Differences Of Gene Expression In Paraquat Poisoning Patients With Different Outcomes

Background and objectiveParquat(PQ)is an efficient and contact herbicide,it is widely used in agricultural production due to its low cost and rapid inactivation upon contact with soil.PQ is high toxic,it can usually result in multiple organ dysfunction after poisoned.The mortality of PQ poisoning can reach up to 80%.At present,there is no specific antidote for paraquat poisoning.Treatments for PQ poisoning include simple support treatment,anti-oxidation treatment,hemoperfusion,hemodialysis,immune regulation,and lung transplantation.However,PQ poisoning remains a high mortality rate.Although PQ has been banned from sale in china,PQ poisoning happens sometimes.It is an urgent problem to find a new effective method for PQ poisoning and explore the internal mechanism of paraquat poisoning.We found significantly difference in PQ poisoning patients with similar basic situation.We speculated that there may be differences in gene expression among different patients,which may lead to different outcomes of PQ poisoning patients.Previous studies have analyzed the gene expression of PQ poisoning in cells and animal models.The results indicated that the organ damage and death caused by PQ may be related to susceptibility genes.Tomita M et al found elevation of gene related to the early lung injury in paraquat poisoned rats,the results of Goldman SM et al suggest that there are susceptibility genes in parkinson's disease caused by PQ.Thus we predict that different prognosis of paraquat poisoning patients may be related to susceptible genes or protective genes in PQ poisoning patients.But there has been no study on the gene expression levels in PQ poisoning patients with different outcomes,previous studies just compared the differences in gene expression between the normal group and the exposed group.We attempted to elucidate the molecular mechanism of death caused by PQ poisoning through analyzing the difference of gene expression between the survival group and the death group using RNA sequencing analysis.The genomic information we obtained will provide useful information for research on the mechanism of death caused by PQ poisoning and help to explore new therapeutic targets and pathways.MethodsBlood samples of paraquat poisoning patients in our hospital from December 2015 to December 2018 were collected.Meanwhile,clinical data of the patients were collected,including age,gender,dosage,time of administration,previous medical history,personal history,concentration of paraquat in urine,length of stay,outcome,etc.Total RNA was extracted and send to Beijing CapitalBio Technology Limited Company for Afymetrix expression spectrum chip analysis.Sequencing results of survival group and death group were compared to find out the difference of genes expression.Gene Ontology(GO)enrichment analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis and new gene prediction were carried out to analysis biological processes and related pathways that differentially expressed genes involved.Realtime fluorescence quantitative PCR(q-PCR)was carried out to test the mRNA expression of TSG-6 in PQ poisoning patients,the difference of mRNA expression of TSG-6 in PQ poisoning patients with different outcomes were analyzed.Enzyme linked immunosorbent assay(ELISA)was used to detect the level of TSG-6 protein in serum of PQ poisoning patients,and the difference of TSG-6 protein expression between the survival group and the death group was analyzed.ResultsA total of 21 PQ poisoning patients were enrolled in this study,including 11 male and 10 female,of these 21 patients,there were 9 died patients and 12 surviving patients.The does of PQ,the Apache 2 score and white blood cell(WBC)in the death group are higher than those in survival group,the hospitalization time of death group was shorter than that of survival group,PaO2 of death group was lower than that of survival group,and the difference between the two groups was statistically significant.We found 27901 differential genes,27732 of which are known protein coding genes,169 of which are new differential genes not mentioned in any database.Compared with the survival group,240 difference genes were statistically significant,including 154 down-regulated genes and 86 up-regulated genes in the death group.Among 86 up-regulated genes,33 genes are involved in the regulatory signaling pathway,while among 154 down regulated genes,36 genes are involved in the regulatory signaling pathway.The results of GO analysis showed that these genes participated in 25 biological processes,17 cell components and 12 molecular functions.The 5 top rich factor of GO enrichments were immunoglobulin receptor binding,chemokine receptor activity,immunoglobulin complex,clathrin-coated endocytic vesicle,and blood microparticle.KEGG analysis showed that the most significant pathway related to paraquat poisoning outcome include:cytokine-cytokine receptor interaction,chemokine signaling pathway,and PI3K-Akt signaling pathway.Metabolism,DNA damage,gene repair,cellular immunity and other signaling pathways are involved in the progress of paraquat poisoning.Analysis of differential gene in patients with different PQ does and out come showed that mRNA expression of TSG-6 in high does and survival group were higher than that in low does survival group,the most significant change was in the expression of inflammatory factors.Compared with high dose death group,expression of TSG-6 decreased 3 times in low dose death group.Result of qPCR was consistent with transcriptome sequencing in survival group,while it is opposite in the death group.We found the expression of TSG-6 in the survival group was lower than that in the death group,and it increased with the increase of trypsin level.The results of TSG-6 protein expression showed that there are no significant difference between survival group and death group,also among pancreatin normal,mild elevation,and significant elevation group.ConclusionsThere were differences in gene transcription between the survival and the death group in PQ poisoning patients.Biological processes related to the lethality of paraquat poisoning include immunoglobulin receptor binding,chemokine receptor activity,immunoglobulin complex,clathrin-coated endocytic vesicle,and blood microparticle;signal pathways related to the lethality of paraquat poisoning include cytokine-cytokine receptor interaction,chemokine signaling pathway,and PI3K-Akt signaling pathway,Metabolism,DNA damage and gene repair,and cellular immunity.TSG-6 may play an anti-inflammatory role in the process of acute paraquat poisoning.