| Cerebral stroke is caused by the obstacle of intracranial blood circulation, leading to brain tissue damage. It is generally divided into hemorrhagic and ischemic stroke(cerebral infarction). And the ischemic stroke accounts for about 80% of cerebrovascular diseases. Acute ischemic stroke caused by cerebral artery occlusion is the main reason for the high morbidity and mortality among the aged population. When acute cerebral ischemia happens, a series of events, such as cells oxidative stress, inflammatory cytokines release, excitatory amino acid release, intracellular calcium overload, and mitochondrial dysfunction lead to pyroptosis, apoptosis or necrosis of neural cells. Exploring the effective neuroprotective drugs is a very important issue to be addressed in the neuroscience field. Cinepazide maleate(CM) is calcium channel blocker of piperazine. A previous research showed that CM could dilate blood vessels and promote cells metabolism. In our latest clinical trial, we showed that CM was as effective as mildronate to exert a neuroprotective effect. However, the mechanisms underlying its effects are still unclear. Therefore, in this thesis we examined the protective effects and possible mechanisms of CM on ischemic stroke-induced neuronal insult in rats.Part I The effects of CM on experimental cerebral ischemia in ratsObjectives: To investigate the neuroprotective effects of CM against ischemia/reperfusion-induced cerebral injury in SD rats.Methods:(1) To explore the best therapeutic doses. Healthy adult male SD rats were used in the research subjects of this experiment, and the neuroprotective effects of CM in an animal model of focal cerebral ischemia were examined. Paired-Samples T Test were used to deal with the experimental data. The matched-paired methods were as follows: NBS difference ≤ 3, BW difference ≤ 20 g, MRI axial scan maximum infarction area percentage ≤ 10%. The rats were divided into three treatment groups(10mg/kg, 20mg/kg, 30mg/kg CM) and a normal saline(NS) control group. Each group contained 15 SD rats, which were dealt with intraperitoneal injection respectively by CM and NS. The changes of NBS on post-operative day(POD) 7 in each rat were observed.(2) To investigate the neuroprotective effects of CM against ischemia/reperfusion-induced cerebral injury. MCAO models and paired rats were performed as above. The experiment contained a CM treatment group(choose the best therapeutic doses of CM) and a NS control group. Each group had 15 MCAO rats. The changes of the mortality, BW, and NBS on POD1, 3 and 7 were observed. Additonally, the volumes of cerebral infarction on POD7 of these 2 groups were compared.Results:(1) NBS scores of(NBS7d- NBS12h) of three CM treatment groups(10mg/kg, 20mg/kg, 30mg/kg) on POD7 were higher than NS control group. However, compared with NS control group(1.25 ± 0.35), only 20mg/kg CM showed statistical significance(2.05 ± 0.19, P < 0.05).(2) 20mg/kg CM not only reduced the total mortality but also apparently decrease the mortality on POD1(CM treatment group: 33.33%, NS control group 85.71%; P < 0.05). CM also promoted the neurological functional recovery(NBS7d- NBS12h)(CM treatment group: 2.00 ± 0.24%, NS control group: 1.19 ± 0.31; P < 0.05), reduced the cerebral infarction volume on POD7(CM treatment group: 24.72 ± 4.36%, NS control group: 32.04 ± 4.01%; P < 0.05), and had a tendency to promote the recovery of body weigh(CM treatment group: 252.06 ± 8.51 g, NS control group: 245.09 ± 8.77 g, P < 0.05).Conclusions: CM protects the brain tissue against cerebral ischemia/reperfusion-induced injury in rats, and 20mg/kg is the most effective concentration.Part II The effects of CM on the types of cell death induced by focal cerebral ischemiaObjectives: To explore whether the pyroptosis is present in the cerebral ischemia tissue of the MCAO modal rat, and the effects of CM on the types of cell death induced by focal cerebral ischemia.Methods: MCAO rats were randomly divided into one CM treatment group(20 mg/kg) and one NS control group. The changes of apoptosis in the cerebral ischemia tissue on POD1, 3 and 7 by TUNEL staining were observed. Pyroptosis indicated by double immunofluorescent staining of TUNEL and IL-1β was examined. The changes in the expression of caspase-1, caspase-3 and IL-1β were also observed.Results:(1) TUNEL+ and IL-1β+ pyroptosis cells were present in CM treatment group(20 mg/kg) and NS control group on POD1, 3 and 7.(2) Compared with NS control group, CM significantly inhibited apoptosis, pyroptosis and decreased the expression of inflammatory cytokine IL-1β on POD1, 3 and 7.(3) Compared with NS control group, CM significantly inhibited MCAO-induced increased expression of caspase-1, caspase-3 and IL-1β.Conclusions:(1) Pyroptosis is evident in the injured brain tissue after cerebral ischemia;(2) CM may exert its neuroprotective effects by inhibiting the apoptosis and pyroptosis caused by cerebral ischemia injury.Part III The effects of CM on ischemia/reperfusion-induced imbalance of redoxObjectives: To study the antioxidative effect of CM on ischemia/reperfusion-induced imbalance of redox in rats.Methods: MCAO rats were randomly divided into one CM treatment group(20 mg/kg) and one NS control group. The tissue homogenates of the infarction area on POD7 were extracted, and then the concentration changes of ROS, SOD, MDA, GSH and LDH were detected. In order to minimize the experimental errors, we used the difference between the value of affected side minus uninjured to make statistical analysis.Results:(1) Compared with NS control group, CM significantly improved the cell total antioxidant capacity: CM significantly increaed the level of SOD(CM treatment group: 5.83 ± 7.38 U/mgpro, NS control group:-3. 41 ± 6.15 U/mgpro; P < 0.05), and lowered the level of ROS(CM treatment group: 26.90 ± 36.49 U/mgpro, NS control group: 65.68 ± 33.87 U/mgpro; P < 0.05);(2) Compared with NS control group, CM enhanced GPX activity, and reduced the oxidative stress damage of cell membranes: CM significantly increased the level of GSH, which is the reaction substrate of GPX(CM treatment group:-1.45 ± 1.14 μmol/mgpro, NS control group: 0.29 ± 1.81 μmol/mgpro; P < 0.05), and decreaed the level of ROS(CM treatment group: 26.90 ± 36.49 U/mgpro, NS control group: 65.68 ± 33.87 U/mgpro; P < 0.05);(3) Compared with NS control group, CM increased the survival of the brain cells after cerebral ischemia: CM obviously increased the LDH level of the infarction area tissue(CM treatment group: 2.43 ± 9.18 U/gprot, NS control group:-10.70 ± 14.98 U/gprot; P < 0.05).Conclusions: CM increases the oxidation resistance of the brain cells, and reduces the damage caused by the oxidative stress. |
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