Therapeutic benefit of oregano oil in the acute idiosyncratic hepatotoxicity induced by carbon tetrachloride in rats: Adverse effects of high dose of oreganum.

Toxicity caused by carbon tetrachloride (CCl4) can lead to serious liver injury. The aim of the study is to investigate the protective effects of oregano oil (Origanum minutiflorum extract oil) against CCl4-induced liver injury. Two doses of oregano oil were used in the experiment: a low dose (LD; 20 mg/kg) and a high dose (HD; 60 mg/kg) during 2 weeks. CCl4 caused severe liver damage, nucleolus destruction in hepatocytes and cytogenetic changes in the nucleus. Indirectly, CCl4 causes decreased protein synthesis and significantly high creatinine and urea values. Hematological disorders have been recorded, such as decreased RBC and hemoglobin concentration, increased WBC and deformability of the erythrocyte membrane. Both doses of oregano oil had protective effects. Improved protein synthesis and high globulins level, creatinine and urea were found in both groups. Cytogenetic changes in the nucleus of hepatocytes were reduced. A high dose of oregano oil had maximal protective effects for RBC, but a very weak effect on hemoglobin synthesis. Also, WBC and lymphocyte values were low. Origanum stimulates protein synthesis and recovery of hepatocytes after liver injury, reduces the deformability of the erythrocyte membrane. High doses of oregano oil decreased WBC and lymphocytes which may lead to a weakening of the immune response. However, high doses are more effective against severe platelet aggregation than low doses, suggesting an effective treatment against thrombocytosis.

[Determination of six halogenated solvent residues in olive oil by headspace gas chromatography].

The residual amount of halogenated solvents in olive oil is an important indicator of its quality. The National Olive Oil Quality Standard GB/T 23347-2021 states that the residual amount of individual halogenated solvents in olive oil should be ≤0.1 mg/kg and that the total residual amount of halogenated solvents should be ≤0.2 mg/kg. COI/T.20/Doc. No. 8-1990, which was published by the International Olive Council, describes the standard method used for the determination of halogenated solvents in olive oil. Unfortunately, this method is cumbersome, has poor repeatability and low automation, and is unsuitable for the detection and analysis of residual halogenated solvents in large quantities of olive oil. At present, no national standard method for determining residual halogenated solvents in olive oil is available in China. Thus, developing simple, efficient, accurate, and stable methods for the determination of residual halogenated solvents in olive oil is imperative. In this paper, a method based on automatic headspace gas chromatography was established for the determination of residual halogenated solvents, namely, chloroform, carbon tetrachloride, 1,1,1-trichloroethane, dibromochloromethane, tetrachloroethylene, and bromoform, in olive oil. The samples were processed as follows. After mixing, 2.00 g (accurate to 0.01 g) of the olive oil sample was added into a 20 mL headspace injection bottle and immediately sealed for headspace gas chromatography analysis. Blank virgin olive oil was used to prepare a standard working solution and the external standard method for quantification. The solvents used in the preparation of halogenated solvent standard intermediates were investigated and methanol was selected as a replacement for N,N-dimethylacetamide to prepare a halogenated solvent standard intermediate owing to its safety. The effects of different injection times (1, 2, 3, 4, 5, 6 s), equilibration temperatures (60, 70, 80, 90, 100, 110, 120 ℃), and equilibration times (4, 5, 8, 10, 20, 30, 40 min) of the headspace sampler on the detection of the residual amounts of the six halogenated solvents were investigated. The optimal injection time and equilibration temperature were 3 s and 90 ℃, respectively. The method demonstrated good analytical performance for the six halogenated solvents when the equilibration time was 30 min. A methodological study was conducted on the optimized method, and the results showed that the six halogenated solvents exhibited good linear relationships in the range of 0.002-0.200 mg/kg, with correlation coefficients of ≥0.9991. The limits of detection (LODs) and quantification (LOQs) of 1,1,1-trichloroethane and bromoform were 0.0006 and 0.002 mg/kg, respectively. The LODs and LOQs of chloroform, carbon tetrachloride, dibromochloromethane, and tetrachloroethylene were 0.0003 and 0.001 mg/kg, respectively. The average recoveries under different spiked levels were 85.53%-115.93%, and the relative standard deviations (n=6) were 1.11%-8.48%. The established method was used to analyze 13 olive oil samples available in the market. Although no halogenated solvents were detected in these samples, a limited number of samples does not represent all olive oils. Hence, monitoring residual halogenated solvents in olive oil remains necessary for its safe consumption. The LOQs of the method for the six halogenated solvents were significantly lower than that of the COI/T.20/Doc. No. 8-1990 standard method (0.02 mg/kg). In addition, the developed method can be conducted under short operation times with high precision and degree of automation as well as good accuracy. Thus, the proposed method is suitable for the determination and analysis of the residues of the six halogenated solvents in large batches of olive oil samples.

