| BackgroundStroke is a leading cause of morbidity and mortality. Ischemic stroke constitutes 85 percents of strokes. One of the major goals in cerebral ischemia study has been to develop neuroprotectants that would reduce or delay ischemic damage, thereby increasing the time available for thrombolysis. Neuropeptide is a large number of active peptides, affecting neuronal growth, differentiation, and death. The majority of neuropeptides are derived from larger protein precursors known as prohormones or proneuropeptides. Proprotein convertase 1(PC1) is known as the significant endoproteolytic enzyme which is ubiquitously expressed in the nervous and endocrine system cells. The study is aimed to determine the possible expression changes of PC1 and its substrates after neural cell ischemic injury in vitro and in vivo and provide novel insight to develop effective neuroprotective agents.The thesis is included in three parts:Chapterl Expression of proprotein convertase 1 and neuropeptide Y after focal cerebral ischemic injury in mice.Chapter2Expression of chromogranin A after focal cerebral ischemic injury in mice.Chapter3Expression of proprotein convertase 1 and chromogranin A in cultured neural NS20Y cells exposed to oxygen-glucose deprivation.All data were handled by SPSS 13.0 ststistical software. Normal distribution measurement data was expressed with mean±standard deviation (χ±S). Statistical comparisons were conducted using 1-way ANOVA followed by SNK tests for intergroup comparisons if the data was homogenitiy of viarience, otherwise by Dunnett’T3 tests. The difference was statistically with P<0.05.Chapter 1 Expression of proprotein convertase 1 and neuropeptide Y after focal cerebral ischemic injury in miceObjectivesTo study the expression changes of PC1 and its substrate neuropeptide Y (NPY) in the cerebral cortex nerve cells after focal cerebral ischemia in mice and to investigate the effect of PC1 in neuronal ischemic injury.MethodsMale C57 mice were randomly allocated into a sham-operation group, an ischemia-reperfusion 4-or 24-hour group. A model of middle cerebral artery occlusion in mice was induced by the intraluminal suture method. Infarct volume quantification in mouse focal cerebral ischemia was determined by triphenyltetrazolium chloride (TTC) method. The mice which manifest neurological deficits were included into the latter experiment. The total mRNA or protein of brain cortex of mice was extracted using commercial kits. Western blot and real-time quantitative nucleic acid amplification were used to detect the expression changes of PC1, NPY, and mRNA in mouse cortical neurons. PC1 expression was also demonstrated by immunofluorescent (IF) study of mouse brain tissue frozen sections.Results1. TTC staining reveals white (unstained) as infarcted regions of cerebral cortex and striatum.The injury volume was calculated in arbitrary units (pixels), and 26±2 of the contralateral non-lesioned area percent in the 24-hour group. The sham-operation, 4-hour groups did not detect infarct volume.2. The mice cortex brain tissue sections IF showed PC1 was detected in the neuron cell body or dendrites. The PC1-positive neurons was 22.661±1.928 percent in the sham-operation group,40.464±2.928 percent the ischemia-reperfusion 4-hour group, and 32.881±1.944 percent the 24-hour group.There is significant difference between groups(F=16.707, P=0.004, n=3). The percentage of the PC1-positive neurons significantly increased after 4,24h of focal ischemia-reperfusion (P<0.05).3. Western blotting demonstrated that the relative expression levels of proPC1(the precursor of PC1) were 0.112±0.010 in the control group,0.356±0.074 in the ischemia-reperfusion 4-hour group, and 0.228±0.048 in the 24-hour group. There was significant difference between the groups(F=5.609, P=0.026, n=4).Compared with the sham-operation group, the expression of proPC1 increased after 4 hours of ischemia reperfusion, and started to decrease after 24 hours.4. Real time PCR demonstrated that the expression of PC1 mRNA of ischemic cortex brain tissue in the ischemia-reperfusion 4-hour group increased 2.660±0.244 folds and in the 24-hour group 2.068±0.226 folds compared with the control group. There was