INTRODUCTION: Endoscopic biliary brushing is the most common method used for sampling in patients with malignant biliary strictures (MBSs); however, its sensitivity is relatively low. There is still no consensus on endoscopy-based biliary brushing, although brushing 10 times in 1 specimen is routinely performed. This study was designed to compare the sensitivity of brush cytology for 10, 20, or 30 brushing times of a pass in 1 specimen in patients with MBSs. METHODS: In this multicenter, prospective, randomized controlled study, patients who underwent endoscopic retrograde cholangiopancreatography for suspected MBSs were enrolled. Patients were randomly assigned to receive 10, 20, and 30 brushing times of a pass. The primary outcome was to compare the sensitivity of brush cytology among the 3 groups. Patients were prospectively followed up for 6 months after endoscopic brushing for malignancy diagnosis. RESULTS: A total of 443 patients were enrolled for intention-to-treat analysis (147, 148, and 148 patients in the 10-times, 20-times, and 30-time groups, respectively). The 3 groups were similar in baseline characteristics. The sensitivity of brush cytology was 38%, 47%, and 57% in the 10-times, 20-times, and 30-times groups, respectively, and the 30-times group showed significantly higher sensitivity than the 10-times group (P = 0.001). The multivariate analysis revealed that stricture length and the number of brushing passes were significant factors for the detection of biliary malignancy. No significant differences were observed in procedure-related complications among the 3 groups. DISCUSSION: Brushing 30 times could increase the diagnostic sensitivity without increasing complications and seems to be preferred for the endoscopic sampling and diagnosis of MBSs (chictr.org.cn, identifier: ChiCTR1800015978).

Liver non-parenchymal cells (NPCs) play a critical role in the progression of non-alcoholic steatohepatitis (NASH). We aimed to explore the heterogeneity of NPCs and identify NASH-specific subpopulations contributing to NASH progression. Through single-cell RNA sequencing, we uncovered a proinflammatory subpopulation of Itgad^hi/Fcrl5^hi macrophages with potential function of modulating macrophage accumulation and promoting NASH development. We also identified subpopulations of Egr1^hi and Ly6a^hi liver sinusoidal endothelial cells (LSECs), which might participate in pathological angiogenesis and inflammation regulation. The Itgad^hi/Fcrl5^hi macrophages, Egr1^hi LSECs, and Ly6a^hi LSECs emerged in the early stage and expanded significantly along with pathological progression of liver injury during NASH. Cell-cell interactions between hepatic stellate cells (HSCs) and Itgad^hi/Fcrl5^hi macrophages, Egr1^hi LSECs or Ly6a^hi LSECs were enhanced in NASH liver. Our results revealed that expansion of Itgad^hi/Fcrl5^hi macrophages, Egr1^hi LSECs or Ly6a^hi LSECs was strongly associated with NASH severity, suggesting these subpopulations might be involved in NASH progression.

Chronic liver disease (CLD) is currently a major health problem worldwide, which is accompanied by chronic liver injury and lack of clinically effective treatment; however, systematic characterization of chronic liver injury procedures at single-cell resolution is lacking. In the present study, we established chronic liver injury mouse models and conducted single-cell RNA sequencing (scRNA-seq), including choline-deficient, ethionine-supplemented (CDE) and 3,5-diethoxycarbonyl 1,4-dihydrocollidinen (DDC) mouse models. We captured in total 16,389 high-quality cells and identified 12 main cell types in scRNA-seq data. Macrophages and endothelial cells are the largest cell populations in our dataset. Transcriptional trajectory analysis revealed different expression patterns of cells between CDE and DDC models and identified potential liver injury markers, such as Ets1, Gda, Itgam, and Sparc. Differential analysis identified 25 and 152 differentially expressed genes in CDE and DDC macrophages, respectively. In addition, 413 genes were detected to exclusively express in specific pseudotime states of macrophages. These genes were found to participate in immune-related biological processes. Further cell-cell communication analysis found extensive receding of cell-cell interactions between different cell types in the liver injury process, especially in the DDC model. Our study characterized the single-cell transcriptional landscape in the process of chronic liver injury, promoting the understanding of the underlying molecular mechanisms and providing candidate clinical strategy for effective intervention of chronic liver diseases.

