Inflammaging is a process of cellular dysfunction associated with chronic inflammation, which plays a significant role in the onset and progression of liver diseases. Research on its mechanisms has become a hotspot. In viral hepatitis, inflammaging primarily involve oxidative stress, cell apoptosis and necrosis, as well as gut microbiota dysbiosis. In non-alcoholic fatty liver disease, inflammaging is more complex, involving insulin resistance, fat deposition, lipid metabolism disorders, gut microbiota dysbiosis, and abnormalities in NAD⁺ metabolism. In liver tumors, inflammaging is characterized by weakening of tumor suppressive mechanisms, remodeling of the liver microenvironment, metabolic reprogramming, and enhanced immune evasion. Therapeutic strategies targeting inflammaging have been developing recently, and antioxidant therapy, metabolic disorder improvement, and immunotherapy are emerging as important interventions for liver diseases. This review focuses on the mechanisms of inflammaging in liver diseases, aiming to provide novel insights for the prevention and treatment of liver diseases.
Humans ; Liver Diseases/pathology* ; Inflammation ; Oxidative Stress ; Non-alcoholic Fatty Liver Disease ; Liver Neoplasms ; Gastrointestinal Microbiome

OBJECTIVE: To investigate the therapeutic potential of pseudolaric acid B (PAB) on non-alcoholic fatty liver disease (NAFLD) and its underlying molecular mechanism in vitro and in vivo. METHODS: Eight-week-old male C57BL/6J mice (n=32) were fed either a normal chow diet (NCD) or a high-fat diet (HFD) for 8 weeks. The HFD mice were divided into 3 groups according to a simple random method, including HFD, PAB low-dose [10 mg/(kg·d), PAB-L], and PAB high-dose [20 mg/(kg·d), PAB-H] groups. After 8 weeks of treatment, glucose metabolism and insulin resistance were assessed by oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). Biochemical assays were used to measure the serum and cellular levels of total cholesterol (TC), triglycerides (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). White adipose tissue (WAT), brown adipose tissue (BAT) and liver tissue were subjected to hematoxylin and eosin (H&E) staining or Oil Red O staining to observe the alterations in adipose tissue and liver injury. PharmMapper and DisGeNet were used to predict the NAFLD-related PAB targets. Peroxisome proliferator-activated receptor alpha (PPARα) pathway involvement was suggested by Kyoto Encyclopedia of Genes and Genomes (KEGG) and search tool Retrieval of Interacting Genes (STRING) analyses. Luciferase reporter assay, cellular thermal shift assay (CETSA), and drug affinity responsive target stability assay (DARTS) were conducted to confirm direct binding of PAB with PPARα. Molecular dynamics simulations were applied to further validate target engagement. RT-qPCR and Western blot were performed to assess the downstream genes and proteins expression, and validated by PPARα inhibitor MK886. RESULTS: PAB significantly reduced serum TC, TG, LDL-C, AST, and ALT levels, and increased HDL-C level in HFD mice (P<0.01). Target prediction analysis indicated a significant correlation between PAB and PPARα pathway. PAB direct target binding with PPARα was confirmed through luciferase reporter assay, CETSA, and DARTS (P<0.05 or P<0.01). The target engagement between PAB and PPARα protein was further confirmed by molecular dynamics simulations and the top 3 amino acid residues, LEU321, MET355, and PHE273 showed the most significant changes in mutational energy. Subsequently, PAB upregulated the genes expressions involved in lipid metabolism and mitochondrial biogenesis downstream of PPARα (P<0.05 or P<0.01). Significantly, the PPARα inhibitor MK886 effectively reversed the lipid-lowering and PPARα activation properties of PAB (P<0.05 or P<0.01). CONCLUSION PAB mitigates lipid accumulation, ameliorates liver damage, and improves mitochondrial biogenesis by binding with PPARα, thus presenting a potential candidate for pharmaceutical development in the treatment of NAFLD.
Animals ; PPAR alpha/metabolism* ; Non-alcoholic Fatty Liver Disease/pathology* ; Male ; Mice, Inbred C57BL ; Lipid Metabolism/drug effects* ; Diterpenes/therapeutic use* ; Organelle Biogenesis ; Diet, High-Fat ; Humans ; Mice ; Liver/metabolism* ; Insulin Resistance ; Mitochondria/metabolism* ; Molecular Docking Simulation

