Miglustat, a glucosylceramide synthase inhibitor, mitigates liver fibrosis through TGF-ß/Smad pathway suppression in hepatic stellate cells.

Transforming growth factor (TGF)-ß/Smad pathway is implicated in the pathogenesis of liver fibrosis, a condition characterized by excessive deposition of extracellular matrix (ECM) proteins such as collagen in response to chronic inflammation. It has been reported that ceramide regulates collagen production through TGF-ß/Smad pathway activation. In this study, we examined whether miglustat, an inhibitor of glucosylceramide synthase, can suppress liver fibrosis by reducing TGF-ß/Smad pathway activity. Human hepatic stellate cells (HHSteCs) were cultured with TGF-ß and multiple miglustat concentrations to examine dose-dependent effects on the expression levels of ECM-related genes and Smad proteins. To evaluate the efficacy of miglustat for fibrosis mitigation, C57BL/6 mice were treated with carbon tetrachloride (CCl4) for 4 weeks to induce liver fibrosis, followed by combined CCl4 plus miglustat for a further 2 weeks. To examine if miglustat can also prevent fibrosis, mice were treated with CCl4 for 2 weeks, followed by CCl4 plus miglustat for 2 weeks. Miglustat dose-dependently downregulated expression of α-smooth muscle actin and ECM components in TGF-ß-treated HHSteCs. Both phosphorylation and nuclear translocation of Smad2 and Smad3 were also suppressed by miglustat treatment. Sirius-Red staining and hydroxyproline assays of model mouse liver samples revealed that miglustat reduced fibrosis, an effect accompanied by decreased expression of ECM. Our findings suggest that miglustat can both prevent and reverse liver fibrosis by inhibiting TGF-ß/Smad pathway.

m6A methylation-induced NR1D1 ablation disrupts the HSC circadian clock and promotes hepatic fibrosis.

The roles of nuclear receptor subfamily 1 group d member 1 (NR1D1) and the circadian clock in liver fibrosis remain unclear. Here, we showed that liver clock genes, especially NR1D1, were dysregulated in mice with carbon tetrachloride (CCl4)-induced liver fibrosis. In turn, disruption of the circadian clock exacerbated experimental liver fibrosis. NR1D1-deficient mice were more sensitive to CCl4-induced liver fibrosis, supporting a critical role of NR1D1 in liver fibrosis development. Validation at the tissue and cellular levels showed that NR1D1 was primarily degraded by N6-methyladenosine (m6A) methylation in a CCl4-induced liver fibrosis model, and this result was also validated in rhythm-disordered mouse models. In addition, the degradation of NR1D1 further inhibited the phosphorylation of dynein-related protein 1-serine site 616 (DRP1S616), resulting in weakened mitochondrial fission function and increased mitochondrial DNA (mtDNA) release in hepatic stellate cell (HSC), which in turn activated the cGMP-AMP synthase (cGAS) pathway. Activation of the cGAS pathway induced a local inflammatory microenvironment that further stimulated liver fibrosis progression. Interestingly, in the NR1D1 overexpression model, we observed that DRP1S616 phosphorylation was restored, and cGAS pathway was also inhibited in HSCs, resulting in improved liver fibrosis. Taken together, our results suggest that targeting NR1D1 may be an effective approach to liver fibrosis prevention and management.

Gomphrena globosa L. extract alleviates carbon tetrachoride-induced liver injury in mice by activating antioxidant signaling pathways and promoting autophagy.

BACKGROUND: Carbon tetrachloride (CCl4) is highly toxic to animal liver and is a major contributor to liver injury. Gomphrena globosa L. (GgL) is an edible plant with anti-inflammation and antioxidation properties. The aim of this study was to investigate the potential therapeutic effects of GgL on liver injury. METHODS AND RESULTS: A model of chronic liver injury in mice was established by intraperitoneal injection of CCl4 (0.4 mL/kg) for 3 weeks, and the mice were treated intraperitoneally with different concentrations of GgL crude extract (GgCE; 100, 200, 300 mg/kg) or Bifendatatum (Bif; 20 mg/kg) in the last 2 weeks. The results showed that GgCE treatment alleviated the liver injury, improved the pathological changes caused by CCl4 on the mice liver, and enhance the antioxidant capacity. We also found that GgCE increased the expression of antioxidant stress related proteins, decreased the phosphorylation levels of autophagy related proteins PI3K and mTOR, and decreased the expression of LC3 II and P62 proteins. CONCLUSION: These results suggest that GgCE alleviated CCl4-induced chronic liver injury in mice by activating antioxidant signaling pathways and promoting autophagy, indicating a potential therapeutic effect of GgCE on liver injury.

