XBP1 activation elevates abnormal synthesis of polyunsaturated fatty acids in hepatocytes to promote liver injury during anti-tuberculosis drug metabolism

doi:10.21203/rs.3.rs-4941415/v1

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XBP1 activation elevates abnormal synthesis of polyunsaturated fatty acids in hepatocytes to promote liver injury during anti-tuberculosis drug metabolism

https://doi.org/10.21203/rs.3.rs-4941415/v1

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Objective

This study aimed to explore new mechanistic insight into the link between abnormal lipid metabolism and ATB-DILI.

Methods

We performed integrative analyses of RNA-Seq, ChIP-Seq, lipids mass spectrometry, in vivo and in vitro experiments, and clinical samples to identify the key pathways and molecules involved in the process of ATB-DILI.

Results

Our study revealed that the cellular polyunsaturated fatty acids (PUFAs) synthesis was abnormally activated in hepatocytes during anti-TB drug metabolism. The levels of phosphatidylethanolamine substrates, ferroptosis-related arachidonic acid, and key enzyme Acyl-CoA synthetase long-chain family member 4 (ACSL4) were significantly up-regulated in ATB-DILI. Further exploration indicated that this phenomenon was linked to the endoplasmic reticulum stress factor X-box binding protein 1 (XBP1). XBP1 activation significantly enhanced the synthesis of PUFAs, thereby increasing the level of lipid peroxidation and ferroptosis, ultimately resulting in ATB-DILI. Moreover, serum Apolipoprotein levels in A-IV (APOA4) and triglyceride were elevated and may serve as early warning biomarkers for ATB-DILI.

Conclusions

These results systematically revealed the importance of XBP1 as a therapeutic target, and clarified the feasibility of using APOA4 and triglyceride as novel early warning biomarkers for ATB-DILI.

Anti-tuberculosis drug-induced liver injury (ATB-DILI)

Polyunsaturated fatty acid (PUFA)

APOA4

XBP1

Ferroptosis

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis ¹. In 2022, there were 10.6 million new tuberculosis cases worldwide, according to the World Health Organization's "Global Tuberculosis Report 2023". With 748,000 new cases of tuberculosis, China ranked third in the world and accounted for 7.1% of the global incidence ². A combination of anti-tuberculosis drugs such as isoniazid (INH) and rifampicin (RIF) is used as first-line therapy for TB, but hepatotoxicity of the drug combination increased significantly ^3,4. Some patients will develop anti-tuberculosis drug-induced liver injury (ATB-DILI), which severely affects the effectiveness of TB treatment.

Anti-tuberculosis drugs are metabolized mainly in the liver and consume a large amount of intracellular reducing substances such as glutathione during their metabolism, significantly increasing oxidative activity ^5,6. This intracellular peroxidation will affect the normal hepatocellular metabolism, produce various effects including endoplasmic reticulum stress, lipid metabolism disorders, and cause liver cell death ^7–10. In the previous study, we found that lipid peroxidation and ferroptosis are linked to ATB-DILI ¹¹. However, the role of lipid peroxidation and ferroptosis in the process of ATB-DILI and its specific regulatory mechanisms need to be further elucidated.

X-box binding protein 1 (XBP1) is a transcription factor that plays a pivotal role in the regulation of cellular responses to endoplasmic reticulum (ER) stress. During ER stress, the ER-resident protein inositol-requiring enzyme-1 splices the XBP1 mRNA to produce a potent transcriptional transactivator termed XBP1s ¹². Other ER stress transcription factors ATF4 and ATF6 can also activate XBP1 transcription and enhance the activity of IRE1α-XBP1 pathway ¹³. XBP1 is widely expressed in the hepatic tissues and is upregulated throughout the development of the liver as well as during the healing of liver injury ¹⁴. During liver lipid synthesis and secretion, the expression level of XBP1 increases, which subsequently increases the concentrations of fatty acids, lecithin, and phosphatidylethanolamine ¹⁵. Liver-specific knockout XBP1 mice had decreased levels of serum cholesterol, triglycerides, and free fatty acids ¹⁶. Studies have shown that ER stress-related mechanisms are strongly correlated with ATB-DILI ⁵. Therefore, Systematic identification of the role of XBP1 in hepatotoxicity induced by anti-TB drugs is highly important.

