Geniposide dosage and administration time: Balancing therapeutic benefits and adverse reactions in liver disease treatment.

Gardenia jasminoides Ellis, a staple in herbal medicine, has long been esteemed for its purported hepatoprotective properties. Its primary bioactive constituent, geniposide, has attracted considerable scientific interest owing to its multifaceted therapeutic benefits across various health conditions. However, recent investigations have unveiled potential adverse effects associated with its metabolite, genipin, particularly at higher doses and prolonged durations of administration, leading to hepatic injury. Determining the optimal dosage and duration of geniposide administration while elucidating its pharmacological and toxicological mechanisms is imperative for safe and effective clinical application. This study aimed to evaluate the safe dosage and administration duration of geniposide in mice and investigate its toxicological mechanisms within a comprehensive dosage-duration-efficacy/toxicity model. Four distinct mouse models were employed, including wild-type mice, cholestasis-induced mice, globally farnesoid X-activated receptor (FXR) knock out mice, and high-fat diet-induced (HFD) NAFLD mice. Various administration protocols, spanning one or four weeks and comprising two or three oral doses, were tailored to each model's requirements. Geniposide has positive effects on bile acid and lipid metabolism at doses below 220 mg/kg/day without causing liver injury in normal mice. However, in mice with NAFLD, this dosage is less effective in improving liver function, lipid profiles, and bile acid metabolism compared to lower doses. In cholestasis-induced mice, prolonged use of geniposide at 220 mg/kg/day worsened liver damage. Additionally, in NAFLD mice, this dosage of geniposide for four weeks led to intestinal pyroptosis and liver inflammation. These results highlight the lipid-lowering and bile acid regulatory effects of geniposide, but also warn of potential negative impacts on intestinal epithelial cells, particularly with higher doses and longer treatment durations. Therefore, achieving optimal therapeutic results requires a decrease in treatment duration as the dosage increases, in order to maintain a balanced approach to the use of geniposide in clinical settings.

Salvianolic acid A attenuates non-alcoholic fatty liver disease by regulating the AMPK-IGFBP1 pathway.

Non-alcoholic fatty liver disease (NAFLD) affects approximately a quarter of the population and, to date, there is no approved drug therapy for this condition. Individuals with type 2 diabetes mellitus (T2DM) are at a significantly elevated risk of developing NAFLD, underscoring the urgency of identifying effective NAFLD treatments for T2DM patients. Salvianolic acid A (SAA) is a naturally occurring phenolic acid that is an important component of the water-soluble constituents isolated from the roots of Salvia miltiorrhiza Bunge. SAA has been demonstrated to possess anti-inflammatory and antioxidant stress properties. Nevertheless, its potential in ameliorating diabetes-associated NAFLD has not yet been fully elucidated. In this study, diabetic ApoE-/- mice were employed to establish a NAFLD model via a Western diet. Following this, they were treated with different doses of SAA (10 mg/kg, 20 mg/kg) via gavage. The study demonstrated a marked improvement in liver injury, lipid accumulation, inflammation, and the pro-fibrotic phenotype after the administration of SAA. Additionally, RNA-seq analysis indicated that the primary pathway by which SAA alleviates diabetes-induced NAFLD involves the cascade pathways of lipid metabolism. Furthermore, SAA was found to be effective in the inhibition of lipid accumulation, mitochondrial dysfunction and ferroptosis. A functional enrichment analysis of RNA-seq data revealed that SAA treatment modulates the AMPK pathway and IGFBP-1. Further experimental results demonstrated that SAA is capable of inhibiting lipid accumulation through the activation of the AMPK pathway and IGFBP-1.

Xiezhuo Tiaozhi formula inhibits macrophage pyroptosis in the non-alcoholic fatty liver disease by targeting the SIRT1 pathway.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a challenging disease to interfere with and represents a potential long-term risk factor for hepatic fibrosis and liver cancer. The Xiezhuo Tiaozhi (XZTZ) formula, a water extract from crude herbs, has been widely used as an anti-NAFLD agent through clinical observation. However, the underlying pharmacological mechanisms of the XZTZ formula and its impact on the potential pathways against NAFLD have not been elucidated. PURPOSE: Our study aims to investigate the pharmacological effects and underlying regulatory mechanisms of the XZTZ formula to treat NAFLD. METHODS: The possible active components and pharmacological mechanisms of the XZTZ formula against NAFLD were identified using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) and molecular docking. To further explore the potential mechanisms, forty-eight 6-week-old male C57BL/6 J mice were given individual attention with high-fat and high-sugar diet (HFHSD) or relevant control (Ctrl) diets for 16 weeks to successfully construct a NAFLD mouse model. Subsequently, the levels of serum biochemicals, pathological changes in the liver, and pyroptosis levels were assessed in mice to investigate the therapeutic effects of the XZTZ formula. Further, LPS-induced RAW264.7 cells and Immortalized Mouse Kupffer cells (ImKC) were used to verify the potential mechanisms of the XZTZ formula against NAFLD in vitro. RESULTS: We identified 7 chemical compounds and 2 potential therapeutic targets as plausible therapeutic points for the treatment of NAFLD using the XZTZ formula. Subsequent histopathological analysis revealed marked hepatic steatosis and lipid accumulation in the HFHSD mice liver, while conditions were effectively ameliorated by administration of the XZTZ formula. Additionally, our work demonstrated that the XZTZ formula could attenuate M1 polarization, promote M2 polarization, and suppress pyroptosis via the SIRT1 pathway in tissue samples. Moreover, validation performed through LPS-induced RAW264.7 and ImKC cells by showing that silencing SIRT1 weaken the effects of the XZTZ formula on relative pyroptosis affirmed that its role was associated with the SIRT1 pathway in macrophage. CONCLUSION: These findings suggest that the XZTZ formula alleviated hepatic steatosis and lipid accumulation in NAFLD mice. These ameliorations are associated with mechanisms involving the attenuation of M1 polarization, promotion of M2 polarization, and anti-pyroptosis effects through the SIRT1 pathway.

