Pathogenesis Of Nonalcoholic Steatohepatitis Research Articles

Pharmacological inhibition of p300 ameliorates steatosis, inflammation, and fibrosis in mice with non-alcoholic steatohepatitis

The histone acetyltransferase p300 plays a pivotal role in regulating gene expression and cellular phenotype through epigenetic mechanisms. It significantly influences lipid metabolism, which is a key factor in the pathogenesis of non-alcoholic steatohepatitis (NASH), by modulating the transcription of genes involved in lipid synthesis and accumulation. This study aimed to investigate the protective potential of inhibiting p300 in NASH. Male C57BL/6J mice were subjected to a methionine- and choline-deficient (MCD) diet for 4 weeks to induce NASH, and during this period, the p300 inhibitor C646 (10 mg/kg) was administered three times a week. C646 treatment reduced the elevation of p300 expression and histone H3 acetylation, leading to a decrease in liver injury markers in the serum and an improvement in the histological abnormalities observed in MCD diet-fed mice. C646 also reduced lipid accumulation by modulating de novo lipogenesis and suppressed inflammation, including cytokine overproduction and macrophage infiltration. Furthermore, C646 mitigated liver fibrosis and myofibroblast accumulation. This protective effect was achieved through the inhibition of apoptosis by reducing p53 and Bax expression and the suppression of ferroptosis by decreasing lipid peroxidation while enhancing antioxidant defenses. Additionally, C646 alleviated endoplasmic reticulum stress, as evidenced by the downregulation of unfolded protein response signaling molecules. These results highlight the potential of p300 as a therapeutic target for NASH.

Carboxylesterase-mediated imaging tool for diagnosis of liver injury and surgical resection navigation of pancreatic cancer

The liver and pancreas are both very important digestive and metabolic organs. Once a patient's liver and pancreas show lesions, such as accompanied by tumor proliferation or acute inflammation, their digestive function will be significantly damaged. The malfunction of carboxylesterase (CE) would indicate various metabolic diseases. Monitoring and utilizing the activity changes of CE of liver and pancreas related metabolic diseases will help to discover the pathological mechanisms and interaction between different organs, which would further provide indications for diagnosis and treatment. In this work, we developed a turn-on red fluorescent probe MG-CE with lysosome targeting for CE sensing in vitro and in vivo imaging showed a decrease of CE level in a hepatitis cell model and in non-alcoholic steatohepatitis (NASH) mouse. The fluorescence intensity was higher in pancreatic ductal adenocarcinoma (PDAC) than that in both cancerous and normal cells. Our results show that CE could promote the prodrug irinotecan activation and cause more significant damage to PDAC cells. Therefore, this work would provide a new molecular tool for the diagnostic imaging of NASH and PDAC, which is helpful in understanding the pathogenesis of NASH and the early diagnosis of NASH and PDAC.

Development of non-alcoholic steatohepatitis is associated with gut microbiota but not with oxysterol enzymes CH25H, EBI2, or CYP7B1 in mice

Liver steatosis is the most frequent liver disorder and its advanced stage, non-alcoholic steatohepatitis (NASH), will soon become the main reason for liver fibrosis and cirrhosis. The “multiple hits hypothesis” suggests that progression from simple steatosis to NASH is triggered by multiple factors including the gut microbiota composition. The Epstein Barr virus induced gene 2 (EBI2) is a receptor for the oxysterol 7a, 25-dihydroxycholesterol synthesized by the enzymes CH25H and CYP7B1. EBI2 and its ligand control activation of immune cells in secondary lymphoid organs and the gut. Here we show a concurrent study of the microbial dysregulation and perturbation of the EBI2 axis in a mice model of NASH.We used mice with wildtype, or littermates with CH25H−/−, EBI2−/−, or CYP7B1−/− genotypes fed with a high-fat diet (HFD) containing high amounts of fat, cholesterol, and fructose for 20 weeks to induce liver steatosis and NASH. Fecal and small intestinal microbiota samples were collected, and microbiota signatures were compared according to genotype and NASH disease state.We found pronounced differences in microbiota composition of mice with HFD developing NASH compared to mice did not developing NASH. In mice with NASH, we identified significantly increased 33 taxa mainly belonging to the Clostridiales order and/ or the family, and significantly decreased 17 taxa. Using an Elastic Net algorithm, we suggest a microbiota signature that predicts NASH in animals with a HFD from the microbiota composition with moderate accuracy (area under the receiver operator characteristics curve = 0.64). In contrast, no microbiota differences regarding the studied genotypes (wildtype vs knock-out CH25H−/−, EBI2−/−, or CYP7B1−/−) were observed.In conclusion, our data confirm previous studies identifying the intestinal microbiota composition as a relevant marker for NASH pathogenesis. Further, no link of the EBI2 – oxysterol axis to the intestinal microbiota was detectable in the current study.

