Effects Of Inflammatory Stimuli On Mitochondria In Glioma Cells

BackgroundGlioblastoma(GBM)are the most common and most fatal malignancies in brain tumors,accounting for 17% of all brain tumors.More than 10,000 new GBM cases are diagnosed in the United States each year.According to the literature,The incidence of glioma is about 3 to 6 people / 10 million,and the incidence of nearly three decades increased year by year,in the 35-year-old cancer patients ranked second in mortality.Glioblastoma,as with other brain tumors,can also lead to epilepsy,nausea,vomiting,headache and mild hemiplegia,the most important of which is the deteriorating memory,personality or neurodegeneration.GBM poor prognosis,with a high mortality rate,the main reason is due to glioma cells with high invasive and metastatic.According to statistics,51% of the parenchyma brain tumors and 20% of the intracranial brain tumors are GBM.In recent years,although the treatment of GBM in surgery,chemotherapy and radiation therapy has made great progress,but because of tumor cell invasion,blood brain barrier and the tumor cell source and its growth mechanism is not clear,Limited the effectiveness of treatment.Inflammatory reactions play a key role in tumorigenesis,cancer cell proliferation,angiogenesis,tumor cell invasion and metastasis.Tumor-associated inflammation is typically characterized by infiltration of innate immune cells(such as microglia and macrophages)as well as the production of cytokines and chemokines,as well as tissue remodeling and angiogenesis.Inflammation on the one hand induces oxygen and nitrogen free radicals,destruction of cell DNA and membrane structure;the other hand,activation and cancer cell growth,development and invasion of cell signaling pathway.Mitochondria is the only organism that contains genetic information in mammals except cells.They are constantly through the division,fusion,budding and sub-structural changes,genetic material exchange,shape and quantity changes.The study found that mtDNA mutations in the GBM cause abnormal mitochondrial electron transport chain function,so that tumor cells can use aerobic glycolysis(ie,glycolysis and low level of oxidative phosphorylation of the combination)to promote its rapid proliferation and tumor growth,thus Visible mitochondria in the GBM energy supply and metabolism in the important role.And mitochondrial splitting and fusion of the steady state with the proliferation of tumors is closely related to,it can be seen,mitochondria in the development of glioma plays an indispensable role in the development process.In this study,the relationship between the changes of GBM mitochondrial biological function and inflammation was studied from the aspects of cell culture,protein imprinting,immunohistochemistry,immunofluorescence,flow cytometry,plasmid transfection and transmission electron microscopy.To observe the changes of mitochondrial morphology,function and autophagy under the stimulation of inflammatory factors,to further reveal the pathogenesis of GBM,to provide experimental basis for seeking effective treatment.Materials and Method 1: Morphological changes of mitochondria in glioma cells stimulated by inflammatory factors1.Ibal immunohistochemistry staining was performed on all sections of glioma tissue,and the morphology and distribution of microglia in gliomas were observed.2.Immunohistochemistry staining of IL-6 was performed on all sections of glioma tissue to observe the expression of inflammatory factors in gliomas at all levels.3.Inflammation factors LPS and IFN-? stimulated U87-MG cells,divided into 0h,4h,8h,24 h group,Western Blotting and cell immunofluorescence detection of Drp1,Mfn2,Hsp60 to observe mitochondrial changes.2: Changes of mitochondrial function in glioma cells stimulated by inflammation factors1.Detection of reactive oxygen species in glioma cells by stimulation with inflammatory factors using active oxygen(ROS)assay kit.2.Lipid peroxidation was observed by using 4-Hydroxynonenal(HNE)to observe the changes of lipid peroxidation in glioma cells stimulated by inflammatory factors.3.The mitochondrial membrane potential changes in glioma cells were measured using the mitochondrial membrane potential detection kit(JC-1)under the stimulation of inflammatory factors.4.Using NAC(N-Acetyl-L-cysteine)as a ROS inhibitor,act on glioma cells stimulated by inflammatory factors to observe changes in mitochondrial morphology.3: Quality control of mitochondria in glioma cells stimulated by inflammatory factors1.The use of LC3 B labeled mitochondrial autophagy,the use of Western Blotting and cell patch immunofluorescence assay in inflammatory factors stimulated glioma cells in the level of autophagy changes2.Plasmid transfection was performed using GFP-LC3 to label autophagy in the cells and to detect changes in autophagy levels in glioma cells stimulated by inflammatory factors.3.Specific fluorescence co-localization using LC3 B and MitoTracker to detect co-localization of autophagosome and mitochondria.4.Using LAMP1-labeled glioma cell lysosomes,MitoTracker labeled mitochondria to detect co-localization of lysosomes and mitochondria.Results1.Iba1 staining clearly shows that with the increase in the level of glioma,microglia cell body increased and tentacles shorter,indicating that high-grade glioma in the formation of microglia;2.The staining of IL-6 showed that the expression of IL-6 was positively correlated with the glioma level.3.The expression of reactive oxygen species in glioma cells was significantly higher than that in the control group(P <0.05),and the expression of reactive oxygen species in the glioma cells was significantly higher than that in the control group(P <0.05)Enhanced mitochondria did not find lipid peroxidation;3)glioma cells mitochondrial membrane potential after inflammatory factor stimulated significantly decreased;4.NAC inhibition of reactive oxygen production experiments found that glioma cells mitochondrial morphology was reversed,restored into tubular structure;5.The level of autophagy was significantly increased in the 4h group and the 8h group,and the autophagy was increased in the 24 h group,but the expression of GFP-LC3 was increased by immunogenicity.6.Mitochondrial(MitoTracker)and autophagosome fluorescence co-localization marker,no mitochondria and autophagy were found common co-localization;7.Lysosomes and mitochondrial co-localization experiments found that more serious damage to the mitochondria slightly through the autophagosome directly into the lysosome.Conclusions1.Inflammation factors LPS and IFN-? can affect the morphological structure of mitochondria in glioma cells.2.Inflammatory factors LPS and IFN-? may have some effect on mitochondrial membrane potential.