significant difference between the groups(F= 19.230, P=0.001, n=4). The PC1 mRNA expression increased after 4 hours and 24 hours of reperfusion time in ischemic cortices.5.Western blotting demonstrated that the relative expression levels of proNPY (precursor of NPY) were 0.165±0.040 in the control group,0.915±0.092 in the ischemia-reperfusion 4-hour group, and 0.802±0.040 in the 24-hour group.There was significant difference between the groups(F=42.079, P<0.005, n=4). Compared with the sham-operation group, the expression of proNPY increased after 4 hours or 24 hours of ischemia reperfusion.6. Real time PCR demonstrated that the expression of NPY mRNA of ischemic cortex brain tissue in the ischemia-reperfusion 4-hour group was 2.310±0.267 and in the 24-hour group 2.385±0.313. There was significant difference between the groups(F= 10.749, P=0.004, n=4). The expression of NPY mRNA in the ischemia-reperfusion 4-hour group increased significantly compared with the control group.ConclusionsCerebral ischemic injury induced PC1 mRNA expression upregulation and caused accumulation of precursor of PC1 (proPC1).The proPC1 expression changes weakened the PC1 enzyme activity, resulting in its substrate NPY increase with the form of precursor. The loss of active neuropeptide such as NPY may exacerbate ischemic injury.Chapter 2 Expression of chromogranin A after focal cerebral ischemic injury in miceObjectivesTo study the expression changes of chromogranin A (CgA) in the cerebral cortex nerve cells after focal cerebral ischemia in mice.MethodsMale C57 mice were randomly allocated into a sham-operation group, an ischemia-reperfusion 4-or 24-hour group. A model of middle cerebral artery occlusion in mice was induced by the intraluminal suture method. The mice which manifest neurological deficits were included into the experiment. The total mRNA or protein of brain cortex of mice was extracted using commercial kits. Western blot and real-time quantitative nucleic acid amplification were used to detect the expression changes of CgA protein and mRNA in mouse cortical neurons.Results1. In the experiment, western blotting identified an approximately 50 kDa molecular mass CgA fragment. The relative expression levels of CgA were 0.267±0.008 in the control group,0.199±0.032 in the ischemia-reperfusion 4-hour group, and 0.146±0.005 in the 24-hour group. There was significant difference between the groups (F=17.121, P=0.003, n=3). Compared with the sham-operation group, the expression of active CgA decreased after 4 hours or 24 hours of ischemia reperfusion.2.Real time PCR demonstrated that the expression of CgA mRNA of ischemic cortex brain tissue in the ischemia-reperfusion 4-hour group was 1.214±0.295 and in the 24-hour group 1.129±0.061. There was no significant difference between the groups(F=0.381, P=0.698, n=3). The expression of CgA mRNA after 4 hours or 24 hours of ischemia-reperfusion remained unchanged compared with the control group.ConclusionsThe expression of active CgA decreased after 4 hours or 24 hours of ischemia-reperfusion, while its mRNA expression remained unchanged. The processing of CgA from its precursor to mature peptide was impaired due to the defective enzyme activity of PC1 during the ischemic injury.Chapter 3 Expression of proprotein convertase 1 and chromogranin A in cultured neural NS20Y cells exposed to oxygen-glucose deprivationObjectivesTo investigate the changes in PC1, CgA expression and the role of PC1 in CgA maturation in NS20Y cells exposed to oxygen-glucose deprivation.MethodsThe NS20Y cells was cultured in Dulbecco Modified Eagle Medium (DMEM) containing 10% fetal bovine serum.When cells were grown to 60 to 70% confluence in dishes, NS20Y cells were treated with 10-4 M cyclic AMP analogs (ctp-cAMP) to induce synthesis, processing of neuropeptide. The NS20Y cells were differentiated with ctp-cAMP for 24h, and incubated in glucose-free DMEM. Then, the cultures were transferred to an anaerobic jar (AnaeroPack system, Japan). At 2h,4h,6h of the OGD treatment, NS20Y cells were transferred into high-glucose DMEM and normoxic enviroment. Cultures exposed to OGD were compared with normoxic controls. The total mRNA and protein of NS20Y cells were extracted by