Protective Effects And Mechanisms Of Total Flavones Of Rhododendra On Cerebral Ischemia Reperfusion Injury

Cerebrovascular ischemic disease is a source of mortality and profound morbidity that remains pervasive in the modern world.Neuroprotective agents reduce the injuries in ischemic penumbra and promote the recovery of brain function.Finding more effective neuroprotective agents with fewer risks has been a challenge.Some traditional Chinese medicines have been proven to be effective in alleviating symptoms that are similar to those induced by cerebral ischemia.It was found that some traditional Chinese medicines,especially those contained flavones,have protective effects against cerebral ischemic injury.Rhododendra is one of the plants that is rich in flavones,such as quercetin,hyperin and rutin,and has been used for treating patients with bronchitis for hundreds of years in China.Recent studies have shown that most flavones have protective effects against cerebral ischemic injury.Also,total flavones of rhododendra (TFR) have protective effects against myocardial ischemic injury by scavenging oxygen fiee radicals and inhibiting nitric oxide.However,there is very limited information about the protective effect of TFR on animal models of cerebral ischemic reperfusion injury.To determine the protective effect and mechanism of TFR on cerebral ischemic reperfusion injury,the models of local cerebral ischemia reperfusion in mice and rats, global ischemia reperfusion in rats and anoxia and reoxygenation in rat hippocampal neurons were used in this study.Influence of TFR on expression of NOS mRNA,ERK and JNK signal pathway,Ca²⁺ and EDHF were analyzed to clarify the possible mechanism. Purpose:In the experiments in vivo,it was determined whether TFR has protective effects on injury induced by local cerebral ischemia reperfusion and by global cerebral ischemia reperfusion.In the experiments in vitro,it was determined whether TFR protects neuron from injury induced by anoxia and reoxygenation in rat primary hippocampal neurons. To clarify the possible mechanism,the effects of TFR on lipid peroxidation,NOS mRNA expression intracellular calcium and EDHF were observed.Methods:1.Middle cerebral artery occlusion(MCAO) was performed to induce local brain ischemia reperfusion model by an intraluminal filament in mice and rats.MCA was occluded for 2h and the brain was reperfused for 24h at that moment neurologic deficiency scores were assessed,and infarct volume was measured by TTC staining. Brain water content,plasma and brain levels of malondialdehyde,nitric oxide contents, lactate dehydrogenase activity,brain levels of superoxide dismutase and glutathione peroxidase activities were evaluated.Expression levels of inducible nitric oxide synthase mRNA,neuronal nitric oxide synthase mRNA and endothelial nitric oxide synthase mRNA were detected by RT-PCR at reperfusion 24h after cerebral ischemia.2.Cerebral ischemia reperfusion was induced by 4-vessel occlusion(4-VO) in rat. Changes in electroencephalograph was recorded before and after ischemia 10min and after reperfusion 5min,10min,15min,30min,45min and 60min.HE staining was used to observe brain pathologic changes.Measuring the releases of lactate dehydrogenase and NSE content.The activation extracellular-signal regulated kinase1/2 and c-Jun N-terminal protein kinase1/2 were investigated by western blot.3.The injury of hippocampai neurons after anoxia and reoxygenation and the protective effects of TFR were observed on cultured neonatal rat primary hippocampal neurons. Cell viability of hippocampal neurons was quantified by measuring MTT staining,the releases of LDH and the content of NO and MDA were measured.The intracellular calcium indicated by the fluorescence in neurons was detected.The effect of brain vessle or cerebral micorvessel endothelial cell on anoxia injury of hippocampal neurons was observed by using MTT staining method and measurement of LDH and NO.Results:1.On MCAO model in mice,TFR(30,60,120 mg/kg) significantly ameliorated the neurological deficit(P＜0.05 or P＜0.01) and reduced the brain water content (P＜0.05).The activity of LDH and the contents of MDA and NO in plasma were decreased(P＜0.05 or P＜0.01).2.On MCAO model in rats,TTC staining showed infarct volume of rats was significantly smaller in the TFR(15,30,60 mg/kg) group after 24 h reperfusion compared with NS control group(P＜0.05).The activities of LDH,SOD and GSH-PX in brain were enhanced,while the contents of MDA and NO in brain were decreased (P＜0.05 or P＜0.01).3.On 4-VO model in rats,compared with sham-operate group,EEG amplitude decrease significantly after ischemia and recovery after reperfusion in NS control and TFR(15, 30,60 mg/kg) groups.At 5 min after reperfusion,there was no difference between the EEG amplitude in treated groups of 15,30,60 mg/kg TFR and that in untreated group (NS control)(P＞0.05).EEG amplitud of TFR(15,30,60 mg/kg) groups was higher than that of NS control at 10 min,15 min and 30 min after reperfusion,however it did not reach the level of statistical significance.At 45 min and 60 min,EEG amplitud of TFR(15,30,60 mg/kg) groups was significantly higher than that of NS control(P＜0.05, P＜0.01).TFR(15,30,60 mg/kg) could significantly improve brain pathologic changes, and inhibite the increases of LDH relasing and NSE content. 4.On primarily cultured hippocampal neurons hypoxia and reoxygenation model,TFR (10,20,40,80,160 mg/L) significantly reduced MTT staining of rat primary hippocampal neurons compared with NS control group(P＜0.01).TFR mardedly inhibited A/R-induced increases of LDH and NO release(P＜0.05)5.TFR(30,60 mg/kg) significantly suppressed the expression of iNOS and nNOS mRNA.Meanwhile,the expression of eNOS mRNA in brain was up-regulated in the TFR(30,60 mg/kg) group(P＜0.05 or P＜0.01).6.On the global cerebral ischemia reperfusion in rats,TFR(30,60 mg/kg) could not change total protein level of ERK 1/2 and JNK1/2,but TFR could increase the activation of ERK and inhibit the ctivation of JNK in ischmeia reperfusion.7.After hippocampal neurons anoxia and reoxygenation,TFR(10,20,40,80,160 mg/L) markedly decreased the intracellular calcium from 499.02±53.87(control group) to 397.28±94.82,335.89±75.09,238.51±38.40,147.56±17.76 and 88.50±7.03, respectively.8.Before anoxia and reoxygenation hippocampal neurons pretreatment with cerebral vessel or cerebral micorvessel endothelial cell respectively,TFR(20,40,80,160 mg/L) significantly increase the neuron viability and reduce the activity of LDH furtherly,but NO content don't change in culture medium.When adding with Indo and L-NAME, neuron viability and LDH don't change furtherly,but NO content was reduced in culture medium.Conclusion:1.TFA has remarkable protective effects on local cerebral ischemic reperfusion injury in mice and rats.2.TFA has remarkable protective effects on global cerebral ischemic reperfusion injury in rats. 3.TFR protects neuron from anoxia and reoxygenation injury in vitro.4.The mechanisms that TFR protect cerebral ischemia and reperfusion injury include anti-lipid peroxidation,up-regulating eNOS mRNA expression and down-regulating iNOS mRNA and nNOS mRNA expression,inceasing the activation of ERK1/2 and inhibiting the activation of JNK1/2,inhibiting the neurons calcium overload.Vascular endothelium,especially non-NO-non-PGI₂(supposed EDHF),might involve the protection of TFR partially.5.TFR may be of value as a new drug against cerebral ischemia reperfusion for clinical application.