Activation of the NOX4/NLRP3 inflammasome pathway has been associated with fibrosis in other organs. An imbalance in intestinal bacteria is an important driving factor of liver fibrosis through the liver-gut axis. This study aimed to explore whether the effect of ursolic acid (UA) on liver fibrosis was associated with the NOX4/NLRP3 inflammasome pathways and intestinal bacteria. Wild-type (WT), NLRP3^−/-, and NOX4^−/- mice and AP-treated mice were injected with CCI4 and treated with or without UA. The intestinal contents of the mice were collected and analyzed by 16S rRNA sequencing. UA alleviated liver fibrosis, which manifested as decreases in collagen deposition, liver injury, and the expression of fibrosis-related factors, and the expression of NOX4 and NLRP3 was significantly inhibited by UA treatment. Even after CCI4 injection, liver damage and fibrosis-related factors were significantly decreased in NLRP3^−/-, NOX4^−/-, and AP-treated mice. Importantly, the expression of NLRP3 was obviously inhibited in NOX4^−/- and AP-treated mice. In addition, the diversity of intestinal bacteria and the abundance of probiotics in NLRP3^−/- and NOX4^−/- mice was significantly higher than those in WT mice, while the abundance of harmful bacteria in NLRP3^−/- and NOX4^−/- mice was significantly lower than that in WT mice. The NOX4/NLRP3 inflammasome pathway plays a crucial role in liver fibrosis and is closely associated with the beneficial effect of UA. The mechanism by which the NOX4/NLRP3 inflammasome pathway is involved in liver fibrosis may be associated with disordered intestinal bacteria.

Liver fibrosis is a reversible wound‐healing response that occurs after liver injury. NADPH oxidases (NOXs) and reactive oxygen species (ROS) which are expressed in hepatocytes (HCs), hepatic stellate cells (HSCs), and Kupffer cells (KCs) play an important role in the development of hepatic fibrosis. In in vitro studies, we had shown that ursolic acid (UA) could reverse liver fibrosis by inhibiting the activation of NOX‐mediated fibrotic signaling networks in HSCs. In this study, we verified that UA could alleviate CCl4‐induced liver fibrosis by reducing the expression of NOXs/ROS in HCs, HSCs, KCs. Meanwhile, the phagocytic index α and clearance index K which represent phagocytosis of KCs were unchanged. Together, all our data demonstrated that UA induced the proliferation of HCs, promoted apoptosis in HSCs, and prevented activation of KCs in vivo by reducing the expression of NOXs/ROS in HCs, HSCs, KCs. Besides, UA had no effect on the host defense function.

Pyroptosis has an important role in liver inflammation and fibrosis. The role of KC pyroptosis in liver fibrosis was unclear. Ursolic acid (UA) has antifibrotic effects, but study on the effect and mechanism of UA on KC pyroptosis in liver fibrosis has not been reported. Therefore, we induced KC pyroptosis using Lipopolysaccharide (LPS) and nigericin (Nig) in vitro. C57BL/6J mice were intraperitoneally injected with carbon tetrachloride (CCl₄) to establish a liver fibrosis model. We demonstrated that UA attenuated CCl₄-induced liver fibrosis, liver damage, and KC pyroptosis of liver tissue. Moreover, KCs were treated with UA and small interfering RNA of NOX2, which showed that inhibiting the NOX2/NLRP3 inflammasome signaling pathway attenuated KC pyroptosis and UA abrogated this effect via suppressing this pathway in vitro. Furthermore, mice were treated with UA, GSK2795039 (a specific inhibitor of NOX2) or MCC950 (a specific inhibitor of NLRP3). Compared with inhibiting NOX2 alone, inhibiting NOX2 in the presence of UA did not markedly ameliorate KC pyroptosis of liver tissue in CCl₄-induced liver fibrosis. In addition, when NLRP3 was silenced or inhibited, the result was similar to that of knocking down NOX2 in vivo and in vitro. These results indicate that UA attenuates liver fibrosis in mice via inhibiting KC pyroptosis, which may be through the suppression of NOX2/NLRP3 inflammasome signaling pathway. It may be a new target for treating liver fibrosis and provide a new theoretical basis for the use of UA to treat liver fibrosis.

Liver fibrosis, driven by chronic injury, hinges on hepatic stellate cell (HSC) activation. Microfibrillar-associated protein 4 (MFAP4), an extracellular matrix protein critical for elastic fiber assembly, is upregulated in hepatic fibrosis, yet its mechanistic role remains unclear. Liver fibrosis was induced in wild-type and Mfpa4 knockout mice using CCl₄ and TAA, while LX-2 cells were activated with TGF-β1. Bioinformatics analysis, histopathology, double immunofluorescence, flow cytometry, Transwell co-culture systems, Western blot, and quantitative PCR were employed to identify the primary intrahepatic cell types expressing MFAP4 and assess its effects on HSC activation and apoptosis. MFAP4 is upregulated in cirrhotic livers and is actively expressed in HSC. Single-cell RNA sequencing analysis and Transwell co-culture experiments revealed that the pro-fibrotic effects of MFAP4 were primarily mediated through HSCs rather than hepatocytes. Inhibition of MFAP4 significantly reduces the expression of fibrosis markers in HSC, inhibits their proliferation and migration, while overexpression of MFAP4 results in the opposite effect, accompanied by enhanced apoptosis resistance. In mouse models, global knockout of Mfap4 significantly alleviates CCl₄- and TAA-induced liver fibrosis. Mechanistic analysis reveals that MFAP4 binds to integrin αvβ3 on the HSC membrane, activating the FAK/PI3K/NFκB signaling pathway, which promotes HSC activation and survival, ultimately exacerbating liver fibrosis. Moreover, MFAP4 mediates a self-sustaining feedback loop via integrin αvβ3, maintaining HSC activation and further promoting fibrosis progression. MFAP4 governs HSC activation and apoptosis resistance via integrin αvβ3-dependent FAK/PI3K/NFκB signaling. Targeting MFAP4 mitigates fibrosis by altering HSC fate.

Acute-on-chronic liver failure (ACLF) is a syndrome characterized by systemic inflammation, leading to high short-term mortality. The lymphocyte to high-density lipoprotein ratio (LHR) has been introduced as a novel marker of inflammation. However, its role as a prognostic inflammatory biomarker in the context of hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) has received limited attention. We retrospectively included 272 patients with HBV-ACLF who met the definition of APALC. Data on clinical features and laboratory tests were collected from medical records within 24 h. Logistic regression was used to identify risk factors for poor short-term prognosis, and LHR-based prediction (LHRB) models were constructed based on risk factors. Furthermore, the accuracy of the LHRB model was validated through rigorous testing. In the survival and death groups, there were statistical differences in their CTP, MELD, MELD-Na, COSSH-ACLF II scores, and LHR. Multivariate logistic regression identified seven predictors significantly associated with 28-day mortality. Furthermore, statistically significant differences in short-term mortality and certain clinical laboratory tests for poor prognosis were observed between the high and low LHR groups. To assess the predictive performance of various models in terms of short-term mortality, the area under the receiver operating characteristic curve (AUROC) was calculated. The AUROC values for the CTP, MELD, MELD-Na, COSSH-ACLF II, and LHRB models were found to be 0.725, 0.788, 0.772, 0.871, and 0.877, respectively. The results in the validation group were similar to those in the training group, and the validation results suggested excellent performance of the LHRB model. LHR levels have the potential to serve as indicators for the prognosis of HBV-ACLF. Additionally, the recently developed LHRB model offers an accessible risk assessment tool.