Role of HNF4α-cMyc Interaction in CDE Diet-Induced Liver Injury and Regeneration.

Hepatocyte nuclear factor 4 alpha (HNF4α) is a nuclear factor essential for liver function that regulates the expression of cMyc and plays an important role during liver regeneration. This study investigated the role of the HNF4α-cMyc interaction in regulating liver injury and regeneration using the choline-deficient and ethionine-supplemented (CDE) diet model. Wild-type (WT), hepatocyte-specific HNF4α-knockout (KO), cMyc-KO, and HNF4α-cMyc double KO (DKO) mice were fed a CDE diet for 1 week to induce subacute liver injury. To study regeneration, normal chow diet was fed for 1 week after CDE diet. WT mice exhibited significant liver injury and decreased HNF4α mRNA and protein expression after CDE diet. HNF4α deletion resulted in significantly higher injury with increased inflammation, fibrosis, proliferation, and hepatic progenitor cell activation compared with WT mice after CDE diet but indicated similar recovery. Deletion of cMyc lowered liver injury with activation of inflammatory genes compared with WT and HNF4α-KO mice after CDE diet. DKO mice had a phenotype comparable to that of the HNF4α-KO mice after CDE diet and a complete recovery. DKO mice exhibited a significant increase in hepatic progenitor cell markers both after injury and recovery phase. Taken together, these data show that HNF4α protects against inflammatory and fibrotic changes after CDE diet-induced injury, which is driven by cMyc.

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.

Hop-derived Humulinones Reveal Protective Effects in in vitro Models of Hepatic Steatosis, Inflammation and Fibrosis.

Nonalcoholic fatty liver disease (NAFLD) is emerging as leading cause of liver disease worldwide. Specific pharmacologic therapy for NAFLD is a major unmet medical need. Recently, iso-alpha acids, hop-derived bitter compounds in beer, have been shown to beneficially affect NAFLD pathology. Humulinones are further hop derived bitter acids particularly found in modern styles of beer. So far, biological effects of humulinones have been unknown. Here, we investigated the effect of humulinones in in vitro models for hepatic steatosis, inflammation and fibrosis. Humulinones dose-dependently inhibited fatty acid induced lipid accumulation in primary human hepatocytes. Humulinones reduced the expression of fatty acid uptake transporter CD36 and key enzymes of (de novo) lipid synthesis. Conversely, humulinones increased the expression of FABP1, CPT1 and ACOX1, indicative for increased lipid combustion. Furthermore, humulinones ameliorated steatosis induced pro-inflammatory gene expression. Furthermore, humulinones significantly reduced the expression of pro-inflammatory and pro-fibrogenic factors in control as well as lipopolysaccharide treated activated hepatic stellate cells, which play a key role in hepatic fibrosis. In conclusion, humulinones beneficially affect different pathophysiological steps of NAFLD. Our data suggest humulinones as promising therapeutic agents for the prevention and treatment of NAFLD.

Yes-associated protein promotes the proliferation and differentiation of liver progenitor cells during liver fibrosis.

Liver fibrosis is closely related to the proliferation and differentiation of liver progenitor cells (LPCs). Yes-associated protein (YAP) is a key effector molecule of the Hippo signaling pathway and plays an important role in regulating cell proliferation and liver homeostasis. However, its role in LPCs proliferation and differentiation during liver fibrosis are not well understood. Using immunohistochemistry, immunofluorescence staining, quantitative PCR and Western blotting, we discovered that LPCs expansion and enhanced YAP expression in LPCs in either choline-deficient, ethionine-supplemented (CDE) diet or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced fibrotic mice, as well as in patients with liver fibrosis. By injecting adeno-associated virus vectors under the transcriptional control of Lgr5 promoter, we found that targeted knockdown of YAP in LPCs attenuated the CDE/DDC diet-induced ductular reaction and liver fibrosis. Using EdU incorporation and Cell Counting Kit-8 assays, we demonstrated that YAP can modulate LPCs proliferation. Importantly, spleen transplantation of YAP-overexpressing LPCs improved their ability to differentiate into hepatocytes and alleviated carbon tetrachloride-induced liver fibrosis. Collectively, our findings indicate that LPCs expansion and differentiation during liver fibrosis could be modulated by YAP, further suggesting the possibility of manipulating YAP expression in LPCs as a potential treatment for chronic liver diseases.

