Si-Wu-Tang attenuates hepatocyte PANoptosis and M1 polarization of macrophages in non-alcoholic fatty liver disease by influencing the intercellular transfer of mtDNA.

ETHNOPHARMACOLOGICAL RELEVANCE: Non-alcoholic fatty liver disease (NAFLD) represents a burgeoning challenge for public health with potential progression to malignant liver diseases. PANoptosis, an avant-garde conceptualization of cell deaths, is closely associated with mitochondrial damage and linked to multiple liver disorders. Si-Wu-Tang (SWT), a traditional Chinese herbal prescription renowned for regulating blood-related disorders and ameliorating gynecological and hepatic diseases, has been demonstrated to alleviate liver fibrosis by regulating bile acid metabolism and immune responses. AIM OF THE STUDY: However, the mechanisms by which mtDNA is released from PANoptotic hepatocytes, triggering macrophage activation and hepatitis and whether this process can be reversed by SWT remain unclear. MATERIALS AND METHODS: Here, sophisticated RNA-sequencing complemented by molecular approaches were applied to explore the underlying mechanism of SWT against NAFLD in methionine/choline-deficient diet (MCD)-induced mice and relative in vitro models. RESULTS: We revealed that SWT profoundly repaired mitochondrial dysfunction, blocked mitochondrial permeability transition and mtDNA released to the cytoplasm, subsequently reversing hepatocyte PANoptosis and macrophage polarization both in MCD-stimulated mice and in vitro. Mechanically, loaded lipids dramatically promoted the opening of mPTP and oligomerization of VDAC2 to orchestrate mtDNA release, which was combined with ZBP1 to promote hepatocyte PANoptosis and also taken by macrophages to trigger M1 polarization via the FSTL1 and PKM2 combination. SWT effectively blocked NOXA signaling and reversed all these detrimental outcomes. CONCLUSION: Our findings show that SWT protects against hepatitis-mediated hepatocyte PANoptosis and macrophage M1 polarization by influencing intrahepatic synthesis, release and intercellular transfer of mtDNA, suggesting a potential therapeutic strategy for ameliorating NAFLD.

Alisol B regulates AMPK/mTOR/SREBPs via directly targeting VDAC1 to alleviate hyperlipidemia.

BACKGROUND: The occurrence of hyperlipidemia is significantly influenced by lipid synthesis, which is regulated by sterol regulatory element binding proteins (SREBPs), thus the development of drugs that inhibit lipid synthesis has become a popular treatment strategy for hyperlipidemia. Alisol B (ALB), a triterpenoid compound extracted from Alisma, has been reported to ameliorate no-nalcoholic steatohepatitis (NASH) and slow obesity. However, the effect of ALB on hyperlipidemia and mechanism are unclear. PURPOSE: To examine the therapeutic impact of ALB on hyperlipidemia whether it inhibits SREBPs to reduce lipid synthesis. STUDY DESIGN: HepG2, HL7702 cells, and C57BL/6J mice were used to explore the effect of ALB on hyperlipidemia and the molecular mechanism in vivo and in vitro. METHODS: Hyperlipidemia models were established using western diet (WD)-fed mice in vivo and oleic acid (OA)-induced hepatocytes in vitro. Western blot, real-time PCR and other biological methods verified that ALB regulated AMPK/mTOR/SREBPs to inhibit lipid synthesis. Cellular thermal shift assay (CETSA), molecular dynamics (MD), and ultrafiltration-LC/MS analysis were used to evaluate the binding of ALB to voltage-dependent anion channel protein-1 (VDAC1). RESULTS: ALB decreased TC, TG, LDL-c, and increased HDL-c in blood, thereby ameliorating liver damage. Gene set enrichment analysis (GSEA) indicated that ALB inhibited the biosynthesis of cholesterol and fatty acids. Consistently, ALB inhibited the protein expression of n-SREBPs and downstream genes. Mechanistically, the impact of ALB on SREBPs was dependent on the regulation of AMPK/mTOR, thereby impeding the transportation of SREBPs from endoplasmic reticulum (ER) to golgi apparatus (GA). Further investigations indicated that the activation of AMPK by ALB was independent on classical upstream CAMKK2 and LKB1. Instead, ALB resulted in a decrease in ATP levels and an increase in the ratios of ADP/ATP and AMP/ATP. CETSA, MD, and ultrafiltration-LC/MS analysis indicated that ALB interacted with VDAC1. Molecular docking revealed that ALB directly bound to VDAC1 by forming hydrogen bonds at the amino acid sites S196 and H184 in the ATP-binding region. Importantly, the thermal stabilization of ALB on VDAC1 was compromised when VDAC1 was mutated at S196 and H184, suggesting that these amino acids played a crucial role in the interaction. CONCLUSION: Our findings reveal that VDAC1 serves as the target of ALB, leading to the inhibition of lipid synthesis, presents potential target and candidate drugs for hyperlipidemia.

Vitamin D Mitigates Hepatic Fat Accumulation and Inflammation and Increases SIRT1/AMPK Expression in AML-12 Hepatocytes.