Here, we revealed that PUFAs synthesis was abnormally activated in hepatocytes during anti-TB drug metabolism by using multi-omics sequencing. Ferroptosis-related arachidonic acid and phosphatidylethanolamine and the key enzyme Acyl-CoA synthetase long-chain family member 4 (ACSL4) were significantly up-regulated occurring in ATB-DILI. The levels of serum Apolipoprotein A-IV (APOA4) and triglyceride were also elevated and may serve as early warning biomarkers for ATB-DILI. Furthermore, we demonstrated that XBP1 played a key role in this abnormal lipid metabolism, which promotes the development of lipid peroxidation and ferroptosis, resulting in liver injury. Taken together, our study reveals the mechanism of XBP1-mediated activation of PUFAs metabolism caused by anti-TB drugs, and the great potential of APOA4 and triglyceride as early warning markers of ATB-DILI, which are expected to become specific early warning markers of ATB-DILI. Our study provided a new perspective on the prevention and treatment of ATB-DILI.

2.1 Animal Experiments

Eight-week-old C57BL/6 mice were obtained and fed at Soochow University (Suzhou, China). All animal experiments were approved by the Committee on the Use of Live Animals of Soochow University (D-2021-011). The mice were evenly divided into groups according to body weight and state. One group was the control group that received an equivalent volume of 1×PBS, and experimental groups were given a combination of rifampicin (Shenyang Hongqi Pharmaceutical Co., Ltd. China) and isoniazid (Shenyang Hongqi Pharmaceutical Co., Ltd.) by intragastric administration to induce ATB-DILI as described in a previous study ¹¹. The mice of Xbp1 liver-specific knockdown were generated by adeno-associated virus (AAV) injected via the tail vein. The status and weight of the mice were observed and recorded every day. At the indicated time points, the mice were sacrificed with cervical dislocation, and the livers were immediately harvested.

2.2 RNA-seq data analysis

Fresh livers were stripped and ground and the total amount and integrity of the RNA were assessed using an RNApure Tissue and Cell Kit (CWBIO, China). The total RNA was sequenced via the 150 bp paired method (Novogene, China). The HISAT2, SAMtools and HTseq-count packages were used to align 150 bp paired-end reads with hg19 (UCSC, USA). The expression of genes and transcripts was quantified using Cufflinks tools. Pathway enrichment was performed using the GO analysis.

2.3 ChIP-seq data analysis

A chromatin immunoprecipitation (ChIP) assay was performed as previously described ¹⁷. Briefly, 50 mg of liver tissue was crosslinked with 1% formaldehyde (Sigma‒Aldrich, USA), after which the reaction was stopped by ice-cold PBS (Gibco, USA) containing 0.125 M glycine (AmericanBio, USA). Nuclei were then isolated, and the chromatin was immunoprecipitated with protein A/G magnetic beads (Thermo Fisher, USA) conjugated with anti-IgG (Cell Signaling Technology, USA) and anti-H3K27ac (Cell Signaling Technology) overnight at 4°C. The DNA was eluted from the beads and subjected to sequencing and analysis.

2.4 Flow cytometry

The levels of lipid peroxidation and Fe ions in liver tissues were detected by flow cytometry. Firstly, fresh liver tissues were minced and washed with PBS. Subsequently, the tissues were placed in a centrifuge tube containing 1ml of 0.1% collagenase III (Solarbio, China) and incubated at 37°C for 1 hour. Next, the fresh tissues were filtered through a 300-mesh filter (Solarbio) to obtain single cells. 2 ml of Red Blood Cell Lysis Buffer (Biotime, China) was added for lyzing red blood cell, followed by incubating with C11-BODIPY^581/591 (Thermo Fisher) or Phen Green SK (Thermofisher) for 30 min at 37°C. The cellular levels of lipid peroxidation and Fe ions were analyzed by flow cytometer (FACS Canto II, BD Biosciences). Flow cytometry data was analyzed by FlowJo Software.