Repolarization Precedes Oval Cell-mediated Hepatocytic Regeneration in the CDE Diet Mouse Model.

The liver has a unique ability to recover from injury unlike any other organ. A poorly understood aspect of liver regeneration is the role of hepatocellular polarization. Neighbor of Punc E11 (Nope) is an oncofetal stem/progenitor cell marker, which is expressed by depolarized adult hepatocytes after cholestatic liver injury and in hepatocellular carcinoma. Liver injury induced by a choline-deficient and ethionine-supplemented diet is reversible if followed by an additional dietary stop interval and enabled us to study the expression of Nope during the induction of chronic liver injury and during subsequent liver regeneration. We could show by quantitative RT-PCR, Western blotting, and immunohistochemistry that the expression of Nope is induced in depolarized adult hepatocytes during injury. However, after another 2 weeks of a normal diet, the polarization of hepatocytes was almost completely restored and the expression of Nope remained limited to bile ducts and oval cells. Using an inducible CK19-lineage tracing model, we could demonstrate that oval cell-mediated hepatocyte regeneration is rare and was preceded by repolarization of hepatocytes. In conclusion, polarization of hepatocytes is an important part of liver regeneration and precedes oval cell-mediated regeneration of the liver. This process can be visualized by a characteristic expression pattern of Nope.

Jinlida granules ameliorate the high-fat-diet induced liver injury in mice by antagonising hepatocytes pyroptosis.

CONTEXT: Jinlida (JLD) as a traditional Chinese medicine formula has been used to treat type 2 diabetes mellitus (T2DM) and studies have shown its anti-obesity effect. OBJECTIVE: To investigate the therapeutic effects of JLD in a mouse model of non-alcoholic fatty liver (NAFL). MATERIALS AND METHODS: C57BL/6J mice were divided into three groups and fed a low-diet diet (LFD), high-fat diet (HFD), or HFD + JLD (3.8 g/kg) for 16 weeks, respectively. The free fatty acids-induced lipotoxicity in HepG2 cells were used to evaluate the anti-pyroptotic effects of JLD. The pharmacological effects of JLD on NAFL were investigated by pathological examination, intraperitoneal glucose and insulin tolerance tests, western blotting, and quantitative real-time PCR. RESULTS: In vivo studies showed that JLD ameliorated HFD-induced liver injury, significantly decreased body weight and enhanced insulin sensitivity and improved glucose tolerance. Furthermore, JLD suppressed both the mRNA expression of caspase-1 (1.58 vs. 2.90), IL-1ß (0.93 vs. 3.44) and IL-18 (1.34 vs. 1.60) and protein expression of NLRP3 (2.04 vs. 5.71), pro-caspase-1 (2.68 vs. 4.92) and IL-1ß (1.61 vs. 2.60). In vitro, JLD inhibited the formation of lipid droplets induced by 2 mM FFA (IC50 = 2.727 mM), reduced the protein expression of NLRP3 (0.74 vs. 2.27), caspase-1 (0.57 vs. 2.68), p20 (1.67 vs. 3.33), and IL-1ß (1.44 vs. 2.41), and lowered the ratio of p-IKB-α/IKB-α (0.47 vs. 2.19). CONCLUSION: JLD has a protective effect against NAFLD, which may be related to its anti-pyroptosis, suggesting that JLD has the potential as a novel agent in the treatment of NAFLD.

Optimization of the Extraction of Proanthocyanidins from Grape Seeds Using Ultrasonication-Assisted Aqueous Ethanol and Evaluation of Anti-Steatosis Activity In Vitro.

