Studies On The Role Of Serum Exosomal MiR-30d-5p In Immune Liver Injury Induced By Trichloroethylene

BackgroundThe organic solvent trichloroethylene(TCE)is commonly used in industry for cleaning metal parts in various sectors such as electroplating and electronics.A small percentage of workers exposed to TCE may develop skin damage,which can mimic drug eruptions and is clinically referred to as occupational medicamentosa-like dermatitis due to Trichloroethylene(OMDT).In addition to skin damage,some OMDT patients may also experience liver function abnormalities,which in severe cases can lead to acute liver failure,a major cause of death among OMDT patients.Therefore,studying the mechanisms of TCE-induced liver damage has significant occupational health implications.miRNAs are a type of conserved non-coding single-stranded RNA containing 19-22 nucleotides,which can bind to potential mRNA targets via complementarity,preventing them from being expressed and ultimately leading to their degradation.miRNAs are mainly found in the double-layered lipid membrane structures of exosomes,which are chemically stable and widely present in various body fluids involved in intercellular communication and play important roles in many aspects of cell growth,apoptosis,and differentiation.Studies have found that changes in miRNA expression profiles exist in many diseases and miRNAs are considered a biomarker with potential for predicting the occurrence and development of various diseases.As OMDT is a composite immune reaction mediated by T lymphocytes,it is worth further investigation whether the expression profile of miRNAs in serum exosomes will change and what role the differentially expressed miRNAs play in it.ObjectiveThe main purpose of this research is to investigate whether miR-30d-5p is involved in the process of immune liver injury caused by TCE,and through what pathway it participates.The population study first collected serum samples from OMDT patients during the onset peak,recovery,and healthy controls,extracted extracellular vesicles and identified them,performed miRNA gene chip analysis,screened differentially expressed miRNAs,experimentally verified miR-30d-5p in the results,correlated it with patient liver function indicators,and finally predicted the target genes of miR-30d-5p and their potential involvement in bioinformatics.In the in vivo experiments,a BALB/c female mouse model of TCE hypersensitivity was first established,and changes in mouse liver function indicators and liver damage were detected,and the expression level of miR-30d-5p was detected.Target indicators predicted to be related to liver apoptosis damage were experimentally verified and correlated with mouse liver function indicators to investigate the specific role of miR-30d-5p in TCE-induced immune liver injury in mice,providing evidence-based support for population studies.Methods1.Six patients with TCE drug rash-like dermatitis admitted to the Guangdong Provincial Institute of Occupational Disease Prevention and Treatment in 2020 were selected.Serum samples were collected during the onset peak and recovery periods,and six healthy volunteers were selected as controls.Extracellular vesicles were extracted from the serum,and identified by transmission electron microscopy,nanoparticle tracking analysis,and protein immunoblotting.Gene chip analysis was used to detect differential expression of miRNAs,and qRT-PCR was used to verify the results and correlate with patient liver function indicators.Finally,bioinformatics analysis was used to explore potential physiological and pathological processes involved,providing clues for research on TCE-induced immune liver injury.2.Based on the research foundation of the project team,a TCE hypersensitivity BALB/c mouse model was established.Thirty-two female BALB/c mice were adaptively fed and randomly divided into a blank control group(six mice),solvent control group(six mice),and TCE treatment group(20 mice)according to body weight.The TCE treatment group was divided into a positive sensitization group and a negative sensitization group based on the degree of back skin lesions.Serum and liver samples were collected.A kit was used to detect the levels of liver function indicators aspartate transaminase(AST)and alanine transaminase(ALT)in mouse serum,and qRT-PCR was used to detect the expression level of miR-30d-5p in mouse serum.The liver damage was detected by HE staining.qRT-PCR was used to detect the expression levels of miR-30d-5p target genes.Western blotting and immunohistochemical methods were used to detect the expression levels of miR-30d-5p target proteins.Results1.The identification results of serum exosomes show that the exosomes under electron microscope are round or oval-shaped,with a bilayered lipid membrane structure.Moreover,the immunoblotting test shows positive expression of characteristic exosomal membrane proteins CD9,Flotollin-1,and HSP70.The nanoparticle tracking analysis indicates that their particle size is about 200 nm,which is consistent with the characteristics of exosomes.2.The gene chip results show that compared with the healthy control group,119 miRNAs are upregulated and 51 miRNAs are downregulated during the peak period of illness,while 36 miRNAs are upregulated and 12 miRNAs are downregulated during the recovery period of illness comparing to the peak period.Furthermore,compared with the healthy control group,69 miRNAs are upregulated and 44 miRNAs are downregulated during the recovery period of illness.3.qRT-PCR detection of serum exosomes validates that miR-30d-5p expression level of OMDT patients during the peak period of illness is significantly reduced compared to the recovery period and healthy control group.4.During the peak period of illness,OMDT patients have significantly increased liver function indices AST,ALT,and GGT,which is negatively correlated with miR-30d-5p expression level of serum exosomes.5.In the miRWalk database,535 candidate target genes of miR-30d-5p are selected.In the miRBD database,736 candidate target genes of miR-30d-5p are identified.The intersection of the two databases shows 46 candidate target genes.GO analysis indicates that miR-30d-5p mainly participates in 73 molecular functions,81 cell components,and 172 biological processes.KEGG analysis shows that most candidate target genes are enriched in various immune-related molecular signaling pathways,such as Apelin,Wnt,and Oxytocin.6.The skin of mice in the blank and solvent groups did not show any significant changes.The sensitization rate of mice treated with TCE was 30.00%,and there was damage such as redness on the back skin.7.Hematoxylin-eosin staining results showed that the liver structure in the blank,solvent,and TCE sensitization-negative groups was normal,while in the TCE sensitization-positive group,damage to the liver structure,cell swelling,and cytoplasm loss could be observed.8.Compared with the blank,solvent,and TCE sensitization-negative groups,the expression levels of serum liver function indicators AST and ALT in the TCE sensitization-positive group of mice were significantly elevated.9.qRT-PCR results showed that the expression level of serum miR-30d-5p in the TCE sensitization-positive group of mice was significantly decreased,and the gene expression levels of TIEG-1,Gα13,RHOB,and Blimp-1 in liver tissues were significantly increased.10.Compared with the blank,solvent,and TCE sensitization-negative groups,immunohistochemistry results showed that the liver structure of TCE sensitization-positive group was damaged,and a large amount of protein deposits of TIEG-1,Gα13,RHOB,Blimp-1 were observed in liver cells.Protein immunoblotting results showed that the expression levels of TIEG-1,Gα13,RHOB,Blimp-1 in the liver tissues of the TCE sensitization-positive group were significantly increased.11.Correlation analysis results showed that the expression levels of liver function indicators AST and ALT were negatively correlated with miR-30d-5p and positively correlated with target genes TIEG-1 and RHOB.12.Compared with the blank,solvent,and TCE sensitization-negative groups,WB and immunohistochemistry results showed that the expression level of Caspase-3 protein in the liver tissues of the TCE sensitization-positive group of mice was significantly increased.ConclusionsDuring the peak period of OMDT disease,the expression level of serum exosomal miR-30d-5p is significantly decreased,which is negatively correlated with liver function indicators,and may participate in TCE-induced immune liver injury by targeting multiple apoptosis-related indicators such as TIEG-1,Gα13,RHOB,and Blimp-1.