2.5 Quantitative real-time PCR (qPCR)

Isolated RNA was used as templates for reverse transcription reactions using PrimeScript™ RT Master Mix kit (Takara, Japan). Quantitative real-time PCR analysis was performed using SYBR Green Real-Time PCR Master Mixes (Thermo Fisher) on a LightCycler 480 Ⅱ Real-Time PCR Detection System (Thermo Fisher). Each sample was repeated three times, and cycle threshold (Ct) values were normalized to Gapdh levels using the 2^−ΔΔCt method. The primer sequences used are listed in Supplementary Table 1.

2.6 Western blot analysis

Total cellular proteins were obtained with a protein lysis buffer (Beyotime, China) and measured the concentration of protein by BCA protein assay kit (Beyotime). Subsequently, the protein samples were separated by 10% SDS-PAGE gel (Beyotime) and transferred to PVDF membrane (Merck Millipore, USA). Then membranes were blocked with 5% non-fat milk (Beyotime) for 1 hour at room temperature and incubated with primary antibodies Xbp1s, Fads1, Elovl5, Acsl4, Apoa4, Fasn, Acly, Acc and Gapdh (all purchased from Proteintech, China) at 4°C overnight, followed by incubation with secondary antibodies (goat anti-rabbit IgG, Proteintech). Enhanced chemiluminescence (ECL, Tanon, China) was used to generate the signals and captured by gel imager (Tanon 5200).

2.7 Liquid chromatography tandem mass spectrometry (LC-MS/MS)

LC-MS/MS-based lipidomics was performed as described previously with modifications¹⁷. Briefly, the total cellular lipids were extracted with methyl tert-butyl ether (MTBE) (Sigma Aldrich) from fresh liver tissues and dried in a SpeedVac concentrator (Thermo Scientific). Lipid samples were resuspended in 50% isopropanol/50% methanol and analyzed by LC-MS/MS. LC-MS/MS analyses were performed using an UHPLC system (1290, Agilent Technologies), equipped with a Kinetex C18 column (2.1 * 100 mm, 1.7 µm, Phenomen). The QE mass spectrometer was used for its ability to acquire MS/MS spectra on data-dependent acquisition (DDA) mode in the control of the acquisition software (Xcalibur 4.0.27, Thermo). The acquired raw files were analyzed using LipidSearch (v1.4) (Thermo Scientific) for sample alignment, MS2 identification, and MS1 peak area calculation. Statistical analysis was conducted using the Perseus (v1.6.6.0) software, wherein the p values were calculated by two-tailed Student’s t-test and corrected for multiple hypothesis testing via the Benjaminin-Hochberg method. Volcano plots were generated using the ggplot2 in the R environment (R Development Core Team; https://www.r-project.org/) (v3.5.0).

2.8 Immunofluorescence Microscopy

Fresh liver tissue was immediately inserted into a universal tissue fixative (Servicebio, China). The tissues were subsequently placed on an embedding table and embedded in OCT embedding compound. The embedding table was placed on the quick-freezing table of the frozen slicer, and the tissue was sectioned at 8 µM after the OCT compound hardened. Frozen liver sections were stained with 0.1% LipidTOX (Thermo Fisher) solution, and the nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI, Beyotime). Fluorescence images were captured using a confocal microscopy (Carl Zeiss, Germany).

2.9 Immunohistochemistry (IHC) and Hematoxylin and eosin (H&E) staining

This study was approved by the Ethics Committee of the Institute at the Affiliated Infectious Diseases Hospital of Soochow University (K-2021-011-01). Liver biopsy tissue was obtained in patients with liver injury caused by antituberculosis drugs combined with isoniazid and rifampicin. The immunohistochemical staining assay was performed with the XBP1s antibody (ab37152, Abcam, USA) and H&E staining were performed as described previously¹¹.

