| BACKGROUNDCerebrovascular disease is one of the highest morbidity,disability and mortality diseases in the world.It is a serious threat to people’s health and quality of life.Ischemic cerebrovascular disease accounts for 70～80%of cerebrovascular disease,which is a common acute disease of nervous system.The basic strategy for the treatment of ischemic cerebrovascular disease is to make the reperfusion recover as soon as possible,but the blood flow after reperfusion may further aggravate the injury of brain,lead to more severe brain dysfunction.This phenomenon is known as cerebral ischemia/reperfusion(IR)injury.Protection of brain tissue from ischemia reperfusion injury is one of the focuses in the treatment of ischemic cerebrovascular disease.The critical role of immune-mediated mechanisms in cerebral I/R injury has been increasingly recognized in the last decades and the important contribution of Toll-like receptors(TLRs)in the induction of inflammatory responses has been demonstrated.In addition,it has been reported that TLR4/NF-κB signaling pathway takes part in the damaging inflammatory process of cerebral I/R injury.At present,lots of studies focus on exploring anti-inflammatory and neuroprotective agents that may alleviate cerebral I/R injury.Daphnetin(DAP),a coumarin derivative extracted from Daphne odora var,is a natural anti-inflammatory product.However,the neuroprotective effect of DAP on cerebral I/R injury is not yet clear and the ability of DAP to antagonize the inflammatory signaling pathway in brain remains to be determined.This study chose mice as the research object.The middle cerebral artery occlusion and reperfusion(MCAO/R)injury model was made.We aimed to study the effect of daphnetin on cerebral I/R injury in mice and explore the potential mechanisms from three aspects,including neuroinflammatory reaction,apoptosis and the effect of TLR4/NF-and B signaling pathway.PART ONEThe establishment of middle cerebral artery occlusion and reperfusion injury model in miceObjective To explore the establishment of middle cerebral artery occlusion and reperfusion injury model in mice and sum up experience.Methods Mice were randomly divided into operation group and sham operation group,16 mice in each group.Suture method was used to establish the middle cerebral artery occlusion and reperfusion injury model in operation group.The middle cerebral artery occlusion suture was removed after 1 hours,following 24 hours of reperfusion.While the sham operation group was operated with only the separation and ligation of common carotid artery,but without inserting the suture.After 24 hours of reperfusion,the neurological deficit was evaluated by neurological score,the percentage of cerebral infarction volume was evaluated by TTC staining,and the percentage of water content in brain tissue was measured by wet dry weighing method.Results The mice in sham operation group did not show postoperative neurologic deficit and the neurological score was zero.15 mice in operation group survived and the survival rate was 93.8%(15/16),2 of which had no neurologic deficit,the remaining 13 showed obvious neurological deficits and the neurological score was 2.43±0.65.There was significant difference between the two groups(P<0.001).The TTC staining of the sham operation group showed uniformly dark red,with no pale infarct,and the percentage of cerebral infarct volume was zero.While the TTC staining of the operation group appeared left cerebral cortex pale infarction,and the percentage of cerebral infarct volume was 36.64±5.12%.There was significant difference between the two groups(P<0.001).The percentage of brain water content in the sham operation group was 63.32±7.85%,while the operation group brain tissue volume increases and the water content percentage was 89.17±8.38%.There was significant difference between the two groups(P<0.05).The total success rate of model establishment was 81.3%(13/16).Conclusion The suture method was simple and easy to operate for the establishment of middle cerebral artery occlusion and reperfusion injury model in mice.It has the advantage of less trauma and high survival rate.The key to the success of the model is to choose mice with appropriate weight,make good anesthesia,shorten the operation time and control the direction and depth of the