Identification of Senkyunolide I as a novel modulator of hepatic steatosis and PPARα signaling in zebrafish and hamster models.

ETHNOPHARMACOLOGICAL RELEVANCE: Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver-related morbidity and mortality, with hepatic steatosis being the hallmark symptom. Salvia miltiorrhiza Bunge (Smil, Dan-Shen) and Ligusticum striatum DC (Lstr, Chuan-Xiong) are commonly used to treat cardiovascular diseases and have the potential to regulate lipid metabolism. However, whether Smil/Lstr combo can be used to treat NAFLD and the mechanisms underlying its lipid-regulating properties remain unclear. PURPOSE: To assess the feasibility and reliability of a short-term high-fat diet (HFD) induced zebrafish model for evaluating hepatic steatosis phenotype and to investigate the liver lipid-lowering effects of Smil/Lstr, as well as its active components. METHODS: The phenotypic alterations of liver and multiple other organ systems were examined in the HFD zebrafish model using fluorescence imaging and histochemistry. The liver-specific lipid-lowering effects of Smil/Lstr combo were evaluated endogenously. The active molecules and functional mechanisms were further explored in zebrafish, human hepatocytes, and hamster models. RESULTS: In 5-day HFD zebrafish, significant lipid accumulation was detected in the blood vessels and the liver, as evidenced by increased staining with Oil Red O and fluorescent lipid probes. Hepatic hypertrophy was observed in the model, along with macrovesicular steatosis. Smil/Lstr combo administration effectively restored the lipid profile and alleviated hepatic hypertrophy in the HFD zebrafish. In oleic-acid stimulated hepatocytes, Smil/Lstr combo markedly reduced lipid accumulation and cell damage. Subsequently, based on zebrafish phenotypic screening, the natural phthalide senkyunolide I (SEI) was identified as a major molecule mediating the lipid-lowering activities of Smil/Lstr combo in the liver. Moreover, SEI upregulated the expression of the lipid metabolism regulator PPARα and downregulated fatty acid translocase CD36, while a PPARα antagonist sufficiently blocked the regulatory effect of SEI on hepatic steatosis. Finally, the roles of SEI on hepatic lipid accumulation and PPARα signaling were further verified in the hamster model. CONCLUSIONS: We proposed a zebrafish-based screening strategy for modulators of hepatic steatosis and discovered the regulatory roles of Smil/Lstr combo and its component SEI on liver lipid accumulation and PPARα signaling, suggesting their potential value as novel candidates for NAFLD treatment.

Comparison of serum Alanine Aminotransferase and Aspartate Aminotransferase among Obese and Non-obese Adults.

Obesity is a major global health issue and WHO points out that 1 in 3 people globally is clinically obese. Obesity is a condition of having an excessive amount of body fat and is linked with several health disorders that include metabolic syndrome, cardiovascular diseases as well as liver diseases. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are usually used to assess liver function. It is established that obesity results in many cases of liver disorders known as steatosis or non-alcoholic fatty liver disease (NAFLD). It is the excessive deposit of fat within the liver cells that may result in inflammation, fibrosis and cirrhosis. Studies on obesity have established that obese subjects significantly tend to have higher liver enzymes like ALT and AST than non-obese adults. This study aims to compare these two liver enzymes between these two groups in the hopes of shedding light on how obesity impacts the liver and offers these biomarkers for NAFLD. The study was designed to assess the status of liver function in adult obese individuals by examining key markers, including serum Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST), and to compare them with non-obese adults. The present cross-sectional study was carried out in the Department of Biochemistry, Mymensingh Medical College, Mymensingh, in collaboration with the Department of Endocrinology and Medicine, Mymensingh Medical College Hospital, from Octy 2023 to June 2024. The subjects were selected based on inclusion and exclusion criteria by purposive non-random sampling. A total of 200 participants took part in this study. Out of them, 100 were selected as case (obese adults) and another 100 non-obese adults were selected as control. In this study, serum ALT and AST levels were measured for analytical study. All the values were expressed as mean±SD. Statistical analysis was done using by using SPSS Windows package version 26.0. The statistical significance of the difference between the case and control was calculated using Student's unpaired 't'- test. Pearson's correlation is done to see the level of significance. After careful evaluation, the mean±SD values of serum ALT were 37.14±15.18 U/L and 21.92±5.10 U/L in case and control groups respectively, and mean±SD values of serum AST were 41.15±15.24 U/L and 25.01±6.65 U/L in case and control group respectively. This study revealed that mean serum ALT and AST levels were significantly increased in obese adults. There was a significant positive correlation found between BMI with serum ALT and AST levels. Analyzing the findings of this cross-sectional study, significant alterations in serum ALT and AST levels were observed among obese adults.