2.10 Statistical analysis

All the statistical analyses were performed using SPSS 24.0 software (SPSS, Inc., USA). The experimental results were statistically evaluated using Student’s t-test for comparisons between two groups or one-way ANOVA for comparisons between more than two groups. Differences were considered significant if p < 0.05.

3.1 Remarkable activation of the hepatic PUFA synthesis pathway occur during the metabolism of anti-TB drugs.

To explore the mechanism of lipid peroxidation and ferroptosis in the hepatic injury induced by the combination of anti-TB drugs, RNA-seq was performed on the liver tissues from ATB-DILI mice with 7 days and 14 days of treatment combined with 100 mg/kg INH and 150 mg/kg RFP. We analyzed the heterogeneity within every group and found that the consistency of sequencing was very high (Fig. 1A). Then we explored the differentially expressed genes (DEGs) genes using RNA sequencing data and found several lipid metabolisms related genes were upregulation upon ATB-DILI (Fig. 1B). Interestingly, as the degree of liver injury intensifies, the expression of PUFA-related genes becomes higher. Unbiased pathway enrichment analysis of DEGs (fold change > 1) from RNA-seq data upon ATB-DILI further confirmed that the lipid metabolism pathways such as arachidonic acid metabolism pathway and linoleic acid metabolism pathway were among the top 10 pathways according to pathway analysis (Fig. 1C). Besides, the amount of intracellular lipid droplets increased in the ATB-DILI liver tissues (Fig. 1D).

Moreover, the volcano plot of RNA-seq upon ATB-DILI with 14 days of treatment showed that the upregulation genes (446 genes) were much more than the downregulation genes (243 genes, Fig. 1E). Next, we examined the expression changes of several important lipid metabolism genes in ATB-DILI. There was a significant increase in the expression of the PUFA synthesis pathway, which involves Scd1, Fads1, and Elovl5 (Fig. 1F and 1G). In contrast, genes involved in fatty acid de novo synthesis enzymes, such as Fasn, Acc, and Acly, were unaffected by the anti-TB drugs (Fig. 1G).

3.2 Phosphatidylethanolamine and triglyceride are abnormally increased in ATB-DILI

The above results indicated the existence of large-scale genes activation during liver injury, suggesting that it may be regulated at the transcriptome level. H3K27ac ChIP-seq was performed in the negative control group and ATB-DILI model with 14 days of anti-TB drugs treatment. The ChIP-seq data illustrated that the overall transcriptome intensity of H3K27ac, the transcriptional activating histone marker, was significantly increased in the promoter regions of genes in ATB-DILI liver tissues (Fig. 2A). As shown in Fig. 2B, the heatmap showed that there was enhanced expression of H3K27ac in the promoter regions of a large number of genes, confirming that the genome level of gene transcription was increased significantly in liver cells after ATB-DILI. The IGV plot of H3K27ac ChIP-seq data also showed elevations in the promoter and enhancer regions of these PUFA synthetase Scd1, Fads1, and Elovl5 genes (Fig. 2C).

To better clarify the specific lipid changes during liver injury, we next performed lipidomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It can be seen from the results of the OPLS-DA score chart that the difference between the two groups of samples is very significant (Fig. 2D). A total of 1,106 lipids were identified and quantified (Fig. 2E). As shown in Fig. 2E, 873 lipid species was increased and 232 was reduced. These results further demonstrated that the intracellular lipid synthesis was significantly and abnormally activated when the hepatocyte metabolized the anti-tuberculosis drugs. Specifically analyzing the abnormally activated lipid classes, we found that the expression of phosphatidylethanolamine (PE) and triglyceride (TAG) increased tens of thousands of times comparing to the negative control (Fig. 2F and 2G). Finally, we used a schematic diagram to integrate multi-omics data of RNA-Seq, ChIP-Seq and lipidomics and identify the abnormal genes and associated lipid substrates in the process of ATB-DILI (Fig. 2H).