Xie Zhuo Tiao Zhi formula ameliorates chronic alcohol-induced liver injury in mice.

This study aimed to evaluate the protective role and potential mechanisms of Xie Zhuo Tiao Zhi decoction (XZTZ) on alcohol-associated liver disease (ALD). XZTZ significantly alleviated alcohol-induced liver dysfunction, based on histological examinations and biochemical parameters after 4-week administration. Mechanically, alcohol-stimulated hepatic oxidative stress was ameliorated by XZTZ, accompanied by the improvement of Nrf2/Keap1 expression and alcohol-activated phosphorylation of pro-inflammatory transcription factors, including JNK, P38, P65, and IκBα, were rescued by XZTZ. In conclusion, XZTZ demonstrates potential in alleviating alcohol-induced liver injury, oxidative stress, and inflammation possibly through modulation of Nrf2/Keap1 and MAPKs/NF-κB signaling pathways, suggesting its potential as a therapeutic option for patients with alcoholic liver disease.

Astragaloside IV ameliorates cisplatin-induced liver injury by modulating ferroptosis-dependent pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: The use of antineoplastic drugs, such as cisplatin, in clinical practice can cause adverse effects in patients, such as liver injury, which limits their long-term use. Therefore, there is an urgent need to develop alternative therapeutic strategies or drugs to minimize cisplatin-induced liver injury. Huangqi, the root of Astragalus membranaceus, is extensively used in traditional Chinese medicine (TCM) and has been employed in treating diverse liver injuries. Astragalus membranaceus contains several bioactive constituents, including triterpenoid saponins, one of which, astragaloside IV (ASIV), has been reported to have anti-inflammatory and antioxidant stress properties. However, its potential in ameliorating cisplatin-induced liver injury has not been explored. AIM OF THE STUDY: The objective of this study was to examine the mechanism by which ASIV protects against cisplatin-induced liver injury. MATERIALS AND METHODS: This study established a model of cisplatin-induced liver injury in mice, followed by treatment with various doses of astragaloside IV (40 mg/kg, 80 mg/kg). In addition, a model of hepatocyte ferroptosis in AML-12 cells was established using RSL3. The mechanism of action of astragaloside IV was investigated using a range of methods, including Western blot assay, qPCR, immunofluorescence, histochemistry, molecular docking, and high-content imaging system. RESULTS: The findings suggested a significant improvement in hepatic injury, inflammation and oxidative stress phenotypes with the administration of ASIV. Furthermore, network pharmacological analyses provided evidence that a major pathway for ASIV to attenuate cisplatin-induced hepatic injury entailed the cell death cascade pathway. It was observed that ASIV effectively inhibited ferroptosis both in vivo and in vitro. Subsequent experimental outcomes provided further validation of ASIV's ability to hinder ferroptosis through the inhibition of PPARα/FSP1 signaling pathway. The current findings suggest that ASIV could function as a promising phytotherapy composition to alleviate cisplatin-induced liver injury. CONCLUSIONS: The current findings suggest that astragaloside IV could function as a promising phytotherapy composition to alleviate cisplatin-induced liver injury.

The micro-743a-3p-GSTM1 pathway is an endogenous protective mechanism against alcohol-related liver disease in mice.

