| Objective:To observe whether a new transmembrane tripeptide drug,proline-glycinephenylalanine(Pro-Gly-Phe,PGF),can penetrate the blood-brain barrier of middle cerebral artery infarcted rats,then hydrolyze at ischemia-injured area to perform its role of neuroprotection.Methods:(1)OGD(Oxygen And Glucose Deprivation)model,randomly divided into sham group,OGD+200μM PGF group,OGD+400μM PGF group,OGD+600μM PGF group,OGD+800μM PGF group,CCK8(Cell Counting kit-8)was used to compare the survival rate of PGF treatment neurons at different doses.(2)32 MCAO(Middle Cerebral Artery Occlusion)rats were randomly divided into Ischemia+FITC(Fluorescein Isothiocyanate)-PGF group,Ischemia+FITC group,Sham+FITC-PGF group,and Sham+FITC group,with8 rats in each group.Immunofluorescence method was used to compare different concentrations of FITC-PGF distribution in the brain and the ratio of cells co-localized with Neu N(Neuronal Nuclei)at 3h,6h,and 9h after ischemia-reperfusion in rats.(3)24 MCAO rats were randomly divided into I/R(Ischemia/Reperfusion)+PGF group,sham group and I/R+Vehicle group.The contents of Pro,Gly and Phe in the brain were compared by high performance liquid chromatography.(4)16 MCAO rats were randomly divided into two groups.I/R+PGF group and I/R+Vehicle group,8 rats in each group,the survival rate and cerebral infarction volume of rats with ischemia-reperfusion at different times and concentrations of PGF were compared by TTC(2,3,5-triphenyltetrazolium Chloride)staining.(5)24 MCAO rats were randomly divided into I/R+Vehicle group,I/R+50mg/kg PGF,I/R+100mg/kg PGF and I/R+200mg/kg PGF group,with 8 rats in each group.Using TTC staining to compare the effect of different concentrations of PGF on infarct volume.Results:(1)Compared with the sham group,the survival rate of neurons in OGD modle with different doses of PGF was significantly higher in the OGD+200μM PGF group,OGD+400μM PGF group,OGD+600μM PGF group,and OGD+800μM PGF group,with statistical significance(p<0.05);compared with the OGD group,the survival rate of neurons in the OGD+200μM PGF group,OGD+400μM PGF group,OGD+600μM PGF group,and OGD+800μM PGF group was significantly increased,with statistical significance(p < 0.05).(2)The distribution of FITC-PGF in the brain and the co-staining rate of FITC-PGF with Neu N at different time and different concentrations in rats after ischemia-reperfusion.The Ischemia+FITC-PGF group had the highest co-staining rate with Neu N,followed by the sham+FITC-PGF group,and then the I/R+FITC group.While there was almost no costaining in Sham+FITC group,the difference was statistically significant(p<0.05).The costaining rate of FITC-PGF and Neu N after 3h,6h,9h,12 h,and 24 h of ischemia was significantly higher than that of FITC,and the difference was statistically significant(p<0.05).The specific labeling rate of FITC-PGF and the rate of FITC-PGF in the ischemianonperfusion were similar(p>0.05).The fluorescence signal of the FITC-PGF group was strong in the field of view under a 4x microscope,while the fluorescence signal of the FITC group was weak.(3)Compared with the amino acid Proline,Glycine and Phenylalanine content,the content in the I/R+PGF group was significantly higher than that in the sham group and the control group,with statistical significance(p<0.05).(4)Comparing the survival rates of the two groups of rats at different ischemia times,there was no significant difference in the survival rates of the PGF group and the control group after 3.0h,6.0h,and 9.0h of ischemia.After 12.0h and 24.0h of ischemia,the survival rates of the PGF group were higher than the survival rates of the control group..Comparing the volume of cerebral infarction between the two groups at different time of ischemia,infarction started to appear at 3.0 h of ischemia in the two groups,and there was no significant difference in the volume of cerebral infarction between the control group and the PGF group at 3 h of ischemia(p>0.05).However,the volume of cerebral infarction in the PGF group was significantly smaller than that in the control group after 6.0h,9.0h,12.0h,and 24.0h of ischemia,the difference between the two groups was significant and statistically significant(p < 0.05).Conclusions:(1)PGF can enter the brain through the blood-brain barrier and localize in specific ischemic areas,reducing neuronal mortality,reducing the volume of cerebral infarction,and playing a role in brain protection.(2)In cerebral ischemic injury,PGF can improve the permeability of the blood-brain barrier in both reperfusion and non-perfusion conditions,and is hydrolyzed into amino acids,thereby playing a role in brain protection.It can be used as a potential neuroprotective agent in the brain.Early treatment of ischemia. |
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