Efficacy And Mechanism Of Shuxuening Injection In The Prognosis Of Ischemic Stroke

Objectives:Ischemic stroke is the main type of stroke,which can be divided into acute phase,subacute phase and recovery phase and so on,seriously endangers human life and health.At present,intravenous thrombolysis with recombinant tissue plasminogen activator（rt PA）or endovascular thrombectomy are mainly used to treat ischemic stroke in clinical practice,but both of them have disadvantages such as hemorrhagic transformation,treatment time window narrowing,reperfusion injury and high disability rate.There is an urgent need to develop a more effective prevention and treatment approach that is broadly applicable to different phases of stroke.Ginkgo biloba is a kind of traditional Chinese herbal medicine used to prevent and treat cardiovascular and cerebrovascular diseases.Therefore,this study constructed a prognosis model of stroke based on the acute phase of stroke in our laboratory,and explored the efficacy and key pharmacological mechanism of Shuxuening Injection（SXNI）,a Ginkgo biloba extract preparation,on this mouse model.Finally,in vitro,the hippocampal neuron cell line HT-22 was used for oxygen glucose deprivation/reoxygenation（OGD/R）to simulate the occurrence and development of ischemic stroke in vivo and make the experimental results more reliable.This study is expected to provide a theoretical basis for the clinical application and prescription optimization of SXNI.Methods:1.The middle cerebral artery occlusion（MCAO）method was used to construct a prognosis model of stroke,that is,blood flow reperfusion was achieved after 30 min of ischemia,resulting in ischemia/reperfusion injury（CIRI）.CIRI mice were then randomly divided into Model group,Minocycline group and SXNI group,and Sham group was set at the same time.From the time of reperfusion,the drug or saline was injected intraperitoneally every day once a day until day 28.The efficacy of SXNI was evaluated by cerebral edema,blood-brain barrier leakage,cerebral infarct volume,water maze,passive avoidance task,neurological deficit score,and automatic gait analysis.2.Brain tissues of mice from SXNI group and Model group were selected for transcriptome sequencing（RNA-seq）to obtain the differentially expressed genes（DEGs）.Then the key DEGs（log₂Fold Change≥1&P≤0.05）were performed on the Ingenuity Pathway Analysis（IPA）core analysis to obtain the key mechanism and key targets of SXNI in the treatment of post-stroke.The results of IPA were verified by real-time reverse transcription-polymerase chain reaction（RT-PCR）,hematoxylin eosin（H&E）,NISSL staining,Western blotting（WB）,enzyme linked immunosorbent assay（ELISA）,and immunofluorescence（IF）.3.In vitro,purchased HT-22 cells were first identified and the IC₅₀of HT-22 was determined via CCK8 assay.HT-22 cells were then used for oxygen-glucose deprivation for 4h and reoxygenation for different times（12h,24h,36h and 48h）to finally select an appropriate time point that could be used to simulate post-stroke in vivo.Finally,at selected OGD/R time points,SXNI was added during reoxygenation,and the expression of key target proteins was detected by immunocytochemistry（ICC）.At the same time,the cell apoptosis and the m RNA and protein expression levels of related factors were explored.Results:1.On day 7 after stroke,significant cerebral infarction,cerebral edema,and blood-brain barrier leakage were observed in the Model group,and on day 28,significant brain atrophy was observed on the affected side.Minocycline and SXNI administration significantly improved these tissue lesions.The results showed that Minocycline and SXNI improved the latency or mistake times of forced swimming and passive avoidance in CIRI Model mice at different time points,and reduced the severity of neurological impairment and gait impairment,such as base of support,mean intensity,print area and stride length.2.In the RNA-seq of brain tissues from Model and SXNI groups,565 DEGs with log₂Fold Change≥1 and P≤0.05 were obtained,including 221 up-regulated and 344down-regulated genes.The top 20 signaling pathways were identified by IPA core analysis of the 565 DEGs.The third Neurotrophin/Trk Signaling pathway is the one most associated with brain disease.Similarly,No.5 on our list of the top 20 biological functions is Neurological Disease.IPA protein-protein interaction（PPI）analysis of DEGs closely related to Neurotrophin/Trk Signaling and Neurological Disease overlapped to obtain 15 key genes.They were Bdnf,Mapk1（Erk）,Map2k3（Mek3）,C-fos,Creb1,Map2k7,Map3k5,Mapk8（Jnk1）,Ngfr p75,Ntf4,Ntrk1,Ntrk2（Trk B）,Pik3r1,Spry2,and Akt1.RT-PCR results confirmed that 3 m L/kg SXNI could significantly increase the m RNA expression levels of Bdnf,Mapk1,Mek3,Creb1,Ntrk1,Trk B,Shh and Vegfa in brain tissues,while decrease the m RNA expression levels of C-fos,Map2k7,Jnk1,Pik3r1,Csf3 and Gfap.H&E and NISSL staining showed that SXNI could improve the injury and neuronal apoptosis in hippocampus CA3 region.WB results showed that SXNI could significantly increase the protein expressions of BDNF,Trk B and Mek3,but decrease the protein expressions of Jnk1 after stroke.ELISA results showed that SXNI could significantly increase the expression of Creb and p-Erk protein levels after stroke.If showed that SXNI could significantly increase the expression of BDNF and Trk B protein levels in hippocampus CA3 region.3.IF co-staining of HT-22 with Tuj1 and NeuN showed that the two marker proteins were100%expressed in HT-22 cell line,which indicated that HT-22 cell line was pure.CCK8assay showed that the IC₅₀of SXNI to HT-22 was 496.3μg/m L.50μg/m L and 200μg/m L were selected as the concentrations for cell administration.Through the exploration of the OGD/R conditions through nuclear counting methods,it was found that the degree of damage to the cells was moderate at 4h of oxygen glucose deprivation,24h and 36h of reoxygenation.In order to better correspond to post-stroke in vivo,OGD/R for 4h/36h was finally selected for follow-up studies.The expression of BDNF and Trk B in HT-22 cells was significantly decreased in OGD/R group by IF dual labeling of BDNF and Trk B,the most critical pair of ligand proteins in the Neurotrophin/Trk Signaling pathway.Although 50μg/m L of SXNI did not significantly increase BDNF expression,200μg/m L did.On Trk B,compared with OGD/R group,both 50μg/m L and 200μg/m L SXNI significantly increased the expression of Trk B protein.SXNI at 200μg/m L can regulate the expression of apoptosis molecules Bax/Bcl-2 and Caspase-3 after OGD/R.Conclusions:1.On the 7th day after stroke,SXNI can effectively improve the size of cerebral infarction,cerebral edema and blood-brain barrier leakage.On the 28th day after stroke,SXNI can reduce brain atrophy volume,cognitive dysfunction and motor deficiencies.2.In vivo and in vitro studies have jointly revealed and verified that BDNF-mediated Neurotrophin/Trk Signaling is a key pathway that SXNI promotes the recovery of cognitive dysfunction and motor deficiencies in post-stroke mice.