| Human brain glioma originates from neuroepithelial tissue and represents the most common primary intracranial tumor. Brain glioma has high degree of alignancy which often invades blood vessels and the connection between glial cells to infiltrate, oppresses and destructs brain tissue. For there is no clear boundary between glioma and normal brain tissue, surgery is difficult to complete resection with easily postoperative relapse. Glioma is characterized by its high invasiveness and resistance to traditional radio-/chemo-therapy, so the patient prognosis is poor and mortality is high. At present the treatment of glioma is still a difficult problem, which causes serious threat to human life and health. Together, these facts leave effective therapy much to be desired.Diabetes mellitus is easily complicated with cerebral vascular disease and sustained hyperglycemia causes severe neurotoxicity, impaired neurological function and poor prognosis. According to statistics, the sick rate, recurrence rate and mortality of patients with diabetic cerebral ischemia were significantly higher than that of non diabetic patients with cerebral ischemia, which bring heavy burden to society and family and have become major public health problems that seriously damage the citizens’health and life. In addition, even without diabetes, blood glucose level in patients with ischemic stroke is often elevated, increasing brain damage. China’s acute ischemic stroke guidelines 2014 pointed out:"there are about 40% patients with hyperglycemia after stroke, cause adverse prognosis." Therefore, in addition to lower the blood glucose level, there is an urgent need to look for effective measures to inhibit hyperglycemia toxicity, improve the nerve lesion, promote the recovery of neurological function and improve the clinical treatment effect.Stroke is the human catastrophic disease, with high incidence, mortality and mutilation rate, which occupies one of the three major causes of death worldwide and the first cause of adult acquired dysfunction by single diseases in the world. The third national death cause investigation in 2008 showed that cerebral apoplexy has become the first cause of death in China. In various reasons, people died of a cerebral apoplexy in our country were more 4～5 times than that of Europe and the United States. Cerebral ischemia-reperfusion injury mechanism is complex, and the destruction of the redox balance is the important pathological features. Therefore, it is of great significance and clinical value to explore the improvement and treatment of cerebral ischemia reperfusion injury by high effective antioxidant neuroprotective agents.Selenium is an essential trace element showing versatile biological effects including antioxidant, antitumor and immunity-enhancement. It has been confirmed that selenium plays an important role to maintain normal nerve function, and selenium deficiency can affect neurocognitive function and lead to neurodegeneration, such as Alzheimer’s disease and Parkinson’s disease. A large number of clinical and animal researches show that high dose of selenium can aggravate oxidative stress, induce the accumulation of ROS, play a pro apoptotic effect; low dose of selenium show antagonistic effects of oxidative stress and inhibition effect of apoptosis. So high dose of selenium is often used as a cancer chemotherapeutic drug in order to achieve anti-cancer purposes, and low dose selenium is used as a cytoprotective agent to fight against damage induced by oxidative stress. Selenocystine (SeC), a naturally available selenoamino acid, is accepted as the twenty-first essential amino acids for human. SeC has been widely used because of its multiple pharmacological effects, especially the redox pathway regulation attracted many researchers’ attention. DsePA, a derivative of SeC with stable property, high safety, and high antioxidant activity, is widely used for antioxidant studies and shows protective effects on acute toxicity and chronic neurological degenerative diseases both in vitro and in vivo.Clinical evidences show that selenium deficiency can greatly increase the risk of cancer, and selenium supplementation can reduce the risk of some cancers. For its broad spectrum of anti-tumor activity, SeC can inhibit the growth of variety of human tumor cells in vitro and in vivo through induction of apoptosis such as liver cancer, lung cancer, breast cancer and melanoma cell tumor, so it occupies potential value of clinical application. In recent years, studies also find that selenium compounds with anti-cancer drugs can enhance the sensitivity of anti-cancer drugs, which can be used as a sensitive agent for cancer chemotherapy. However, whether SeC has inhibitory effect on the growth of glioma cells and the mechanism have not been reported.As a high oxygen consumption organ, the high neuronal glucose consumption must be accompanied by a large number of free radicals generated in brain, but hyperglycemia will destroy intracellular antioxidant enzyme systems, weaken their scavenging ability, so that a large number of free radicals cannot be cleared in time. Sustained hyperglycemia can affect and damage the inner membrane of mitochondria, start the dissipation of mitochondrial membrane potential, which lead to a large number of free radicals. The depositions of excessive free radicals cause the oxidation of the plasma membrane and the destruction of synapses, disrupt the connections between neurons, and even lead to neuronal apoptosis. The experiment confirmed that antagonism of oxidative stress damage induced by hyperglycemia can significantly reduce the nerve damage/toxicity and improve the nerve lesion. However, there is no report about the mechanism of the effect of organic selenium on hyperglycemia induced neurotoxicity.The pathological mechanism of cerebral ischemia reperfusion injury is complex in acute ischemic stroke, and the damage of redox imbalance is an important pathological feature. Oxidative stress plays a key role in cerebral ischemia reperfusion resulting in lipid peroxidation and DNA damage via ROS overload. Therefore, it is of great significance and clinical value to explore the improvement and treatment of cerebral ischemia reperfusion injury by high effective antioxidant neuroprotective agents. The direct protective effect of selenium on cerebral ischemia reperfusion injury has not been reported in the study.So this paper carried out the study of SeC and its derivatives DSePA in diseases of nervous system and explored the effects and mechanism of organic selenium on brain glioma, hyperglycemia