OBJECTIVES: To investigate the regulatory role of the NLRP3 signaling pathway in hepatocyte pyroptosis in nonalcoholic steatohepatitis (NASH) under hypoxia. METHODS: Twenty-four male C57BL/6 mice were randomized equally into hypoxic control (A), hypoxic NASH model (B), hypoxic NASH+NLRP3 inhibitor (C), and hypoxic NASH+caspase-1 inhibitor (D) groups. In groups B-D, the mice were fed a methionine choline-deficient (MCD) diet under hypoxic conditions (to simulate a 5000 m altitude) for 6 weeks; the mice in groups C and D received intraperitoneal injections of the respective inhibitors every other day. RESULTS: Compared with those in group A, the mice in group B showed significantly elevated serum levels of FBG, TC, TG, ALT and AST, increased liver lipid content, inflammatory cell infiltration and collagen fiber deposition, and enhanced hepatic expressions of NLRP3, caspase-1, IL-1β and GSDMD proteins, with obvious swelling, cristae breakage, vacuolization, and outer membrane disruption of the mitochondria, ribosome loss in the cytoplasm, destruction of the nuclear membrane, and pathological changes of the rough endoplasmic reticulum. Treatment with NLRP3 inhibitor and caspase-1 inhibitor both significantly lowered serum levels of TC, TG, ALT and AST (but without significantly affecting FBG) in the mouse models, and reduced liver lipid content, inflammatory cell infiltration, collagen deposition, and expression levels of NLRP3, caspase-1, GSDMD and IL-1β. The treatments also significantly improved pathological changes in the mitochondria, ribosomes and endoplasmic reticulum in liver tissues of the mice. CONCLUSIONS NLRP3 signaling pathway plays a key role in promoting hepatocyte pyroptosis in NASH mice under hypoxic condition, and inhibiting this pathway can effectively reduce liver inflammation, suggesting its potential as a therapeutic target for NASH treatment.
Animals ; Non-alcoholic Fatty Liver Disease/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Pyroptosis ; Mice, Inbred C57BL ; Male ; Hepatocytes/pathology* ; Signal Transduction ; Mice ; Hypoxia/metabolism* ; Caspase 1/metabolism* ; Interleukin-1beta/metabolism* ; Liver/metabolism*

OBJECTIVES: To identify cuproptosis- and ferroptosis-related genes involved in nonalcoholic fatty liver disease and to determine the diagnostic value of hub genes. METHODS: The gene expression dataset GSE89632 was retrieved from the Gene Expression Omnibus database to identify differentially expressed genes (DEGs) between the non-alcoholic steatohepatitis (NASH) group and the healthy group using the 'limma' package in R software and weighted gene co-expression network analysis. Gene ontology, kyoto encyclopedia of genes and genomes pathway, and single-sample gene set enrichment analyses were performed to identify functional enrichment of DEGs. Ferroptosis- and cuproptosis-related genes were obtained from the FerrDb V2 database and available literatures, respectively. A combined signature for cuproptosis- and ferroptosis-related genes, called CRF, was constructed using the STRING database. Hub genes were identified by overlapping DEGs, WGCNA-derived key genes, and combined signature CRF genes, and validated using the GSE109836 and GSE227714 datasets and real-time quantitative polymerase chain reaction. A nomogram of NASH diagnostic model was established utilizing the 'rms' package in R software based on the hub genes, and the diagnostic value of hub genes was assessed using receiver operating characteristic curve analysis. In addition, immune cell infiltration in NASH versus healthy controls was examined using the CIBERSORT algorithm. The relationships among various infiltrated immune cells were explored with Spearman's correlation analysis. RESULTS: Analysis of GSE89632 identified 236 DEGs between the NASH group and the healthy group. WGCNA highlighted 8 significant modules and 11,095 pivotal genes, of which 330 genes constituted CRF. Intersection analysis identified IL6, IL1B, JUN, NR4A1, and PTGS2 as hub genes. The hub genes were all downregulated in the NASH group, and this result was further verified by the NASH validation dataset and real-time quantitative polymerase chain reaction. Receiver operating characteristic curve analysis confirmed the diagnostic efficacy of these hub genes with areas under the curve of 0.985, 0.941, 1.000, 0.967, and 0.985, respectively. Immune infiltration assessment revealed that gamma delta T cells, M1 macrophages, M2 macrophages, and resting mast cells were predominantly implicated. CONCLUSIONS Our investigation underscores the significant association of cuproptosis- and ferroptosis-related genes, specifically IL6, IL1B, JUN, NR4A1, and PTGS2, with NASH. These findings offer novel insights into the pathogenesis of NASH, potentially guiding future diagnostic and therapeutic strategies.
Non-alcoholic Fatty Liver Disease/pathology* ; Humans ; Ferroptosis/genetics* ; Copper/metabolism* ; Gene Ontology ; Gene Expression Profiling