Attenuation of phenobarbital-induced cytochrome P450 expression in carbon tetrachloride-induced hepatitis in mice models.

Certain pathological conditions, such as inflammation, are known to affect basal cytochrome P450 (CYP) expression by modulating transcriptional regulation, and the pharmacokinetics of drugs can vary among patients. However, changes in drug-induced CYP expression under pathological conditions have not been elucidated in detail. Here, we investigated the effects of hepatic inflammation and injury on phenobarbital-induced expression of CYP isoforms in mice. Phenobarbital was administered once as a CYP inducer in the carbon tetrachloride-induced hepatitis model mice. The mRNA expression levels of Cyp3a11 and Cyp2b10 in the liver and small intestine were measured using reverse transcription polymerase chain reaction. The enzymatic activity of CYP3A in liver S9 was evaluated using midazolam as the substrate. Phenobarbital increased the mRNA expression of Cyp3a11 and Cyp2b10 in the liver of healthy mice, but not in the small intestine. Increased mRNA expression of hepatic Cyp3a11 and Cyp2b10 by phenobarbital was significantly suppressed in the hepatitis model mice. Hepatitis also suppressed the increased CYP3A enzymatic activity induced by phenobarbital in liver S9, consistent with the results of Cyp3a11 mRNA expression. These results suggest that the inducibility of CYP by phenobarbital may vary in patients with hepatitis, indicating that pharmacokinetic drug-drug interactions can be altered under certain pathological conditions.

In Vitro/In Vivo Hepatoprotective and Antioxidant Effects of Defatted Extract and a Phenolic Fraction Obtained from Phlomis Tuberosa.

An in vitro/in vivo hepatotoxicity and hepatoprotection evaluation of a defatted extract and a phenolic fraction from Phlomis tuberosa, administered alone and in a carbon tetrachloride (CCl4)-induced metabolic bioactivation model, was performed. The extract and the phenolic fraction were analysed by high performance liquid chromatography (HPLC) to determine the total flavonoid content, to identify flavonoids and to quantify verbascoside. In addition, total polyphenolics in the samples were expressed as gallic acid equivalents. Applied alone, the extract and the fraction (5, 10 and 50 µg/mL) did not show a statistically significant hepatotoxic effect on isolated rat hepatocytes in vitro. In a CCl4-induced hepatotoxicity model, the samples exhibited a concentration-dependent, statistically significant hepatoprotective effect, which was most pronounced at 50 µg/mL for both. The phenolic fraction exhibited a more pronounced hepatoprotective effect compared to the extract. Data from the in vitro study on the effects of the extract were also confirmed in the in vivo experiment conducted in a CCl4-induced hepatotoxicity model in rats. A histopathological study showed that the animals treated with CCl4 and the extract had an unaltered histoarchitecture of the liver. The effects of the extract were the same as those of silymarin.

Synergistic Effects of Vitis vinifera L. and Centella asiatica against CCl4-Induced Liver Injury in Mice.

