BACKGROUND: Hepatic inflammatory cell accumulation and the subsequent systematic inflammation drive acute-on-chronic liver failure (ACLF) development. Previous studies showed that the vagus nerve exerts anti-inflammatory activity in many inflammatory diseases. Here, we aimed to identify the key molecule mediating the inflammatory process in ACLF and reveal the neuroimmune communication arising from the vagus nerve and immunological disorders of ACLF. METHODS: Proteomic analysis was performed and validated in ACLF model mice or patients, and intervention animal experiments were conducted using neutralizing antibodies. PNU-282987 (acetylcholine receptor agonist) and vagotomy were applied for perturbing vagus nerve activity. Single-cell RNA sequencing (scRNA-seq), flow cytometry, immunohistochemical and immunofluorescence staining, and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology were used for in vivo or in vitro mechanistic studies. RESULTS: The unbiased proteomics identified C-X-C motif chemokine ligand 9 (CXCL9) as the greatest differential protein in the livers of mice with ACLF and its relation to the systematic inflammation and mortality were confirmed in patients with ACLF. Interventions on CXCL9 and its receptor C-X-C chemokine receptor 3 (CXCR3) improved liver injury and decreased mortality of ACLF mice, which were related to the suppressing of hepatic immune cells' accumulation and activation. Vagus nerve stimulation attenuated while vagotomy aggravated the expression of CXCL9 and the severity of ACLF. Blocking CXCL9 and CXCR3 ameliorated liver inflammation and increased ACLF-associated mortality in ACLF mice with vagotomy. scRNA-seq revealed that hepatic macrophages served as the major source of CXCL9 in ACLF and were validated by immunofluorescence staining and flow cytometry analysis. Notably, the expression of CXCL9 in macrophages was modulated by vagus nerve-mediated cholinergic signaling. CONCLUSIONS Our novel findings highlighted that the neuroimmune communication of the vagus nerve-macrophage-CXCL9 axis contributed to ACLF development. These results provided evidence for neuromodulation as a promising approach for preventing and treating ACLF.
Animals ; Mice ; Chemokine CXCL9/metabolism* ; Vagus Nerve/physiology* ; Acute-On-Chronic Liver Failure/metabolism* ; Humans ; Male ; Mice, Inbred C57BL ; Proteomics ; Flow Cytometry ; Receptors, CXCR3/metabolism*

OBJECTIVES: To explore the role of the cGAS-STING signaling pathway in the therapeutic mechanism of Liangxue Jiedu Huayu Formula (LXJDHYF) for acute-on-chronic liver failure (ACLF) in mice. METHODS: Thirty C57BL/6 mice were randomly divided into blank control group, model group, low- and high-dose LXJDHYF groups, and H151 (a specific cGAS-STING pathway inhibitor) group (n=6). In all but the control group, the mice were treated with CCl₄ to induce liver cirrhosis followed by intraperitoneal injections of lipopolysaccharide and D-amino galactose to establish mouse models of ACLF. After the treatments, the mouse livers were collected for HE and TUNEL staining, and serum levels of ALT, AST and TBil were determined. In bone marrow-derived macrophages (BMDMs) and liver tissues of ACLF mice, the expressions of cGAS-STING signaling pathway-related mRNAs including IFN‑β, ISG15, IL-6 and TNF-α were determined with RT-qPCR, and the phosphorylation levels of IRF3 and STING proteins were investigated using Western blotting. RESULTS: Compared with the mice in the model group, the LXJDHYF-treated mice exhibited milder hepatocyte necrosis and inflammatory cell infiltration in the liver with significantly reduced hepatocyte apoptosis. LXJDHYF treatment also significantly lowered serum levels of ALT, AST, TBil, IL-6 and TNF-α in ACLF mice and effectively suppressed the expressions of cGAS-STING signaling pathway-related mRNA in both the BMDMs and the liver tissues and the phosphorylation of IRF3 and STING proteins in the BMDMs. CONCLUSIONS LXJDHYF can significantly improve liver function and attenuate inflammation in ACLF mice possibly by inhibiting excessive activation of the cGAS-STING signaling pathway.
Animals ; Signal Transduction/drug effects* ; Mice ; Nucleotidyltransferases/metabolism* ; Mice, Inbred C57BL ; Acute-On-Chronic Liver Failure/etiology* ; Membrane Proteins/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Liver/metabolism* ; Disease Models, Animal ; Interferon Regulatory Factor-3/metabolism* ; Interleukin-6/metabolism* ; Male

