| Stroke is the third most fatal disease in the world,second only to cardiovascular disease and cancer.In China,stroke has become the leading cause of death,and also contributes to severe and chronic adult disability,it seriously disturbes human life and health.According to its clinical characteristics,stroke can be divided into two categories,ischemic stroke and hemorrhagic stroke.Ischemic stroke patients account for about 70%of all stroke patients worldwide.Statistics show that ischemic stroke patients account for about 85%of all stroke cases in China and has a high mortality rate of 50%,with about 3/4 in ischemic stroke patients who survive lose work ability to different extent or have paralysis,aphasia and other serious sequelae.In recent years,researchers have been focusing on the study of ischemic brain injury in the acute phase of nerve injury cascade,and in animal models have found a lot of neuroprotective compounds,but the clinical efficacy after conversion are not ideal.Therefore,elucidating the exact pathogenesis of ischemic brain injury,put forward the new post-stroke neuroprotective strategies and R&D efficacy and allow more patients benefit from the treatment of drugs is still the urgent problem to be solved.Autophagy and apoptosis are two hot fields of stroke research.Autophagy is an effective mechanism for the regulation of homeostasis in the body,the intracellular misfolding or unfolded proteins caused by ischemia and hypoxia,and the damaged organelles need to be transported by autophagy to lysosomal degradation,the pathological factors that are cleared in time will lead to apoptosis or death.At the same time,there is a mutual regulation between autophagy and apoptosis,and autophagy can promote and inhibit apoptosis under the influence of different factors.Neurological protection and repair in the late stage of ischemic brain injury is also an important area of current treatment.Because of the limited number of patients who can receive treatment in ischemic brain injury "golden hours",the majority of patients are treated in the acute phase.Conservative treatment relies on subsequent neurotrophic and neurological repairs to restore the patient's consciousness,sensation,cognition and movement as much as possible by promoting the body's inherent healing ability,and this has become the key to post-stroke care and treatment.Ginkgo biloba is a kind of natural plant with early medicinal record and important medicinal value.It has been used as medicine for more than 5,000 years.It has long been used in the prevention and treatment of cardiovascular diseases,anti-aging,anti-cancer and enhancing autoimmunity and so on.Studies have shown that ginkgo biloba leaves and fruits contain a variety of medicinal ingredients can be used for the prevention and treatment of a variety of diseases,the main ingredients are ginkgolides A,B,C,J,M and ginkgo lactone(bilobalide,BB).Among them,bilobalide is the only sesquiterpene lactone extracted from Ginkgo biloba.It has the effects of anti-excitotoxicity,oxidative stress injury.The current ginkgolide content of the preparation is used for the brain,peripheral blood circulation disorders Ginkgo biloba extract injection Kinna(ginkgo lactone content of about 2.9%of the standard extract),the other is Chengdu Baoyu Pharmaceutical Co.,Ltd.independent research and development of the ginkgolide injection,which bilobalide content of up to 48%,mainly for ischemic stroke recovery of cerebral infarction indications treatment.Although the clinical application shows that bilobalide-containing mixed preparations have a good effect on stroke,the effect and exact mechanism of bilobalide monomer in acute and convalescent stage of ischemic stroke is not clear which therefore limited its application in the treatment of ischemic brain injury and indications.Based on previous studies,this study aims to further study the effect of bilobalide on acute and recovery phase of cerebral ischemic brain injury in mice with transient middle cerebral artery occlusion(tMCAO),and its role in the recovery of nerve function,and explore its related mechanism of action.Part I The Protective Effects and Mechanisms of Bilobalide on Ischemia-Induced Acute Brain Injury in MiceAim:Ischemia/Reperfusion(I/R)model was established by tMCAO to study the protective effects of bilobalide on acute injury of brain tissue and to explore the changes of autophagy and apoptosis in neurons,and the possible interaction and mechanism,in order to expand its clinical application and mechanism of the mechanism of further study to provide experimental basis.Methods:(1)The mouse tMCAO model was prepared according to the Longa method.After 6 h of ischemia and reperfusion,the mice were treated with ginaton(10 mg/kg)and bilobalide(BB,3.5,7 and 14 mg/kg ip,bid).