Vitamin A, C and E levels in patients with Non-Alcoholic Fatty Liver Disease: An Indian Cohort Study.

This study aimed to investigate the levels of vitamins A, C, and E in patients with Non-Alcoholic Fatty Liver Disease (NAFLD) compared to healthy controls and to explore the correlation between these vitamin levels and various other parameters, including bone mineral density (BMD), adiposity (fat storage), insulin resistance and subclinical inflammation. The study involved 50 participants diagnosed with NAFLD and 50 healthy controls. Blood samples were collected to measure vitamin A, C and E levels, along with other parameters like insulin, inflammatory markers, and liver function tests. Additionally, participants underwent DEXA scans to assess BMD and body composition. Vitamin levels: The study found no significant deficiencies in vitamin A or C levels in either group. However, vitamin E levels were significantly higher in the NAFLD group compared to controls, although only one case of vitamin E deficiency was observed in the NAFLD group. No significant correlations were found between vitamin levels and BMD, adiposity parameters, insulin resistance, or subclinical inflammation markers in either group. The study acknowledges the limited data available on the association between NAFLD, vitamin levels and BMD in the Asian Indian population. The findings regarding vitamin A and C levels are consistent with some previous studies, whereas the higher vitamin E levels in the NAFLD group contradict other research. This discrepancy might be due to factors like sample size, dietary habits, or vitamin fortification programs. The lack of significant correlations between vitamin levels and other parameters suggests that further research is needed to understand the complex interplay between these factors in NAFLD development and progression.

TGF-ß/Smad signaling pathway in fatty liver disease: a case-control study.

BACKGROUND: Fatty liver disease is a metabolic disorder that recently has been classified into two categories: metabolic dysfunction-associated fatty liver disease (MAFLD) and non-MAFLD. TGF-ß signaling pathway is likely a significant factor in the pathogenesis of this condition, exerting its effects through its downstream signaling proteins, Smad2/3. Accordingly, this study aimed to investigate the TGF-ß signaling pathway in the white blood cells (WBCs) of patients with MAFLD compared to those with non-MAFLD and control groups. METHODS AND RESULTS: In this study, 41 patients with fatty liver were evaluated, comprising 22 patients with MAFLD and 19 patients with non-MAFLD, and compared to 22 healthy controls. Gene expression of TGF-ß1, TGF-ß3, and CTGF were quantified using qRT-PCR, and the protein expressions of Smad2/3 and P-Smad2/3 were analyzed using western blotting. Gene expression analysis revealed a significant decrease in the gene expressions of the TGF-ß1 and TGF-ß3 and an increase in CTGF gene expression in patients with MAFLD and non-MAFLD compared to the control group. Notably, the Smad2/3 protein expression was significantly higher in the non-MAFLD group compared to the control group (P < 0.05). On the other hand, the P-smad2/3 protein expression was significantly elevated in the MAFLD group compared to the control group (P < 0.001). CONCLUSIONS: TGF-ß signaling pathway in WBCs of patients with fatty liver are affected by a complex signaling pathway. However, metabolic factors most probably affect TGF-ß1 gene expression and its downstream signaling proteins more than TGF-ß3.

Identification of necroptosis genes and characterization of immune infiltration in non-alcoholic steatohepatitis.

BACKGROUND: The most common progressive form of non-alcoholic fatty liver disease (NAFLD) is non-alcoholic steatohepatitis (NASH), which is characterized by the development of cirrhosis, and requires liver transplantation. We screened for the differentially expressed necroptosis-related genes in NASH in this study, and analyzed immune infiltration through microarray and bioinformatics analysis to identify potential biomarkers, and explore the molecular mechanisms involved in NASH. METHODS: The GSE24807 microarray dataset of NASH patients and healthy controls was downloaded, and we identified the differentially expressed genes (DEGs). Necroptosis-related differential genes (NRDEGs) were extracted from these DEGs, and functionally annotated by enrichment analyses. The core genes were obtained by constructing gene co-expression networks using weighted gene co-expression network analysis (WGCNA). Finally, the transcription factor (TF) regulatory network and the mRNA-miRNA network were constructed, and the infiltrating immune cell populations were analyzed with CIBERSORT. RESULTS: We identified six necroptosis-related genes (CASP1, GLUL, PYCARD, IL33, SHARPIN, and IRF9), and they are potential diagnostic biomarkers for NASH. In particular, PYCARD is a potential biomarker for NAFLD progression. Analyses of immune infiltration showed that M2 macrophages, γδ T cells, and T follicular helper cells were associated with the immune microenvironment of NASH, which is possibly regulated by CASP1, IL33, and IRF9. CONCLUSIONS: We identified six necroptosis-related genes in NASH, which are also potential diagnostic biomarkers. Our study provides new insights into the molecular mechanisms and immune microenvironment of NASH.

