| Background:Cerebrovascular disease(CVD) has been considered to be a kind of vital disease for its high incidence rate and high disabilitiy rate. Amongst them is ischemic cerebrovascular disease seen frequently, it threatens people's life and health severely, gives heavy burden to the society and families. Because the mechanism of ischemic/reperfusion damage has not been known, it is the importance and difficulity of researches in many countries. With the development of mechanism after focal cerebral ischemic/reperfusion, some reports suggest that lots of nerves commitment to apotosis. Therefore, it is necessary and important to investigate the ischemic/reperfusion-induced cell apoptosis. Inhibition of neural apoptosis is one of perspective therapeutic means in interfering in ischemic/reperfusion injury.Neclear factor-κB is an important intranuclear transcription factor. It regulates the transcription and expression of a series of cell factor and erzymes. After focal ischemic/reperfusion, upregulation of NF-κB is actived and translocates into nuclear, is involved in apoptosis and inflammation course. C-Rel is a member of NF-κB protein. It is considered a inhibitior of apoptosis. Bcl-2 family proteins is the founding members of a new category of proto-oncogenes that modulates programmed cell death, as opposed to promoting or inhibiting cell division. Bcl-xl protein is an antiapoptotic member of Bcl-2 family proteins. In order to find a way to reduce injury after focal ischemic/reperfusion, it is important to study C-Rel and Bcl-xl. PDTC can selectively inhibite the activation of C-Rel, reduce expression of targeted genes .Objective : (1)To investigate the expression temporal and spatial changes of C-Rel protein in Wistar rat after focal ischemic/reperfusion, (2)To investigate the expression temporal and spatial changes of Bcl-xl protein in Wistar rat after focal ischemic/reperfusion, (3)To investigate that different NF-κB dimers plays different roles in the process of focal ischemic/reperfusion in rats, and accumulate experience for clinical treatment.Methods:Animal model was made by ligating external carotid artery and inserting a piece of nylon thread into the internal carotid artery among male Wistar rats. The rats were randomly divided into four groups : normal control group (NC group), sham operation control group (SC group), ischemic/reperfusion group (I/R group), ischemic/reperfusion treated with PDTC 100 mg. kg﹣1 group (I/RP group). Each group which has 6 rats contains six time points, such as 2h, 6h, 12h,24h,48h and 72h, expect for NC group which has only 3 rats. We use the HE staining method to observe the pathology variety, neurological impaire score method to observe neuro impairement, immunohistochemistry method to detect the expression of C-Rel, Bcl-xl protein, use RT-PCR detect the expression of Bcl-xlmRNA.Results: (1) After cerebral ischemia in rats showed neurological deficit, reperfusion 2h, 6h, 12h, the decreasing trend in ratings, but the comparison at each time point there was no significant difference ( p> 0.05); with the ischemia/reperfusion time extended, showing significant motor dysfunction;I / R group compared with the SC group, there are significant differences (p <0.05). PDTC intervention group score at each time point than the SC group, there are significant differences (p <0.05)(2) Light microscope and the SC group NS in rat brain nerve cells, glial cells, hippocampal pyramidal cells arranged in an orderly manner, the organizational structure of a complete, gap-free abnormal cell nucleus clear. I/R group can be seen in brain tissue changes in ischemic injury, reperfusion 2h, MCA blood supply to areas of cerebral cortex, thalamus, striatum neurons showed mild ischemic changes in ischemic brain tissue can be seen with mild osteoporosis, glial cell nuclear deep slightly stained, loose surrounding tissue cells, hippocampal pyramidal cell layer of normal structure; Reperfusion 6h, 12h, a further increase ischemic injury in the ischemic zone in the strong eosinophilic cytoplasm, karyopyknosis deeply stained, in the ischemic penumbra apoptosis and can be seen seemingly normal neurons; reperfusion 24h, ischemic zone blood extended to the entire MCA area was pale and clear boundaries and surrounding tissue, the central area of ischemic neurons, glial cells and cells surrounding both the medium edema, ischemic marginal zone as well as edema. Reperfusion 48h, 72h, clear the central area of ischemic neurons, glial cell line-dissolved necrosis, vacuolar structures appear, but the entire ischemic area decreased over 24h. PDTC group, we can see right MCA ischemic brain lesions, and the I/R group, reperfusion 2h, 6h, 12h, 24h, 48h, 72h, less ischemic cell edema, degeneration and necrosis of the small number of ischemic area than those in I/R group reduced accordingly.(3) The result of C-Rel protein expression by immunohistochemistry method: NC group found only a little amount of weakly positive C-Rel-labeled cell, the cell nuclear were not stained. In I/R group: C-Rel-labeled cell started to increase at 2h, and reach a peaked 12h, Comparing SC group and NC group with I/RP group, the differences were significant (p<0.05), then, C-Rel-labeled cell started to decrease at 48h, Comparing SC group and NC group with I/RP group 48h, 72h, there were not significant differences (p>0.05). In I/RP group: positive C-Rel-labeled cell weakly increased, there were not significant differences between SC group, NC group and I/RP group (p>0.05).(4) The result of Bcl-xl protein expression by immunohistochemistry method: NC group found only a little amount of weakly positive Bcl-xl-labeled cell. In I/R group: Bcl-xl-labeled cell started to increase at 2h, and reach a peaked 12h, Comparing SC group and NC group with I/R group, the differences were significant (p<0.05), then, Bcl-xl -labeled cell started to decrease at 24,48h,72h, Comparing SC group and NC group with I/R group 48h, 72h, there were not significant differences (p>0.05). In I/RP group : positive Bcl-xl -labeled cell weakly increased, there were not significant differences between SC group, NC group and I/RP group (p>0.05).(5) The result of Bcl-xl mRNA by RT-PCR method: In I/R group: The expression of Bcl-xl mRNA started to increase at 2h, and reach a peaked 12h, Comparing SC group and NC group with I/R group, the differences were significant (p<0.05), then, the expression of Bcl-xlmRNA started to decrease at 24,48h,72h. Comparing SC group and NC group with I/R group 48h, 72h, there were not significant differences (p>0.05). In I/RP group: positive In I/RP group: positive Bcl-xl -labeled cell weakly increased, there were not significant differences between SC group, NC group and I/RP group (p>0.05) weakly increased, there were not significant differences between SC group, NC group and I/RP group (p>0.05).Conclusion:(1)The protein expression of C-Rel in Wistar rat after focal ischemic/reperfusion increased and was actived, peaked at 12h, returned to based levels at 48,72h, the result demonstrated that the expression and activation inhibite neural apoptosis in Wistar rat after focal ischemic/reperfusion; (2)The protein expression of Bcl-xl in Wistar rat after focal ischemic/reperfusion increased, peaked at 12h, returned to based levels at 48h,72h, the expression temporal and spatial changes of Bcl-xl in Wistar rat after focal ischemic/reperfusion is the same as C-Rel;(3) PDTC inhibite the activation of C-Rel, reduce the expression of Bcl-xl protein, the result demonstrated that the expression and activation of C-Rel induced and upregulate the expression of Bcl-xl protein;(4) By inducing expression of Bcl-xl protein, C-Rel may inhibite neural apoptosis in Wistar rat after focal ischemic/reperfusion.
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