The Study On The Role Of N-acetyl-p-benzenequinoneimine In The Pathogenesis Of Primary Biliary Cholangitis

Background and Purpose Primary biliary cholangitis（PBC）is one of the common autoimmune liver diseases,which usually occurs in women over the age of 40.Genes and environmental factors mainly determine the occurrence of PBC.Anti-mitochondrial antibody（AMA）is the serological marker for diagnosing primary biliary cholangitis（PBC）.And 95% of PBC patients can be found AMA-positive in peripheral blood.The epitopes mainly recognized by AMA are primarily distributed in the inner lipoyl domain of the E2 subunits of the pyruvate dehydrogenase complex（PDC-E2）（containing the amino acid sequence 177-314）.ILD).PDC-E2 mainly catalyzes oxidative decarboxylation reactions during energy metabolism in mitochondria.The lipoic acid（LA）acyl group of PDC-E2 ILD achieves electron transfer and acyl transfer by opening and closing the dithiolane disulfide bond.The process plays a vital role in the decarboxylation reaction.Compounds（drug and biological factors）in environmental factors all lead to the intolerant of PDC-E2 LD and the production of AMA by modifying PDC-E2 LD.Acetaminophen（acetyl-para-aminophenol,APAP）is a common clinical antipyretic and analgesic drug,and the conventional treatment dose rarely causes liver damage.However,excessive APAP can cause serious liver damage and even liver failure.In clinical practice,AMA can be detected in the peripheral blood of 40.6% of patients with overdoses of APAP.The metabolite of APAP,N-Acetyl-p-benzoquinone Imine（NAPQI）,may generate new antigens by modifying PDC-E2 LD.However,not all patients with APAP-induced acute liver injury develop AMA.Therefore,genes may be the internal factors that determine whether patients with excessive oral APAP develop AMA.In addition,N-Acetyl-p-benzoquinone Imine(NAPQI,a metabolite of APAP,may produce new antigens by modifying PDC-E2 LD.As an electrophilic compound,NAPQI can modify the construction of PDC-E2 LD by binding LA’s dithiolane,which may induce immune intolerance to PDC-E2 LD.NAPQI The specific mechanism is not yet clear.This study used high-throughput sequencing and bioinformatics to explore common differentially expressed genes in NAPQI-induced AMA-positive PBC patients.On the other hand,we explored the possible mechanism of modifying the construction of PDC-E2 LD via NAPQI in the pathogenesis of PBC.MethodOur methods contain two major parts into two parts:Part 1: We selected 6 PBC patients and six age-and sex-matched healthy controls,extracted exosomes to detect m RNA in plasma,and sequenced them after reverse transcription to determine the different genes between PBC patients and healthy controls.In addition,we obtained two GSE raw matrix files（GSE74000 and GSE13430）related to APAP-induced liver failure from the public gene expression omnibus（GEO）.The genes with significant differences（| log FC | > 1 ＆ p < 0.05）in the two obtained GSE files were chosen.Then the common differential expression genes（CDEGs）in the three GSE files are sorted with the degree of interaction between proteins.STING database was explored for the form of a protein-protein network.Molecular complex detection（Molecular Complex Detection,MCODE）in Cytoscape 15.0 software was to identify tightly connected modules.The top ten genes of CDEGs were identified based on the degree of interaction.The expression of these ten genes was detected in the serum of PBC patients via real-time quantitative reverse transcription（q RT-PCR）.We finally identified the five upregulated genes in the three datasets based on expression level.We also analyzed the correlation between expression level and clinical parameters.Based on the collected clinical data,we conducted a nomogram to predict the possibility of ascites generation in PBC patients with a high level of TFG-CDEGsPart2:1.The recombinant protein PDC-E2 protein fragment containing amino acid residues 177-314 was obtained by intein-mediated protein ligation.The recombinant peptide（PDC-228）containing amino acid residues 177-252 was connected with the synthetic polypeptide（PP）（253 to 314）containing（ILD）in PDCE2.The constructed fragment is called PPL.Firstly,the compounds LA,2OA,and NAPQI were connected with PP to form PP-LA,PP-2OA and