| Background:Ischemic stroke(IS)is one of the major diseases that threaten human health and survival.It is a neurological disorder characterized by ischemic necrosis or softening of brain tissue caused by blood circulation disorders in the brain,leading high morbidity and mortality.At present,the main clinical treatment is to restore the blood reperfusion and oxygen supply in the ischemic area as soon as possible.However,when the blood flow supply is restored,the cerebral ischemia reperfusion injury(CIRI)will be also caused,which will further aggravate the pathological damage of ischemic tissue and nervous system and worsen the clinical symptoms.The pathological process of CIRI is complex,and multiple mechanisms are involved,thus CIRI is an urgent problem to be solved in the current clinical treatment of IS.Network pharmacology is gradually emerging,which combines systems biology,bioinformatics and pharmacology related methods to explore the mechanism of drug action from multiple components,multiple targets and multiple pathways.As an emerging discipline,network pharmacology has been widely used in the prediction and screening of is therapeutic drug targets and pathways,which has effectively promoted the discovery and development of relevant new drugs and the understanding of therapeutic mechanism.Geraniin is a polyphenol active compound extracted and isolated from Phyllanthus amarus Linn.(Euphorbiaceae),a characteristic medicinal plant native to Yunnan province.It has been proved to have antioxidant,anti-inflammatory,anti-platelet aggregation and other functions.Recently,geraniin has been reported to have a protective effect on nerve cell damage caused by Alzheimer’s disease in vitro,but the mechanism of this neuroprotective effect remains unclear.These results suggest that geraniin may have a neuroprotective effect.Objective:To study the improvement effect and protective mechanism of geraniin on CIRI in rats.Based on network pharmacology,the potential targets and pathways of geraniin improving CIRI is explored,and whether geraniin plays a neuroprotective role by regulating PI3K/Akt/mTOR signal pathway to inhibit autophagy is further verified.Our study provides theoretical and experimental basis for further analysis of geraniin in the treatment of IS by improving CIRI.Methods:1.The neuroprotective effects of geraniin in vivo and in vitro1.1 In vivo study:effects of geraniin on middle cerebral artery occlusion/reperfusion(MCAO/R)in rats(1)The model of rat MCAO/R was duplicated,and the male SPF SD rats were randomly divided into sham group,MCAO/R model group,geraniin(5,10,20 mg/kg)group and nimodipine(1 mg/kg)as positive drug group.Except for sham group,MCAO/R model was duplicated.2 h after ischemia,geraniin(5,10,20 mg/kg)and nimodipine(1 mg/kg)were injected intraperitoneally for the first time,and given once at 24 h,48 h,72 h after reperfusion,respectively.Neurological scores and open field behavior experiments were performed on rats in each group at 12 h after the last intraperitoneal injection.Then the rats were euthanized,and the rat brain tissue was collected for the follow-up experiments.(2)Rat brain tissues were stained with 2,3,5-triphenyltetrazolium chloride(TTC)and hematoxylin and eosin(HE).(3)Rat serum and brain tissue homogenate were used.The activity of superoxide dismutase(SOD)was detected by water-soluble tetrazolium-1(WST-1),the content of lactate dehydrogenase(LDH)was detected by microplate method,and the content of malondialdehyde(MDA)was detected by thiobarbituric acid(TBA),the contents of nitric oxide(no)and neuronal nitric oxide synthase(nNOS)were detected by enzymelinked immunosorbent assay.(4)Immunofluorescence staining was performed for terminal deoxynucleotidyl transferase-mediated D-UTP Nick end labeling(TUNEL).The apoptosis of nerve cells in cerebral cortex and hippocampus was observed.