Screening And Verification Of Differential Proteins Related To Clinical Viral Encephalitis

Objective:Label-free proteomics was used as a screening tool to analyze differential proteins(DEPs)in cerebrospinal fluid of patients with viral encephalitis(VE).The biological processes and signaling pathways involved in these DEPs were analyzed by bioinformatics techniques such as GO、KEGG pathways,and the DEPs related to VE were preliminarily screened.Further expand the sample size to verify and analyze its clinical relevance using the enzyme-linked immunosorbent assay(ELISA)to explore whether it could be a biomarker for diagnostic VE.Methods:From November 2020 to February 2021,the CSF was collected from 11patients with VE and 5 patients with non-intracranial infection matched with age,gender and other basic information in the Affiliated Hospital and the Third Affiliated Hospital of Zunyi Medical University within 24h after admission.Using Label-free proteomics screening technique,all the proteins in cerebrospinal fluid were detected by quality inspection,enzymatic hydrolysis,mass spectrometry and data analysis.Then the DEPs was GO、IPR and KEGG pathway enriched by Interproscan software,analysis and preliminary screening of DEPs in important functions(Preliminary screening of functional proteins).Then 20 cases of VE(VE group)and non-cranial infection(control group)were collected at the same time,and the expression of the important function DEPs is used to calculate the sample size by ELISA.Further expanding the sample,selected 73 VE and 53control group patients,again used ELISA to detect the expression changes of important function DEPs,analyzed the correlation between different functional protein and clinical indicators,and explore whether it could be a diagnostic marker of VE.SPSS18.0 was used for statistical analysis of all data.If the measurement data obeys normal distribution,the mean soil standard deviation is applied and the T-test is adopted.If the measurement data is non-normal distribution,the median and the interquartile are adopted and the non-parameter test is adopted.The counting data were checked byχ~2.Pearson Correlation Analysis was used to analyze the correlation between important functional DEPs of cerebrospinal fluid and clinical parameters.(P≤0.05 is considered statistically significant)Results:(1)A total of 301 DEPs,including 80 up-regulated DEPs and 221down-regulated DEPs,were identified by difference analysis of co-expressed Proteins(Fold-change<1.2)in two groups of CSF samples using Label-free proteomics as a screening tool.1)GO analysis in BP,CC,MF process showed that DEPs was mainly associated with organic substance metabolic process、catalytic activity、cellular process and synapse organization.2)KEGG pathway analysis found that the main DEPs was cell adhesion molecular pathway enrichment.3)Bioinformatics analysis of GO,subcellular localization and KEGG enrichment analysis showed that cell adhesion molecules may play an important role in VE,among which NLGN2,NFASC,NRXN3,LRRC4B and NLGN1play the most important role.Further literature searches speculated that protein NLGN2,NRXN3,NFASC associated with inflammation and axons may be a diagnostic marker of VE.(2)The expression volume of NLGN2,NRXN3,NFASC protein screened in the cerebrospinal fluid of VE and non-intracranial infected patients was detected by ELISA method.First,NLGN2,NRXN3,NFASC protein was detected using ELISA for 20 CSF samples in each group and calculated the sample size required for this experiment.To further expand the sample size of 73 cases VE group and 53 control group samples to detect NLGN2 protein,the final two results showed that the expression of NLGN2 protein was significantly up-regulated and statistically significant(P<0.05),and NRXN3、NFASC protein expression was not obvious,suggesting that NLNG2 protein may be a potential biomarker for the diagnosis of VE.(3)To further clarify,the correlation analysis of NLGN2 protein with clinical CSF Pro,CSF pressure,WBC,LY,NEUT and other indicators.We found that NLGN2 protein in CSF was positively correlated with CSF pro(r=0.228),but not statistical difference with CSF pressure,WBC,WBC,LY,NEUT,Hospitalization days(P>0.05).Since the study showed that the expression of NLGN2 was up-regulated and down-regulated in different symptoms,there was no statistical difference in age,sex and NLGN2 protein of different symptoms in VE group.Conclusion:The cell adhesion molecular proteins associated with axonal injury NLGN2up-regulated and CSF Pro positively correlated in VE and may be potential biomarkers for diagnostic VE.