Molecular Mechanisms Of The Effects Of Metformin On Glioma-related Brain Edema

Research background and objective: Intracranial tumor is a very common type of the disease in clinical neurology. In all brain tumors, primary glioma accounts for75-80%, which is the most common malignant brain tumor. Glioma-related cerebral edema can cause compression and damage of brain tissue and increase intracranial pressure, which eventually lead to cerebral ischemia, brain herniation, and death. In the early 1960 s, a variety of studies have attempted to use drugs, such as glucocorticoids, to reduce the cerebral edema. Gradually, the pharmacological mechanisms and side effects of these drugs have been clinically recognized. Therefore,we plan to find a new drug that is safer and has higher efficacy to treat brain edema in order to reduce mortality and morbidity in patients with glioma, and improve the prognosis of patients.Cerebral edema can be broadly divided into four categories: vasogenic edema,cytotoxic edema, osmotic edema, and interstitial edema. The brain edema caused by a brain tumor is more directly related to the first two categories. The mechanism of in brain tumor-induced vasogenic edema is mainly due to the mass effect of tumor,which causes oppressive effects on peritumoral brain tissue, leads to the damage and destruction of BBB(Blood Brain Barrier, BBB), and increases capillary permeability,water infiltration, and accumulation of water in perivascular and intercellular space.Meanwhile, due to the leakage of proteins, osmotic pressure is increased leading to water leakage, thereby increasing the degree of brain edema. Additionally, brain tumors induced cytotoxic edema is due to tumor compression of the brain tissue,causing brain ischemia, hypoxia, nerve cell dysfunction, and reduction in mitochondrial adenosine triphosphate(ATP) generation. The activities of sodium-potassium ATP enzyme and calcium-magnesium ATP enzyme on nerve cell membranes are decreased. The disorders of intracellular and extracellular ion exchanges of sodium, potassium, calcium and magnesium can lead to high intracellular sodium and potassium concentrations with intracellular calcium overload.This will cause cell swelling and intracellular edema. In general, these two types of brain edema often coexist and evolve with each other, creating a vicious cycle.Under normal physiological conditions, the BBB consists of endothelial cells,which form tight junctions. Tight junction molecule, including Claudin 3, Claudin Claudin 5 and 12, and other transmembrane proteins such as occludin, play a crucial role in endothelial cell tight junctions. Intact BBB can prevent fluid within the blood vessels into the brain tissue. However, the rapid growth of the brain tumor will cause oppression against surrounding normal brain tissue, resulting in ischemia and hypoxia,which not only induce cytotoxic edema around the tumor cells, but also cause structural damage on the surrounding BBB. Studies have shown that glioma can release large amounts of vascular endothelial growth factor(VEGF) causing abnormal expression of vascular endothelial tight junction molecule, resulting in leakage of fluid from blood vessels and the formation of brain edema. In addition, the aquaporin4 protein(AQP4) plays an important role in the formation of brain tumor induced cytotoxic edema. Under physiological conditions, AQP4 is densely expressed at the perivascular astrocytes foot. However, in glioma tissue, normal localization of AQP4 disappears, and AQP4 is widely dispersed on the cell membrane. AQP4 upregulation in glial cells can increase the cell permeability, which is highly correlated with the degree of brain edema.Metformin is a widely used to treat type 2 diabetes and has high potency and fewer side effects. A lot of studies have suggested that, in addition to lowering blood sugar, metformin may have a protective effect on blood vessel permeability. A recent study even showed that metformin could suppress astrocyte swelling in vitro.Therefore, the goal of this project was to study the effects of metformin on glioma-related edema, using in vitro endothelial cell culture and in vivo animal models of glioma-induced edema, and assess the mechanisms underlying the therapeutic effects of metformin on glioma-induced edema.Research methods: In order to study the effects of metformin on the regulation of vasogenic brain edema, we first established in vitro model of the BBB using cultured endothelial cell monolayer b End3. By measuring b End3 endothelial cell monolayer resistance, we evaluated the effects of metformin on changes of endothelial cell permeability induced by hypoxia and VEGF treatment. At the same time, using Western blot, we observed the effects of metformin on the expression of Occluding,Claudin-5, ZO-1, and ZO-2 in b End3 cells. In order to study the mechanisms underlying the effects of metformin on cytotoxic edema, we established astrocytes culture, and further observed the effects of metformin on AMPK activation, regulation of NF-κB, and the expression of AQP4 in astrocytes. Finally, we established an in vivo animal model of glioma-related edema. We evaluated the in vivo effects of metformin on glioma-induced changes in vascular permeability and its impact on the expression of AQP4 in the peritumoral tissues in order to interpret the putative therapeutic mechanisms of metformin in treating glioma-related brain edema.Research results: The resistance value of this b End3 cell culture model is 341 ± 36?.cm2. Therefore, our b End3 cell culture model met the permeability requirements as in vitro BBB model. Furthermore, we found that metformin reduced the hypoxia or VEGF-induced permeability of the endothelial cells. Additionally, we found that hypoxia or VEGF incubation reduced the expression of tight junction proteins such as Occluding, Claudin-5, ZO-1, and ZO-2 in b End3 cells. However, 0.5 m M metformin reversed such an effect. Thus, our results suggested that metformin might reduce the increase in endothelial cell permeability caused by hypoxia and VEGF exposure by enhancing the protein expression of tight junction molecules. When in the growth,glioma cells can secrete large amounts of VEGF and cause local hypoxia environment,which can damage BBB and induce vasogenic brain edema. Thus, our results indicate that metformin can prevent vasogenic edema induced by the growth of glioma.However, the growth of glioma may also induce cytotoxic edema. Our experiments further elucidated the pharmacological effects of metformin on the regulation of protein expression of AQP4 in astrocytes. The regulatory effects of metformin may be dependent on activation of AMPK, thereby inhibiting the activity of NF-κB to reduce the expression of VEGF-induced upregulation of AQP4. Since the expression of AQP4 is positively correlated with the formation of cytotoxic brain edema, our results indicated that metformin might reduce cytotoxic brain edema induced by the growth of glioma. Finally, we have established an in vivo animal model of glioma-related edema. We found that metformin could reduce the enhancement of vascular permeability induced by the growth of C6 glioma and decrease the expression of AQP4 in the peritumoral tissues in vivo, thereby reducing the amount of fluid in the brain tissue and preventing the occurrence of glioma-related edema.Research conclusion: Our study found and verified that metformin could reduce the vasogenic and cytotoxic by using in vitro cell culture and in vivo animal model. Our results indicate that metformin can reduce the formation of glioma-induced peritumoral edema. These results may have positive influence on the clinical treatment of glioma in patients.