| BackgroundIschemic cerebrovascular disease is one of the causes that result in human death and disability. Although a large number of researchers in this field have made unremitting studies, in addition to thrombolytic therapy, there are not any kind of treatment measures or medicines that their therapeutic effect have been proved reliable in clinic. As the time window of thrombolytic therapy is short, to find some effective neuroprotective agents become more urgently.We always focuse on finding and screening the drugs and measures which may have brain protection. Because cerebral ischemia-reperfusion injury is a cascade that include a series of insult factors involving several pathological aspects and previous studies have shown that almost all of the neuroprotective agents for a single insult factor are not effective in clinical research, the combination of neuroprotective agents or to develop multi-target drugs have become the new approach of cerebral protection. The role of nitric oxide (NO) is a hot issue in the study of cerebral ischemia-reperfusion injury. The majority view is that the role of endogenous NO depends on the nitricoxide synthase (NOS) type. the NO generated from the endothelium-nitricoxide synthase (eNOS) has the neuroprotective effect, that generated from induced-nitricoxide synthase (iNOS) and neuronal-nitricoxide synthase (nNOS) have neurotoxic effects. Several current studies suggests that exogenous NO can inhibit the harmful effects of endogenous NO, promote angiogenesis and neurogenesis to play a neuroprotective effect. The multiple harmful effects of the large number of OFR produced by cerebral ischemia-reperfusion injury is confirmed by a number of researches, so we designed and synthesized the double-target drug named acetyl ferulaic isosorbide using OFR scavenger FA and exogenous NO donor ISMN, and investigate it's neuroprotective effect and the related mechanisms.Chinese traditional medicine play an important role in ischemic stroke treatment. Monomer ginsenoside Rd is a new drug for the treatment of ischemic cerebrovascular disease and it's clinical phase III study is ongoing. The neuroprotective effects of tetramethylpyrazine and puerarin have been demonstrated, whether the combination of these three drugs have more stronger neuroprotective effects is worthy to further study.This study include two parts. Part I: To investigate the cerebral protective effect and related mechanism of the double-target drugs: acetyl ferulaic isosorbide (AFI); Part II: To study the whether the combination of these three drugs have more stronger neuroprotective effects than singles.Part I the neuroprotective effect of AFI: a novel ROS-eliminating and NO-releasing drug ObjectiveTo investigate the neuroprotective effect of AFI and related mechanism in rat.Method1 The neuroprotection of AFISixty male SD rats weighing 280 - 320 g were randomly assigned into 6 groups ( n = 10, each group): control group, AFI group, ISMN group, SF group, SF + ISMN group and Edavarone group. rats were subjected to the right middle cerebral artery occlusion (MCAO) for 2 h, followed by reperfusion for 72 h. 10 min before reperfusion, the drug or vehicle were respectively infused intraperitoneally. The neurological behavior scores (NBS) were assessed at 72 h after reperfusion. The percentage of infarct volume was determined at 72 h after reperfusion by using TTC stain.2 the dose-effect relationship of AFIForty male SD rats weighing 280 - 320 g were randomly assigned into 4 groups ( n = 10, each group): control group, AFI-1mg/kg group, AFI-3mg/kg group, AFI-9mg/kg group. Rats were subjected to the right middle cerebral artery occlusion (MCAO) for 2 h, followed by reperfusion for 72 h. 10 min before reperfusion, the AFI(1,3,9mg/kg) or vehicle were respectively infused intraperitoneally. The neurological behavior scores (NBS) were assessed at 72 h after reperfusion. The percentage of infarct volume was determined at 72 h after reperfusion by using TTC stain.3 The effect of AFI on caspase-3 activityTwenty four male SD rats weighing 280 - 320 g were randomly assigned into 3 groups ( n = 8, each group): sham group, control group, AFI-3mg/kg group. rats were subjected to the right middle cerebral artery occlusion (MCAO) for 2 h, followed by reperfusion for 24 h. 10 min before reperfusion, the AFI(3mg/kg) or vehicle were respectively infused intraperitoneally. 