Patients with cirrhosis who are hospitalized for an acute decompensation (AD) and also have organ failure(s) are at high risk of short-term death. These patients have a syndrome called Acute-on-Chronic Liver Failure (ACLF). ACLF is now considered as a new syndrome that it is distinct from "mere" AD not only because of the presence of organ failure(s) and high short-term mortality but also because of younger age, higher prevalence of alcoholic etiology of cirrhosis, higher prevalence of some precipitants (such as bacterial infections, active alcoholism), and more intense systemic inflammatory response. ACLF is a new syndrome also because severe sepsis or severe alcoholic hepatitis do not account for 100% of the observed cases; in fact, almost 50% of the cases are of "unknown" origin. In other words, severe sepsis, severe alcoholic hepatitis and ACLF of "unknown origin" are subcategories of the syndrome.
Acute-On-Chronic Liver Failure/complications/mortality/*pathology ; Age Factors ; Cytokines/metabolism ; Hepatitis, Alcoholic/complications ; Humans ; Liver Cirrhosis/*complications/diagnosis ; Sepsis/complications ; Severity of Illness Index ; Survival Rate

OBJECTIVETo determine the differential protein and mRNA expressions of mitochondria fusion protein-2 (Mfn2) in hepatic tissues in conditions of cirrhosis and acute on chronic liver failure using rat model systems,and to determine the correlative effects on production of adenosine triphosphate (ATP) and reactive oxygen species (ROS).

METHODSA liver cirrhotic rat model (LC rats) was established by intraperitoneal injection of carbon tetrachloride (CCl4,in vegetable oil),and these mice were subsequently used (10 weeks later) to establish the acute on chronic liver failure rat model (LF rats) by injecting lipopolysaccharide and D-amino-galactose.Control groups (normal controls,NC rats) were established for each model by intraperitoneal injection of vegetable oil only.Protein expression of Mfn2 in liver was quantified by western blotting with fluorescence densitometry and immunofluorescence staining,and mRNA expression was measured by real-time fluorescence quantitative PCR.ROS levels in liver were measured by fluorescence spectrophotometry,and ATP content was measured by bioluminescence assay.Significance of inter-group differences was assessed by one-way ANOVA,and correlations were determined using bivariate statistical modeling.

RESULTSMfn2 protein expression was significantly lower in the liver tissues from modeled rats than that from the control rats (LC:0.051+/-0.004 and LF:0.037+/-0.007 vs.NC:0.254+/-0.008;F=444.98,P less than 0.05).The mRNA expression followed the same trend of lower expression (LC:21.21+/-0.93 and LF:24.35+/-0.85 vs.NC:19.09+/-0.69; F=66.941,P less than 0.05).The ATP content in liver tissues was also significantly lower in the modeled rats (LC:2.07+/-0.05 mol/L and LF:1.81+/-0.11 mol/L vs.NC:3.24+/-0.08 mol/L; F =574.21,P less than 0.05).Lower Mfn2 expression was correlated with lower ATP content (r =0.982) and higher ROS content (r =0.803).

CONCLUSIONReduced Mfn2 expression in liver tissue may cause a decrease in ATP synthesis and increase in ROS generation,thereby disrupting metabolism and increasing oxidative stress in the liver under conditions of cirrhosis and liver failure.

Acute-On-Chronic Liver Failure ; metabolism ; Animals ; Carbon Tetrachloride ; GTP Phosphohydrolases ; metabolism ; Liver Cirrhosis ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Mitochondrial Proteins ; metabolism ; Rats

Acute-On-Chronic Liver Failure ; blood ; pathology ; Case-Control Studies ; Cytochrome Reductases ; metabolism ; Humans ; Prospective Studies ; alpha-Fetoproteins ; metabolism

OBJECTIVETo evaluate the levels of high mobility group box 1 protein (HMGB1) in serum of patients with hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) and investigate its potential relation to the clinical features of these patients.

