| BackgroundSepsis an organ dysfunction caused by a host’s malfunctioning response to infection.A lot of inflammatory mediators release,oxidative stress,mitochondrial dysfunction,calcium imbalance,cell apoptosis caused by microglia and astrocyte dysfunction,neuronal dysfunction,neurotransmitter disorder,blood-brain barrier damage when sepsis occurs.This leads to neurological dysfunction.Microglia and astrocytes are the main immune cells in central nervous system.The inflammatory factors in the brain are mainly derived from microglia.Inflammatory factors will inhibit nerve repair and aggravate it damage.Astrocytes and microglia secrete a variety of cytokines and inflammatory mediators to regulate the inflammatory response.Astrocytes play multiple roles in inflammation in the central nervous system.It not only restrict peripheral immune cells from entering the central nervous system,but also produce neurotrophic factors which promote tissue repair.In addition,astrocytes can promote neurodegeneration and inflammation by recruiting surrounding inflammatory cells and activating microglias in the central nervous system.Non-coding RNA is a type of RNA molecules which cannot encode proteins.MicroRNAs(miRNAs)and long non-coding RNAs(Long non-coding RNA,lncRNAIncRNAs)have tissue specificity and expression timing,which can directly or indirectly regulate RNA and affect the function of genes and proteins.Then regulate the occurrence and development of diseases.miRNA is highly expressed in central nervous system.It not only plays an important role in the development and plasticity in the nervous system,but is also closely related to the occurrence and development of diseases related to the nervous system.Current research shows that miRNAs play an important role in central nervous system diseases regulatory,such as Alzheimer’s disease,Parkinson’s syndrome,and multiple sclerosis.IncRNA was originally considered to be the "noise" in genome transcription.It is a by-product of RNA polymerase Ⅱ transcription and no biological function.However,recent studies have shown that IncRNA regulation is abnormal in many diseases.With the deepening of research,it is found that IncRNA is widely involved in transcription level,apparent modification level and post-transcription level.And its function is more complicated than miRNA and protein.The tissue specificity of IncRNA is more prominent in the central nervous system,and it occupies a very important position in central nervous system damage diseases(traumatic brain injury,subarachnoid hemorrhage,cerebral hemorrhage,ischemic stroke).However,the role of miRNA and IncRNA in the neuroinflammatory response caused by sepsis is still unknown.Therefore,this study mainly explores how IncRNA XIST and miRNA-122-5p regulate the neuroinflammatory response caused by the activation of microglia and astrocytes in the peripheral white matter during sepsis.Objectives1.Explore the expression of IncRNA XIST and miR-122-5p in the periventricular white matter of sepsis rats;2.Explore the specific mechanism of miR-122-5p regulating the inflammatory response of microglia and astrocytes;3.Explore the correlation between miR-122-5p and inflammation in the cerebrospinal fluid of patients with intracranial infection.MethodsRat sepsis model was established using cecal ligation and puncture(CLP).Real-time fluorescence quantitative PCR(RT-PCR)technology was used to detect the expression levels of IncRNA XIST and miR-122-5p in the peripheral white matter of the sepsis rat.Using RNAhybrid and other bioinformatics analysis software to predict the correlation among IncRNA XIST,miR-122-5p,and protein kinase C eta(PKCη).Using dual luciferase reporter gene experiments to verify the three Verify the relationship.The expression of PKCη,p65,phosphorylated p65 and inflammatory factors in microglia and astrocytes was detected by western blot experiment.Over express miR-122-5p by microinjecting adeno-associated virus AAV into the lateral ventricle.Combine immunofluorescence staining and western blot to verify that miR-122-5p could modulate sepsis-induced neuroinflammation in the peripheral white matter of sepsis rat.At the same time,using the siRNA of IncRNA XIST,siRNA of PKCη,and mimics of miR-122-5p further to verify that IncRNA XIST can regulate inflammation by targeting miR-122-5p/PKCη in microglia and astrocytes.In addition,the expression level of miR-122-5p and inflammatory factors in the cerebrospinal fluid of patients with intracranial infection was detected.The correlation between miR-122-5p and inflammatory factors were analysized.Results1.The expression level of miR-122-5p in the peripheral white matter of the sepsis rat group was significantly lower than that in the control group.Starbase predicts that there are 101 targeted lncRNAs in the upstream of miR-122-5p,and IncRNA XIST is one of the targeted IncRNA.Starbase and miRanda predictes the downstream target genes for miR-122-5p and found that there is a targeting relationship between miR-122-5p and PKCη.The double luciferase reporter gene experiment show that the wild group luciferin decreased but the mutant group did not change which proved that there is a targeting relationship between miR-122-5p and PKCη.At the same time,the expression levels of lncRNA XIST and PKCη in the peripheral white matter of sepsis rats were detected by qPCR,and it was found that the expression levels of both were significantly higher than that in the control group.2.Using cecal ligation and perforation(CLP)to construct a sepsis rat model,and detect the expression levels of inflammatory factors IL-1β and TNF-α in the peripheral white matter by qPCR,ELISA and western bolt.The results show that the levels of inflammatory factors were higher than those in the control group after CLP 6h,lday,3day,and 7day.miR-122-5p adeno-associated virus was constructed and observed the neuroinflammation reaction in the peripheral white matter.qPCR,ELISA and western bolt results all showed that the expression levels of inflammatory factors IL-1β and TNF-α in the white matter were decreased after overexpression miR-122-5p.3.Exploring the expression of IncRNA XIST,miR-122-5p,and PKCη in microglia and astrocytes,we construct miR-122-5p mimics to over-express miR-122-5p and the results showed that overexpression of miR-122-5p can significantly reduce the expression levels of inflammatory factors IL-1β and TNF-α in microglia and astrocytes.The expression levels of PKCη and phosphorylated p65 decreased after overexpression of miR-122-5p.The siRNA fof lncRNA XIST and PKCη were used to obverse the levels of inflammatory factors IL-1β,TNF-α,and protein kinase C decreased.After knocking down PKCη and IncRNA xist,the expression levels of PKCη,phosphorylated p65,IL-1β,and TNF-α decreased.4.The level of miR-122-5p in the cerebrospinal fluid of patients with intracranial infection decreased significantly.Compared with normal patients,patients in the intracranial infection group had a longer stay in the ICU,and there were statistical differences in cerebrospinal fluid microalbumin,white blood cells and Paneth test.The expression level of miR-122-5p in the cerebrospinal fluid of patients with intracranial infection decreased,and the expression levels of inflammatory factors IL-1β and TNF-α increased in the cerebrospinal fluid,and there was a negative correlation between miR-122-5p and inflammatory factors.ConclusionsLncRNA XIST can act as an endogenous sponge to adsorb miR-122-5p which lead to activate PKCη and NF-kB signaling pathway by activating microglia and astrocytes in sepsis-associated neuroinflammatory.The level of miR-122-5p is negatively correlated with IL-1β and TNF-α in the cerebrospinal fluid of intracranial infection patients. |
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