Conventional extraction methods of proanthocyanidins (PAC) are based on toxic organic solvents, which can raise concerns about the use of extracts in supplemented food and nutraceuticals. Thus, a PAC extraction method was developed for grape seeds (GS) and grape seed powder using food-grade ethanol by optimizing the extraction conditions to generate the maximum yield of PAC. Extraction parameters, % ethanol, solvent: solid (s:s) ratio, sonication time, and temperature were optimized by the central composite design of the response surface method. The yields of PAC under different extraction conditions were quantified by the methylcellulose precipitable tannin assay. The final optimum conditions were 47% ethanol, 10:1 s:s ratio (v:w), 53 min sonication time, and 60 °C extraction temperature. High-performance liquid chromatography analysis revealed the presence of catechin, procyanidin B2, oligomeric and polymeric PAC in the grape seed-proanthocyanidin extracts (GS-PAC). GS-PAC significantly reduced reactive oxygen species and lipid accumulation in the palmitic-acid-induced mouse hepatocytes (AML12) model of steatosis. About 50% of the PAC of the GS was found to be retained in the by-product of wine fermentation. Therefore, the developed ethanol-based extraction method is suitable to produce PAC-rich functional ingredients from grape by-products to be used in supplemented food and nutraceuticals.

Combination of molecular docking and liver transcription sequencing analysis for the evaluation of salt-processed psoraleae fructus-induced hepatotoxicity in ovariectomized mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Salt-processed Psoraleae fructus (SPF) is widely used as a phytoestrogen-like agent in the treatment of osteoporosis. However, SPF-associated hepatotoxicity is a known health hazard. Cholestasis is often associated with SPF-induced hepatotoxicity. Notably, clinical liver injury is a common side effect of SPF in the treatment of osteoporosis; however, the exact mechanism underlying this phenomenon is unclear. AIM OF THE STUDY: To evaluate SPF-induced hepatotoxicity in an ovariectomized murine model of estrogen deficiency and examine the mechanisms underlying this process. MATERIALS AND METHODS: To explore the molecular mechanism of SPF-induced cholestatic liver injury, different concentrations of SPF (5 and 10 g/kg) were intragastrically administered to ovariectomized and non-ovariectomized female ICR mice for 30 days. RESULTS: SPF-treated mice showed noticeably swollen hepatocytes, dilated bile ducts, and elevated levels of serum biochemical markers. Compared to ovariectomized mice, these changes were more prominent in non-ovariectomized mice. According to the sequence data, a total of 6689 mRNAs were identified. Compared with the control group, 1814 differentially expressed mRNAs were identified in the group treated with high SPF doses (SPHD), including 939 upregulated and 875 downregulated mRNAs. Molecular docking and Western blot experiments showed that liver injury was closely related to the estrogen levels. Compared with the negative control group, the expression levels of FXR, Mrp2, CYP7a1, BSEP, SULT1E1, HNF4a, and Nrf2 decreased in the estradiol-treated (E2), low-dose SPF-treated (SPLD), and SPHD groups. Interestingly, the expression levels of FXR, CYP7a1, SULT1E1, and HNF4α were significantly higher in the ovariectomized groups than in the non-ovariectomized groups (#P < 0.05; ###P < 0.001). CONCLUSIONS: Overall, this study demonstrates that SPF downregulates key enzymes involved in cholesterol and bile acid biosyntheses, posing a risk for cholestatic liver injury. SPF also regulates the FXR-SULT1E signaling pathway via HNF4α, which is an important causative factor of cholestasis. Moreover, the severity of liver damage was significantly lower in the ovariectomized groups than in the non-ovariectomized group. These results suggest that the estrogen level is the most critical factor determining liver injury.

Effects and Mechanism of Oxymatrine Combined with Compound Yinchen Granules on the Apoptosis of Hepatocytes through the Akt/FoxO3a/Bim Pathway.