Elucidating the hepatoprotective mechanisms of cholic acid against CCl4-Induced acute liver injury: A transcriptomic and metabolomic study.

ETHNOPHARMACOLOGICAL RELEVANCE: Cholic acid (CA) is one of the main active ingredients in Calculus Bovis, a traditional Chinese medicine, which helps to regulate the heart and liver meridians, clearing the heart, opening the mouth, cooling the liver and calming the wind. However, the molecular mechanism of its liver protective effect is still unclear. AIM OF THE STUDY: Growing attention has been directed towards traditional Chinese medicine (TCM), particularly Calculus Bovis, as a potential solution for liver protection. Despite this interest, a comprehensive understanding of its hepatoprotective mechanisms remains lacking. This research seeks to explore the potential protective properties of cholic acid (CA) against CCl4-induced acute liver injury (ALI) in mice, while also examining the mechanisms involved. MATERIALS AND METHODS: In the experiment, a mouse model was employed to ALI using CCl4, and the potential therapeutic effects of orally administered CA at varying doses (15, 30, and 60 mg/kg) were assessed. The study employed a multi-faceted approach, integrating liver transcriptomics with serum metabolomics, and conducting thorough analyses of serum biochemical markers and liver histopathological sections. RESULTS: Oral CA administration markedly reduced the organ indices of the liver, spleen, and thymus in comparison with the model group. It also elevated the expression of superoxide dismutase (SOD) in serum while diminishing the concentrations of ALT, AST, MDA, IL-6, and TNF-α. Moreover, CA ameliorated the pathological damage induced by CCl4. Integrated metabolomic and transcriptomic analyses indicated that the hepatoprotective action of CA on ALI is mediated through the modulation of lipid metabolic pathways-specifically, metabolisms of glycerophospholipid, arachidonic acid, as well as linoleic acid-and by altering the expression of genes such as Ptgr1, PLpp1, Tbxas1, and Cyp2c37. CONCLUSIONS: The current investigation offers insights into the hepatoprotective mechanisms by which CA mitigates ALI caused by CCl4 exposure, thus supporting the further evaluation and development of CA-based therapeutics for ALI.

Boldine protects against carbon tetrachloride-induced chronic liver injury by regulating NF-κB signaling pathway.

Sustained liver injuries predominantly promote oxidative stress and inflammation that lead to the progression of chronic liver disease (CLD), including fibrosis, cirrhosis, and hepatocellular carcinoma. Boldine, an alkaloid isolated from Peumus boldus, has been shown to have antioxidant and anti-inflammatory effects. Currently, there is no definitive treatment option available for CLD. Therefore, we investigated the hepatoprotective effect of boldine against carbon tetrachloride (CCl4 )-induced chronic liver injury in rats. CCl4 (2 mL/kg., b.w., i.p.) was administered twice weekly for 5 weeks to induce chronic liver injury in rats. Separate groups of rats were given boldine (20 mg/kg b.w., and 40 mg/kg b.w.) and silymarin (100 mg/kg b.w.) orally, daily. Serum transaminases, lipid peroxidation, and antioxidant levels were measured, and nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (cox-2), interleukin-1 ß (IL-1ß), and α-smooth muscle actin (α-SMA) gene and protein expressions were evaluated. CCl4 administration increased liver marker enzymes of hepatotoxicity in serum and oxidative stress markers, inflammatory genes and α-smooth muscle actin expression in liver tissue. Boldine concurrent treatment suppressed CCl4 -induced elevation of transaminase levels in serum, restored enzymic and non-enzymic antioxidants, and downregulated NF-κB, TNF-α, Cox-2 and IL-1ß expressions, thereby suppressing hepatic inflammation. Boldine administration also repressed α-SMA expression. The results of this study demonstrate the antioxidant, anti-inflammatory, and antifibrotic properties of boldine, and it can be a potential therapeutic candidate in the treatment of CLD.