PP-NAPQI-LA.Through the technology of intein mediated protein connection,we obtained NAPQI-PPL by modification of LA.2.The ILD fragment（r PDC-E2 ILD）of recombinant PDC-E2（recombinant-PDC-E2 LD,r PDCE2LD）was obtained by expressing E.coli containing p GEX4 T as a positive control.3.We selected one hundred and fifty-eight patients with PBC,thirty-eight patients with rheumatoid arthritis（RA）,and seventy-two healthy controls.The levels of PDCE2 antibody,anti-LA-PDC-E2 antibody,anti-2OA antibody,and anti-NAPQI antibody were detected by ELISA.Thirty PBC patients with positive anti-LA-PDC-E2 antibody and NAPQI-BSA antibody were selected.The inhibited ELISA test was used to detect neoantigen epitope-specific antibodies identifying NAPQI modified PDC-E2 LD in the serum of PBC patients.4.PBC patients with positive NAPQI-PPL were selected to purify specific antibodies against NAPQI-PPL.Western blot analysis identified the characteristics of purified antibodies: BSA was added as a negative control,r PDC-E2 was added as the positive control,and the purified antibody was added as the primary antibody.Western blotting analysis was performed to detect the reactivity of the new antibody to different antigens（including NAPQI-PPL,NAPQI-BSA,LA-PPL,r PDC-E2,and BSA）.The cross immunoreactivity between NAPQI-PPL and LA-PPL was detected by Western blot analysis.5.The antibody types（Ig G/Ig M）of NAPQI-PPL in different PBC clinical stages were analyzed by ELISA.Results1.We obtained the differential genes between PBC and healthy controls in the exosome sequencing.We identified 102 CDGEs in the selected three data sets.The top 10 most strongly interacting genes identified through the STRING database were:MYC,CKS2,PCNA,PTTG1,MAOA,CEBPD,CDKN2 D,CDKN1C,UBE2 S,and KLF2.2.The expression levels of the top 10 most strongly interacting genes in the plasma of PBC patients were detected by q RT-PCR.We finally determined that the expression levels of CKS2,PCNA,PTTG1,UBE2 S,and KLF2 were up-regulated in the datasets（GSE74000 and GSE13420）and in the plasma of PBC patients.3.The expression levels of KLF2 in the serum exosomes of PBC patients were negatively correlated with serum alkaline phosphatase and transglutaminase（R =-0.729,p = 0.017;R =-0.875,p < 0.001,respectively）.Univariate logistic regression analysis found that UBE2 S and KLF2 were associated with ascites formation in PBC patients（p = 0.036,p = 0.031）.The nomogram that included screened TFG-CDEGs and other clinical parameters（age,gender,UBE2 S,and KLF2）was valuable for predicting ascites’ production in PBC（C-index: 0.980）.4.We successfully obtained the conjugated LA-PPL,2OA-PPL,and NAPQI-PPL by intein-mediated protein linkage.They have the same antigenicity as r PDCE2,which can react to AMA in the serum of PBC patients.5.The response rates to r PDC-E2,LA-PPL,2OA-BSA,NAPQI-BSA and BSA with serum were 94.93%（150 / 158）,90.5%（143 / 158）,71.43%（112 / 158）,60.26%（95 /158）and 0%（0 / 158）.The positive rate of the antibodies mentioned above in the PBC group was significantly higher than that in the healthy control group and RA group.However,there was no significant difference between the RA and healthy control groups.6.Correlation analysis showed no correlation between anti-lipoic acid and antiNAPQI antibodies（r=0.2,P=0.069）.7.The NAPQI-modified PDC-E2（NAPQI-PPL）antibody exists in the peripheral blood in 40% of PBC patients,and this antibody mainly exists in early PBC patients.8.The antibody of NAPQI-PPL purified from the serum of PBC patients identified by inhibition ELISA only produces an antigen-antibody reaction to NAPQI-PPL,which is specific.9.The main type of antibodies against NAPQI-BSA and NAPQI-PPL in the serum of PBC patients is Ig M,and the main type of antibodies against LA-PPL is Ig G.The Ig G/Ig M ratio of NAPQI-PPL was less than one and was between NAPQI-BSA and LAPPL.Conclusion1.PCNA,CKS2,PTTG1,UBE2 S,and KLF2 may be potential genes that lead to PBC in patients with an oral overdose of APAP.2.Electrophilic compounds induce immunity first when enter the body.Then,the immune response was generated from the side chain to main chain of the PDC-E2 LD modified by the compound.Finally,the range of the immune was extended to the LAlinked PDC-E2 LD immune response,leading to the production of AMA.