Apoptosis Of Cells Surrounding Hematoma Mediated By AQP4 RNAi In Rat Hemorrhage

Backgrounds and objectives:Cerebral hemorrhage is a common disease in older patients. With high rate of mortality and morbidity, it seriously threats to human life. It is about 20% for the patients dead due to cerebral hemorrhage among all disease mortality. There is no effective prevention and treatment for the damage by brain hemorrhage, and the two type of damages, hematoma occupation effect and directly injuries on surrounding brain tissues, are the two main mechanisms of hemorrhagic brain injury.Actually the secondary injury is an important factor to influence the prognosis. It was validated that brain tissue around hematoma had incomplete ischemic area, which resulted in secondary damage by inducing cell apoptosis. Cell apoptosis was involved in the secondary injury of brain hemorrhage. Apoptosis processes included inducible initiation, intracellular regulation, implementation and phagocytosis. One of the features of apoptosis is water loss and cell shrinkage. The water was lost by simple diffusion of hydrophobic phospholipid bilayer or by help from some aquaporins. It was reported that cell apoptosis around hematoma was associated with aquaporins.Aquaporin(AQP) is one kind of molecules for water channel. More than 13 kinds of aquaporin(AQP0 ~ AQP12) have been found in mammalian tissues, among which aquaporin 4(AQP4) expressed richly in brain tissue. AQP4 participated in the processes of the formation and regression of brain edema, which played important regulatory roles in motion of water across the cell membrane during apoptosis. In this study, we tried to down-regulate AQP4 expression by RNAi technology, which relieved hematoma and apoptosis of brain cells and protected brain tissues surrounding hematoma to reduce secondary damage. Objectives: This study aims to investigate the influence by aquaporin-4(AQP4) si RNA on apoptosis of cells surrounding hematoma in rat hemorrhage models. Methods: Total 30 SD rats were divided into 3 groups: rat hemorrhage model group, blank plasmid group and AQP4 si RNA group, with 10 rats in each group. The caudate putamen brain hemorrhage model was induced by Thrombin Ⅶ injection. After three days, a modified Longa grading method was applied to classify neurological function. The content of water in brain was determined and TUNEL assay was used to detect apoptosis in brain tissue. AQP4 expression was detected by RT-PCR, and the expressions of matrix metalloprotei-2(MMP-2), matrix metalloprotein-9(MMP-9), Caspase-3, and Bcl-2 were detected by Western Blot. Results: The estimated scores about neurological function were significantly lower in AQP4 si RNA group than in blank plasmid group and hemorrhage model group(P < 0.05). Rat brain tissue bleeds obviously and hematoma was observed in brain tissues in blank plasmid group and hemorrhage model group. In AQP4 si RNA group, the content of water in brain tissue was about 76.7%. TUNEL results showed that the stained brown areas of surrounded tissues of hematoma were observed in blank plasmid group and hemorrhage model group, while stained color was lighter in AQP4 si RNA group. AQP4 RNA expression was significantly higher in blank plasmid group and hemorrhage model group than in AQP4 si RNA group. Compared with the blank plasmid group and hemorrhage model group, the expressions of MMP-2/MMP-9 and Caspase-3 were decreased significantly in AQP4 si RNA group, while Bcl-2 expression was significantly increased(P < 0.05). Conclusion: AQP4 down-regulation by si RNA can significantly relieve the damage of neurological function after hemorrhage of rat. AQP4 si RNA also represses the apoptosis of cells around hematoma. These may be associated with the decrease of MMP-2, MMP-9, and Caspase-3, and the increase of Bcl-2 induced by down-regulation of AQP4.