| Objective:Gliomas account for about 80%of the central nervous system(CNS)malignant tumors.Standard methods for treating gliomas include surgical resection and postoperative radiotherapy.The prognosis of glioma after treatment is very poor.To improve the prognosis of patients,it is important to choose the proper anesthetics that is not aggravate or even reverse the trend of proliferation and invasion of gliomas.Oxidative stress plays a key role in tumor angiogenesis and cancer progression,and it can promote the development of tumors.What’s more,the imbalance of metal ion metabolism aggravates tumor oxidative stress,such as high levels of intracellular Fe2+may lead to oxidative stress and protein aggregation,and even cause neuronal death.Divalent metal transporter 1(DMT1)is the main Fe2+ transporter in the human body and it is responsible for the regulation of intracellular and extracellular iron metabolism.Propofol is commonly used in clinical sedative as a general anesthetic drug,with anti-tumor and antioxidant effects.DMTI is regulated by neuronal N-methyl-D-aspartic acid(NMDA)receptors.However,the main glutamate(Glu)receptor expressed in glioma cells is a-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid(a-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid,AMPA receptors.The absence of GluR2 subunit in AMPA receptors in glioma cells is indicative of permeability to Ca2+,named as Ca2+ permeable AMPA receptors(CPARs).CPARs-mediated Ca2+ influx induces intracellular ROS upregulation,thereby aggravating oxidative stress in tumor cells.The purpose of this study was to investigate the effect of propofol on the proliferation and invasion of gliomas from the perspective of oxidative stress in tumor,and to further explore the molecular mechanism of this effect,as well as the relationship between CPARs and DMT1.Methods:This study was divided into three parts.Part Ⅰ:Explore the antitumor effects of propofol on the proliferation and invasion of gliomas in vivo.Adult male Wistar rats were selected and randomly divided into 4 groups(n=30/group):sham operation group(S group),glioma model group(G group),and propofol infusion dose of 20 mg·kg-1·h-1 group(P1 group)and propofol infusion dose of 40 mg·kg-1·h-1 group(P2 group).C6 brain glioma cells were injected into the caudate nucleus of rat brain using a stereotaxic apparatus to establish an animal glioma model.After 10 d the model was established,propofol was infused in 2 groups through the rat tail vein at a rate of 20 mg·kg-1·h-1 or 40 mg·kg-1·h-1 for 6 h.The following indicators were observed on 18 d after establishing model:the weight of gliomas;HE staining and GFAP immunohistochemistry were used to detect the establishment of glioma models;Western blot was used to determine the expression of VEGF in glioma periphery(within 2 mm diameter of the tumor periphery)and core.By immunofluorescence,the number of MMP-2 and MMP-9 positive cells in GFAP expression glioma cells were counted.The changes of blood-brain barrier structure were observed under transmission electron microscope.Part Ⅱ:The effect of propofol on GluR2 and DMT1 in gliomas was studied from the perspective of oxidative stress.The group was divided as same as Part Ⅰ(n=18/group).Propofol was infused in group PI and P2 for 6 h 10 d latter.On 18 d after model establishment,the glioma tissues were taken from the periphery and core of gliomas to measure the GluR2 expression level by Western blot.Immunofluorescence was used to observe the number of glioma cells positive for DMT1 expression.The cerebrospinal fluid of each group was extracted to detect glutathione content by glutathione assay kit.Part Ⅲ:AMPA receptor activator(R,S)-AMPA was performed to treat glioma cells in vitro,to explore GluR2-DMT1 pathway,and the effects of propofol in this pathway.The cultured cells were randomly divided into 6 groups:glioma group(G group),propofol concentration of 1.2 μg/mL group(P1 group)and propofol concentration of 4.4 μg/mL group(P2 group),CPARs activator(R,S)-AMPA treated glioma cells group(A+G group),CPARs activator(R,S)-AMPA and propofol 1.2 μg/mL co-treated group(A +P1 group),(R,S)-AMPA and propofol 4.4 μg/mL co-treated group(A+P2 group).Immunofluorescence was used to observe DMT1 expression in glioma cells in each group.ROS assay kit was used to detect ROS production in glioma cells.Flow cytometry was used to analyze the cell cycle of glioma cells after treatment with propofol or(R,S)-AMPA.Results:In Part Ⅰ,the overall structure of glioma was observed by HE staining.The positive expression of GFAP in glioma was detected by immunohistochemistry.It was confirmed that the rat C6 glioma model was established successfully.In gliomas weighing,we found that compared with group G,infusion of propofol could make tumors weight decreased(P<0.01).Compared with S group,the expression of VEGF in the tumor-bearing group(G,P1 and P2)increased significantly(P<0.01).Yet propofol inhibited glioma VEGF Expression(P<0.01),and the effect was more obvious in the periphery zone.Mainwhile,the expression of MMP-2 and MMP-9 after propofol treatment observed by immunofluorescence was similar to that of VEGF,there was no statistical difference between P1 and P2 groups.Through transmission electron microscope,the infusion of propofol can reduce the destruction of the glioma blood-brain barrier by tumor cells invasion,and maintain the integrity of the structure and function of it.Part Ⅱ:GluR2 and DMT1 were oppositely affected by propofol.Compared with S group,the expression of GluR2 was significantly decreased in group G(P<0.01),and propofol could raise the levels of GluR2 in group P1 and P2 in comparison with non-treated glioma cells(P<0.01),that is,the expression of CPARs in gliomas was decreased by propofol.Besides,propofol infusion group(P1 group and P2 group)decreased DMT1 expression and glutathione content compared with G group(P<0.05).Part Ⅲ:After propofol treatment on glioma cells,DMT1 expression and ROS levels decreased significantly(P<0.01),and the cell cycle was arrested at the G1 phase.Whereas,(R,S)-AMPA incubation reversed this effect(P<0.01),indicating that DMT1 is a downstream of CPARs.Conclusion:Propofol can improve the oxidative stress state,inhibit the proliferation and invasion of tumor by regulating the CPARs-DMT1 pathway of gliomas,which shows anti-tumor neuroprotective effect. |
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