Study On The Effect And Mechanism Of Liquidambar-Glycyrrhiza And Its Effective Components Against Ischemic Stroke

Objective:Based on the analysis of the anti-ischemic stroke(IS)effect of LipuidambariGlycyrrhiza,combined with the nano-preparation technology,it was further clarified that the effective component of Lipuidambaris-Glycyrrhizae,namely,Betulonic acid-Glycyrrhetinic acid nanoparticles(B A-GA NPs),was the material basis for the treatment of ischemic stroke,and further explore the possible molecular mechanisms of BA-GA NPs in the treatment of ischemic stroke.Methods:1.Middle cerebral artery occlusion(MCAO)was used to establish focal ischemic stroke model of ICR mice.Bilateral common carotid occlusion(BCCAO)was used to construct a gerbil model of transient global ischemic stroke.The experimental animals were randomly divided into Sham group(Sham group),model group(MCAO/BCCAO group),lg/kg LLT-GC group and 5g/kg LLT-GC group.TTC staining was used to evaluate cerebral infarction volume,behavioral score was used to evaluate neural function,Morris water maze test was used to evaluate learning and memory ability of gerbils,immunohistochemical staining and Nissl staining were used to observe the changes in the number of immune-activated cells of NeuN and Nissl bodies in hippocampal CA1 region of mice and gerbils after ischemia reperfusion.2.Nanoparticles(NPs)were infused with Betulonic acid and Glycyrrhetinic acid(1:1 combination)using emulsified solvent evaporation method.After that,high performance liquid chromatography was used to determine the content of nanoparticles(BA-GA NPs)and in vitro slow-release performance.Malvern Particle Analyzer was used to measure the average particle size,Zeta potential and stability of nanoparticles,and Scanning Electron Microscopy was used to observe the morphological characteristics of nanoparticles.3.Intraperitoneal injection of BA-GA NPs after ischemic stroke was performed in mice and gerbils respectively,and TTC staining was used to observe and compare the differences in cerebral infarction volume among the groups.Neurobehavioral scores were used to observe the recovery of nerve function,immunohistochemical staining(NeuN,Iba-1,GFAP)and Nissl staining to observe neuronal injury and glial cell activation.Western Blot assay was used to detect the expression levels of inflammatory proteins,MEK/ERK and cAMP/PKA/CREB signaling pathways in the ischemic hippocampus of mice and gerbils.Results:1.LLT-GC can significantly reduce the volume of cerebral infarction induced by focal ischemic stroke in mice,reduce the death of hippocampal CA1 region and cortical neurons,promote neurological rehabilitation,and improve the symptoms of neurological impairment.In addition,LLT-GC can significantly reduce the death of neurons in hippocampal CA1 region of gerbils,promote neural rehabilitation,and improve learning and memory ability.2.The characterization results show that BA-GA NPs is spherical with a particle size of 243.6nm and a surface charge of-1.94mV.Stability test and in vitro sustained release test show that BA-GA NPs has good stability and slow release characteristics.3.BA-GA NPs intervention can significantly reduce the volume of cerebral infarction in mice after MCAO modeling,reduce neuronal defects,promote the recovery of neural function,and inhibit the expression of MEK/ERK signaling pathway proteins.BA-GA NPs intervention can significantly reduce NeuN cell death after BCCAO modeling,reduce the activation of Iba-1 and GFAP,improve learning and memory and motor coordination in gerbils,and reduce the expression of pro-inflammatory factors TNF-α,IL-1β,NF-κB and MEK/ERK signaling pathway proteins.The expression of cAMP/PKA/CREB signaling pathway was activated.Conclusion:1.LLT-GC had neuroprotective effects on focal ischemic stroke and transient global ischemic stroke.2.BA-GA NPs prepared by emulsifying solvent volatilization have good stability and slow release characteristics.3.BA-GA NPs play a neuroprotective role in ischemic stroke through multiple pathways and multiple targets,among which MEK/ERK and cAMP/PKA/CREB signaling pathways may be the potential mechanism of BA-GA NPs in the treatment of CIS.