Emerging evidence has demonstrated a strong correlation between vitamin D status and fatty liver disease. Aberrant hepatic fat infiltration contributes to oxidant overproduction, promoting metabolic dysfunction, and inflammatory responses. Vitamin D supplementation might be a good strategy for reducing hepatic lipid accumulation and inflammation in non-alcoholic fatty liver disease and its associated diseases. This study aimed to investigate the role of the most biologically active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), in hepatic fat accumulation and inflammation in palmitic acid (PA)-treated AML-12 hepatocytes. The results indicated that treatment with 1,25(OH)2D significantly decreased triglyceride contents, lipid peroxidation, and cellular damage. In addition, mRNA levels of apoptosis-associated speck-like CARD-domain protein (ASC), thioredoxin-interacting protein (TXNIP), NOD-like receptor family pyrin domain-containing 3 (NLRP3), and interleukin-1ß (IL-1ß) involved in the NLRP3 inflammasome accompanied by caspase-1 activity and IL-1ß expression were significantly suppressed by 1,25(OH)2D in PA-treated hepatocytes. Moreover, upon PA exposure, 1,25(OH)2D-incubated AML-12 hepatocytes showed higher sirtulin 1 (SIRT1) expression and adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. A SIRT1 inhibitor alleviated the beneficial effects of 1,25(OH)2D on PA-induced hepatic fat deposition, IL-1ß expression, and caspase-1 activity. These results suggest that the favorable effects of 1,25(OH)2D on hepatic fat accumulation and inflammation may be, at least in part, associated with the SIRT1.

Jaceosidin attenuates the progression of hepatic fibrosis by inhibiting the VGLL3/HMGB1/TLR4 signaling pathway.

BACKGROUND: Jaceosidin (JA) is a natural flavone extracted from Artemisia that is used as a food and traditional medicinal herb. It has been reported to possess numerous biological activities. However, the regulatory mechanisms underlying amelioration of hepatic fibrosis remain unclear. HYPOTHESIS/PURPOSE: We hypothesized that jaceosidin acid (JA) modulates hepatic fibrosis and inflammation. METHODS: Thioacetamide (TAA) was used to establish an HF mouse model. In vitro, mouse primary hepatocytes and HSC-T6 cells were induced by TGF-ß, whereas mouse peritoneal macrophages received a treatment lipopolysaccharide (LPS)/ATP. RESULTS: JA decreased serum transaminase levels and improved hepatic histological pathology in TAA-treated mice stimulated by TAA. Moreover, the expression of pro-fibrogenic biomarkers associated with the activation of liver stellate cells was downregulated by JA. Likewise, JA down-regulated the expression of vestigial-like family member 3 (VGLL3), high mobility group protein B1 (HMGB1), toll-like receptors 4 (TLR4), and nucleotide-binding domain-(NOD-) like receptor protein 3 (NLRP3), thereby inhibiting the inflammatory response and inhibiting the release of mature-IL-1ß in TAA-stimulated mice. Additionally, JA suppressed HMGB1 release and NLRP3/ASC inflammasome activation in LPS/ATP-stimulated murine peritoneal macrophages. JA decreases the expression of pro-fibrogenic biomarkers related to liver stellate cell activation and inhibits inflammasome activation in mouse primary hepatocytes. It also down-regulated α-SMA and VGLL3 expressions and also suppressed inflammasome activation in HSC-T6 cells. VGLL3 and α-SMA expression levels were decreased in TGF-ß-stimulated HSC-T6 cells following Vgll3 knockdown. In addition, the expression levels of NLRP3 and cleaved-caspase-1 were decreased in Vgll3-silenced HSC-T6 cells. JA enhanced the inhibitory effects on Vgll3-silenced HSC-T6 cells. Finally, Vgll3 overexpression in HSC-T6 cells affected the expression levels of α-SMA, NLRP3, and cleaved-caspase-1. CONCLUSION: JA effectively modulates hepatic fibrosis by suppressing fibrogenesis and inflammation via the VGLL3/HMGB1/TLR4 axis. Therefore, JA may be a candidate therapeutic agent for the management of hepatic fibrosis. Understanding the mechanism of action of JA is a novel approach to hepatic fibrosis therapy.

GITRL impairs hepatocyte repopulation by liver progenitor cells to aggravate inflammation and fibrosis by GITR+CD8+ T lymphocytes in CDE Mice.

