| Objective: Central Nervous System Infection is a common neurological disease with high morbidity and mortality.The main cause of the blood-brain barrier is the pathogenic microbial invasion of inflammatory changes,leading to structural damage and decreased function,there is evidence that the adhesion of protein family involved in the process,but the specific mechanism is not clear.In this study,we ithe function of P120cn(P120ctn)in lipopolysaccharide-mediated blood-brain barrier disorder and inflammatory response in vitro model.Methods: We mainly use human brain microvascular endothelial cells(human brain microvascular endothelial cells HBMECs)as the object of study.HBMEC(2×104)was incubated with Fluoro Blok colored tissue culture(pore size 3 ?m),and FITC-labeled dextran was added to the medium at a final concentration of 100 ug/ml.Cells were stimulated with a concentration of 1 ug / ml.The corresponding time for stimulation was 0 hours,3 hours,6 hours,12 hours,24 hours,samples were collected from the recipient chamber and samples were measured by fluorescence spectrophotometer and microplate reader The fluorescein-labeled dextran concentration in the sample was calculated from the standard curve of fluorescein-labeled dextran in PBS.The protein level of P120 ctn was detected by Western blot analysis.At the same time,the content of FITC dextran was correlated with the protein content of P120 ctn.In order to further verify that the change of P120 ctn protein level was related to the permeability of blood-brain barrier,we knocked down P120 ctn by RNA interference technique or overexpressed P120 ctn with adenovirus infection in HBMECs.The expression of P120 ctn protein was detected by immunoblotting.Real time PCR was used to detect the level of P120 ctn messenger RNA.The permeability of endothelial cells in each group was also detected by FITC dextran labeling experiments,and their changes were observed in the case of lipopolysaccharide LPS(1ug/ml)for 24 hours.Immunoblotting method was used to detect experimental samples The protein level of the close connective protein occludin,cldudin-5.P120ctn affects not only the integrity of the tight connection but may also the migration of monocytes.The mononuclear cells isolated from PBMC were set up to knock down the target protein P120 ctn group or the overexpressed protein P120 ctn group,and the two control groups were stimulated with LPS to detect the migration of monocytes.At the same time,the HBMECs were set up to knock down the target protein P120 ctn group or the overexpressed protein P120 ctn group,and the two control groups were stimulated with LPS to detect the m RNA levels of MCP-1,IL-1?,CXCL-1,ICAM-1,VCAM-1 and E-selectin were detected by real-time quantitative PCR to check whether P120 ctn affected the secretion of cytokine.P120ctn affects not only the integrity of the tight connection but may also the inflammatory response of cells.The mononuclear cells isolated from PBMC were set up to knock down the target protein P120 ctn group or the overexpressed protein P120 ctn group,and the two control groups were stimulated with LPS to detect the migration of monocytes.At the same time,the m RNA levels of MCP-1,IL-1?,CXCL-1,ICAM-1,VCAM-1 and E-selectin were detected by real-time quantitative PCR to check whether P120 ctn affected the secretion of cytokine.The luciferase reporter gene expression of P120 catenin protein and instantaneous transfection of NF-?B,We transiently transfected with NF-?B luciferase reporter gene seted up in knock down the target protein P120 ctn group or the overexpressed protein P120 ctn group,and the two control groups.After 48 hours,LPS was added for 24 hours to monitor the luciferase activity in each group.How does it affect the activation of NF-?B?The expression of P65 and P50 in the nucleus of the epithelial cells and the control group were detected by immunoblotting.The levels of pi?b?,ikk? and ikk? in the cytoplasm were detected by immunoblotting,and the correlation was analyzed.Results: We found that the level of P120 catenin in human brain microvascular endothelial cells was significantly reduced by lipopolysaccharide and decreased to 40% at 24 hours,and the content of FITC dextran was increased with the prolongation of lipopolysaccharide stimulation And the increase of cell permeability was negatively correlated with the decrease of P120 ctn protein level r = 0.8682,p <0.05,which indicated that the change of cell permeability in endothelial cells was related to the P120 ctn protein.The P120 ctn protein level was significantly up-regulated at the time of MOI = 100 by adenovirus-mediated overexpression of the target protein P120 ctn.The target protein P120 ctn was knocked down by small interfering RNA,and if it was transferred to 20 nm,it could be knocked down at the protein level Low-level protein P120 ctn level.After treatment with LPS,the occludin and claudin-5 levels of P120 ctn and tight junctions decreased with time,and the change was linear.Indated in the case the of lipopolysaccharide stimulationed,P120 ctn interferes with the stability of blood-brain barrier by influencing the expression of tightly linked protein.The migration ability of the mononuclear cells isolated from PBMC treated with lipopolysaccharide alone was stronger than that of the two control groups,which increased the migration ability by about 50%.Suggesting that P120 ctn can affect monocyte migration.Similar results were seen from adenovirus-mediated overexpression in the P120 ctn group compared to the two control groups.After knocking down the P120 chain protein,some cytokine: MCP-1,interleukin-1?,CXCL-1 were significantly increased,suggesting that P120 ctn affects the secretion of cytokine.The luciferase reporter gene of NF-?B was transfected and the activity of luciferase was significantly enhanced after the treatment of lipid polysaccharide,indicating that the endothelial cells were activated by lipid polysaccharide and activated NF-?B signaling pathway.It was found that cells overexpressing P120 ctn were significantly lower than those of the two control groups.After treatment,the activity of luciferase was significantly enhanced,which also well demonstrated that endothelial cells were activated by lipopolysaccharide NF-?B signaling pathway,also well showed that P120 ctn affected the activation of NF-?B.And P65 and P50 were significantly enhanced by LPS treatment.The above experimental results showed that P120 ctn can be transported to the nucleus by inhibiting the NF-?B subunit P65 and P50.Thereby inhibiting the activation of the NF-?B signaling pathway.In further study,we found that pikba increased in the cytoplasm after LPS treatment,but the level of the P120 ctn group was significantly lower compaired with the control group.It was also found that the protein level of i?b? was significantly decreased after LPS treatment,but in the P120 ctn overexpression group,the degree of decline was significantly lower than that of the control group,so we can see that overexpression of P120 catenin can inhibit the phosphorylation of i?b?,Thereby inhibiting its degradation by proteasomes,resulting in accumulation of i?b? levels in overexpression groups.The phosphorylation of ikba was mainly through ikk kinase,so that we detected the protein level of ikk? and found there was no significant difference in the overall protein.Continuing to track,we found the phosphorylation of ikk? was significantly enhanced with the stimulation of lipid polysaccharides,but the P120 ctn group was significantly lower than the control group.From this all,we can conclude P120 ctn can inhibit ikk? activation so that inhibiting the phosphorylation of i?b?,whiah inhibits the degradation of the proteasome ikba mediated,leading to decrease the activation of NF-?B.Similarly,by knocking down P120 catenin protein,we detected the protein levels of ikk? and pi?b?,and found that pikk? was significantly higher than the control group in the lower group.So that,we can say that the low P120 protein can promote the phosphorylation of ikk? and i?b?,thus promoting the activation of the downstream NF-?B pathway.Conclusion: P120 catenin can protect the blood-brain barrier through tight junction;which also can inhibit the activation of NF-?B in brain microvascular endothelial cells during the pathogen invased,,so that the inflammatory response was inhibited.It is reveals that verexpression of P120 ctn can induced the brain-brain barrier disorder and inflammatory response,as a new strategy for the treatment central nervous system infection. |
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