Identification of common signature genes and pathways underlying the pathogenesis association between nonalcoholic fatty liver disease and heart failure.

Background: Non-alcoholic fatty liver disease (NAFLD) and heart failure (HF) are related conditions with an increasing incidence. However, the mechanism underlying their association remains unclear. This study aimed to explore the shared pathogenic mechanisms and common biomarkers of NAFLD and HF through bioinformatics analyses and experimental validation. Methods: NAFLD and HF-related transcriptome data were extracted from the Gene Expression Omnibus (GEO) database (GSE126848 and GSE26887). Differential analysis was performed to identify common differentially expressed genes (co-DEGs) between NAFLD and HF. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) were conducted to explore the functions and regulatory pathways of co-DEGs. Protein-protein interaction (PPI) network and support vector machine-recursive feature elimination (SVM-RFE) methods were used to screen common key DEGs. The diagnostic value of common key DEGs was assessed by receiver operating characteristic (ROC) curve and validated with external datasets (GSE89632 and GSE57345). Finally, the expression of biomarkers was validated in mouse models. Results: A total of 161 co-DEGs were screened out in NAFLD and HF patients. GO, KEGG, and GSEA analyses indicated that these co-DEGs were mainly enriched in immune-related pathways. PPI network revealed 14 key DEGs, and SVM-RFE model eventually identified two genes (CD163 and CCR1) as common key DEGs for NAFLD and HF. Expression analysis revealed that the expression levels of CD163 and CCR1 were significantly down-regulated in HF and NAFLD patients. ROC curve analysis showed that CD163 and CCR1 had good diagnostic values for HF and NAFLD. Single-gene GSEA suggested that CD163 and CCR1 were mainly engaged in immune responses and inflammation. Experimental validation indicated unbalanced macrophage polarization in HF and NAFLD mouse models, and the expression of CD163 and CCR1 were significantly down-regulated. Conclusion: This study identified M2 polarization impairment characterized by decreased expression of CD163 and CCR1 as a common pathogenic pathway in NAFLD and HF. The downregulation of CD163 and CCR1 may reflect key pathological changes in the development and progression of NAFLD and HF, suggesting their potential as diagnostic and therapeutic targets.

Structure-function analysis of time-resolved immunological phases in metabolic dysfunction-associated fatty liver disease (MASH) comparing the NIF mouse model to human MASH.

Metabolic dysfunction-associated steatohepatitis (MASH) is a common but frequently unrecognized complication of obesity and type 2 diabetes. The association between these conditions is multifaceted and involves complex interactions between metabolic, inflammatory, and genetic factors. Here we assess the underlying structural and molecular processes focusing on the immunological phase of MASH in the nonobese inflammation and fibrosis (NIF) mouse model and compare it to the human disease as well as other murine models. Histopathology together with synchrotron-radiation-based x-ray micro-computed tomography (SRµCT) was used to investigate structural changes within the hepatic sinusoids network in the NIF mouse in comparison to patients with different severities of MASH. A time-course, bulk RNA-sequencing analysis of liver tissue from NIF mice was performed to identify the dynamics of key processes associated with the pathogenesis. Transcriptomics profiling of the NIF mouse revealed a gradual transition from an initially reactive inflammatory response to a regenerative, pro-fibrotic inflammatory response suggesting new avenues for treatment strategies that focus on immunological targets. Despite the lack of metabolic stress induced liver phenotype, a large similarity between the NIF mouse and the immunological phase of human MASH was detected. The translational value was further supported by the comparative analyses with MASH patients and additional animal models. Finally, the impact of diets known to induce metabolic stress, was explored in the NIF mouse. An obesogenic diet was found to induce key physiological, metabolic, and histologic changes akin to those observed in human MASH.

Efficacy of the Mediterranean diet in treating metabolic dysfunction-associated steatotic liver disease (MASLD) in children and adolescents: a systematic review and meta-analysis.