(2)Preparation of tMCAO model in mice,and the minimum effective dose of BB(7 mg/kg)was administered after 6h of reperfusion.The mice were sacrificed 72h after reperfusion,and the expressions of autophagy-related proteins LC3,p62 and apoptosis-related protein in neurons were detected by immunofluorescence.The surval of neurons were detected by NeuN immunohistochemistry.The expressions of LC3,p62,Beclinl,Bcl-2,Bax and Cleaved Caspase-3 in ischemic penumbra were detected by Western Blot.(3)In vitro,the expression of Cleaved Caspase-3 and neurons were detected by Western Blot after reoxygenation(0,3,6,12 and 24h,respectively)following three hours of oxygen glucose deprivation(OGD).(4)After N2a cells were treated with OGD for 3h,the levels of autophagy and apoptosis levels were measured at different time points.(5)After N2a cells were treated with OGD for 3h,the levels of LC3,p62,Beclinl,BcL-2,Bax and Cleaved Caspase-3 were detected.(6)N2a cells were treated with OGD for 3h,and then treated with complete culture medium containing 1 ?M BB for 24h,Western Blot was used to detect the autophagy-related proteins LC3,p62 and Cleaved Caspase-3,respectively.(7)After the cells were treated with OGD for 3h,the cells were treated with 2.5 mM autophagy inhibitor 3MA and cell apoptosis was detected by flow cytometry and Western Blot.Results:(1)BB(7 and 14 mg/kg)could reduce the infarct volume and reduce the neurological function score and brain water content.(2)BB(7 mg/kg)could reduce the loss of neurons in ischemic penumbra 72h after tMCAO model.(3-4)BB(7.0 mg/kg)could increase the cell LC3B and inhibit the expression of p62 and Cleaved Caspase-3 in 72h ischemic penumbra neurons.(5)The level of autophagy in N2a cells after OGD/R was higher than that in control group.(6)BB promoted the autophagy level of N2a cells after 24h of reoxygenation and inhibited the apoptosis;(7)The autophagy was Inhibitor 3MA was able to inhibit the inhibitory effect of BB on cell apoptosis after OGD/R.Conclusion:Autophagy is associated with the inhibition of apoptosis by BB in acute stage of ischemic brain injury.Part II The Protective Effects and Mechanisms of Bilobalide on Neurogenesis after Ischemic Injury in MiceAims:To identified the protective effects and mechanisms of bilobalide on neurogenesis after ischemic injury in mice,mice I/R model was prepared by tMCAO method.The effects of BB on the proliferation and differentiation of stem cells were studied,to provide a reliable basis for elucidating the pharmacological mechanism of its convalescence medication and expanding its application.Methods:The model of I/R injury was established according to Longa method.After 1 hour of ischemia and 24h after reperfusion,the model mice were randomly divided into model group,bilobalide 3.5 mg/kg,7 mg/kg and 14 mg/kg administration groups for 14 and 42 days.(1)The mortality and body weight of each group were statistically analyzed.Neurological score was performed at 1,3,7,14,28 and 42 days after operation.(2)The effect of BB on the motor function of mice with I/R injury recovery period.(3)Morris water maze test was performed on the mice for 42 days to evaluate the effect of BB on the spatial learning and memory ability of mice.(4)Using the animal MRI-T2 weighted imaging technique at 24h,7d,14d and 42d after model and 14 mg/kg BB treatment group of mice brain injury.(5)intraperitoneal injection of BrdU(5-Bromo-2-deoxy Uridine,Sigma,50 mg/kg,continuous administration of 4 times,the interval of 12h interval)was performed on mice 12-13 days after operation to labele S phase proliferation cells and the effects of BB on the neuronal damage induced by I/R model were evaluated.14 days after reperfusion,brain slices were used to test BrdU immunohistochemistryto evaluation of stem cell proliferation,and 42 days after reperfusion the defferentaion of stem cell.(5)In vitro culture of adult neural stem cells(ANSCs),and to investigate the effect of stem cells on the differentiation of stem cells into the neurons.The effects of OGD/R and different concentrations of BB(0.01,0.1,1,10 and 50 ?M)on the viability and self-renewal of neural stem cells were observed by MTT and cell counting.