| Ischemic stroke (ischemic stroke, IS) refers to disorders of the blood supply of the brain. Ischemia and hypoxia of brain lead to brain tissue necrosis, softening, and focal or complete neurological deficit symptoms. In modern medicine, ischemic stroke includes cerebral thrombosis, cerebral embolism, transient ischemic attack (TIA). Cerebral ischemia, a common disease with great hazard to human life and health, because of its high incidence, mortality, morbidity and health economic burden, has attracted more and more attention in the field of drug discovery. The role of apoptosis in cerebral ischemic injury is receiving increasing attention. Studies suggest that both acute focal cerebral ischemia and delayed neuron death after transient cerebral ischemia have the participation of neuron apoptosis, which has an important role in the delayed neuron death and the expansion of the penumbra. These discoveries offer new avenues for the treatment of cerebral ischemia, so the application of drugs to prevent neuron apoptosis has become hot issue in the currenly domestic and international research.Patented new drug Tongluo Huatan Capsule for stroke, derived from the theory of Injury of Brain Col laterals by Toxins put forwarded by WANG Yongyan, an academician of China National Engineering Resarch Institute, based on Tongluo Huatan Soup from Series Recipe of Stroke, added to bear bile powder, which has special neuroprotective effect, by writer’s supervisor FAN Jiping to make an scientifical and optimal formation, has the effects of detoxication, relaxing collaterals and reducing phlegm. Previous pharmacological studies and clinical studies also show that Tongluo Huatan Capsule has advanced effects for ischemic stroke. This project, focusing on apoptosis in the ischemic penumbra after cerebral ischemic injury,aims at exploring the neuroprotective effects and mechanisms of Tongluo Huatan Capsule on cerebral ischemic injury in the overall, cellular, molecular levels in vivo and vitro models, which will provide further evidence and mechanisms for traditional Chinese medicine’s treatment for ischemic stroke.1. Research of Neuroprotect ive Effects and Mechanisms of Tongluo Huatan Capsule on Rats with Cerebral Ischemic Injury1.1Influences of Tongluo Huatan Capsule on Neural Function and Neuron Pathological Damage of Rats with Cerebral Ischmic InjuryObjective To investigate the influences of Tongluo Huatan Capsule on neural function and neuron pathological damage of rats with cerebral ischemic injury Methods The rats were s t rat i f ied randomly di vided into seven groups as nor ma1group, sham group, model group, nimodi pine group, high dose TLHT capsule group, medium dose TLHT capsule group and low dose TLHT capsule group. Cercbral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO).The rats were respectively given distilled wa ter, niniodipine and Tongluohua tan Capsule6hours after operation. The neuroprotecive effect was detected by Narrow-Alley corner Tes t at the1,2,3,5and7days after the opera t i on respect i vel y. The HE s ta in ing and Nissl’s staining was applied to observe the pathologic change of ischemic cerebral cortex.Results (1)The scores of Narrow-Alley corner Test of the model group at the1st,2nd,3rd,5th,7th days after opration were significantly increased compared wi th the normal group and sham group (P<0.01). The score of the nimodipine group at the1st,2nd,3rd,5th,7th days after opration were reduced compared with the model group (P<0.01). The score of High Dose Tongluo Huatan Capsule at the1st,2nd,5th,7th days after operation had no statistical significance compared with the model group (P>0.05). The score of High Tongluo Huatan Capsule at the3rd day after operation was reduced compared with the model group (P<0.01). The score of Middle Dose Tongluo Huatan Capsule at the1st,2nd,3rd,5th,7th days after operation were reduced compared wi th the model group (P<0.01, P<0.05). The score of Low Dose Tongluo Huatan Capsule at the1st day after operation was decreased compared with the model group (P<0.05).The score of Low Dose Tongluo Huatan Capsule at the2nd,3rd,5th7th days after operation had no s tatisttcal signi f icance compared with the model group (P>0.05).(2) In the ischemic area of the rats of the model group, we can see interstitial vacuoles of varying sizes, nuclear condensation, dissolution, loose cytoplasm, unclear cell boundaries, many apoptotic cells; while in that of the nimodipine and every Tongluo Huatan Capsule, we can see necrotic area were decreased and the neurons in the ischemic penumbra can still distinguish the structural integrity.