| Objective: Ischemic stroke is devastating to society.As one of the leading causes of death worldwide,its neurological disability significantly reduces the quality of life of patients compared to its lethality.There are many types of microbial communities in the human intestine,and their symbiotic relationship with humans always affects the development and maturity,energy metabolism,and immune regulation processes in the host.Many neurological diseases such as multiple sclerosis,Parkinson's disease,depression,Alzheimer's disease,and amyloid lateral sclerosis are all affected by the function and structural composition of the intestinal flora to varying degrees.Intestinal flora can regulate important processes of the central nervous system through the braingut axis mechanism,such as myelination,neurogenesis and regeneration,immune cell activation,blood-brain barrier integrity,etc.In a healthy body,the intestinal flora is interdependent in an appropriate ratio.However,after an ischemic brain injury event,the host's intestinal flora homeostasis is destroyed,and the imbalance of the flora can eventually cause a series of changes in the intestinal and brain immune cells directly related to the prognosis of stroke,thereby increasing neurological function defect.The immune response of the central nervous system plays an indispensable role in the process of brain injury repair.A variety of cells in the body,such as T lymphocytes,B lymphocytes,and microglia,are involved in the process.Short-chain fatty acids are the final product of the fermentation and metabolism of the intestinal flora using dietary fiber,which can protect the integrity of the intestinal epithelium,help reduce the immune inflammatory response of the body,and act as a bridge between the intestinal flora and the central nervous system.In recent years,researchers hope to modify the intestinal flora through different external interventions.They hope that by changing the composition of the flora,the immune response and metabolites of the intestinal flora will be affected,and finally the central system immunity can be regulated.The role of cells.Studies have pointed out that the depletion effect of antibiotics on the intestinal flora can affect the key regulators of neuroinflammatory response and reduce cerebral blood deficiency damage in mice.In healthy individuals,the immune response is in a balanced state,and the immune system should be strictly controlled.However,under pathological conditions,this balance is lost,and the immune response is over or under activated,which can lead to inflammation or infection,respectively.Trying to infiltrate the damaged brain by inflammatory factors produced by peripheral immune cells may be a reasonable way to reduce neuroinflammation and peripheral immune disorders after stroke.Interventions that usually focus solely on neuroinflammation often cost the rest of the body's immune suppression and infection.Based on this,we will apply a 4-week broad-spectrum antibiotic cocktail treatment to change the structure of rat intestinal flora and promote the recovery of neurological function in rats after stroke.This study aims to explore the internal connection and related mechanisms of intestinal flora and neurological recovery after ischemic stroke,explore the great potential of intestinal flora to improve the outcome of stroke,and provide intervention methods for intestinal flora important scientific data.Methods:1.In this experiment,we divided the rats into the following three groups: sham operation group(SHAM group),ischemic group(ISC group),ischemic infarction +five-linkage long-term broad-spectrum antibiotics treatment group(ISCAB group).We first used 16 s RNA sequencing technology to identify and analyze the flora of the rat's ileum and predict its function.2.Quantitative targeted metabolic analysis of the main target short-chain fatty acids(acetic acid,propionic acid,butyric acid,caproic acid),and analyze the correlation relationship with the flora.3.Evaluate the behavioral performance of each group of animals through the cylinder experiment,the paste removal experiment,and the balance beam experiment to observe the recovery of nerve function in each group of rats.4.Quantitatively analyze the phenotype and morphology of microglia in each group by immunofluorescence.5.Nissl staining was used to compare the influence of infarct volume post-stroke before and after the application of broad-spectrum antibiotics treatment.6.Using BDA antegrade tracing,immunofluorescence staining,and protein quantification methods to observe the axon regeneration,neurogenesis,and apoptosis of animals.7.In the study of molecular mechanism involved in the process,we first use the GEO database on NCBI to analyze the differentially expressed genes in peripheral blood through the method of biological information analysis,and use GO and KEGG pathway analysis to further analyze the enrichment of the differentially expressed proteins.Then we use RT-q PCR and ELISA to investigate the expression change of TLR2,TLR4,NF-?B,IL-10,IL-6,TNF-? and other inflammatory marker in peripheral blood and ileum tissue,to preliminary analysis of the role of NF-k B signaling pathway in regulating immune inflammation response.8.Finally,protein quantification,RT-q PCR,immunohistochemistry,Sholl analysis and immunofluorescence quantitative analysis of brain tissue are used to detect the changes in microglia function and whether other immune cells are involved in this process.To verify whether the NF-k B signaling pathway in brain tissue is regulated by antibiotics at the transcriptome level and the change in the phenotype and morphology of microglia.To detect whether other immune cells in the center(astrocytes,CD4+Foxp3+Treg lymphocytes)are involved in the process.Results: The results of bacterial population sequencing indicated that in ISC rats,Firmicutes had the highest relative abundance(67.09%±3.34%).After the use of broad-spectrum antibiotics,the Proteobacteria in the ISCAB group dominates the intestinal flora(68.13%±4.82%),and the change in flora structure is reflected in the decreasing abundance of the flora and the changing diversity of ? and ?.In the antibiotic-treated rats,some probiotics such as Lactococcus,Paracoccus Coprococcus,Bacillus,Streptococcus,Enterococcus,and Faecalibacterium increased in abundance.The results of intestinal flora metabolism showed that the content of acetate was significantly up-regulated,while the level of butyrate showed a downward trend.In the behavioral test of rats,the error rate of the balance beam was lower,the frequency of use of the upper limbs was more frequent,the voluntary movement increased,the time of sticking removal was shorter,and the contact sticking Earlier.Ni's staining showed that antibiotics significantly reduced the proportion of infarct volume.Peripheral blood GEO database life information analysis indicated that changes in peripheral blood after stroke are related to TNF-like receptor signaling pathways and positive regulation of cytokines.Following results of RT-q PCR and immunofluorescence showed that the level of inflammatory factors in the intestinal tract of the ISCAB group was reduced,and the peripheral blood pro-inflammatory factor TNF-beta was significantly downregulated.The RT-q PCR and immunofluorescence results of brain tissue showed that the use of antibiotics can reduce nerve apoptosis,promote microglia activation,change the ratio of different phenotypes of microglia in the brain,and lead to the level of inflammation in the brain tissue decline.Conclusion:1.The regulation of the intestinal flora of rats through 4 weeks of broad-spectrum drinking water combined with antibiotics reduces the number and abundance of the overall flora of rats after stroke,and promotes the production of some intestinal probiotics.The decrease in the number of pathogenic bacteria in the intestines will eventually lead to an increase in the content of short-chain fatty acids in the intestinal contents.2.Antibiotics can reduce the level of systemic inflammatory response by regulating nerve apoptosis,promote the recovery of neurological function,reduce the infarct volume,and neurogenesis and axon regeneration are not involved in this process.3.The immune cells that participate in this process and play a major role are microglia.Antibiotics can regulate the activation and inflammatory response of microglia after stroke.It promotes changes in the ratio of different phenotypes of microglia,regulates the function of microglia,affects the level of secreted cytokines,and ultimately leads to a decrease in the inflammatory response in the brain. |
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