At present, cerebral ischemic disease has become one of the main diseases that causing death and disability. Cerebrovascular disease has become one of the three major causes of death in Chinese cities, and the mortality and morbidity are still increasing year by year. While during the perioperative period, cardiopulmonary bypass, continuous intraoperative hypotension and damages to cerebrovascular from the neurosurgical can all lead to cerebral ischemia-reperfusion injury, and in severe cases, affect the prognosis of patients. Cerebral ischemia can not only lead to necrosis, but also trigger inflammatory response, causes a lot of apoptosis, while a large number death of nerve cell will lead to the loss of nerve function. Therapeutic hypothermia is controlled lowering the body temperature to achieve therapeutic purposes. Hypothermia is one of the complex modal responses, it can reduce the damage of neurons, blocks the activation of glial cells, reduce dysfunction of endothelial cells and blood brain barrier and reduce neuronal cell death induced by cerebral ischemia which associated with neuroinflammation. At present, therapeutic hypothermia is the standard treatment for HIE. The international medical community recognized hypothermia classifies into mild hypothermia (33–35℃), moderate hypothermia (28–32℃), deep hypothermia (17–27℃) and ultra-deep hypothermia (2–16℃). Studies have found that the temperature between 28℃ and 35℃ has a good protective effect on the brain, with few adverse reactions. This temperature segment is named as mild hypothermia. This paper studied 32℃ hypothermia and 27℃ hypothermia to protect hypoxia and glucose deficiency injury mechanism. The study of the brain protection mechanism of hypothermia can provide evidence for the development of drugs on brain protection.
PI3K/AKT signaling pathway is involved in cell proliferation, apoptosis, transcription, migration, glucose metabolism and other processes. The PI3K/AKT pathway, as one of the key signal axes for cell survival, is the key factor in reducing apoptosis of PC12 cells which was dealt with OGD/R[13, 14]. Activation of PI3K/AKT signaling pathway not only plays a role in the protection of brain injury, but also in other tissue systems. For example, hypothermia can improve the activity of AKT in myocardial tissue, thereby protecting myocardial cells and reducing cell apoptosis. In addition, relevant studies have shown that the PI3K/AKT/mTOR signaling pathway mediates the pathological injury process after cerebral ischemia-reperfusion is a target of ischemic brain injury protection. Activate this pathway can alleviate neonatal hypoxic-ischemic brain injury, repair neurological function after stroke, and provide neuroprotection[15, 16]. Activation of PI3K/AKT signaling pathway can reduce the expression of inflammatory factors and protect vascular function[17]. AKT can rapidly activate molecular functions such as mTOR, which serves as a multifunctional collection point, it involved in regulating cellular nutrition, energy supply, and promoting protein synthesis. In addition, we usually concern about PI3K/AKT/mTOR mediated apoptosis, it also regulates autophagy in the bilateral mode[18].
In this study, established OGD/R model for PC12 cells and give LY294002 inhibitor to selectively block the signaling pathway of PI3K/AKT/mTOR, and culture at 37℃, 32℃ and 27℃, aiming to observe the effects of hypothermia intervention on PI3K/AKT/mTOR signaling pathway and autophagy. The results showed that hypothermia treatment could effectively improve the cell viability of OGD/R treated PC12 cells, and the cell viability increased with the decrease of temperature (P < 0.05). OGD/R significantly inhibited the expression levels of phosphorylated PI3K and AKT in PC12 cells (P < 0.05). Hypothermia intervention promoted the phosphorylation of AKT and PI3K protein in cells (P < 0.05), and the phosphorylation of PI3K and AKT at 27℃ was higher than that at 32℃ (P < 0.05). In addition, use flow cytometry to measure the apoptosis rate of different treatments, and the results showed that hypothermia could inhibit the apoptosis induced by OGD/R.