(5)Immunofluorescence method was used to observe neuron-specific marker proteins in the cerebral cortex and hippocampus of rats:neuron specific nucleoprotein(NeuN)and microtubule associated protein-2(Map-2);and autophagy related proteins:coiledcoil,myosin-like Bcl-2 interacting protein(Beclin-1),Sequestosome-1(P62),microtubule-associated protein 1 light chain 3(Map1LC3)Ⅱ/Ⅰ.(6)Autophagosomes and autophagolysosomes in rat brain tissue were observed by transmission electron microscopy.(7)Western blot was used to detect the expressions of apoptosis-related proteins Bax,Bcl-2,caspase3,neuron-related proteins NeuN,Map-2,autophagy related proteins Beclin-1,P62,LC3Ⅱ/Ⅰ.1.2 In vitro study:effects of geraniin on PC12 cells injured by glucose deprivation hypoxia/reoxygenation(OGD/R)(1)CCK8 chemical method was used to detect the safe concentration range of geraniin on PC 12 cells and the effect of OGD/R on the viability of PC 12 cells.Detection of safe concentration range of geraniin:PC 12 cells were cultured aseptically,and the cells were divided into control group,DMSO group and different concentration groups of geraniin(0.01μmol/L,0.1 μmol/L,1 μmol/L,10 μmol/L,100μmol/L),24 h after incubation of geraniin,the cell viability was measured by CCK8 method.Detection of the effective concentration range of geraniin:the cells were divided into control group,OGD/R model group and different concentration groups of geraniin(0.1μmol/L,1μmol/L,10μmol/L),nimodipine group(10 μmol/L).In addition to the control group,each group was treated with OGD for 2 h,and then treated with glucose and reoxygenation for 24 h.Cell viability was determined as above.(2)The cell culture supernatant was obtained.The activity of SOD was detected by WST-1 method,the content of LDH was detected by microplate method,the content of MDA was detected by TBA method,and the contents of no and nNOS were detected by enzyme-linked immunosorbent assay.(3)The apoptosis rate of PC 12 cells was detected by flow cytometry.(4)Western blot was used to detect the expressions of apoptosis-related proteins Bax,Bcl-2,caspase3,neuron-related proteins NeuN,Map-2,autophagy related proteins Beclin-1,P62,LC3Ⅱ/Ⅰ.2.Network pharmacology analysis of potential targets and pathways of geraniin on improving cerebral ischemia-reperfusion injuryIn this part,geraniin drug targets and IS disease targets were obtained through TCMSP,GeneCards and OMIM databases,PPI network map was constructed by STRING database and Cytoscape software,and biological process and signal pathway enrichment analysis of potential targets were conducted by DAVID database.The enriched targets were analyzed by molecular docking.The occurrence of IS will be accompanied by CIRI.In this part,through data mining of the targets and pathways of geraniin in the treatment of IS,we hope to obtain the potential mechanism of geraniin in improving CIRI and provide basis for subsequent experimental research.3.Molecular experiments confirmed that geraniin could inhibit autophagy through PI3K/Akt/mTOR pathway mediating a neuroprotective role(1)Western blot was used to detect the expression of PI3K/Akt/mTOR signal pathway related proteins and the changes of phosphorylated proteins in vivo and in vitro.(2)After pretreatment with rapamycin(Rapa)(20 μmol/L),the effects of geraniin on PI3K/AKT/mTOR signaling pathway,autophagy and neuroprotection of OGD/R injured PC 12 cells were observed.Experimental groups:control group,control+Rapa group,OGD/R group,OGD/R+Rapa group,OGD/R+geraniin group,OGD/R+Rapa+geraniin group.Western blot was used to detect the expression of PI3K/AKT/mTOR signaling pathway proteins and their phosphorylation levels,autophagy related proteins Beclin-1 and P62,and neuron related proteins NeuN and Map-2.SOD activity was detected by WST-1 method and MDA content was detected by TBA method.