In metabolomics research, derivatization methods, particularly stable isotope derivatization, are commonly employed to enhance the coverage and qualitative and quantitative accuracy of analyte compounds. In our previous work, we adopted a homolog derivatization approach combined with our in-house developed mass spectrometry triple-dimensional derivatization filter (MS-TDF) software to achieve low-cost metabolomics studies. However, differential derivatization efficiencies across homologs led to an increased false positive rate. In this study, we introduce a natural isotope triple-dimensional combinatorial derivatization (NITCD) strategy that overcomes these limitations. The approach employs 4-bromo-2-hydrazinopyridine, which provides a characteristic 1:1 isotopic doublet pattern ( 79 Br/ 81 Br) for intelligent metabolite identification via MS-TDF software. Combined with 2-hydrazinopyridine as a structurally matched internal standard, the system not only reduces false-positive identifications but also enables reliable relative quantification. This strategy was successfully applied to a metabolomics study on rhein treatment in inflammatory bowel disease (IBD). The experimental results demonstrate that, by using NITCD strategy, 564 target compounds can be detected in mouse plasma, with 148 in colon tissue, and 81 in spleen tissue. More importantly, these metabolites were identified, along with their dynamic changes during rhein treatment. It was found that rhein might reverse the IBD-induced alterations in arachidonic acid metabolism, tyrosine metabolism, primary bile acid biosynthesis, and tryptophan metabolism.
Background This study aimed to investigate the roles and mechanisms of tenofovir alafenamide fumarate (TAF)/tenofovir disoproxil fumarate (TDF) in treating liver fibrosis. Methods The effects of TAF/TDF on carbon tetrachloride (CCl 4 )-induced liver fibrosis in C57BL/6 wild-type or nonstructural protein 5A transactivated protein 9 (NS5ATP9) knockout mice were studied. The differentiation, activation, and proliferation of LX-2 cells after TAF/TDF treatment were tested in vitro. The expression of NS5ATP9 and activities of transforming growth factor-β1 (TGFβ1)/Sekelsky mothers against decapentaplegic homolog 3 (Smad3) and NF-κB/NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome signaling pathways were detected in TAF/TDF-treated mice and LX-2 cells. The genes related to extracellular matrix accumulation were detected in vivo and in vitro after NS5ATP9 silencing or knockout. Results TAF/TDF significantly inhibited CCl 4 -induced liver fibrosis in mice, and regulated the differentiation, activation, and proliferation of hepatic stellate cells (HSCs). Furthermore, TAF/TDF suppressed the activities of TGFβ1/Smad3 and NF-κB/NLRP3 inflammasome signaling pathways in vivo and in vitro. NS5ATP9 inhibited liver fibrosis through TGFβ1/Smad3 and NF-κB signaling pathways. TAF/TDF upregulated the expression of NS5ATP9 in vivo and in vitro. Finally, TAF/TDF could only show marginal therapeutic effects when NS5ATP9 was silenced and knocked out in vivo and in vitro. Conclusions TAF/TDF prevented progression and promoted reversion of liver fibrosis through assembling TGFβ1/Smad3 and NF-κB/NLRP3 inflammasome signaling pathways via upregulating the expression of NS5ATP9. TAF/TDF also regulated the differentiation, activation, and proliferation of HSCs. The findings provided strong evidence for the role of TAF/TDF as a new promising therapeutic strategy in liver fibrosis.
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