BACKGROUND: There are limited treatment options for metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic Fatty Liver Disease (MASLD) in children and adolescents. AIM: To evaluate the effectiveness of the Mediterranean diet in improving liver function in children and adolescents with MASLD. METHODS: In this systematic review and meta-analysis, we searched PubMed, Scopus, Embase, CINAHL, and Cochrane CENTRAL for interventional studies investigating the effect of Mediterranean diet on MASLD in children and adolescents. The primary outcome was a change in liver function measured using these liver enzymes; Alanine Transaminase (ALT), Aspartate Transaminase (AST) and Gamma-glutamyl transferase (GGT). The secondary outcomes were lipid profile, body weight, and insulin resistance. The risk of bias was assessed using the MASTER scale. Bias-adjusted inverse variance heterogeneity models were used to synthesize overall weighted mean differences for the treatment effect (WMD) and their 95% confidence intervals. Heterogeneity and publication bias were evaluated using the I2 statistics, Tau-squared and Doi plots, respectively. RESULT: Out of 5915 study records identified from database searches, five studies with 308 participants, two randomized controlled trials, and three quasi-experimental studies, met the inclusion criteria. In overall synthesis, the Mediterranean diet was associated with moderate improvements in liver function as shown by reductions in the liver enzymes [ALT - WMD - 10.85 U/L, 95% CI -20.03 to -1.68, I2 = 42, T2 = 38.8, AST - WMD - 9.26 U/L, 95% CI -17.14 to -1.38, I2 = 70.7, T2 = 42.7, and GGT - WMD - 1.99 95% CI -5.09 to 1.11)], but changes in body weight, lipid profile and insulin resistance were small and insignificant. CONCLUSION: The Mediterranean diet may improve liver function in children with MASLD. More randomized controlled trials are needed to develop high-certainty evidence on these findings. REGISTRATION: This protocol was registered on the International Prospective Register of Systematic Reviews (PROSPERO) CRD42023426939. 31/05/2023.

The mediating role of insulin resistance in the association between inflammatory score and MAFLD: NHANES 2017-2018.

BACKGROUND: The association between inflammatory score, insulin resistance (IR), and metabolic-associated fatty liver disease (MAFLD) is inconclusive. OBJECTIVE: The objective of this study was to examine the relationship between the inflammatory score and MAFLD and investigate the potential mediating effect of IR (evaluated by triglyceride-glucose index) in this association. METHODS: Calculating inflammatory score was performed based on white blood cells and high-sensitivity C-reactive protein. The association between the inflammatory score and MAFLD was evaluated based on the weighted multifactor logistic regression model. Restricted cubic splines (RCS) were used to visualize the dose-response relationship between the inflammatory score and MAFLD. We also conducted a mediation analysis to assess the extent to which IR mediates this association. RESULTS: Among the 1090 participants, 563 were ultimately diagnosed with MAFLD. Multivariate logistic regression results indicated a close positive association between inflammatory score and MAFLD (odds ratio = 1.235, 95% confidence interval 1.069-1.427, p = .007). The RCS results indicated a linear dose-response relationship between the inflammatory score and the risk of MAFLD after adjusting for potential confounding factors. Furthermore, the mediation analysis results showed that IR partially mediated the association between the inflammatory score and MAFLD (percent mediation = 33%). CONCLUSION: Our research results indicate that the inflammatory score is positively associated with the risk of MAFLD, and IR plays a partial mediating effect in this association.

Diagnostic accuracy of exhaled nitric oxide for the non-invasive identification of patients with fibrotic metabolic dysfunction-associated steatohepatitis.

BACKGROUND: Fibrotic metabolic dysfunction-associated steatohepatitis (MASH) is a condition at risk of progressing to advanced liver disease. We examined whether an innovative exhaled nitric oxide (eNO) breath test (BT) can accurately diagnose fibrotic MASH without requiring blood tests. METHODS: One hundred and forty-seven patients with MASH were recruited, and all tests were undertaken within 1 week of recruitment. With fibrotic MASH (NAS ≥ 4 and fibrosis stage ≥ 2) as the main outcome indicator, the diagnostic efficacy of eNO in identifying fibrotic MASH was compared to other validated models for advanced fibrosis requiring venesection, namely FAST, Agile 3+, and FIB-4 scores. RESULTS: The mean age was 40.36 ± 12.28 years, 73.5% were men. Mean body mass index was 28.83 ± 4.31 kg/m2. The proportion of fibrotic MASH was 29.25%. The area under the receiver operating curve for eNO in diagnosing fibrotic MASH was 0.737 [95% CI 0.650-0.823], which was comparable to FAST (0.751 [0.656-0.846]), Agile 3+ (0.764 [0.670-0.858]), and FIB-4 (0.721 [0.620-0.821]) (all DeLong test p > 0.05). A cut-off of eNO <8.5 ppb gave a sensitivity of 86.0% and a negative predictive value of 88.5% for ruling-out fibrotic MASH. A cut-off of eNO >13.5 ppb provided a specificity of 91.3% and a positive predictive value of 65.4% for ruling-in fibrotic MASH. Sensitivity analyses demonstrated that the diagnostic efficacy of eNO was similar across characteristics such as age. Moreover, adding vibration-controlled transient elastography-LSM (liver stiffness measurement) reduced the uncertainty interval from 46.9% to 39.5%. CONCLUSIONS: The eNO-BT is a promising simple test for non-invasively identifying fibrotic MASH, and its performance is further improved by adding LSM measurement.