(3)After pretreatment with 3 Methyladenine(3MA)(20 μmol/L),the effects of geraniin on PI3K/AKT/mTOR signaling pathway,autophagy and neuroprotective effect of OGD/R injured PC12 cells were observed.Experimental groups:control group,control+3MA group,OGD/R group,OGD/R+3MA group,OGD/R+geraniin group,OGD/R+3MA+geraniin group.Western blot was used to detect the expression of PI3K/AKT/mTOR signaling pathway proteins and their phosphorylation levels,autophagy related proteins Beclin-1 and P62,and neuron related proteins NeuN and Map-2.SOD activity was detected by WST-1 method and MDA content was detected by TBA method.(4)After pretreatment with LY294002(20 μmol/L),the effects of geraniin on PI3K/AKT/mTOR signaling pathway,autophagy and neuroprotective effect of OGD/R injured PC 12 cells were observed.Experimental groups:control group,control+LY294002 group,OGD/R group,OGD/R+LY294002 group,OGD/R+geraniin group,OGD/R+LY294002+geraniin group.Western blot was used to detect the expression of PI3K/AKT/mTOR signaling pathway proteins and their phosphorylation levels,autophagy related proteins beclin-1 and P62,and neuron related proteins NeuN and Map-2.SOD activity was detected by WST-1 method and MDA content was detected by TBA method.Results:1.Geraniin could improve the neurological injury of MCAO/R rats by antioxidation,anti-apoptosis and inhibition of autophagy(1)Results of behavioral experiment:Compared with sham group,the neurological score of model group was significantly increased(P<0.01);compared with model group,scores of geraniin(20 mg/kg)and nimodipine(1 mg/kg)groups were significantly decreased(P<0.01).The results of open field experiment showed that compared with sham group,the movement distance and movement speed of model group were significantly reduced within 15 min(P<0.01);compared with model group,geraniin(20 mg/kg)and nimodipine(1 mg/kg)significantly increased movement distance and movement speed(P<0.01).(2)TTC staining results showed that,compared with sham operation group,cerebral infarction volume in model group was significantly increased(P<0.01).Compared with model group,geraniin(10,20 mg/kg)and nimodipine(1 mg/kg)significantly reduced infarct volume(P<0.01).(3)HE pathological examination showed that compared with sham group,nerve cells in cortex and hippocampus of model group were disordered,with widened cell space,wrinkled cell body,nuclear pyknosis,nucleolus disappearance and chromatin hyperchromatin.Compared with model group,geraniin(20 mg/kg)and nimodipine(1 mg/kg)had neat arrangement of nerve cells,tight cell space,full cell body and clear nucleoli.(4)Expression of neuron marker protein:immunofluorescence results showed that compared with sham group,positive expression of Map-2 and NeuN in cerebral cortex and hippocampus of model group was significantly decreased(P<0.01).Compared with model group,positive expression of Map-2 in cerebral cortex and hippocampus of geraniin(20 mg/kg)groups were increased(P<0.05);positive expressions of Map-2 and NeuN in cerebral cortex and hippocampus of geraniin(20 mg/kg)and nimodipine(1 mg/kg)groups were increased(P<0.01).Western Blot results showed that compared with sham group,the expressions of Map-2 and NeuN in model group were significantly decreased(P<0.01).Compared with model group,the expressions of Map-2 and NeuN in geraniin(20 mg/kg)and nimodipine(1 mg/kg)groups were significantly increased(P<0.05,P<0.01).(5)Oxidative stress test results showed that compared with sham group,the contents of LDH,MDA,NO and nNOS in serum and brain tissue of model group were significantly increased,and SOD activity was significantly decreased(P<0.01).Compared with model group,contents of LDH and MDA in serum and LDH in tissue homogenate of geraniin(10 mg/kg)group were significantly decreased(P<0.05,P<0.01);contents of LDH,MDA,NO and nNOS in geraniin(20 mg/kg)and nimodipine(1 mg/kg)groups were significantly decreased,and SOD activity was significantly increased(P<0.01).