Mouse Liver Research Articles

Genome-wide DNA methylation sequencing reveals the involvement of ferroptosis in hepatotoxicity induced by dietary exposure to food-grade titanium dioxide

BackgroundFollowing the announcement by the European Food Safety Authority that the food additive titanium dioxide (E 171) is unsafe for human consumption, and the subsequent ban by the European Commission, concerns have intensified over the potential risks E 171 poses to human vital organs. The liver is the main organ for food-grade nanoparticle metabolism. It is increasingly being found that epigenetic changes may play an important role in nanomaterial-induced hepatotoxicity. However, the profound effects of E 171 on the liver, especially at the epigenetic level, remain largely unknown.MethodsMice were exposed orally to human-relevant doses of two types of E 171 mixed in diet for 28 and/or 84 days. Conventional toxicology and global DNA methylation analyses were performed to assess E 171-induced hepatotoxicity and epigenetic changes. Whole genome bisulfite sequencing and further ferroptosis protein detection were used to reveal E 171-induced changes in liver methylation profiles and toxic mechanisms.ResultsExposed to E 171 for 28 and/or 84 days resulted in reduced global DNA methylation and hydroxymethylation in the liver of mice. E 171 exposure for 84 days elicited inflammation and damage in the mouse liver, whereas 28-day exposure did not. Whole-genome DNA methylation sequencing disclosed substantial methylation alterations at the CG and non-CG sites of the liver DNA in mice exposed to E 171 for 84 days. Mechanistic analysis of the DNA methylation alterations indicated that ferroptosis contributed to the liver toxicity induced by E 171. E 171-induced DNA methylation changes triggered NCOA4-mediated ferritinophagy, attenuated the protein levels of GPX4, FTH1, and FTL in the liver, and thereby caused ferroptosis.ConclusionsLong-term oral exposure to E 171 triggers hepatotoxicity and induces methylation changes in both CG and non-CG sites of liver DNA. These epigenetic alterations activate ferroptosis in the liver through NCOA4-mediated ferritinophagy, highlighting the role of DNA methylation and ferroptosis in the potential toxicity caused by E 171 in vivo.

The protective role of RACK1 in hepatic ischemia‒reperfusion injury-induced ferroptosis.

Although ferroptosis plays a crucial role in hepatic ischemia‒reperfusion injury (IRI), the molecular mechanisms underlying this process remain unclear. We aimed to explore the potential involvement of the receptor for activated C kinase 1 (RACK1) in hepatic IRI-triggered ferroptosis. Using hepatocyte-specific RACK1 knockout mice and alpha mouse liver 12 (AML12) cells, we conducted a series of in vivo and in vitro experiments. We found that RACK1 has a protective effect on hepatic IRI-induced ferroptosis. Specifically, RACK1 was found to interact with AMPKα through its 1-93 amino acid (aa) region, which facilitates the phosphorylation of AMPKα at threonine 172 (Thr172), ultimately exerting an antiferroptotic effect. Furthermore, the long noncoding RNA (lncRNA) ZNFX1 Antisense 1 (ZFAS1) directly binds to aa 181-317 of RACK1. ZFAS1 has a dual impact on RACK1 by promoting its ubiquitin‒proteasome-mediated degradation and inhibiting its expression at the transcriptional level, which indirectly exacerbates hepatic IRI-induced ferroptosis. These findings underscore the protective role of RACK1 in hepatic IRI-induced ferroptosis and showcase its potential as a prophylactic target for hepatic IRI mitigation.

Gender-specific alteration of steroid metabolism and its impact on viral replication in a mouse model of hepatitis B virus infection

ABSTRACT Hepatitis B virus (HBV) is a sex-specific pathogen that is more severe in males than in females. Sex disparities in HBV infection have been attributed to hormonal differences between males and females. However, whether HBV infection affects the metabolic signatures of steroid hormones and how these influences viral replication remains unclear. In this study, we investigated whether HBV infection alters steroid metabolism and its effects on HBV replication. Serum samples from male and female mice obtained after the hydrodynamic injection of replication-competent HBV plasmids were subjected to quantitative steroid profiling. Serum steroid levels in mice were analyzed using an in vitro metabolism assay with the mouse liver S9 fraction. The alteration of steroids by HBV infection was observed only in male mice, particularly with significant changes in androgens, whereas no significant hormonal changes were observed in female mice. Among the altered steroids, dehydroepiandrosterone (DHEA) levels increased the most in male mice after HBV infection. An in vitro metabolism assay revealed that androgen levels were significantly reduced in HBV-infected male mice. Furthermore, the genes involved in DHEA biosynthesis were significantly upregulated in HBV-infected male mice. Interestingly, reduced dihydrotestosterone in male mice significantly inhibits viral replication by suppressing HBV promoter activity, suggesting a viral strategy to overcome the antiviral effects of steroid hormones in males. Our data demonstrated that HBV infection can cause sex-specific changes in steroid metabolism.