The association of liver fibrosis and chronic kidney disease in patients with metabolic associated fatty liver disease: A cross-sectional study.

OBJECTIVES: To examine the relation between liver fibrosis and chronic kidney disease (CKD) in metabolic-associated fatty liver disease (MAFLD) patients and its risk factors. METHODS: The current study was carried out at Tanta University Hospital, Tanta, Egypt, from May 2021 to January 2023 and included 84 MAFLD patients with CKD and 80 MAFLD patients without CKD. All participants had been examined by abdominal ultrasonography and transient elastography with controlled attenuation parameter. RESULTS: Chronic kidney disease patients exhibited a greater incidence of fibrosis compared to patients without CKD (75.6% vs. 24.4%). Logistic analysis demonstrated that the presence of multiple health conditions, such as MAFLD, diabetes mellitus, hypertension, and cardiovascular disease, were individually linked to CKD. Gender and body mass index were not independent factors related to CKD. Additionally, factors such as age, hyperuricemia, hypertriglyceridemia, hypercholesterolemia, hypoalbuminemia, hyperbilirubinemia, and viral hepatitis, apart from MAFLD comorbidities, were independently linked to CKD. CONCLUSION: Chronic kidney disease may represent a potential risk influence for liver fibrosis development in MAFLD patients.

Chitinase 1: a novel therapeutic target in metabolic dysfunction-associated steatohepatitis.

Background: Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by persistent inflammatory cascades, with macrophage activation playing a pivotal role. Chitinase 1 (CHIT1), produced by activated macrophages, is a key player in this cascade. In this study, we aimed to explore the role of CHIT1 in MASH with progressive liver fibrosis. Methods: Fibrotic liver tissue and serum from distinct patient groups were analyzed using nCounter MAX, flow cytometry, immunohistochemistry, and enzyme-linked immunosorbent assay. A MASH mouse model was constructed to evaluate the effectiveness of OATD-01, a chitinase inhibitor. Macrophage profiling was performed using single-nuclei RNA sequencing and flow cytometry. Results: CHIT1 expression in fibrotic liver tissues was significantly correlated with the extent of liver fibrosis, macrophages, and inflammation. Single-nuclei RNA sequencing demonstrated a notable increase in macrophages numbers, particularly of lipid-associated macrophages, in MASH mice. Treatment with OATD-01 reduced non-alcoholic fatty liver disease activity score and Sirius red-positive area. Additionally, OATD-01-treated mice had lower CHIT1, F4/80, and α-smooth muscle actin positivity, as well as significantly lower levels of inflammatory markers, pro-fibrotic genes, and matrix remodeling-related mRNAs than vehicle-treated mice. Although the population of F4/80+CD11b+ intrahepatic mononuclear phagocytes remained unchanged, their infiltration and activation (CHIT1+MerTK+) significantly decreased in OATD-01-treated mice, compared with that observed in vehicle-treated mice. Conclusions: Our study underscores the pivotal role of CHIT1 in MASH. The observed significant improvement in inflammation and hepatic fibrosis, particularly at higher doses of the CHIT1 inhibitor, strongly suggests the potential of CHIT1 as a therapeutic target in MASH accompanied by progressive liver fibrosis.

Tangerine Peel-Derived Exosome-Like Nanovesicles Alleviate Hepatic Steatosis Induced by Type 2 Diabetes: Evidenced by Regulating Lipid Metabolism and Intestinal Microflora.