As an alternative pathway for liver regeneration, liver progenitor cells and their derived ductular reaction cells increase during the progression of many chronic liver diseases. However, the mechanism underlying their hepatocyte repopulation after liver injury remains unknown. Here, we conducted progenitor cell lineage tracing in mice and found that fewer than 2% of hepatocytes were derived from liver progenitor cells after 9 weeks of injury with a choline-deficient diet supplemented with ethionine (CDE), and this percentage increased approximately three-fold after 3 weeks of recovery. We also found that the proportion of liver progenitor cells double positive for the ligand of glucocorticoid-induced tumour necrosis factor receptor (GITRL, also called Tnfsf18) and SRY-related HMG box transcription 9 (Sox9) among nonparenchymal cells increased time-dependently upon CDE injury and reduced after recovery. When GITRL was conditionally knocked out from hepatic progenitor cells, its expression in nonparenchymal cells was downregulated by approximately fifty percent, and hepatocyte repopulation increased by approximately three folds. Simultaneously, conditional knockout of GITRL reduced the proportion of liver-infiltrating CD8+ T lymphocytes and glucocorticoid-induced tumour necrosis factor receptor (GITR)-positive CD8+ T lymphocytes. Mechanistically, GITRL stimulated cell proliferation but suppressed the differentiation of liver progenitor organoids into hepatocytes, and CD8+ T cells further reduced their hepatocyte differentiation by downregulating the Wnt/ß-catenin pathway. Therefore, GITRL expressed by liver progenitor cells impairs hepatocyte differentiation, thus hindering progenitor cell-mediated liver regeneration.

Gypenoside XIII regulates lipid metabolism in HepG2 hepatocytes and ameliorates nonalcoholic steatohepatitis in mice.

Gypenoside XIII is isolated from Gynostemma pentaphyllum (Thunb.) Makino. In mice, G. pentaphyllum extract and gypenoside LXXV have been shown to improve non-alcoholic steatohepatitis (NASH). This study investigated whether gypenoside XIII can regulate lipid accumulation in fatty liver cells or attenuate NASH in mice. We used HepG2 hepatocytes to establish a fatty liver cell model using 0.5 mM oleic acid. Fatty liver cells were treated with different concentrations of gypenoside XIII to evaluate the molecular mechanisms of lipid metabolism. In addition, a methionine/choline-deficient diet induced NASH in C57BL/6 mice, which were given 10 mg/kg gypenoside XIII by intraperitoneal injection. In fatty liver cells, gypenoside XIII effectively suppressed lipid accumulation and lipid peroxidation. Furthermore, gypenoside XIII significantly increased SIRT1 and AMPK phosphorylation to decrease acetyl-CoA carboxylase phosphorylation, reducing fatty acid synthesis activity. Gypenoside XIII also decreased lipogenesis by suppressing sterol regulatory element-binding protein 1c and fatty acid synthase production. Gypenoside XIII also increased lipolysis and fatty acid ß-oxidation by promoting adipose triglyceride lipase and carnitine palmitoyltransferase 1, respectively. In an animal model of NASH, gypenoside XIII effectively decreased the lipid vacuole size and number and reduced liver fibrosis and inflammation. These findings suggest that gypenoside XIII can regulate lipid metabolism in fatty liver cells and improve liver fibrosis in NASH mice. Therefore, gypenoside XIII has potential as a novel agent for the treatment of NASH.

Ginsenoside Rg1 attenuates lipopolysaccharide-induced chronic liver damage by activating Nrf2 signaling and inhibiting inflammasomes in hepatic cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginseng (Panax ginseng C. A. Meyer) is a precious traditional Chinese medicine with multiple pharmacological effects. Ginsenoside Rg1 is a main active ingredient extracted from ginseng, which is known for its age-delaying and antioxidant effects. Increasing evidence indicates that Rg1 exhibits anti-inflammatory properties in numerous diseases and may ameliorate oxidative damage and inflammation in many chronic liver diseases. AIM OF THE STUDY: Chronic inflammatory injury in liver cells is an important pathological basis of many liver diseases. However, its mechanism remains unclear and therapeutic strategies to prevent its development need to be further explored. Thus, our study is to delve the protective effect and mechanism of Rg1 against chronic hepatic inflammatory injuries induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: The chronic liver damage model in mice was build up by injecting intraperitoneally with LPS (200 µg/kg) for 21 days. Serum liver function indicators and levels of IL-1ß, IL-6 and TNF-α were examined by using corresponding Kits. Hematoxylin and Eosin (H&E), Periodic acid-Schiff (PAS), and Masson stains were utilized to visualize hepatic histopathological damage, glycogen deposition, and liver fibrosis. The nuclear import of p-Nrf2 and the generation of Col4 in the liver were detected by IF, while IHC was employed to detect the expressions of NLRP3 and AIM2 in the hepatic. The Western blot and q-PCR were used to survey the expressions of proteins and mRNAs of fibrosis and apoptosis, and the expressions of Keap1, p-Nrf2 and NLRP3, NLRP1, AIM2 inflammasome-related proteins in mouse liver. The cell viability of human hepatocellular carcinoma cells (HepG2) was detected by Cell Counting Kit-8 to select the action concentration of LPS, and intracellular ROS generation was detected using a kit. The expressions of Nuclear Nrf2, HO-1, NQO1 and NLRP3, NLRP1, and AIM2 inflammasome-related proteins in HepG2 cells were detected by Western blot. Finally, the feasibility of the molecular interlinking between Rg1 and Nrf2 was demonstrated by molecular docking. RESULTS: Rg1 treatment for 21 days decreased the levels of ALT, AST, and inflammatory factors of serum IL-1ß, IL-6 and TNF-α in mice induced by LPS. Pathological results indicated that Rg1 treatment obviously alleviated hepatocellular injury and apoptosis, inflammatory cell infiltration and liver fibrosis in LPS stimulated mice. Rg1 promoted Keap1 degradation and enhanced the expressions of p-Nrf2, HO-1 and decreased the levels of NLRP1, NLRP3, AIM2, cleaved caspase-1, IL-1ß and IL-6 in livers caused by LPS. Furthermore, Rg1 effectively suppressed the rise of ROS in HepG2 cells induced by LPS, whereas inhibition of Nrf2 reversed the role of Rg1 in reducing the production of ROS and NLRP3, NLRP1, and AIM2 expressions in LPS-stimulated HepG2 cells. Finally, the molecular docking illustrated that Rg1 exhibits a strong affinity towards Nrf2. CONCLUSION: The findings indicate that Rg1 significantly ameliorates chronic liver damage and fibrosis induced by LPS. The mechanism may be mediated through promoting the dissociation of Nrf2 from Keap1 and then activating Nrf2 signaling and further inhibiting NLRP3, NLRP1, and AIM2 inflammasomes in liver cells.