Liver-specific Coxsackievirus and adenovirus receptor deletion develop metabolic dysfunction–associated fatty liver disease

Metabolic dysfunction–associated fatty liver disease (MAFLD) is a common liver disease associated with obesity and is caused by the accumulation of ectopic fat without alcohol consumption. Coxsackievirus and adenovirus receptor (CAR) are vital for cardiac myocyte-intercalated discs and endothelial cell-to-cell tight junctions. CAR has also been reported to be associated with obesity and high blood pressure. However, its function in the liver is still not well understood. The liver of obese mice exhibit elevated CAR mRNA and protein levels. Furthermore, in the liver of patients with non-alcoholic steatohepatitis, CAR is reduced in hepatocyte cell–cell junctions compared to normal levels. We generated liver-specific CAR knockout (KO) mice to investigate the role of CAR in the liver. Body and liver weights were not different between wild-type (WT) and KO mice fed a paired or high-fat diet (HFD). However, HFD induced significant liver damage and lipid accumulation in CAR KO mice compared with WT mice. Additionally, inflammatory cytokines transcription, hepatic permeability, and macrophage recruitment considerably increased in CAR KO mice. We identified a new interaction partner of CAR using a protein pull-down assay and mass spectrometry. Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3C (APOBEC3C) demonstrated a complex relationship with CAR, and hepatic CAR expression tightly regulated its level. Moreover, Apolipoprotein B (ApoB) and Low-density lipoprotein receptor (LDLR) levels correlated with APOBEC3C expression in the liver of CAR KO mice, suggesting that CAR may regulate lipid accumulation by controlling APOBEC3C activity. In this study, we showed that hepatic CAR deficiency increased cell-to-cell permeability. In addition, CAR deletion significantly increased hepatic lipid accumulation by inducing ApoB and LDLR expression. Although the underlying mechanism is unclear, CARs may be a target for the development of novel therapies for MAFLD.

Deubiquitinase JOSD1 tempers hepatic proteotoxicity

Derangements in protein homeostasis and associated proteotoxicity mark acute, chronic, and drug-induced hepatocellular injury. Metabolic dysfunction-associated proteasomal inhibition and the use of proteasome inhibitors often underlie such pathological hepatic proteotoxicity. In this study, we sought to identify a candidate deubiquitinating enzyme (DUB) responsible for reversing the proteotoxic damage. To this end, we performed a siRNA screening wherein 96 DUBs were individually knocked down in HepG2 cells under proteasomal inhibitor-induced stress for dual readouts, apoptosis, and cell viability. Among the putative hits, we chose JOSD1, a member of the Machado-Josephin family of DUBs that reciprocally increased cell viability and decreased cell death under proteotoxicity. JOSD1-mediated mitigation of proteotoxicity was further validated in primary mouse hepatocytes by gain and loss of function studies. Marked plasma membrane accumulation of monoubiquitinated JOSD1 in proteotoxic conditions is a prerequisite for its protective role, while the enzymatically inactive JOSD1 C36A mutant was conversely polyubiquitinated, does not have membrane localisation and fails to reverse proteotoxicity. Mechanistically, JOSD1 physically interacts with the suppressor of cytokine signalling 1 (SOCS1), deubiquitinates it and enhances its stability under proteotoxic stress. Indeed, SOCS1 expression is necessary and sufficient for the hepatoprotective function of JOSD1 under proteasomal inhibition. In vivo, adenovirus-mediated ectopic expression or depletion of JOSD1 in mice liver respectively protects or aggravates hepatic injury when challenged with proteasome blocker Bortezomib. Our study thus unveils JOSD1 as a potential candidate for ameliorating hepatocellular damage in liver diseases.

