| Background:Sepsis is one of the leading causes of death in critically ill patients.Although the guidelines of the "Surviving Sepsis Campaign" recommend a series of classic treatment strategies for sepsis,there are still problems of low cure rate and high morbidity and mortality.Moreover,the mortality of fungal sepsis is often higher than that of bacterial sepsis,seriously endangering the clinical prognosis of patients.Sepsis associated encephalopathy(SAE)refers to the cognitive dysfunction of survivors of sepsis,which is directly related to higher mortality,and the long-term cognitive dysfunction and physical functional disability pose new challenges to nursing needs and social health care.It is worth noting that sepsis can change the diversity of intestinal microorganisms and the content of their metabolites short chain fatty acids(SCFAs).In recent years,these changes may be related to cognitive dysfunction in survivors of sepsis have attracted more and more attention.Since gut microbiota and metabolites play an important role in the occurrence and development of sepsis and SAE,interventions by regulating gut microbiota and SCFAs are of great significance for improving sepsis and sepsis-induced cerebral dysfunction.Mitogen activated protein kinase(MAPK)can be activated by different extracellular stimuli and is involved in the regulation of cell growth,differentiation and inflammatory response.Extracellular signal regulated kinase(ERK)includes ERK1 and ERK2.ERK1/2 is involved in a variety of cognitive processes such as emotion,spatial memory,social behavior and so on.However,the role and mechanism of SCFAs in sepsis and related brain injury through MAPK/ERK pathway are still unclear.Therefore,this study aims to explore the potential mechanism of SCFAs on sepsis-associated encephalopathy at the animal and cellular levels,and to provide a theoretical basis for finding effective treatment methods for sepsis-associated encephalopathy.Methods:1.In animal experiments,SAE mouse model was established by CLP in C57BL/6j mice pretreated with SCFAs mixture and ERK pathway inhibitor SCH772984.On the7 th day after modeling,open field test and water maze test were performed.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of pro-inflammatory and anti-inflammatory cytokines in hippocampal tissue homogenate.The ultrastructure of hippocampus was observed by HE staining.The changes of tight junction proteins Occludin and ZO-1 in blood brain barrier(BBB)were detected by immunohistochemistry.qPCR was used to detect the transcriptional changes of MAP2,SYP and PSD95 in hippocampus.Finally,Western Blot was used to determine the changes in the expression of CD-11 b,Iba-1,p38,P-p38,ERK,and P-ERK proteins to determine whether SCFAs had protective effect on the brain of mice with SAE caused by CLP modeling.2.In cell experiments,microglia cells(BV2)and neurons(HT22)of mouse were selected as the research subjects and the microglia and neurons were damaged by LPS induced sepsis.In order to further determine whether SCFAs can play a neuroprotective role via MAPK/ERK pathway.The methods of CCK8 assay for detecting the cell viability and ELISA was used to detect the levels of inflammatory mediators in BV2 cells after treatment,then the appropriate concentration of SCFAs mixture was selected as no more than 800 u M.Subsequently,BV2 cells and HT22 cells were co-cultured and divided into control group,LPS group,LPS+SCFAs group and LPS+SCFAs+SCH772984 group.The protein expressions of p38,ERK,P-p38 and P-ERK were detected by Western Blot.qPCR was used to detect the transcription of MAP2,SYP and PSD95 in neurons.Finally,the morphological changes of microglia and neurons were detected by transmission electron microscopy to determine whether SCFAs could alleviate LPS-induced neuroinflammation and neuronal and synaptic damage by regulating the activity of MAPK/ERK pathway.Results:1.The levels of pro-inflammatory cytokines IL-1β,IL-6,and TNF-α in the hippocampus were significantly increased in the CLP-induced SAE mouse model.The activation of microglia,nuclear shrinkage and apoptosis of neuronal cell,and other ultrastructural changes,the decreased expression of BBB tight junction proteins as Occludin and ZO-1,and the decreased transcription of neuronal cytoskeletal protein MAP2 and synaptic plasticity-related proteins SYP and PSD95 are the key links in the development of sepsis-induced brain injury.2.SCFAs can significantly inhibit the activity of MAPK/ERK pathway,improve the neuroinflammatory response induced by CLP and promote the transformation of microglia from pro-inflammatory phenotype to anti-inflammatory phenotype to play an anti-inflammatory role.At the same time,SCFAs can help to maintain the integrity of BBB and improve the synaptic plasticity of neurons.Moreover,the combination of SCFAs and ERK inhibitor can produce more significant anti-inflammatory effect and brain protection.3.LPS-induced neuronal cell damage models associated with sepsis were used in cell experiments.SCFAs could significantly reduce the neuroinflammatory response of BV2 cells,inhibit the activity of MAPK/ERK pathway,and increase the transcription of MAP2,SYP and PSD95 in HT22 cells.The number of autophagosomes in the neurons was significantly reduced and the autophagy and apoptosis were improved under the electron microscope,which played a neuroprotective role.Moreover,the combination of SCFAs and ERK inhibitors has synergistic effects.Conclusion:This study confirmed that SCFAs inhibited neuroinflammation by inhibiting the excessive activation of MAPK/ERK pathway,promoting the transformation of microglia from pro-inflammatory phenotype to anti-inflammatory phenotype,maintaining BBB integrity,improving synaptic plasticity and neuronal autophagy and apoptosis at the animal and cellular levels.It also plays a protective role in sepsis-induced nerve injury,which provides a theoretical basis for the brain protection of SCFAs in patients with sepsis-associated encephalopathy. |
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