Sesamol as a potential candidate for the treatment of hepatic fibrosis, based on its regulation of FXR/LXR axis-mediated inhibition of autophagy through crosstalk between hepatic cells and macrophage.

BACKGROUND: Sesamol (SEM), a natural lignan compound isolated from sesame, has strong anti-oxidant property, regulating lipid metabolism, decreasing cholesterol and hepatoprotection. However, its anti-hepatic fibrosis effect and mechanisms have not been comprehensively elucidated. HYPOTHESIS/PURPOSE: This study aims to investigate the anti-hepatic fibrosis of SEM and its underlying mechanisms. METHOD: C57BL/6 mice with hepatic fibrosis were induced by TAA, then administrated with SEM or curcumin, respectively. HSCs were stimulated by TGF-ß or conditioned medium, and then cultured with SEM, GW4064, GW3965, Rapamycin (RA) or 3-methyladenine (3-MA), respectively. Mice with hepatic fibrosis also were administrated with SEM, RA or 3-MA to estimate the effect of SEM on autophagy. RESULTS: In vitro, SEM significantly inhibited extracellular matrix deposition, P2 × 7r-NLRP3, and inflammatory cytokines. SEM increased FXR and LXRα/ß expressions and decreased MAPLC3α/ß and P62 expressions, functioning as 3-MA (autophagy inhibitor). In vivo, SEM reduced serum transaminase, histopathology changes, fibrogenesis, autophagy markers and inflammatory cytokines caused by TAA. LX-2 were activated with conditioned medium from LPS-primed THP-1, which resulted in significant enhance of autophagy markers and inflammatory cytokines and decrease of FXR and LXRα/ß expressions. SEM could reverse above these changes and function as 3-MA, GW4064, or GW3965. Deficiency of FXR or LXR attenuated the regulation of SEM on α-SMA, MAPLC3α/ß, P62 and IL-1ß in activated LX-2. In activated THP-1, deficiency of FXR could decrease the expression of LXR, and vice versa. Deficiency of FXR or LXR in activated MΦ decreased the expressions of FXR and LXR in activated LX-2. Deficiency FXR or LXR in activated MΦ also attenuated the regulation of SEM on α-SMA, MAPLC3α/ß, P62, caspase-1 and IL-1ß. In vivo, SEM significantly reversed hepatic fibrosis via FXR/LXR and autophagy. CONCLUSION: SEM could regulate hepatic fibrosis by inhibiting fibrogenesis, autophagy and inflammation. FXR/LXR axis-mediated inhibition of autophagy contributed to the regulation of SEM against hepatic fibrosis, especially based on involving in the crosstalk of HSCs-macrophage. SEM might be a prospective therapeutic candidate, and its mechanism would be a new direction or strategy for hepatic fibrosis treatment.