OBJECTIVES: Over 25% of the global population is affected by metabolic dysfunction-associated fatty liver disease (MAFLD), yet its pathogenesis remains unclear. Endoplasmic reticulum stress (ERS) may be involved in the onset and progression of MAFLD. Adenosine 5'-monophosphate-activated protein kinase α2 (AMPKα2), a key regulator of hepatic energy metabolism, may influence MAFLD development via ERS modulation. This study aims to investigate the role of AMPKα2 in a high-fat diet-induced MAFLD mouse model and its regulatory effect on the inositol-requiring enzyme 1 alpha (IRE1α)-c-Jun N-terminal kinase (JNK) signaling pathway. METHODS: Liver-specific AMPKα2 knockout mice on a C57BL/6 background were generated and subjected to MAFLD induction. Mice were divided into four groups: wild-type control (WT+Chow, basic diet for 12 weeks), wild-type high-fat diet (WT+HFD, high-fat diet for 12 weeks), AMPKα2 knockout control (AMPKα2 KO+Chow), and AMPKα2 knockout high-fat diet (AMPKα2 KO+HFD). Blood glucose, lipid levels, and liver function were assessed post-treatment. Liver histology was analyzed using Oil Red O, hematoxylin-eosin, Masson, and Sirius Red staining. Western blotting was used to evaluate the expression of AMPKα2, ERS markers, autophagy, apoptosis, and ferroptosis-related proteins. RESULTS: Compared with the WT+Chow group, the WT+HFD group showed significantly elevated blood glucose, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels (all P<0.01); histological analyses revealed hepatic steatosis, vacuolization, and fibrosis, with a significantly increased non-alcoholic steatohepatitis activity score (NAS) (P<0.001). Phosphorylated IRE1α and the autophagy marker microtubule-associated protein light chain (LC) 3II/LC3I were markedly upregulated, while apoptotic proteins (Cleaved-Caspase 3, BAX, Bcl-2) and ferroptosis markers (SLC7A11, GPX4) showed no significant change (P>0.05). In the AMPKα2 KO+HFD group, blood glucose, ALT, and AST levels were significantly reduced compared to the WT+HFD group. Histological improvements were observed with reduced vacuolization and lipid accumulation. Expression of p-IRE1α, JNK, and LC3II/LC3I was significantly decreased (P<0.05). CONCLUSIONS Hepatic AMPKα2 knockout alleviates high-fat induced MAFLD, potentially by inhibiting the IRE1α-JNK pathway and reducing autophagy.
Animals ; AMP-Activated Protein Kinases/physiology* ; Protein Serine-Threonine Kinases/metabolism* ; Mice, Knockout ; Diet, High-Fat/adverse effects* ; Mice, Inbred C57BL ; Mice ; Endoplasmic Reticulum Stress ; Endoribonucleases/metabolism* ; Male ; Liver/pathology* ; Non-alcoholic Fatty Liver Disease/metabolism* ; MAP Kinase Signaling System/physiology* ; Fatty Liver/metabolism* ; Signal Transduction

