| Objective: Subarachnoid hemorrhage(SAH) is an acute severe cerebrovascular disease with high mortality and morbidity. The main reason of the high mortality rate was believed to be cerebral vascular spasm(CVS), however little effects were achieved after plenty of studies. Present studies suggest that early brain injury(EBI) after SAH was the main reason leading to high mortality and poor prognosis. In this study, SAH models in rats were established, the expression of peroxisome proliferator activated receptor(PPARβ/δ) was exogenously regulated to detect its effects on EBI and to explore the mechanism of it after EBI.Method:1. SAH model was built on 128 male SD rats by injecting non-heparinized arterial blood into prechiasmatic cistern. Rats were randomly divided into two parts, Part I was injected with over-expressed adenovirus(Ad-PPARβ/δ) into lateral ventricle to up-regulate the expression of PPARβ / δ. Part II was injected with interference RNA(si-PPARβ/δ) into lateral ventricle to down-regulate the expression ofPPARβ / δ. Part I was devided into Control group, SAH group, SAH +Ad-GFP group and SAH + Ad-PPARβ / δ group. Part II was devided into Control group, SAH group, SAH + si-CON group and SAH + si-PPARβ / δgroup. Neurological function scoring and balance beam test were applied to detect the behavioral change in each group at 6h, 12 h and 24 h after SAH.Changes in cerebral blood flow(CBF) of each group were monitored at 1h,6h, 12 h and 24 h after SAH. Changes in permeability of blood brain barrier(BBB) and brain edema were detected in each group at 24 h after SAH by Evans Blue(EB) exudation and wet-dry weight method respectively.Neuronal apoptosis in rats’ hippocampuses at 24 h after SAH was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL). The expressions of tight junction protein Claudin,Claudin-5, Zonula occludens- 1(ZO-1) and apoptosis related protein Bax,Bcl-2, Cleaved-caspase3 in hippocampuses were detected by Western Blot.2. Animal model establishment and grouping were the same as above.The expression of PPARβ / δ protein in hippocampus was detected by immunohistochemistry and Western Blot. Activity changes of matrix metalloproteinase-9(MMP-9) in rats’ hippocampuses were detected by Gelatin zymography method. And the expressions of MMP-9, of NF-κB,c-Fos, TGF-β1 were determined using Western Blot.Result:1. The mortality rate of rats being constructed SAH model in 24 h was33.3%, and significant neurological disorders and reduce in cerebral blood flow were observed. The application of Ad-PPARβ / δ showed no significant change in 12 h after SAH, however it obviously improved the rats’ behavioral function, promoted the recovery of CBF and reduced mortality at 24 h. The ameliorative symptoms mentioned above disappeared after the use of si-PPARβ/δ, aggravated neurological dysfunction was even found compared with SAH group, and the recovery of cerebral blood flow at 24 h after SAH was also slower.2. BBB rupture and severe cerebral edema were obvious in rats at 24 h after SAH. The overexpression of PPARβ/δ was able to reduce EB exudation and up-regulate expression of ZO-1, but has no effect on expressions of Occludin and Claudin-5. In PPARβ /δ silence group, the further increase in permeability of BBB was observed, the loss of ZO-1was more serious compared with SAH group, but no significant change was found in Occludin and Claudin-5, showing no statistical difference with SAH group.3. Neuronal cell apoptosis in rats’ hippocampuses were distinct after SAH according to TUNEL assay, the expressions of apoptosis-related proteins- Bax, Bcl-2 and Cleaved-caspase3- increased significantly.Ad-PPARβ / δ treatment after SAH reduced the expression of Bax and Cleaved-caspase3 and increased the expression of Bcl-2. Compared with SAH group, si-PPARβ/δ treatment increased the expression of Bax andCleaved-caspase3 while reduced the expression level of Bcl-2.4. PPARβ /δ mainly expressed in the cytoplasm of nerve cells in the hippocampuses of rats with little expression in the nucleus. The expression of PPARβ /δ reduced at 24 h after SAH, and the treatment of Ad-PPARβ/δand si-PPARβ/δ could up-regulate and down-regulate the expression levels of PPARβ /δ separately. Western Blot test results showed that the activity and expression levels of MMP-9 increased after SAH in rats. The overexpression of PPARβ /δ significantly could reduce the activity and expression level of MMP-9, and the adverse results were obtained after PPARβ /δ silence with increased expression and activity of MMP-9. The expression of NF-κB showed consistent trends with MMP-9. The increased expressions of c-Fos, and TGF-β1 were only detected after SAH, showing no correlation with PPARβ /δ expression.Conclusion:1. PPARβ/δ can reduce the mortality of rats with SAH by promoting the recovery of CBF and improve neurological dysfunction after EBI.2. PPARβ/δ may promote the expression of ZO-1 to improve the BBB damage and reduce the extent of brain edema.3. PPARβ/δ can reduce neuron cell apoptosis after SAH and improve early brain injury.4. The neuroprotective role of PPARβ/δ in EBI may be realized by down-regulating the NF-κB / MMP-9 pathway... |
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