Study On The Regulatory Effect Of Methyltransferase DOT1L On P53-Bax/Bcl-2 Pathway In Anti-tuberculosis Drug-induced Liver Injury

Objectives This study aims to explore the changes in the expression level of the methyltransferase disruptor of telomeric silencing 1-like(DOT1L)in serums of patients with anti-tuberculosis drug-induced liver injury(ADLI),and explore the potential mechanism of DOT1 L in regulating the p53-Bax/Bcl-2 apoptosis pathway of mice.Methods The exploration was carried out from two levels of humen and animals.Firstly,basic clinical data and blood samples of newly-treated pulmonary tuberculosis patients who were hospitalized in the Fifth Hospital of Shijiazhuang City from July 2019 to January2020 were obtained.According to the inclusion and exclusion criteria,188 study subjects were included and were followed up at three time points,T1,T2,and T3.Real-time fluorescence quantitative PCR(RT-q PCR)and enzyme-linked immunosorbent assay(ELISA)were used to detect the levels of DOT1 L,tumor protein p53(p53),Bcl-2associated X protein(Bax),B-cell lymphoma-2(Bcl-2)m RNA and protein in serums of ADLI patients.Secondly,three anti-tuberculosis drugs isoniazid,rifampicin and pyrazinamide were administered in combination to establish ADLI model of mice.Mice were randomly divided into blank group,drug group,drug-solvent group,drug-inhibitor group,blank-solvent group,blank-inhibitor group.Mice were sacrificed on 0 d,7 d,14 d,21 d and 28 d.Blood samples and liver tissues were obtained from mice.Hematoxylineosin staining(HE staining)method was used to observe pathological changes of the liver tissues of mice in each group.Microplate method was used to detect the levels of liver function indexes alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serums of mice in each group.RT-q PCR was used to detect m RNA expression levels of DOT1 L,p53,Bax and Bcl-2 in serums and liver tissues of mice.ELISA was used to detect protein expression levels of DOT1 L,p53,Bax and Bcl-2 in serums of mice.Western blot(WB)was used to detect protein expression levels of DOT1 L and H3K79 in liver tissues of mice.Immunohistochemical staining(IHC)was used to detect the cellular location of DOT1 L.Chromatin immunoprecipitation(Ch IP)method was used to detect the methylation levels of H3K79 in the promoter region of p53 gene.Results The results included two parts,including tuberculosis patients and animal model of mice.In serums of tuberculosis patients,m RNA and protein levels of DOT1 L,p53,and Bax in the ADLI group from T1 to T3 were increased first and then decreased,while Bcl-2was decreased first and then increased(all P<0.05).The m RNA and protein levels of DOT1 L,p53,Bax,and Bcl-2 in ADLI group were higher than those in Non-ADLI group at T2 and T3(all P<0.05).The results of correlation analysis and interaction analysis showed that levels of DOT1 L in the groups were positively correlated with ALT,AST,p53 and Bax,and negatively correlated with Bcl-2 in serums.The changes in m RNA and protein of DOT1 L,p53,Bax,and Bcl-2 at three time points of two groups all had interactions between time and group(all P<0.05).In animal model of mice,after 7 d of administration,the liver tissues of mice showed pathological changes and liver function indexes increased,suggesting that the ADLI model of mice were successfully constructed.From 7 d to 28 d after administration,the changes in m RNA and protein expression levels of DOT1 L,p53,Bax,and Bcl-2 in serums and liver tissues of drug-inhibitor group were consistent with those in serums of tuberculosis patients.The expression levels of DOT1 L,p53,and Bax were increased first and then decreased,and were higher than the levels at 0 d.The expression levels of Bcl-2 was decreased first and then increased,and was lower than the levels at 0 d(all P<0.05).In terms of regulatory mechanism,IHC results confirmed that DOT1 L in liver tissue was mainly located in nucleus,and as the degree of liver damage deepens,nuclear necrosis and lysis increase,and its expression trend were basically the same as that of H3K79 protein.By 28 d,compared with the control group,the enrichment factor of the p53 gene promoter region in the drug group increased significantly(P<0.05),while the enrichment factor of the blank-inhibitor group were significantly reduced(P<0.05).The enrichment factor of the p53 gene promoter region in the drug-inhibitor group was lower than that of the drug-solvent group,but higher than that of the blanksolvent group(all P<0.05).Conclusions 1 The methyltransferase DOT1 L is highly expressed in the serum of ADLI patients and is closely related to the p53-Bax/Bcl-2 apoptosis pathway.2 DOT1 L is highly expressed in ADLI mouse serum and liver tissue.The inhibition of DOT1 L can reduce the degree of liver injury through inhibiting p53-Bax/Bcl-2 apoptotic pathway by reducing the H3K79 methylation level in the promoter region of p53 gene.Figure 24;Table 24;Reference 120...