BACKGROUND AND AIMS: Epidemiological evidence suggests that the phenotype of glutathione S-transferase mu 1 (GSTM1), a hepatic high-expressed phase II detoxification enzyme, is closely associated with the incidence of alcohol-related liver disease (ALD). However, whether and how hepatic GSTM1 determines the development of ALD is largely unclear. This study was designed to elucidate the role and potential mechanism(s) of hepatic GSTM1 in the pathological process of ALD. METHODS: GSTM1 was detected in the liver of various ALD mice models and cultured hepatocytes. Liver-specific GSTM1 or/and micro (miR)-743a-3p deficiency mice were generated by adenoassociated virus-8 delivered shRNA, respectively. The potential signal pathways involving in alcohol-regulated GSTM1 and GSTM1-associated ALD were explored via both genetic manipulation and pharmacological approaches. RESULTS: GSTM1 was significantly upregulated in both chronic alcohol-induced mice liver and ethanol-exposed murine primary hepatocytes. Alcohol-reduced miR-743a-3p directly contributed to the upregulation of GSTM1, since liver specific silencing miR-743a-3p enhanced GSTM1 and miR-743a-3p loss protected alcohol-induced liver dysfunctions, which was significantly blocked by GSTM1 knockdown. GSTM1 loss robustly aggravated alcohol-induced hepatic steatosis, oxidative stress, inflammation, and early fibrotic-like changes, which was associated with the activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK), and p38. GSTM1 antagonized ASK1 phosphorylation and its downstream JNK/p38 signaling pathway upon chronic alcohol consumption via binding with ASK1. ASK1 blockage significantly rescued hepatic GSTM1 loss-enhanced disorders in alcohol-fed mice liver. CONCLUSIONS: Chronic alcohol consumption-induced upregulation of GSTM1 in the liver provides a feedback protection against hepatic steatosis and liver injury by counteracting ASK1 activation. Down-regulation of miR-743a-3p improves alcohol intake-induced hepatic steatosis and liver injury via direct targeting on GSTM1. The miR-743a-3p-GSTM1 axis functions as an innate protective pathway to defend the early stage of ALD.

Loss of macrophage TSC1 exacerbates sterile inflammatory liver injury through inhibiting the AKT/MST1/NRF2 signaling pathway.

Tuberous sclerosis complex 1 (TSC1) plays important roles in regulating innate immunity. However, the precise role of TSC1 in macrophages in the regulation of oxidative stress response and hepatic inflammation in liver ischemia/reperfusion injury (I/R) remains unknown. In a mouse model of liver I/R injury, deletion of myeloid-specific TSC1 inhibited AKT and MST1 phosphorylation, and decreased NRF2 accumulation, whereas activated TLR4/NF-κB pathway, leading to increased hepatic inflammation. Adoptive transfer of AKT- or MST1-overexpressing macrophages, or Keap1 disruption in myeloid-specific TSC1-knockout mice promoted NRF2 activation but reduced TLR4 activity and mitigated I/R-induced liver inflammation. Mechanistically, TSC1 in macrophages promoted AKT and MST1 phosphorylation, and protected NRF2 from Keap1-mediated ubiquitination. Furthermore, overexpression AKT or MST1 in TSC1-knockout macrophages upregulated NRF2 expression, downregulated TLR4/NF-κB, resulting in reduced inflammatory factors, ROS and inflammatory cytokine-mediated hepatocyte apoptosis. Strikingly, TSC1 induction in NRF2-deficient macrophages failed to reverse the TLR4/NF-κB activity and production of pro-inflammatory factors. Conclusions: Macrophage TSC1 promoted the activation of the AKT/MST1 signaling pathway, increased NRF2 levels via reducing Keap1-mediated ubiquitination, and modulated oxidative stress-driven inflammatory responses in liver I/R injury. Our findings underscore the critical role of macrophage TSC1 as a novel regulator of innate immunity and imply the therapeutic potential for the treatment of sterile liver inflammation in transplant recipients. Schematic illustration of macrophage TSC1-mediated AKT/MST1/NRF2 signaling pathway in I/R-triggered liver inflammation. Macrophage TSC1 can be activated in I/R-stressed livers. TSC1 activation promotes phosphorylation of AKT and MST1, which in turn increases NRF2 expression and inhibits ROS production and TLR4/NF-κB activation, resulting in reduced hepatocellular apoptosis in I/R-triggered liver injury.

Preventive effect of low-carbohydrate high-fat dietary pattern on liver disease caused by alcohol consumption via a 6pgd-involved mechanism in mice.

A low-carbohydrate high-fat (LCHF) dietary pattern has been reported to improve chronic metabolic diseases. However, whether and how the LCHF diet affects the pathological progression in patients with alcohol-related liver diseases (ALD) is largely unknown. This study was conducted to evaluate the effect of the LCHF diet on ALD and clarify its potential mechanism(s). The ALD model was established by feeding C57BL/6N mice with a Lieber-DeCarli liquid alcohol diet with a modified carbohydrate/fat ratio under an isoenergetic pattern. After an eight-week intervention, we observed that the LCHF diet significantly reduced alcohol-induced hepatic steatosis and liver injury, along with improved lipid metabolic-related gene disorders and redox imbalance. The alcohol-stimulated increase in pro-inflammatory cytokine cytokines expression, including TNF-α, IL-1ß, and IL-6, was markedly reversed by the LCHF diet. Liver transcriptome sequencing and qPCR validation showed that twenty-four alcohol-disturbed genes were significantly reversed by LCHF-diet intervention. The top differentially expressed genes were selected for further investigation. Among them, 6-phosphogluconate dehydrogenase (6PGD) was significantly up-regulated by alcohol treatment in both the liver and cultured hepatocytes. Spearman correlation analysis revealed that 6PGD was positively associated with hepatic steatosis, liver injury, and oxidative stress indexes. In vitro, the 6PGD knockdown ameliorated alcohol-induced hepatotoxicity and intracellular lipid accumulation, as well as lipid metabolic-related gene disorders, implying the involvement of 6PGD in LCHF-protected ALD. In conclusion, LCHF diet intervention alleviated chronic alcohol consumption-induced liver dysfunction in mice. 6PGD is a potential novel target for ALD prevention that contributes to LCHF-improved ALD. A LCHF diet might be a promising choice for ALD management.