3.3 Serum APOA4 and triglyceride may be early warning biomarkers for ATB-DILI.

Here, through multi-omics, we found that the expression of APOA4 and triglyceride in hepatocytes was increased significantly during ATB-DILI. The liver is the main organ of lipid metabolism and determines the level of lipids in the circulatory system. So, we measured the concentration of APOA4 and triglyceride in serum during liver injury to evaluate the value in predicting ATB-DILI. We used a negative ATB-DILI mice model, which was treated with a low dose of anti-TB drug (25 mg/kg INH and 50 mg/kg RFP) ¹¹. Furthermore, we collected clinical serum samples from 10 patients who received anti-TB drug treatment but had not yet developed liver injury. These results revealed that the concentration of serum APOA4 and triglyceride elevated after drug treatment, while the other indicators remained unchanged, indicating that serum APOA4 and triglyceride levels may be early warning biomarkers for predicting ATB-DILI. (Fig. 3A and 3B).

3.4 The endoplasmic reticulum stress molecule XBP1 is activated in ATB-DILI.

To explore the mechanism underlying the regulation of abnormal lipid metabolism, we focused on core transcription factors of fatty-acid metabolism ¹⁷. Because protein processing in the ER pathway was enriched among the top 3 according to the pathway analysis, we screened the transcription factors associated with endoplasmic reticulum and liver lipid metabolism in the multi-omics data (Fig. 1C). The transcription activation marker H3K27ac was enriched in the promoter and enhancer areas of Xbp1 (Fig. 4A). qPCR analysis showed significantly elevated expression levels of Xbp1 (Fig. 4B). And Western blot analysis revealed the spliced form of Xbp1 (Xbp1s) was also increased in ATB-DILI tissues (Fig. 4C). Moreover, we detected XBP1s expression in liver biopsies from patients with liver injury through immunohistochemistry (IHC) and found that the expression of XBP1 in areas with liver injury was significantly higher than that in areas without liver injury (Fig. 4D).

3.5 Liver-specific knockdown of XBP1 can reduce abnormal synthesis of polyunsaturated fatty acids and ATB-DILI.

We employed an adeno-associated virus that specifically knocked down hepatocellular XBP1 of mice to assess the role of XBP1 in ATB-DILI. The mice treated with the combination INH and RFP (as the negative control group) were 50% dead after 8 days of treatment (Fig. 5A). The survival of mice was significantly improved by injecting adeno-associated virus (AAV-Xbp1-KD group) into the tail vein. After Xbp1 knockdown, the liver weight to body weight ratio in the mice was reduced compared with the negative control group (Fig. 5B). The concentration of serum ALT, ALP, and AST were also decreased following Xbp1 knockdown (Fig. 5C). It can be seen in H&E sections that the vacuoles in liver cells have been significantly improved (Fig. 5D). And the lipid droplet number in the frozen sections from the knockdown group was decreased compared with that in the negative control group (Fig. 5E).

Besides, flow cytometry analysis revealed that the level of lipid peroxidation (C11-BODIPY^581/591) and Fe ion concentration (Phen Green SK, PGSK) were decreased after Xbp1 knockdown (Fig. 5F). The expression of the PUFA synthase genes Scd1, Fads1, Elovl5, Apoa4 and especially, the ferroptosis marker Acsl4 was dramatically downregulated in AAV-Xbp1-KD group (Fig. 5G and 5H). Taken together, these data uncovered the key role of XBP1 in the synthesis of PUFAs and the occurrence of ferroptosis in ATB-DILI. Given that XBP1 regulates abnormal lipid metabolism in DILI, it is conceivable that downregulation of XBP1 might alleviate the hepatotoxicity of anti-tuberculosis drugs.

Recent studies have found that lipid metabolism changes and abnormal accumulation of lipids occur in the liver during the metabolism of anti-tuberculosis drugs. RIF promotes lipid synthesis and accumulation in the liver by activating the expression of PPARG ¹⁸. The combined use of INH and RFP resulted in a significant increase in cholesterol, triglycerides, and total lipids in the liver ¹⁹. The serum levels of high-density lipoprotein and apolipoprotein A are elevated after RFP treatment ²⁰. And combination of RFP and INH can increase serum cholesterol and phospholipid levels ^19,21. However, these studies only focus on the changes in lipid classes during the metabolism of anti-tuberculosis drugs, lacking systematic mechanisms of hepatic lipid metabolism induced by anti-tuberculosis drugs and their role in the process of ATB-DILI.

