Background

Stroke causes significant disabilities and cognitive impairment in afflicted individuals throughout the world [1]. The basic pathophysiology of cerebral ischemic stroke is complex, involving the interplay of autophagy, apoptosis, oxidative stress, inflammation, and energy attenuation [2, 3]. Recently, evidence has been presented highlighting the role of miRNAs in cerebral ischemia-reperfusion injury, identifying miRNAs as potential therapeutic targets [4, 5].

Dexmedetomidine (DEX) is a α2-adrenoceptor agonist that exhibits sedative, anxiolytic, and analgesic functions [6]. DEX is known to exert positive effects (in comparison with other sedatives), including mitigation of respiratory depression and hypotension, alleviation of lung and kidney damage, and decreased neuronal apoptosis [7]. DEX also has a long-term neuroprotective influence on cognitive dysfunction and brain injury [8].

The role of microRNAs (miRs), short non-coding RNA molecules, is to bind to mRNAs and inhibit the expression of target genes. The downregulation of miRs in neuronal cells is intricately linked to neurodegenerative disease [9]. Approximately 70% of all known miRs are expressed in the brain (either locally or tissue wide), and these are critical to the functionality of the nervous system [10]. Oxygen-glucose deprivation/reperfusion (OGD/R) miRs are reported to suppress apoptosis of develo** hippocampal astrocytes in rodents, thus affording protection against hepatic ischemia/ reperfusion injury [11, 12]. An interaction between miR-223-3p and TIAL1 has been demonstrated to contribute to the neuroprotective effects of DEX in hippocampal neuronal cells in vitro [13]. Hence, DEX may regulate OGD/R-induced inflammation and apoptosis through miRs. Hao et al. (2017) report that miR-17-5p is pro-apoptotic, and that miR-17-5p overexpression induces neuronal death and apoptosis [19]. DEX preconditioning is reported to protect the heart from apoptosis following ischemic/ reperfusion injury in a diabetic rat model (both in vivo and in vitro) by activating PI3K/ Akt signaling [20]. Furthermore, DEX preconditioning protects the heart from apoptosis in ischemic/ reperfusion injury in diabetic rats by activating PI3K/ Akt signaling in vivo and in vitro [21]. However, the mechanisms by which DEX regulates these effects remain to be elucidated.

miRNAs participate in a range of essential biological processes, including neuronal apoptosis during ischemic stroke and nervous system dysfunction [22]. Here, we explored the underlying biological mechanism of DEX attenuation of OGD/R-induced neurotoxicity, and we investigated the involvement of miR-17-5p and potential molecular factors. miR-17-5p is significantly upregulated in the early stage of cerebral ischemia-reperfusion injury (within 4 h). Moreover, onset of miR-17-5p upregulation is earlier than the observed changes in urea nitrogen level and neutrophil gelatinase-related lipocalin (NGAL) concentration [23]. miR-17-5p is known to be induced by p53 and to protect from renal ischemia-reperfusion injury by targeting death receptor 6 [24]. In addition, miR-17-5p is down-regulated by the Act A/ Smads signaling loop, thus enhancing the neuroprotective effect after ischemic injury [25]. Although these effects of miR-17-5p may modulate the therapeutic efficacy of DEX on OGD/R-induced neurotoxicity, no reports to this effect have been published thus far.

TLR4 belongs to the Toll-like receptor family. These innate pattern recognition receptors mediate the host response to pathogen infection [26]. TLR4 activation promotes the production of inflammatory cytokines, such as IL-1β, TNF-α, and IL-6 [27]. Aberrant IL-1β and IL-6 responses induced by TLR4 have been observed in patients. TLR4 mRNA is reported to have a binding site for miR-17-5p [28]. Thus, miR-17-5p may regulate inflammation induced by oxygen and glucose deprivation/ reperfusion via TLR4/ NF-κB [29].

In our present study, we report that miR-17-5p was downregulated in the OGD/R group, and that miR-17-5p mediated OGD/R-induced inflammation and apoptosis. DEX treatment increased miR-17-5p expression in a dose-dependent manner in PC12 cells. Moreover, miR-17-5p overexpression suppressed the inflammatory response by inhibiting NF-κB. Conversely, miR-17-5p downregulation produced the opposite phenotype. To explore the mechanisms underlying these effects, the TargetScan V7.2 database was employed to identify miR-17-5p regulated genes. miR-17-5p was predicted to bind to TLR4 mRNA. Our results reveal that miR-17-5p levels were negatively correlated with TLR4 levels, and that miR-17-5p binding to the 3′-UTR of TLR4 suppressed expression in a luciferase gene reporter assay. However, the role of miR-17-5p remained undefined.

To further investigate the role of miR-17-5p in OGD/R-induced inflammation and apoptosis in PC12 cells, miR-17-5p mimic and miR-17-5p inhibitor were separately transfected into each group. miR-17-5p mimic was observed to inhibit OGD/R-induced inflammation and apoptosis. Moreover, we identified NF-κB signaling as a potential mediator of miR-17-5p inhibition, and we subsequently demonstrated that miR-17-5p mimic could inhibit TLR4/ NF-κB signaling. For many drugs such as soy isoflavones and genistein, phosphorylation of IκBα and P65 is necessary for their neuroprotective effects [30]. From a consideration of these results, we propose that DEX upregulates miR-17-5p, and that miR-17-5p inhibits NF-κB subsequently reducing OGD/R-induced inflammation and apoptosis. In addition, we observed that inhibition of OGD/R-induced inflammation and apoptosis was suppressed following TLR4 overexpression or miR-17-5p silencing, which suggests that DEX attenuates oxygen-glucose deprivation/ reperfusion-induced inflammation and apoptosis in PC12 cells through an effect on the miR-17-5p/ TLR4/ NF-κB axis. Together, our data demonstrate the potential of DEX as a novel intervention strategy for cerebral ischemia-reperfusion injury. These findings require further verification in human stroke patients.

Our study had several limitations worth noting. First, the optimal concentration of DEX in vivo was not investigated. Therefore, DEX dosage should be further investigated in in vivo experiments. Second, DEX has only been observed thus far to up-regulate the expression of miR-17-5p in PC12 cells. Further studies are required to confirm these effects in vivo. On the side, in this study, DEX mainly exerts its protective effects through its anti-inflammatory activity, which is an ancillary activity compared to its main property (hypnotic drug). Would glucocorticoids, the reference anti-inflammatory drugs, provide the same protection while avoiding undesired central effects? Several studies in recent years have reported on this, Wang et al. [31] found knockout in studies glucocorticoid-regulated kinase 1 (SGK1) knockdown upregulated beclin-1 and LC-3 expression mediated by Intracarotid cold saline infusion (ICSI), This suggests that ICSI has a neuroprotective effect on ischemic stroke after reperfusion through up-regulation of SGK1 and inhibition of autophagy. The role of glucocorticoid signaling was also reported in the study of Perović et al. [32], They found Food restriction (FR) applied prior to TBI significantly changes p-GR levels, and it’s transcriptional activity during the recovery period after TBI. Moreover, as a pretreatment, FR modulates other protective factors in response to TBI, such as 11β-HSD1, NF-κB (p65) and HSP70 that act in parallel with GR in it’s anti-inflammatory and neuroprotective effects in the rat model of brain injury. That’s what we’re going to do next. Conclusion.

DEX inhibits OGD/R-induced inflammation and apoptosis in PC12 cells by increasing miR-17-5p expression, downregulating TLR4, and inhibiting NF-κB signaling. These results preliminarily explain the neuroprotective mechanism of DEX in ischemic stroke, and provide a direction for further searching for therapeutic targets for ischemic stroke.