| Background:Advanced glycation end products?AGEs?is an important pathogenic substance of diabetes mellitus?DM?,and also plays an important role in the promotion of Alzheimer's disease?AD?.It can promote the excessive phosphorylation of microtubule-associated protein Tau protein and the deposition of amyloid??A??.The macrophage migration inhibitory factor?MIF?,another pathogenic factor in DM,was also found to be significantly higher in cerebrospinal fluid?CSF?in AD patients and mild cognitive impairment?MCI?patients than in normal patients of the same age.Loss of MIF can also reduce the excessive phosphorylation of Tau protein.Objective:1.Study on the effect of AGEs on MIF and its molecular mechanism.2.The protective effect of MIF inhibitor ISO-1 on AD cell model induced by AGEs was studied.Methods:1.PC12 cells were cultured in vitro and treated with AGEs.Real-time fluorescence quantitative PCR?qRT-PCR?and Western blot?Western Blot?were used to detect the expression of MIF mRNA and protein in PC12 cells.Blank control group and BSA control group were set up.2.PC12 cells were cultured in vitro.PC12 cells were treated with AGEs.Nuclear factor-k-gene binding?NF-?B?inhibitor,mitogen-activated protein kinase?MAPK?inhibitor,phosphoinositide 3 kinase?PI3K?inhibitor,phospholipase C?PLC?inhibitor,the receptor of advanced glycation endproducts?RAGE?inhibitor were added respectively and qRT-PCR assay was used to detect mRNA expression of MIF.Blank control group and AEGs control group were set up.3.The AD cell model was established.Screening the optimum concentration of AGEs and the concentration of A?1-40-40 in the AD cell model by the CCK-8 assay and the MTT method.The cells number and state changes were observed under the inverted microscope through the action of AGEs and MIF inhibitor iso-1 on PC12cells induced by A?1-40.The expression of mRNA in interleukin 1 beta?IL-1??,interleukin 6?IL-6?and tumor necrosis factor-alpha?TNF-??was detected by RT-PCR.cells were observed under inverted microscope.The mRNA expression of IL-1?and IL-6,TNF-?was detected by qRT-PCR.Results:1.There was no significant difference in BSA between the control group and the blank control group.However,compared with the control group,the AGEs concentration of 100?g/ml,200?g/ml showed statistically significant differences?P<0.05?.The MIF mRNA expression was significantly higher than that in the control group.Cells treated with 100?g/ml or 300?g/ml AGEs produced statistically significant differences,with significantly higher levels of MIF protein detected in these groups than in the blank control group.2.Compared with the AEGs control group,the mRNA and protein expression of MIF were decreased by adding NF-?B inhibitor,MAPK inhibitor,RAGE inhibitor and PLC inhibitor to some extent?P<0.05?.3.Under inverted microscope?100×?,the PC12 cells in the control group have many and long dendrites.The cells tightly adhered to the bottom of the bottle,with dense,clear structure and visible nucleoli,and rapid proliferation.Compared with the control group,the proliferation of cells in A?1-40 modeling group was inhibited,with the number of cells decreased,the intercellular junctions were relaxed and some dendrites were retraction.After AGEs treatment,cell proliferation was further inhibited,a large number of cells floating round,more cell fragments.Loss of normal neuronal features of adherent cells.After pretreatment with ISO-1,the inhibition of cell proliferation was significantly decreased.The number of cells increased,floating cells decreased and adherent cells formed well,intercellular junctions increased,dendrites became longer and more.In the AD model group,percent inhibition of cell growth was significantly increased,and the expressions of the IL-1?,IL-6 and TNF-?mRNAs were increased in the AD model group.Compared with the AD model group treated with AGEs,combination AGEs with ISO-1 significantly improved the cell survival rate and downregulated the expression of inflammatory mediators in the AD cell model?P<0.05?. |
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