| Objective:To explore the protective mechanism of Notoginsenoside R1 on acute focal cerebral ischemia in rats.Method:1.Healthy and clean male SD rats(260-280g)are randomly divided into six groups,sham operation group;model group;low,middle,and high dose Notoginsenoside R1 intervention groups and n-butylphthalide positive control group.Rats in the model group,intervention groups and positive control group are molded with permanent middle cerebral artery occlusion(pMCAo).Rats in the sham operation group only undergo artery-separating then suture.All rats are returned to cages after operation.Rats in sham operation group are not be treated.Rats in intervention groups and positive control group are given low(10mg/kg/d),middle(20mg/kg/d),high(40mg/kg/d)dose of Notoginsenoside R1 and 20mg/kg/d n-butylphthalide by intraperitoneal injection.Rats in model group are injected intraperitoneally with the same amount of PBS after operation.The frequency of administration is detailed in each section.2.Post-operation by 12,24 and 72 hours,rats are treated as follows:twelve hours later,rats in each group are tested for Bederson score,then infused with 4%Evans Blue solution through the jugular vein and circulating for 1 hour.Take out brains after that and detect the leakage of blood-brain barrier with the help of in vivo imaging system(IVIS).In situ zymography of brain frozen sections is used to detect the activity and distribution of MMPs;Western blot of cerebral ischemic penumbra tissue is used to detect the expression of MMP2/9 and tight junction proteins.Twenty-four hours after operation,changes of infarct volume are observed by TTC staining of rats' brain slices.At the same time,the expression of glucose transporter,lactate transporter,citrate synthase and ATP content are detected to assess the level of energy metabolism in brains.Seventy-two hours after surgery,the Garcia JH rat neurological score is used to check changes in motor and sensory function;frozen brain sections through immunofluorescent staining to observe neurogenesis in hippocampus of ischemic side;Brain tissues in sham group,model group,high dose Notoginsenoside R1 intervention group and n-butylphthalide positive drug control group are submitted for proteomics detection.3.In vitro,brain endothelial bEnd.3 and neuron N2a cell lines are respectively stimulated by oxygen and glucose deprivation(OGD).Notoginsenoside R1 and n-butylphthalide are used for intervention.An endothelial barrier transwell model is constructed by bEnd.3,OGD time and effective concentration of drugs are based on the degree of dextran leakage.Then,cells in each group extract subcellular proteins,and detect the expression of tight junctions and caveolin1 in each component by Western blot;With a reference of cell viability,the concentration of Notoginsenoside R1 and n-butylphthalide in N2a cells under the stimulation of OGD is screened.The mitochondrial morphology is observed by live cell imaging.The mitochondrial expression and membrane potential are detected by flow cytometry.The DNA and RNA of N2a cells are extracted respectively,and Real-Time PCR is used to detect the copy number of mitochondrial DNA and the expression of mRNA array related to mitochondrial energy metabolism,find out the differential genes and back to brain tissue for verification.4.Inductively analyze the results of brain tissue proteomics,focusing on the interpretation of protein function annotations,protein difference analysis,and functional analysis of differential proteins.Find out the possible pathway mechanisms in post-stroke as well as Notoginsenoside R1 and n-butylphthalide involved in cerebral ischemia.Results:1.Notoginsenoside R1 significantly reduces the Bederson score and blood-brain barrier leakage in rats with acute cerebral ischemia injury,and restrains the activation of MMPs in the striatum and cortex of infarct side,as well as MMP2/9 in ischemic penumbra;Increases the expression of zo1 and claudin5.In vitro experiments show that Notoginsenoside R1 suppresses the leakage of brain endothelial cells stimulated by OGD,recovers the expression of zol and claudin5,and activates the redistribution of occludin mediated by caveolinl.2.Notoginsenoside R1 significantly reduces the infarct volume of rats with ischemic brain injury,up-regulates the expression of glucose transporters 1 and 3,lactate transporter 1,and citrate synthase,with increases ATP content in penumbra.In vitro experiments have shown that Notoginsenoside R1 increases cell viability of N2a with OGD,improves their mitochondrial morphology,promotes mitochondrial fluorescence expression,mitochondrial DNA copy number,and ameliorates mitochondrial membrane potential.Analyzes the expression of mRNA arrays related to mitochondrial energy metabolism,compares with OGD group,Notoginsenoside R1 group have 3 significantly up-regulated genes and 11 significantly down-regulated genes.It is verified that atp12a and atp6vlg3 also significantly up-regulate in Notoginsenoside R1 intervened penumbra tissues.3.Notoginsenoside R1 significantly improves the neurological function of rats after seventy-two hours of cerebral ischemic injury,increases the expression of GFAP+/Nestin+neural stem cells in hippocampus of infarcted side,and promotes transformation of DCX+neural precursor cells into mature NeuN+.4.Brain tissue proteomics analysis results show that seventy-two hours' permanent MCAo model can activate the coagulation cascade,which may play the role of procoagulant and anti-fibrinolysis,and induce cells to produce ROS to participate ferroptosis;Notoginsenoside R1 may inhibit the PI3K/AKT/Erk cascade through relaxin signals,reducing the production of MMP2/9 and NO after ischemic stroke,activating JNK to act as a neuromodulator;There are many target pathways involve in n-butylphthalide intervention,and its anti-cerebral ischemia mechanism may also contain the promotion of tissue anaerobic glycolysis,as well as anti-inflammatory and anti-apoptotic effects of IL-4 and COX2.Conclusion:The study shows that Notoginsenoside R1 has a good protective effect on rats with acute focal cerebral ischemia injury,and its mechanism may be related to intervening degradation and redistribution of tight junction proteins through Cav1/MMP2/9 pathway,thus reducing the leakage of blood-brain barrier,and promotes glucose metabolism and the expression of mitochondrial energy metabolism related genes atp12a and atp6v1g3 in brain,together with hippocampus neurogenesis. |
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