induced neurotoxicity and ischemic stroke. First of all, this study selected U251 and U87 human glioma cell lines as the research object, studied effects of SeC on cell cycle of U87 and U251 and analyzed the possible mechanism from important signaling pathways to provide an experimental basis for the treatment of human brain glioma. Secondly, this study selected PC 12 cells as models of neuron, used DSePA to intervene in the hyperglycemia toxicity, and discussed its antagonism to hyperglycemia neurotoxicity. Finally, this study established mice models with transient focal cerebral ischemia/eperfusion (tFCI/R) injury, and the protective effect and mechanism of SeC on the injury of ischemia reperfusion injury were discussed.Objective1. Explore the inhibition effect of SeC on the growth of U251 and U87 human glioma cell lines and the potential molecular mechanism.2. Explore the effect and mechanism of DSePA on hyperglycemia neurotoxicity in PC 12 cells.3. Explore the protective effect and mechanism of SeC on mice with tFCI/R injury.Methods1. U251 and U87 cells in logarithmic growth phase were selected and randomly divided into normal control group and SeC-treated group. After treated with 5,10,20 μM SeC respectively for 24 h or 48 h, the phase contrast microscope was used to observe cell morphology; and MTT colorimetric method was used to determine the cell survival rate, to understand the inhibition effect of SeC on U251 and U87 cell growth; flow cytometry (FCM) was applied to analyize the influence of SeC on U251 and U87 cell cycle distribution; and TUNEL-DAPI method was used to observe the DNA damage on U251 cell; the DCFH-DA probe and ultra oxygen anion kits were applied to check ROS and superoxide anion generation; Western blotting was used to detect the influence of SeC on cell cycle regulatory protein cyclin A, apoptosis related proteins p21, p53 and cell signal pathway proteins MAPKs and AKT in U251. At the same time, SeC was combined with multiple pathway protein inhibitors to study its effects on cell cycle distribution.2. PC 12 cells in the logarithmic growth phase were cultured in vitro and randomly divided into normal control group and DSePA-treated group, add 100 mM glucose for 48 h to produce high glucose damage cell model, then DSePA was added into the cells. Cell apoptosis were analized and observed by flow cytometry and TUNEL-DAPI costaining; detection of caspase-3/-8/-9 activation and Western blotting were used to determine the apoptosis pathway; JC-1 probe was used to evaluate the mitochondrial membrane potential and the DCFH-DA to detect intracellular ROS level.3. TFCI/R injury mice models were made and qualified mice were selected by Doppler flow monitoring and neurological score. Then the mice were randomized to SeC-treated group and vehicle group, and chose the mice experienced the whole operation but without the middle cerebral artery occlusion as sham operated group. Intraperitoneal injection of SeC solution (2 mg/30 g) was given to mice in SeC-treated group 1 time daily for 3 consecutive days after operation. Vehicle group and Sham group were given the same way of injection of saline. In postoperative 24 h, 48 h and 72 h, a neural function defect score and neurobehavioral test were carried out; TTC staining was used to measure the volume of cerebral infarction and dry-wet weight method was used to measure brain edema changes, immunofluorescence staining was used to observe the expression of AQP4, TUNEL-DAPI staining was used to observe the apoptosis of neurons, and Western blotting to detect the activity of caspase-3 and caspase-9 expression.Results1. MTT results showed a time/dose dependent inhibition of SeC on U251 and U87 cell growth, and the cells were observed rounding, synaptic reduction, and the cell number was significantly reduced, the difference was statistically significant compared with normal cells (P<0.05); flow cytometry showed that SeC induced S-phase arrest significantly in U251 and U87, which is consistent with the reduction of cyclin A expression; TUNEL-DAPI analysis and ROS test showed that SeC treatment induced DNA damage by ROS increasing in U251 cells; Western blotting showed that SeC treatment significantly increased DNA damage marker p21 and p53 expressions, with increasing expressions of pJNK, p38 and pAKT while decreasing of pERK.2. DSePA pretreatment can effectively reduce the toxic effect of high glucose on PC 12 cells. Flow cytometry showed that DSePA pretreatment can significantly inhibit the apoptosis of PC 12 cells induced by high glucose with a reduction of the sub-G1 peak; fluorescence staining results showed that, the pretreatment of DSePA can effectively inhibit high glucose induced chromatin fragmentation and nuclear enrichment. Caspase activity assay showed DSePA pretreatment inhibited high glucose induced caspase activity and Western blotting detected from the protein levels found that DSePA significantly inhibited PARP cleavage and caspase-3/-7/-9 activation induced by high glucose; mitochondrial membrane potential and structure detection confirmed that DSePA blocking the mitochondrial damage induced by high glucose. DSePA inhibits the production of superoxide anion and ROS accumulation induced by high glucose in PC12 cells.3. The performance of mice in neurological deficits scoring was improved at 24h, 48h and 72h after tFCI/R operation, while the improvement was significant in SeC-treated group compared with that of vehicle group. The SeC-treated mice showed ameliorating functions of motor and sensory, reduced brain infarct volume and edema degree. Mechanism studies revealed that SeC reverted AQP4 changes, suppressed oxidative stress and inhibited neuronal apoptosis. In summary, these results indicate that SeC exerts novel neuroprotective effects against focal cerebral ischemia reperfusion injury through inhibition of oxidative stress and apoptosis, which validated the potential application in treatment of ischemia stroke.Conclusion1. SeC has the potential to inhibit human glioma cell growth by inducing S-phase arrest through triggering ROS-mediated DNA damage and regulating the MAPKs and AKT signaling pathway.2. DSePA can effectively restrain high glucose-induced PC 12 cytotoxicity in vitro, the strategy of using DSePA could be a highly effective way in prevention or therapy of diabetic neuropathy.3. Intraperitoneal injection of SeC has protective effect on cerebral ischemia reperfusion injury in mice via inhibiting edema and resisting the apoptosis of neurons induced by oxidation. |
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