(6)The effect of BB on the proliferation and cell cycle of stem cells was evaluated by BrdU immunofluorescence labeling and flow cytometry.(7)The effect of BB(0.1,1 and 10?M)on the differentiation of stem cells to neurons(Tuj-1)or astrocytes(GFAP)was assessed by immunofluorescence.(8)The effect of BB on the expressions of p-Akt,p-p38 and p-JNK in the cells was determined by the method of Western blot.(9)Western blot was used to detect the total protein and phosphorylated protein levels of Akt,p38 and JNK after incubation of JNK agonist anisomycin(Anis)for 24h.(10)The effect of BB on the differentiation of neural stem cells into Tuj-1 neurons was observed by immunofluorescence after incubation of JNK agonist anisomycin(Anis)for 7 days.Results:(1)The long-term survival rate of mice in 7 and 14 mg/kg BB treatment group was higher than that in I/R model mice,and BB could relieve the weight loss of mice induced by I/R injury.(2)Compared with I/R model group mice,7 and 14 mg/kg BB treatment improved the neurological function,decreased the right turn rate in the corner test,longer latency to drop in rotarod test,and less foot fault times in the foot fault test.(3)Morris water maze positioning navigation test,BB can reduce the time to find the underwater platform in mice,while BB can increase the times of mice to cross the platform and increase the distance and time of mice in the target quadrant in spatial probe test.(4)MRI showed that there was a large area of high-intensity signal in the injured brain tissue of 24 hours after operation.After 7 days,the signal became weak and the brain tissue defect appeared.At 14 days,the high signal appeared on the edge of the tissue defect.14 mg/kg BB was able to decrease the size of the 14 and 42 days after the model of the mice.(5)7 and 14 mg/kg BB increased the amount and distribution of neural stem cells in SVZ and SGZ 14 days after reperfusion.(6)7 and 14 mg/kg BB increased the number of BrdU+-NeuN+ cells in SVZ and SGZ 42 days after reperfusion.(7)0.1,1,10 and 50 ?M BB could increase the viability of ANSCs after OGD/R and increase the diameter and number of neurons after 7 days of growth.(8)BB increases the number of BrdU+-ANSCs after OGD/R,and increases the number of cells in S phase.(9)BB promotes the differentiation of ANSCs to Tuj-1+ neurons after OGD/R 7 days.(10)BB inhibits OGD/R induced upregultion of p-p38 and p-JNK,and increased the expression of p-Akt.(11)10?M JNK agonist anisomycin abolished promotion of BB to the differentiation of ANSCs into Tuj-1+ neurons.Conclusion:BB can promote the repair of ischemic brain injury by inhibiting the proliferation of neural stem cells and the differentiation of neurons into neurons by inhibiting JNK MAPK pathway.The major contributions of the present study:1.Autophagy is associated with the inhibition of apoptosis by bilobalide in I/R model mice.Bilobalide significantly improved the acute injury of ischemic brain injury,including neurological disorders,cerebral infarction and brain edema,and bilobalide can promote neuronal autophagy and inhibit neuronal apoptosis.Meanwhile,in vitro studies confirmed that bilobalide could inhibit neurons apoptosis by promoting autophagy.The study revealed the neuroprotective effects and mechanisms of bilobalide on ischemic brain injury,and accumulated the academic foundation for expanding its clinical application in the acute stage of ischemic brain injury.2.Bilobalide portects central nervous system against ischemia injury by promoting neurogenesis in late phase of I/R model,and the neuroprotection is associated with inhibition of JNK MAPK pathway.It was found that bilobalide could improve the neurological function of ischemic brain injury and could promote endogenous nerve regeneration.At the same time,it was confirmed that bilobalide could promote the survival,self-renewal,proliferation and differentiation into neurons and these properties may be associated with the inhibition of JNK MAPK pathway in neural stem cells.Our study provides new experimental evidences for elucidating the protective effect and mechanisms of bilobalide in the early stage therapy of ischemic brain injury,and provides experimental basis and new ideas to promote the application of bilobalide combined preparation and bilobalide monomer preparation in the recovery stage of ischemic brain injury. |
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