| Stroke is one of the major causes of disability and death in humans worldwide,and ischemic stroke is the most common subtypes.However,thrombolysis as an effective treatment is limited by the treatment window.In contrast,the repair stage can provide a treatment window of up to several months.The repair and plasticity of brain tissue after stroke include neurogenesis,axonal regeneration,and remyelination,which are closely related to the behavioral recovery.Therefore,to indicate an effective treatment method may enhance the plasticity of brain tissue after stroke and further promote the functional recovery after stroke.Gut microbiome influences neurophysiology and behavior,and gut bacteria may influence the central nervous system(CNS)function by regulating host metabolites.Circulating metabolites can enter the CNS through the blood brain barrier(BBB)and directly affect neural activity.Gut nutrition support has been applied in patients with cerebral stroke.Long-term gut nutrition can shape the intestinal microbiome community and affect the gut immune of the host,which may exert a positive role in the prognosis of cerebral stroke.The High fiber diet(HFD),one of the most widely used gut nutrition interventions at present,has been fully proved to be beneficial in clinical trials of hypertension,diabetes,inflammatory bowel disease(IBD)and other diseases.HFD can promote the growth of probiotics,is conducive to improve the gastrointestinal environment and maintain the balance of the gut microbiome.Two of the most important probiotics,lactobacillus,and bifidobacterium,have been shown in clinical research that could reduce anxiety and depression,ameliorate symptoms in Autism,multiple sclerosis(MS),Alzheimer's Disease(AD)and Parkinson's Disease(PD).The HFD is fermented by the gut microbiome to produce short-chain fatty acids(SCFA).SCFA as a bridge between microbiome and immunity to mediate the occurrence and development of diseases,such as MS,AD,PD.Studies have shown that SCFAs could regulate CNS immunity,and directly promote the differentiation of T cells into Tregs or DCS.These effects are mediated by the receptors of SCFAs G protein-coupled receptor(GPCR)and/or histone deacetylase(HDAC)in the host.Therefore,we applied HFD,gut microbiome transplantation,SCFAs drinking water and probiotics intervention to ischemic rats to explore the effects of the gut microbiome and its metabolite on neurogenesis,apoptosis and behavioral function during chronic recovery after ischemia.Methods: 1.In this study,we use 16 s r RNA method to analyze intestinal contents of SHAM group,ISC group,ISC+HFD group,ISC+SCFAs group,ISC+PB group,ISC+TRANS group.2.With use of SCFAs targeting metabolomics,we quantitative analyse the level of SCFAs of cecum contents in each group.3.Animal behavioral performance was evaluated by tapered/ledged beam-walking test,cylinder test,and sticky label test.4.The phenotype and morphology of microglia were analyzed by immunofluorescence,immunohistochemistry and flow cytometry.5.Neurogenesis and apoptosis were detected by immunofluorescence,immunohistochemistry and flow cytometry.6,The expressions of inflammatory cytokines of microglia and nervous regeneration factor in SVZ were screened using PCRArray technique,the expressions of the related signaling pathway molecules such as Toll-like receptor-2(TLR2),TLR4,nuclear factor kappa-B(NF-kB),Ras,c AMP-response element-binding protein(CREB),p-CREB,extracellular signal-regulated kinase 1/2(ERK1/2),p-ERK1/2,P38,P-p38,c-Jun N-terminal kinase(JNK),p-JNK were validated by western blot.7,In in vitro experiments,the expressions of TLR2,TLR4,NF-kB were measured in microglia with the use of western blot after SCFAs intervention to analyze the activation of NF-kB signaling pathway.Then ERK1/2 and JNK pathway inhibitor were added and RT-q PCR was used to detect the expression of inflammatory factors,as well as the protein level of ERK1/2 and JNK pathways with western blot to explore the role of Mitogen-activated protein kinase(MAPK)pathway in regulating the inflammatory response of microglia.8.Finally,the supernatant of microglia was used for the conditioned culture of neural stem cells(NSCs).The concentration of brain-derived neurotrophic factor(BDNF)and Neurotrophins-3(NT3)protein in the supernatant of microglia cells was detected by enzyme-linked immunosorbent assay(ELISA).Subsequently,neurogenesis and differentiation were detected by immunofluorescence.Results: 1.After the application of HFD,Lactobacillus was significantly up-regulated,accounting for more than 90% of the bacterias,the proportion of Proteobacteria and Bacteroidetes was up-regulated while that of Firmicutes was down-regulated in the phylum level.Probiotics such as Lactobacillus and Bifidobacterium were significantly increased in ISC+HFD group.In the ISC+PB group,Bifidobacteria,Lactobacillus,and Streptococcus were orally administered,16 s results showed that Lactobacillus and Streptococcus increased significantly compared with the ISC group.The proportion of Clostridium,Prevotella,and Ruminococcaceae increased significantly.The microbiome composition of ISC rats was similar to that of ISC+PB group rats,while that of ISC+TRANS and ISC+HFD group rats were similar.2,Combined with the metabolic results,levels of SCFAs in the ISC+TRANS group were not significantly up-regulated,while that of ISC+SCFAs and ISC+PB group rats were significantly up-regulated.3,Behavioral results showed that the application of SCFAs and probiotics significantly improved the motor and sensory function recovery after stroke.4,Flow cytometry results showed that SCFAs promoted the activation of microglia,and the proportion and number of M2-type microglia were increased significantly,however,the morphology of microglial cells was not significantly changed.5,SCFAs increased the proportion and number of Nestin+ cells and promoted the neurogenesis in SVZ,and have no significant effect on nervous apoptosis.6,TLR and neurogenesis signaling PCR array results showed that the expression of related genes in the MAPK signaling pathway in microglia were significantly up-regulated after the application of SCFAs,and KEGG analysis showed that the expression of IL1R1,TNF,MAP2K3,JUN,MAP3K1,and TRAF6 genes were significantly up-regulated,neurogenic molecules such as BDNF,NTF3,DCX and Olig2 were up-regulated in SVZ.7,SCFAs inhibited NF-kB signaling pathway in microglia,SCFAs induce the production of cytokine and,these processes are mediated by the JNK signaling pathway but independent on the mediation of ERK1/2.8.In in vitro experiments showed that SCFAs promoted neurogenesis by promoting secretion of BDNF and NT3 by microglia.Conclusion: 1.After HFD treatment in ischemic rats,probiotics were up-regulated in the intestine and levels of SCFAs significantly increased.2.SCFAs promotes behavioral function recovery by promoting neurogenesis rather than regulating apoptosis.3.SCFAs promotes the activation and inflammation of microglia.4.SCFAs inhibited NF-kB signaling pathway in microglia,SCFAs induce the production of cytokine and,these processes are mediated by the JNK signaling pathway but independent on the mediation of ERK1/2. |
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