Study On The Protective Effect Of Panax Notoginseng Supercritical Extract Against Ischemic Brain Injury

Ischemic stroke,with high incidence,high death rate and high disability rate,is one of the most serious diseases having threaten human’s health.It is extremely significant to research novel and effective drugs and seek impactful strategies for prevention and treatment of ischemic stroke.In recent years,Panax Notoginseng and its preparations,which has function of promoting blood circulation,have been proverbially used for the prophylaxis and cure based on the understanding of the pathogenesis of blood stasis.Although the research group had discovered that it could have restrained the excessive production of intracellular calcium and exerted the neuroprotective effect on apoptosis after cerebral ischemia,the mechanism is still not specific.It is of great importance to elucidate the active targets of SFE of Panax Notoginseng and investigate its neuroprotective mechanism.Objective:To study the effect of SFE of Panax Notoginseng on cerebral ischemia-reperfusion injury on rats and glutamate damaged PC12 cells,the expression of PICK1 protein,GluR2 protein and PICK1/GluR2 fusion protein were observed,which indicated the neuroprotective effect and mechanism of SFE of Panax Notoginseng.Methods:The rat model of cerebral ischemia-reperfusion injury was constructed by using a modified Longa line plug method.The neuroprotective effect of SFE of Panax Notoginseng was studied by examining changes in brain tissue water content,cerebral infarct volume,and intracellular calcium concentration in hippocampal tissue.The expression effect of it on PICK1 protein,GluR2 protein and PICK1/GluR2 fusion protein were investigated by RT-PCR,Western blot and coimmunoprecipitation methods,respectively.The glutamate-injured PC12 cells model was established to research the neuroprotective effect of it by inspecting varieties in cell morphology,cell viability,and intracellular calcium concentration.The expression effect of it on PICK1 protein,GluR2protein and PICK1/GluR2 fusion protein were severally observed by RT-PCR,Western blot and coimmunoprecipitation methods.Molecular docking technology was applied to screen the active ingredients of SFE of Panax Notoginseng against PICK1 protein.Results:1.The protective effect of SFE of Panax Notoginseng on MCAO injure in rats（1）The brain tissue water content and the volume measurement of cerebral infarction results showed that,compared with the sham group,the brain tissue water content of the MCAO model group increased significantly（P<0.01）,and the cerebral infarction volume improved significantly（P<0.01）.Compared with the model group,brain tissue water content of SFE of Panax Notoginseng groups(1.875,3.75,7.5 g·kg^-1)were lower significantly（P<0.05,P<0.01）,and the volume of cerebral infarction were increased extremely（P<0.05,P<0.01）（2）The Fluo-3/AM fluorescence staining results indicated that,compared with the sham group,the intracellular Ca²⁺concentration in the MCAO model group augmented significantly（P<0.01）.Compared with the model group,the concentration of intracellular Ca²⁺of SFE of Panax Notoginseng groups(1.875,3.75,7.5 g·kg^-1)decreased notably（P<0.01）.（3）The RT-PCR and Western blot results indicated that,compared with the sham group,the expression of PICK1 mRNA and GluR2 mRNA in the MCAO model group were decreased significantly（P<0.01）,and the expression of PICK1 and GluR2 protein were reduced distinctly（P<0.01）.Compared with the model group,PICK1 mRNA expression was decreased evidently（P<0.01）,GluR2 mRNA expression was raised markedly（P<0.01）,PICK1 protein expression was decreased remarkably（P<0.01）and GluR2protein expression of SFE of Panax Notoginseng groups(1.875,3.75,7.5 g·kg^-1)was observaly added（P<0.01）.（4）The coimmunoprecipitation results explained that,compared with sham group,the expression of PICK1/GluR2 fusion protein in MCAO model group was increased evidently（P<0.01）.Compared with the model group,PICK1/GluR2 protein expression of SFE of Panax Notoginseng groups(1.875,3.75,7.5 g·kg^-1)was observably declined（P<0.01）.2.The protective effect of SFE of Panax Notoginseng on glutamate-damaged PC12cells（1）Inverted microscope showed that PC12 cells were mainly spindle or polygonal shape,with strong adhesion ability,and similar nerve synapses.In the Glu model group,the majority of cells became smaller,the adhesion ability greatly decreased,and the cell neurites significantly declined.Compared with the model group,the density of cells of SFE of Panax Notoginseng groups(25,50,100 mg·L^-1)groups increased,and rounder cells decreased remarkably.