Xie Zhuo Tiao Zhi formula modulates intestinal microbiota and liver purine metabolism to suppress hepatic steatosis and pyroptosis in NAFLD therapy.

BACKGROUND: Current evidence indicates a rising global prevalence of Non-Alcoholic Fatty Liver Disease (NAFLD), which is closely associated to conditions such as obesity, dyslipidemia, insulin resistance, and metabolic syndrome. The relationship between the gut microbiome and metabolites in NAFLD is gaining attention understanding the pathogenesis and progression of dysregulated lipid metabolism and inflammation. The Xie Zhuo Tiao Zhi (XZTZ) decoction has been employed in clinical practice for alleviating hyperlipidemia and symptoms related to metabolic disorders. However, the pharmacological mechanisms underlying the effects of XZTZ remain to be elucidated. PURPOSE: The objective of this study was to examine the pharmacological mechanisms underlying the hypolipidemic and anti-inflammatory effects of XZTZ decoction in a mouse model of NAFLD, as well as the effects of supplementing exogenous metabolites on PO induced cell damage and lipid accumulation in cultured hepatocytes. METHODS: A high-fat diet (HFD) mouse model was established to examine the effects of XZTZ through oral gavage. The general condition of mice and the protective effect of XZTZ on liver injury were evaluated using histological and biochemical methods. Hematoxylin and eosin staining (H&E) staining and oil red O staining were performed to assess inflammatory and lipid accumulation detection, and cytokine levels were quantitatively analyzed. Additionally, the study included full-length 16S rRNA sequencing, liver transcriptome analysis, and non-targeted metabolomics analysis to investigate the relationship among intestinal microbiome, liver metabolic function, and XZTZ decoction. RESULTS: XZTZ had a significant impact on the microbial community structure in NAFLD mice. Notably, the abundance of Ileibacterium valens, which was significantly enriched by XZTZ, exhibited a negative correlation with liver injury biomarkers such as, alanine transaminase (ALT) and aspartate transaminase (AST) activity. Moreover, treatment with XZTZ led to a significant enrichment of the purine metabolism pathway in liver tissue metabolites, with inosine, a purine metabolite, showing a significant positive correlation with the abundance of I. valens. XZTZ and inosine also significantly enhanced fatty acid ß-oxidation, which led to a reduction in the expression of pro-inflammatory cytokines and the inhibition of liver pyroptosis. These effects contributed to the mitigation of liver injury and hepatocyte damage, both in vivo and vitro. Furthermore, the utilization of HPLC fingerprints and UPLC-Q-TOF-MS elucidated the principal constituents within the XZTZ decoction, including naringin, neohesperidin, atractylenolide III, 23-o-Acetylalisol B, pachymic acid, and ursolic acid which are likely responsible for its therapeutic efficacy. Further investigations are imperative to fully uncover and validate the pharmacodynamic mechanisms underlying these observations. CONCLUSION: The administration of XZTZ decoction demonstrates a protective effect on the livers of NAFLD mice by inhibiting lipid accumulation and reducing hepatocyte inflammatory damage. This protective effect is mediated by the upregulation of I.valens abundance in the intestine, highlighting the importance of the gut-liver axis. Furthermore, the presesnce of inosine, adenosine, and their derivatives are important in promoting the protective effects of XZTZ. Furthermore, the in vitro approaching, we provide hitherto undocumented evidence indicating that the inosine significantly improves lipid accumulation, inflammatory damage, and pyroptosis in AML12 cells incubated with free fatty acids.

Farnesoid X receptor is an important target for the treatment of disorders of bile acid and fatty acid metabolism in mice with nonalcoholic fatty liver disease combined with cholestasis.

