent mechanism that inhibited this pathway, and that SYB has potential use in reducing or possibly preventing I/R-induced neuronal injury.

1. Introduction

Stroke is an important cerebrovascular disease that afflicts many people worldwide and frequently causes death or long-term disability [1]. Ischemic stroke is the most common type, accounting for about 80% of all strokes [2, 3]. Brain injury is caused by disruption of blood flow to the brain and is characterized by oxidative stress. In addition, reoxygenation resulting from the restoration of blood flow exacerbates tissue damage [4].
Pathophysiologically, ischemia and reperfusion can inhibit the activity of endogenous antioxidant enzymes and promote the overproduction of reactive oxygen species (ROS) [5–7]. Previous studies have shown that antioxidants significantly reduce ischemic damage through the inhibition of ROS production [8–11]. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a reduction-oxidation- (redox-) sensitive transcription factor that binds to antioxidant response elements (ARE) and activates the transcription of antioxidant enzymes. Studies have shown that cysteine residues on protein Keap1 are oxidized by ROS, leading to the release and activation of Nrf2 [12, 13]. Thus, Nrf2 is a useful therapeutic target for reducing or preventing ROS damage in the brain following ischemia/reperfusion (I/R) injury. Cyclic AMP (cAMP) response element-binding protein (CREB) is a leucine zipper transcription factor that inhibits ROS generation and suppresses severe ischemic injury by upregulating brain-derived neurotrophic factor (BDNF) and Bcl-2 [14, 15].
MicroRNAs (miRNAs) are endogenous, short (≈22 nucleotides), noncoding single-strand RNAs that regulate gene expression at the posttranscriptional level by influencing the translation of specific target mRNAs. Recent research revealed that a variety of miRNAs play important roles in ischemic injury through the modulation of cellular redox reactions and mitochondrial function [16]. For example, downregulation of miR-134 enhances Bcl-2 expression and alleviates ischemic injury by regulating CREB activity [17].
Long noncoding RNAs (lncRNAs) play key roles in various cellular contexts under both physiological and pathological conditions, and they are involved in diverse biological processes such as RNA processing, modulation of apoptosis and invasion, and chromatin modification [18–20]. AK046177 is a 606-base pair (bp) noncoding RNA sequence derived from a gene sequence (from 116850844 to 116851448) located on chromosome 13.
Safflower yellow is the flavonoid compound extracted from Carthamus tinctorius L., which includes the components hydroxysafflor yellow A (HSYA) and safflor yellow B (SYB). It has been shown to effectively reduce oxidative stress-mediated damage [21, 22]. A study by Wang et al. demonstrated that HSYA significantly increases antioxidant enzyme activity by activating the cAMP/PKA signaling pathway [23]. SYB (Figure 1(a)) is a yellow amorphous powder with a purity of more than 98% by HPLC, and it is water soluble and has demonstrated protective effects in neuronal injury models induced by oxidative stress [24, 25]. However, its effect on brain injury induced by I/R remains to be investigated. This study tested whether SYB reduces I/R-mediated brain injury, and evaluated its potential mechanisms by studying changes in the expression of AK046177, miR-134, Nrf2, and CREB.

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Figure 1 

Effect of safflor yellow B on neurological deficit score, infarction area, total motor score, and cAMP level. (a) Chemical structure of safflor yellow B. As shown in (b)–(e), rats were divided into six groups: sham, ischemia/reperfusion (I/R), AK046177 siRNA, AK046177 siRNA+miR-134 agomir, SYB, SYB+miR-134 agomir. Except for the sham group, all rats in the other groups had established ischemia for 1 h followed by reperfusion for 23 h. SYB and saline were administrated by tail vein continuously for three days before treatment with I/R, and miR-134 agomir and AK046177 siRNA were given via intracerebroventricular injection. The neurological deficit score of each rat was obtained according to Longa’s method. Infarct volumes were measured by staining brain sections with 2,3,5-triphenyltetrazolium chloride. (a) represents pathological changes of cerebral infarction ((a) sham; (b) I/R; (c) AK046177 siRNA; (d) AK046177 siRNA+miR-134 agomir; (e) SYB; (f) SYB+miR-134 agomir). (b) represents neurological deficit scores. (c) represents the infarction area. (d) represents total motor scores (n=8). As shown in (f)–(g), cAMP levels in the cerebral cortex and primary fetal cortical cells were detected using a ¹²⁵I-radioimmunoassay according to the method described by the manufacturer. Data are presented as mean ±S.D (n=8 in tissues;n=3 in cells). One-way ANOVA test was used to determine statistical significance.**P<.01 vs the sham group or the control group, ^## P<0.01 vs. the I/R group or the OGD/R group,^ΔP<0.05 or ^ΔΔP<0.01 vs. the AK046177 siRNA group, and ⁺P<0.05 or ⁺⁺P<0.01 vs. the SYB group.