(6)The results of the apoptosis assay showed that compared with sham group,the positive expression of TUNEL was significantly increased in model group(P<0.01).Compared with model group,the positive expression of TUNEL in geraniin(20 mg/kg)and nimodipine(1 mg/kg)groups was significantly decreased(P<0.01).Western blot results showed that compared with sham group,the expression of Bax and caspase-3 in MCAO/R group was significantly increased,and the expression of Bcl-2 was significantly decreased(P<0.05,P<0.01).Compared with model group,the expressions of Bax and caspase-3 in geraniin(20 mg/kg)and nimodipine(1 mg/kg)groups were significantly decreased,and the expressions of Bcl-2 were significantly increased(P<0.05,P<0.01).(7)The results of transmission electron microscopy showed that autophagosomes and lysosomes were significantly increased in model group compared with sham group.Compared with model group,the numbers of autophagosomes and autophagolysosomes in geraniin(20 mg/kg)and nimodipine(1 mg/kg)groups were decreased.(8)Autophagy protein expression:immunofluorescence results showed that compared with sham group,the positive expression of Beclin-1 in the cerebral cortex and hippocampus of model group was significantly increased,and the positive expression of P62 was significantly decreased(P<0.05,P<0.01).Compared with model group,the positive expression of Beclin-1 in cortex and hippocampus of geraniin(20 mg/kg)and nimodipine(1 mg/kg)groups was significantly decreased,and the positive expression of P62 was significantly increased(P<0.05,P<0.01).Western Blot results showed that compared with sham group,the expression of Beclin-1 and LC3II/I in model group was significantly increased,and the expression of P62 was significantly decreased(P<0.01).Compared with model group,the expressions of Beclin-1 and LC3Ⅱ/Ⅰ in geraniin(20 mg/kg)and nimodipine(1 mg/kg)groups were significantly decreased,and the expression of P62 was significantly increased(P<0.01).2.Geraniin could improve OGD/R injury of PC12 cells by anti-oxidation,antiapoptosis and inhibition of autophagy(1)Cell viability test:the test results of the safe concentration range of geraniin showed that compared with the control group,geraniin(100 μmol/L)group significantly decreased cell viability(P<0.01)and toxic reaction occurred;the detection results of the effective concentration range of geraniin showed that compared with the control group,the cell viability of the model group decreased significantly(P<0.01),and compared with the model group,geraniin(10 μmol/L)group,nimodipine(10 μmol/L)group significantly increased cell viability(P<0.01).(2)Expression of neuronal marker protein:Compared with control group,the expression of Map-2 and NeuN in model group was significantly decreased(P<0.01).Compared with model group,the expressions of Map-2 and NeuN in geraniin(10μmol/L),nimodipine(10 μmol/L)groups were significantly increased(P<0.01).(3)Oxidative stress test results showed that compared with control group,the contents of LDH,MDA,NO and nNOS in model group were significantly increased,and the activity of SOD was significantly decreased(P<0.01).Compared with model group,the contents of LDH,MDA,NO and nNOS in geraniin(10 μmol/L)and nimodipine(10 μmol/L)groups were significantly decreased,and the activity of SOD was significantly increased(P<0.01).(4)Apoptosis detection:flow cytometry results showed that compared with the normal group,the apoptosis rate of geraniin(10 μmol/L)and nimodipine(10 μmol/L)groups was significantly increased(P<0.01),compared with the model group,the apoptosis rate of geraniin(0.1,1,10 μmol/L)and nimodipine(10 μmol/L)groups was significantly decreased(P<0.05,P<0.01).Western blot results showed that compared with normal group,the expression of Bax and caspase-3 in model group was significantly increased,and the expression of Bcl-2 was significantly decreased(P<0.01).Compared with model group,the expressions of Bax and caspase-3 in geraniin(1,10 μmol/L)and nimodipine(10 μmol/L)groups were significantly decreased,and the expressions of Bcl-2 were significantly increased(P<0.05,P<0.01).