Based On AKT/NF-κB/iNOS Pathway To Study The Mechanisms Of Shenfu Injection In The Treatment Of Vascular Dementia

ChapterⅠNetwork pharmacology investigation into the mechanisms of the Shenfu Injection on VDObjective:To establish the interaction network between Shenfu Injection（SFI）and the target of Vascular dementia（VD）by network pharmacological methods and predict the potential target and mechanism of Shenfu injection on vascular dementia.Methods:1.Collection of active ingredients and targets of SFI:The TCMSP databases were used to identify the active ingredient and targets of SFI.2.Collection of VD-related targets:Gene Cards、Drug Bank、OMIM database were used to download VD-related targets.3.Protein-protein interaction（PPI）network construction and topology analysis of PPI network:the intersection target imports String database to construct PPI network.Cytoscape3.7.2 Software topology analysis to screen core targets in PPI networks.4.GO functional enrichment and KEGG pathway enrichment analysis:DAVID and Matescape database obtained core gene enrichment analysis results and then carried out visualization processing.Results:1.17 active components of ginseng and 6 active components of aconite in SFI were obtained.Removing the repeated sequences of ginseng and aconite,105 bioactive targets were collected.Among 1551 potential VD targets.57 potential intersection targets overlapped with SFI and VD.2.The topology analysis of the PPI network shows that the network consists of 57nodes and 454 edges.High-degree targets AKT1,TNF,PTGS2,IL-1β,CASP3,JUN,PPARG,NF-κB and NOS were considered as the key targets.3.The results of GO functional analysis found 341 items of biological process（BP）,which mainly involved drug response,response to external stimuli and lipopolysaccharide,positive regulation of nitric oxide biosynthesis,and response to stress and hypoxia.44 cell components（CC）were mainly concentrated in membrane raft,presynaptic membrane component and postsynaptic membrane component.The 73items of molecular function（MF）results were mainly involved to enzyme binding,identical protein binding,and protein homologous dimerization activity.4.The enrichment analysis of the KEGG pathway showed that there were 127pathways,which were mainly enriched with lipid and atherosclerosis,AGE-RAGE signaling pathway,TNF signaling pathway,fluid shear stress,atherosclerosis,and tumor necrosis factor signaling pathway in diabetes complications.Conclusion:Based on the information of core targets and potential pathways,it can be preliminarily predicted that SFI may treat VD by regulating the targets of AKT1,TNF,NF-κB and cancer signaling pathway,PI3K-AKT and other related signaling pathways.PPI network and KEGG results enrichment analysis predicted that SFI may improve neuroinflammation,oxidative stress,apoptosis and other pathological changes in VD.ChapterⅡThe mechanism of Shenfu injection in the treatment of vascular dementia model mice through AKT/NF-κB/i NOS pathwayObjective:To observe the therapeutic effect of Shenfu injection on cognitive dysfunction of VD model mice with vascular dementia,and to explore whether the mechanisms are related to protein kinase B（Akt1）/nuclear factor kappa-B（NF-κB）/inducible nitric oxide synthase（i NOS）pathway.Methods:1.50 male Kunming mice with a weight range of 24-28g were selected and the VD mouse model was established by the bilateral common carotid artery occlusion and reperfusion method.The mice were scored after anesthesia.Randomly divided the mice into VD model group,SFI low-dose group(5m L·kg^-1),SFI medium-dose group(10m L·kg^-1)and SFI high-dose group(20m L·kg^-1),with 10 mice in each group.A sham operation control group was reserved before operation.The mice in each group were given the same feeding conditions,different doses of SFI were injected through the tail vein in each drug group,and the sham operation group and VD model group were given the same amount of normal saline for 21 consecutive days.2.Behavioristics:The Morris water maze experiment began 14 days after the mice were administrated.Record the latency time of each group of mice in the water maze navigation test and the times of crossing the target platform in the space exploration experiment.To evaluate the cognitive function of learning and memory in mice.3.Cerebral morphology:The whole brains of mice in each group