Pathogenesis and complications of non-alcoholic steatohepatitis and its management by nutraceuticals

Non-Alcoholic Steatohepatitis (NASH) is a real health problem that is commonly associated with obesity and diabetes. Cardiovascular diseases, liver carcinoma, and liver failure might develop from NASH if not well managed. The present article deals with the pathogenesis of non-alcoholic steatohepatitis and its complications in addition to exploring the importance of using nutraceuticals in its prevention and treatment.

Machine learning-based integration identifies the ferroptosis hub genes in nonalcoholic steatohepatitis

BackgroundFerroptosis, is characterized by lipid peroxidation of fatty acids in the presence of iron ions, which leads to cell apoptosis. This leads to the disruption of metabolic pathways, ultimately resulting in liver dysfunction. Although ferroptosis is linked to nonalcoholic steatohepatitis (NASH), understanding the key ferroptosis-related genes (FRGs) involved in NASH remains incomplete. NASH may be targeted therapeutically by identifying the genes responsible for ferroptosis.MethodsTo identify ferroptosis-related genes and develop a ferroptosis-related signature (FeRS), 113 machine-learning algorithm combinations were used.ResultsThe FeRS constructed using the Generalized Linear Model Boosting algorithm and Gradient Boosting Machine algorithms exhibited the best prediction performance for NASH. Eight FRGs, with ZFP36 identified by the algorithms as the most crucial, were incorporated into in FeRS. ZFP36 is significantly enriched in various immune cell types and exhibits significant positive correlations with most immune signatures.ConclusionZFP36 is a key FRG involved in NASH pathogenesis.

BRP39 Regulates Neutrophil Recruitment in NLRP3 Inflammasome-Induced Liver Inflammation

Background and AimsBRP-39 (Chi3L1) and its human homolog YKL-40, is an established biomarker of liver fibrosis in nonalcoholic steatohepatitis (NASH) patients, but its role in NASH pathogenesis remains unclear. We have recently identified Chi3L1 as one of the top upregulated genes in mice with inducible gain-of-function NOD-like receptor protein 3 (NLRP3) activation that mimics several liver features of NASH. This study aimed to investigate the effects of BRP39 deficiency on NLRP3-induced liver inflammation using Tamoxifen-inducible Nlrp3 knock-in mice sufficient (Nlrp3A350V CRT) and deficient for BRP39 (Nlrp3A350V /BRP-/- CRT). MethodsUsing Tamoxifen-inducible Nlrp3A350V knock-in mice (Nlrp3A350V CRT) mice and Tamoxifen-inducible Nlrp3A350V knock-in BRP39 deficient mice (Nlrp3A350V BRP-/- CRT), we investigated the consequence of BRP39 deficiency influencing NLRP3 induced liver inflammation. ResultsOur results showed that BRP39 deficiency in NLRP3 induced inflammation improved body weight and liver weight. Moreover, liver inflammation, fibrosis and HSC activation were significantly reduced, corresponding to significantly decreased Ly6C+ infiltrating macrophages, CD68+ OPN+ hepatic LAMs (lipid associated macrophages), and activated Ly6G+ and H3Cit+ neutrophils accumulation in the liver. Further investigation revealed that circulatory neutrophils from NLRP3 induced BRP39 deficient mice have impaired chemotaxis and migration ability, and this was confirmed by RNA-bulk sequencing showing reduced immune activation, migration and signaling responses in neutrophils. ConclusionThese data showcase the importance of BRP39 in regulating the NLRP3 inflammasome during liver inflammation and fibrotic NASH by altering cellular activation, recruitment and infiltration during disease progression, and revealing BRP39 to be a potential therapeutic target for future treatment of inflammatory NASH and its associated diseases.