In order to explore the protective mechanism of hypothermia intervention on hypoxia and glucose deficiency injury, we set up LY294002 in this experiment which is a protease inhibitor of PI3K, compared the phosphorylation levels of PI3K, AKT and mTOR proteins in PC12 cells which under different treatments (control, OGD/R, LY294002 inhibitor) and different temperatures (37℃, 32℃, 27℃). The results showed that after added LY294002 inhibitor, the phosphorylation levels of PI3K, AKT and mTOR proteins were all significantly decreased at different temperatures (P < 0.05). The lower the temperature was, the phosphorylation levels of PI3K, AKT and mTOR proteins were increased in each group (P < 0.05). Those indicate that hypothermia intervention protects hypoxia and glucose deficiency injury by activating PI3K/AKT/mTOR signaling pathway.
Autophagy plays an important role in the growth of normal cell and the development and pathophysiological process of diseases, and it plays an important part in aging, tumor, cardiovascular disease, neurodegenerative diseases, resistance of microbial infection, slowing down aging and prolonging life expectancy. Autophagy is a catabolic mechanism, that is the autophagy vesicles with double membrane structure package part of the cytoplasm, damaged organelles and formation of autophagy with protein. And connotation with the body to form the so-called autophagy connotation, transferred to the lysosome in the form of dynamic dependencies and form with the fusion of autophagy-lysosome, degradation the contents of the package, complete the turnover and metabolism of intracellular substances[19]. As PI3K/AKT/mTOR and PI3K III/Beclin-1/Bcl-2 are important signaling pathways involved in autophagy. Activate the signaling pathway of PI3K/AKT/mTOR can inhibits the autophagy of a variety of cells[20–23]. While Beclin-1 is a specific autophagy gene in the PI3K III/Beclin-1/Bcl-2 signaling pathway, beclin-1 protein can form a compound with PI3K III to induce related autophagy proteins locate on the autophagosome membrane, thus promote the formation of autophagosomes[24]. Activation of regulated autophagy can not only provide energy through degrading proteins, but also protect cells by degrading damaged proteins and synthesizing new proteins[25]. Some other studies have found that oxygen-glucose deprivation can lead to autophagy in PC12 cells, and inhibition of autophagy can decrease the cell viability and aggravate the damage[26]. But studies have found that autophagy has dual character in determining the fate of damaged nerve cells: appropriate activation is beneficial to maintain the energy and the survival of cell homeostasis under the ischemic stress, while excessive or prolonged activation of autophagy can cause neuronal necrosis and apoptosis[27]. Microtubule-associated protein light chain (LC3) is one of the specific components of autophagosome membrane, and is a specific component of mammalian autophagosome membrane. Therefore, the detection of autophagy can be observed by the immunofluorescence point of LC3. When autophagy is induced to be up-regulated, detect the autophagosome through transmission electron microscopy and detect the immunofluorescence of LC3, and the results prove that hypothermia intervention can activate autophagy to protect hypoxia and glucose deficiency injury. In this study, we only use transmission electron microscopy and immunofluorescence to observe autophagy, and it may be necessary to further detect the expression changes of autophagy related markers.
Although therapeutic hypothermia is the standard treatment for neonatal HIE, it is associated with extensive physiological changes, almost affect all organ systems and functions, such as the effect on injury-induced aseptic inflammatory response and bilirubin metabolism, etc.[28]. In addition, approximately 50% newborns have disabilities at 12–18 months after treatment[29, 30]. So, the research is still needed on effective preventive measures, combination therapy with neuroprotective reagents and other richer combinations treatment. For example, the research of xiao-ya Gao and other studies demonstrated that human urokinase (HUK), neuroglobulin (Ngb) or dizoxipine (MK-801) combined with mild hypothermia had a significant protective effect on OGD/ R-induced cell death[31].
In conclusion, this study proved that hypothermia intervention can not only improve the cell viability and reduce the apoptosis rate of PC12 cells which injured by hypoxia and glucose deficiency reperfusion, but also promote the phosphorylation levels of PI3K, AKT and mTOR proteins, and up-regulate the autophagy level. Therefore, hypothermia intervention may protect hypoxia and glucose deficiency injury by activating PI3K/AKT/mTOR signaling pathway and autophagy.