were removed and made into paraffin wax or frozen section for follow-up H&E and Nissl staining to observe the changes of hippocampal neurons.4.ELISA detection of Interleukin-1（IL-1β）、Interleukin-6（IL-6）and Tumour necrosis factor-α（TNFα）in the serum of mice in each group.5.Griess assay:The content of nitric oxide in the serum was detected.6.Gene expression analysis:RT-PCR detection of Akt1,NF-κB and i NOS m RNA expression changes in the hippocampus tissue of mice in each group.7.Protein level analysis:The expression of Akt1、p-Akt1、NF-κB、p-NF-κB and i NOS in the hippocampus tissue of mice in the sham group,VD model group and SFI low,medium and high groups were measured by western blot.Results:1.Compared with the sham operation group,the Morris water maze navigation time of mice in VD model group was significantly increased,and the number of finding the platform was significantly decreased（P<0.01）.Compared with the VD model group,the positioning navigation time of SFI groups decreased,and the time and number of target platform stay increased significantly（P<0.01）.2.HE staining results revealed that the cells in the hippocampal CA1 region of mice in VD model group were sparsely arranged,structural evacuation,and cell layer becomes thinner.In the SFI group,the nucleus of neurons in the hippocampal CA1region showed light coloring,shallow shrinkage,less polygon of cell body,and certain thickening of cell layer.3.Nissl staining results showed that the number of neurons in the hippocampal CA1 area of VD model group mice decreased,the cells contracted,the cytoplasm was deeply stained,the Nissl bodies decreased,and the number of positive cells decreased.In contrast,the SFI groups at different doses showed an increase in the number of hippocampal CA1 neurons,with most cells having a clear and normal morphology,and an increase in the number of Nissl bodies,with the middle dose group being the most significant.4.Compared with the sham operation group,the serum NO content of VD model group mice increased significantly.Serum NO content of mice was decreased by the low,medium and high doses of SFI（P<0.01）.5.The IL-1β,IL-6,and TNFαlevels in the serum of VD model group mice were significantly higher than those in the sham operation group.While the SFI groups significantly decreased the serum IL-1β,IL-6 and TNF-αcontents in VD model mice（P<0.01）.especially in high-dose group.6.The m RNA expression of NF-κB and i NOS（P<0.05）in the hippocampus of VD model group mice was significantly increased,while the m RNA expression of NF-κB and i NOS（P<0.05）in the hippocampus tissue of SFI-treated VD mice decreased to varying degrees.7.Western blot results showed that compared with the sham operation group,the protein expression of p-Akt1,NF-κB,p-NF-κB,and i NOS in the hippocampus of VD model group mice significantly increased,while the SFI low,medium,and high dose groups reduced the protein expression of p-Akt1,NF-κB,p-NF-κB,and i NOS（P<0.05）in the hippocampus tissue of VD mice.Conclusion:1.Each doses of SFI can improve the learning and memory ability of VD mice,and improved the pathological injury of the hippocampus of VD model mice in different degrees,indicating that SFI may improve the cognitive dysfunction of VD mice and protect the brain tissue of VD mice.2.SFI significantly decreased the levels of serum inflammatory cytokines TNF-α,IL-1βand IL-6 in VD mice,suggesting that the protective effect of SFI on VD mice may be related to its improvement of nervous system inflammation.3.The protective effect of SFI on VD mice may be related to the decrease of gene and protein expression of p-Akt1,p-NF-κB and i NOS in the hippocampus of mice,and the decrease of the serum NO content in mice.ChapterⅢThe mechanism of Shenfu Injection to protect nerve and glial cells damaged by oxygen-glucose deprivation through AKT/NF-κB/i NOS pathwayObjective:To explore the protective effects of SFI on neurons and glial cells induced by oxygen-glucose deprivation（OGD）in vitro VD model,to evaluate whether it involves Akt1/NF-κB/i NOS pathway.Methods:1.Suckling mice within 24h were sterilized and removed the brain tissue.Isolate the hippocampus and the cerebral cortex.Cut up the hippocampus and cerebral cortex for digestion.Centrifugally suspended cell blast suspension plate.2.Hippocampal neurons and glial cells were identified by immunofluorescence（MAP2 