(5)Autophagy protein expression:compared with normal group,the expression of Beclin-1 and LC3Ⅱ/Ⅰ in model group was significantly increased,while the expression of P62 was significantly decreased(P<0.01).Compared with model group,the expressions of Beclin-1 and LC3Ⅱ/Ⅰ in geraniin(1,10 μmol/L)and nimodipine(10μmol/L)groups were significantly decreased,and the expression of P62 was significantly increased(P<0.01).3.Network pharmacology results(1)Related targets of geraniin were searched in the database,and 130 related targets were collected,including 40 Swiss Target Prediction and 90 Pharm Mapper.A total of 3096 targets associated with ischemic stroke were identified based on Genecard and TTD databases.(2)Geraniin was mapped to ischemic stroke disease targets,and 92 key targets were obtained by cross mapping,and the PPI network diagram of related targets was constructed.The PPI network diagram shows that there are 92 nodes,567 edges,the clustering coefficient is 0.557,and the average degree value is 12.3.Degree sorted and obtained the top 20 protein kinase B1(AKT1),epidermal growth factor receptor 2(ERBB2),oncogene homologues(HRAS),matrixmetalloproteinase9(MMP-9),cell division cycle 42(CDC42)and other targets.(3)A total of 50 pathways were obtained by GO and KEGG enrichment analysis.In order of p-value,the pathway with the highest correlation of geraniin in the treatment of ischemic stroke was PI3K/AKT/mTOR signaling pathway.Signaling pathway map was constructed and the key downstream targets were found to be related to autophagy.(4)Molecular docking analysis showed that geraniin had strong binding ability with PI3K,ATK,mTOR,Beclin-1 and P62,with the binding ability of-7.86,-7.46,-6.69,8.62 and-7.02 kJ/mol,respectively.4.Geraniin could inhibit autophagy mediated neuroprotection through PI3K/AKT/mTOR pathway(1)In vivo experiment PI3K/AKT/mTOR pathway protein expression detection:western blot results showed that compared with the sham group,the expression of pPI3K,p-AKT and p-mTOR in the model group was significantly decreased(P<0.01).Compared with model group,the expressions of p-PI3K,p-Akt and p-mTOR in geraniin(20 mg/kg)group were significantly increased(P<0.05,P<0.01).(2)In vitro experiment PI3K/AKT/mTOR pathway protein expression detection:western blot results showed that compared with the normal group,the expression of pPI3K,p-AKT and p-mTOR in the model group was significantly decreased(P<0.01).Compared with model group,the expressions of p-PI3K,p-AKT and p-mTOR in geraniin(10 μmol/L)group were significantly increased(P<0.01).(3)Rapa inhibitor pretreatment results:western blot results showed that compared with normal group,the expression of p-mTOR,P62,NeuN and Map-2 in normal+Rapa group and model group was significantly decreased,and the expression of Beclin-1 was significantly increased(P<0.05,P<0.01).Compared with model group,the expression of p-mTOR,P62,NeuN and Map-2 in model+Rapa group was significantly decreased,while the expression of Beclin-1 was significantly increased(P<0.05,P<0.01).Compared with model group,the expression of p-mTOR,P62,NeuN and Map2 in model+geraniin(10μmol/L)group was significantly increased,and the expression of Beclin-1 was significantly decreased(P<0.05,P<0.01).Compared with model+geraniin(10μmol/L)group,the expression of p-mTOR,NeuN,MAP-2 and P62 in model+Rapa+geraniin(10μmol/L)group was significantly decreased,and the expression of Beclin-1 was significantly increased(P<0.05,P<0.01).Compared with normal group,SOD activity in normal+Rapa group and model group was significantly decreased,and MDA content was significantly increased(P<0.01).Compared with model group,SOD activity was significantly decreased and MDA content was significantly increased in model+Rapa group(P<0.05,P<0.01).Compared with model group,SOD activity in model+geraniin(10 μmol/L)group was significantly increased,and MDA content was significantly decreased(P<0.01).Compared with model+geraniin(10 μmol/L)group,SOD activity in model+Rapa+geraniin(10 μmol/L)group was significantly decreased,and MDA content was significantly increased(P<0.05,P<0.01).