BACKGROUND AND AIM: The prevalence of nonalcoholic fatty liver disease (NAFLD) has been rising globally. NAFLD patients combined with cholestasis have more obvious liver fibrosis, impaired bile acid (BA), and fatty acid (FA) metabolism and severer liver injury; however, its therapeutic options are limited, and the underlying metabolic mechanisms are understood. Here, we aimed to investigate the effects of farnesoid X receptor (FXR) on BA and FA metabolism in NAFLD combined with cholestasis and related signaling pathways. METHODS: A mouse model of NAFLD combined with cholestasis was established by joint intervention with high-fat diet (HFD) and alpha-naphthylisothiocyanate. The effects of FXR on BA and FA metabolism were evaluated by serum biochemical analysis. Liver damage was identified by histopathology. The expression of nuclear hormone receptor, membrane receptor, FA transmembrane transporter, and BA transporter protein in mice were measured by western blot. RESULTS: NAFLD mice combined with cholestasis developed more severe cholestasis and dysregulated BA and FA metabolism. Meanwhile, the expression of FXR protein was decreased in NAFLD mice combined with cholestasis compared to the controls. Fxr-/- mice showed liver injury. HFD aggravated the liver injury with decreased BSEP expression, increased expression of NTCP, LXRα, SREBP-1c, FAS, ACC1, and CD36, and significantly increased BA and FA accumulation. CONCLUSION: All the results suggested that FXR plays a key role in both FA and BA metabolism in NAFLD combined with cholestasis and thus may be a potential target for the treatment of disorders of BA and FA metabolism in NAFLD combined with cholestasis.

Alcoholic Setdb1 suppression promotes hepatosteatosis in mice by strengthening Plin2.

BACKGROUND AND AIMS: Hepatosteatosis is one of the early features of alcoholic liver disease (ALD) and pharmaceutical or genetic interfering of the development of hepatosteatosis will efficiently alleviate the progression of ALD. Currently, the role of histone methyltransferase Setdb1 in ALD is not yet well understood. METHOD: Lieber-De Carli diet mice model and NIAAA mice model were constructed to confirm the expression of Setdb1. The hepatocyte-specific Setdb1-knockout (Setdb1-HKO) mice was established to determine the effects of Setdb1 in vivo. Adenovirus-Setdb1 were produced to rescue the hepatic steatosis in both Setdb1-HKO and Lieber-De Carli mice. The enrichment of H3k9me3 in the upstream sequence of Plin2 and the chaperone-mediated autophagy (CMA) of Plin2 were identified by ChIP and co-IP. Dual-luciferase reporter assay was used to detect the interaction of Setdb1 3'UTR and miR216b-5p in AML12 or HEK 293 T cells. RESULTS: We found that Setdb1 was downregulated in the liver of alcohol-fed mice. Setdb1 knockdown promoted lipid accumulation in AML12 hepatocytes. Meanwhile, hepatocyte-specific Setdb1-knockout (Setdb1-HKO) mice exhibited significant lipid accumulation in the liver. Overexpression of Setdb1 was performed with an adenoviral vector through tail vein injection, which ameliorated hepatosteatosis in both Setdb1-HKO and alcoholic diet-fed mice. Mechanistically, downregulated Setdb1 promoted the mRNA expression of Plin2 by desuppressing H3K9me3-mediated chromatin silencing in its upstream sequence. Pin2 acts as a critical membrane surface-associated protein to maintain lipid droplet stability and inhibit lipase degradation. The downregulation of Setdb1 also maintained the stability of Plin2 protein through inhibiting Plin2-recruited chaperone-mediated autophagy (CMA). To explore the reasons for Setdb1 suppression in ALD, we found that upregulated miR-216b-5p bound to the 3'UTR of Setdb1 mRNA, disturbed its mRNA stability, and eventually aggravated hepatic steatosis. CONCLUSIONS: Setdb1 suppression plays an important role in the progression of alcoholic hepatosteatosis via elevating the expression of Plin2 mRNA and maintaining the stability of Plin2 protein. Targeting hepatic Setdb1 might be a promising diagnostic or therapeutic strategy for ALD.

Novel Nomograms Based on Gamma-Glutamyl Transpeptidase-to-Lymphocyte Ratio Predict Prognosis of Hepatocellular Carcinoma Patients After Hepatectomy.

Background: The prediction of prognosis of hepatocellular carcinoma (HCC) is of great significance in improving disease outcome and optimizing clinical management, while reliable prognostic indicators are lacking. This study was conducted to develop readily-to-use nomograms for prognosis prediction of HCC after hepatectomy. Materials and Methods: Data of eligible patients were collected and analyzed retrospectively. Independent prognostic factors were identified by Cox regression, and nomograms for the prediction of disease-free survival (DFS) and overall survival (OS) were developed. The performance of the nomograms was evaluated by receiver operating characteristics (ROC) curves, C-indexes and calibration curves and was verified by the validation cohort. The predictive value of the nomograms was also compared with the 8th edition of American Joint Committee on Cancer (AJCC) Tumor-Node-Metastasis (TNM) and the Barcelona Clinic Liver Cancer (BCLC) staging systems. Results: In total, 599 patients were enrolled in the analysis: 420 in the training cohort and 179 in the validation cohort. The optimal cut-off value of Gamma-Glutamyl Transpeptidase-to-Lymphocyte Ratio (GLR) was 19.5. GLR contributed significantly to the nomograms with good predictive power. In ROC analyses, the areas under curve (AUCs) of the nomograms for 1-, 3- and 5-year DFS and OS prediction were 0.758, 0.756, 0.734 and 0.810, 0.799, 0.758, respectively. The C-indexes of the DFS nomogram were 0.697 (95% CI 0.665-0.729) in the training cohort and 0.710 (95% CI 0.664-0.756) in the validation cohort. For OS prediction, the C-indexes were 0.741 (95% CI 0.704-0.778) and 0.758 (95% CI 0.705-0.811) in the training and validation cohorts, respectively. The calibration curves demonstrated satisfactory agreement between nomogram predictions and actual observations. The nomograms demonstrated superior predictive performance to the TNM and the BCLC staging systems. Conclusion: Our novel nomograms showed adequate performance in the prediction of HCC prognosis after hepatectomy, which may facilitate the risk stratification and individualized management of HCC patients.

