The effects of rapamycin on the symptoms of cerebral ischemia due to changing the expression of miR-1 and its target genes, Bad and Bcl-w

Introduction: Stroke is the major cause of long-term disability in adults. The precise role of the mTOR signaling pathway in neural viability due to rapamycin effect in the animal model of middle cerebral artery occlusion (MCAO) remained elusive. Since the relationship between mTOR and miR-1, especially in neurons, is unknown, we have evaluated the effect of rapamycin as a post-ischemic treatment on improving stroke symptoms. Methods: Rats were divided into three groups including sham, control and rapamycin treatment group. Each contains four subgroups (n=7). One hour after MCAO, rats were received intravenously 0.1ml normal saline or 0.1ml rapamycin in the control and treatment groups, respectively. After 24 hours, neurologic deficit score, infarct volume, brain edema, and blood-brain barrier (BBB) permeability were measured in the control and treatment group. The expression of miR-1, Bcl-w and Bad were analyzed using quantitative RT-PCR in all groups. Results: Our results indicate that post-treatment with rapamycin, significantly reduces neurological deficits, infarct volume, brain edema and BBB permeability. It also decreases the level of miR-1 and Bad expression and increases the level of Bclw expression. Conclusion: According to our findings, post-ischemic treatment with rapamycin can be effective in improving symptoms of stroke using changing in the expression of the miR-1 gene and consequently, a changing in the expression of the target genes of this miRNA (i.e., Bad and Bcl-w). In summary, we unravel for the first time a link between mTOR, miRNA-1, Bcl-w and Bad in brain ischemia. D ow nl oa de d fr om p pj . p hy ph a. ir at 1 :0 6 + 03 30 o n W ed ne sd ay O ct ob er 3 0t h 20 19 173 | Physiol Pharmacol 22 (2018) 172-182 Talebi et al. administration of current treatment for stroke has been limited because of its narrow therapeutic window (Bansal et al., 2013). Rapamycin is an important mediator of the mammalian target of rapamycin (mTOR) pathway and could be an effective cure for many types of neuronal injuries and related diseases (Fletcher et al., 2013). In addition to, the mTOR signaling pathway role in regulating different processes such as immune responses and cell growth, it also has a critical role in the nervous system. Studies have illustrated rapamycin enhances neuronal viability and decreases damages in several animal models (Foster and Fingar, 2010; Hailer, 2008). Dysfunction of the mTOR pathway has been detected in several neurological diseases including brain stroke and neurodegenerative diseases (Lu et al., 2014). Rapamycin has a protective role in the CNS by inhibiting mTOR signaling pathway results in autophagy. As limited autophagy leads to brain injuries, rapamycin administration reduces neurological deficits and apoptosis in a rat model of subarachnoid hemorrhage (Jing et al., 2012). Lu et al. (2014) have demonstrated inhibition of mTOR pathway modulates the immune response and also improves neurobehavioral deficit in the model of intracerebral hemorrhage. Conversely, many studies have revealed mTOR pathway ameliorates neural damages and improves memory function (Gong et al., 2006) and synaptic plasticity (Ma et al., 2010) in Alzheimer disease and also makes a long-term memory in the amygdala (Parsons et al., 2006) Many studies confirm the mechanism of microRNAsderived rapamycin. Micro(mi)RNAs are small noncoding molecules (containing about 22 nucleotides) play a vital role in many biological processes such as proliferation, apoptosis and cell differentiation using changing in the expression of target genes (Negrini et al., 2009). Evaluating proteins profile after brain ischemia in numerous studies indicate proteins involvement in ischemic damage (Dharap and Vemuganti, 2010; Lee et al., 2010; Lusardi et al., 2010). Therefore, clarifying the function of the miRNAs could be useful to understand the pathogenesis of cerebrospinal ischemia. MicroRNAs, such as miR-1, are gene expression regulators and control many physiological processes by changing the expression of their target genes (Negrini et al., 2009). It has been reported miR-1 has a critical role in many disorders including ischemia results in cardiomyocytes (Bigdeli et al., 2007) and neuronal hypoxia due to apoptosis (Bigdeli et al., 2008). Although the role of microRNAsderived rapamycin has been confirmed in different biological processes (Jin et al., 2013; Martin et al., 2014; Sun et al., 2010), its neuroprotective function still needs to be clarified. On the other hand, the role of rapamycin as an apoptosis inhibitor or inducer has been challenged in many studies (Foster and Fingar, 2010). Bcl-w and Bad are anti-apoptotic and pro-apoptotic genes, respectively, are regulated with rno-miR-1-3p. Members of the bcl2 family can act either as antiapoptotic or pro-apoptotic molecules based on the presence of their conserved regions which termed bcl2 homology domains (Van Delft and Huang, 2006). Thus in the future, investigating the pathway of apoptosis could be an appropriate target to be studied in evaluation the possible effect of miR-1derived rapamycin during brain ischemia. According to what has been mentioned so far and also the effect of Bcl-w and Bad derivedmiR-1 in the apoptotic pathway in many studies (Tang et al., 2009; Ouyang and Giffard, 2014; Wang et al., 2010), as well as the rapamycin role in stroke improvement due to changes in various pathways including apoptosis, the goal of this study was to investigate the unknown effect of miR-1-derived rapamycin on the improvement of stroke due to changing of Bcl-w and Bad expression. Our results indicated that posttreatment with rapamycin significantly ameliorates neurological deficits and also reduces brain edema, blood-brain barrier permeability and infarct volume. The expression of miR-1 and Bad are decreased while the expression of Bcl-w is increased during our investigation. Hence, according to the results of this study in the mechanism of micro-RNA-derived rapamycin, it is hoping that a step forward would be taken to improve stroke. Materials and methods Animals All laboratory methods performed in this study have been approved by the Bioethics Committee of Shahid Beheshti University, Iran. All male rats (250–350g) were purchased from Pastor Institute and were provided with standard food and water. All animals D ow nl oa de d fr om p pj . p hy ph a. ir at 1 :0 6 + 03 30 o n W ed ne sd ay O ct ob er 3 0t h 20 19 Rapamycin and the expression of miR-1, Bcl-w and Bad Physiol Pharmacol 22 (2018) 172-182 | 174 were kept in the similar condition of 12:12 hours of a dark-light cycle at 25°C.