Abstract PR-12: The circadian rhythm gene Dec2 regulates multiple components of the antigen presentation pathway to promote pancreatic cancer dormancy by immune evasion

Abstract Metastatic recurrence following curative-intent pancreatic surgery is a major clinical problem as nearly all die of this at both early and latent periods following surgery. While latent recurrences are due to the reactivation of disseminated dormant tumor cells, the mechanisms that regulate dormancy remain unclear. Using a novel model of early-stage resectable pancreatic cancer that mimics early and latent outcomes in patients, we identified Dec2 (bhlh41) a transcriptional repressor and circadian regulator, as upregulated in dormant tumor cells harvested from the liver. Disseminated tumor cells harvest from the livers of latent recurrent mice were Dec2 hi/Ki67lo indicating that these cells are dormant. We confirmed the relationship of Dec2 and Ki67 in human pancreatic cancer using liver tissue specimens from patients with metastatic disease whereby small clusters of metastatic cells were Dec2hi/Ki67lo and larger metastatic tumors were Dec2lo/Ki67hi. To understand the mechanism of Dec2 in dormancy we deleted Dec2 from the tumor cells evaluated them in the resection model and observed a substantial increase in survival after resection due to an immune mediated mechanism as the survival benefit was abrogated in immunodeficient conditions indicating that Dec2 promotes tumor progression through immune mediated mechanism. Dec2 promotes immune evasion by downregulating multiple components of the antigen presentation pathway including proteosome and immunoproteosome genes, MHCI and MHCI chaperones. As Dec2 is a circadian regulator, we evaluated several components of the antigen presentation pathway including proteosome and surface MHCI and found these to display oscillation that was not only anti-phase with Dec2 but resulted in differential T-cell mediated tumor cell killing depending on the time of day. These studies elucidate a new mechanism by which Dec2 regulates cancer dormancy through immune evasion and identifies several novel therapeutic strategies to restore anti-tumor immunity. They also provide the first evidence that antigen presentation is under circadian regulation in pancreatic cancer. Citation Format: Chris Harris, Crissy Dudgeon, Lan Wang, Orjola Prela, Juliana Cazarin de Menezes, Brian Altman, Brian Haab, Chongfeng Gao, Zachary Klamer, Anna Repesh, Subhajyoti De, Darren R Carpizo. The circadian rhythm gene Dec2 regulates multiple components of the antigen presentation pathway to promote pancreatic cancer dormancy by immune evasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-12.

Knockdown of the Clock gene in the liver aggravates MASLD in mice via inhibiting lipophagy.

The increased global prevalence of metabolic dysfunction-associated steatohepatitis (MASLD) has been closely associated with chronic disorders of the circadian clock. Herein, we investigate the role of Clock, a core circadian gene, in the pathogenesis of MASLD. Wild-type (WT) and liver-specific Clock knockdown (Clock-KD) mice were fed a Western diet for 20weeks to induce MASLD. A cellular MASLD model was established by treating AML12 cells with free fatty acids and the effects of Clock knockdown were examined following transfection with Clock siRNA. Increased lipid deposition and more severe steatohepatitis and fibrosis were observed in the livers of Western diet-fed but not normal chow diet-fed Clock-KD mice after 20weeks compared to WT mice. Moreover, the Clock gene was found to be significantly downregulated in WT MASLD mice. The Clock gene was shown to regulate the expression of lipophagy-related proteins (LC3B, P62, RAB7, and PLIN2) in vivo and in vitro. Knockdown of Clock was found to inhibit lipophagy resulting in increased accumulation of lipid droplets in the mouse liver and AML12 cells. Interestingly, the CLOCK protein was shown to interact with P62. However, knockdown of the Clock gene did not promote transcription of the P62 gene but suppressed degradation of the P62 protein during lipophagy in AML12 cells. The hepatic Clock gene regulates lipophagy and affects lipid droplet deposition in liver cells, and thus plays a critical role in the development of MASLD induced by a Western diet.