Liver injury can be acute or chronic, resulting from a variety of factors, including viral hepatitis, drug overdose, idiosyncratic drug reaction, or toxins, while the progression of pathogenesis in the liver rises due to the involvement of numerous cytokines and growth factor mediators. Thus, the identification of more effective biomarker-based active phytochemicals isolated from medicinal plants is a promising strategy to protect against CCl4-induced liver injury. Vitis vinifera L. (VE) and Centella asiatica (CE) are well-known medicinal plants that possess anti-inflammatory and antioxidant properties. However, synergism between the two has not previously been studied. Here, we investigated the synergistic effects of a V. vinifera L. (VE) leaf, C. asiatica (CE) extract combination (VCEC) against CCl4-induced liver injury. Acute liver injury was induced by a single intraperitoneal administration of CCl4 (1 mL/kg). VCEC was administered orally for three consecutive days at various concentrations (100 and 200 mg/kg) prior to CCl4 injection. The extent of liver injury and the protective effects of VCEC were evaluated by biochemical analysis and histopathological studies. Oxidative stress was evaluated by measuring malondialdehyde (MDA) and glutathione (GSH) levels and Western blotting. VCEC treatment significantly reduced serum transaminase levels (AST and ALT), tumor necrosis factor-α (TNF-α), and reactive oxygen species (ROS). CCl4- induced apoptosis was inhibited by VCEC treatment by reducing cleaved caspase-3 and Bcl2-associated X protein (Bax). VCEC-treated mice significantly restored cytochrome P450 2E1, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) expression in CCl4-treated mice. In addition, VCEC downregulated overexpression of proinflammatory cytokines and hepatic nuclear factor kappa B (NF-κB) and inhibited CCl4-mediated apoptosis. Collectively, VCEC exhibited synergistic protective effects against liver injury through its antioxidant, anti-inflammatory, and antiapoptotic ability against oxidative stress, inflammation, and apoptosis. Therefore, VCEC appears promising as a potential therapeutic agent for CCl4-induced acute liver injury in mice.

Structural characterization and hepatoprotective activity of exopolysaccharide from Bacillus velezensis SN-1.

BACKGROUND: Exopolysaccharide biopolymers produced by microorganisms are crucial to the environment. They contribute to areas such as the health and bionanotechnology sectors, food and cosmetic industries as gelling agents, and environmental sector as flocculants owing to their biodegradability and non-toxic nature. The current study aimed to isolate the fraction of released exopolysaccharide (rEPS) by Bacillus velezensis SN-1 from Chinese Da-Jiang. RESULTS: The weighted average molecular weight of the major isolated component, rEPS-2, was 202 kDa, and its monosaccharide composition included mannose, glucose, and galactose at a molar ratio of 0.38:0.30:0.32. Further, the rEPS-2 was characterized using methylation analysis and one-dimensional/two-dimensional nuclear magnetic resonance (1D/2D NMR) spectroscopy. In vivo hepatoprotective effects indicated that rEPS-2 could alleviate carbon tetrachloride (CCl4 )-induced liver injury in mice by lowering the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and the malondialdehyde (MDA) levels. Furthermore, rEPS-2 can increase the expression of antioxidant genes HO-1, GCLC and NQO1 in the Nrf2/ARE signaling pathway, thereby increasing the activity of antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and reduced catalase (CAT) in liver cells. Furthermore, the rEPS-2 can be used and modulate the gut microbiota of mice with liver injury caused by CCl4 . CONCLUSIONS: These results suggest that rEPS-2 has promising potential to serve as hepatoprotective agents. © 2022 Society of Chemical Industry.

[Effects of triterpenoids in fatty products on liver condition of laboratory animals with acute toxic hepatitis].

