| Objective: Clinically brain cell injury after intracerebral hemorrhage (ICH) always causes dysfunction or death on such patients. Studies had identified that the mass of thrombin generated in the process of blood clotting may contribute to the brain cell injury after ICH. Thrombin, a key enzyme in the "waterfall reaction" of blood clotting, is also known as the multifunction serine protease enzyme in other field. There are great deals of thrombin-activated receptors- protease-activated receptors (PARs) on the brain astrocytes. Thrombin might participate in the apoptotic injury of astrocytes through PARs. Aprotinin, a natural serine protease inhibitor derived from bovine lung, had been reported that it could accommodate the cell reactions when PARs were activated in our previous study. However, it remains unclear whether aprotinin had protective functions to the injury of astrocytes correlated with thrombin. In this study, Astrocytes were cultured primarily to study the action of thrombin in vitro. Different precondition dosages were compared to observe the protective effect of aprotinin as an agonist of thrombin and to explore its possible mechanism.Materials and Methods: Astrocytes were isolated from the cortex of less than 24-hour-old healthy neonatal inbred KM mouse. Immunocytochemistry (ICC) method was employed to determine the purity of astrocytes, and only when it more than 95% the cells were used in the experiment. This study comprised two parts, dosage-dependent thrombin experiment (Group T) to investigate the effect of thrombin at different dosages on the cells, and aprotinin precondition experiment (Group A) aiming to explore the protective effect of different-dosaged aprotinin on the cell injury. In Group T, following 5 dosages of thrombin were applied to observe the dosage-dependent fashion: C (culture medium control), T10 (thrombin 10 U/ml), T20 (thrombin 20 U/ml), T50 (thrombin 50 U/ml), and T100 (thrombin 100 U/ml). In Group A, 100 U/ml thrombin was added to the cell culture 15min after aprotinin treatment. Aprotinin were applied at 6 different dosages to observe the effect of aprotinin preconditioning at different dosages, i.e., C (culture medium control), A0 (only culture medium preconditioning), A100 (100 KI U/ml aprotinin preconditioning), A300 (300 KI U/ml aprotinin preconditioning), A500 (500 KI U/ml aprotinin preconditioning), and A700 (700 KI U/ml aprotinin preconditioning). Flow cytometric analysis and AO-EB staining method were used to measure the apoptosis of astrocytes. The expression of apoptosis related genes were detected by ICC. Then the damage of DNA was detected by comet assay. LDH in the culture medium was measured with Beckman biochemistry clinical system for the permeability of cell membrane. Modified staining method with Coomassie brilliant blue R-250 was used to observe the shape of cellular skeleton. Finally, the ultrastructure of the cultured cells was observed by transmission electron microscopy.Results and Conclusions: In this study: â‘ Primarily cultured astrocytes grew well and were identified with specific protein of astrocytes-GFAP by ICC. Only the cells with purity over 95% were used in the drug action model. â‘¡After thrombin application, astrocytes lost their polarity and aggregated in mass with whose apophysis retracted markedly. In some cells, the nuclei were observed concentrated and marginated. The higher the dosage of thrombin was, the more severe changes were observed. Precondition with aprotinin could lessen such changes, but even A700 couldn't wipe out all these changes absolutely. â‘¢Flow cytometry revealed : The apoptotic ratio elevated with the increasing of thrombin dosage. Aprotinin attenuated the apoptosis markedly but the difference became milder when the dosage became higher. â‘£AO-EB staining method showed many yellow spots around the nuclei of astrocytes. As the dosage of thrombin increasing, more yellow spots were seen and deeper in color. Aprotinin reduced the quantity and color of the yellow spots, and ameliorated the aggregation of cell...
|
PDF Full Text Request