The aim of the present study was to investigate the effects and mechanism of oxymatrine (OMT) combined with compound yinchen granules (CYG) on the apoptosis of hepatocytes through the Akt/FoxO3a/Bim pathway in rats with acute liver failure. The rat model of acute liver failure was established using lipopolysaccharide/D-galactosamine (LPS/D-GalN). The expression of proteins in rat liver tissues was detected by western blot analysis. The mRNA expression of FoxO3a, Bim, Bax, Bcl-2, and caspase-3 in rat liver tissues was detected by RT-qPCR. The apoptosis rate of rat hepatocytes was determined by flow cytometry. Western blots showed that when compared with the normal group, the expression of p-Akt and p-FoxO3a in the model group was decreased (P < 0.05), while the expression of Bim was increased (P < 0.01). Compared with the model group, the expression of p-Akt and p-FoxO3a in the OMT group and the OMT combined with CYG groups was increased (P < 0.05 or P < 0.01), while the expression of Bim was decreased (P < 0.05). The Bax/Bcl-2 ratio and caspase-3 protein expression in the model group were significantly higher than those in the normal group (P < 0.01). The Bax/Bcl-2 ratio and the expression of caspase-3 protein in the OMT group and the OMT combined with CYG groups were significantly lower than those in the model group (P < 0.01). The results of RT-qPCR were consistent with those of western blot. The results of flow cytometry showed that the apoptosis rate of hepatocytes in the OMT group and the OMT combined with CYG groups was significantly lower than that in the model group (P < 0.05 or P < 0.01). We concluded that LPS/D-GalN can induce apoptosis of hepatocytes in rats with acute liver failure through the Akt/FoxO3a/Bim pathway. OMT combined with CYG inhibits apoptosis of hepatocytes in rats with acute liver failure via the Akt/FoxO3a/Bim pathway.

Qingluotongbi formula regulates the LXRα-ERS-SREBP-1c pathway in hepatocytes to alleviate the liver injury caused by Tripterygium wilfordii Hook. f.

ETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium wilfordii Hook. f. (TW) is widely used to treat autoimmune and inflammatory diseases; however, its development and application is limited by its significant association with liver injury. The compound formula Qingluotongbi (QLT) employs TW as its main component and is used to treat rheumatoid arthritis with no adverse reactions, suggesting that QLT may reduce the liver toxicity of TW. AIM OF THE STUDY: We examined whether TW interferes with lipid metabolism to induce liver injury, and evaluated the protective effect of QLT in in vivo and in vitro experiments. MATERIALS AND METHODS: After administration of QLT and its ingredients, HepaRG cells and SD rats were tested for biochemical indicators, hepatocytes lipid changes, and rat liver pathological changes, and then we analyzed for the gene expression of liver X receptor α (LXRα), endoplasmic reticulum stress (ERS) key proteins, sterol regulatory element binding protein-1c (SREBP-1c), and lipid-synthesizing enzymes. In HepaRG cells, the protein expression of glucose-regulated protein 78 kDa (GRP78) and LXRα was detected after addition of an LXRα inhibitor, LXRα agonist, and ERS inhibitor. RESULTS: TW caused significant elevation of biochemical indicators and lipid droplet deposition in hepatocytes, as well as upregulated the gene expression of LXRα, ERS key proteins, SREBP-1c, and lipid-synthesizing enzymes in both in vitro and in vivo settings, and caused liver injury in rats. QLT can alleviate the lipotoxic liver injury caused by TW. LXRα agonist further activated ERS induced by TW, whereas LXRα inhibitor significantly reduced ERS and lipotoxic injury induced by TW in HepaRG cells. CONCLUSIONS: TW upregulated LXRα to activate ERS and increased the gene expression of SREBP-1c and lipid-synthesizing enzymes, leading to increased lipid synthesis in hepatocytes to result in liver injury. QLT inhibited the LXRα-ERS-SREBP-1c pathway and reduced abnormal lipid synthesis in hepatocytes and the hepatotoxicity of TW.

