| Objective The purpose of this experiment was to clarify the effect of specific mode electroacupuncture（2/100Hz,3mA,6s-6s,40min）stimulated Baihui and Shuigou points of rats to improve BBB permeability,and the regulation mechanism of SHH-Glil-TJs signal pathway.Methods The experiment for the present study was conducted in two parts.For the first part of the experiment,rats were divided into 2 groups of 15 each:a control group and a group which received EA（2/100 Hz,3 mA,6s-6s,40 min）as specified parameters.The rats in both groups were injected with 2%EB（2mL/kg）and fluorescein isothiocyanate（FITC）dextran saline solution via an indwelling needle in the tail vein.Rats in the EA group underwent the insertion of acupuncture needles at Baihui（GV20,the center of the parietal bone）,and a 16-mm long,0.07-mm diameter needle was inserted at Shuigou（GV26,cleft lip in the middle of 1mm below the tip of the nose）.The needles were then stimulated using an acupuncture point nerve stimulator at an intensity of 3 mA for 40 min.The rats in the control group did not receive any treatment.The mixed solution of EB and FICF in the EA group was started immediately after the injection,while the control group was circulated for the same time as the EA（40min）after the injection.The rats in both groups were killed immediately after the end of the time.All experiments were conducted between 8:30 a.m.and 8:30 p.m.China Standard Time.The EB content in the brain tissue of eight rats from each group was evaluated.The expression of FITC-dextran and EB was observed in four rats using an optical confocal microscope,and the cerebrovascular ultrastructure in three rats was evaluated using a transmission electron microscope.For the second part of the experiment,rats were divided into three groups of 28:Control group（group A）,EA group,and EA+PUR group.Group EA and group EA+PUR underwent the same EA procedure described above.30 min before the start of EA,groups Control and EA were intraperitoneally injected with 5 mL/kg solvent,and group EA+PUR was intraperitoneally injected with 5 mL/kg solution of PUR.From each group,eight rats were randomly selected and injected with 2%EB normal saline solution through the tail vein prior to EA to determine the content of EB in brain tissue;four were injected with 2%EB and four were injected with FITC-dextran saline solution through the tail vein to evaluate the expression of FITC-dextran and EB under an optical confocal microscope;eight underwent western blotting and quantitative real-time polymerase chain reaction（qRT-PCR）to detect the gene and protein expression of Shh,Gli1,and TJ proteins;and four were observed with a confocal microscope to evaluate the fluorescence expression of TJ proteins.Results Experiment 1:1.Under naked observation,after specific mode of EA intervention,compared with the Control group,EB infiltration into the brain of rats in EA group was obvious;The content of EB in cerebral cortex of rats was detected by multifunctional microplate analyzer.Compared with Control group,there was more EB penetration in EA group,and the difference was statistically significant（***P<0.001）;2.By comparing the fluorescence expression of EB and FITC-Dextran in the brain tissues of rats in the control group and the EA group,the fluorescence expression of EB and FITC-Dextran in the cerebral parenchyma of the cerebral cortex of rats was not found in the blank group.However,EB and FITC-Dextran fluorescence could be observed in the cortex of rats after specific mode of EA intervention,and traces of penetrating BBB into the brain parenchyma could be observed in the EA group.3.Under transmission electron microscope,the cerebral blood vessels in control group were full and normal,and the endothelial cells were smooth,stable and compact without obvious gaps;After the intervention of specific mode of EA,the structure of the junction of endothelial cells in the cerebral blood vessels of rats in the EA group was changed.Experiment 2:1.Compared with the control group,the expression level of Shh mRNA in the frontal cortex of rats was significantly decreased after specific mode of EA intervention（*P<0.05）,and injection of Shh signaling pathway agonist PUR before EA could up-regulate the expression of Shh mRNA（##P<0.01）.Compared with the control group,the expression level of Gli1 mRNA in the frontal cortex of rats was significantly decreased after specific mode of EA（*P<0.05）,and injection of Shh signaling pathway agonist PUR before EA could up-regulate Gli1 mRNA expression（###P<0.001）,the level of Shh protein in frontal cortex of rats in EA group decreased compared with that in control group（*P<0.05）,the use of PUR up-regulated Shh protein expression（#P<0.05）;The level of Glil protein in the frontal cortex of rats in EA group was significantly lower than that in control group（***P<0.001）,and the use of PUR up-regulated Gli1 protein expression（#P<0.05）;2.In the control group,tight junction protein Claudin5,Occludin and ZO-1 showed a dense and continuous and stable structure without obvious traces of fracture,and Claudin5 overlapped closely with Occludin and ZO-1.Compared with the control group,Occludin and ZO-1 in the EA group after specific mode of EA intervention showed signs of fracture to varying degrees compared with Claudin5,which could not completely cover the fluorescence imprint of Claudin5.However,the rats injected with PUR before EA maintained the stability of tight junction protein compared with the EA group.WB results showed that Occludin in the frontal cortex of rats decreased after EA intervention,but there was no statistical difference,while the expression of Occludin in the rats injected with PUR before EA was higher than that of the EA group（#P<0.05）.Zo-1 protein expression was significantly decreased in the frontal cortex of rats after EA intervention（**P<0.01）,while ZO-1 expression in the EA+PUR group increased compared with the EA group,but there was no statistical difference.It suggested that EA downregulated the expression of tight junction protein Occludin and ZO-1 by inhibiting Shh signaling pathway,while the use of PUR reversed the effect of EA.3.EB permeation was observed in EA group but not in control group.EB also appeared in EA+PUR group,but the blue area was significantly less than EA group.Shh signaling pathway inhibitor PUR was injected into male SD rats in EA+PUR group 30 min before electrostimulation,and then electrical stimulation was performed according to the above specific parameters.We measured the content of EB in cerebral cortex of each group with multifunctional microplate analyzer,and found that the content of EB in EA group was higher than that in control group（***P<0.001）,while PUR reversed this effect and reduced the content of EB in brain（#P<0.05）.By comparing the fluorescence expression of EB and FITC-Dextran in the brain tissues of rats in each group,it was found that both EB and FITC-Dextran had obvious infiltration in the frontal cortex of rats after EA intervention in a specific mode,while PUR injection before EA could reduce the infiltration of the two dyes.Conclusion（1）Specific stimulation mode electroacupuncture can improve the permeability of BBB in rats.The verification of this effect provides experimental basis for electroacupuncture to help macromolecule drugs enter brain tissue and play a therapeutic role;（2）The mechanism of increasing BBB permeability by electroacupuncture in specific stimulation mode is related to the inhibition of Shh-Gli1 signaling pathway and the downregulation of TJs. |
PDF Full Text Request