The anti-hyperlipidemic effects of Poria cocos (Schw.) Wolf extract: Modulating cholesterol homeostasis in hepatocytes via PPARα pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Poria cocos (Schw.) Wolf (Polyporaceae, P.cocos), which is born on the pine root, has a history of more than two thousand years of medicine in China. P.cocos was first recorded in the Shennong's Herbal Classic, studies have proved its lipid-lowering effect. AIM OF STUDY: The aim of study was to investigate the underlying mechanism of P.cocos extract on hyperlipidemia. MATERIALS AND METHODS: Male Sprague-Dawley (SD) rats aged 9-12 weeks were intraperitoneally (IP) injected with Triton-WR 1339 to establish an acute hyperlipidemia model. At 0 h and 20 h after the model was established, low and high doses of P.cocos extract or simvastatin were given twice. After 48 h, the rats were sacrificed, and liver and serum samples were collected for analysis. The cell model was constructed by treating L02 cells with 1% fat emulsion-10% FBS-RPMI 1640 medium for 48 h. At the same time, low and high doses of P.cocos extract and simvastatin were administered. Oil red O staining was used to evaluate the lipid accumulation in the cells, and H&E staining was used to evaluate the liver lesions of rats. Real-time quantitative PCR and western blotting were used to detect the expressions of lipid metabolism-related genes. RESULTS: P.cocos extract relieved lipid accumulation in vitro and alleviated hyperlipidemia in vivo. Both gene and protein expressions of peroxisome proliferator-activated receptor α (PPARα) were shown to be up-regulated by P.cocos extract. Additionally, P.cocos extract down-regulated the expressions of fatty acid synthesis-related genes sterol regulatory element-binding protein-1 (SREBP-1), Acetyl-CoA Carboxylase 1 (ACC1) and fatty acid synthase (FAS), while up-regulated the expressions of cholesterol metabolism-related genes liver X receptor-α (LXRα), ATP-binding cassette transporter A1 (ABCA1), cholesterol 7alpha-hydroxylase (CYP7A1) and low density lipoprotein receptor (LDLR), which were reversed by the treatment with the PPARα inhibitor GW6471. CONCLUSION: P.cocos extract ameliorates hyperlipidemia and lipid accumulation by regulating cholesterol homeostasis in hepatocytes through PPARα pathway. This study provides evidence that supplementation with P.cocos extract could be a potential strategy for the treatment of hyperlipidemia.

Low concentrations of ethylene bisdithiocarbamate pesticides maneb and mancozeb impair manganese and zinc homeostasis to induce oxidative stress and caspase-dependent apoptosis in human hepatocytes.

The worldwide and intensive use of phytosanitary compounds results in environmental and food contamination by chemical residues. Human exposure to multiple pesticide residues is a major health issue. Considering that the liver is not only the main organ for metabolizing pesticides but also a major target of toxicities induced by xenobiotics, we studied the effects of a mixture of 7 pesticides (chlorpyrifos-ethyl, dimethoate, diazinon, iprodione, imazalil, maneb, mancozeb) often detected in food samples. Effects of the mixture was investigated using metabolically competent HepaRG cells and human hepatocytes in primary culture. We report the strong cytotoxicity of the pesticide mixture towards hepatocytes-like HepaRG cells and human hepatocytes upon acute and chronic exposures at low concentrations extrapolated from the Acceptable Daily Intake (ADI) of each compound. Unexpectedly, we demonstrated that the manganese (Mn)-containing dithiocarbamates (DTCs) maneb and mancozeb were solely responsible for the cytotoxicity induced by the mixture. The mechanism of cell death involved the induction of oxidative stress, which led to cell death by intrinsic apoptosis involving caspases 3 and 9. Importantly, this cytotoxic effect was found only in cells metabolizing these pesticides. Herein, we unveil a novel mechanism of toxicity of the Mn-containing DTCs maneb and mancozeb through their metabolization in hepatocytes generating the main metabolite ethylene thiourea (ETU) and the release of Mn leading to intracellular Mn overload and depletion in zinc (Zn). Alteration of the Mn and Zn homeostasis provokes the oxidative stress and the induction of apoptosis, which can be prevented by Zn supplementation. Our data demonstrate the hepatotoxicity of Mn-containing fungicides at very low doses and unveil their adverse effect in disrupting Mn and Zn homeostasis and triggering oxidative stress in human hepatocytes.

Branched-chain fatty acids affect the expression of fatty acid synthase and C-reactive protein genes in the hepatocyte cell line.

Fatty acids (FAs) are known to play an important role in human metabolism; however, still little is known about the functions of certain FA classes present in blood at relatively low concentrations. Examples of such compounds include branched-chain fatty acids (BCFAs). Recently, lowered BCFAs blood concentration was noticed in obese patients. An inverse correlation was found between serum concentrations of BCFAs and triglyceride levels, as well as C-reactive protein (CRP) concentration. Obesity is the most frequently observed component of metabolic syndrome and both disorders are accompanied by the dysregulation of FAs metabolism. However, not all of them are well understood. Our study is the first attempt at presenting the opposite effects of an iso-BCFA (14-methylpentadecanoic acid, 14-MPA) and an anteiso-BCFA (12-methyltetradecanoic acid, 12-MTA) on selected genes related to fatty acid synthesis and inflammation: FASN, SREBP1, CRP, and IL-6 in the HepG2 cell line. We observed lowered expression of FASN, SREBP1, CRP, and IL-6 in cells treated with 14-MPA in comparison with control cells. In contrast, supplementation with 12-MTA caused opposite effects: increased mRNA levels of FASN, CRP, and IL-6. 12-MTA did not influence SREBP1 expression. The results of our preliminary study may suggest potential benefits of the supplementation of iso-BCFAs in obese patients, for inflammation and hypertriglyceridemia prevention.

Insulin-induced gene 2 protects against hepatic ischemia-reperfusion injury via metabolic remodeling.