Antifungal activities of Equol against Candida albicans in vitro and in vivo

ABSTRACT Candida albicans is an opportunistic fungal pathogen that can cause systemic infections in immunocompromised individuals. Morphological transition and biofilm formation are major virulence factors of C. albicans. Moreover, biofilm enhances resistance to antifungal agents. Therefore, it is urgent to identify new and effective compounds to target the biofilm of C. albicans. In the present study, the antifungal activities of equol against C. albicans were investigated. In vitro, the microdilution analysis and spot assay result showed that equol exhibited potent inhibitory activities against C. albicans. Further investigations confirmed that the antifungal effects of equol involved interference with the transition from yeast to hypha and biofilm formation of C. albicans. In addition, transcriptome sequencing and reverse transcription-quantitative PCR (qRT-PCR) analysis showed that equol significantly downregulated the expression of several genes in the Ras1-cAMP-PKA pathway related to hyphae and biofilm formation, and significantly upregulated the expression of the negative transcriptional repressors RFG1 and TUP1. Moreover, equol effectively reduced the production of cAMP, a key messenger in the Ras1-cAMP-PKA pathway, while supplementation with cAMP partly rescued the equol-induced defects in hyphal development. Furthermore, in a mouse model of systemic candidiasis (SC), equol treatment significantly decreased the fungal burden (liver, kidneys, and lung) in mice and local tissue damage, while enhancing the production of interleukin-10 (IL-10). Together, these findings confirm that equol is a potentially effective agent for treatment of SC.

Role of Deoxyribonucleic Acid Origami for Alleviating Kidney and Liver Injury in Diabetic Sepsis.

Treating diabetic sepsis (DS) can be challenging because of the persistent infection of multiple organs. To address this complicated pathological condition, it is necessary to develop advanced materials and gain a better understanding of their roles. In this study, we developed a two-dimensional planar material with a rectangular deoxyribonucleic acid origami nanostructure (termed Rec-DON). Rec-DON was used to improve liver and renal function in DS mice, as it preferentially accumulates in these organs, and has superior anti-inflammatory activity and the ability to scavenge reactive oxygen species. The role of Rec-DON in the treatment of DS mice was investigated via quantitative proteomics. This study revealed that Rec-DON can regulate key proteins located primarily in the cytoplasm and mitochondrion, involved in protein transport, antigen processing and presentation, and steroid metabolic process, and can also bind to various proteins to restore liver and renal function in DS mice. This study presented Rec-DON as a liver and kidney targeting material and revealed its role in alleviating multiorgan injury in DS.

SIRT1 and FOXO1 role on MASLD risk: effects of DHA-rich n-3 PUFA supplementation and exercise in aged obese female mice and in post-menopausal overweight/obese women.

Sirtuins 1 (SIRT1) and Forkhead box protein O1 (FOXO1) expression have been associated with obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). Exercise and/or docosahexaenoic acid (DHA) supplementation have shown beneficial effects on MASLD. The current study aims to assess the relationships between Sirt1, Foxo1 mRNA levels and several MASLD biomarkers, as well as the effects of DHA-rich n-3 PUFA supplementation and/or exercise in the steatotic liver of aged obese female mice, and in peripheral blood mononuclear cells (PBMCs) of postmenopausal women with overweight/obesity. In the liver of 18-month-old mice, Sirt1 levels positively correlated with the expression of genes related to fatty acid oxidation, and negatively correlated with lipogenic and proinflammatory genes. Exercise (long-term treadmill training), especially when combined with DHA, upregulated hepatic Sirt1 mRNA levels. Liver Foxo1 mRNA levels positively associated with hepatic triglycerides (TG) content and the expression of lipogenic and pro-inflammatory genes, while negatively correlated with the lipolytic gene Hsl. In PBMCs of postmenopausal women with overweight/obesity, FOXO1 mRNA expression negatively correlated with the hepatic steatosis index (HSI) and the Zhejiang University index (ZJU). After 16-weeks of DHA-rich PUFA supplementation and/or progressive resistance training (RT), most groups exhibited reduced MASLD biomarkers and risk indexes accompanying with body fat mass reduction, but no significant changes were found between the intervention groups. However, in PBMCs n-3 supplementation upregulated FOXO1 expression, and the RT groups exhibited higher SIRT1 expression. In summary, SIRT1 and FOXO1 could be involved in the beneficial mechanisms of exercise and n-3 PUFA supplementation related to MASLD manifestation.

