| Cerebral vascular disease has become one of the most important reasons causing death and disability in the world. At present, the treatment methods for cerebral ischemia are not satisfactory. In clinical practice, it is urgent to find the nerve protective agent of high efficiency and low toxicity, which promotes the blood to be re-through and reduces the brain injury caused by ischemia and reperfusion.Vitexin is the main active ingredient of traditional Chinese medicine Flostrolliichinensis, belongs to the class of Flavonoids from carbon dioxide. This research group showed that Vitexin has good ant oxidation and protective effects on aging mice hippocampus nerve cell structure. Research on protective effect of Vitexin on brain injury in rats with cerebral ischemia reperfusion injury has not been reported. In this experiment, SD male rats as experimental subjects, the use of modified Longa suture method of manufacturing cerebral ischemia and reperfusion(middle cerebral artery occlusion, MCAO) model, for drug intervention to Vitexin, edaravone regarded as a positive control. Study Vitexin on cerebral ischemia reperfusion rats damage protection and to explore its action mechanism. Method 1. The establishment of MCAO modelLonga line switch method is used to Build the model, ischemia reperfusion after 2 h, treated rats neurobehavioral scores after waking up to make sure that we built the success model. 2. Grouping and drug deliveryThe 228 SD rats were randomly divided into six groups, namely sham operation group(normal saline + solvent), model group(saline + solvent), model + edaravone positive control group(3.24μmol·kg-1), model + vitexin(1.62, 3.24, 6.48μmol·kg-1) different doses, all groups were intraperitoneal injection after 1 h of reperfusion, treatment time was 1d and 3d. 3. Effect of Vitexin on cerebral infarct volume in MCAO ratsAfter treatment of 1d on ischemia reperfusion rats, the rats were sacrificed after anesthetized and each of their the brain coronary was cut into five slices. Then we measured the infarct volume by the TTC staining. 4. The protective of Vitexin on blood brain barrier and the effects of antioxidant in MCAO ratsAfter treatment of 1d and 3d on ischemia reperfusion rats, they were sacrificed after anesthetized. We measured the brain water content by wet-dry weighting method,The numbers of aquaporin-4(AQP-4) positive ce lls by the immunohistochemical staining method. Corresponding reagent kit measured the activity of superoxide dismutase(SOD), the content of reactive oxygen species(ROS) and malondialdehyde(MDA). 5. The effects of Vitexin on the anti-inflammatory in MCAO ratsAfter treatment of 1d and 3d on ischemia reperfusion rats, they were sacrificed after anesthetized. We measured the numbers of toll like receptor 4(TLR-4) and NF-κB(P65) positive cells by the immunohistochemical staining method and measured the expression of TLR-4 m RNA, NF-κB(P65) m RNA by the real-time quantitative polymerase chain reaction(RT-q PCR) method and measured the tumor necrosis factor-α(TNF-α) content by the enzyme-linked immuno sorbent assay(ELISA) method. 6. The effects of Vitexin on the non- antioxidant in MCAO ratsAfter treatment of 1d and 3d on ischemia reperfusion rats, they were sacrificed after anesthetized. We measured the nitric oxide(NO) content and the activity of nitric oxide(NOS), Na+-K+-ATPs by the corresponding kits. We measured the content of glutamate(Glu), aspartate(Asp), glycine(Gly) by the automatic analyzer for amino acids. 7. The effects of Vitexin on cerebral histomorphology in MCAO ratsAfter treatment of 1d and 3d on ischemia reperfusion rats, they were sacrificed after anesthetized. HE staining was used to observe the morphological changes of nerve cells in the ischemic area of rat, and to observe the ultrastructure of the neurons in the ischemic area of the rat by transmission electron microscope. Results: 1. Preparation of MCAO modelCompared with sham group, the rat couldn’t fully extend his left forepaw, fall down or circle on the contralateral side. This proved that we built the successful MCAO model. 2. Vitexin reduce the volume of cerebral infarction in MCAO ratsCompared with sham group, the model group rats could be observed the pale infarcts and the cerebral infarction volume had a significant difference(P < 0.01); Compared with model group, after treatment of 1d on ischemia reperfusion rats, all treatment group could reduce the cerebral infarction volume in a dose and time dependent manner, there’s significant difference(P < 0.05, P < 0.01); Compared with edaravone group, the ability of reducing the cerebral infarction volume on middle and low dose group of Vitexin was lower than edaravone(P < 0.05, P < 0.01), and the high dose group of Vitexin and edaravone had no significant difference. 3. The protective of Vitexin on blood brain barrier and the effects of antioxidant in MCAO rats 3.1 Vitexin reduce the brain water content in MCAO ratsCompared with sham group, the model group rats revealed marked edema and had a significant difference(P < 0.05, P < 0.01); Compared with model group, after treatment of 1d and 3d on ischemia reperfusion rats, all treatment group could reduce the cerebral edema in a dose and time dependent manner, there’s significant difference(P < 0.05, P < 0.01); Compared with edaravone group, the ability of reducing the cerebral infarction volume on middle and low dose group of Vitexin was lower than edaravone(P < 0.05, P < 0.01), and the high dose group of Vitexin and edaravone had no significant difference. 3.2 Vitexin decrease the numbers of AQP-4 positive cells in MCAO ratsCompared with sham group, the model group rats showed lots of AQP-4 positive cells and had a significant difference(P < 0.05, P < 0.01); Compared with model group, after treatment of 1d and 3d on ischemia reperfusion rats, all treatment group could reduce the numbers of AQP-4 positive cells in a dose and time dependent manner, there’s significant difference(P < 0.05, P < 0.01); Compared with edaravone group, the ability of reducing the cerebral infarction volume on middle and low dose group of Vitexin was lower than edaravone(P < 0.05, P < 0.01), and the high dose group of Vitexin and edaravone had no significant difference. 