BACKGROUND: Hepatic ischemia-reperfusion (IR) injury is the primary reason for complications following hepatectomy and liver transplantation (LT). Insulin-induced gene 2 (Insig2) is one of several proteins that anchor the reticulum in the cytoplasm and is essential for metabolism and inflammatory responses. However, its function in IR injury remains ambiguous. METHODS: Insig2 global knock-out (KO) mice and mice with adeno-associated-virus8 (AAV8)-delivered Insig2 hepatocyte-specific overexpression were subjected to a 70% hepatic IR model. Liver injury was assessed by monitoring hepatic histology, inflammatory responses, and apoptosis. Hypoxia/reoxygenation stimulation (H/R) of primary hepatocytes and hypoxia model induced by cobalt chloride (CoCl2) were used for in vitro experiments. Multi-omics analysis of transcriptomics, proteomics, and metabolomics was used to investigate the molecular mechanisms underlying Insig2. RESULTS: Hepatic Insig2 expression was significantly reduced in clinical samples undergoing LT and the mouse IR model. Our findings showed that Insig2 depletion significantly aggravated IR-induced hepatic inflammation, cell death and injury, whereas Insig2 overexpression caused the opposite phenotypes. The results of in vitro H/R experiments were consistent with those in vivo. Mechanistically, multi-omics analysis revealed that Insig2 is associated with increased antioxidant pentose phosphate pathway (PPP) activity. The inhibition of glucose-6-phosphate-dehydrogenase (G6PD), a rate-limiting enzyme of PPP, rescued the protective effect of Insig2 overexpression, exacerbating liver injury. Finally, our findings indicated that mouse IR injury could be attenuated by developing a nanoparticle delivery system that enables liver-targeted delivery of substrate of PPP (glucose 6-phosphate). CONCLUSIONS: Insig2 has a protective function in liver IR by upregulating the PPP activity and remodeling glucose metabolism. The supplementary glucose 6-phosphate (G6P) salt may serve as a viable therapeutic target for alleviating hepatic IR.

Liver Extracellular Matrix-Based Nanofiber Scaffolds for the Culture of Primary Hepatocytes and Drug Screening.

Immortalized liver cell lines and primary hepatocytes are currently used as in vitro models for hepatotoxic drug screening. However, a decline in the viability and functionality of hepatocytes with time is an important limitation of these culture models. Advancements in tissue engineering techniques have allowed us to overcome this challenge by designing suitable scaffolds for maintaining viable and functional primary hepatocytes for a longer period of time in culture. In the current study, we fabricated liver-specific nanofiber scaffolds with polylactic acid (PLA) along with a decellularized liver extracellular matrix (LEM) by the electrospinning technique. The fabricated hybrid PLA-LEM scaffolds were more hydrophilic and had better swelling properties than the PLA scaffolds. The hybrid scaffolds had a pore size of 38 ± 8 µm and supported primary rat hepatocyte cultures for 10 days. Increased viability (2-fold increase in the number of live cells) and functionality (5-fold increase in albumin secretion) were observed in primary hepatocytes cultured on the PLA-LEM scaffolds as compared to those on conventional collagen-coated plates on day 10 of culture. A significant increase in CYP1A2 enzyme activity was observed in hepatocytes cultured on PLA-LEM hybrid scaffolds in comparison to those on collagen upon induction with phenobarbital. Drugs like acetaminophen and rifampicin showed the highest toxicity in hepatocytes cultured on hybrid scaffolds. Also, the lethal dose of these drugs in rodents was accurately predicted as 1.6 g/kg and 594 mg/kg, respectively, from the corresponding IC50 values obtained from drug-treated hepatocytes on hybrid scaffolds. Thus, the fabricated liver-specific electrospun scaffolds maintained primary hepatocyte viability and functionality for an extended period in culture and served as an effective ex vivo drug screening platform to predict an accurate in vivo drug-induced hepatotoxicity.

Attenuation of binuclear hepatocytes in the paracancerous liver tissue is associated with short-term recurrence of hepatocellular carcinoma post-radical surgery.

Short-term recurrence of hepatocellular carcinoma (HCC) after radical resection leads to dismal outcomes. To screen high-recurrence risk patients to provide adjuvant treatment is necessary. Herein, based on our previous research, we further focused on the changes in the abundance of binuclear hepatocytes (ABH) in the paracancerous liver tissue to discuss the relationship between the attenuation of binuclear hepatocytes and postoperative short-term recurrence, by combining with the assessment of the value of a reported independent early recurrence risk factor in HCC, protein induced by vitamin K absence or antagonist-II (PIVKA-II). A cohort of 142 paracancerous liver tissues from HCC patients who received radical resection was collected. Binuclear hepatocytes were reduced in the paracancerous liver tissues, compared with the liver tissues from normal donors. ABH was negatively correlated with clinical features such as tumor size, TNM stages, tumor microsatellite formation, venous invasion, and Alpha-fetoprotein (AFP) level, as well as the expression of E2F7 and Anillin, which are two critical regulators concerning the hepatocyte polyploidization. According to the short-term recurrence information, ABH value was laminated, and univariate and multivariate logistic regression was performed to analyze the relationship between paracancerous ABH and short-term tumor relapse. Simultaneously, the predictive effectiveness of the ABH value was compared with the preoperative PIVKA-II value. As observed, the paracancerous ABH value below 1.5% was found to be an independent risk factor for recurrence. In conclusion, the paracancerous ABH is a credible indicator of short-term recurrence of HCC patients after radical resection, and regular assessment of ABH might help to prevent short-term HCC recurrence.