SYK promotes the formation of neutrophil extracellular traps by inducing PKM2 nuclear translocation and promoting STAT3 phosphorylation to exacerbate hepatic ischemia-reperfusion injury and tumor recurrence

BackgroundAt present, hepatic ischemia-reperfusion injury (IRI) is an important complication of partial hepatectomy and liver transplantation, and it is an important cause of poor prognosis. Spleen tyrosine kinase(SYK) plays an important role in a variety of signaling pathways in the liver, but its role in hepatic IRI is still unclear. This study aims to investigate the role and mechanism of SYK in hepatic IRI and tumor recurrence.MethodsWe first observed the activation of SYK in the liver of mice in response to hepatic IRI. Subsequently, Pharmacological inhibitions of SYK were used to evaluated the effect of SYK on neutrophil recruitment and NETosis, and further explored the effect of SYK on IRI and tumor recurrence.ResultsOur study shows that SYK is activated in response to hepatic IRI and aggravates liver injury. On the one hand, neutrophils SYK during the early stage of liver reperfusion increases neutrophil extracellular traps (NETs) production by promoting Pyruvate kinase M2(PKM2) nuclear translocation leading to upregulation of phosphorylated STAT3, thereby exacerbating liver inflammation and tumor recurrence. On the other hand, macrophages SYK can promote the recruitment of neutrophils and increase the activation of NLRP3 inflammasome and IL1β, which further promotes the formation of NETs.ConclusionsOur study demonstrates that neutrophil and macrophage SYK synergistically promote hepatic IRI and tumor recurrence, and SYK may be a potential target to improve postoperative hepatic IRI and tumor recurrence.

Liver adrenoceptor alpha-1b plays a key role in energy and glucose homeostasis in female mice.

The liver plays a major role in glucose and lipid homeostasis and acts as a key organ in the pathophysiology of metabolic diseases. Intriguingly, increased sympathetic nervous system (SNS) activity to the liver has been associated with the development and progression of type 2 diabetes and obesity. However, the precise mechanisms by which the SNS regulates hepatic metabolism remain to be defined. While liver alpha1-adrenoceptors were suggested to play a role in glucose homeostasis, the specific subtypes involved are unknown mainly because of the limitations of pharmacological tools. Here we generated and validated a novel mouse model allowing tissue-specific deletion of alpha-1b adrenoceptor (Adra1b) in hepatocytes to investigate the role of liver ADRA1B in energy and glucose metabolism. We found that selective deletion of Adra1b in mouse liver has limited metabolic impact in lean mice. However, loss of Adra1bin hepatocytes exacerbated diet-induced obesity, insulin resistance and glucose intolerance in female, but not male mice. In obese females, this was accompanied by reduced hepatic gluconeogenic capacity and reprogramming of gonadal adipose tissue with hyperleptinemia. Our data highlight sex-dependent mechanisms by which the SNS regulates energy and glucose homeostasis through liver ADRA1B.

Insulin-like growth factor-binding protein7 exacerbates inflammatory response and lipid metabolism imbalance in alcohol-associated liver disease

Alcohol-associated liver disease(ALD), caused by excessive alcohol consumption, are often associated with inflammatory outbreaks and lipid deposition in the liver. The role of Insulin-like growth factor-binding protein 7 (IGFBP7), an important metabolic regulator, in ALD, its underlying regulatory mechanism, and its potential implication in anti-ALD therapies remain unknown. We investigated the effects of IGFBP7 on hepatic inflammation and lipid metabolism disruption in a mouse model of ALD. Mice were fed by chronic ethanol feeding plus a single binge of ethanol feeding(chronic-plus-single-binge model). In addition, ethanol exposure modeling studies were performed on cultured hepatocytes to verify molecular correlations. The results showed that IGFBP7 expression was significantly elevated in the livers of mice and hepatocytes after chronic ethanol exposure. Subsequently, the results of a study by specific knockout of IGFBP7(IGFBP7-cKO) in mouse hepatocytes and lentiviral silencing of IGFBP7 in vivo suggested that IGFBP7 deletion could improve liver function levels in alcohol-fed mice; It also attenuated the outbreak of hepatitis factor and the disorder of lipid metabolism in mice.Using RNA-seq sequencing of mouse liver tissue, we found that IGFBP7 affects several downstream metabolic signaling pathways, including PPAR, MAPK, FoxO, etc. Then, we used the PPARα plasmid in hepatocytes and discovered that overexpressing PPARα reversed the impact of IGFBP7 on lipid metabolism disorders in hepatocytes. In conclusion, IGFBP7 deficiency in alcohol-associated liver disease alleviates the decline in liver function and the imbalance of lipid metabolism in mice, attenuates the inflammatory outbreak, and affects a variety of downstream lipid metabolism factors by regulating PPARα. Hence, IGFBP7 may be an effective therapeutic target in the treatment of ALD.