1-Methylnicotinamide promotes hepatic steatosis in mice: A potential mechanism in chronic alcohol-induced fatty liver disease.

Alcohol abuse and its related diseases are the major risk factors for human health. Alcohol-related liver disease (ALD) is a leading cause of morbidity and mortality worldwide. Although the mechanism of ALD has been widely investigated, liver metabolites associated with long-term alcohol intake-induced hepatic steatosis have not been well explored. In this study, we aimed to investigate the role and mechanisms of 1-methylnicotinamide (1-MNA), a metabolite during nicotinamide adenine dinucleotide (NAD+) metabolism, in the pathogenesis of ALD. C57BL/6 wild-type mice were subjected to chronic alcohol feeding with or without 1-MNA (50 mg/kg/day). Our data showed that 1-MNA administration significantly enhanced chronic alcohol consumption-induced hepatic steatosis. Mechanistic studies revealed that alcohol-increased hepatic protein levels of sterol regulatory element-binding transcription factor (SREBP-1c), a key enzyme that regulates lipid lipogenesis, were enhanced in mice administered with 1-MNA, regardless of alcohol feeding. Consistently, alcohol-increased mRNA and protein levels of hepatic diacylglycerol o-acyltransferase 2 (DGAT2) and very low-density lipoprotein receptor (VLDLR) were also exacerbated by 1-MNA administration. Alcohol-induced hepatic endoplasmic reticulum (ER) stress was enhanced by 1-MNA administration, which was evidenced by increased protein levels of binding immunoglobulin protein (BIP), phosphorylated- protein kinase r-like ER kinase (PERK), activating transcription factor 4 (ATF4), and C/EBP-homologous protein (CHOP) in the mouse liver. Overall, this study demonstrated that 1-MNA serves as a pathogenic factor in the development of ALD. Targeting liver 1-MNA levels may serve as a promising therapeutic approach for improving hepatic steatosis in ALD.

CFDP1 promotes hepatocellular carcinoma progression through activating NEDD4/PTEN/PI3K/AKT signaling pathway.

BACKGROUND AND AIMS: It is being increasingly reported that the Cranio Facial Development Protein 1 (CFDP1) plays a significant role in the onset and progression of tumors. Nonetheless, the underlying mechanisms associated with CFDP1 that contribute to hepatocellular carcinoma (HCC) and the specific biological role of CFDP1 remain vague. METHODS: The Gene Expression Omnibus (GEO) database was analyzed to obtain the gene expression profiles as well as the matching clinical data of HCC patients. The gene co-expression network was developed by means of weighted gene co-expression network analysis (WGCNA) to screen for possible biomarkers that could be used for the purpose of predicting prognosis. The Cancer Genome Atlas (TCGA) and Gene Expression Profile Interaction Analysis (GEPIA) databases were used to assess the relationship between survival and expression. In addition, we identified the underlying mechanism associated with CFDP1 by analyzing the KEGG pathway database, applying the GSEA and GeneCards analysis method. We performed a sequence of experiments (in vivo and in vitro) for the purpose of investigating the specific function of CFDP1 in liver cancer. RESULTS: The obtained results revealed high expression of CFDP1 in HCC tissues and cell lines. A positive correlation between the overexpression of CFDP1 and the adverse clinicopathological features was observed. Moreover, we observed that the low recurrence-free survival and overall survival were associated with CFDP1 overexpression. In addition, GeneCards and GSEA analysis showed that CFDP1 may interact with NEDD4 and participate in PTEN regulation. Meanwhile, CFDP1 can promote the malignant development of liver cancer in vivo and in vitro. The western blotting technique was also employed so as to examine the samples, and the findings demonstrated that CFDP1 enhanced the malignancy of HCC via the NEDD4-mediated PTEN/PI3K/AKT pathway. CONCLUSION: We highlighted that CFDP1 played an oncogenic role in HCC and was identified as a possible clinical prognostic factor and a potential novel therapeutic target for HCC.