Genome editing without nucleases confers proliferative advantage to edited hepatocytes and corrects Wilson disease.

Application of classic liver-directed gene replacement strategies is limited in genetic diseases characterized by liver injury due to hepatocyte proliferation, resulting in decline of therapeutic transgene expression and potential genotoxic risk. Wilson disease (WD) is a life-threatening autosomal disorder of copper homeostasis caused by pathogenic variants in copper transporter ATP7B and characterized by toxic copper accumulation, resulting in severe liver and brain diseases. Genome editing holds promise for the treatment of WD; nevertheless, to rescue copper homeostasis, ATP7B function must be restored in at least 25% of the hepatocytes, which surpasses by far genome-editing correction rates. We applied a liver-directed, nuclease-free genome editing approach, based on adeno-associated viral vector-mediated (AAV-mediated) targeted integration of a promoterless mini-ATP7B cDNA into the albumin (Alb) locus. Administration of AAV-Alb-mini-ATP7B in 2 WD mouse models resulted in extensive liver repopulation by genome-edited hepatocytes holding a proliferative advantage over nonedited ones, and ameliorated liver injury and copper metabolism. Furthermore, combination of genome editing with a copper chelator, currently used for WD treatment, achieved greater disease improvement compared with chelation therapy alone. Nuclease-free genome editing provided therapeutic efficacy and may represent a safer and longer-lasting alternative to classic gene replacement strategies for WD.

Evidence for the Metabolic Activation of Deferasirox In Vitro and In Vivo.

Deferasirox (DFS) is used for the treatment of iron accumulation caused by the need for long-term blood transfusions, such as thalassemia or other rare anemia. Liver injury due to exposure to DFS has been documented, and the toxic mechanisms of DFS are unknown. The present study aimed to investigate the reactive metabolites of DFS in vitro and in vivo to help us understand the mechanisms of DFS hepatotoxicity. Two hydroxylated metabolites (5-OH and 5'-OH) were identified during incubation of DFS-supplemented rat liver microsomes. Such microsomal incubations fortified with glutathione (GSH) or N-acetylcysteine (NAC) as capture agents offered two GSH conjugates and two NAC conjugates. These GSH conjugates and NAC conjugates were also detected in bile and urine of rats given DFS. CYP1A2 and CYP3A4 were found to dominate the metabolic activation of DFS. Administration of DFS induced decreased cell survival in cultured primary hepatocytes. Pretreatment with ketoconazole and 1-aminobenzotrizole made hepatocytes less susceptible to the cytotoxicity of DFS.

Image-based quantification of histological features as a function of spatial location using the Tissue Positioning System.

BACKGROUND: Tissues such as the liver lobule, kidney nephron, and intestinal gland exhibit intricate patterns of zonated gene expression corresponding to distinct cell types and functions. To quantitatively understand zonation, it is important to measure cellular or genetic features as a function of position along a zonal axis. While it is possible to manually count, characterize, and locate features in relation to the zonal axis, it is labor-intensive and difficult to do manually while maintaining precision and accuracy. METHODS: We addressed this challenge by developing a deep-learning-based quantification method called the "Tissue Positioning System" (TPS), which can automatically analyze zonation in the liver lobule as a model system. FINDINGS: By using algorithms that identified vessels, classified vessels, and segmented zones based on the relative position along the portal vein to central vein axis, TPS was able to spatially quantify gene expression in mice with zone specific reporters. INTERPRETATION: TPS could discern expression differences between zonal reporter strains, ages, and disease states. TPS could also reveal the zonal distribution of cells previously thought to be positioned randomly. The design principles of TPS could be generalized to other tissues to explore the biology of zonation. FUNDING: CPRIT (RP190208, RP220614, RP230330) and NIH (P30CA142543, R01AA028791, R01CA251928, R01DK1253961, R01GM140012, 1R01GM141519, 1R01DE030656, 1U01CA249245). The Pollack Foundation, Simmons Comprehensive Cancer Center Cancer & Obesity Translational Pilot Award, and the Emerging Leader Award from the Mark Foundation For Cancer Research (#21-003-ELA).

Melatonin Promotes Mitochondrial Biogenesis and Mitochondrial Degradation in Hepatocytes During Sepsis.