FABP4 deficiency ameliorates alcoholic steatohepatitis in mice via inhibition of p53 signaling pathway

Fatty acid-binding protein 4 (FABP4) plays an essential role in metabolism and inflammation. However, the role of FABP4 in alcoholic steatohepatitis (ASH) remains unclear. This study aimed to investigate the function and underlying mechanisms of FABP4 in the progression of ASH. We first obtained alcoholic hepatitis (AH) datasets from the National Center for Biotechnology Information–Gene Expression Omnibus database and conducted bioinformatics analysis to identify critical genes in the FABP family. We then established ASH models of the wild-type (WT) and Fabp4-deficient (Fabp4−/−) mice to investigate the role of FABP4 in ASH. Additionally, we performed transcriptional profiling of mouse liver tissue and analyzed the results using integrative bioinformatics. The FABP4-associated signaling pathway was further verified. FABP4 was upregulated in two AH datasets and was thus identified as a critical biomarker for AH. FABP4 expression was higher in the liver tissues of patients with alcoholic liver disease and ASH mice than in the corresponding control samples. Furthermore, the Fabp4−/− ASH mice showed reduced hepatic lipid deposition and inflammation compared with the WT ASH mice. Mechanistically, Fabp4 may be involved in regulating the p53 and sirtuin-1 signaling pathways, subsequently affecting lipid metabolism and macrophage polarization in the liver of ASH mice. Our results demonstrate that Fabp4 is involved in the progression of ASH and that Fabp4 deficiency may ameliorate ASH. Therefore, FABP4 may be a potential therapeutic target for ASH treatment.

Oncogenic plasmid DNA and liver injury agent dictates liver cancer development in a mouse model.

Primary liver cancer is an increasing problem worldwide and is associated with significant mortality. A popular method of modelling liver cancer in mice is plasmid hydrodynamic tail vein injection (HTVI). However, plasmid-HTVI models rarely recapitulate the chronic liver injury which precedes the development of most human liver cancer. We sought to investigate how liver injury using thioacetamide contributes to the pathogenesis and progression of liver cancer in two oncogenic plasmid-HTVI-induced mouse liver cancer models. Fourteen-week-old male mice received double-oncogene plasmid-HTVI (SB/AKT/c-Met and SB/AKT/NRas) and then twice-weekly intraperitoneal injections of thioacetamide for six weeks. Liver tissue was examined for histopathological changes, including fibrosis and steatosis. Further characterization of fibrosis and inflammation was performed with immunostaining and real-time quantitative PCR. RNA sequencing with pathway analysis was used to explore novel pathways altered in the cancer models. Hepatocellular and cholangiocellular tumors were observed in mice injected with double-oncogene plasmid-HTVI models (SB/AKT/c-Met and SB/AKT/NRas). Thioacetamide induced mild fibrosis and increased alpha smooth muscle actin-expressing cells. However, the combination of plasmids and thioacetamide did not significantly increase tumor size, but increased multiplicity of small neoplastic lesions. Cancer and/or liver injury upregulated profibrotic and proinflammatory genes while metabolic pathway genes were mostly downregulated. We conclude that the liver injury microenvironment can interact with liver cancer and alter its presentation. However, the effects on cancer development vary depending on the genetic drivers with differing active oncogenic pathways. Therefore, the choice of plasmid-HTVI model and injury agent may influence the extent to which injury promotes liver cancer development.

An antifibrotic compound that ameliorates hyperglycaemia and fat accumulation in cell and HFD mouse models.