One of the principles of prevention and non-medicamentous treatment of liver diseases, including hepatitis of different etiology, is the normalization of the diet through the consumption of food with physiologically active ingredients, in particular betulin, which helps to eliminate the causes of metabolic and oxidative disorders within liver cells. The aim of the research was to assess in vivo the influence of triterpene alcohol betulin extracted from Betula pendula Roth. birch bark in fat-containing products (for example mayonnaise) on the blood biochemical parameters and liver morphological structure of rats with initiated acute toxic hepatitis. Material and methods. Hepatoprotective and antioxidant activities of betulin as part of mayonnaise samples has been investigated in vivo on the model of toxic hepatitis initiated by carbon tetrachloride in male Wistar rats weighing 210-265 g. The animals were divided into 4 groups of 10 animals each: CG-1 - intact, CG-2 and MG - with carbon tetrachloride initiated toxic hepatitis. rats of the main groups were orally administered mayonnaise once a day at a dosage of 1 ml for 21 days after the formation of the model pathology: OG-1 with the added betulin (1 mg per 1 kg of body weight), OG-2 without betulin. Disorders of metabolic and oxidative processes in liver cells of animals were evaluated by biochemical indicators of blood plasma: the level of glucose, albumin, total cholesterol, triglycerides and urea and the activity of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and γ-glutamyltransferase. Oxidative stress in rats was estimated by the activity of catalase and superoxide dismutase in blood hemolysate (at a dilution of 1:200 and 1:10, respectively); the total prooxidant (in blood plasma) and total antioxidant (in blood hemolysate at a dilution of 1:10) activity were determined spectrophotometrically (colored complexes of TWIN-80 oxidation products with thiobarbituric acid). The morphological structure of rats' liver was estimated by microscopy of prepared cuts of hepatic tissue. Results. Based on biochemical parameters of rat blood plasma, it has been established that the administration of mayonnaise with betulin prevents the development of cytolic syndrome and suppresses the process of peroxidation by directly neutralizing free radicals. Aspartate aminotransferase and alkaline phosphatase activity in blood plasma of the experimental animals of the main group MG-1 reduced by 20.7 and 35.2% compared with indicators of the rats of the main group MG-2. Glucose concentration normalized to the level of the control group CG-1. The concentration of bilirubin and triglycerides decreased by 22.9 and by 48.1%, which indicates a significant reduction in the indicators of cholestatic syndrome in the group of animals OG-1 compared to OG-2. The total prooxidant activity and the concentration of thiobarbiturate-reactive products decreased compared to the CG-2 and MG-2 groups, which indicates the suppression of oxidative stress and, as a result, an improvement in liver conditions of animals with toxic hepatitis even when taking a fat-containing product. In liver histopeparates of animals receiving mayonnaise with betulin, necrobotic changes were less pronounced in comparison with the group MG-2. They were estimated at 1 point: small-drip dystrophy spots were found, haemorrhages in the interregional septum with inflammatory infiltration in the course of hemorrhages against the presence of necrosis hepatocytes with pronounced adipose dystrophy in the centres of the lobules, step necrosis with signs of replacing the damaged hepatocytes of the connective tissue, accompanied by centrolobular hemorrhages in MG-2 rats. Conclusion. Introduced into the composition of mayonnaise betulin, reduces the development of cytolic syndrome in toxic hepatitis and suppresses the process of peroxidation, on the basis of which fat-containing foods with betulin can be recommended for clinical examination as specialized products in acute and chronic liver diseases, including complicated cholestasis.

LncRNA Airn maintains LSEC differentiation to alleviate liver fibrosis via the KLF2-eNOS-sGC pathway.

BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as important regulators in a variety of human diseases. The dysregulation of liver sinusoidal endothelial cell (LSEC) phenotype is a critical early event in the fibrotic process. However, the biological function of lncRNAs in LSEC still remains unclear. METHODS: The expression level of lncRNA Airn was evaluated in both human fibrotic livers and serums, as well as mouse fibrotic livers. Gain- and loss-of-function experiments were performed to detect the effect of Airn on LSEC differentiation and hepatic stellate cell (HSC) activation in liver fibrosis. Furthermore, RIP, RNA pull-down-immunoblotting, and ChIP experiments were performed to explore the underlying mechanisms of Airn. RESULTS: We have identified Airn was significantly upregulated in liver tissues and LSEC of carbon tetrachloride (CCl4)-induced liver fibrosis mouse model. Moreover, the expression of AIRN in fibrotic human liver tissues and serums was remarkably increased compared with healthy controls. In vivo studies showed that Airn deficiency aggravated CCl4- and bile duct ligation (BDL)-induced liver fibrosis, while Airn over-expression by AAV8 alleviated CCl4-induced liver fibrosis. Furthermore, we revealed that Airn maintained LSEC differentiation in vivo and in vitro. Additionally, Airn inhibited HSC activation indirectly by regulating LSEC differentiation and promoted hepatocyte (HC) proliferation by increasing paracrine secretion of Wnt2a and HGF from LSEC. Mechanistically, Airn interacted with EZH2 to maintain LSEC differentiation through KLF2-eNOS-sGC pathway, thereby maintaining HSC quiescence and promoting HC proliferation. CONCLUSIONS: Our work identified that Airn is beneficial to liver fibrosis by maintaining LSEC differentiation and might be a serum biomarker for liver fibrogenesis.

