| Ischemic cerebrovascular disease seriously affects the health of tens of millions of patients. Its morbidity and disability rate is very high. Yet it has been another difficulty of reperfusion injury when improve the cerebral blood supply in the intervention of ischemic cerebrovascular disease. Cerebral ischemia-reperfusion injury mechanism is unclear, there are many factors involved in the damage process. oxidative stress destroy the oxidative and antioxidative balance of tissues and cells,lead to too much free radical generation and insufficient removal, Therefore, the neural protection strategy for oxidative stress can significantly improve the effectiveness of the intervention of stroke.Recent discovery suggests that Antioxidant response element (ARE) exist in almost all antioxidant genes with protective effect. Various factors including oxidative stress can lead to Nrf2 nuclear translocation after action on ARE, Adjust the antioxidant gene expression to protect cells against oxidative stress damage, therefore the activation of Nrf2 pathway to start the body’s resistance to oxidative stress become feasible direction of reducing the cerebral ischemia reperfusion injury caused by oxidative stress.Nrf2/ARE pathway has been proved to be the most important way of antixidation.Some medicine can strengthen the capability of antioxidation by regulating the expression of Nrf2.Phloretin are dihydrochalcone which are members of the flavonoid family. We take SOD,GSH, GSH-PX and MDA as oxidative stress indicator to show the Oxidative stress injury and the capability of antioxidation. Phloretin is in apple and apple derived products are rich in content, a wide range of sources.Other researchs has proved that phloretin has Neuroprotective effect and antioxidation,and can protect tissues by regulate Nrf2.So it can be expected that phloretin has the substantial protective intervention on cerebral ischemia/reperfusion injury caused by oxidative stress. Therefore, we conducted this experiment to research the brain injury in the course of cerebral ischemia and reperfusion, the relationship between oxidative stress indicator enzymes and Nrf2 expression.,the index changes after phloretin intervention.The purpose of this study is to suggestthat phloretin probably play a protective role against oxidative stress after cerebral schemia/reperfusion by up-regulating the expression of Nrf2.Objective To investigate the protective effect and the mechanism of phloretin against oxidative stress on cerebral ischemia reperfusion injury in rats through up-regulation of Nrf2 pathway, so as to provide treatment of cerebral ischemia reperfusion injury in a new direction.Methods1. Healthy SD rats (weighing 220-280mg) were chosen as the research object, all animals fasted for 12 h before operation, all experimental procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of LaboratoryAnimals. The protocol was approved by the Animal Ethics Committee of Shandong University.Rats were randomly assigned to five experimental groups:sham-surgery group, I/R group, and high-, medium-and low-dose phloretin treatment groups (20,40, and 80 mg·Kg·d-1 respectively). The phloretin groups were treated drugs with intragastric administration for 14 days and other groups were given the same amount of normal saline.1h after the last administration, the focal cerebral ischemia-reperfusion rat model was established with the method of intraluminal vascular occlusion as previously reported with slight modification.2. Experimental animals were anesthetized by intraperitoneal injection of 10% chloral hydrate of 35 mg/Kg,Rats in the I/R group and phloretin treatment group were buildedas cerebral ischemia reperfusion models by modified thread occluding right middle cerebral artery (MCA), the judgment standard of successful model:the rats were unstableafter they wake up from anesthesia, tumbling, left hemiparesis, left paw cannot extend,Chasing its own tail, walking along the right side, moving around a circle when grab the tails. After 2h of occlusion, withdraw the filament out carefully to achieve reperfusion.Twenty-four hours after the reperfusion, the following experiment was continued. The sham-surgery rats were subjected to the same surgical procedures,except that the insertion depth ofbrain line bolt was 8-10 mm,avoided to occludethe middle cerebral arteries,During thesurgical procedure, rectal temperature was monitored and maintained at 37-38℃ with a heating pad. The room temperature was controlled in the range of 25-27℃.3. Twenty four hours after reperfusion,accordingto the five-grade marking system blinding method of Runge et,rats were marked Neurological Severity Scores(NSS):no neurologic deficit mark 0 point; can not fully extend opposite forepaw mark 1 point; contralateral circling mark 2 points; contralateral dumping mark 3 points; no spontaneous walking with depressed consciousness level mark 4 points. The higher score, the more severe neurological damage.After finish Neurological Severity Scores, the rats were sacrificed and brain tissues were isolated and stained with 2,3,5-triphenyltetrazolium chloride (TTC), image was analysed with image analysis software, cerebral infarction area was calculated to evaluate the degree of cerebral infarction.4. After the rats were sacrificed, Determination of the degree of brain edema with wet dry method, remove and weight the two hemispheres of the brain (wet weight), dry tissue under 100 degrees celsius for 24 hours, determine dry weight. Formula:water content=(wet weight -dry weight)/wet weight×100%.5. Superoxide dismutase, Glutathione, Glutathione Peroxidase is an important indicator enzyme to reflect oxidative stress, malondialdehyde can indirectly reflect the damage caused by oxidative stress, so we selected Superoxide dismutase,Glutathione, Glutathione Peroxidase,Malondialdehyde as the indicator enzyme of oxidative stress, we assayed Superoxide dismutase activity with the xanthine