| Stroke causes serious damage to human health, and influences people’slife quality. Over the years, researchers have done a lot of work in theprevention and treatment of stroke, such as positive anticoagulation,thrombolysis, emergency intervention as well as the methods of minimallyinvasive surgery, and greatly improved the prognosis of patients with stroke.Still, about75%of the patients suffer from varying degrees of sequelaebecause of severe or complicated conditions or seeing a doctor not in time.Therefore, the study of stroke is still a long way. Recanalization happens inabout30%of ischemic stroke patients because of thrombolysis therapy orspontaneous to the distal embolus. Recanalization will be accompanied by theoccurrence of ischemia/reperfusion injury, so further study ofischemia/reperfusion injury mechanisms and development of new drugsagainst ischemic damage or finding the new role of traditional drugs againstischemic damage become a research hotspot in recent years, in order toprovide theoretical basis and clinical treatment for stroke.The events that occur subsequent to cerebral artery occlusion are termedcerebral ischemia/reperfusion denoting that these are distinct phases of cellularinjury with ATP depletion, lactate accumulation and acidosis observed duringischemia and the production of reactive oxygen and nitrogen species duringreperfusion. Neurons are very sensitive to changes in the supply of glucoseand oxygen that occurs during ischemic conditions. In-depth researchaccording to these pathological process and intervention become the key tosave the ischemic brain.In recent years, based on the research of the neuroprotective agent, somenatural or synthetic materials have been gradually discovered and confirmedto be protective against cerebral ischemia injury. Though these roles were amazing, more in-depth studies of their mechanism of action need to be done.Only by way of exploration and comprehensive understanding of theseneuroprotective agents, we can provide strong theoretical basis for thedevelopment of new drugs.Putative receptor protein related to the angiotensin receptor AT1(APJ) isa G protein coupled receptor first identified in human genes in1993, andknown as orphans G protein coupled receptor. In1998, human extracted andpurified APJ endogenous ligand from bovine stomach secretions using thereverse pharmacology method, and called it apelin. In humans, apelin mainlyexists in the heart, adrenal glands, kidney, lung vascular endothelium and largevascular endothelial cells. It is also expressed in the lung, heart muscle cells,kidney, adrenal gland cells, vascular smooth muscle cells, nerve cells, adiposetissue and connective tissue. Preproapelin is composed of77amino acidresidues, and its carboxyl C end is sequence for synthetic peptide and can bebroken down into a variety of relative molecular weight of different matureapelin active peptides by peptide enzyme, including apelin-17, apelin-36,apelin-12and apelin-13. Biological activity of apelin-13may be the strongest.More recent studies demonstrated that apelin-13is protective againstischemia-induced injury in cardiomyocytes. Apelin/APJ has been proved to bemyocardial protective against ischemia/reperfusion injury. Exogenousapelin-13or apelin-36could reduce myocardial infarction size. Apelin-13inhibited myocardial cells endoplasmic apoptosis induced byischemia/reperfusion. In addition, early myocardial ischemia and ischemicendogenous induced apelin/APJ expression increase, which protected theischemic myocardial cell damage and increased myocardial contraction force.Currently, research on apelin/APJ’s role in cerebrovascular disease is very few.Does apelin play a protective role against cerebral ischemia/reperfusion invivo? What is the mechanism of apelin? These problems need to further study.Therefore we evaluated the role