（2）MTT and LDH results illuminated that,compared with the normal group,the cell survival rate of the Glu model group was significantly reduced（P<0.01）,and the LDH activity was markedly increased（P<0.01）.Compared with the model group,the cell survival rate increased significantly（P<0.05,P<0.01）and the LDH activity of SFE of Panax Notoginseng groups(25,50,100 mg·L^-1)decreased significantly（P<0.05,P<0.01）and showed a dose-effect relationship in a certain extent.（3）The Hoechst 33342 staining indicated that the apoptosis of normal group was less,and the nucleus distributed homogeneous blue fluorescence.In the Glu model group,some cells were stained like fragments and showed high brightness fluorescence（P<0.01）.Compared with the model group,the nuclear pyknosis and apoptotic cells of SFE of Panax Notoginseng groups(25,50,100 mg·L^-1)decreased significantly（P<0.05,P<0.01）.（4）The Fluo-3/AM fluorescence staining results indicated that,compared with the normal group,the intracellular Ca²⁺concentration in the Glu model group augmented significantly（P<0.01）.Compared with the model group,the concentration of intracellular Ca²⁺of SFE of Panax Notoginseng groups(25,50,100 mg·L^-1)observably decreased（P<0.05,P<0.01）.（5）The RT-PCR and Western blot results indicated that,compared with the normal group,the expression of PICK1 mRNA and GluR2 mRNA in the Glu model group were significantly decreased（P<0.01）,and the expression of PICK1 and GluR2 protein were significantly reduced（P<0.01）.Compared with the model group,PICK1 mRNA expression was evidently decreased（P<0.05,P<0.01）,GluR2 mRNA expression was markedly raised（P<0.05,P<0.01）,PICK1 protein expression was remarkably decreased（P<0.05,P<0.01）and GluR2 protein expression of intracellular Ca²⁺of SFE of Panax Notoginseng groups(25,50,100 mg·L^-1)was observaly added（P<0.05,P<0.01）.There was no significant difference in the expression of PICK1 mRNA and GluR2 mRNA between the SFE+FSC231 group and the FSC231 inhibitor group（P>0.05）,and there was no remarkable difference in the expression of PICK1 and GluR2 protein between the SFE+FSC231 group and the FSC231 inhibitor group（P>0.05）.（6）The co-immunoprecipitation results explained that,compared with normal group,the expression of PICK1/GluR2 fusion protein in Glu model group was evidently increased（P<0.01）.Compared with the model group,PICK1/GluR2 protein expression of SFE of Panax Notoginseng groups(25,50,100 mg·L^-1)was observably declined（P<0.05,P<0.01）.There was no significant difference in the expression of PICK1/GluR2 fusion protein between the SFE+FSC231 group and the FSC231inhibitor group（P>0.05）.3.To screen the active ingredients of SFE of Panax Notoginseng against PICK1protein by GC-MS and Molecular Docking technologyA total of 26 components in SFE of Panax notoginseng were identified by GC-MS.Then,Six chemical components with high scores were screened by Molecular Docking technology.6 components more than the FSC231（scoring threshold-15.87）were screened.The components were in turn according to the scores:Spathulenol（-18.33）,Falcarinol（-18.01）,Telfairic acid（-17.89）,AO1（-16.07）,E,E,Z-1,3,12-Nonadecatriene-5,14-diol（-16.07）and Traumatic acid（-16.02）.The results of GC-MS identification and Molecular Docking showed that the relative contents of six chemical components of more than 1.5%were Spathulenol,Falcarinol,Telfairic acid.The hydrogen bonding and hydrophobic interaction were predominant between Spathulenol,Falcarinol,Telfairic acid and PICK1 protein.Conclusions:1.SFE of Panax Notoginseng has neuroprotective effect on both ischemia-reperfusion injury on rats and Glu-damaged PC12 cells.2.It may be related to the inhibition of the interaction between PICK1 and GluR2proteins,the down-regulation of PICK1 protein expression and the up-regulation of GluR2 protein expression.3.Spathulenol,Falcarinol and Linoleic acid are the main active components against PICK1 protein.