Long noncoding RNA SNHG4 promotes the malignant progression of hepatocellular carcinoma through the miR-211-5p/CREB5 axis.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is one of the main death-leading malignant tumors which deserve in-depth explorations to uncover the underlying molecular mechanisms. Plenty of proofs have revealed that long noncoding RNAs (lncRNAs) participate in malignancy and progression of HCC. Nevertheless, the definite role of lncRNA-SNHG4 in HCC remains vague. METHODS: To figure out the role of SNHG4 in HCC, the bioinformatics analysis and functional assays and in vivo assay were performed. RESULTS: Our findings demonstrated that the data from The Cancer Genome Atlas (TCGA) displayed that the higher expression of lncRNA SNHG4 was detected in HCC tissues, which predicted the poor prognosis. The upregulation of SNHG4 was positively associated with worse clinicopathological characteristics. The functional experiments were performed to identify the role of SNHG4 in HCC. We found that SNHG4 enhanced the proliferative, migratory and invasive capacities of HCC cell line, and facilitated the tumor growth in vivo. A series of follow-up studies have shown that SNHG4 promoted the progression and malignancy of HCC through upregulating CREB5 via sponging miR-211-5p. CONCLUSION: Collectively, the above findings suggest that SNHG4 promotes HCC malignancy through the SNHG4/miR-211-5p/CREB5 axis, providing potential therapeutic targets and prognostic factors for HCC. Highlights SNHG4 is overexpressed in HCC and correlated with the poor clinical characteristics SNHG4 promotes the malignant progression of HCC by reducing miR-211-5p expression MiR-211-5p inhibits CREB5 expression in HCC The oncogenic effect of SNHG4 in HCC can be reversed by CREB5 silencing.

miR-488-5p mitigates hepatic stellate cell activation and hepatic fibrosis via suppressing TET3 expression.

BACKGROUND AND AIMS: Numerous studies have demonstrated that hepatic fibrosis, a progressive condition as an endpoint of multiple chronic hepatic diseases, is largely characterized with the extensive activation of hepatic stellate cells (HSCs). The precise effect of miR-488-5p in HSCs during hepatic fibrosis has not been elucidated. METHODS: In our study, qRT-PCR was applied to assess the level of miR-488-5p in activated HSCs stimulated by TGF-ß1. We built murine liver fibrosis models with carbon tetrachloride (CCl4), high-fat diet (HFD) and bile duct ligation (BDL). In vitro, the effects of miR-488-5p in HSCs were examined through cell proliferation assay and apoptosis. Luciferase reporter assay was applied to identify the underlying target of miR-488-5p. In vivo, the effects of miR-488-5p were explored through mouse liver fibrosis models. RESULTS: The reduction of miR-488-5p in the activated HSCs induced by TGF-ß1 and three mouse hepatic fibrosis models were identified. The in vitro functional experimentations verified that miR-488-5p restrained expression of fibrosis-related markers and proliferative capacity in HSCs. Mechanically, we identified that miR-488-5p inhibited tet methylcytosine dioxygenase 3 (TET3) expression via straightly binding onto the 3' UTR of its mRNA, which sequentially restrained the TGF-ß/Smad2/3 pathway. TET3 inhibition induced by the overexpression of miR-488-5p reduced extracellular matrix deposition, which contributed to mitigating mouse liver fibrosis. CONCLUSION: We highlight that miR-488-5p restrains the activation of HSCs and hepatic fibrosis via targeting TET3 which is involved in the TGF-ß/Smad2/3 signaling pathway. Collectively, miR-488-5p is identified as a potential therapeutic target for hepatic fibrosis.

Design and Synthesis of Novel Indole Ethylamine Derivatives as a Lipid Metabolism Regulator Targeting PPARα/CPT1 in AML12 Cells.

Peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1 (CPT1) are important targets of lipid metabolism regulation for nonalcoholic fatty liver disease (NAFLD) therapy. In the present study, a set of novel indole ethylamine derivatives (4, 5, 8, 9) were designed and synthesized. The target product (compound 9) can effectively activate PPARα and CPT1a. Consistently, in vitro assays demonstrated its impact on the lipid accumulation of oleic acid (OA)-induced AML12 cells. Compared with AML12 cells treated only with OA, supplementation with 5, 10, and 20 µM of compound 9 reduced the levels of intracellular triglyceride (by 28.07%, 37.55%, and 51.33%) with greater inhibitory activity relative to the commercial PPARα agonist fenofibrate. Moreover, the compound 9 supplementations upregulated the expression of hormone-sensitive triglyceride lipase (HSL) and adipose triglyceride lipase (ATGL) and upregulated the phosphorylation of acetyl-CoA carboxylase (ACC) related to fatty acid oxidation and lipogenesis. This dual-target compound with lipid metabolism regulatory efficacy may represent a promising type of drug lead for NAFLD therapy.

miR-345-5p curbs hepatic stellate cell activation and liver fibrosis progression by suppressing hypoxia-inducible factor-1alpha expression.