Mediator MED23 regulates inflammatory responses and liver fibrosis.

Liver fibrosis, often associated with cirrhosis and hepatocellular carcinomas, is characterized by hepatic damage, an inflammatory response, and hepatic stellate cell (HSC) activation, although the underlying mechanisms are largely unknown. Here, we show that the transcriptional Mediator complex subunit 23 (MED23) participates in the development of experimental liver fibrosis. Compared with their control littermates, mice with hepatic Med23 deletion exhibited aggravated carbon tetrachloride (CCl4)-induced liver fibrosis, with enhanced chemokine production and inflammatory infiltration as well as increased hepatocyte regeneration. Mechanistically, the orphan nuclear receptor RAR-related orphan receptor alpha (RORα) activates the expression of the liver fibrosis-related chemokines C-C motif chemokine ligand 5 (CCL5) and C-X-C motif chemokine ligand 10 (CXCL10), which is suppressed by the Mediator subunit MED23. We further found that the inhibition of Ccl5 and Cxcl10 expression by MED23 likely occurs because of G9a (also known as euchromatic histone-lysine N-methyltransferase 2 [EHMT2])-mediated H3K9 dimethylation of the target promoters. Collectively, these findings reveal hepatic MED23 as a key modulator of chemokine production and inflammatory responses and define the MED23-CCL5/CXCL10 axis as a potential target for clinical intervention in liver fibrosis.

Metformin versus silymarin as hepatoprotective agents in mice fibrotic model caused by carbon tetrachloride.

OBJECTIVES: To study metformin hepatoprotective effects compared to silymarin on hepatic fibrosis caused by carbon tetrachloride (CCl4) in mice. MATERIAL AND METHODS: Liver fibrosis in mice was achieved by intraperitoneal injection of 2mL/kg of CCl4 dilution in olive oil [1:9 (v/v)] twice a week for 7 weeks followed by oral treatment with metformin (250mg/kg/day) or silymarin (100mg/kg/day) (a standard hepatoprotective drug). The changes that follow liver fibrosis were assessed by measurement of hepatic enzymes (ALT, AST and ALP), histopathological examination using hematoxylin and eosin stain, special stains, and α-smooth muscle actin (α-SMA) immunostaining, measuring oxidative stress markers (MDA, GSH, NOx and MnSOD) and transforming growth factor-beta 1 (TGF-ß1) in liver. RESULTS: Liver fibrosis was obviously developed in mice after intraperitoneal injection with CCl4 for 7 weeks. Both silymarin and metformin treatment exhibited a significant decrease in the fibrotic changes and similarly an increase in endogenous antioxidants. Interestingly there is a significant difference between silymarin and metformin regarding both efficacy and potency. CONCLUSION: These findings highlight the anti-inflammatory, antioxidant and antifibrotic effects of metformin in CCl4-induced hepatic fibrosis in mice, but silymarin is more beneficial.

Saikosaponin-d alleviates hepatic fibrosis through regulating GPER1/autophagy signaling.

