| Background and Objective: cardiac arrest(CA)is a common acute severe disease with high mortality.Even if patients survive after recovery,there are often multiple sequelae,among which neurological function injury is the most common,and it is easy to become the burden of society and family.CA/CPR(cardiopulmonary resuscitation)may lead to hypoxic ischemic brain injury.Cognitive impairment is one of the major clinical consequences.The most common cognitive impairment is impaired memory,reduced attention,and impaired information processing ability.Therefore,the current clinical treatment of CA should not only pay attention to survival,but also pay attention to the prognosis of survivors.Ginsenoside Rg1 is a tetracyclic triterpenoid compound derived from panax notoginseng and ginseng rhizomes,which can effectively improve psychiatric disorders.The purpose of this study was to investigate whether ginsenoside Rg1 has a protective effect on cognitive impairment caused by cardiac arrest and cardiopulmonary resuscitation(CA/CPR)and its possible protective mechanism.Methods:(1)Rat CA/CPR model was established.56 SD rats were randomly divided into Sham group(n= 16)and model group(n=40).The model group was divided into CA/CPR group(n= 20)and ginsenoside-Rg1 intervention group(CA/CPR+G-Rg1 group(n= 20).Model group received anesthesia,tracheal intubation,arteriovenous catheterization,asphyxia induced cardiac arrest,cardiopulmonary resuscitation and other operations.The rats in CA/CPR+G-Rg1 group were intraperitoneally injected 40 mg/kg ginsenoside Rg1 dissolved with 10 mg/ml normal saline every day for 14 consecutive days.The CA/CPR group was also given intraperitoneal injection of the same amount of normal saline.The sham group was given the same procedure except cardiac arrest and CPR(2)In vivo and In vitro experiment.After 14 days,the learning and memory function and synaptic plasticity of the rats were evaluated using LTP(long-term potentiation)recorded by electrophysiological experiment and Morris water maze test,respectively.(3)Morphological and molecular biology experiments.The rats were killed 14 days after modeling.After anesthesia,the rats were irrigated with pre-cooled normal saline at the pulsation of the heart apex,and the left and right cerebral hemispheres of the rats were taken after spinal dislocation and decapitation.The left cerebral hemispheres of the rats were harvested,and the activation of microglia and astrocytes in the hippocampus was observed by immunohistochemical(IHC)analysis and immunofluorescence(IF)analysis under the microscope.Deoxyribonucleotide terminal transferase mediated notch end labeling(TUNEL)was used to evaluate neuronal cell apoptosis.The density of dendritic spines was evaluated by Golgi staining.On the other hand,the hippocampus in the right hemisphere was used for other molecular biology experiments.The ultrastructure of hippocampal neurons and synapses was observed by transmission electron microscopy(TEM).Glutamic acid content in hippocampus was detected by enzyme-linked immunosorbent assay(ELISA).Reverse transcription polymerase chain reaction(RTPCR)and Western blot,WB)detected glial fibrillary acidic protein(GFAP),ionized calcium-binding adapter molecule 1(Ionized calcium-binding Adapter Molecule 1),respectively.IBal1),(excitatory amino acid transporter 1,EAAT1),(excitatory amino acid transporter 2,(Excite amino acid transporter 1,ea AT1),(excitatory amino acid transporter2,EAAT2),Glutamine Synthetase(GS),Glu N2 B,Synaptotagmin-1,syt-1),postsynaptic density Protein-95(PSD-95),c AMP-responsive element binding protein(CAMPResponsive Element Binding protein),CREB),brain-derived neurotrophic factor(BDNF),interleukin-1β,Expression of m RNA and protein levels of IL-1β,tumour necrosis factor-α(TNF-α).(4)Statistical analysis: Unless otherwise stated,all data in this article are expressed as mean ± SEM.In addition to the non-normally distributed or heterogeneous variance data analyzed by Kruskal Wallis test,other data between groups were subjected to multiple comparisons by one-way analysis of variance and LSD post hoc test using SPSS version16.0 software(IBM,NY,USA).Use the Graph Pad Prism 8.0 software(Graph Pad Software,CA,USA)to convert data into various intuitive graphics.Representative preand post-tetanus traces were produced by Igor Pro software(Wave Metricsis Software,OR,USA).All images were quantitated with Image J software(Scion,MD,USA).A pvalue < 0.05 was considered statistically significant.Results:(1)There were no significant differences in the basic parameters of all groups before and after resuscitation(p>0.05).In vivo Morris water maze display: The latency of escape in CA/CPR rats was significantly longer than that in Sham group(p < 0.05),and the latency of escape in CA/CPR rats was significantly shortened by ginsenoside Rg1treatment(p < 0.05),indicating that learning and memory function of rats was impaired by CA/CPR,and ginsenoside Rg1 could alleviate behavioral disorders caused by CA/CPR.Electrophysiological display: Basal synaptic transmission and long-term enhancement on the Schaffer side of CA1 were impaired in the model group compared with the control group(p < 0.01),while ginsenoside Rg1 treatment improved partially damaged LTP in rats(p < 0.01),suggesting that ginsenoside Rg1 improved neuroplasticity induced by CA/CPR.