The Neuroprotective Effect And Mechanism Of Shenzhi Jiannao Recipe On Pseudovascular Dementia Cell Model

As one of the common types of dementia,vascular dementia(VD)is closely related to cerebrovascular disease,with progressive memory decline,cognitive dysfunction and emotional and other mental changes as the main clinical manifestations.The pathological mechanism of VD is closely related to excitatory amino acid toxicity,synaptic plasticity,calcium overload,oxidative stress and apoptosis.Early prevention,early detection and early treatment of VD can significantly improve the survival rate and quality of life of patients,and reduce mortality.Based on " toxin damaging brain collaterals" TCM innovative etiology and pathogenesis theory and combined with many years of clinical experience,we established Shenzhi Jiannao prescription(SZJN),which has the effect of detoxication and dredging collaterals.The experimental results in vivo showed that SZJN could improve the cognitive function in VD rats,repair the damaged hippocampal neurons,and play a neuroprotective role by regulating the endocytosis of NMDA receptor mediated by clathrin.However,most of the studies only evaluate the effects of SZJN against VD from a single target or pathway,and do not fully explain its pharmacodynamic mechanism.Besides,SZJN has not been further analyzed and verified by in vitro cell experiments,and its molecular mechanism is worthy of further exploration and researchObjective:Based on the theory of " toxin damaging brain collaterals " of VD,(1)to explore the pharmacological effect and molecular mechanism of SZJN in treating VD through network pharmacology;(2)through the experiment in vitro,to explore the effect of SZJN on the proliferation and toxicity of PC12 cells;(3)to study the effect of SZJN on the cell cycle,apoptosis and intracellular Ca2+ and ROS/Superoxide of PC 12 cells injured by glutamate.To explore the neuroprotective effect of SZJN on VD cell model,verify the therapeutic effectiveness of SZJN and the reliability of network pharmacology results.(4)to study the effect of SZJN on the expression of clathrin,RAB5B and NMDAR1 in PC 12 cells injured by glutamate.To explore its influence on the endocytosis of NMDA receptors mediated by clathrin and its potential mechanism and verify the therapeutic effectiveness of SZJN.Finally,to verify the reliability of network pharmacology results.Methods:Part 1:Based on multiple databases,the blood chemical components and targets of SZJN were retrieved.VD targets were collected by different databases,and the therapeutic targets of SZJN were obtained by integrating the data.The network diagram of SZJN-active compound-treatment target was constructed by using Cytoscape 3.7.1,and the PPI network was constructed by using STRING database.Moreover,GO function and KEGG pathway enrichment analysis were carried out by using David database.Part 2:The injury of PC 12 cells induced by glutamate was used as VD cell model.CCK-8 method and IncuCyte dynamic cell imaging technology were used to determine the appropriate dose of glutamate for modeling and the low,medium and high dose of SZJN.The proliferation and toxicity of VD cells were detected and morphological observation was carried out.Part 3:Glutamate induced PC 12 cell injury was used as the VD cell model.SZJN with different doses of low,medium and high and MH cultured VD cell model respectively.Flow cytometry was used to detect cell cycle and apoptosis.Fluo-4 AM was used to detect intracellular Ca2+and ROS/Superoxide kit was used to detect intracellular ROS and superoxide.qRT-PCR was used to detect the relative expression of caspase-3 mRNAPart 4:Glutamate induced PC 12 cell injury was used as the VD cell model.Different doses of SZJN and MH were used to culture VD cell model.Immunofluorescence and Western blot techniques were used to detect the distribution and protein expression of clathrin,NMDAR1 and RAB5B,respectively.qRT-PCR was used to detect the mRNA expression of clathrin and NMDAR1.Results:Part 1:The network pharmacology computer prediction results:(1)we found 3 single herbs,18 active compounds and 154 therapeutic targets in the network diagram of SZJN-active compounds-treatment targets.Among them,the top three active compounds were Paeoniflorin,Albiflorin and Neomangiferin,and the targets with highest degree values were FGF1 and FGF2.(2)PPI network containd 154 target proteins,and the key proteins were INS?ALB?AKT1?CASP3?JUN?PTGS2,etc.(3)There were 4960 items in GO enrichment analysis,including 4176 items related to biological process,306 items related to cell composition and 478 items related to molecular function.(4)There were 30 pathways involved in the enrichment analysis of KEGG,including the MAPK signaling pathway,HIF-1 signaling pathway,apoptosis,interaction pathway of neuroactive ligand receptor and calcium signaling pathway.Part 2:The results of experiments in vitro showed that:(1)drug intervention dose screening:the suitable dosage of glutamate was 22.5 mM,and the suitable dosage of SZJN was 0.05,0.1 and 0.2 mg/mL.The appropriate intervention dose of MH was 10 ?M.