Appropriate management of blood glucose levels and the prevention of complications are important in the treatment of diabetes. We have previously reported on a compound named HPH-15 that is not only antifibrotic but also AMP-activated protein kinase (AMPK)-activating. In this study, we evaluated whether HPH-15 is useful as a therapeutic medication for diabetes. We examined the effects of HPH-15 on AMPK activation, glucose uptake, fat accumulation and lactic acid production in L6-GLUT4, HepG2 and 3T3-L1 cells, as a model of muscle, liver and fat tissue, respectively. Additionally, we investigated the glucose-lowering, fat-accumulation-suppressing, antifibrotic and AMPK-activating effect of HPH-15 in mice fed a high-fat diet (HFD). HPH-15 at a concentration of 10 µmol/l increased AMPK activation, glucose uptake and membrane translocation of GLUT4 in each cell model to the same extent as metformin at 2 mmol/l. The production of lactic acid (which causes lactic acidosis) in HPH-15-treated cells was equal to or less than that observed in metformin-treated cells. In HFD-fed mice, HPH-15 lowered blood glucose from 11.1±0.3 mmol/l to 8.2±0.4 mmol/l (10 mg/kg) and 7.9±0.4 mmol/l (100 mg/kg) and improved insulin resistance. The HPH-15 (10 mg/kg) group showed the same level of AMPK activation as the metformin (300 mg/kg) group in all organs. The HPH-15-treated HFD-fed mice also showed suppression of fat accumulation and fibrosis in the liver and fat tissue; these effects were more significant than those obtained with metformin. Mice treated with high doses of HPH-15 also exhibited a 44% reduction in subcutaneous fat. HPH-15 activated AMPK at lower concentrations than metformin in vitro and in vivo and improved blood glucose levels and insulin resistance in vivo. In addition, HPH-15 was more effective than metformin at ameliorating fatty liver and adipocyte hypertrophy in HFD-fed mice. HPH-15 could be effective in preventing fatty liver, a common complication in diabetic individuals. Additionally, in contrast to metformin, high doses of HPH-15 reduced subcutaneous fat in HFD-fed mice. Presumably, HPH-15 has a stronger inhibitory effect on fat accumulation and fibrosis than metformin, accounting for the reduction of subcutaneous fat. Therefore, HPH-15 is potentially a glucose-lowering medication that can lower blood glucose, inhibit fat accumulation and ameliorate liver fibrosis.

Deciphering the mechanism of Chaihu Shugan San in the treatment of nonalcoholic steatohepatitis using network pharmacology and molecular docking.

In China, there is a long history and rich clinical experience in treating nonalcoholic steatohepatitis (NASH) with traditional Chinese herbal medicines, including Chai Hu Shu Gan San. This study aims to investigate the potential regulatory effects of Chaihu Shugan San (CSS) on liver lipid metabolism and inflammatory damage in mice with experimental nonalcoholic steatohepatitis (NASH) induced by a choline-deficient high-fat diet (CDHFD). Utilizing network pharmacology, we systematically explore the mechanisms of action and therapeutic potential of CSS against NASH. Potential targets in CSS and targets for NASH were identified using online databases. Functional enrichment and protein-protein interaction analyses were conducted to identify hub-targeted genes and elucidate the underlying molecular mechanisms. The affinities of active compounds in CSS with hub-targeted genes were evaluated using molecular docking. Finally, hub-targeted genes were validated through real-time polymerase chain reaction, western blotting, and immunofluorescence in choline-deficient high-fat diet mice, both with and without CSS treatment. CSS reduces serum ALT and AST levels in NASH mice(P < 0.05) and ameliorates ballooning degeneration in the livers of NASH mice, thereby lowering the NAS score(P < 0.05). Including naringenin, high-performance liquid chromatography/mass spectrometrys identified 12 chromatographic peaks. Based on network pharmacology analysis, CSS contains a total of 103 active compounds and 877 target genes. Transferase activity represents a potential mechanism for therapeutic intervention of CSS in NASH. The transcriptional levels and protein expression of the SIRT1 gene in NASH mice are significantly increased by CSS (P < 0.05). Naringenin is probable active compound in CSS and SIRT1 is the hub gene by which CSS is involved in NASH treatment.

Evidence that extracellular HSPB1 contributes to inflammation in alcohol-associated hepatitis.

