Exploration Of The Pharmacological Effects And Mechanism Of Action Of Pinocembrin In Promoting Therapeutic Angiogenesis In Vitro After Cerebral Ischemic Injury

Background:Stroke is the second leading cause of death worldwide and is characterized by high morbidity and mortality.Currently,less than 10%of patients have access to pharmacological treatment in clinical settings due to a single treatment approach.Therefore,the task of expanding the drug pool for ischemic stroke treatment for clinical patients is urgent.Pinocembrin(PCB)is a natural small molecule flavonoid with positive pharmacological effects against ischemic stroke and is currently in phase II clinical trials.It has been shown that PCB has beneficial therapeutic angiogenesis in the brain in MCAO rats,but its mechanism of action has not yet been elucidated.Objective:To establish a glyoxylate deprivation/reoxygenation(OGD/R)Model of human brain microvascular endothelial cells(h BMECs)to investigate the pro-angiogenic effects of PCB in vitro.Based on cellular pharmacokinetics,we investigated the targets of PCB action in h BMECs and its mechanism of promoting therapeutic angiogenesis.Methods:In vitro experiments were used h BMECs to establish OGD/R Models,which were divided into control,Model,and 20/40/80μM PCB groups.The ability of PCB to promote angiogenesis of h BMECs induced by OGD/R in vitro was investigated by Brdu staining experiment,scratching experiment and tube forming experiment.Combined with live cell imaging and laser confocal technology,the absorption and distribution patterns of PCBs in h BMECs were investigated to find the possible targets of PCB action in h BMECs.To determine whether PCB can affect mitochondrial structure and function in h BMECs,mitochondrial fluorescent probes,transmission electron microscopy techniques,JC-1 staining,ATP assay kits and NAD~+/NADH assay kits were used to probe.The mechanism of PCB in vitro angiogenesis was investigated by Western-Blot assay.Then,the MCAO SD male rat Model was used,which were grouped into Sham-operation group,Model group and PCB administration group(i.v.,5 mg/kg,7 d).The ischemic semi-dark areas of rat brain tissue were taken to verify the mechanism of promoting by Western-Blot experiments.Results:(1)PCB dose-dependently promoted the proliferation,migration and tube-forming ability of OGD/R-induced h BMECs in vitro within 20-80μM concentrations(P﹤0.05).(2)In h BMECs,the uptake of 20-80μM PCB was dose-and time-dependent;after entering the cells,the drug continuously entered the mitochondria at 5-60 min and reached equilibrium in the mitochondria at 90 min.(3)PCB in the concentration range of 20-80μM increased the number of mitochondria in h BMECs after OGD/R,restored the normal reticular structure and internal cristae structure of mitochondria,decreased the degree of swelling,dose-dependently increased the membrane potential level(P﹤0.01),and enhanced the ATP content and NAD~+/NADH ratio in h BMECs(P﹤0.05).(4)For in vitro cell experiments,the results of Western-Blot assays demonstrated that PCB(20/40/80μM)increased the expression of VEGFR2 protein by enhancing the expression levels of NAMPT and SIRT proteins and decreasing the expression levels of NOTCH/DLL4 and HES1,HEY1 proteins in the nucleus of h BMECs after OGD/R.Meanwhile,PCB enhanced the expression of VEGFA protein and its downstream effectors in h BMECs after OGD/R.(5)Through in vivo experiments,PCB(5 mg/kg)was also found to enhance the expression levels of NAMPT and SIRT proteins and decrease the expression levels of NOTCH/DLL4 and HES1,HEY1 proteins in the ischemic hemispheric cortex of MCAO rats in order to increase the expression of VEGFR2 protein.Meanwhile,PCB enhanced the expression of VEGFA protein and its downstream effectors in the cerebral ischemic hemispheric cortex of MCAO rats,and the experimental results were consistent with the in vitro experiments.Conclusion:PCB has in vitro pro-therapeutic angiogenesis pharmacological effects.It can rapidly enter h BMECs,distribute in mitochondria,and exert pharmacological effects in mitochondria,restore normal mitochondrial morphology and function.PCB also reverse-regulated NOTCH/DLL4 signaling pathway in mitochondria through NAMPT-NAD~+-SIRT1 cascade and increased VEGFR2expression.PCB promoted VEGFA protein expression,enhanced VEGFA/VEGFR2binding to form dimer and activated VEGFA downstream effectors to promote therapeutic angiogenesis.