Hepatocyte FBXW7-dependent activity of nutrient-sensing nuclear receptors controls systemic energy homeostasis and NASH progression in male mice

Nonalcoholic steatohepatitis (NASH) is epidemiologically associated with obesity and diabetes and can lead to liver cirrhosis and hepatocellular carcinoma if left untreated. The intricate signaling pathways that orchestrate hepatocyte energy metabolism and cellular stress, intrahepatic cell crosstalk, as well as interplay between peripheral tissues remain elusive and are crucial for the development of anti-NASH therapies. Herein, we reveal E3 ligase FBXW7 as a key factor regulating hepatic catabolism, stress responses, systemic energy homeostasis, and NASH pathogenesis with attenuated FBXW7 expression as a feature of advanced NASH. Multiomics and pharmacological intervention showed that FBXW7 loss-of-function in hepatocytes disrupts a metabolic transcriptional axis conjointly controlled by the nutrient-sensing nuclear receptors ERRα and PPARα, resulting in suppression of fatty acid oxidation, elevated ER stress, apoptosis, immune infiltration, fibrogenesis, and ultimately NASH progression in male mice. These results provide the foundation for developing alternative strategies co-targeting ERRα and PPARα for the treatment of NASH.

Lactobacillus plantarum ameliorates NASH-related inflammation by upregulating l-arginine production

Lactobacillus is a probiotic with therapeutic potential for several diseases, including liver disease. However, the therapeutic effect of L. plantarum against nonalcoholic steatohepatitis (NASH) and its underlying mechanisms remain unelucidated. Therefore, we delineated the L. plantarum-mediated NASH regulation in a mouse model to understand its therapeutic effect. We used a choline-deficient high-fat diet (CD-HFD)-induced murine model that recapitulated the critical features of human metabolic syndrome and investigated the effect of L. plantarum on NASH pathogenesis using transcriptomic, metagenomic, and immunohistochemistry analyses. Validation experiments were performed using liver organoids and a murine model fed a methionine-choline-deficient (MCD) diet. L. plantarum treatment in mice significantly decreased liver inflammation and improved metabolic phenotypes, such as insulin tolerance and the hepatic lipid content, compared with those in the vehicle group. RNA-sequencing analysis revealed that L. plantarum treatment significantly downregulated inflammation-related pathways. Shotgun metagenomic analysis revealed that L-arginine biosynthesis-related microbial genes were significantly upregulated in the L. plantarum group. We also confirmed the elevated arginine levels in the serum of the L. plantarum group. We further used liver organoids and mice fed an MCD diet to demonstrate that L-arginine alone was sufficient to alleviate liver inflammation. Our data revealed a novel and counterintuitive therapeutic effect of L. plantarum on alleviating NASH-related liver inflammation by increasing circulating L-arginine.

Molecular mechanisms of metabolic disease-associated hepatic inflammation in non-alcoholic fatty liver disease and non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is the leading chronic liver disease worldwide, with a progressive form of non-alcoholic steatohepatitis (NASH). It may progress to advanced liver diseases, including liver fibrosis, cirrhosis, and hepatocellular carcinoma. NAFLD/NASH is a comorbidity of many metabolic disorders such as obesity, insulin resistance, type 2 diabetes, cardiovascular disease, and chronic kidney disease. These metabolic diseases are often accompanied by systemic or extrahepatic inflammation, which plays an important role in the pathogenesis and treatment of NAFLD or NASH. Metabolites, such as short-chain fatty acids, impact the function, inflammation, and death of hepatocytes, the primary parenchymal cells in the liver tissue. Cholangiocytes, the epithelial cells that line the bile ducts, can differentiate into proliferative hepatocytes in chronic liver injury. In addition, hepatic non-parenchymal cells, including liver sinusoidal endothelial cells, hepatic stellate cells, and innate and adaptive immune cells, are involved in liver inflammation. Proteins such as fibroblast growth factors, acetyl-coenzyme A carboxylases, and nuclear factor erythroid 2-related factor 2 are involved in liver metabolism and inflammation, which are potential targets for NASH treatment. This review focuses on the effects of metabolic disease-induced extrahepatic inflammation, liver inflammation, and the cellular and molecular mechanisms of liver metabolism on the development and progression of NAFLD and NASH, as well as the associated treatments.

Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice.

Nonalcoholic steatohepatitis (NASH) is an advanced stage of fatty liver disease characterized by liver damage, inflammation, and fibrosis. Although neutrophil infiltration is consistently observed in the livers of patients with NASH, the precise role of neutrophil-recruiting chemokines and infiltrating neutrophils in NASH pathogenesis remains poorly understood. Here, we aimed to elucidate the role of neutrophil infiltration in the transition from fatty liver to NASH by examining hepatic overexpression of interleukin-8 (IL8), a major chemokine responsible for neutrophil recruitment in humans. Mice fed a high-fat diet (HFD) for 3 months developed fatty liver without concurrent liver damage, inflammation, and fibrosis. Subsequent infection with an adenovirus overexpressing human IL8 for an additional 2 weeks increased IL8 levels, neutrophil infiltration, and liver injury in mice. Mechanistically, IL8-induced liver injury was associated with the upregulation of components of the NADPH oxidase 2 complex, which participate in neutrophil oxidative burst. IL8-driven neutrophil infiltration promoted macrophage aggregate formation and upregulated the expression of chemokines and inflammatory cytokines. Notably, IL8 overexpression amplified factors associated with fibrosis, including collagen deposition and hepatic stellate cell activation, in HFD-fed mice. Collectively, hepatic overexpression of human IL8 promotes neutrophil infiltration and fatty liver progression to NASH in HFD-fed mice.

Macrophage metabolism in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) and its inflammatory and often progressive subtype nonalcoholic steatohepatitis (NASH), have emerged as significant contributors to hepatic morbidity worldwide. The pathophysiology of NAFLD/NASH is multifaceted, variable, and remains incompletely understood. The pivotal role of liver-resident and recruited macrophages in the pathogenesis of NAFLD and NASH is widely acknowledged as a crucial factor in innate immunity. The remarkable plasticity of macrophages enables them to assume diverse activation and polarization states, dictated by their immunometabolism microenvironment and functional requirements. Recent studies in the field of immunometabolism have elucidated that alterations in the metabolic profile of macrophages can profoundly influence their activation state and functionality, thereby influencing various pathological processes. This review primarily focuses on elucidating the polarization and activation states of macrophages, highlighting the correlation between their metabolic characteristics and the transition from pro-inflammatory to anti-inflammatory phenotypes. Additionally, we explore the potential of targeting macrophage metabolism as a promising therapeutic approach for the management of NAFLD/NASH.

Metabolic crosstalk between skeletal muscle cells and liver through IRF4-FSTL1 in nonalcoholic steatohepatitis

Inter-organ crosstalk has gained increasing attention in recent times; however, the underlying mechanisms remain unclear. In this study, we elucidate an endocrine pathway that is regulated by skeletal muscle interferon regulatory factor (IRF) 4, which manipulates liver pathology. Skeletal muscle specific IRF4 knockout (F4MKO) mice exhibited ameliorated hepatic steatosis, inflammation, and fibrosis, without changes in body weight, when put on a nonalcoholic steatohepatitis (NASH) diet. Proteomics analysis results suggested that follistatin-like protein 1 (FSTL1) may constitute a link between muscles and the liver. Dual luciferase assays showed that IRF4 can transcriptionally regulate FSTL1. Further, inducing FSTL1 expression in the muscles of F4MKO mice is sufficient to restore liver pathology. In addition, co-culture experiments confirmed that FSTL1 plays a distinct role in various liver cell types via different receptors. Finally, we observed that the serum FSTL1 level is positively correlated with NASH progression in humans. These data indicate a signaling pathway involving IRF4-FSTL1-DIP2A/CD14, that links skeletal muscle cells to the liver in the pathogenesis of NASH.