BACKGROUND: Liver biopsy for the diagnosis of non-alcoholic steatohepatitis (NASH) is limited by its inherent invasiveness and possible sampling errors. Some studies have shown that cytokeratin-18 (CK-18) concentrations may be useful in diagnosing NASH, but results across studies have been inconsistent. We aimed to identify the utility of CK-18 M30 concentrations as an alternative to liver biopsy for non-invasive identification of NASH. METHODS: Individual data were collected from 14 registry centers on patients with biopsy-proven non-alcoholic fatty liver disease (NAFLD), and in all patients, circulating CK-18 M30 levels were measured. Individuals with a NAFLD activity score (NAS) ≥5 with a score of ≥1 for each of steatosis, ballooning, and lobular inflammation were diagnosed as having definite NASH; individuals with a NAS ≤2 and no fibrosis were diagnosed as having non-alcoholic fatty liver (NAFL). RESULTS: A total of 2571 participants were screened, and 1008 (153 with NAFL and 855 with NASH) were finally enrolled. Median CK-18 M30 levels were higher in patients with NASH than in those with NAFL (mean difference 177 U/L; standardized mean difference [SMD]: 0.87 [0.69-1.04]). There was an interaction between CK-18 M30 levels and serum alanine aminotransferase, body mass index (BMI), and hypertension ( P  < 0.001, P  = 0.026 and P  = 0.049, respectively). CK-18 M30 levels were positively associated with histological NAS in most centers. The area under the receiver operating characteristics (AUROC) for NASH was 0.750 (95% confidence intervals: 0.714-0.787), and CK-18 M30 at Youden's index maximum was 275.7 U/L. Both sensitivity (55% [52%-59%]) and positive predictive value (59%) were not ideal. CONCLUSION This large multicenter registry study shows that CK-18 M30 measurement in isolation is of limited value for non-invasively diagnosing NASH.
Humans ; Non-alcoholic Fatty Liver Disease/diagnosis* ; Keratin-18 ; Biomarkers ; Biopsy ; Hepatocytes/pathology* ; Apoptosis ; Liver/pathology*

Metabolic associated fatty liver disease (MAFLD) is a liver disease with hepatocyte steatosis caused by metabolic disorders, which is closely related to obesity, diabetes, metabolic dysfunction, and other factors. Its pathological process changes from simple steatosis, liver inflammation to non-alcoholic steatohepatitis (NASH), and then leads to liver fibrosis, cirrhosis, and liver cancer. At present, no specific therapeutics are available for treatment of MAFLD targeting its etiology. Celastrol is the main active component of the traditional Chinese medicine Celastrus orbiculatus Thunb. In recent years, it has been found that celastrol shows important medicinal value in regulating lipid metabolism, reducing fat and weight, and protecting liver, and then ameliorates MAFLD. This article reviews the related research progress of celastrol in the prevention and treatment of MAFLD, so as to provide a reference for the comprehensive development and utilization of celastrol.
Humans ; Non-alcoholic Fatty Liver Disease/metabolism* ; Liver/pathology* ; Pentacyclic Triterpenes/metabolism* ; Obesity

As a member of the apolipoprotein C (ApoC) family with a relatively high content, ApoC3 plays a major role in the regulation of triglyceride metabolism, and plays an important role in the occurrence and development of cardiovascular diseases, glucose and lipid metabolism disorders. Nonalcoholic fatty liver disease (NAFLD) refers to the accumulation of a large amount of fat in the liver in the absence of a history of chronic alcohol consumption or other damage to the liver. A large number of previous studies have shown that there is a correlation between the gene polymorphism and high expression of ApoC3 and NAFLD. In the context of hypertriglyceridemia (HTG), this article reviews the relationship between ApoC3 and NAFLD, glucose and lipid metabolism, and islet β cell function, showing that ApoC3 can not only inhibit lipoprotein lipase (LPL) and hepatic lipase (HL) activity, delay the decomposition of triglyceride in plasma to maintain the body's energy metabolism during fasting, but also be significantly increased under insulin resistance, prompting the liver to secrete a large amount of very low-density lipoprotein (VLDL) to induce HTG. Therefore, targeting and inhibiting ApoC3 might become a new approach to treat HTG. Increasing evidence suggests that ApoC3 does not appear to be an independent "contributor" to NAFLD. Similarly, our previous studies have shown that ApoC3 is not an independent factor triggering islet β cell dysfunction in ApoC3 transgenic mice, but in a state of excess nutrition, HTG triggered by ApoC3 high expression may exacerbate the effects of hyperglycemia and insulin resistance on islet β cell function, and the underlying mechanism remains to be further discussed.
Apolipoprotein C-III/genetics* ; Non-alcoholic Fatty Liver Disease/pathology* ; Glucose/metabolism* ; Lipid Metabolism ; Humans ; Animals ; Hypertriglyceridemia/metabolism* ; Islets of Langerhans/metabolism*

