| Research Background:Stroke is due to the blood vessels that supply the brain blood blocking or rupture occurred,resulting in brain blood disorder or cause damage to the brain of an acute cerebrovascular disease,because of the different pathogenesis of stroke,generally can be divided into hemorrhagic stroke(also known as intracranial hemorrhage)and ischemic cerebralstroke(cerebral infarction).Ischemic stroke(IS)has a higher incidence and disability rate than hemorrhagic stroke,and can cause more severe damage to the brain after multiple treatments are used to restore blood supply.Clinically known as Cerebral ischemia reperfusion injury(CIRI).However,due to the complex pathogenesis of CIRI,it has not been fully elucidated at present,resulting in limited drugs and methods for the clinical treatment of CIRI.Therefore,it is urgent to further study CIRI and its protective mechanism,and find a new targeted drug that can assist or treat CIRI.Mangiferin is extracted from roots of rhizomaanemarrhenae and mango leaves out a double benzene acetone kind of flavonoids,has good antioxidation,antitumor,resistance to diabetes,anti-inflammatory and immune regulation,protect liver,bravery and various pharmacological activities,can be in the central nervous system,cardiovascular system and respiratory system play a positive role in treatment of various diseases.However,whether MGF has a protective effect on CIRI and the specific molecular mechanism of how it plays a protective role have not been fully proved and need further study.Silent Information Regulator 1(SIRT1)is a member of Sirtuin family.Studies have shown that a variety of natural drugs can play a protective role in central nervous system by regulating the expression of SIRT1.Peroxisome proliferators-Activated receptor-γcoactivator 1α(PGC-1α)is the target protein of SIRT1.It has also been proved to play an anti-injury role in neurological diseases such as stroke.It is noteworthy that SIRT1 has been confirmed to play a variety of protective roles by regulating PGC-1α.However,whether SIRT1 plays a protective role in CIRI by regulating PGC-1α signaling pathway remains unclear and needs further clarification.Research Objectives:Mouse derived neuron/nerve cell model N2 a cells and Hypoxia/Reoxygenation(HR) model are classic in vitro models for studying cerebral ischemia-reperfusion injury.Therefore,at the cellular level,a series of experimental methods and detection methods will be used to explore :(1)whether MGF has a protective effect on nerve cell HR injury;(2)Whether MGF plays a protective role in neuronal HR injury by regulating SIRT1/PGC-1α signaling pathway.Research methods:1.Taking N2 a cells as the research object,the HR injury model was constructed,which was mainly divided into the following three parts :(1)MGF toxicity test;(2)MGF preprotection experiment;(3)MGFanti-HR injury experiment.Muse instrument,microscope and Western Blotwere used to detect related indexes in each group,and further elucidate the protective effect of MGF on HR injury of N2 a cells at the cellular level.2.Taking N2 a cells as the research object,SIRT1 gene was interfered by siRNA technology to construct the HR injury model,which was mainly divided into the following three parts :(1)SIRT1 siRNA toxicity experiment;(2)SIRT1 siRNA interference fragment identification experiment;(3)SIRT1 siRNA1269 induced HR injury in MGF anti-N2 A cells.A series of instruments and methods were used to detect and compare the related indexes in each group,to explore the protective effect of MGF on N2 a cell HR injury,and further confirm the key role of SIRT1/PGC-1α pathway activation in the protection of CIRI by MGF.Results:1.(1)compared with the Control group,the morphology and viability of N2 a cells treated with MGF at different concentrations(5,10,20,50 and 100μM)for 24 h in each group had no significant change,indicating that MGF had no toxic effect on N2 a cells within the concentration range.(2)Compared with the Control group,the expression of SIRT1 and PGC-1α in N2 a cells treated with MGF at different concentrations(5,10,20,50 and 100μM)for 24 h in MGF group was significantly up-regulated,suggesting that MGF could activate SIRT1/PGC-1αsignaling pathway in normal N2 a cells within the concentration range.(3)Compared with Control group,the cells in HR group were severely damaged(P <0.05);Compared with HR group,the cell morphology and cell viability of 10 mpa group are improved obviously.The apoptosis rate decreased significantly.The LDH level of culture medium decreased.ROS production decreased(P < 0.05),indicating that MGF had a significant protective effect on HR injury of N2 a cells.(4)Compared with the Control group,the protein expression levels of SIRT1,PGC-1α,Nrf2,NQO1,HO-1,NRF1,UCP2 and Bcl2 in HR group were significantly decreased(P <0.05);Compared with HR group,the expression levels of above proteins in MGF+HR group were significantly up-regulated(P < 0.05).2.(1)Compared with the Control siRNA group,the cell growth state of SIRT1 siRNA508,SIRT1 siRNA800 and SIRT1 siRNA1269(100p M,24h)did not change significantly.The results showed that SIRT1 siRNA508,SIRT1 siRNA800 and SIRT1 siRNA1269 at this concentration had no toxic effect on N2 a cells and could be used in the next experiment.(2)Compared with the Control siRNA group,SIRT1 siRNA1269 significantly reduced SIRT1 mRNA(P < 0.05),so SIRT1 siRNA1269 was selected for subsequent experiments.(3)Compared with Control siRNA+MGF+HR group,cells in SIRT1 siRNA+MGF+HR group shrank and adhered less;Cell viability decreased significantly.The apoptosis rate increased significantly.The LDH level of culture medium increased.ROS production increased(P < 0.05),suggesting that SIRT1 siRNA could reverse the protective effect of MGF on HR-induced N2 a cells.(4)Compared with the Control siRNA+MGF+HR group,the expression levels of SIRT1,PGC-1α,Nrf2,NQO1,HO-1,NRF1,UCP2 and Bcl2 in SIRT1 siRNA+MGF+HR group were significantly decreased(P < 0.05).These results suggest that SIRT1 and its related signaling pathways play a key role in MGF anti-N2 A cell HR injury.Research Conclusions:(1)MGF had a significant protective effect on HR injury of nerve cells.(2)MGF plays a protective role against neuronal HR injury by activating SIRT1/PGC-1αpathway,inhibiting oxidative stress response and mitochondrial dysfunction,and is expected to be a candidate drug for treatment of CIRI. |
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