Purpose: Non-alcoholic fatty liver disease (NAFLD) represents a significant global health burden, exhibiting a strong correlation with insulin resistance, obesity, and type 2 diabetes (T2DM). Despite the severity of hepatic steatosis in T2DM patients, no specific drugs have been approved for clinical treatment of the disease. Tangerine peel is one kind of popular functional food and reported to possess hypoglycemic and lipid-lowering potential. In this study, we investigated the effects of Tangerine-peel-derived exosome-like nanovesicles (TNVs) on hepatic lipotoxicity associated with T2DM. Methods: The TNVs was prepared by differential centrifugation of the aqueous extract of Tangerine and chemical properties were characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and LC-MS/MS. The hypoglycemic and lipid-lowering potential of TNVs were possessed by biochemical measurement, RT-PCR, 16S rRNA sequencing, GC/MS, UHPLC-MS/MS, in vivo small animal imaging assay and HE staining. Subsequently, effects of TNVs on lipid accumulation and glycolysis were investigated on 3T3-L1 and AML-12 cells. Results: TNVs significantly inhibited insulin resistance, reduced hepatic lipid accumulation, facilitate intestinal mucosal repair, rescued gut microbiota dysbiosis, regulated colonic SCFA and liver bile acid metabolism in db/db mice. Furthermore, TNVs restored the expression of key genes in glucose and lipid metabolism (ACC, AMPK, CD36, LXRα, PPAR-γ, SREBP-1) while activating the expression of genes related to glycolysis (G6Pase, GLUT2, PCK1, PEPCK) in db/db mice. Further cell-based mechanistic studies revealed that TNVs reduced lipid accumulation in 3T3-L1 and AML-12 cells via regulation of glucose and lipid metabolism-related genes (UCP1, FGFR4, PRDM16, PGC-1α, Tmem26, Cpt1, Cpt2 and PPAR-α). Conclusion: We for the first time demonstrated that TNVs could significantly improve glucose and lipid metabolism via activating the expression of genes related to fatty acid ß-oxidation and glycolysis.

The multifaceted roles of B lymphocytes in metabolic dysfunction-associated steatotic liver disease.

Recent evidence suggests that adaptive immune cells are important contributors to metabolic dysfunction-associated steatotic liver disease (MASLD, formerly non-alcoholic fatty liver disease, NAFLD). In liver biopsies from MASLD patients, the accumulation of intrahepatic B cells is positively correlated with the MASLD activity score. Hepatic B-cell infiltration is observed in experimental models of metabolic dysfunction-associated steatohepatitis (MASH, formerly non-alcoholic steatohepatitis, NASH). Intrahepatic B2 cells have been shown to contribute to MASLD/MASH by activating T cells, macrophages and hepatic stellate cells, and by producing pathogenic IgG antibodies. In mice fed a MASH diet, selective depletion of B2 cells reduces steatohepatitis and fibrosis. Intestinal B cells are metabolically activated in MASH and promote T-cell activation independently of TCR signaling. In addition, B cells have been shown to contribute to liver fibrosis by activating monocyte-derived macrophages through the secretion of IgA immunoglobulins. Furthermore, our recent study indicates that certain B cell subsets, very likely regulatory B cells, may play a protective role in MASLD. This review summarizes the molecular mechanisms of B cell functions and discusses future research directions on the different roles of B cells in MASLD and MASH.

[Clinical value of multiparameteric quantitative ultrasound for assessing high-risk steatohepatitis].

Objective: To investigate the clinical value of multiparameteric quantitative ultrasound combined with a non-invasive prediction model for assessing high-risk steatohepatitis. Methods: One hundred and ninety-four cases with metabolic-associated fatty liver disease (MAFLD) who underwent liver biopsy in Huashan Hospital, Fudan University, from June 2021 to September 2022 were selected. Shear wave elastography (SWE), shear wave dispersion (SWD) imaging, and attenuation imaging (ATI) examinations were conducted in all patients before biopsy. High-risk steatohepatitis was defined as a total activity score of ≥4 in patients with steatohepatitis, hepatocellular ballooning, and liver lobular inflammation based on pathological hepatic steatosis, inflammatory activity, and fibrosis scoring system (SAF), and fibrosis stage≥F2. Binary logistic regression analysis was used to identify the factors influencing high-risk steatohepatitis. A predictive model for diagnosing high-risk steatohepatitis was constructed using R language. The DeLong test was used to compare the area under the curve between groups. Measurement data was compared between groups using the t-test or rank-sum test, and count data were compared between groups using the χ2 test. Results: There were 46 cases (23.7%) with high-risk steatohepatitis. The quantitative ultrasound parameters included elastic modulus (OR=2.958, 95%CI: 1.889-4.883, P<0.001), dispersion coefficient (OR=1.786, 95%CI: 1.424-2.292, P<0.001) and attenuation coefficient (OR=42.642, 95%CI: 3.463-640.451, P=0.004). Serological indexes of fasting blood glucose (OR=1.196, 95%CI: 1.048-1.392, P=0.011), alanine aminotransferase (OR=1.012, 95%CI: 1.006-1.019, P<0.001), aspartate aminotransferase (OR=1.027, 95%CI: 1.014-1.042, P<0.001), γ-glutamyl transferase (OR=1.008, 95%CI: 1.001-1.017, P=0.041) and HDL cholesterol (OR=0.087, 95%CI: 0.016-0.404, P=0.003) were the factors influencing its progression. The AUCs of elastic modulus, dispersion coefficient, attenuation coefficient, multiparametric ultrasound model, serological index model, and ultrasound combined with serology model for the diagnosis of high-risk steatohepatitis were 0.764, 0.758, 0.634, 0.786, 0.773 and 0.825, respectively. The results of the DeLong test showed that the ultrasound combined with the serological model was significantly better than the serological index model and the elastic modulus, dispersion coefficient, and attenuation coefficient alone (P=0.024, 0.027, 0.038 and <0.001). Conclusion: The combination of multiparametric quantitative ultrasound is helpful for the non-invasive diagnosis of high-risk steatohepatitis and possesses great clinical significance.