Here, with the advent of high-throughput sequencing technology, we have been able to uncover and analyze how anti-TB drugs affect the transcriptome and metabolome of hepatocytes ²². By using RNA-seq, H3K27ac ChIP-seq, and lipid LC-MS/MS on the liver tissues from the ATB-DILI model, our present study demonstrated that the expression of members in the PUFA synthesis pathway upregulated both the transcriptional and protein levels. Interestingly, unlike the enhancement of the PUFA synthesis pathway, the expression levels of fatty acid de novo synthetases were not altered. We found that the cellular content of phosphatidylethanolamine and triglyceride increased tens of thousands of times compared to anti-TB metabolism. This was because the expression of genes such as SCD1, FADS1, and ELOVL5 was up-regulated during ATB-DILI, which enhanced the synthesis of PUFA and provided sufficient raw materials for the elevation of phosphatidylethanolamine and triglyceride levels. Of note, some of these up-regulated lipids, such as phosphatidylethanolamine (PE), can react with arachidonic acid (AA) by ACSL4 to form AA-PE which was shown to be one of the most important causes of lipid peroxidation and ferroptosis ^23,24. These results clearly illustrated that the abnormal accumulation of PUFAs, rather than saturated fatty acids, is the key link between ferroptosis and ATB-DILI, which further confirmed our previously discovered and other studies ^11,25.

Currently, standard serum liver biomarkers, such as alanine transaminase, alkaline phosphatase, and aspartate aminotransferase are used to diagnose and monitor ATB-DILI ²⁶. However, these parameters are less effective in the early diagnosis of ATB-DILI. Hence, we screened related lipid-related biomarkers for predicting ATB-DILI based on our results and found that APOA4 and triglyceride are potential serum early warning biomarkers in early diagnosis of ATB-DILI. As a plasma protein that modulates several metabolism processes and participates in various physiological functions, the plasma APOA4 concentration is positively correlated with the HDL-H level ²⁷. Studies have reported that APOA4 is a biomarker for the early diagnosis of liver fibrosis ²⁸. The detection of serum triglyceride is a simple and cheap detection method. Our study suggested that the occurrence of liver injury could be predicted through a common indicator. In the future, a larger clinical sample size is needed to further analyze the accuracy of APOA4 and triglyceride as early predicted biomarkers of ATB-DILI compared to that of other classical biomarkers.

Emerging evidence indicates that various toxic substances of anti-TB drugs induce the unfolding of proteins, thereby triggering ER stress ²⁹. INH may cause hepatotoxicity by increasing ROS levels, which weakens antioxidant capacity, leading to ER stress and liver injury ³⁰. RFP-induced hepatoxicity is closely related to ER stress by activating bile acid accumulation or the PXR-CYP enzyme-XBP1-IRE1 signaling pathway, which may affect changes in the stress pathway mediated by activation of ATF4, ATF6, and XBP1 transcription factors ^31,32. Moreover, 4-phenylbutyrate (4-PBA) has been reported to attenuate RFP-induced liver cell injury by inhibiting ER stress and the ubiquitination and degradation of MRP2 ³³. In this study, we found that the expression of the endoplasmic reticulum stress factor XBP1 was significantly higher in ATB-DILI than in the control group. The activation form XBP1s was overexpressed in the liver biopsy tissues of ATB-DILI patients, indicating that the activation of XBP1 is closely related to the ATB-DILI. However, current studies targeting XBP1 have yielded mixed results, with some showing that hepatocyte-specific XBP1 deletion worsened liver injury ^12,34. Other studies have shown that inhibition of XBP1 could play a protective role in liver injury and other liver diseases ^35–37. Consistent with the latter, in our experiments, we explored the molecular link between XBP1, the abnormal hepatocyte lipid metabolism and ferroptosis in ATB-DILI. We demonstrated that the knockdown of XBP1 significantly reduces the ATB-DILI by inhibiting PUFAs synthesis. Lipid peroxidation and ferroptosis were also significantly restrained, which provides new insight into the role of XBP1 in ATB-DIL. Based on our findings, we speculate that pharmacological inhibition of XBP1 may represent a promising strategy for treating ATB-DILI.