suture.PART TWOThe effect of daphnetin on cerebral ischemia/reperfusion injury in miceObjective To explore the effect of daphnetin on cerebral ischemia/reperfusion injury in mice.Methods Mice were randomly divided into control group and daphnetin low,middle and high dose group,12 mice in each group.The daphnetin group was injected daphnetin respectively with the dose of 5mg/kg,10mg/kg and 20mg/kg 30 minutes before modeling through intraperitoneal injection,while the control group was injected saline through intraperitoneal injection.Suture method was used to establish the middle cerebral artery occlusion and reperfusion injury model in operation group.The middle cerebral artery occlusion suture was removed after 1 hours,following 24 hours of reperfusion.After 24 hours of reperfusion,the neurological deficit was evaluated by neurological score and the percentage of cerebral infarction volume was evaluated by TTC staining.Results Compared with the control group,the 5mg/kg group had no statistically significant difference in neurological score and infarct volume percentage(P>0.05),while the 10mg/kg and 20mg/kg group showed statistically significant difference(P<0.05).Meanwhile,there was no statistically significant difference in neurological score and infarct volume percentage between 10mg/kg and 20mg/kg group(P>0.05).Conclusion Daphnetin showed neuroprotective effect on cerebral ischemia/reperfusion injury in mice,which can lower the neurological behavioral score and reduce the cerebral infarct volume.Both 10mg/kg and 20mg/kg showed neuroprotective effect on cerebral ischemia/reperfusion injury in mice,but there was no statistically significant difference between the two groups.10mg/kg was chosed as the optimal dose in the following study.PART THREEThe potential mechanisms of the neuroprotective effect of daphnetin on cerebral ischemia/reperfusion in miceObjective To explore the potential mechanisms of the neuroprotective effect of daphnetin on cerebral ischemia/reperfusion in mice.Methods Mice were randomly divided into sham group,MCAO/R group,MCAO/R+vehicle group and MCAO/R+DAP group,12 mice in each group.The sham group was operated with only the separation and ligation of common carotid artery,but without inserting the suture.The MCAO/R group was operated with suture method.The MCAO/R+vehicle group was injected saline through intraperitoneal injection 30 minutes before modeling.The MCAO/R+DAP group was injected daphnetin with the dose of 10mg/kg 30 minutes before modeling through intraperitoneal injection.After 24 hours of reperfusion,the expression levels of TNF-α and IL-1β were quantified using ELISA assay,the neural cells apoptosis was analyzed by the TUNEL assay,and the TLR4,NF-κB p65 and IκBα protein levels were measured by Western blot analysis.Results The ELISA assay results showed that:compared with the sham group,the expression levels of TNF-α and IL-1β in the MCAO/R group were obviously higher(P<0.001);compared with the MCAO/R+vehicle group,the expression levels of TNF-a and IL-1β in the MCAO/R+DAP group decreased(P<0.05).The TUNEL assay results showed that:compared with the sham group,the number of apoptotic cells in the MCAO/R group was obviously higher(P<0.001);compared with the MCAO/R+vehicle group,the number of apoptotic cells in the MCAO/R+DAP group reduced(P<0.05).The Western blot analysis results showed that:compared with the sham group,the protein levels of TLR4 and NF-κB p65 in the MCAO/R group were abundantly increased(P<0.05,P<0.01),whereas TLR4 and NF-κB p65 in the MCAO/R+DAP group were dramaticly decreased compared with the MCAO/R+vehicle group(P<0.05,P<0.05).Meanwhile,compared with the sham group,the level of IκBαin the MCAO/R group was decreased(P<0.01),whereas IκBα in the MCAO/R+DAP group was dramaticly increased compared with the MCAO/R+vehicle group(P<0.05).Conclusion Daphnetin decreased the overexpression of inflammatory cytokines(TNF-a,IL-1β,attenuated neural cells apoptosis and down-regulated the expression levels of TLR4 and NF-κB after cerebral ischemia/reperfusion.These results indicated that the underlying mechanism of the neuroprotective effects of daphnetin on cerebral ischemia/reperfusion injury was involved with the inhibition of the TLR4/NF-κB signaling pathway. |
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