Antitumor and hepatoprotective effect of Cuscuta reflexa Roxb. in a murine model of colon cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Cuscuta reflexa Roxb. (C. reflexa) is a well-known traditional herbal plant, with numerous inherent therapeutic potentials including anticancer, antitumor, antibacterial, analgesic, anthelmintic, laxative and others. Moreover, the anticancer and antitumor potentials of this herb are ongoing with several trails, thus an attempt was made to assess the anticancer and hepatoprotective potentials of traditional C. reflexa herbs. METHOD: The dried ethanolic extract of C. reflexa was tested for acute oral toxicity in the treated animals subsequently their behavioral, neurological, and autonomic profiles changes were observed. The preliminary anti-cancer effects of extracts against 1, 2- Dimethyl hydrazine (DMH) induced animals were assessed through barium enema X-ray, colonoscopy, and Aberrant crypt foci (ACF) studies. The blood samples of the animals (treated and untreated) were collected and their in-vitro histological parameters were evaluated by the experienced technician. RESULTS: It was observed that C. reflexa significantly reduced Disease activity indexing (DAI) level and ACF counting, as well as demonstrated similar activity as of the standard drug 5-Fluorouracil (5-FU). Histopathological results revealed that the apoptotic bodies decreased in the DMH-induced group (group II) during cancer progression while in 5-FU treated (group III) and C. reflexa treated (group IV and V) animals the apoptotic bodies were increased. Inversely, the mitotic bodies increased in group II animals and reduced in group III, IV, and V animals. In the colonic section, DMH-induced cancer assay exhibited significant effects on the levels of hemoglobin, Packed cell volume (PCV), Red blood cell (RBC) counts, Mean corpuscular hemoglobin concentration (MCHC), Mean corpuscular volume (MCV), and Mean cell hemoglobin (MCH), and was found to be less in group II animals whereas administration of C. reflexa efficiently recovered back the loss probably by healing the colon damage/depletion of cancer progression. Moreover, compared to the group II animals, the neutrophil count was within the normal range in C. reflexa administered group. CONCLUSIONS: In the present study, the major hematological parameters significantly increased within DMH treated animals and exhibited extensive damage in the hepatic regions. Moreover, the histopathological findings demonstrated that the C. reflexa extracts potentially reduced the cell proliferation, with no toxicity. The C. reflexa extracts exhibited impending anti-cancer activity as well as protected the hepatic cells and thus could be potentially used in the management of colon or colorectal cancer and hepatic impairments.

Sorafenib attenuates liver fibrosis by triggering hepatic stellate cell ferroptosis via HIF-1α/SLC7A11 pathway.

OBJECTIVES: Evidences demonstrate that sorafenib alleviates liver fibrosis via inhibiting HSC activation and ECM accumulation. The underlying mechanism remains unclear. Ferroptosis, a novel programmed cell death, regulates diverse physiological/pathological processes. In this study, we aim to investigate the functional role of HSC ferroptosis in the anti-fibrotic effect of sorafenib. MATERIALS AND METHODS: The effects of sorafenib on HSC ferroptosis and ECM expression were assessed in mouse model of liver fibrosis induced by CCl4 . In vitro, Fer-1 and DFO were used to block ferroptosis and then explored the anti-fibrotic effect of sorafenib by detecting α-SMA, COL1α1 and fibronectin proteins. Finally, HIF-1α siRNA, plasmid and stabilizers were applied to assess related signalling pathway. RESULTS: Sorafenib attenuated liver injury and ECM accumulation in CCl4 -induced fibrotic livers, accompanied by reduction of SLC7A11 and GPX4 proteins. In sorafenib-treated HSC-T6 cells, ferroptotic events (depletion of SLC7A11, GPX4 and GSH; accumulation iron, ROS and MDA) were discovered. Intriguingly, these ferroptotic events were not appeared in hepatocytes or macrophages. Sorafenib-elicited HSC ferroptosis and ECM reduction were abrogated by Fer-1 and DFO. Additionally, both HIF-1α and SLC7A11 proteins were reduced in sorafenib-treated HSC-T6 cells. SLC7A11 was positively regulated by HIF-1α, inactivation of HIF-1α/SLC7A11 pathway was required for sorafenib-induced HSC ferroptosis, and elevation of HIF-1α could inhibit ferroptosis, ultimately limited the anti-fibrotic effect. CONCLUSIONS: Sorafenib triggers HSC ferroptosis via HIF-1α/SLC7A11 signalling, which in turn attenuates liver injury and fibrosis.

Protective effect of hawthorn vitexin on the ethanol-injured DNA of BRL-3A hepatocytes.