OBJECTIVES: This study aimed to explore the functions and potential regulatory targets of local macrophages in nonalcoholic fatty liver combined with Porphyromonas gingivalis (P. gingivalis)infection. METHODS: Single-cell RNA sequencing was used to analyze the phenotypes and functional changes in various cells in the liver tissue of nonalcoholic steatohepatitis (NASH) mice fed with P. gingivalis. Real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay, and immunofluorescence staining were applied to observe the inflammation and expression levels of macrophage antigen presenting functional markers in the NASH liver. Oil red staining was performed to observe the accumulation of local adipose tissue in the NASH liver. Results were verified through RT-PCRand RNA sequencing using P. gingivalis-lipopolysaccharide treated mouse peritoneal macrophages. RESULTS: In comparison with healthy livers with Kupffer cells, the NASH liver combined with P. gingivalis infection-related macrophages showed significant heterogeneity. C1qb, C1qc, Mafb, Apoe, and Cd14 were highly expressed, but Cd209a, H2-Aa, H2-Ab1, and H2-DMb1, which are related to the antigen presentation function, were weakly expressed. Further in vivo and in vitro investigations indicated that the activation and infiltration of these macrophages may be due to local P. gingivalis-lipopolysaccharide accumulation. CONCLUSIONS P. gingivalis-lipopolysaccharide induces a local macrophage immunotolerance phenotype in nonalcoholic fatty liver, which may be the key mechanism of periodontitis pathogen infection that promotes NASH inflammation and pathogenesis. This study further clarifies the dysfunction and regulatory mechanisms of macrophages in the pathogenesis of P. gingivalis-infected NASH, thereby providing potential therapeutic targets for its clinical treatment.
Mice ; Animals ; Non-alcoholic Fatty Liver Disease/pathology* ; Kupffer Cells/pathology* ; Porphyromonas gingivalis ; Lipopolysaccharides/metabolism* ; Inflammation/pathology* ; Macrophages/metabolism* ; Mice, Inbred C57BL

The intestinal mucus layer is a barrier that separates intestinal contents and epithelial cells, as well as acts as the "mucus layer-soil" for intestinal flora adhesion and colonization. Its structural and functional integrity is crucial to human health. Intestinal mucus is regulated by factors such as diet, living habits, hormones, neurotransmitters, cytokines, and intestinal flora. The mucus layer's thickness, viscosity, porosity, growth rate, and glycosylation status affect the structure of the gut flora colonized on it. The interaction between "mucus layer-soil" and "gut bacteria-seed" is an important factor leading to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Probiotics, prebiotics, fecal microbiota transplantation (FMT), and wash microbial transplantation are efficient methods for managing NAFLD, but their long-term efficacy is poor. FMT is focused on achieving the goal of treating diseases by enhancing the "gut bacteria-seed". However, a lack of effective repair and management of the "mucus layer-soil" may be a reason why "seeds" cannot be well colonized and grow in the host gut, as the thinning and destruction of the "mucus layer-soil" is an early symptom of NAFLD. This review summarizes the existing correlation between intestinal mucus and gut microbiota, as well as the pathogenesis of NAFLD, and proposes a new perspective that "mucus layer-soil" restoration combined with "gut bacteria-seed" FMT may be one of the most effective future strategies for enhancing the long-term efficacy of NAFLD treatment.
Humans ; Non-alcoholic Fatty Liver Disease/therapy* ; Gastrointestinal Microbiome ; Probiotics ; Prebiotics ; Fecal Microbiota Transplantation ; Bacteria ; Liver/pathology*