Comparison of the Effect of 8- and 12-Week Aerobic Exercise with Calorie Restriction on the Bax and Bcl-2 Expression in Rats with Non-alcoholic Fatty Liver Disease

Background: In addition to oxidative stress, the apoptosis of liver cells plays a main role in non-alcoholic steatohepatitis pathogenesis. Nevertheless, the main mechanisms of the response of liver cell apoptosis in non-alcoholic steatohepatitis (NASH) models caused by a high-fat diet, as well as the effects of exercise with and without calorie restriction on these mechanisms, have not been assessed to date. Objectives: The present study aimed to investigate the effects of two exercise protocols with and without calorie restriction on apoptosis and liver damage in rats with non-alcoholic fatty liver disease (NAFLD). Methods: Sixty-four male Wistar rats were subjected to a high-fat diet for eight weeks and were divided randomly into eight groups: Control, calorie restriction (CR), aerobic exercise (AE), and aerobic exercise + calorie restriction (AC) for eight and twelve weeks. Also, two groups of rats that had normal and free access to food were named sham groups. The training groups exercised on the treadmill for five sessions a week for eight and twelve weeks. Two-way ANOVA was utilized for data analysis at a significance level of 0.05. Results: According to the findings, in both 8- and 12-week protocols, the expression of Bax proteins in the exercise and exercise + calorie restriction groups showed a significant decrease compared to the control group (P < 0.05). Also, Bcl-2 protein expression significantly increased in the exercise and exercise + calorie restriction groups than the control group (P < 0.05). The training groups showed no significant difference between the 8- and 12-week protocols. Conclusions: It was revealed that exercise with and without caloric restriction and independent of the protocol duration can improve the apoptosis of hepatocytes in NAFLD.

MicroRNAs and Nonalcoholic Steatohepatitis: A Review.

Non-alcoholic fatty liver disease (NAFLD) is a clinicopathologic syndrome caused by fat deposition in hepatocytes. Patients with nonalcoholic steatohepatitis (NASH), an advanced form of NAFLD with severe fibrosis, are at high risk for liver-related complications, including hepatocellular carcinoma (HCC). However, the mechanism of progression from simple fat deposition to NASH is complex, and previous reports have linked NAFLD to gut microbiota, bile acids, immunity, adipokines, oxidative stress, and genetic or epigenetic factors. NASH-related liver injury involves multiple cell types, and intercellular signaling is thought to be mediated by extracellular vesicles. MicroRNAs (miRNAs) are short, noncoding RNAs that play important roles as post-transcriptional regulators of gene expression and have been implicated in the pathogenesis of various diseases. Recently, many reports have implicated microRNAs in the pathogenesis of NALFD/NASH, suggesting that exosomal miRNAs are potential non-invasive and sensitive biomarkers and that the microRNAs involved in the mechanism of the progression of NASH may be potential therapeutic target molecules. We are interested in which miRNAs are involved in the pathogenesis of NASH and which are potential target molecules for therapy. We summarize targeted miRNAs associated with the etiology and progression of NASH and discuss each miRNA in terms of its pathophysiology, potential therapeutic applications, and efficacy as a NASH biomarker.

Lysosomal cholesterol overload in macrophages promotes liver fibrosis in a mouse model of NASH.