commercial kits after 12h of OGD treatment. Expression of PC1, CgA was detected by western blotting and real time PCR. For the NS20Y cell immunocytochemistry, the OGD-cell and the control dish was washed two times with PBS for 10min, and then fixed 4% paraformaldehyde buffered by PBS (1 mL) for 15min, and subsequently used as PC1 immunocytochemistry and TUNEL analysis.Results1. With TUNEL staining, the apoptosis rate in the control cells was 7.00±1.15%, in the OGD-2-hour cells 17.67±1.45%, in the OGD-4-hour cells 28.67±0.88%, in the OGD-6-hour cells 33.00±0.58%. There was significant difference between groups (F=119.57, P<0.005, n=3).The results showed that OGD induced substantial cell apoptosis in a dose-dependent manner.2. PC1 immunofluorescence appeared to be enhanced by OGD, and it reached the highest level in OGD-6-hour cells, approximately 3.667 folds more than the control cells.The relative PC1 immunofluorescence in control cells was 0.015±0.003, in OGD-2-hour cells 0.023±0.005, in OGD-4-hour cells 0.038±0.006, in OGD-6-hour cells 0.055±0.011. There was significant difference between groups (F=7.049, P=0.005, n=3). The post-hoc LSD test revealed that the upregulation of PC1 levels was significant in OGD-4-hour and OGD-6-hour cells compared with the control cells (P<0.05), in OGD-6-hour cells compared with OGD-2-hour cells (P<0.05).3. OGD caused the accumulation of precursor peptide of PC1.Western blot analysis demonstrated that the proPCl expression in control cells was 0.433±0.058, in OGD-2-hour cells 0.713±0.060, in OGD-4-hour cells 0.774±0.034, in OGD-6-hour cells 0.735±0.020.There was significant difference between groups(F=11.389, P=0.001, n=3). The upregulation of proPC1 was significant in OGD-2-hour, OGD-4-hour and OGD-6-hour cells compared with the control cells (P<0.05).There was no significant difference within the OGD-2-hour, OGD-4-hour and OGD-6-hour groups by post-hoc LSD test.4. Real time PCR showed that the PC1 mRNA was upregulated following OGD injury. The PC1 mRNA relative expression in OGD-2-hour cells was 1.730±0.293, in OGD-4-hour cells 2.223±0.176, in OGD-6-hour cells 2.373±0.274.There was significant difference between groups (F=7.989, P=0.009, n=3). The upregulation of PC1 mRNA was significant in OGD-2-hour, OGD-4-hour and OGD-6-hour cells compared with the control cells (P<0.05). There was no significant difference within the OGD-2-hour, OGD-4-hour and OGD-6-hour groups by post-hoc LSD test.5. CgA protein level was downnregulated by OGD injury.Western blot analysis demonstrated that the mature CgA expression in control cells was 0.893±0.018, in OGD-2-hour cells 0.763±0.009, in OGD-4-hour cells 0.250±0.026, in OGD-6-hour cells 0.123±0.008. There was significant difference between groups (F=489.381, P<0.005, n=3). The down regulation of mature CgA was significant in OGD-2-hour,OGD-4-hour and OGD-6-hour cells compared with the control cells(P<0.05).6. The expression of CgA in NS20Y cells remained unchanged following OGD-induced injury. The CgA mRNA relative expression in OGD-2-hour cells was 0.714±0.084, in OGD-4-hour cells 0.804±0.159, in OGD-6-hour cells 0.904±0.052.There was no significant difference between groups(F=1.741, P =0.191, n=6).ConclusionsThe upregulation of expression of proPC1 and mRNA was observed in NS20Y cells exposed to OGD insult. Although the expression of mRNA of CgA remained unchanged, the mature CgA expression in NS20Y cells decreased following OGD injury. Thus the reduction of mature CgA may be correlated with defective processing ability of PC1.Full Text Conclusions1. The upregulation of expression of proPCl and mRNA is induced by ischemic injury in vitro and in vivo.2. Although the expression of mRNA of CgA remaines unchanged, the mature CgA expression decreases following ischemia reperfusion. The reduction of mature CgA may be correlated with defective processing ability of PC1.3. Ischemic injury weakenes the PCI enzyme activity in mice, resulting in its substrate NPY increase with the form of precursor.4. There is a defective neuropeptide processing of PC1. The loss of active neuropeptide and accumulation of precursor peptide may exacerbate ischemic injury. |
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