Study On Anti-oxidative Avtivity And Protective Effect Of Scutellarin Methyl Ester-4’-Dipeptide Conjugates For The Treatment Of Hypoxic-Ishemic Encephalopathy(HIE)in Neonatal Rats

Objective:The biological activities of a series of designed and synthesized scutellarin methyl ester-4’-dipeptide conjugates were studied in order to explore whether the above derivatives have better antioxidant activity than scutellarin（Scu）and the protective effect against hypoxic-ischemic encephalopathy in neonatal rats,so as to provide theoretical and practical basis for the further study of structural modification of Scu.Methods:（1）In anti-oxidative assay:Antioxidant activity of target compounds were evaluated by using H₂O₂-induced PC12 cells（rat adrenal pheochromocytoma cells）oxidative damage model.Then the compounds with better activity were screened by the leakage rate of lactate dehydrogenase（LDH）and the content of malondialdehyde（MDA）in cells.The pharmacological activity of the selected compounds was further determined by Hoecst33342 staining,the content of reactive oxygen species（ROS）,the（MMP）,cell cycle of mitochondrial membrane potential,Annexin V-FITC staining and the expression of Caspase-3,Bax and Bcl2proteins,and further confirmed whether the pharmacological activity of the tested compounds was significantly better than that of Scutellarin.（2）Studying on the protective effects against hypoxic-Ischemic encephalopathy in neonatal Rats:300seven-day neonatal SD rats were randomly divided into 5 groups:Sham group,HIE group,（HIE+Scu）group（2.8mg/kg）,（HIE+1a）group（1.3mg/kg）,（HIE+1a）group（4.4mg/kg）.TTC staining,neurobehavioral score,brain water content,HE staining and the expression of APP and BACE-1 protein were measuerd at 48 hours after HIE modeling.Nissl staining was used to observe the brain tissue loss area as an index at 4weeks after HIE modeling,to evaluate whether the protective effect of the tested compound against neonatal hypoxic-ischemic encephalopathy was better than that of Scutellarin.Results:（1）The preliminary results of antioxidant activity showed that compound 1a,1b,1c and 2d could significantly increase the survival rate of PC12cells compared with Scu group and further results showed that compound 1a could significantly reduce the content of LDH and intracellular MDA in cell supernatant compared with Scu group.The further results showed that compared with Scu group,compound 1a could significantly reduce apoptotic bodies,reduce the content of ROS,the level of MMP and the rate of apoptosis,it could increase the ratio of Bcl-2/Bax protein,and allieviate the activity of Caspase-3 protein.（2）The protective effect of compound 1a on neonatal hypoxic-ischemic encephalopathy showed that,compared with Scu group,compound 1a could significantly reduce the volume of cerebral infarction,improve the degree of neurological impairment in HIE model neonatal rats,reduce brain water content,brain water content,brain tissue loss area and brain tissue APP,BACE-1 protein expression level.Conclusion:In this study,the antioxidant activity of twelve compounds was studied by PC12 cell oxidative damage model in vitro,and the antioxidant activity of compound 1a was significantly better than that of Scu in vitro.Then the HIE model was established to study protection against hypoxic-ischemic encephalopathy of compound 1a in neonatal rats.The results showed that the protective effect of compound 1a against neonatal hypoxic-ischemic encephalopathy was significantly better than that of Scu,suggesting that the modification of scutellarin with carbamate dipeptide strategy could significantly enhance the protective effect of compound 1a on hypoxic-ischemic encephalopathy and reduce brain damage.This will lay a theoretical and practical foundation for the development of scutellarin derivatives for the treatment of hypoxic-ischemic encephalopathy.