(2)In vitro:(1)IHC&IF analysis showed that compared with Sham group,glial cell activation was significantly increased in CA/CPR group,which was manifested by enlargement of cell body and contraction of branch,increased percentage of stained areas and number of positive cells in Iba1-1(p < 0.001)and GFAP(p < 0.001).Ginsenoside Rg1 significantly decreased glial cell activation after resuscitation(p < 0.01),suggesting that ginsenoside Rg1 could alleviate the pro-inflammatory polarization of microglia and astrocytes after CA/CPR(2)TUNEL analysis showed that the apoptosis index of CA/CPR group was significantly higher than that of Sham group(p < 0.001),and ginsenoside Rg1 could significantly inhibit the widespread apoptosis of neurons induced by CA/CPR(p < 0.001).These results indicated that ginsenoside-Rg1 could reduce neuronal apoptosis induced by CA/CPR.(3)TEM showed that compared with Sham group,hippocampal neuron organelles of CA/CPR rats were reduced,nuclei were irregularly broken,and chromatin was aggregated and marginalized.Ginsenoside Rg1 significantly alleviated morphological changes related to apoptosis mediated by CA/CPR.(4)Golgi staining showed that the density of dendritic spines per micron in CA/CPR was significantly decreased compared with Sham group(p < 0.001),and these CA/CPR induced changes were significantly improved after ginsenoside Rg1 administration(p <0.001).(5)ELISA showed that compared with Sham group,hippocampal glutamate level of CA/CPR rats was significantly increased,and ginsenoside Rg1 significantly decreased glutamate content after resuscitation(p < 0.05).(6)RT-PCR&WB display,a.Compared with Sham,m RNA abundance of EAAT2,EAAT1,GS and Glu N2 B,the major regulators of glutamate signaling,were significantly decreased in the hippocampus of CA/CPR rats(p < 0.05,p < 0.05,p < 0.05,p < 0.05,p < 0.05,p < 0.05).Ginsenoside-Rg1 treatment restored the expression of these damaged mrnas mediated by CA/CPR(p< 0.05,p < 0.05,p < 0.05,p < 0.05,p < 0.05,p < 0.05,p < 0.05,p < 0.05,p < 0.05).Western blot analysis of the protein expression levels of these major regulatory factors also supported the above results,indicating that ginsenoside Rg1 improved hippocampal glutamate signaling dysfunction after CA/CPR.b.Compared with Sham group,microglia marker Iba1-1(p < 0.001)and astrocyte marker GFAP(p < 0.001)in CA/CPR group were significantly higher than those in sham group.c.Compared with Sham group,Synaptatagmin-1,PSD-95,CREB,BDNF in CA/CPR group were significantly lower than those in sham group(Synaptatagmin-1: p < 0.001;PSD-95: p < 0.001;CREB: p < 0.05;pro-BDNF: p < 0.001;m BDNF: p < 0.05),ginsenoside Rg1 significantly up-regulated these markers(Synaptatagmin-1: p < 0.001;PSD-95: p < 0.001;CREB: p < 0.05;BDNF: p < 0.01).d.Compared with Sham group,m RNA expressions of pro-inflammatory cytokines IL-1β(p < 0.05)and TNF-α(p < 0.001)were significantly up-regulated in CA/CPR group,and IL-6 was also increased(p = 0.08).Ginsenoside-Rg1 treatment down-regulated these inflammatory cytokines to Sham group(p > 0.05).Conclusions:(1)Memory loss and impaired LTP in hippocampal CA1 were observed in rats after CA/CPR,confirming that CA/CPR leads to cognitive impairment and that ginsenoside Rg1 mitigated these changes.(2)Ginsenoside Rg1 inhibited neuroinflammation by improving hippocampal glial cell activation and decreasing the expression of related pro-inflammatory cytokines after CA/CPR.(3)Ginsenoside Rg1 can improve neuroplasticity by alleviating neuronal apoptosis,dendritic spines and synaptic ultrastructural defects after CA/CPR,and up-regulate the expression of synaptogene-related proteins,which may be related to the activation of CREB-BDNF related signaling pathways.(4)Ginsenoside Rg1 inhibits glutamate-induced excitatory toxicity by improving the expression of the main regulatory factors of glutamic acid signaling,and improves cognitive function through neuroprotective effects,providing a new idea for the treatment and intervention of CA/ CPR related cognitive disorders. |
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