BACKGROUND: Hepatic fibrosis is the final pathway of chronic liver disease characterized by excessive accumulation of extracellular matrix (ECM), which eventually develop into cirrhosis and liver cancer. Emerging studies demonstrated that Saikosaponin-d (SSd) exhibits a protective role in liver fibrosis. However, the mechanism underlying anti-liver fibrosis of SSd in vivo and in vitro remains unclear. METHODS AND RESULTS: Transforming growth factor (TGF)-ß and carbon tetrachloride (CCl4) were used for creating liver fibrosis model in vitro and in vivo, respectively. The role of SSd in regulating liver fibrosis was assessed through Sirius red and Masson staining, and IHC assay. We found that SSd attenuated remarkably CCl4-induced liver fibrosis as evidenced by decreased collagen level, and decreased expression of fibrotic markers Col 1 and α-SMA. Meanwhile, SSd repressed autophagy activation as suggested by decreased BECN1 expression and increased p62 expression. Compared with HSCs from CCl4-treated group, the primary HSCs from SSd-treated mice exhibited a marked inactivation of autophagy. Mechanistically, SSd treatment enhanced the expression of GPER1 in primary HSCs and in TGF-ß-treated LX-2 cells. GPER1 agonist G1 repressed autophagy activation, whereas GPER1 antagonist G15 activated autophagy and G15 also damaged the function of SSd on suppressing autophagy, leading to subsequent increased levels of fibrotic marker level in LX-2 cells. CONCLUSIONS: Our findings highlight that SSd alleviates hepatic fibrosis by regulating GPER1/autophagy pathway.

Canagliflozin Improves Liver Function in Rats by Upregulating Asparagine Synthetase.

INTRODUCTION: Canagliflozin (CANA) is a sodium-glucose cotransporter 2 inhibitor that was recently approved for treating diabetes. However, its effects on liver function are not well understood. The function of asparagine synthetase (ASNS) has been studied in several cancers but not in liver injury. Therefore, we investigated the connection between CANA and ASNS in alleviating damage (i.e., their hepatoprotective effect) in a rat liver injury model. METHODS: The rat model of liver injury was established using carbon tetrachloride treatment. Rats with liver injury were administered CANA orally for 8 weeks daily. After week 8, peripheral blood was collected to measure serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase levels. Liver histopathology was examined using hematoxylin and eosin staining to determine the degree of liver injury. Protein expression in the rat livers was examined using Western blotting. RESULTS: CANA treatment decreased serum alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase levels compared with those of the untreated group, demonstrating diminished liver injury. Mechanistically, CANA treatment activated AMP-activated protein kinase (AMPK), leading to increased nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and activating transcription factor 4 (ATF4), which upregulated ASNS expression in liver-injured rats. CONCLUSION: CANA significantly alleviated liver injury by activating the AMPK/Nrf2/ATF4 axis and upregulating ASNS expression, indicating its potential for treating patients with type 2 diabetes mellitus with impaired liver function.

Smad7 Deficiency in Myeloid Cells Does Not Affect Liver Injury, Inflammation or Fibrosis after Chronic CCl4 Exposure in Mice.

Myeloid cells play an essential role in the maintenance of liver homeostasis, as well as the initiation and termination of innate and adaptive immune responses. In chronic hepatic inflammation, the production of transforming growth factor beta (TGF-ß) is pivotal for scarring and fibrosis induction and progression. TGF-ß signalling is tightly regulated via the Smad protein family. Smad7 acts as an inhibitor of the TGF-ß-signalling pathway, rendering cells that express high levels of it resistant to TGF-ß-dependent signal transduction. In hepatocytes, the absence of Smad7 promotes liver fibrosis. Here, we examine whether Smad7 expression in myeloid cells affects the extent of liver inflammation, injury and fibrosis induction during chronic liver inflammation. Using the well-established model of chronic carbon tetrachloride (CCl4)-mediated liver injury, we investigated the role of Smad7 in myeloid cells in LysM-Cre Smadfl/fl mice that harbour a myeloid-specific knock-down of Smad7. We found that the chronic application of CCl4 induces severe liver injury, with elevated serum alanine transaminase (ALT)/aspartate transaminase (AST) levels, centrilobular and periportal necrosis and immune-cell infiltration. However, the myeloid-specific knock-down of Smad7 did not influence these and other parameters in the CCl4-treated animals. In summary, our results suggest that, during long-term application of CCl4, Smad7 expression in myeloid cells and its potential effects on the TGF-ß-signalling pathway are dispensable for regulating the extent of chronic liver injury and inflammation.

Hepatoprotective Effects of Bioflavonoid Luteolin Using Self-Nanoemulsifying Drug Delivery System.