METHODSSixty patients with HBV-related ACLF, 30 patients with chronic hepatitis B (CHB), and 24 healthy individuals (controls) were enrolled in the study. Markers of liver function, such as aspartate aminotransferase (AST), were measured by routine biochemical methods. Imaging studies, such as abdominal computed tomography or magnetic resonance imaging, were used for disease staging. Serum levels of HMGB1 were measured by ELISA. Deaths within the 2-month follow-up after serum collection were used for the survival analysis. Patients who developed peritonitis, pneumonia, or other bacterial and fungal infections during the 2-month follow-up after serum collection were classified as the infected group. Pairwise comparisons were carried out by t-test, and multiple comparisons were carried out by analysis of variance.

RESULTSPatients with HBV-related ACLF had significantly higher serum levels of HMGB1 than CHB patients or controls (P = 0.003). Among the patients with HBV-related ACLF, those in the late stage (n = 20) had significantly higher levels of HMGB1 than those in the early stage (n = 20) (P = 0.005). The serum levels of HMGB1 correlated well with AST level in patients with HBV-related ACLF (P = 0.006). In addition, patients with HBV-related ACLF who developed infection or died during follow-up also had significantly higher levels of HMGB1 (P = 0.028 or P = 0.017, respectively).

CONCLUSIONEnhanced serum level of HMGB1 is associated with development of HBV-related ACLF in CHB patients. The strong correlation between HMGB1 and AST levels suggest that HMGB1 may be useful as a prognostic marker for development of ACLF.

Adolescent ; Adult ; Aspartate Aminotransferases ; metabolism ; Case-Control Studies ; Female ; HMGB1 Protein ; blood ; Hepatitis B virus ; Hepatitis B, Chronic ; blood ; Humans ; Liver Failure ; blood ; Liver Failure, Acute ; blood ; Male ; Middle Aged ; Young Adult

OBJECTIVETo investigate the expression and role of augmenter of liver regeneration (ALR) in hepatic failure.

METHODSALR polyclonal antibody was prepared and purified. Serum ALR in patients with hepatic failure, chronic hepatitis B and healthy persons were quantified by ELISA, ALR mRNA in hepatic tissues were quantified by real-time PCR.

RESULTSDifferent serum ALR levels foreshowed different outcomes for hepatic failure patients: The liver function was restored in 6 patients with higher ALR level [(1613.5+/-369.6) pmol/ml], and the liver function was deteriorated in 12 patients with lower ALR level [(462.3+/-235.8) pmol/ml]. ALR level in patients with chronic hepatitis B [(969.2+/-332.5) pmol/ml] was similar to that in healthy persons [(806.9+/-240.8) pmol/ml]. ALR mRNA level in hepatic failure patients receiving OLT (103.45 copies/microl) was lower than that in chronic hepatitis B patients (104.37 copies/microl) and healthy persons (104.31 copies/microl), ALR mRNA level in chronic hepatitis B and healthy persons was similar.

CONCLUSIONThese findings suggest serum ALR level reflected ALR mRNA level in liver and is helpful in estimating the survival time of patients with hepatic failure.

Animals ; Case-Control Studies ; Enzyme-Linked Immunosorbent Assay ; Female ; Gene Expression ; Hepatitis B, Chronic ; blood ; metabolism ; pathology ; Hepatocytes ; metabolism ; Humans ; Liver ; metabolism ; pathology ; Liver Failure, Acute ; blood ; metabolism ; pathology ; Liver Regeneration ; Mice ; Mice, Inbred BALB C ; Polymerase Chain Reaction ; methods ; Prognosis ; Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

Animals ; Biotransformation ; Cell Line, Tumor ; Cell Proliferation ; Culture Media ; Diazepam ; pharmacokinetics ; Fibroblasts ; metabolism ; pathology ; Hepatitis, Chronic ; blood ; complications ; Humans ; Liver Failure, Acute ; blood ; etiology ; Liver Neoplasms ; metabolism ; pathology ; Plasma