| Objective: In cerebral ischemia, vascular occlusion cause to ischemicpathological process. Ischemia and subsequent reperfusion injury is themain factor of vascular endothelial cell injury. And the damage andnecrosis of endothelial cells trigger the damage of intercellular junctionproteins and the connection between endothelial cells and basementmembrane, which results in the destruction of blood brain barrier integrity.At the same time, the stabilitu of endothelial cells or internal environmentcan influence the morphology of nervous system. Therefor, it is the key ofacute ischemia therpy to maintain the stability of endothelial cells.Fibulin-5is an extracellular matrix (ECM) protein crucial for stabilizationof macromolecular ECM complexes. This protein plays a key role inphysiological process such as tissue development, remodeling and repair.Fibulin-5acts as a multifunctional protein involved in cell-cell andmatrix-cell connection. It can influence vascular cell proliferation, adhesion,and migration. The expression of fibulin-5was strongly up-regulated in theembryonic vasculature and neural crest, but was down-regulated in adulttissue. And it was dramaticly activated and up-regulated in re-builted vessles or re-generation tissue. Experimental researches found thatfibulin-5was lowly expressed in adult vascular endothelia cells, butup-regulated in injured vessels and endothelia cells under ischemia. Toinvestigate the role of fibulin-5in cerebral ischemia reperfusion (IR), ourstudy observed the expression of fibulin-5in rats after cerebral ischemiareperfusion, and the correlation among fibulin-5expression and the bloodbrain barrier (BBB) permeability. At the same time, we investigated theeffect of fastigial nucleus stimulation (FNS) on the expression of fibulin-5in rats after stroke to explore new field of vision in treat of ischemiccerebral vascular disease.Methods:Part one：108male Sprageue-Dawley rats were randomly divided into6groups：control，sham-operation，and6h,24h,48h,72h after ischemiareperfusion groups. The focal middle cerebral artery occlusion (MCAO)model was induced by ligation with nylon monofilament. Evan Blue (EB)was used to judge BBB permeability. Laser confocal was used to fix thelocation of fibulin-5in brain tissue. The expression of fibulin-5mRNA andprotein were assessed by real-time quantitative PCR and western blot,respectively.Part two:100male SD rats were divided into5groups：sham-operation，ischemia reperfusion group (24h), and FNS group,sham-FNS group, PI3K/Akt pathway inhibitor LY294002group. To investigate the effect of FNS on the expression of fibulin-5after cerebralischemia and the relationship among fibulin-5expression and BBBpermeability, the focal MCAO model was induced by ligation with nylonmonofilament. FNS and PI3K/Akt pathway inhibitor LY294002weretreated after2h of ischemia reperfusion. EB content was used to assay thepermeability of BBB. The expression of fibulin-5mRNA and protein wereassessed by real-time quantitative PCR and western blot respectively.Results:Part one:①The fibulin-5was mainly expressed in ECM, and alsoexpressed in endothelia cells.②EB contents at different time pointsfollowing IR showed that, slightly increased EB content was observed at6h（8.63±1.03）in the infracted brain tissues, and the peak value weredetected at24h（13.86±1.33） and48h （13.06±1.77）after cerebral IR.EB content decreased at72h（10.66±1.47）following perfusion. Thedifference in EB content was significant when each individual group wascompared with sham group (P<0.01) or compared with each other (P<0.05).③Quantitative analysis was used to calculate the expression of fibinlin-5mRNA at different time following reperfusion. Quantitative analysisshowed that the expression of fibinlin-5mRNA increased6h afterreperfusion and the increase was significant compared with sham group(1.22±0.03, P<0.01). The expression of fibinlin-5mRNA kept increasing6-24h after IR, and then decreased gradually48h (1.19±0.02）after perfusion. The different time points group all showed significant differencecompared with the sham group (P<0.01).④Since we observed similarchanges in EB content and fibinlin-5mRNA level after IR, next we attemptto address their association. Based on the statistical analysis, the correlationcoefficient r between EB content and fibinlin-5mRNA level was0.748,and it was statistically significant (P<0.01). The data suggests that mRNAexpression of fibulin-5is positively correlated with BBB permeability.⑤Western blot showed that, compared with sham group, the level of fibulin-5protein increased at6h after ischemia（1.26±0.46）, reached the maximumat24h（1.78±0.48） and then gradually decreased to a low level which wasstill higher than the sham group at72h (0.89±0.27）. There was nosignificant difference between normal group and sham group (P>0.05).Part two:①Compared with IR24h group (14.19±0.89), EB contentwas significantly lower in FNS group (9.42±0.57, P<0.01) while there wasno obviously difference between IR group and sham FNS group(13.47±1.44， P>0.05), suggesting that FNS could decrease BBBpermeability follow ischemia. In contrast, inhibitor group （1.27±0.03）evidently increased EB content compared with IR group （P<0.01）. EBcontent was significantly up-regulated in IR group and intervention groupcompared with sham-operated group, respectively.②Compared with IRgroup, FNS significantly promoted the expression of fibulin-5mRNA at24h （1.62±0.02，P<0.01） after cerebral ischemia while the level of fibulin-5 mRNA had no evidently change in sham FNS group(1.46±0.02, P>0.05).The expression of fibulin-5mRNA was significantly decreased in inhibitorgroup in our investigation（1.27±0.03, P<0.01）.③The protein expressionof fibulin-5had the same trend. Fibulin-5protein expression was obviouslyincreased in the FNS group（0.66±0.07） with IR group（0.45±0.03） andsham FNS group（0.43±0.05）. In addition, the expression of fibulin-5wassignificantly down-regulated in inhibitor group after perfusion（0.28±0.03）.Conclusion: The expression of fibulin-5was obviously up-regulatedafter cerebral ischemia reperfusion, and this change had evident positivecorrelation with BBB permeability. FNS could increase the expression offibulin-5and lighten the damage of BBB permeability after reperfusion.And PI3K/Akt pathway inhibitor LY294002could decrease the expressionof fibulin-5and aggravate the damage of BBB integrity. Therefor, fibulin-5maybe had important role in protectting BBB integrity damage afterischemia reperfusion. |
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