Alcohol-associated hepatitis (AH) is the most life-threatening form of alcohol-associated liver disease (ALD). AH is characterized by severe inflammation attributed to increased levels of ethanol, microbes or microbial components, and damage-associated molecular pattern (DAMP) molecules in the liver. HSPB1 (Heat Shock Protein Family B (Small) Member 1; also known as Hsp25/27) is a DAMP that is rapidly increased in and released from cells experiencing stress, including hepatocytes. The goal of this study was to define the role of HSPB1 in AH pathophysiology. Serum HSPB1 was measured in a retrospective study of 184 heathy controls (HC), heavy alcohol consumers (HA), patients with alcohol-associated cirrhosis (AC), and patients with AH recruited from major hospital centers. HSPB1 was also retrospectively evaluated in liver tissue from 10 HC and AH patients and an existing liver RNA-seq dataset. Finally, HSPB1 was investigated in a murine Lieber-DeCarli diet model of early ALD as well as cellular models of ethanol stress in hepatocytes and hepatocyte-macrophage communication during ethanol stress. Circulating HSPB1 was significantly increased in AH patients and levels positively correlated with disease-severity scores. Likewise, HSPB1 was increased in the liver of patients with severe AH and in the liver of ethanol-fed mice. In vitro , ethanol-stressed hepatocytes released HSPB1, which then triggered TNFα-mediated inflammation in macrophages. Anti-HSPB1 antibody prevented TNFα release from macrophages exposed to media conditioned by ethanol-stressed hepatocytes. Our findings support investigation of HSPB1 as both a biomarker and therapeutic target in ALD. Furthermore, this work demonstrates that anti-HSPB1 antibody is a rational approach to targeting HSPB1 with the potential to block inflammation and protect hepatocytes, without inactivating host defense. HSPB1 is significantly increased in serum and liver of patients with alcohol-associated hepatitis.Ethanol consumption leads to early increases in HSPB1 in the mouse liver.Hepatocytes subjected to ethanol stress release HSPB1 into the extracellular environment where it activates TNFα-mediated inflammation in macrophages.Anti-HSPB1 antibody blocks hepatocyte-triggered TNFα in a model of hepatocyte-macrophage communication during ethanol stress.

A ferrous fluorescence lifetime response probe for monitoring changes in lipid droplets during ferroptosis and imaging in liver disease model

Ferrous ions (Fe2⁺) accumulation and abnormal alterations in lipid droplets (LDs) are closely associated with ferroptosis. In the liver, excessive iron accumulation promotes oxidative stress and exacerbates lipid droplet accumulation, while the disruption of iron homeostasis may also affect the formation and size of lipid droplets, their increased number and size can exacerbate the severity of disease under fatty liver conditions. The leads to hepatocyte damage, further triggering liver inflammation, fibrosis, and ultimately resulting in cirrhosis and hepatocellular carcinoma. Therefore, real-time monitoring of iron ion and lipid droplet changes is crucial for assessing the severity of liver disease, disease progression, and understanding the mechanisms of ferroptosis. We have developed a fluorescent probe, NRFep, for real-time monitoring of iron ion fluctuations and visualization of lipid droplet changes in ferroptosis and liver disease models. NRFep is specific and sensitive to iron ions and exhibits excellent stability in both cells and animal models. In addition, NRFep can be used to monitor changes in iron ions and lipid droplets in mouse liver injury and fatty liver models. Through fluorescence lifetime imaging technology, NRFep can also study the dynamic changes of intracellular iron ion content. NRFep provides a powerful tool for studying ferroptosis and related diseases, and its unique dual-monitoring function opens up new possibilities for developing new diagnostic and therapeutic strategies.

Improved Liver Intravital Microscopic Imaging Using a Film-Assisted Stabilization Method.

Intravital microscopy (IVM) is a valuable method for biomedical characterization of dynamic processes, which has been applied to many fields such as neuroscience, oncology, and immunology. During IVM, vibration suppression is a major challenge due to the inevitable respiration and heartbeat from live animals. In this study, taking liver IVM as an example, we have unraveled the vibration inhibition effect of liquid bridges by studying the friction characteristics of a moist surface on the mouse liver. We confirmed the presence of liquid bridges on the liver through fluorescence imaging, which can provide microscale and nondestructive liquid connections between adjacent surfaces. Liquid bridges were constructed to sufficiently stabilize the liver after abdominal dissection by covering it with a polymer film, taking advantage of the high adhesion properties of liquid bridges. We further prototyped a microscope-integrated vibration-damping device with adjustable film tension to simplify the sample preparation procedure, which remarkably decreased the liver vibration. In practical application scenarios, we observed the process of liposome phagocytosis by liver Kupffer cells with significantly improved image and video quality. Collectively, our method not only provided a feasible solution to vibration suppression in the field of IVM, but also has the potential to be applied to vibration damping of precision instruments or other fields that require nondestructive ″soft″ vibration damping.