of apelin in cerebral ischemia and its potentialmechanism. Part Ⅰ Apelin’s effect against focal cerebral ischemia/reperfusion injuryin miceObjective：This study is to evaluate the effect of apelin on cerebralischemia/reperfusion injury in mice.Methods：Focal transient cerebral ischemia was induced in male ICRmice using modified suture occlusion technique. The mice were randomlydivided into6groups: Sham group, Vehicle group, ischemia/reperfusion (I/R)group, apelin-low dose (APLN-L) group, apelin-middle dose (APLN-M)group and apelin-high dose (APLN-H) group.Sham group: with the same operation to I/R group except not insertingnylon line. Apelin-13treatment groups:5μl apelin-13was injected into ateralcerebral ventricle45min after ischemia, namely,15min before reperfusion.Vehicle group: equal volume of2%DMSO was injected into ateralcerebral ventricle45min after ischemia, namely,15min before reperfusion.Apelin-13concentration was10μg/kg in APLN-L group,50μg/kg inAPLN-M group and100μg/kg in APLN-H group. Mice were reanesthetizedand killed at24h after stroke, and neurological function, infarct volume, brainedema and apoptosis were measured. RT-qPCR and western blotting wereused to analyze the expression of Bax, Bcl-2, caspase-3and cleaved caspase-3.Cleaved caspase-3activity was also tested.Results：①Neurological function score of I/R group was higher than that of Shamgroup. Neurological function scores were significant reduced in APLN-Hgroup compared with Vehicle group (P＜0.05). There was no significantdifference between APLN-M and Vehicle group and between APLN-L groupand Vehicle group (P>0.05).②After TTC staining, the normal tissue was stained deep red while theinfarct area pale gray. No infarction was observed in Sham group. Apelin-13treated mice demonstrated smaller infarct volumes compared with Vehicle andI/R mice.③Percentage of brain water content of I/R group (84.68±0.929)%was higher than that of Sham group (78.33±0.625)%(P＜0.05). Percentage ofbrain water content of APLN-H group (82.70±1.172)%and APLN-M group(83.70±1.048)%decreased compared with that Vehicle group (84.51±1.401)%. There was no significant difference between APLN-L group (84.81±0.919)%and Vehicle group (84.51±1.401)%(P＞0.05).④TUNEL staining assays demonstrated that I/R group and Vehicle groupexhibited many stained, condensed nuclei, either isolated or within thecytoplasm of cells (400×light microscopy). Apoptotic cells decreased inAPLN-H group (28.00±2.280per field) and APLN-M group (32.40±3.262per field) compared with Vehicle group (42.80±3.544per field)(P<0.05).There was no significant difference between APLN-L group (41.40±1.200per field) and Vehicle group (42.80±3.544per field)(P＞0.05).⑤RT-qPCR results showed that elevations in Bax, Bcl-2and caspase-3mRNA expression were seen in stroke brains with minimal changes seen insham brains (P＜0.05). I/R group presented increase of Bax, Bcl-2andcaspase-3compared with Sham group (P<0.05). Bax and caspase-3mRNAexpression of APLN-H,-M,-L groups was lower than that of Vehicle group.Bcl-2mRNA expression of APLN-H,-M groups was higher than that ofVehicle group (P＜0.05). There was no significant difference of Bcl-2mRNAexpression between APLN-L group and Vehicle group (P＞0.05).⑥Western-blotting results showed that elevations in Bax, Bcl-2andcaspase-3were seen in stroke brains with minimal changes seen in shambrains (P＜0.05). APLN-H,-M and-L groups presented decrease of Bax, andcaspase-3and increase of Bcl-2compared with Vehicle group (P <0.05).⑦Western-blotting results showed that elevations in cleaved caspase-3were seen in stroke brains with minimal changes seen in sham brains (P＜0.05). APLN-H,-M,-L groups presented decrease of cleaved caspase-3compared with Vehicle group (P <0.05).