| Part 1 100?M Propofol pretreatment reverses glutamate-induced astroglial excitotoxicity in rats via p90RSK signaling pathwayObjective:To detect the semi inhibitory concentration of glutamic acid on primary astrocytes of rats.We build on the toxic astrocytes model by administration of the semi inhibitory concentration of glutamic acid.The effect of pretreatment with propofol at 100?M for 6 hours on the co-expression of GFAP-Caspase-3 in astrocytes and the effect of pretreatment with propofol for 6hours on the primary astrocytes induced by glutamic acid were observed.Method:One day after the newborn SD rats were anesthetized with chloral hydrate,their heads were cut off,their skulls and meninges were peeled off under aseptic operation,bilateral cerebral cortex were separated passively,cortex tissues were taken and put into DMEM-F12 medium,then they were cut up,filtered in 200/400 mesh,digested and centrifuged and incubated at 37?for 10 minutes,following with digestion.Cell suspension was taken and transferred to 25cm~2jet culture flask in equal amount.The primary cultured cells were transferred to 37?shaking table overnight for removing the cortical neurons and microglia.when observing the confluence of cells under 200×light microscope,the cells were divided into 6 groups and the following drug function experiments of glutamate and propofol were carried out to identify by GFAP immunohistochemistry(1:400)and immunofluorescence(1:100).experiment is divided into six groups:control group,propofol group,propofol+Glu group,glutamate group,propofol+glutamate+inhibitor PD9805 group,inhibitor PD9805 group.The effect of propofol on the apoptosis of astrocytes were detected by GFAP-caspase-3 co-expression immunofluorescence.the survival rate of propofol cells and the concentration of glutamic acid semi inhibition were analyzed by CCK8 cell counts kits,the protein expression levels of p90RSK,Bcl-2,Bax and cleave caspase-3 in p90RSK signaling pathway were detected by Western Blot,and the apoptosis rate of cells in each group were detected by flow cytometry.Result:1.Compared with normal astrocytes,100?M propofol decreased the expression of Caspase-3,but it has not effect on the expression of GFAP.Glutamate induced astrocytes excitotoxicity by semi inhibitory concentration 3.5m M.Its neurotoxicity may be related to the inhibition of cystine uptake,the increase of oxidative stress,and the promotion of mitochondrial apoptosis.The preliminary experiment showed that 12.5?M to 100?M propofol had no significant toxic effect on the activity of astrocytes,and the concentration of 100?M propofol did not significantly reduce the OD value of astrocytes in primary culture for 6 hours.2.Glutamic acid inhibited the expression of Bcl-2 in astrocytes,up regulated mitochondrial apoptosis protein cleave caspase-3 and pro apoptosis protein Bax,After pretreatment of astrocytes with propofol,the ratio of Bcl-2/Bax increased,and the first target downstream substrate p90RSK of ERK1/2 also increased after pretreatment with propofol.After the upstream inhibitor of ERK1/2(PD9805),the ratio of Bcl-2/Bax decreased.RT-PCR confirmed that the expression of Bcl-2 m RNA of antiapoptosis gene increased after pretreatment with propofol,and increase of annexin also confirmed that propofol can effectively reverse the neurotoxicity of glutamate and improve the activity of astrocytes.Conclusion:100?M propofol pretreatment of astrocytes has no toxic effect on astrocytes,activation of p90RSK in astrocytes can promote cell survival and inflammatory repair pathway;Glutamate directly inhibited the survival rate of astrocytes and the expression level of total protein p90RSK/Bcl-2,as well as activation of the mitochondrial apoptosis pathway related proteins(cleave-caspase 3 and Bax);propofol pretreatment of astrocytes reversed the excitatory toxicity of glutamate through the p90RSK/Bcl-2 signal pathway,and reduced the death of astrocytes.Part 2 The effect of propofol on the calcium homeostasis of astrocytes induced by glutamateObjective:To study the effect of calcium signal on astrocytes induced by glutamate pretreated with propofol and the effect of propofol and glutamate on CD38/calcium signal pathway.Methods:one day after newborn SD rats were anesthetized with chloral hydrate,their heads were severed,their skulls and meninges were peeled off under aseptic operation,bilateral cerebral cortex were separated passively,cortex tissues were taken and put into DMEM-f12 medium for culture,then they were cut up,filtered in 200/400 mesh,digested and centrifuged,incubated at37?for 10 minutes,digested again,cell suspension was taken and transferred to25cm~2jet culture flask in equal amount,the primary cells cultured in incubator were transferred to shaking table at 37?after the third day of culture,and the fluid was changed overnight for removing the cortical neurons and microglia.When cells were confluence at 80%,the cells were