In our study, it was found that the viability of PC-12 cells decreased significantly, while the degree of apoptosis and autophagy increased significantly, when PC-12 cells were treated with OGD. After the use of Remifentanil, the OGD damage of cells was alleviated, which was manifested by the recovery of cell viability and the decrease of apoptosis and autophagy. The expression of apoptosis and autophagy-related proteins also changed accordingly. The following mechanism analysis showed that miR-124 was positively regulated by Remifentanil, and the cytoprotective effect of Remifentanil was reversed by knocking down miR-124.
In the context of neurosurgery operations, patients often experience hypoxic and ischemic brain injury. Cerebral ischemia leads to neuronal death and eventually leads to neurological dysfunction. The ischemia-reperfusion (I/R) process of the brain puts the brain tissue in a state of hypoxia and ischemia, thus activating various cell-death processes, such as necrosis, apoptosis or autophagy-related cell death [23-25]. Apoptosis, a form of cell death, plays a vital role in normal development and tissue homeostasis. By contrast, inappropriate or excessive apoptosis is associated with many types of neurodegenerative disease, including cerebral ischemia [26]. In addition, autophagy is an evolutionarily conservative process of massive degradation and recycling of cytoplasmic proteins and organelles [27]. As a degradation / recirculation system, autophagy appears in the pathological changes of many organs and plays an important role in cerebral ischemia [28-29]. Our results showed that the neuroprotective effect of Remifentanil on PC-12 cells was indeed achieved through apoptosis and autophagy, which was consistent with the above studies.
Opioid agonists evinced neuroprotective effects in relieving hypoxia injury. Remifentanil was a new type of opioid drug, which had the following characteristics: quick action, fast clearance rate and short blood-brain balance time. Previous studies had shown that Remifentanil plays a role in the cardio-protection of ischemic cardiac injury through opioid receptors [29]. In addition, recent reports had found that Remifentanil preconditioning had a neuroprotective effect against cerebral ischemic injury. For example, Remifentanil exerted a neuroprotective effect on global cerebral ischemia-reperfusion injury, by improving spatial learning and memory in rats. It was related to the inhibition of neuronal apoptosis in the hippocampal CA1 region, and the regulation of signal-pathway and apoptosis-related genes. Remifentanil treatment enhanced the expression of the anti-apoptotic gene Bcl-2 and inhibited the expression of the pro-apoptotic gene Bax in the hippocampal CA1 region [30]. Animal experiments showed that Remifentanil preconditioning significantly reduced the infarct size and neurological damage after focal cerebral ischemia in rats. Remifentanil had an anti-apoptotic effect in vitro, but had no effect on necrotizing death. Conversely, it significantly decreased the activity of caspase-3 and the level of cleaved caspase-3, inhibited the expression of Bax protein and the activity of caspase-9 in the cortex, and maintained the integrity of mitochondria. Mechanism studies had found that this involved opioid and N-methyl-D-aspartate (NMDA) receptors, as well as mitochondrial-dependent apoptosis pathways [31].
These results suggested that Remifentanil had the potential effect of reducing hypoxia injury and protecting the survival of nerve cells. In a manner similar to our experimental results, OGD significantly decreased the viability of PC-12 cells, and deepened the degree of apoptosis and autophagy. Nonetheless, after pre-treatment with Remifentanil, this situation was reversed, which fact was reflected in the obvious enhancement of cell viability, the decrease in the number of apoptotic cells and the expression of intracellular cleaved caspase-3 and Bax proteins. Meanwhile, there was an increase of p62 expression and a decrease in the LC-I/LC-II ratio, which meant that the process of apoptosis and autophagy was weakened. This was consistent with the conclusions obtained by our predecessors.
MiRNAs is one of the main types of non-coding RNA derived from most genes, and it plays an important role in mRNAs transcription and protein synthesis [32]. MiR-124 is the key regulator of microglia in the central nervous system [33]. It has been reported that up-regulation of miR-124 may regulate apoptosis and autophagy in patients with Parkinson's disease (PD), thereby reducing the loss of dopaminergic neurons [14]. More importantly, miR-124 also played a dual role by regulating the apoptosis of cerebral ischemic cells [34]. Sun et al. reported that high levels of miR-124 in ischemic penumbra can enhance the expression of anti-apoptosis proteins Bcl-2 and Bcl-xl, while the up-regulated expression of miR-124 protects neurons from ischemic apoptosis [35].
All these studies suggest that miR-124 is an important factor involved in the process of anoxic injury or brain injury of nerve cells, and that high levels of miR-124 contribute to the survival of nerve cells. In this study, Remifentanil inhibited the damaging effect of OGD on PC-12 cells, while increasing the level of intracellular miR-124. Further studies have shown that the low expression of miR-124 weakens the protective effect of Remifentanil on PC-12 cells induced by OGD. This was mainly reflected in the enhancement of apoptosis and autophagy, and the decrease of cell viability. Therefore, we believe that Remifentanil reduced OGD damage by up-regulating miR-124, and miR-124 was an important participant in the mechanism of Remifentanil cytoprotective function. This was also the first time that the pharmacological effects of miR-124 and Remifentanil were directly discussed.
Earlier studies had confirmed that I/R injury in brain tissue promoted the phosphorylation of JAK2 and its downstream signal transducers, which exerted an anti-apoptotic effect and promoted cell viability [36]. In addition, mTOR activity was regulated by a variety of intracellular and extracellular factors; in turn, mTOR affected the rates of translation, transcription, protein degradation, signal transduction, metabolism and cytoskeleton dynamics [36]. Meanwhile, mTOR played a key role in the OGD damage of cells. Conversely, circZNF292 targeted BNIP3 by activating the mTOR signalling pathway, thereby alleviating OGD-induced damage in H9c2 cells [37].
‘Regulated in development and DNA damage responses 1’ (aka REDD1) regulated hypoxia-induced neuronal oxidative stress damage, by mediating the mTOR autophagy signal. The down regulation of REDD1 also interfered with the increase in apoptosis caused by OGD/R, as well as the overexpression of Bax and caspase-3. In addition, blocking the mTOR pathway attenuated the protective effects of the REDD1 inhibitor on OGD-induced neuronal injury and oxidative stress [38].
More importantly, the activity of JAK2/mTOR was regulated by miR-124. For example, miR-124 inhibited apoptosis by activating a series of JAK2 pathways, and by promoting the expression of its downstream anti-apoptotic protein Bcl-2, thus protecting CIRI. In LPS-stimulated microglia-BV-2, acetylcholine (Ach) activated the JAK2 pathway deactivated by LPS, while the levels of miR-124 and downstream target C/EBP α were significantly increased, indicating the regulatory relationship between miR-124 and the JAK2 pathway [39]. Huang et al. confirmed that the inhibitory effect of miR-124-3p on neuronal inflammation was achieved by regulating the activity of mTOR signal transduction. MiR-124-3p affected the activity of the mTOR pathway by targeting PDE4B [40]. MiR-124 acted as a growth inhibitory miRNA, and played an important role in inhibiting tumorigenesis by targeting mTOR [41]. Similarly, hypoxia led to the inactivation of the JAK2/mTOR pathway in OGD-stimulated PC-12 cells, and Remifentanil pre-treatment relieved this inhibitory effect. Further mechanism studies showed that miR-124 silencing partially eliminated the function of Remifentanil, and reduced the activity of the signal pathway. MiR-124 mediated the activation of the JAK2/mTOR pathway by Remifentanil.
In conclusion, Remifentanil activated the JAK2/mTOR pathway by up-regulating miR-124 in PC-12 cells, which played a protective role in the response of these PC-12 cells to OGD stimulation.