⑧Cleaved caspase-3activity was obviously lower in APLN-L,-M and-H groups than that in Vehicle group (P <0.05).Conclusions： Mice appeared obvious neurologic deficits and brain edema after focal cerebral ischemia/reperfusion. Apelin-13treatmenteffectively ameliorated neurological defect, brain infarct volume and brainedema. Apelin-13also significantly attenuated apoptosis by reducingTUNEL-positive cells, down-regulating Bax, caspase-3and cleaved caspase-3and up-regulating Bcl-2. It provided the evidence that apelin-13protectedneurons against cerebral ischemia insults and anti-apoptosis was one of itsactions.Part Ⅱ Research in correlation between ERK1/2signal pathway andapelin-13’s anti-apoptosis effectObjective： This study is to detect the mechanism of apelin-13’sneuroprotection, and evaluate whether ERK1/2signal pathway was involvedin it.Methods：Focal transient cerebral ischemia was induced in male ICRmice using modified suture occlusion technique. Experiment Ⅰ: The micewere randomly divided into6groups: Sham group, Vehicle group, I/R group,APLN-L group, APLN-M group and APLN-H group. Experiment Ⅱ: Themice were randomly divided into5groups: Sham group, Vehicle group,APLN-M group, APLN-M+PD98059(APLN+PD) group and PD98059+I/R(PD) group.Sham group: with the same operation to I/R group except not insertingnylon line.Apelin-13treatment groups: in experiment Ⅰ,5μl of apelin-13wasinjected into ateral cerebral ventricle45min after ischemia, namely,15minbefore reperfusion; in experiment Ⅱ,5μl of2%DMSO was injected intoateral cerebral ventricle15min before ischemia and5μl of apelin-13wasinjected into ateral cerebral ventricle15min before reperfusion.Vehicle group: in experiment Ⅰ,5μl of2%DMSO was injected intoateral cerebral ventricle45min after ischemia, namely,15min beforereperfusion; in experiment Ⅱ:5μl of2%DMSO was injected into ateralcerebral ventricle15min before ischemia and15min before reperfusionrespectively. PD98059treatment groups: in experiment Ⅱ in APLN+PD group,5μlof PD98059was injected into ateral cerebral ventricle15min before ischemia,and then5μl of apelin-13was injected into ateral cerebral ventricle15minbefore reperfusion; in PD group,5μl of PD98059was injected into ateralcerebral ventricle15min before ischemia, and then5μl of2%DMSO wasinjected into ateral cerebral ventricle15min before reperfusion.Mice were reanesthetized and killed at24h after stroke. RT-qPCR andwestern blotting were used to analyze the expression of Bax, Bcl-2, caspase-3,cleaved caspase-3and ERK1/2. Cleaved caspase-3activity was also tested.Results：Experiment Ⅰ①RT-qPCR results showed that ERK1/2mRNA expression was seen inall groups. There was no significant difference among all groups (P＞0.05).②Western-blotting results showed that ERK1/2protein expression wasseen in all groups. There was no significant difference among all groups (P＞0.05). P-ERK1/2protein expression was seen in all groups. P-ERK1/2increased in I/R group compared with Sham group (P＜0.05). P-ERK1/2increased APLN-H,-M,-L groups compared with Vehicle group (P＜0.05).Experiment Ⅱ①RT-qPCR results showed that ERK1/2mRNA expression was seen inall groups. There was no significant difference among all groups (P＞0.05).②Western-blotting results showed that ERK1/2protein expression wasseen in all groups. There was no significant difference among all groups (P＞0.05). P-ERK1/2protein expression was seen in all groups. P-ERK1/2decreased in PD group compared with Vehicle group(P＜0.05). P-ERK1/2decreased in APLN+PD group compared with APLN-M group (P＜0.05).③RT-qPCR results showed that Bax, Bcl-2and caspase-3mRNAexpression were seen in all groups. There was no significant differencebetween PD group and Vehicle group (P＞0.05). Bax and caspase-3mRNAdecreased and Bcl-2mRNA increased in APLN-M group compared withVehicle group (P＜0.05). Bax and caspase-3mRNA increased and Bcl-2 mRNA decreased in APLN+PD group compared with APLN-M group (P＜0.05). There was no significant difference of Bax、Bcl-2mRNA betweenAPLN+PD group and PD group (P＞0.05). Caspase-3mRNA decreased inAPLN+PD group compared with PD group (P＜0.05).