In the present study, the infarction volume in the MCAO/R model rats decreased with the prolongation of the reperfusion time (P < 0.05). Meanwhile, the miR155 expression obviously increased in both the ischemic core and the IP area of the model rats, but this trend weakened as the reperfusion time increased. Meanwhile, the expression of mRNAs of key molecules in the Rheb/mTOR signaling pathway, including Rheb, mTOR, S6kb1, and 4Ebp1, seemed to increase in both the ischemic core and the IP area of the model rats, but statistically significant difference was observed only in the 48-h group (P < 0.05). Interestingly, the mRNA expression level of S6kb1 obviously increased in all the 24-, 48-, and 72-h groups in both the ischemic core and the IP area (P < 0.05). In general, no obvious difference was observed in the expression characteristics of these molecules between the ischemic core and the IP area (P > 0.05), indirectly indicating that miR-155 participated in the ischemia–reperfusion injury; however, it showed a significant difference between the groups of different reperfusion times.
IS constitutes almost 80% of the stroke cases, while the remaining 20% are hemorrhage strokes [29]. Once blood flow to the brain is insufficient, the cells experience a series of molecular programs, including the toxicity of excitatory neurotransmitters, dysfunction of mitochondria, acidosis, imbalance of ions, oxidative stress, and inflammation. These molecular programs can result in cell death and nonreciprocal tissue damage[30]. Oxidative stress in the brain results in ischemic injury [31, 32]can eventually initiating programmed cell death pathways, including apoptosis, autophagy, and necroptosis [33–35]. With the wide application of intravenous thrombolysis and arterial embolectomy therapy in clinics at present, blood rebuilding is no doubt the most effective treatment for IS, which has been widely proved by clinical and experimental studies. This is also what exactly the MCAO/R model is based on and widely used in IS studies in vivo. Thus, the longer the blood rebuilding, the lighter the ischemic injury, as demonstrated in the present study (Figure. 1).
Specific stroke-induced miRNA expression profiles have been reported in both the blood and the brain, and in experimental models and patients at different reperfusion times[36–38]. The pattern of circulating miRNA expression suggests an early influence of age in stroke pathology, with a later emergence of sex as a factor for stroke severity [39]. Changed inflammation-related microRNA profiles in plasma following IS have been reported [40]. In addition, the patterns of miRNA expression were used to predict stroke subtypes [38]. MiRNA 155 has been demonstrated to mainly participate in neuroinflammation [41]. Although previous studies have investigated miRNA155 expression in ischemic animal models, only a few studies have focused on miRNA 155 in the IS. Xx et al. demonstrated that miR155 potently induced autophagy under hypoxic conditions by targeting multiple mRNA molecules of the mTOR signaling pathway, thus accelerating cell apoptosis, which suggested that miR155 was likely involved in the ischemic cerebral injury mediated by the mTOR signaling pathway. They proved that miR-155 combined with the 3’-untranslated region of Rheb mRNA and further inhibited the Rheb expression at the posttranscriptional level[26]. Guoping Xing et al. demonstrated that the expression of miR-155 increased in the cerebral tissues of MCAO rats with ischemia. On the contrary, the expression of Rheb and mTOR decreased at the protein level. Exogenous miR-155 inhibitors downregulated miR-155 expression but upregulated Rheb and mTOR expression, showing a protective effect in the injury process of IS. This protective role was characterized by a decrease in the infarct size and decreased apoptosis rate[27]. The results of the present study showed that the expression of miRNA155 increased in ischemia–reperfusion injury, which showed opposite changes with the ischemia–reperfusion injury extent, and this was consistent with the results of the aforementioned studies[26, 27]. Therefore, therapeutic approaches that targeted miR-155 probably worked in IS [42].