Gut microbiota of miR-30a-5p-deleted mice aggravate high-fat diet-induced hepatic steatosis by regulating arachidonic acid metabolic pathway.

BACKGROUND: Patients with non-alcoholic fatty liver disease (NAFLD) often exhibit hepatic steatosis and dyslipidemia. Studies have shown that intestinal microorganisms are closely related to the occurrence of NAFLD and atherosclerosis. Our previous study has underscored the protective role of microRNA-30a-5p (miR-30a-5p) against atherosclerosis. METHODS AND RESULTS: In the present study, we aimed to elucidate the effect and underlying mechanism of the intestinal microorganisms of miR-30a-5p knockout (KO) mice on NAFLD. Our findings demonstrated that KO exacerbated high-fat diet (HFD)-induced hepatic steatosis and disrupted liver function, as evidenced by elevated levels of total cholesterol, low-density lipoprotein, alanine aminotransferase, aspartate transaminase, and total bile acids in serum. Fecal microbiota from HFD-fed KO mice induced hepatic steatosis, dyslipidemia, and higher levels of enzymes indicative of liver damage in wild-type mice. Remarkably, KO mice significantly intensified the above effects. 16s rDNA sequencing and metabolomics of the intestinal microbiota in the HFD-treated KO and WT mice showed that the loss of miR-30a-5p resulted in intestinal microbiota imbalance and was highly related to the arachidonic acid metabolic pathway. Targeted metabolomic in the liver tissues unveiled upregulation of COX-related (PGF2a, 8-iso-PGF2a and PGF2) and LOX-related (LTB4, LTD4, 12S-HETE and 15S-HETE) factors in HFD-treated KO mice. Immunohistochemistry and transcriptional analyses showed that miR-30a-5p affected arachidonic acid metabolism through the LOX/COX pathways. Besides, COX/LOX pathways and hepatic steatosis were reversed after reintroducing miR-30a-5p in HFD-treated KO mice. CONCLUSIONS: This study reveals the pivotal mechanism by which miR-30a-5p and intestinal microbes regulate hepatic steatosis and abnormal lipid metabolism, offering promising avenues for NAFLD and atherosclerosis therapeutics. HIGHLIGHTS: MiR-30a-5p deletion aggravated hepatic steatosis and lipid disorder induced by an HFD in mice. Gut microbiota participated in the regulation of hepatic steatosis in the context of miR-30a-5p. Gut microbiota metabolism-related arachidonic acid metabolic pathway contributed to miR-30a-5p-regulated hepatic steatosis and lipid disorder. Reintroducing miR-30a-5p reversed hepatic steatosis and arachidonic acid metabolism disorder caused by HFD and miR-30a-5p deletion.

Association between phthalates exposure and non-alcoholic fatty liver disease under different diagnostic criteria: a cross-sectional study based on NHANES 2017 to 2018.

