Study Of NLRP3/IL-1β Mediating Effect On Schizophrenia-like Behaviors Induced By Prenatal Lipopolysaccharide Exposure

Objectives:Schizophrenia(SCZ)is a group of chronic,severe,and debilitating psychiatric clinical syndromes,characterized by disconnected sensations,perception and mental activities from reality.In severe cases,the functions aforementioned are completely inconsistent with reality.Most patients develop it in adolescence and early adulthood.It’s difficult to cure the disease,so the illness course is protracted.Besides,many patients develop to suffer from SCZ for lifetime,thereby,it brings huge burdens to the individual,family and society.However,at present,the diagnostic criteria for SCZ still rely on symptomology and the auxiliary markers are limited.In this study,RNA sequencing was performed on the peripheral blood leukocytes from patients with schizophrenia and healthy controls.Systematic bioinfomatic analysis was used to calculate the hub differentially expressed genes(DEGs).Moreover,online peripheral blood RNA-sequencing data of depressive disorder(MDD)and bipolar disorder(BD)were downloaded and reanalysied.The DEGs in the peripheral blood of these three diseases were compared,and those with better specificity were screened out as auxiliary references for SCZ diagnosis and differential diagnosis.Then,the screened DEGs in peripheral blood were compared to the DEGs from SCZ patients’ brain tissue based on the previous research and literatures.Finally,animal models were ultilized to explore that whether the shared DEGs mediate the SCZ-like behaviors.Methods:1.High-throughput RNA sequencing technology was performed respectively on the peripheral leukocytes of 50 SCZ patients and 50 healthy volunteers(HCs).After standardizing the expression data,the R platform limma statistical package was used to analyze the Differentially expressed genes(DEGs)between SCZ and HCs.2.Using the weighted gene co-expression network(WGCNA)statistical package of the R platform,the co-expression networks of DEGs were constructed.The modules with a high degree of disease correlation were screened,and the protein-protein interaction(PPI)network was constructed via String database to screen out candidate hub DEGs.3.Using the online database Gene Expression Omnibus(GEO)platform to screen the MDD and BD peripheral blood RNA sequencing datasets that meet the requirements,and use GEO2 R to calculate the selected datasets to obtain the differentially expressed DEGs.4.Utilizing the WGCNA network to construct the peripheral blood DEGs of MDD and BD patients,screen out modules with high disease correlation,and PPI was used to screen candidate hub DEGs.Pathway enrichment analysis was performed on the candidate hub DEGs of the three diseases to find the common pathways.In the common pathways,SCZ specific hub DEGs were analyzed,and a diagnostic model was constructed via mathematical models.Verifying the performance and accuracy of these models.5.Inject LPS intraperitoneally into mice on the 15 th day of pregnancy to construct an animal model.After the offspring mice enter puberty,behavioral tests were conducted to detect whether abonormal expression of NLRP3/IL-1β in the hippocampus and prefrontal cortex of the mice mediates SCZ-like abnormal behavior.6.The two groups(control and SCZ model group)mices were selected randomly for prefrontal cortex and hippocampus tissue collection.RNA-sequencing analysis was performed on these tissues.The downstream molecules of hub DEGs that mediate SCZ-like abnormal behavior were screend,and SH-SY5 Y cells was used to verify the regulatory relationship.Results:1.Among the functional enrichment pathways of the candidate hub DEGs from those diseases,there are several common pathways: Signaling by Interleukins,Cellular responses to stimuli,Cellular responses to stress and Cytokine Signaling in Immune system pathways.Further analysis found that,among the candidate hub DEGs enriched in these pathways,NLRP3,CASP1,IL1 B and IL-18 may be important in SCZ with high specificity.The interaction enrichment pathway analysis of SCZ found that NLRP3,CASP1,IL1 B and IL-18 were enriched in the response to lipopolysaccharide pathway.It suggests that the up-regulation of m RNA levels of these four genes is related to the lipopolysaccharide exposure,which is consistent with previous studies.At the same time,this pathway has a very high degree of connectivity in the interaction between the pathways enriched by candidate hub DEGs of SCZ,indicating that it may be a key pathway for SCZ.2.Using Lasso and Logistic regression to construct the mathematical models.Besides,receiver operating characteristic curve(ROC curves)was used to evaluate the auxiliary values in the diagnosis of SCZ.Then,comparing the diagnostic efficacy of the four genes in MDD and BD,and the results showed that the two diagnostic models are better for SCZ(AUC and 95%CI of Lasso: BD,0.763(0.592-0.935);MDD,0.6(0.502-0.698),SCZ,0.894(0.831-0.958);AUC and 95%CI of Logistic: BD,0.763(0.592-0.934);MDD,0.605(0.507-0.702);SCZ,0.894(0.829-0.958)).3.By intraperitoneal injection,LPS was injected into mice on the 14.5th pregnancy day.It was shown that NLRP3 and IL-1β in the prefrontal cortex and hippocampus of male offspring mices with pregnancy LPS exposure were significantly upregulated.However,the alterations were not found in the corresponding brain tissues of female offspring mice.In the behavioral tests,only the male offspring mice showed SCZ-like abnormal behaviors,while the behavioral performances of the two group of female offspring mices were not significantly different.4.This study randomly selected two groups of mice for prefrontal cortex and hippocampus tissues collection.In order to screen the downstream molecules of IL-1β,RNA-seq analysis was performed.Comparing with the research group’s previous RNA-seq analysis of SH-SY5 Y cells with overexpressed IL-1β,the results showed that CUX2 may be a key molecule for IL-1β in regulating neurodevelopment.After co-culturing with IL-1β and SH-SY5 Y cells,it was found that the CUX2 m RNA expression was significantly reduced,and after treatment with IL-1RA,the level of CUX2 m RNA was restored.Conclusions:1.The m RNA levels of NLRP3,CASP1,IL1 B and IL-18 are elevated in the peripheral blood of SCZ patients,and the abnormal expression of these four genes has a good auxiliary value for the diagnosis of SCZ.In addition,the expression of these genes helps to differentiate SCZ,MDD and BD2.NLRP3/IL-1β mediated the occurrence of SCZ-like abnormal behavior.3.IL-1β may mediate SCZ-like abnormal behavior by down-regulating CUX2 and thereby causing impaired neurodevelopment.