(3) The number of nissl body in the ischemic penumbra were significantly decreased with the model group and the sham group(P<0.01). That of the nimodipine group were significantly increased compared with the model group (P<0.01).That of every Tongluo Huatan Capsule group (P<0.01, P<0.05)Conclusion Tongluohuatan Capsule can promote the recovery of neurological dysfunction after ischemic injury in rats, alleviate the pathological damage of neurons, and repair the structure, function and metabolism of the corresponding brain areas.1.2Influences of Tongluo Huatan Capsule on Neuron Apoptosis in the Ischemic Penumbra of Rats with Cerebral Ischraic Injury Objective To investigate the influences of Tongluo Huatan Capsule on neuron apoptosis in the ischemic penumbra of rats with cerebral ischmic injuryMethods The rats were stratified randomly divided into seven groups as normal group, sham group, model group, nimodipine group, high dose TLHT capsule group, medium dose TLHT capsule group and low dose TLHT capsule group. Cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO).The rats were respect ively given distilled water, nimodipine and Tongluohua tan Capsu le6hours after operation. The TUNEL technology was used to test the level of neuron apoptosis in the ischemic penumbra.Results The level of neuron apoptosis in the ischemic penumbra of model group was increased compared with normal group and sham group (P<0.01). That of the nimodipine group was decreased compared with model group (P<0.01).That of the High Tongluo Huatan Capsule was decreased compared with model group (P<0.01) That of the Middle Tongluo Huatan Capsule group was decreased compared with model group (P<0.01).That of the Low Tongluo Huatan Capsule was decreased compared with model group (P<0.05)Conclusion Tongluo Huatan Capsule can reduce the level of neuron apoptosis in the ischemic penumbra of rats with cerebral ischemic injury, which maybe related to its effects of promoting the recovery of neurological dysfunction after ischemic injury in rats and alleviat ing the pathological damage of neurons.1.3Influences of Tongluo Huatan Capsule on the Expression of Bax and Bcl-2in the Ischemic Penumbra of Rats with Cerebral Ischemic InjuryObjective To investigate the influences of Tongluo Huatan Capsule on the expression of Bax and Bcl-2in the ischemic penumbra of rats with cerebral ischmic injuryMethods The rats were stratified randomly divided into seven groups as normal group, sham group, model group, nimodipine group, high dose TLHT capsule group, medium dose TLHT capsule group and low dose TLHT capsule group. Cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO). The rats were respect ively given distilled water, nimodipine and Tongluohua tan Capsule6hours after operation. The immunohistochemistry method was applied to detect the expression of Bax and Bcl-2in the ischemic penumbra.Results The expression of Bax and Bcl-2in ischemic penumbra of the model group was increased and Bax/Bcl-2was increased compared with the normal group and the sham group (P<0.01). The express ion of Bax and Bcl-2in ischemic penumbra of the nimodipine group was decreased and Bax/Bcl-2was decreased compared with the model (P<0.01). The expression of Bax and Bcl-2in ischemic penumbra of every Tongluo Huatan Capsule group was decreased and Bax/Bcl-2was decreased compared with the model(P<0.01)Conclusion Tonghio Huatan Capsule can promote the expression of Bcl-2, inhibit the expression of Bax in the ischemic penumbra,and decrease Bax/Bcl-2, which maybe related to its effects of reducing the level of apoptosis in the ischemic penumbra and alleviating the neurodysfunction of rats with cerebral ischemic injury.1.4Influences of Tongluo Huatan Capsule on the Expression of CytC. Caspase-9and Caspase-3in the Ischemic Penumbra of Rats with Cerebral Ischemic InjuryObjective To investigate the influences of Tongluo Huatan Capsule on the expression of CytC, Caspase-9and Caspase-3in the ischemic penumbra of rats with cerebral ischemic injuryMethods The rats were stratified randomly divided into seven groups as normal group, sham group, model group, nimodipine group, high dose TLHT capsule group, medium dose TLHT capsule group and low dose TLHT capsule group. Cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO). The rats were respect ively given distilled water, nimodipine and Tongluohuatan Capsule6hours after operat ion. The immunohistochemistry, western-blot and RT-PCR methods were appl ied to detect the express ion of CytC, Caspase-9and Caspase-3in the ischemic penumbra from the levels of protein and mRNA.Results The expression of CytC protein and the expression