Accumulation of lipotoxic lipids, such as free cholesterol, induces hepatocyte death and subsequent inflammation and fibrosis in the pathogenesis of nonalcoholic steatohepatitis (NASH). However, the underlying mechanisms remain unclear. We have previously reported that hepatocyte death locally induces phenotypic changes in the macrophages surrounding the corpse and remnant lipids, thereby promoting liver fibrosis in a murine model of NASH. Here, we demonstrated that lysosomal cholesterol overload triggers lysosomal dysfunction and profibrotic activation of macrophages during the development of NASH. β-cyclodextrin polyrotaxane (βCD-PRX), a unique supramolecule, is designed to elicit free cholesterol from lysosomes. Treatment with βCD-PRX ameliorated cholesterol accumulation and profibrotic activation of macrophages surrounding dead hepatocytes with cholesterol crystals, thereby suppressing liver fibrosis in a NASH model, without affecting the hepatic cholesterol levels. In vitro experiments revealed that cholesterol-induced lysosomal stress triggered profibrotic activation in macrophages predisposed to the steatotic microenvironment. This study provides evidence that dysregulated cholesterol metabolism in macrophages would be a novel mechanism of NASH.

Advancements in Diagnostic and Therapeutic Interventions of Non-alcoholic Fatty Liver Disease: A Literature Review.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common diseases of the liver globally. Non-alcoholic steatohepatitis (NASH) has a complicated pathophysiology which includes lipid buildup, oxidative stress, endoplasmic reticulum stress, and lipotoxicity. Recently, there has been tremendous improvement in understanding of NASH pathogenesis due to advancements in the scientific field. It is being investigated how non-invasive circulating and imaging biomarkers can help in NAFLD and NASH diagnosis and monitoring the progress. Multiple medications are now undergoing clinical trials for the treatment of NASH, and lifestyle changes have been acknowledged as one of the maintreatment methods. The purpose of this review articleis to discuss the incidence of NAFLD globally,management issues with NASH, and its relation to the metabolic syndrome. Itexplains pathophysiology as well as therapeutic strategies using natural items, dietary changes, and pharmaceutical treatments. While emphasizing the necessity for surrogate endpoints to facilitate medication development for NASH, the study also considers the potential of non-invasive imaging biomarkers including magnetic resonance imaging (MRI) and magnetic resonance elastography (MRE).

Cellular stress in the pathogenesis of nonalcoholic steatohepatitis and liver fibrosis.

The burden of chronic liver disease is rising substantially worldwide. Fibrosis, characterized by excessive deposition of extracellular matrix proteins, is the common pathway leading to cirrhosis, and limited treatment options are available. There is increasing evidence suggesting the role of cellular stress responses contributing to fibrogenesis. This Review provides an overview of studies that analyse the role of cellular stress in different cell types involved in fibrogenesis, including hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells and macrophages.

A Study to Evaluate the Efficacy of Saroglitazar in Non-Alcoholic Steatohepatitis Induced by High Fructose Diet Rat Model

Background Non-alcoholic steatohepatitis (NASH) is a clinical condition with a global prevalence of 25.24%. Peroxisome proliferator-activated receptors (PPAR) have been significantly associated with the pathogenesis of NASH. Aim To evaluate the efficacy of saroglitazar in an animal model of NASH by evaluating the magnitude of changes in liver function tests (LFT) and histopathology. Materials and Methods The baseline parameters of 14 male Sprague–Dawley rats were recorded and then grouped into four groups: treatment groups (high high-dose saroglitazar [HDSG] and low low-dose saroglitazar [LDSG] doses of saroglitazar), normal control, and disease control. Initially, except for the normal control, the other three groups were fed a fructose diet for 5 weeks and then all four groups were fed a standard chow diet for the next 2 weeks during which the two treatment groups were orally gavaged with saroglitazar. Changes in LFT, body weight (BW), lipid profile, oxidative stress, and histopathology were evaluated at different time points. Results A statistically significant reduction was found in the mean serum glutamic-oxaloacetic transaminase (SGOT) ( p = 0.0267) and serum glutamate-pyruvate transaminase (SGPT) ( p = 0.0059) between the groups at the end of treatment. As with BW changes ( p < 0.001), a significant difference was observed between the time points in HDSG and LDSG with respect to all parameters of the lipid profile assessed ( p < 0.05). Amelioration of hepatocellular ballooning and lobular inflammation in histopathology was evident in both treatment groups. Immunohistochemistry revealed loss of cytokeratin CK8/18 in disease control while it was preserved in LDSG and HDSG. Conclusion The study has explicitly illustrated the improvement in the biochemical and pathological changes in the rat model of NASH induced by a high fructose diet.