Objective: This study aimed to investigate the protective mechanisms of melatonin in an in vitro model of sepsis-induced hepatocyte injury, specifically focusing on mitophagy and mitochondrial biogenesis. Methods: In this study, we utilized lipopolysaccharide (LPS)-treated AML12 cells to establish an in vitro model of sepsis-induced hepatocyte injury. The effects of melatonin pretreatment were examined through various analyses, including assessments of oxidative stress, inflammation, mitophagy, mitochondrial biogenesis, and adenosine triphosphate (ATP) levels. Results: The results revealed that LPS-treated AML12 cells exhibited elevated levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 protein, intracellular reactive oxygen species (ROS), and lipid peroxidation, specifically malondialdehyde (MDA). Moreover, the levels of key markers associated with mitophagy, including PTEN-induced putative kinase 1 (PINK1), parkin, and LC3, were significantly increased (P < .05). Similarly, markers of mitochondrial biogenesis, such as peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1 (NRF1), and mitochondrial transcription factor A (TFAM), were also significantly increased (P < .05). Conversely, superoxide dismutase (SOD) activity and ATP levels were significantly decreased in LPS-treated AML12 cells compared to the control group (P < .05). However, melatonin pretreatment led to a significant decrease in TNF-α and IL-6 protein levels, intracellular ROS, and MDA levels (P < .05), along with a significant increase in SOD activity, ATP levels, and markers of mitophagy and mitochondrial. Conclusions: Our findings demonstrate that melatonin plays a role in regulating mitochondrial quality control in sepsis-induced hepatocytes. It achieves this result by promoting mitophagy and inducing mitochondrial biogenesis, thereby selectively eliminating dysfunctional mitochondria.

An aqueous olive leaf extract (OLE) ameliorates parameters of oxidative stress associated with lipid accumulation and induces lipophagy in human hepatic cells.

Fatty liver is a disease characterized by a buildup of lipids in the liver, often resulting from excessive consumption of high-fat-containing foods. Fatty liver can degenerate, over time, into more severe forms of liver diseases, especially when oxidative stress occurs. Olive leaf extract (OLE) is a reliable source of polyphenols with antioxidant and hypolipidemic properties that have been successfully used in medicine, cosmetics, and pharmaceutical products. Using "green" solvents with minimal impact on the environment and human health, which simultaneously preserves the extract's beneficial properties, represents one of the major challenges of biomedical research. In the present study, we assayed the potential antioxidant and lipid-lowering effect of a "green" OLE obtained by a water ultrasound-assisted extraction procedure, on the human hepatic HuH7 cell line, treated with a high concentration of free fatty acids (FFA). We found that high FFA concentration induced lipid accumulation and oxidative stress, as measured by increased hydrogen peroxide levels. Moreover, the activity of antioxidant enzymes, catalase, superoxide dismutase, and glutathione peroxidase, was reduced upon FFA treatment. Coincubation of high FFA with OLE reduced lipid and H2O2 accumulation and increased the activity of peroxide-detoxifying enzymes. OLE ameliorated mitochondrial membrane potential, and hepatic parameters by restoring the expression of enzymes involved in insulin signaling and lipid metabolism. Electron microscopy revealed an increased autophagosome formation in both FFA- and FFA + OLE-treated cells. The study of the autophagic pathway indicated OLE's probable role in activating lipophagy.

Hepatocyte NLRP3 interacts with PKCε to drive hepatic insulin resistance and steatosis.

Hepatic insulin resistance (IR), as a downstream sequela of nonalcoholic fatty liver disease (NAFLD), is strongly associated with liver steatosis. Despite numerous mechanism advancements, the molecular underpinnings and pathogenesis of hepatic IR, especially regarding the pattern recognition receptors in hepatocytes, remain elusive. Here, we identified hepatocyte NLRP3 as a direct and previously-unresolved driver of hepatic IR to promote steatosis response. Under the model of NAFLD, we identified hepatocyte NLRP3 as a crucial inducer of hepatic IR by undertaking multilayer transcriptomic searches and further confirmed that its expression was increased in the liver tissues from NAFLD patients and mouse models (high-fat diet (HFD), leptin-receptor-deficient (db/db) mice), and in palmitic acid (PA)-induced hepatocytes. Loss- or gain-of-function of hepatocyte-specific NLRP3 in HFD-induced mice ameliorated or exacerbated hepatic IR and steatosis, respectively. Mechanistically, NLRP3 directly bound to and promoted protein kinase C epsilon (PKCε) activation to impair insulin signaling and increase liver steatosis, while inhibition of PKCε activation dampened the beneficial effects seen in HFD-induced NLRP3-deficient mice. Moreover, we performed screening and discovered that the transcription factor Yin Yang 1 (YY1) positively controlled NLRP3 expression. In translational potential, adeno-associated virus serotype 8 (AAV8)-mediated NLRP3 knockdown in the liver alleviated hepatic IR and steatosis in db/db mice, and pharmacological inhibition of NLRP3 markedly alleviated diet-induced metabolic disorders. This finding reveals a previously-unexpected regulatory axis from YY1 to PKCε via NLRP3 induction for metabolic diseases and establishes the YY1-NLRP3-PKCε axis as a potential therapeutic target for NAFLD.