| Background and Purpose Drug-induced liver injury is the most common side effect in the course of anti-tuberculous drug therapy,and it is also the main obstacle of tuberculosis management and control in China.Although great progress has been made in the study of anti-tuberculous drug-induced liver injury,the pathogenesis of the disease is still not fully elucidated,leading to the lack of effective risk assessment and early diagnostic markers.In the past decade,for the transcriptome study research is becoming more and more thorough,it is showed that further study of disease related gene expression profile has a pivotal role in exploring the happening and development of the disease,especially in learning the crucial pathogenic biology which can promote the development of new biological markers for risk assessment and disease diagnosis or prognosis judgement.However,transcriptome data on the course of anti-tuberculous drug-induced liver injury are still lacking.Therefore,in this project,we explored the differences in the expression profiles of non-coding RNA and m RNA in hepatocellular injury caused by anti-tuberculosis drugs,so as to comprehensively understand the potential pathogenesis of anti-nodal druginduced liver injury,and then further study the biological processes closely related to the occurrence of the disease based on transcriptional results.Methods(1)Serum samples from patients with liver injury caused by anti-tuberculosis drugs were collected,and the differential gene expression profiles related to diseases in serum were obtained by microarray chip technology.Then,bioinformatics methods were used to analyze the biological functions of differentially expressed genes,and bio-mapping software was used to construct the co-expression network as well as protein interaction network between genes,so as to screen the core genes and signal pathways tightly related to the diseases.Then,RT-q PCR was employed to detect the expression of target genes in the disease group and the control group,respectively.(2)To establish a rifampicin toxicity model using in vitro cultured cells,and use small molecule compounds and si RNA interference technology to detect the role of key signaling pathways in rifampicin hepatotoxicity.Secondly,flow cytometry combined with different fluorescent probes was used to study the key factors of cell injury,and WB technology was used to detect the expression of key signaling targets in cells to clarify the related mechanism of hepatocyte injury.(3)The function and mechanism of key signaling pathways and protein molecules were verified in vivo by constructing a mouse model of rifampicin liver injury,detecting blood biochemical indicators,liver tissue lesions and protein expression changes.Results(1)Through RNA gene expression profiling,we screened 568 m RNAs and 845 non-coding RNAs related to anti-tuberculosis druginduced liver injury.The results of functional enrichment analysis indicated that the mitochondrion organization,regulation of cellular catabolic process and positive regulation of organelle organization et al pathways are likely to be key events in the development of antituberculosis drug-induced liver injury.More importantly,KEGG pathway analysis further revealed that the NCOA4(Nuclear Receptor Coactivator4,NCOA4)-related ferroptosis pathway may be one of the main mechanisms of the disease.In addition,the m RNA content of NCOA4,a key regulator of the ferroptosis pathway,was significantly higher in serum samples from patients with anti-tuberculosis drug-induced liver injury than in controls without liver injury,indicating that NCOA4 is associated with anti-tuberculosis drug-induced liver injury.At the same time,the protein interaction network suggested that the ubiquitin family(such as UBA52,UBB,UBC,etc.)and the heat shock protein family(such as HSPA8,HSP90AB1 and HSP90AA1,etc.)may be the core factors in antituberculosis drug-induced liver injury.(2)The results of the in vitro rifampicin injury model of Hep G2 and AML12 cells show that ferroptosis inhibitors and deferoxamine can effectively alleviate the decline of cell viability caused by rifampicin.Fluorescent probe labeling results showed that high levels of lipid peroxidation occurred in damaged cells,accompanied by a significant increase in intracellular iron ion concentration.More importantly,the mechanistic study found that rifampicin stimulation led to the continuous enhancement of autophagic flux in Hep G2 and AML12 cells,resulting in a significant increase in NCOA4-mediated ferritin autophagy and induced cell death.Inhibition of autophagy or inhibition of NCOA4 gene expression can significantly reduce ferritin-selective autophagy,and at the same time reduce the hepatotoxicity of rifampicin.In addition,Western blotting confirmed that the inhibition of HSPA8 expression played an important role in rifampicininduced autophagy,and induction of HSPA8 expression could reverse rifampicin-induced cell death to a certain extent.(3)In a mouse model,it was verified that the increase of iron content in liver tissue is related to tissue damage,and ferroptosis induced by NCOA4-mediated ferritinselective autophagy is an important pathogenic factor of rifampicininduced liver damage.Furthermore,the treatment of autophagy inhibitors,ferroptosis inhibitors or HSPA8 inducers in animals can effectively improve the abnormal liver function and tissue steatosis caused by rifampicin exposure.Conclusion Ferritin-selective autophagy-induced ferroptosis induced by NCOA4 is one of the important pathogenic mechanisms in the pathogenesis of anti-tuberculosis drug-induced liver injury.Whether using small molecule compounds to inhibit ferritin degradation or using gene intervention technology to attenuate autophagy,all have the effect of alleviating the hepatotoxicity of rifampicin.In short,the NCOA4-mediated ferroptosis pathway may be a new mechanism for the occurrence of antituberculosis drug-induced liver injury,and hopefully provide a basis for the development of new targets for early diagnosis and intervention of the disease. |
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