④Western-blotting results showed that Bax, Bcl-2and caspase-3proteinexpression was seen in all groups. There was no significant difference betweenPD group and Vehicle group (P＞0.05). Bax and caspase-3decreased andBcl-2increased in APLN-M group compared with Vehicle group (P＜0.05).Bax and caspase-3increased and Bcl-2decreased in APLN+PD groupcompared with APLN-M group (P＜0.05). There was no significant differenceof Bax、Bcl-2between APLN+PD group and PD group (P＞0.05). Caspase-3decreased in APLN+PD group compared with PD group (P＜0.05).⑤Western-blotting results showed that cleaved caspase-3proteinexpression was seen in all groups. There was no significant difference betweenPD group and Vehicle group (P＞0.05). Cleaved caspase-3decreased inAPLN-M group compared with Vehicle group (P＜0.05). Cleaved caspase-3increased in APLN+PD group compared with APLN-M group (P＜0.05).There was no significant difference between APLN+PD group and PD group(P＞0.05).⑥Cleaved caspase-3activity decreased in APLN-M group comparedwith Vehicle group (P<0.05). There was no significant difference between PDgroup and Vehicle group (P＞0.05). Cleaved caspase-3activity increased inAPLN+PD group compared with APLN-M group (P<0.05). Cleavedcaspase-3activity decreased in APLN+PD group compared with PD group (P<0.05).Conclusions：P-ERK1/2was up-regulated while total ERK has no change after focalcerebral ischemia/reperfusion, which prompted ERK1/2protein activationmay play an important role in ischemia/reperfusion mechanism. Apelin-13treatment enhanced the phosphorylation level of ERK1/2, which promptedthat apelin-13promoted the activation of ERK1/2protein, and ERK1/2 signaling pathway might be involved in the apelin-13’s action. Afterapplication of PD98059to inhibit ERK1/2activation, apelin-13’s effect onBax, Bcl-2and caspase-3was attenuated or disappeared. It proved thatapelin-13’s effect on Bax, Bcl-2and caspase-3associated with the activationof ERK1/2, namely, ERK1/2signal pathway was involved in the apelin-13anti-apoptosis effect in cerebral ischemia/reperfusion.Part Ⅲ Research in correlation between PI-3K/Akt signal pathway andapelin-13’s anti-apoptosis effectObjective： This study is to detect the mechanism of apelin-13’sneuroprotection, and evaluate whether PI-3K/Akt signal pathway wasinvolved in it.Methods：Focal transient cerebral ischemia was induced in male ICRmice using modified suture occlusion technique. Experiment Ⅰ: The micewere randomly divided into6groups: Sham group, Vehicle group, I/R group,APLN-L group, APLN-M group and APLN-H group. Experiment Ⅱ: Themice were randomly divided into5groups: Sham group, Vehicle group,APLN-M group, APLN-M+LY294002(APLN+LY) group and LY294002+I/R (LY) group.Sham group: with the same operation to I/R group except not insertingnylon line.Apelin-13treatment groups: in experiment Ⅰ,5μl of apelin-13wasinjected into ateral cerebral ventricle45min after ischemia, namely,15minbefore reperfusion; in experiment Ⅱ,5μl of2%DMSO was injected intoateral cerebral ventricle15min before ischemia and5μl of apelin-13wasinjected into ateral cerebral ventricle15min before reperfusion.Vehicle group: in experiment Ⅰ,5μl of2%DMSO was injected intoateral cerebral ventricle45min after ischemia, namely,15min beforereperfusion; in experiment Ⅱ:5μl of2%DMSO was injected into ateralcerebral ventricle15min before ischemia and15min before reperfusionrespectively.LY294002treatment groups: in experiment Ⅱ in APLN+LY group,5μl of LY294002was injected into ateral cerebral ventricle15min beforeischemia, and then5μl of apelin-13was injected into ateral cerebral ventricle15min before reperfusion; in LY group,5μl of LY294002was injected intoateral cerebral ventricle15min before ischemia, and then5μl of2%DMSOwas injected into ateral cerebral ventricle15min before reperfusion.Mice were reanesthetized and killed at24h after stroke. RT-qPCR andwestern blotting were used to analyze the expression of Akt, Bax, Bcl-2,caspase-3and cleaved caspase-3. Cleaved caspase-3activity was also tested.Results：Experiment Ⅰ①RT-qPCR results showed that Akt mRNA expression was seen in allgroups. There was no significant difference among all groups (P＞0.05).