The target of rapamycin (TOR) was first discovered in yeast using a mutant of a protein that caused resistance to the negative effects of rapamycin on growth [43]. Two homologs of TOR are present in yeast, known as TOR1 and TOR2. Interestingly, only one homolog of TOR was found in mammalian cells and was called mTOR, which played an essential role in the initiation of translation, transcription, organization of the cytoskeleton, and cell growth, proliferation, and survival [44–48]. The activity of mTOR was maintained at the degree of its normal function of equilibrium by combining several proteins with mTOR to form two complexes, named as mTORC1 and mTORC2[49], to realize the biological function of mTOR. Compared with mTORC2, mTORC1 has been found to be more sensitive to rapamycin [50]. Phosphorylation on its specific residues at the C-terminal of mTOR regulated its activity, including serine2448, the target of Akt and p70 ribosomal S6 kinase (p70S6K) [51–53], threonine2446, the target of AMP-activated protein kinase and p70S6K [51, 54], and serine2481, a nonsensitive rapamycin autocatalytic site of mTOR [55, 56]. Rheb is a small Guanosine Triphosphate enzyme(GTPase) regulating the survival, growth, and differentiation of cells by enhancing the signaling of the mTORC1 pathway[57]. Moreover, Rheb is the necessary protein in phosphorylating and activating mTOR[24]. Activated Rheb–GTP can directly combine with Raptor and further activate mTORC1 and regulate 4EBP1 to combine with mTORC1[58]. P70S6K and 4EBP1 are the two primary well-demonstrated downstream targets of mTORC1. Active mTORC1 can phosphorylate these two molecules to further activate p70S6K and inactivate 4EBP1. Activated p70S6K accelerates the biogenesis of mRNAs, translation of ribosomal proteins, and growth of cells [59, 60]. When mTORC1 is inhibited, nonphosphorylated 4EBP1 competitively binds with eukaryotic translation initiation factor 4G (eIF4G) to eIF4E. The combination of eIF4G with eIF4E is essential for initiating translation by interacting with the 5′-mRNA cap structure. The mTORC1 phosphorylates 4EBP1 and further causes the latter to dissociate from eIF4E, thus allowing eIF4G to combine with eIF4E and accelerate the initiation of translation [14, 61]. Several studies demonstrated that regulating mTOR activity had the potential of neuroprotection during IS. The application of estradiol to adult female ovariectomized rats before focal cerebral ischemia obviously reduced infarct volumes and apoptosis in the cerebral cortex and, at the same time, prevented the decline in the expression of phosphorylated mTOR and p70S6K induced by ischemia[62]. The downregulation of S6K1 accelerated injury in astrocytes induced by oxygen–glucose deprivation (OGD), an in vitro model of ischemia; on the contrary, the downregulation of S6K1 through adenoviral infection relieved cell injury[63]. Furthermore, the knockout of S6K increased the infarct volume and the mortality of mice with focal cerebral ischemia [63]. Moreover, the downregulated mTOR activity induced by rapamycin made cell survival tough and facilitated apoptotic injury in neural cells enduring OGD [64–66]. Erythropoietin has been demonstrated to defend microglia from OGD by promoting the mTOR activity and inhibiting the release of mitochondrial cytochrome c because mTOR inhibition through rapamycin silences the cell-protective function of erythropoietin [65]. Rapamycin can also enlarge the brain infarct size and increase the neurological deficit score in rats with focal cerebral ischemia, indicating that promoting mTOR activation may lead to lighter ischemic brain injury and better behavior recovery. Previous studies found that Rheb mRNA levels were upregulated in IS, which was consistent with the results of the present study[67]. However, the protein level was downregulated in IS[27], indicating that the Rheb expression was regulated at the posttranscriptional levels and MiR-155 might directly act on Rheb mRNA. Although the protein and phosphorylation levels of the aforementioned molecules of the mTOR signaling pathway were not tested in this study, previous studies showed that the total protein levels of mTOR, S6K1, and 4EBP1were upregulated while the phosphorylation levels were downregulated (please see the complementary data, unpublished).