identified by GFAP immunohistochemistry(1:400)and immunofluorescence(1:100),and then using western blot to dectect the expression of CD38 in astrocytes pretreatedwith propofol,and the intensity of calcium was detected by calcium fluorescence probe.The relationship between CD38,MAPK signal pathway and cell survival was retrieved and analysized by bioinformatics methods such as KEGG and UCSC.The primary astrocytes and astrocytes were cultured respectively,the experimental groups were as follows:control(normal group),propofol(propofol group),propofol+Glu(glutamate intervention group after propofol pretreatment),glutamate(glutamate group)Results:1.There was no significant change in CD38 expression of primary astrocytes induced by glutamic acid for 6 hours.2.Propofol pretreatment enhanced CD38 expression of astrocytes induced by glutamate.3.The release of calcium in mitochondria is accompanied by the release of calcium.Propofol causes the accumulation of calcium in mitochondria by activating CD38 of astrocytes.Conclusion:The expression of CD38 was not affected by the semi inhibitory concentration of glutamate.Propofol activated CD38 to promote calcium accumulation in cell membrane and extracellular release.Part 3 propofol pretreatment attenuates glutamate-induced PC12 neuron toxicity via CD38 signaling pathwayObjective:To study the effect of CD38/calcium signaling pathway in propofol pretreated astrocyte culture medium on the co-cultured neuronal cell line PC12,to explore the role of CD38 in the communication between astrocytes and neurons,and to further explore the power and mechanism of mitochondrial transfer.Method:Experimental group:astrocytes derived from newborn Sprague dawley rats were provided by animal experimental center of Guangxi Medical University.PC12 was purchased from the national experimental cell resource sharing service platform.When the cell culture converged to 80%,CD38 si RNA designed by biotechnology company was mixed with the transfection reagent and put into astrocytes for incubation for 24 hours After the CD38 was knockdown successfully confirmed by RT-PCR,it was divided into four groups:control(normal PC12),glutamate(3.5mm glutamic acid pre dried PC-12 cells for 6h)and astrocyte medium+Glutamate(containing the supernatant of astrocytes pretreated with propofol+glutamic acid to interfere with PC-12 cells);si RNA CD38 medium+glutamate(containing the supernatant of astrocytes pretreated with propofol(CD38 silenced)and glutamic acid to pretreat PC-12cells).Western blot was used to detect the first target protein p90RSK of MAPK signal pathway and mitochondrial apoptosis pathway.After CD 38-PE single staining and annexin PI double staining,the apoptosis rate of each group was detected by flow cytometry.Results:Propofol activated astrocyte culture medium can protect co-cultured neurons PC12 from glutamate induced excitotoxicity.In the first part,it has been proved that 100?m propofol can activate the p90RSK signal pathway expression of astrocytes after 6 hours of pretreatment.Compared with non-astrocytes culture medium,the astrocytes treated with propofol can protect PC12 from glutamate induced excitotoxicity After cell culture,the results showed that exposure to 3.5 m M glutamic acid interfered with the synchronous up regulation of p90RSK/CREB/Bcl-2 in PC12 neurons,and there was no significant expression of mitochondrial apoptotic protein cleave-caspse-3.The results suggested that the excitotoxicity of glutamic acid was also inhibited by p90RSK signal in neurons.The culture medium of astrocytes effectively reduce the neuronal apoptosis of glutamic acid by inhibiting mitochondrial apoptotic pathway In the second part,it is proved that propofol pretreatment can activate the expression of CD38,which is involved in the release of calcium ions and the release of mitochondria which absorbed calcium ions.The results of CD38single staining flow cytometry showed that the expression of glial cell culture medium was significantly higher than that of non-glial cell culture,so the results showed that propofol pretreatment of glial cell culture medium can reduce the apoptosis effect of glutamate on PC12,but propofol pretreatment of glial cell culture medium after si RNA CD38 silencing had no significant anti-apoptosis effect.Conclusion:inhibition of CD38/Ca2+signaling pathway can decrease the protective effect of astrocytes on co-cultured neurons in glutamate induced apoptosis.CD38 promotes the release of calcium ions and drives the release of mitochondria from astrocytes.PC12 internalizes Vesicles and produces agonistic glutamate excitatory neurotoxicity.However,propofol pretreatment with si RNA CD38 could not improve the expression of p90RSK and p-CREB in PC12,which indicated that CD38 played an important role in the regulation of intercellular calcium and Vesicles transfer between PC12 and astrocytes. |
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