ABSTRACT: Vitexin is a natural active ingredient in hawthorn leaves, which has a wide range of anti-tumor effects. This study was conducted to assess the protective effect of hawthorn vitexin on the ethanol-injured DNA of hepatocytes in vitro and to explore its mechanism. The effect of different concentrations of hawthorn vitexin on ethanol-injured hepatocytes was detected via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method to study the protective effect of hawthorn vitexin on ethanol-injured DNA damage in hepatocytes. Single-cell gel electrophoresis was used to observe the effect of hawthorn vitexin on ethanol-induced DNA damage in hepatocytes, and the Olive tail moment was measured. Cell physiological and biochemical indexes, such as superoxide dismutase activity, malonaldehyde content, and glutathione peroxidase activity, were detected with kits. The mRNA expression of the superoxide dismutase gene was measured via real-time quantitative polymerase chain reaction. It was showed that 0.2, 0.4, and 0.8 mg mL-1 hawthorn vitexin could significantly repair hepatocyte growth and ethanol-induced DNA damage. This effect was closely related to the improvement in superoxide dismutase, malonaldehyde, and glutathione peroxidase. Hawthorn vitexin could be used to repair ethanol-injured hepatocytes through antioxidation effects, and showed potential for the treatment of liver injury.

Chemical characters and protective effect of Baqi Lingmao formula on experimental liver injury.

OBJECTIVE: To investigate the chemical characters of water-extract of Baqi Lingmao formula (BQLM formula) and its effects on anti-liver injury in model mice and live cells. METHODS: BQLM formula was composed of ten herbal medicines. We determined the contents of alkaloids, saponins, phenolic acids and flavonoid in BQLM formula by UV spectrophotometry. The active components of alkaloids and phenolic acids in BQLM formula were identified by HPLC chromatography. The anti-hepatic injury effects of BQLM formula were investigated with concanavalin A (ConA)-induced hepatitis model of mice, human liver LO2 and HepG2.2.15 cells. RESULTS: BQLM formula (2 and 10 g/kg, orally) significantly improved the damages of liver tissues and functions caused by ConA in mice, reduced the infiltration of inflammatory cells into liver and inhibited the inflammatory cytokine secretion of interferon-γ, tumor necrosis factor-α and interleukin-6. BQLM formula simultaneously decreased the levels of alanine aminotransferase and aspartate aminotransferase of liver and serum, and recovered the superoxide dismutase and catalase activities of liver to normal levels in ConA-induced hepatic-injury mice. The serum of BQLM formula group stimulated the human liver LO2 cell proliferation in vitro. Further, BQLM formula obviously promoted the proliferation of normal hepatocytes (LO2 cells) and inhibited the hepatocytes death induced by ConA. It also significantly inhibited the proliferation of HepG2.2.15 cells and decreased the secretion of HBsAg and HBeAg in vitro. CONCLUSIONS: BQLM formula has anti-inflammation and anti-hepatitis virus Beffects, and is capable of improving liver injury in vivo and in vitro.

Evaluation of a Three-Dimensional Primary Human Hepatocyte Spheroid Model: Adoption and Industrialization for the Enhanced Detection of Drug-Induced Liver Injury.

Drug-induced liver injury is a leading cause of compound attrition during both preclinical and clinical drug development, and early strategies are in place to tackle this recurring problem. Human-relevant in vitro models that are more predictive of hepatotoxicity hazard identification, and that could be employed earlier in the drug discovery process, would improve the quality of drug candidate selection and help reduce attrition. We present an evaluation of four human hepatocyte in vitro models of increasing culture complexity (i.e., two-dimensional (2D) HepG2 monolayers, hepatocyte sandwich cultures, three-dimensional (3D) hepatocyte spheroids, and precision-cut liver slices), using the same tool compounds, viability end points, and culture time points. Having established the improved prediction potential of the 3D hepatocyte spheroid model, we describe implementing this model into an industrial screening setting, where the challenge was matching the complexity of the culture system with the scale and throughput required. Following further qualification and miniaturization into a 384-well, high-throughput screening format, data was generated on 199 compounds. This clearly demonstrated the ability to capture a greater number of severe hepatotoxins versus the current routine 2D HepG2 monolayer assay while continuing to flag no false-positive compounds. The industrialization and miniaturization of the 3D hepatocyte spheroid complex in vitro model demonstrates a significant step toward reducing drug attrition and improving the quality and safety of drugs, while retaining the flexibility for future improvements, and has replaced the routine use of the 2D HepG2 monolayer assay at GlaxoSmithKline.

