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Background and Aims Characterization of cell specific transcriptional responses to hepatotoxicants is lost in the averages of bulk RNA-sequencing (RNA-seq). Single-cell/nuclei RNA-seq technologies enable the transcriptomes of individual cell (sub)types to be assessed within the context of in vivo models. Methods Single-nuclei RNA-sequencing (snSeq) of frozen liver samples from male C57BL/6 mice gavaged with sesame oil vehicle or 30 μg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4 days for 28 days was used to demonstrate the application of snSeq for the evaluation of xenobiotics. Results A total of 19,907 genes were detected across 16,015 nuclei from control and TCDD-treated livers. Eleven cell (sub)types reflected the expected cell diversity of the liver including distinct pericentral, midzonal, and periportal hepatocyte subpopulations. TCDD altered relative proportions of cell types and elicited cell-specific gene expression profiles. For example, macrophages increased from 0.5% to 24.7%, while neutrophils were only present in treated samples, consistent with histological evaluation. The number of differentially expressed genes (DEGs) in each cell type ranged from 122 (cholangiocytes) to 7625 (midcentral hepatocytes), and loosely correlated with the basal expression level of Ahr, the canonical mediator of TCDD and related compounds. In addition to the expected functions within each cell (sub)types, RAS signaling and related pathways were specifically enriched in nonparenchymal cells while metabolic process enrichment occurred primarily in hepatocytes. snSeq also identified the expansion of a Kupffer cell subtype highly expressing Gpnmb, as reported in a dietary NASH model. Conclusions We show that snSeq of frozen liver samples can be used to assess cell-specific transcriptional changes and population shifts in models of hepatotoxicity when examining freshly isolated cells is not feasible., Graphical abstract

Background Abnormal construction of the extracellular matrix (ECM) is intimately linked with carcinogenesis and the development of solid tumours, especially hepatocellular carcinoma (HCC). As the major component of the ECM, collagen plays a pivotal role in carcinogenesis. P4HA2, the essential enzyme during collagen formation, becomes an important target in HCC treatment. Here, we tried to decipher whether aspirin (ASA), a classic anti-inflammatory drug, could improve the prognosis of HCC through targeting P4HA2. Methods Western blotting, qRT-PCR assay, immunofluorescence staining, luciferase reporter gene assay, and ChIP assay were applied to demonstrate the molecular mechanism of the regulation of P4HA2 expression by aspirin. A mouse xenograft model, cell viability assay, colony formation assay, and immunohistochemistry analysis were used to evaluate the anti-fibrosis effect of aspirin through targeting the NF-κB/P4HA2 axis and LMCD1-AS1/let-7g/P4HA2 axis in vitro and in vivo. The TCGA database was used to evaluate the correlation among P4HA2, let-7g, LMCD1-AS1 and overall survival of HCC patients. Findings In xenograft mice, aspirin was capable of targeting P4HA2 to decrease collagen deposition, resulting in the inhibition of liver tumour growth. TCGA database analysis revealed the close association between a higher P4HA2 concentration in HCC patients and shorter overall survival or a higher cancer stage and the pathological grade. Mechanistically, NF-κB can bind to the promoter of P4HA2 to activate its transcription. Moreover, lncRNA LMCD1-AS1 functions as a molecular sponge of let-7g to post-transcriptionally induce the target gene of let-7g, namely, P4HA2. Interpretation Our findings disclose the novel role and regulatory mechanism of aspirin in the suppression of HCC by disrupting abnormal collagen deposition. Funds 973 Program, National Natural Scientific Foundation of China, Fundamental Research Funds for the Central Universities, Project of Prevention and Control of Key Chronic Non-Infectious Diseases.

Increasing evidence indicates that mitochondrial-associated redox signaling contributes to the pathophysiology of heart failure (HF). The mitochondrial-targeted antioxidant, mitoquinone (MitoQ), is capable of modifying mitochondrial signaling and has shown beneficial effects on HF-dependent mitochondrial dysfunction. However, the potential therapeutic impact of MitoQ-based mitochondrial therapies for HF in response to pressure overload is reliant upon demonstration of improved cardiac contractile function and suppression of deleterious cardiac remodeling. Using a new (patho)physiologically relevant model of pressure overload-induced HF we tested the hypothesis that MitoQ is capable of ameliorating cardiac contractile dysfunction and suppressing fibrosis. To test this C57BL/6J mice were subjected to left ventricular (LV) pressure overload by ascending aortic constriction (AAC) followed by MitoQ treatment (2 µmol) for 7 consecutive days. Doppler echocardiography showed that AAC caused severe LV dysfunction and hypertrophic remodeling. MitoQ attenuated pressure overload-induced apoptosis, hypertrophic remodeling, fibrosis and LV dysfunction. Profibrogenic transforming growth factor-β1 (TGF-β1) and NADPH oxidase 4 (NOX4, a major modulator of fibrosis related redox signaling) expression increased markedly after AAC. MitoQ blunted TGF-β1 and NOX4 upregulation and the downstream ACC-dependent fibrotic gene expressions. In addition, MitoQ prevented Nrf2 downregulation and activation of TGF-β1-mediated profibrogenic signaling in cardiac fibroblasts (CF). Finally, MitoQ ameliorated the dysregulation of cardiac remodeling-associated long noncoding RNAs (lncRNAs) in AAC myocardium, phenylephrine-treated cardiomyocytes, and TGF-β1-treated CF. The present study demonstrates for the first time that MitoQ improves cardiac hypertrophic remodeling, fibrosis, LV dysfunction and dysregulation of lncRNAs in pressure overload hearts, by inhibiting the interplay between TGF-β1 and mitochondrial associated redox signaling., Graphical abstract fx1