(2)Cell morphology:normal PC 12 cells were irregular long fusiform with complete morphology,obvious protrusion and firm adherence;PC 12 cells cultured with glutamate were round,with incomplete structure and shrunk cell body.The protuberances became shorter,decreased and disappeared,and the cell attachment was not firm.With the increase of time,the number of dead cells increased significantly,and the cell death rate increased significantly.Compared with the model group,cells in SZJN-L,SZJN-M and SZJN-H group were relatively complete,with a small number of protrusions and synaptic connection in some cells.Moreover,the cell adherent wall was stronger;the number of dead cells was not obvious,and the cell death rate was not significantly increased.The cell morphology of MH group was slightly improved compared with that of the model group.(3)CCK-8 proliferation experiment:glutamate could significantly reduce the survival rate of PC 12 cells and inhibit cell proliferation(P<0.05);SZJN-L,SZJN-M and SZJN-H and MH could significantly improve the survival rate of PC 12 cells and promote cell proliferation(P<0.01).(4)IncuCyte proliferation experiment:glutamate could continuously increase the total green fluorescent area and the number of dead cells;the death rate increased significantly and the cell viability continued to decline;SZJN could effectively reduce the green fluorescent area,the number of dead cells and the death rate,and the cell viability showed an upward trend.The results of MH were similar to those of glutamate.(5)CFSE proliferation experiment:glutamate could significantly increase the CFSE average fluorescence intensity(P<0.01);the average fluorescence intensity of all doses of SZJN and MH decreased,but there was no significant difference(P>0.05).Part 3:Experiments in vitro results showed that:(1)cell cycle:glutamate could significantly increase the proportion of S-phase cells(P<0.01),SZJN-L,SZJN-M and SZJN-H and MH could significantly reduce the proportion of S-phase cells(P<0.01).(2)Apoptosis:glutamate could significantly increase the apoptosis rate(P<0.01).SZJN-L,SZJN-M and SZJN-H and MH could significantly reduce apoptosis and cytotoxicity(P<0.01).(3)Ca2+ and ROS/Superoxide:glutamate could significantly increase the levels of Ca2+ and ROS/Superoxide(P<0.01).The levels of Ca2+ and ROS/Superoxide in SZJN-L,SZJN-M and SZJN-H group and MH group were lower than those in the model group(P<0.05,P<0.01).(4)qRT-PCR:glutamate could significantly increase the expression level of caspase-3 mRNA(P<0.01).SZJN could significantly reduce the expression level of caspase-3 mRNA(P<0.01).Part 4:Experiments in vitro results showed that:(1)Immunofluorescence:clathrin was mainly expressed in cell membrane and cytoplasm;RAB5B was mainly expressed in early endosomes in cell membrane and cytoplasm;NMDAR1 was mainly expressed in cell membrane and synapse.Glutamate could reduce the expression of clathrin and RAB5B(P<0.01,P<0.01),increase the expression level of NMDAR1(P<0.01).SZJN could up regulate the expression level of clathrin and RAB5B(P<0.05,P<0.05),down regulate the level of NMDAR1(P<0.05).(2)qRT-PCR:glutamate could significantly reduce the expression of clathrin mRNA(P<0.01),and increase the expression level of NMDAR1 mRNA(P<0.01).SZJN could significantly increase the relative expression of clathrin and significantly reduce the expression of NMDAR1(P<0.01).(3)Western blot:glutamate could inhibit the expression of clathrin and RAB5B,and significantly increase the expression level of NMDAR1(P<0.01).SZJN could significantly up regulate the expression level of clathrin and RAB5B,and down regulate the level of NMDAR1(P<0.01).Conclusion:(1)The target and pathway mechanism of SZJN in the treatment of VD are multi angle,multi-path and multi link.In this study,we found that the predicted therapeutic targets of SZJN are mostly related to blood vessel,neuroprotection and synaptic plasticity regulation.The interaction between the targets and pathways suggested that they might be the key to the treatment of VD by SZJN.It provided a new idea for further study and analysis of other specific mechanis:ms of SZJN in the treatment of VD.(2)Appropriate concentration of glutamate could significantly promote the proliferation of PC 12 cells,but high concentration of glutamate would produce cytotoxicity,leading to cell death.Low,medium and high doses of SZJN could obviously promote the proliferation of PC 12 cells injured by glutamate,improve the cell survival rate and the phase object confluence,as well as reduce the green object count and the cytotoxic damage.They had a certain repair effect on cell morphology,and improved the state of cell adhesion(3)SZJN could effectively inhibit cell cycle arrest and down-regulate the expression of caspase-3 to reduce cell apoptosis.It reduced the concentration of free calcium ion in cells,prevented calcium influx and the further increase of glutamate toxicity.It could promote the clear of ROS/Superoxide and reduce oxidative stress damage.SZJN plays a significant neuroprotective role by affecting multiple pathways.It also verified the effectiveness of SZJN in treating VD and the reliability of network pharmacology computer prediction results(4)The accumulation of excitotoxicity could be caused by the abnormal endocytosis of NMDA receptor mediated by clathrin.SZJN could improve the endocytosis of NMDA receptors mediated by clathrin in PC12 cells injured by glutamate and reduce the toxicity of neuroexcitatory amino acids.The potential mechanism of its neuroprotection may be related to up regulating the expression of clathrin and RAB5B and promoting the endocytosis of NMDA receptor mediated by clathrin,which verified the effectiveness of SZJN in treating VD and the research results of network pharmacology.