Purpose: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease. Phthalates have been suggested to influence the development of NAFLD due to their endocrine-disrupting properties, but studies based on nationally representative populations are insufficient, and existing studies seem to have reached conflicting conclusions. Due to changes in legislation, the use of traditional phthalates has gradually decreased, and the phthalates substitutes is getting more attention. This study aims to delve deeper into how the choice of diagnostic approach influences observed correlations and concern about more alternatives of phthalates, thereby offering more precise references for the prevention and treatment of NAFLD. Methods: A cohort of 641 participants, sourced from the National Health and Nutrition Examination Survey (NHANES) 2017-2018 database, was evaluated for NAFLD using three diagnostic methods: the Hepatic Steatosis Index (HSI), the US Fatty Liver Indicator (US.FLI), and Vibration Controlled Transient Elastography (VCTE). The urinary metabolite concentrations of Di-2-ethylhexyl phthalate (DEHP), Di-isodecyl phthalate (DIDP), Di-isononyl phthalate (DINP), Di-n-butyl phthalate (DnBP), Di-isobutyl phthalate (DIBP), Di-ethyl phthalate (DEP) and Di-n-octyl phthalate (DnOP) were detected. The association between NAFLD and urinary phthalate metabolites was evaluated through univariate and multivariate logistic regression analyses, considering different concentration gradients of urinary phthalates. Results: Univariate logistic regression analysis found significant correlations between NAFLD and specific urinary phthalate metabolites, such as Mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), Mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), Mono-2-ethyl-5-carboxypentyl phthalate (MECPP), and Mono-(carboxyisoctyl) phthalate (MCiOP), across different diagnostic criteria. In a multivariate logistic regression analysis adjusting only for demographic data, MEOHP (OR = 3.26, 95% CI = 1.19-8.94, p = 0.029), MEHHP (OR = 3.98, 95% CI = 1.43-11.1, p = 0.016), MECPP (OR = 3.52, 95% CI = 1.01-12.2, p = 0.049), and MCiOP (OR = 4.55, 95% CI = 1.93-10.7, p = 0.005) were positively related to NAFLD defined by HSI and VCTE. The correlation strength varied with the concentration of phthalates, indicating a potential dose-response relationship. Adjusting for all covariates in multivariate logistic regression, only MCiOP (OR = 4.22, 95% CI = 1.10-16.2, p = 0.044), as an oxidative metabolite of DINP, remained significantly associated with NAFLD under the VCTE criterion, suggesting its potential role as a risk factor for NAFLD. Conclusion: This research highlights a significant association between DINP and NAFLD. These findings underscore the need for further investigation into the role of the phthalates substitutes in the pathogenesis of NAFLD and the importance of considering different diagnostic criteria in research.

The effect of probiotic supplementation on non-alcoholic fatty liver disease (NAFLD) fibrosis score in patients attending a tertiary hospital clinic in Cairo, Egypt.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic fat accumulation (> 5% of liver tissue) in the absence of alcohol abuse or other chronic liver diseases. NAFLD can progress to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). This study aimed to assess the efficacy of probiotic (lactobacillus) supplementation on NAFLD fibrosis score. METHODOLOGY: A double-arm randomized controlled trial was conducted in the family medicine clinic of a tertiary hospital, enrolling patients with sonographic evidence of NAFLD. Fifty patients were divided into two groups: the Probiotic group received lifestyle modification instructions along with daily probiotic supplementation for twelve weeks, with regular monthly follow-up visits. The Standard Treatment group received low-fat diet and lifestyle modification instructions only. RESULTS: The mean age of participants was 46.10 years (SD 10.11), with 70% females and 30% males. The study found a statistically significant difference in liver enzymes (ALT and AST) and BMI in the probiotic group before and after intervention. However, there was no significant difference in NAFLD fibrosis score between the two groups. CONCLUSION: Short-term probiotic treatment resulted in improvements in ALT, AST, and BMI in the probiotic group, but did not significantly affect NAFLD fibrosis score. Further research with larger sample sizes and longer follow-up periods is warranted. TRIAL REGISTRATION: The clinical trial was registered at Protocol Registration and Results System with number NCT06074094 (12/09/2021).

CHIP ameliorates nonalcoholic fatty liver disease via promoting K63- and K27-linked STX17 ubiquitination to facilitate autophagosome-lysosome fusion.

The fusion of autophagosomes and lysosomes is essential for the prevention of nonalcoholic fatty liver disease (NAFLD). Here, we generate a hepatocyte-specific CHIP knockout (H-KO) mouse model that develops NAFLD more rapidly in response to a high-fat diet (HFD) or high-fat, high-fructose diet (HFHFD). The accumulation of P62 and LC3 in the livers of H-KO mice and CHIP-depleted cells indicates the inhibition of autophagosome-lysosome fusion. AAV8-mediated overexpression of CHIP in the murine liver slows the progression of NAFLD induced by HFD or HFHFD feeding. Mechanistically, CHIP induced K63- and K27-linked polyubiquitination at the lysine 198 residue of STX17, resulting in increased STX17-SNAP29-VAMP8 complex formation. The STX17 K198R mutant was not ubiquitinated by CHIP; it interfered with its interaction with VAMP8, rendering STX17 incapable of inhibiting steatosis development in mice. These results indicate that a signaling regulatory mechanism involving CHIP-mediated non-degradative ubiquitination of STX17 is necessary for autophagosome-lysosome fusion.