Magnesium isoglycyrrhizinate prevents cadmium-induced activation of JNK and apoptotic hepatocyte death by reversing ROS-inactivated PP2A.

OBJECTIVES: Cadmium (Cd) induces reactive oxygen species (ROS)-mediated hepatocyte apoptosis and consequential liver disorders. This study aimed to investigate the effect of magnesium isoglycyrrhizinate (MgIG) on Cd-induced hepatotoxicity. METHODS: L02 and AML-12 cells were used to study MgIG hepatoprotective effects. Cd-evoked apoptosis, ROS and protein phosphatase 2A (PP2A)/c-Jun N-terminal kinase (JNK) cascade disruption were analysed by cell viability assay, 6-diamidino-2-phenylindole (DAPI) and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, ROS imaging and Western blotting. Pharmacological and genetic approaches were used to explore the mechanisms. KEY FINDINGS: We show that MgIG attenuated Cd-evoked hepatocyte apoptosis by blocking JNK pathway. Pre-treatment with SP600125 or ectopic expression of dominant-negative c-Jun enhanced MgIG's anti-apoptotic effects. Further investigation found that MgIG rescued Cd-inactivated PP2A. Inhibition of PP2A activity by okadaic acid attenuated the MgIG's inhibition of the Cd-stimulated JNK pathway and apoptosis; in contrast, overexpression of PP2A strengthened the MgIG effects. In addition, MgIG blocked Cd-induced ROS generation. Eliminating ROS by N-acetyl-l-cysteine abrogated Cd-induced PP2A-JNK pathway disruption and concurrently reinforced MgIG-conferred protective effects, which could be further slightly strengthened by PP2A overexpression. CONCLUSIONS: Our findings indicate that MgIG is a promising hepatoprotective agent for the prevention of Cd-induced hepatic injury by mitigating ROS-inactivated PP2A, thus preventing JNK activation and hepatocyte apoptosis.

The Jieduan-Niwan (JDNW) Formula Ameliorates Hepatocyte Apoptosis: A Study of the Inhibition of E2F1-Mediated Apoptosis Signaling Pathways in Acute-on-Chronic Liver Failure (ACLF) Using Rats.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a severe, complicated human disease. E2F1-mediated apoptosis plays an important role in ACLF development. Jieduan-Niwan (JDNW) formula, a traditional Chinese medicine (TCM), has shown remarkable clinical efficacy in ACLF treatment. However, the hepatoprotective mechanisms of the formula are barely understood. PURPOSE: This study aimed to investigate the mechanisms of JDNW formula in ACLF treatment by specifically regulating E2F1-mediated apoptotic signaling pathways in rats. METHODS: The JDNW components were determined by high-performance liquid chromatography (HPLC) analysis. The ACLF rat model was established using human serum albumin immune-induced liver cirrhosis, followed by D-galactosamine and lipopolysaccharide joint acute attacks. The ACLF rat was treated with JDNW formula. Prothrombin time activity was measured to investigate the coagulation function. Liver pathological injury was observed by hematoxylin-eosin (HE) and reticular fiber staining. The hepatocyte apoptosis index and apoptosis rate were determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and flow cytometry, respectively. Additionally, the expression of key genes and proteins that regulate E2F1-mediated apoptosis was analyzed by quantitative real-time PCR and Western blot. RESULTS: Seven major components of JDNW formula were detected. The formula ameliorated the coagulation function, decreased the hepatocyte apoptosis index and apoptosis rate, and alleviated liver pathological damage in ACLF rats. The down-regulation of the expression of genes and proteins from p53-dependent and non-p53-dependent apoptosis pathways and the up-regulation of the expression of genes from blocking anti-apoptotic signaling pathways indicated that JDNW formula inhibited excessive hepatocyte apoptosis in ACLF rats via E2F1-mediated apoptosis signaling pathways. CONCLUSION: The findings indicate that JDNW formula protects livers of ACLF rats by inhibiting E2F1-mediated apoptotic signaling pathways, implying that these pathways might be a potential therapeutic target for ACLF treatment.