Luteolin (LUT) is a natural pharmaceutical compound that is weakly water soluble and has low bioavailability when taken orally. As a result, the goal of this research was to create self-nanoemulsifying drug delivery systems (SNEDDS) for LUT in an attempt to improve its in vitro dissolution and hepatoprotective effects, resulting in increased oral bioavailability. Using the aqueous phase titration approach and the creation of pseudo-ternary phase diagrams with Capryol-PGMC (oil phase), Tween-80 (surfactant), and Transcutol-HP (co-emulsifier), various SNEDDS of LUT were generated. SNEDDS were assessed for droplet size, polydispersity index (PDI), zeta potential (ZP), refractive index (RI), and percent of transmittance (percent T) after undergoing several thermodynamic stability and self-nanoemulsification experiments. When compared to LUT suspension, the developed SNEDDS revealed considerable LUT release from all SNEDDS. Droplet size was 40 nm, PDI was <0.3, ZP was -30.58 mV, RI was 1.40, percent T was >98 percent, and drug release profile was >96 percent in optimized SNEDDS of LUT. For in vivo hepatoprotective testing in rats, optimized SNEDDS was chosen. When compared to LUT suspension, hepatoprotective tests showed that optimized LUT SNEDDS had a substantial hepatoprotective impact. The findings of this investigation suggested that SNEDDS could improve bioflavonoid LUT dissolution rate and therapeutic efficacy.

Tissue Repair in the Mouse Liver Following Acute Carbon Tetrachloride Depends on Injury-Induced Wnt/ß-Catenin Signaling.

In the liver, Wnt/ß-catenin signaling is involved in regulating zonation and hepatocyte proliferation during homeostasis. We examined Wnt gene expression and signaling after injury, and we show by in situ hybridization that Wnts are activated by acute carbon tetrachloride (CCl4 ) toxicity. Following injury, peri-injury hepatocytes become Wnt-responsive, expressing the Wnt target gene axis inhibition protein 2 (Axin2). Lineage tracing of peri-injury Axin2+ hepatocytes shows that during recovery the injured parenchyma becomes repopulated and repaired by Axin2+ descendants. Using single-cell RNA sequencing, we show that endothelial cells are the major source of Wnts following acute CCl4 toxicity. Induced loss of ß-catenin in peri-injury hepatocytes results in delayed repair and ultimately injury-induced lethality, while loss of Wnt production from endothelial cells leads to a delay in the proliferative response after injury. Conclusion: Our findings highlight the importance of the Wnt/ß-catenin signaling pathway in restoring tissue integrity following acute liver toxicity and establish a role of endothelial cells as an important Wnt-producing regulator of liver tissue repair following localized liver injury.

Group II muscarinic acetylcholine receptors attenuate hepatic injury via Nrf2/ARE pathway.

Parasympathetic nervous system dysfunction is common in patients with liver disease. We have previously shown that muscarinic acetylcholine receptors (mAchRs) play an important role in the regulation of hepatic fibrosis and that the receptor agonists and antagonists affect hepatocyte proliferation. However, little is known about the impact of the different mAchR subtypes and associated signaling pathways on liver injury. Here, we treated the human liver cell line HL7702 with 10 mmol/L carbon tetrachloride (CCL4) to induce hepatocyte damage. We found that CCL4 treatment increased the protein levels of group I mAchRs (M1, M3, M5) but reduced the expression of group II mAchRs (M2, M4) and activated the Nrf2/ARE and MAPK signaling pathways. Although overexpression of M1, M3, or M5 led to hepatocyte damage with an intact Nrf2/ARE pathway, overexpression of M2 or M4 increased, and siRNA-mediated knockdown of either M2 or M4 decreased the protein levels of Nrf2 and its downstream target genes. Moreover, CCL4 treatment increased serum ALT levels more significantly, but only induced slight changes in the expression of mAchRs, NQO1 and HO1, while reducing the expression of M2 and M4 in liver tissues of Nrf2-/- mice compared to wild type mice. Our findings suggest that group II mAchRs, M2 and M4, activate the Nrf2/ARE signaling pathway, which regulates the expression of M2 and M4, to protect the liver from CCL4-induced injury.