Hepatic fibrosis, as a common stage of multiple liver diseases, currently has no effective drug treatment. Emerging evidence shows that miRNAs participate in the progression of liver fibrosis. However, the potential role of miRNAs in hepatic fibrosis is not yet fully understood. Herein, we first confirmed that miR-345-5p expression was significantly decreased in activated hepatic stellate cells (HSCs) and fibrotic livers. Functional analysis showed that overexpression of miR-345-5p in human LX-2 cells suppressed the expression of profibrotic markers and cellular proliferation in vitro. Using a dual-luciferase assay, we demonstrated that miR-345-5p regulates HSC activation by targeting the 3'UTR of HIF-1α mRNA. In addition, overexpression of miR-345-5p in vivo alleviated murine liver fibrosis induced by carbon tetrachloride (CCl4) injection, high-fat diet (HFD) feeding and bile duct ligation (BDL). Furthermore, overexpression of miR-345-5p downregulated the expression of HIF-1α and fibrosis markers in livers from different fibrosis models. Collectively, we conclude that miR-345-5p mediates the activation of HSCs by targeting HIF-1α, which subsequently modulates TGFß/Smad2/Smad3 signaling. Thus, miR-345-5p may become a novel therapeutic target for the treatment of liver fibrosis.

FSTL1 promotes liver fibrosis by reprogramming macrophage function through modulating the intracellular function of PKM2.

OBJECTIVE: Follistatin-like protein 1 (FSTL1) is widely recognised as a secreted glycoprotein, but its role in modulating macrophage-related inflammation during liver fibrosis has not been documented. Herein, we aimed to characterise the roles of macrophage FSTL1 in the development of liver fibrosis. DESIGN: Expression analysis was conducted with human liver samples obtained from 33 patients with liver fibrosis and 18 individuals without fibrosis serving as controls. Myeloid-specific FSTL1-knockout (FSTL1M-KO) mice were constructed to explore the function and mechanism of macrophage FSTL1 in 3 murine models of liver fibrosis induced by carbon tetrachloride injection, bile duct ligation or a methionine-deficient and choline-deficient diet. RESULTS: FSTL1 expression was significantly elevated in macrophages from fibrotic livers of both humans and mice. Myeloid-specific FSTL1 deficiency effectively attenuated the progression of liver fibrosis. In FSTL1M-KO mice, the microenvironment that developed during liver fibrosis showed relatively less inflammation, as demonstrated by attenuated infiltration of monocytes/macrophages and neutrophils and decreased expression of proinflammatory factors. FSTL1M-KO macrophages exhibited suppressed proinflammatory M1 polarisation and nuclear factor kappa B pathway activation in vivo and in vitro. Furthermore, this study showed that, through its FK domain, FSTL1 bound directly to the pyruvate kinase M2 (PKM2). Interestingly, FSTL1 promoted PKM2 phosphorylation and nuclear translocation, reduced PKM2 ubiquitination to enhance PKM2-dependent glycolysis and increased M1 polarisation. Pharmacological activation of PKM2 (DASA-58) partially countered FSTL1-mediated glycolysis and inflammation. CONCLUSION: Macrophage FSTL1 promotes the progression of liver fibrosis by inducing M1 polarisation and inflammation based on the intracellular PKM2 reprogramming function of macrophages.

Macrophage nuclear factor erythroid 2-related factor 2 deficiency promotes innate immune activation by tissue inhibitor of metalloproteinase 3-mediated RhoA/ROCK pathway in the ischemic liver.

BACKGROUND AND AIMS: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of reactive oxygen species (ROS) and inflammation and has been implicated in both human and murine inflammatory disease models. We aimed to characterize the roles of macrophage-specific Nrf2 in liver ischemia/reperfusion injury (IRI). APPROACH AND RESULTS: First, macrophage Nrf2 expression and liver injury in patients undergoing OLT or ischemia-related hepatectomy were analyzed. Subsequently, we created a myeloid-specific Nrf2-knockout (Nrf2M-KO ) strain to study the function and mechanism of macrophage Nrf2 in a murine liver IRI model. In human specimens, macrophage Nrf2 expression was significantly increased in liver tissues after transplantation or hepatectomy. Interestingly, lower Nrf2 expressions correlated with more severe liver injury postoperatively. In a mouse model, we found Nrf2M-KO mice showed worse hepatocellular damage than Nrf2-proficient controls based on serum biochemistry, pathology, ROS, and inflammation. In vitro, Nrf2 deficiency promoted innate immune activation and migration in macrophages on toll-like receptor (TLR) 4 stimulation. Microarray profiling showed Nrf2 deletion caused markedly lower transcriptional levels of tissue inhibitor of metalloproteinase 3 (Timp3). ChIP-seq, PCR, and luciferase reporter assay further demonstrated Nrf2 bound to the promoter region of Timp3. Moreover, a disintegrin and metalloproteinase (ADAM) 10/ROCK1 was specifically increased in Nrf2-deficient macrophages. Increasing Timp3 expression effectively inhibited ADAM10/ROCK1 expression and rescued the Nrf2M-KO -mediated inflammatory response on TLR4 stimulation in vitro. Importantly, Timp3 overexpression, recombinant Timp3 protein, or ROCK1 knockdown rescued Nrf2M-KO -related liver IRI by inhibiting macrophage activation. CONCLUSIONS: In conclusion, macrophage Nrf2 mediates innate proinflammatory responses, attenuates liver IRI by binding to Timp3, and inhibits the RhoA/ROCK pathway, which provides a therapeutic target for clinical organ IRI.