24 h after reperfusion, the animals were sacrificed and the penumbra cortex and striatum of right hemisphere were dissected for the measurement caspase-3 activity.4 The effect of AFI on neuron apoptosisFor immunohistochemistry and TUNEL analysis, 36 animals were randomly assigned into 3 groups ( n = 12, each group): sham group, control group, AFI-3mg/kg group. Rat were anesthetized with an overdose of 2% sodium pentobarbital and perfusion-fixed via the left ventricle using 4% paraformaldehyde at 4 h and 24 h after MCAO. The brain blocks were embedded in paraffin and cut into 5μm coronal sections and were used for and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. TUNEL-positive cell numbers were counted in a blind fashion in four fields in the penumbra cortex and striatum of right hemisphere at high-power microscopic magnification (×400) and expressed as the number of positive cells per high-power field. Data from six animals in each group at corresponding time point were analyzed.Result1 The AFI induced neuroprotection on neurologic scores and infarct volume.AFI, SF, Edaravone, the combined application of SF and ISMN all can siganifacantly elevate the neurological behavior scores (NBS) and reduce the infart volume of rats after reperfusion than those of control group ( P< 0.05). Edaravone has the best effect (P<0.001) and AFI has the better(P<0.01). There are no siginicant difference between ISMN group and control group on NBS and infart volume 2 The dose-effect relationship of AFICompare with the control group, AFI with the dose of 3 mg/kg and 9 mg/kg both elevate the neurological behavior scores (NBS) and reduced brain infarct volume after reperfusion(P<0.05), AFI with the dose of 1 mg/kg has not produce significant neuroprotection.3 The effect of AFI on caspase-3 activityAFI(3mg/kg) treatment can inhibit the caspase-3 activity in the the penumbra cortex and striatum of right hemisphere as compare with control group(P<0.01).4 The effect of AFI on neuron apoptosisIn the the penumbra cortex and striatum of right hemisphere, the number of apoptotic neurons in the AFI treatment (3 mg/kg) group was significantly lower than that in the control group at 4 h (P<0.05) and 24 h after reperfusion(P<0.01). ConclusionAFI has neuroprotective effect against cerebral ischemia-reperfusion injury. The neuroprotection of AFI may be mediated by it's inhibition of caspase-3 and decrease the number of neuron apoptosis.Part II Neuroprotective effects study of ginsenoside Rd and it's combined application with othersObjectiveTo investigate the neuroprotective effect of ginsenoside Rd and the combined application of ginsenoside Rd with puerarin and(or) tetramethylpyrazine in rats. Compare whether combination of ginsenoside Rd with puerarin and (or) tetramethylpy -razine have more effective protection than ginsenoside Rd.MethodFifty male SD rats weighing 280 - 320 g were randomly assigned into 5 groups ( n = 10, each group). Drugs were respectively infused intraperitoneally as single or combined method for medicine administration groups. The control group received infusions of vehicle. rats were subjected to the right middle cerebral artery occlusion (MCAO) for 2 h, followed by reperfusion for 72 h. The neurological behavior scores (NBS) were assessed at 72 h after reperfusion. The percentage of infarct volume was determined at 72 h after reperfusion. ResultEither ginsenoside Rd or the combined application of ginsenoside Rd with puerarin and(or) tetramethylpyrazine can siganifacantly elevate the NBS and reduce the infart volume after reperfusion than those of control group ( P< 0. 05). No significant difference was found in NBS and infarct volume among drug administration groups.ConclusionGinsenoside Rd and the combined application of ginsenoside Rd with puerarin and(or) tetramethylpyrazine have similar neuroprotective effects. The combined application of ginsenoside Rd with puerarin and (or) tetramethylpyrazine did not generate synergistic effect.
|
PDF Full Text Request