3.3 Vitexin Protecte the activity of SOD and reducing the ROS and MDA content in MCAO ratsCompared with sham group, the activity of SOD was decreased and the ROS and MDA content was increased in the model group and had a significant difference(P < 0.05, P < 0.01); Compared with model group, after treatment of 1d and 3d on ischemia reperfusion rats, all treatment group could upregulate the activity of SOD and reduce the ROS and MDA content in a dose and time dependent manner, there’s significant difference(P < 0.05, P < 0.01); Compared with edaravone group, the ability of reducing the cerebral infarction volume on middle and low dose group of Vitexin was lower than edaravone(P < 0.05, P < 0.01), and the high dose group of Vitexin and edaravone had no significant difference. 4. The effects of Vitexin on the anti-inflammatory in MCAO rats 4.1 Vitexin reduce the numbers of TLR-4 and NF-κB(P65) positive cellsin MCAO ratsCompared with sham group, the model group rats showed lots of TLR-4 and NF-κB(P65) positive cells and had a significant difference(P < 0.05, P < 0.01); Compared with model group, after treatment of 1d and 3d on ischemia reperfusion rats, all treatment group could reduce the numbers of TLR-4 and NF-κB(P65) positive cells in a dose and time dependent manner, there’s significant difference(P < 0.05, P < 0.01); Compared with edaravone group, the ability of reducing the cerebral infarction volume on middle and loe dose group of Vitexin was lower than edaravone(P < 0.05, P < 0.01), and the high dose group of Vitexin and edaravone had no significant difference. 4.2Vitexin reduce the the expression of TLR-4 and NF-κB(P65) m RNAin MCAO ratsCompared with sham group, the model group rats showed a high expression of TLR-4 and NF-κB(P65) m RNA and had a significant difference(P < 0.05, P< 0.01); Compared with model group, after treatment of 1d and 3d on ischemia reperfusion rats, all treatment group could reduce the expression of TLR-4 and NF-κB(P65) m RNA in a dose and time dependent manner, there’s significant difference(P < 0.05, P < 0.01); Compared with edaravone group, the ability of reducing the cerebral infarction volume on middle and low dose group of Vitexin was lower than edaravone(P < 0.05, P < 0.01), and the high dose group of Vitexin and edaravone had no significant difference. 4.3 Vitexin reduce the TNF-α content in MCAO ratsCompared with sham group, the TNF-α content was increased in the model group and had a significant difference(P< 0.05, P < 0.01); Compared with model group, after treatment of 1d and 3d on ischemia reperfusion rats, all treatment group could reduce the TNF-α content in a dose and time dependent manner, there’s significant difference(P < 0.05, P < 0.01); Compared with edaravone group, the ability of reducing the cerebral infarction volume on middle and low dose group of Vitexin was lower than edaravone(P < 0.05, P < 0.01), and the high dose group of Vitexin and edaravone had no significant difference. 5. The effects of Vitexin on the non- antioxidant in MCAO rats 5.1 Vitexin reduce the NO content, inhibition the activity of NOS and protecting the activity of Na+-K+-ATP in MCAO ratsCompared with sham group, the NO content and the activity of NOS were increased, and the activity of Na+-K+-ATP was decreased in the model group and had a significant difference(P < 0.05, P < 0.01); Compared with model group, after treatment of 1d and 3d on ischemia reperfusion rats, all treatment group could reduce the NO content and the activity of NOS and increased the activity of Na+-K+-ATP in a dose and time dependent manner, there’s significant difference(P < 0.05, P < 0.01); Compared with edaravone group, the ability of reducing the cerebral infarction volume on middle and low dose group of Vitexin was lower than the edaravone(P < 0.05, P < 0.01), and the high dose group of Vitexin and edaravone had no significant difference. 5.2 Vitexin reduce the content of the excitatory amino acid(EAA) and the inhibitory amino acid(IAA) in MCAO ratsCompared with sham group, the content of Asp and Glu were increased, and the content of Gly was decreased in the model group and had a significant difference(P < 0.05, P < 0.01); Compared with model group, after treatment of 1d or 3d on ischemia reperfusion rats, all treatment group could reduce the content of Asp and Glu and increased the content of Gly in a dose and time dependent manner, there’s significant difference(P < 0.05, P < 0.01); Compared with edaravone group, the ability of reducing the cerebral infarction volume on middle and low dose group of Vitexin was lower than edaravone(P < 0.05, P < 0.01), and the high dose group of Vitexin and edaravone had no significant difference.6. The effects of Vitexin on the cerebral histomorphology in MCAO ratsIn the sham group, the cells structures and morphology were normal under the light microscopy. Compared with the sham group, the cells shrinkaged and were karyopyknosis in the model group. After treated with Vitexin and edaravone, the cells had been restored in a dose and time dependent manner. The high dose group of Vitexin and edaravone had no significant difference.In the sham group, the neuron structure was complete, almost no edema, and the organelles were visible. Compared with the sham group, the cells were highly edema. The rough endoplasmic reticulum swelled obviously and the ribosomes denuded. After treated with Vitexin and edaravone, the cell organelle integrity were been improve, the cell edema degree was reduced and lots of organelles were recovered. The high dose group of Vitexin and edaravone had no significant difference.Conclusion:Vitexin plays a protective role in MACO rats. Its protection mechanism is mainly through the following three ways: oxidative stress resistance, anti inflammation and toxicity of excitatory amino acids. The middle and low dose groups of Vitexin were lower than edaravone in protecting the MCAO rats and high dose group of Vitexin and edaravone had no significant difference, and ischemia-reperfusion rats for 3d effect is better than that of 1d. |
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