(4)3MA inhibitor pretreatment results:western blot results showed that compared with control group,the expression of p-PI3K,p-AKT,p-mTOR and Beclin-1 in control+3MA group was significantly decreased(P<0.01),and the expression of P62,NeuN and Map-2 was significantly increased(P<0.01).Compared with control group,the expression of p-PI3K,p-AKT,p-mTOR,P62,NeuN and Map-2 in model group was significantly decreased(P<0.01),and the expression of Beclin-1 was significantly increased(P<0.01).Compared with model group,the expressions of p-PI3K,p-AKT,p-mTOR and Beclin-1 in model+3MA group were significantly decreased(P<0.05,P<0.01),and the expressions of P62,NeuN and Map-2 were significantly increased(P<0.05,P<0.01).Compared with model group,the expression of p-PI3K,p-AKT,pmTOR,P62,NeuN and Map-2 in model+geraniin(10 μmol/L)group was significantly increased(P<0.05,P<0.01),and the expression of Beclin-1 was significantly decreased(P<0.05,P<0.01).Compared with model+geraniin(10 μmol/L)group,the expression of p-PI3K,p-AKT,p-mTOR,Beclin-1,NeuN and Map-2 in model+3MA+geranniin(10 μmol/L)group was significantly decreased.The expression of P62 was significantly increased(P<0.05,P<0.01).Compared with control group,SOD activity in control+3MA group was significantly increased and MDA content was significantly decreased(P<0.05,P<0.01).Compared with control group,SOD activity was significantly decreased and MDA content was significantly increased in model group(P<0.01).Compared with model group,SOD activity in model+3MA group and model+geraniin(10 μmol/L)group was significantly increased,and MDA content was significantly decreased(P<0.01).Compared with model+geraniin(10μmol/L)group,SOD activity in model+3MA+geraniin(10 μmol/L)group was significantly increased,and MDA content was significantly decreased(P<0.05).(5)LY294002 inhibitor pretreatment results:western blot results showed that compared with control group,the expression of p-PI3K,p-AKT,p-mTOR,P62,NeuN and Map2 in control LY294002 group and model group was significantly decreased,while the expression of Beclin-1 was significantly increased(P<0.01).Compared with model group,the expression of p-PI3K,p-AKT,p-mTOR,P62,NeuN and Map-2 in model+LY294002 group was significantly decreased,and the expression of Beclin-1 was significantly increased(P<0.01).Compared with model group,the expression of pPI3K,p-AKT,p-mTOR,P62,NeuN and Map-2 in model+geraniin(10 μmol/L)group was significantly increased,and the expression of Beclin-1 was significantly decreased(P<0.05,P<0.01).Compared with model+geraniin(10μmol/L)group,the expressions of p-PI3K,p-AKT,p-mTOR,P62,NeuN and Map-2 in model+LY294002+geraniin(10 μmol/L)group were significantly decreased.Beclin-1 expression was significantly increased(P<0.05,P<0.01).Compared with control group,SOD activity in control+LY294002 group and model group was significantly decreased,and MDA content was significantly increased(P<0.01).Compared with model group,SOD activity in model+LY294002 group was significantly decreased,MDA content was significantly increased(P<0.01);Compared with model group,SOD activity in model+geraniin(10 μmol/L)group was significantly increased,and MDA content was significantly decreased(P<0.01).Compared with model+geraniin(10 μmol/L)group,SOD activity in model+LY294002+geraniin(10 μmol/L)group was significantly decreased,and MDA content was significantly increased(P<0.01).Conclusion:1.Geraniin protects neurons and improves cerebral ischemia reperfusion injury by antioxidation,anti-apoptosis and inhibition of autophagy.2.Geraniin may regulate autophagy by activating the PI3K/AKT/mTOR signaling pathway.3.Geraniin exerts neuroprotective effects by up regulating PI3K/AKT/mTOR signaling pathway and inhibiting autophagy. |
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