②Western-blotting results showed that Akt protein expression was seenin all groups. There was no significant difference among all groups (P＞0.05).P-Akt protein expression was seen in all groups. P-Akt increased in I/R groupcompared with Sham group (P＜0.05). P-Akt increased APLN-H,-M,-Lgroups compared with Vehicle group (P＜0.05).Experiment Ⅱ①RT-qPCR results showed that Akt mRNA expression was seen in allgroups. There was no significant difference among all groups (P＞0.05).②Western-blotting results showed that Akt protein expression was seenin all groups. There was no significant difference among all groups (P＞0.05).P-Akt decreased in LY group compared with Vehicle group(P＜0.05). P-Aktdecreased in APLN+LY group compared with APLN-M group (P＜0.05).③RT-qPCR results showed that Bax, Bcl-2and caspase-3mRNAexpression were seen in all groups. There was no significant differencebetween LY group and Vehicle group (P＞0.05). Bax and caspase-3mRNAdecreased and Bcl-2mRNA increased in APLN-M group compared withVehicle group (P＜0.05). Bax and caspase-3mRNA increased and Bcl-2mRNA decreased in APLN+LY group compared with APLN-M group (P＜0.05). There was no significant difference of Bax、Bcl-2mRNA between APLN+LY group and LY group (P＞0.05). Caspase-3mRNA decreased inAPLN+LY group compard with LY group (P＜0.05).④Western-blotting results showed that Bax, Bcl-2and caspase-3proteinexpression was seen in all groups. There was no significant difference betweenLY group and Vehicle group (P＞0.05). Bax and caspase-3decreased andBcl-2increased in APLN-M group compared with Vehicle group (P＜0.05).Bax and caspase-3increased and Bcl-2decreased in APLN+LY groupcompared with APLN-M group (P＜0.05). There was no significant differenceof Bcl-2between APLN+LY group and LY group (P＞0.05). Bax andcaspase-3decreased in APLN+LY group compard with LY group (P＜0.05).⑤Western-blotting results showed that cleaved caspase-3proteinexpression was seen in all groups. There was no significant difference betweenLY group and Vehicle group (P＞0.05). Cleaved caspase-3decreased inAPLN-M group compared with Vehicle group (P＜0.05). Cleaved caspase-3increased in APLN+LY group compared with APLN-M group (P＜0.05).Cleaved caspase-3decreased in APLN+LY group compared with LY group (P＜0.05).⑥Cleaved caspase-3activity was lower in APLN-M group than that inVehicle group (P＜0.05). There was no significant difference between LYgroup and Vehicle group (P＞0.05). Cleaved caspase-3activity increased inAPLN+LY group compared with APLN-M group (P <0.05). Cleavedcaspase-3activity decreased in APLN+LY group compared with LY group (P＜0.05).Conclusions：P-Akt was up-regulated while total Akt has no change after focal cerebralischemia/reperfusion, which prompted Akt protein activation may play animportant role in ischemia/reperfusion mechanism. Apelin-13treatmentenhanced the phosphorylation level of Akt, which prompted that apelin-13promoted the activation of Akt protein, and PI-3K/Akt signaling pathwaymight be involved in the apelin-13’s action. After application of LY294002toinhibit Akt activation, apelin-13’s effect on Bax, Bcl-2and caspase-3was attenuated or disappeared. It proved that apelin-13’s effect on Bax, Bcl-2andcaspase-3associated with the activation of Akt, namely, PI-3K/Akt signalpathway was involved in the apelin-13anti-apoptosis effect in cerebralischemia/reperfusion. |
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