In conclusion, using comprehensive omics analyses, mouse models, and clinical samples, this study provides new mechanistic insight into the link between abnormal lipid metabolism and ATB-DILI. Moreover, this study identified early warning biomarkers and promising therapeutic targets to address the unmet clinical need for the prevention of tuberculosis and enhancement of current tuberculosis treatment options.

ATB-DILI: anti-tuberculosis drug-induced liver injury; PUFAs: polyunsaturated fatty acids; TB: Tuberculosis; XBP1: X-box binding protein 1; APOA4: Apolipoprotein A4; INH: isoniazid; RIF: rifampicin; ER: endoplasmic reticulum; SCD1: Stearoyl-Coenzyme A desaturase 1; FADS1: Fatty Acid Desaturase 1; ELOVL5: ELOVL Fatty Acid Elongase 5; FASN: Fatty Acid Synthase; ACLY: ATP Citrate Lyase; ACC: Acetyl-CoA Carboxylase Alpha; ACSL4: Acyl-CoA synthetase long-chain family member 4; PE: phosphatidylethanolamine; TAG: triglyceride.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Institute at the Affiliated Infectious Diseases Hospital of Soochow University (K-2021-011-01). All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects and/or their legal guardian(s).

All animal experiments were approved by the Committee on the Use of Live Animals of Soochow University (D-2021-011). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines.

Data availability statement

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

This study was supported by Translational Research Grant of ECHD (GCZ202005), Suzhou Key Medical Center, Science and Technology Project of Suzhou (SLT2021010), Basic Research on Medical and Health Application of the People’s Livelihood Science and Technology Project of Suzhou (SKJY2021139, SKY2022208), Suzhou Basic Research Program (Medical Applied Basic Research) (SKY2023072).

Competing interests

The authors declare that they have no declaration of interest.

Authors’ contribution statement

QYL, YJ, and QS performed the experiments and analyzed the data. YJ and YQG analyzed and interpreted the data. FXZ, JPZ, and MYW provided the necessary materials. YZP and SM conceived and designed the study and drafted the manuscript. YLQ, MYW, QS, and YZP secured financing for the study. All authors read and approved the final manuscript.

Acknowledgments

Not applicable.

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XBP1 activation elevates abnormal synthesis of polyunsaturated fatty acids in hepatocytes to promote liver injury during anti-tuberculosis drug metabolism

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Abstract

Objective

Methods

Results

Conclusions

Figures

1. Introduction

2. Materials and Methods

2.1 Animal Experiments

2.2 RNA-seq data analysis

2.3 ChIP-seq data analysis

2.4 Flow cytometry

2.5 Quantitative real-time PCR (qPCR)

2.6 Western blot analysis

2.7 Liquid chromatography tandem mass spectrometry (LC-MS/MS)

2.8 Immunofluorescence Microscopy

2.9 Immunohistochemistry (IHC) and Hematoxylin and eosin (H&E) staining

2.10 Statistical analysis

3. Results

3.1 Remarkable activation of the hepatic PUFA synthesis pathway occur during the metabolism of anti-TB drugs.

3.2 Phosphatidylethanolamine and triglyceride are abnormally increased in ATB-DILI

3.3 Serum APOA4 and triglyceride may be early warning biomarkers for ATB-DILI.

3.4 The endoplasmic reticulum stress molecule XBP1 is activated in ATB-DILI.

3.5 Liver-specific knockdown of XBP1 can reduce abnormal synthesis of polyunsaturated fatty acids and ATB-DILI.

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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