for neurons,GFAP for astrocytes,DAPI for nuclei）.The cell purity was determined to be more than 90%for follow-up experiments.3.1mmol.L^-1,2mmol.L^-1,5mmol.L^-1,10mmol.L^-1 and 20mmol.L^-1 of sodium disulfite（Na₂S₂O₄）were cultured for 3h,6h,12h and 24h,respectively.Reoxygenated for 24h.The CCK8 method tests the inhibition rate and selects the appropriate Na₂S₂O₄administration concentration and time to replicate OGD model.4.SFI administration group 25μL/m L,75μL/m L,125μL/m L,175μL/m L,225μL/m L,275μL/m L pretreated cells for 3h.After 3h,cells were given 5m M Na₂S₂O₄,and SFI was incubated with Na₂S₂O₄for 12h.Then reoxygenation culture for 24h.CCK8 detect cell viability and determines the appropriate concentration of SFI.5.Griess method was used to detect the NO content in the cell culture supernatant of the control group,OGD model group and low,medium and high SFI groups.6.Gene level analysis:RT-PCR detection of Akt1,NF-κB and i NOS m RNA expression changes in the control group,OGD model group and SFI groups.7.Protein level analysis:The expression of Akt1、p-Akt1、NF-κB、p-NF-κB and i NOS in the control group,OGD model group and SFI low,medium and high groups were measured by western blot.Results:1.Immunofluorescence staining showed green fluorescence in the cytoplasm and blue fluorescence in the nucleus.Hippocampal neurons have normal cell bodies,which are fusiform,triangular or round.The neurite and interweaves with each other.The nucleus are large,round and located in the cell body.The glial cell body is star-shaped and large,which sends out long and branch processes,and the nucleus is round in the cytoplasm.The purity of primary cells was above 90%by calculation.2.Na₂S₂O₄ in the concentration range of 1-20m M was cultured with cells for 24h,12h,6h,and 3h.CCK8 results showed that different times and concentrations of Na₂S₂O₄ culture could reduce the cell activity,and the decrease was most significant when the concentration was 20m M culture for 24h.When the final concentration was5m M and the time was 12h,the cell activity was closest to IC50.Therefore,take 5m M Na₂S₂O₄ culture for 12h and the reoxygenation of resugar for 24h to induce OGD.3.Different concentrations of SFI were pretreated for 3h before OGD injury to detect cell activity.The results showed that different concentrations of SFI could increase the cell survival rate under OGD injury,but cell viability decreased at275μL/m L of SFI,suggesting that excessive concentrations above 275μL/m L may cause cytotoxicity.Therefore,appropriate three SFI concentrations of 75μL/m L,125μL/m L and 175μL/m L were selected as low,medium and high dose groups for subsequent experiments.4.Compared with the control group,the content of NO in the supernatant of OGD group was significantly increased.While the content of NO in cell supernatant was decreased in low,medium and high dose SFI groups（P<0.01）.5.Compared with the control group,the expressions of Akt1,NF-κB and i NOS（P<0.05）m RNA in OGD cells were significantly increased,while in SFI group were decreased in different extents.6.Compared with the control group,the expression of p-Akt1,NF-κB,p-NF-κB and i NOS（P<0.05）protein in OGD cells was significantly increased.The protein expressions were decreased in low-dose,medium-dose and high-dose SFI groups.Conclusion:1.The purity of hippocampal neurons and glial cells identified by immunofluorescence was above 90%,and the cells were in good condition,indicating that the culture of primary hippocampal neurons and glial cells was successfully completed in this experiment.2.The cell activity was closest to IC50 when the final concentration of Na₂S₂O₄was 5m M and the time of co-culture with cells was 12h.Therefore,the experiment selected 5m M Na₂S₂O₄ co-culture with cells for 12h and then reglucosed and reoxygenated for 24h to reproduce OGD damage as the in vitro VD model.3.SFI concentrations of 75μL/m L,125μL/m L and 175μL/m L all increased the cell survival rate of OGD-induced injured neurons and glial cells,and had no toxic effect on cells,suggesting that SFI had protective effects on OGD-induced injured neurons and glial cells.4.The protective effect of SFI on OGD-induced neurons and glial cells may be related to the decrease of gene and protein expression of Akt1,p-Akt1,NF-κB,p-NF-κB and i NOS,and the decrease of NO content in the supernatant of cells.