Indole-3-Carbinol Derivative DIM Mitigates Carbon Tetrachloride-Induced Acute Liver Injury in Mice by Inhibiting Inflammatory Response, Apoptosis and Regulating Oxidative Stress.

3,3'-Diindolylmethane (DIM), a metabolic product of indole-3-carbinol extracted from cruciferous vegetables exhibits anti-inflammatory and anti-cancer properties. Earlier, the product has been demonstrated to possess anti-fibrotic properties; however, its protective effects on liver injury have not been clearly elucidated. In this study, we postulated the effects and molecular mechanisms of action of DIM on carbon tetrachloride (CCl4)-induced liver injury in mice. Acute liver injury was induced by a single intraperitoneal administration of CCl4 (1 ml/kg) into mice. DIM was injected via subcutaneous route for three days at various doses (2.5, 5 and 10 mg/kg) before CCl4 injection. Mice were sacrificed and serum was collected for quantification of serum transaminases. The liver was collected and weighed. Treatment with DIM significantly reduced serum transaminases levels (AST and ALT), tumor necrosis factor-α (TNF-α) and reactive oxygen species (ROS). CCl4- induced apoptosis was inhibited by DIM treatment by the reduction in the levels of cleaved caspase-3 and Bcl2 associated X protein (Bax). DIM treated mice significantly restored Cytochrome P450 2E1, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in CCl4 treated mice. In addition, DIM downregulated overexpression of hepatic nuclear factor kappa B (NF-κB) and inhibited CCl4 mediated apoptosis. Our results suggest that the protective effects of DIM against CCl4- induced liver injury are due to the inhibition of ROS, reduction of pro-inflammatory mediators and apoptosis.

Toxicant-Induced Metabolic Alterations in Lipid and Amino Acid Pathways Are Predictive of Acute Liver Toxicity in Rats.

Liver disease and disorders associated with aberrant hepatocyte metabolism can be initiated via drug and environmental toxicant exposures. In this study, we tested the hypothesis that gene and metabolic profiling can reveal commonalities in liver response to different toxicants and provide the capability to identify early signatures of acute liver toxicity. We used Sprague Dawley rats and three classical hepatotoxicants: acetaminophen (2 g/kg), bromobenzene (0.4 g/kg), and carbon tetrachloride (0.3 g/kg), to identify early perturbations in liver metabolism after a single acute exposure dose. We measured changes in liver genes and plasma metabolites at two time points (5 and 10 h) and used genome-scale metabolic models to identify commonalities in liver responses across the three toxicants. We found strong correlations for gene and metabolic profiles between the toxicants, indicative of similarities in the liver response to toxicity. We identified several injury-specific pathways in lipid and amino acid metabolism that changed similarly across the three toxicants. Our findings suggest that several plasma metabolites in lipid and amino acid metabolism are strongly associated with the progression of liver toxicity, and as such, could be targeted and clinically assessed for their potential as early predictors of acute liver toxicity.

Chemical profiling, in vitro and in vivo bioactivities of leaf extracts of Vitex neugundo.

The Vitex negeundo is a widely used medicinal plant which has not been fully investigated in the past. We assessed the in vivo hepatoprotective and in vitro antioxidant, antibacterial, cytotoxicity and antiproliferative study of leaf extracts of V. Neugundo. The chemically profiled using HPLC, three flavonoids were quantified and GC-MS analysis revealed the presence of two new compounds those were not reported earlier. The animal study was conducted on mice treated with CCl4 using methanolic and chloroform extracts (100, 200 and 300 mg/kg b.w), with silymarin as a positive control. Hepatoprotective effects were determined by analyzing blood for liver marker enzymes, direct bilirubins and hematological parameters (RBC, WBC and platelets). The methanolic extract (300 mg/kg b.w) has shown the stronger hepatoprotective effects against abnormalities produced by CCl4. The in vivo hepatoprotective effects correlated well with the in vitro antioxidant, cytotoxicity and antiproliferative activities and with high levels of flavonoids and other organic compounds analyzed from plant extracts. The leaf extracts of this plant could be good candidates for lead compound required for the development of antioxidant/anticancer drugs.