oxidase hydroxylamine method, checked the activity of GSH-PX and GSH with micro plate method measured Malondialdehyde activity with thiobarbituric acid method, and measured the protein concentration by Bradford.6. To detect mRNA expression and protein level of Nrf2 by Real-Time PCR and Western blot analysis.7. Statistics:Use SPSS 13.0 (Statistical Package statistics for the Social Sciences) statistical software (SPSS, Chicago, IL. USA) for statistical analysis.Experimental values were mean±standard deviation (mean±SD), was used for statistical analyses including one-way ANOVA and Student’s t-test. A probability (P)-value<0.05 was considered statistically significant.Results1.Twenty fourhours after reperfusion following a 2 hour MCA occlusion, rats’model showed that the reaction of rats in the sham operation group was normal, no physical activity obstacle, the gait was stable, No neurologic deficits symptoms; compared with sham operation group, rats in the ischemia/reperfusion groups stoodunstably, left hemiparesis, left claw cannot stretch, Chased its own tail along the left side, circled to the left side when grabbed the tail.The evidences means that cerebral ischemia reperfusion model had been constructed successfully.2.According to the five-grade marking system of neurologic deficits, neurological function score of rats in I/R group were significantly higher than sham operation group (P<0.01),it means that rats in 1/R group had a neurological impairment. Neurological function score of rats in all phloretin treatment groups were higher than sham-surgery group, but significantly lower than I/R group (P<0.05).3.Comparison of cerebral infarction area showed that in the sham-surgery group the red color was uniformly distributed in the brain tissue, we can not detect cerebral infarction, while in the I/R group extensive white infarction lesions was found (P<0.01).Cerebral infarction area in all phloretin treatment groups were reduced than those in I/R groups (P<0.05).4. The changes of brain edema:no cerebral edema in the sham operation group, brain edema was obvious in the I/R group, (P<0.01).Degree of cerebral edema in the phloretin treatment groups were decreased obviously than that in the I/R group (P<0.05).5. Superoxide dismutase (SOD), Glutathione (GSH), Glutathione Peroxidase (GSH-PX), Malondialdehyde (MDA) activity:24 hours after reperfusion, compared with sham operation group, the activities of SOD、GSH、GSH-PXS in the I/R group were significantly decreased while increased of MDA (P<0.01).t in each phloretin treatment group,they are decreased than those in sham operated group, but increased obviously than in I/R group(P<0.05), Malondialdehyde activity in I/R group increased obviously than sham operation group (P<0.01).MDA activity in each phloretin treatment group increased than sham operation group, but decreased significantly than in I/R group (P<0.05).6. Relative mRNA expression of Nrf2 was elevated in the I/R group than in the sham operation group (P<0.05). Treatments with phloretin significantly increased mRNA expression levels of Nrf2 in a dose-dependent manner (P<0.05). In accordance with RT-PCR results, western blot analysis showed that Nrf2 protein level was upregulated in the I/R group, and significantly increased in phloretin pretreatment groups(P<0.05). (Figure 4).Conclusions1. Cerebral ischemia/reperfusion can cause significant neurological damage and brain edema, cerebral infarction.2.Superoxide dismutase,Glutathione and Glutathione Peroxidase induced by cerebral ischemia/reperfusionwere significantly decreased, while MDA level increased significantly, differences in the level of representation was statistically significant, it suggests that ischemia/reperfusion caused obvious oxidative stress injury to the brain tissue.3. Compared with I/R group,the score of the rats’ brain function decreased obviously after phloretin intervention,it suggests that phloretin can reduce neurological damage induced by cerebral ischemia/reperfusion.4.Compared with model group, brain edema was significantly relieved after phloretin intervention. It suggests that phloretin intervention significantly reduce cerebral edema induced by cerebral ischemia/reperfusion in rats.5.After Phloretin intervention, the increaseof Superoxide dismutase, Glutathione and Glutathione Peroxidase levels is obvious than the shaml group.The comparison was statistically significant, it suggests that phloretin can improve the antioxidant capacity of brain tissue and reduce oxidative stress injuryin the ischemia/reperfusion,6.The level of Malondialdehyde decreased significantly when compared with model group after intervention of phloretin.The comparison was statistically significant; it suggests that phloretin can alleviate oxidative stress injury of cerebral ischemia/reperfusion.7.The Nrf2 mRNA expression and protein level of the model group significantly enhanced than the sham-surgery group, the difference was statistically significant, it indicates that cerebral ischemia/reperfusion activates Nrf2 gene expression.8.The Nrf2 mRNA expression and protein level significantly enhanced in the phloretin treatment group than the I/R group, the difference was statistically significant, it suggests that phloretin can enhance the expression of Nrf2 gene.Our results suggest that there is obvious oxidative stress damage in the process of cerebral ischemia/reperfusion, and the intervention of phloretin could inhibit the oxidativestress reaction during cerebral ischemia reperfusion injury, improve the cerebral infarction, cerebral edema and cerebral function score caused by cerebral ischemia reperfusion injury, in this process, Phloretin markedly enhanced the expression of Nrf2.Therefore, our study suggests that in the rats experiment,phloretin protect against oxidative stress injury during cerebral ischemia/reperfusion through the pathway of up-regulating Nrf2. |
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