Restraint stress promotes nonalcoholic steatohepatitis by regulating the farnesoid X receptor/NLRP3 signaling pathway.

Psychological stress promotes nonalcoholic steatohepatitis (NASH) development. However, the pathogenesis of psychological stress-induced NASH remains unclear. This study aims to explore the underlying mechanism of restraint stress-induced NASH, which mimics psychological stress, and to discover potential NASH candidates. Methionine choline deficient diet- and high fat diet-induced hepatosteatotic mice are subjected to restraint stress to induce NASH. The mice are administrated with Xiaoyaosan granules, NOD-like receptor family pyrin domain containing 3 (NLRP3) inhibitors, farnesoid X receptor (FXR) agonists, or macrophage scavengers. Pathological changes and NLRP3 signaling in the liver are determined. These results demonstrate that restraint stress promotes hepatic inflammation and fibrosis in hepatosteatotic mice. Restraint stress increases the expressions of NLRP3, Caspase-1, Gasdermin D, interleukin-1β, cholesterol 7α-hydroxylase, and sterol 12α-hydroxylase and decreases the expression of FXR in NASH mice. Xiaoyaosan granules reverse hepatic inflammation and fibrosis and target FXR and NLRP3 signals. In addition, inhibition of NLRP3 reduces the NLRP3 inflammasome and liver damage in mice with restraint stress-induced NASH. Elimination of macrophages and activation of FXR also attenuate inflammation and fibrosis by inhibiting NLRP3 signaling. However, NLRP3 inhibitors or macrophage scavengers fail to affect the expression of FXR. In conclusion, restraint stress promotes NASH-related inflammation and fibrosis by regulating the FXR/NLRP3 signaling pathway. Xiaoyaosan granules, NLRP3 inhibitors, FXR agonists, and macrophage scavengers are potential candidates for the treatment of psychological stress-related NASH.

CXCL5 promotes lipotoxicity of hepatocytes through upregulating NLRP3/Caspase-1/IL-1β signaling in Kupffer cells and exacerbates nonalcoholic steatohepatitis in mice

Immune-inflammatory responses play a key role in the development of nonalcoholic steatohepatitis (NASH). Previous studies have demonstrated that CXC motif chemokine ligand 5 (CXCL5) correlates positively with obesity and type 2 diabetes. This study is to explore the functional role of CXCL5 in the pathogenesis of NASH. To establish a NASH model, mice were fed with methionine-and choline-deficient high-fat diet for 6 weeks and anti-CXCL5 mAb was injected during the same period. An in vitro NASH model was established by treating palmitic acid (PA), using a trans-well co-culture system of mouse primary hepatocytes and Kupffer cells (KCs), and recombinant mouse (rm) CXCL5 was treated after PA administration. Our data showed that hepatic CXCL5 levels were highly expressed in the NASH mouse model. CXCL5 neutralization significantly alleviated the severity of NASH livers, demonstrated by pathological analysis, decreased biochemicals, and inflammation. Besides, neutralizing CXCL5 reduced lipid accumulation, cell death, and fibrosis in injured livers. In vitro, rmCXCL5 could not affect the activation of hepatic stellate cells. Also, rmCXCL5 exacerbated PA-induced hepatotoxicity and lipid deposition in hepatocytes co-cultured with KCs rather than in single-cultured hepatocytes. Mechanistically, rmCXCL5 not only promoted NOD-like receptor pyrin domain-containing protein 3 (NLRP3) expression, Cleaved caspase-1 expression, and interleukin 1 beta (IL-1β) secretion in single-cultured and co-cultured KCs but also increased lipid deposition in co-cultured hepatocytes. In addition, MCC950, an inhibitor of NLRP3, almost abolished the effects of rmCXCL5 on PA-treated co-culture system. Therefore, CXCL5 could exacerbate NASH by promoting lipotoxicity of hepatocytes via upregulating NLRP3/Caspase-1/IL-1β signaling in KCs.