Background and Aim: Hydronidone (HDD) is a novel pirfenidone derivative developed initially to reduce hepatotoxicity. Our previous studies in animals and humans have demonstrated that HDD treatment effectively attenuates liver fibrosis, yet the underlying mechanism remains unclear. This study aimed to investigate whether HDD exerts its anti‐fibrotic effect by inducing apoptosis in activated hepatic stellate cells (aHSCs) through the endoplasmic reticulum stress (ERS)‐associated mitochondrial apoptotic pathway.Methods: The carbon tetrachloride (CCl₄)‐ and 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC)‐induced liver fibrosis models were used for in vivo studies. In vitro studies were conducted using the human hepatic stellate cell line LX‐2. The apoptotic effect of HDD on aHSCs was examined using TUNEL and flow cytometry assays. The small interfering RNA (siRNA) technique was employed to downregulate the expression of interest genes.Results: HDD treatment significantly promoted apoptosis in aHSCs in both the CCl₄‐ and DDC‐induced liver fibrosis in mice and LX‐2 cells. Mechanistic studies revealed that HDD triggered ERS and subsequently activated the IRE1α‐ASK1‐JNK pathway. Furthermore, the influx of cytochrome c from the mitochondria into the cytoplasm was increased, leading to mitochondrial dysfunction and ultimately triggering apoptosis in aHSCs. Notably, inhibition of IRE1α or ASK1 by siRNA partially abrogated the pro‐apoptotic effect of HDD in aHSCs.Conclusions: The findings of both in vivo and in vitro studies suggest that HDD induces apoptosis in aHSCs via the ERS‐associated mitochondrial apoptotic pathway, potentially contributing to the amelioration of liver fibrosis.

Capillarization of liver sinusoidal endothelial cells (LSECs) is a central event in response to liver injury. In this study, we investigated the role of Yes-associated protein 1 (YAP1, also known YAP) in LSEC capillarization and liver injury. YAP expression was assessed in liver samples from mice injured by CCl4 injection and DDC diet. EC-specific Yap1 conditional knockout mice (Yap1∆end) were generated by breeding Yap1fl/fl mice with Cdh5-CreERT2 mice. HA-PEI/siYap1 nanoparticles were applied to specifically inhibit YAP expression in LSECs. YAP was primarily expressed in LSECs, and its expression was elevated during liver fibrosis. EC-specific Yap1 deficiency significantly increased the fenestrae in LSECs and mitigated hepatocyte death and liver fibrosis. Overexpression of YAP in EC aggravated capillarization, hepatocyte death, and liver fibrosis. Mechanistically, YAP inhibited Gata6 transcription via binding to its promoter and thus resulted in LSEC capillarization. Overexpression of GATA6 in EC alleviated capillarization and liver fibrosis by activating Nos3 transcription. Moreover, specific delivery of HA-PEI-siYap1 nanoparticles to LSEC alleviated liver injury in mice. YAP-GATA6/eNOS signaling is essential in LSEC capillarization and subsequent hepatocyte death. Interventions targeting YAP in LSECs offer a promising strategy for the treatment of liver fibrosis. YAP expression in LSEC is upregulated in injured mice. Deletion/overexpression of Yap1 in LSECs attenuates/aggravates capillarization, hepatocyte death, and liver injury in mice, respectively. YAP inhibits Gata6 transcription via binding to its promoter and results in LSEC capillarization through GATA6/eNOS-dependent pathway. Delivering siYap1 to LSEC via nanoparticles represents a new approach for treating liver fibrosis.