of Caspase-9and Caspase-3protein and mRNA in the ischemic penumbra of the model group were increased compared with the normal group and the sham group (P<0.01). The expression of CytC protein and the expression of Caspase-9and Caspase-3protein and mRNA in the ischemic penumbra of the nimodipine group were decreased compared with model group (P<0.01). The expression of CytC protein and the expression of Caspase-9and Caspase-3protein and mRNA in the ischemic penumbra of every Tongluo Huatan Capsule group were decreased compared with model group (P<0.01, P<0.05)Conclusion Tongluo Huatan Capsule can decrease the expression of CytC proterin, Caspase-9and Caspase-3protein and mRNA in the ischemic penumbra, which maybe related to its effects of reducing the level of apoptosis in the ischemic penumbra and alleviating the neurodysfunction of rats with cerebral ischemic injury.2. Research of the Protective Effects and Mechanisms of Tongluo Huatan Capsule on H2O2-induced apoptosis in PC12cells2.1Influences of Tongluo Huatan Capsule on Viability of PC12Cells Injuried by H2O2Objective To investigate the viability of Tongluo Huatan Capsule on PC12 cells injured by H2O2Methods PC12cells were cultured, and the cell viability of PC12cells injured by different H2O2concentrations and proliferated by different Tongluo Huatan Capsule were observed by MTT assay to determine the concentration that will cause a H2O2-induced apoptosis model of PC12cells and the security concentrations of Tongluo Huatan Capsule for PC12cells. PC12cells in the logarithmic growth phase were divided into the control group, the model group and the low, medium and high dose Tongluo Huatan Capsule treatment groups. Apoptosis of PC12cells were induced by H2O2at the concent rat ion of200μmol·L-1MTT assay was used to detect the cell viability of PC12cells protected by Tongluo Huatan capsule at different concentrations.Results PC12cells were injured by H2O2at200μmol·L-1concent rat ion, whi le Tongluo Huatan Capsule at less than or equal to the concentration of100μ mol·L-1had the effect of proliferation to the cell viability of PC12cells and have no dose relationship. Compared with the model group,6.25,12.5,25,50,100μmol·L-1Tongluo Huatan Capsule groups improved cell viability (P<0.01).Conclusion:Tongluo Huatan Capsule can improve PC12cells viability injured by H2O2.2.2Influences of Tongluo Huatan Capsule on Apoptosis of PC12Cells induced by H202Objective To investigate the protective effect of Tongluo Huatan Capsule on H2O2-induced apoptosis in PC12cellsMethods PC12cells in the logarithmic growth phase were divided into the control group, the model group and the low, medium and high dose Tongluo Huatan Capsule treatment groups. Apoptosis of PC12cells were induced by H2O2at the concentration of200μmol·L-1, Hoechst33342fluorescent staining was used to observe morphological changes of cell apoptosis, the flow cytometry were used to detect the rate of apoptosis in PC12cells.Results Compared with the control group, the level of apoptosis was increased after injured by H2O2(P<0.01), while compared with the model group, the level of apoptos i s of6.25μg·mL-1.25μg·mL-1100μg·mL-1Tongluo Huatan Capsule groups were decreased (P<0.01). In the model group, a lot of obviously pyknosis cells were observed, while compared with the model group, the pyknosis cells were decreased in Tongluo Huatan Capsule group.Conclusion:Tongluo Huatan Capsule can inhibit H2O2-induced apoptosis in PC12cells.2.3Influences of Tongluo Huatan Capsule on the Expression of CytC and Caspase-3in the PC12Cells Induced by H2O2 Objective To investigate the influences of Tongluo Huatan Capsule on the expression of CytC and Caspase-3in the PC12cells induced by H202Methods PC12cel1s in the logarithmie growth phase weredivided into contro1group, model group and low, medium and high dose Tongluo Huatan capsule treatment groups. Apoptosis of PC12cells were induced by H2O2at the concentration of200μmo1? L-1, the Western blot technology was used to detect the expressions of protein CytC and Caspase-3.Results Compared with the control group, the expression of CytC and Caspase-3were increased after in jured by H202(P<0.01), while compared with the model group, the leve1of the express ion of CytC and Caspase-3were decreased in6.25μg·mL25μg? mL-1、100μg?mL-1Tongluo Huatan Capsule groups(P<0.01),and had dose relationship.Conclusion Tongluo Huatan Capsule can decrease the expression of CytC and Caspase-3in PC12cells induced by H2O2, which maybe responsible for its effect of reducing apoptosis of PCI2cells induced by H2O2. |
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