Picroside II Improves Severe Acute Pancreatitis-Induced Hepatocellular Injury in Rats by Affecting JAK2/STAT3 Phosphorylation Signaling.

Picroside II is an important ingredient agent in Traditional Chinese medicine and hoped to reduce hepatocellular injury caused by severe acute pancreatitis (SAP). An SAP-induced hepatocellular injury model was established in rats by using pentobarbital sodium. 27 rats were divided into 3 groups: the sham group (SG), model group (MG), and Picroside groups (PG). SAP-induced hepatocellular injury was assessed using hematoxylin and eosin staining. We measured hepatocellular enzymes (amylase (AMY), alanine aminotransferase (ALT), and aspartate aminotransferase (AST)), oxidative stress factors (superoxidase dismutase (SOD) and malondialdehyde (MDA)), and inflammatory factors (tumor necrosis factor α (TNF-α), interleukin- (IL-) 6, and IL-10), apoptotic factors (BAX and cleaved caspase 3), and inflammatory signaling (Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3), p-JAK2, and p-STAT3) in hepatocellular tissues. The SAP-induced hepatocellular injury model was successfully established. Picroside II treatment repaired hepatocellular injury by reducing the activities of AMY, ALT, and AST; reducing the levels of MDA, TNF-α, IL-1, IL-6, p-JAK2, p-STAT3, BAX, and cleaved caspase 3; and increasing the levels of SOD and IL-10. Picroside II exerted protective function for the SAP-induced hepatocellular injury model. Picroside II improved SAP-induced hepatocellular injury and antioxidant and anti-inflammatory properties by affecting JAK2/STAT3 phosphorylation signaling.

Protective effect of Phaeoporus obliquus polysaccharide against acute liver injury induced by carbon tetrachloride and alcohol in mice.

Studied the optimum extraction process of polysaccharide from Phaeoporus obliquus and the effect of Phaeoporus obliquus polysaccharide on carbon tetrachloride (CCl4)- or alcohol-induced acute liver injury in mice. The main factor in influencing the extraction rate of Phaeoporus obliquus polysaccharide were extraction power and time, which was a kind of pyran glucose by infrared spectroscopy. CCl4 and alcohol were employed respectively to establish CCl4 and alcohol-induced acute liver injury mouse models. Compared with model groups mice, Phaeoporus obliquus polysaccharide treatment at the doses of 100mg/kg and 200mg/kg exhibited an obvious reduction liver index, ALP, ALT, AST levels, MDA content and TNF-α level (p<0.01) and SOD activity was increased, which was in a dose-dependent manner. Compared with the model group, the necrosis degree of hepatocytes was obviously reduced and the small fat droplets were formed in some cytoplasm, especially in high dose group, which the liver cells recovered to the level of normal group. Rt-PCR results showed that the expression of CYP2E1 mRNA in liver tissues of Phaeoporus obliquus polysaccharide groups were significantly reduced, and the difference were statistically significant compared with the model group (p<0.05). These results demonstrated that Phaeoporus obliquus polysaccharide has significantly hepatoprotective effect on CCl4 and alcohol-induced acute liver injury in mice.

Engineering the Vasculature of Stem-Cell-Derived Liver Organoids.

The vasculature of stem-cell-derived liver organoids can be engineered using methods that recapitulate embryonic liver development. Hepatic organoids with a vascular network offer great application prospects for drug screening, disease modeling, and therapeutics. However, the application of stem cell-derived organoids is hindered by insufficient vascularization and maturation. Here, we review different theories about the origin of hepatic cells and the morphogenesis of hepatic vessels to provide potential approaches for organoid generation. We also review the main protocols for generating vascularized liver organoids from stem cells and consider their potential and limitations in the generation of vascularized liver organoids.