Oleuropein, a phenolic component of Olea europaea L. ameliorates CCl4-induced liver injury in rats through the regulation of oxidative stress and inflammation.

OBJECTIVE: This study aimed to assess the hepatoprotective role of oleuropein (Olp), a phenolic compound found in olive, against carbon tetrachloride (CCl4)-induced liver damage in rats. MATERIALS AND METHODS: The research involved male albino rats, which received intraperitoneal injections of 100 mg/kg b.w. of oleuropein for 8 consecutive weeks before being subjected to carbon tetrachloride (CCl4) at a dosage of 1.0 ml/kg b.w. Changes induced by CCl4 in antioxidant and inflammatory marker levels were assessed using ELISA assay kits. Moreover, CCl4-induced liver tissue architecture alteration, fibrosis, and expression pattern of protein were evaluated by performing H&E, Sirius red, Masson trichrome, and immunohistochemistry staining. RESULTS: Increased serum transaminases and massive hepatic damage were observed by this liver toxicant. The hepatic injury was further evidenced by a significant decrease in antioxidant enzyme activity [superoxide dismutase (SOD), glutathione peroxidase (GPx), Glutathione (GSH) and Total Antioxidant Capacity (T-AOC)]. The administration of CCl4 resulted in an increased inflammatory response, which was measured by C-reactive protein, interleukin-6, as well as tumor necrosis factor-alpha. Olp as a curative regimen led to significant attenuation in the inflammatory response and oxidative/nitrosative stress. This polyphenol treatment improved the hepatic tissue architecture and decreased fibrosis. In the CCl4 treatment group, the expression pattern of IL-6 protein was high, whereas expression was decreased after Olp, as evidenced by immunohistochemistry staining. CONCLUSIONS: The study suggests that oleuropein treatment has the potential to reduce liver damage caused by CCl4 induction by inhibiting oxidative stress and inflammation and maintaining liver tissue architecture. This could make it a promising treatment option for liver pathogenesis.

Crosstalk of lysyl oxidase-like 1 and lysyl oxidase prolongs their half-lives and regulates liver fibrosis through Notch signal.

BACKGROUND: Lysyl oxidase (LOX) family members (LOX and LOXL1 to 4) are crucial copper-dependent enzymes responsible for cross-linking collagen and elastin. Previous studies have revealed that LOX and LOXL1 are the most dramatically dysregulated LOX isoforms during liver fibrosis. However, the crosstalk between them and the underlying mechanisms involved in the profibrotic behaviors of HSCs, as well as the progression of liver fibrosis, remain unclear. METHODS: pCol9GFP-HS4,5Tg mice, Loxl1fl/flGfapCre mice, human HSC line, and primary HSCs were enrolled to study the dysregulation pattern, profibrotic roles, and the potential mechanisms of LOX and LOXL1 interaction involved in the myofibroblast-like transition of HSCs and liver fibrogenesis. RESULTS: LOX and LOXL1 were synergistically upregulated during liver fibrogenesis, irrespective of etiology, together orchestrating the profibrotic behaviors of HSCs. LOX and LOXL1 coregulated in HSCs, whereas LOXL1 dominated in the coregulation loop. Interestingly, the interaction between LOXL1 and LOX prolonged their half-lives, specifically enhancing the Notch signal-mediated myofibroblast-like transition of HSCs. Selective disruption of Loxl1 in Gfap+ HSCs deactivated the Notch signal, inhibited HSC activation, and relieved carbon tetrachloride-induced liver fibrosis. CONCLUSIONS: Our current study confirmed the synergistic roles and the underlying mechanisms of LOXL1 and LOX crosstalk in the profibrotic behaviors of HSCs and liver fibrosis progression, providing experimental evidence for further clear mechanism-based anti-LOXL1 strategy development in the therapy of liver fibrosis.

Attenuation of carbon tetrachloride-induced nephrotoxicity by gum Arabic extract via modulating cellular redox state, NF-κB pathway, and KIM-1.

The current study investigated the ameliorating impact of GA water extract (GAE) on CCl4-induced nephrotoxicity in renal cells and tissue by comparing its effectiveness with the Ketosteril (Ks) drug in restoring oxidative stress and necroinflammation. The cell morphology, necrosis, and redox state were evaluated in Vero cells. The influence of GAE on CCl4-induced oxidative stress, inflammation, and necrosis was examined in rats. The predicted inhibitory mechanism of GAE phenolic constituents against COX-2 and iNOS was also studied. The results revealed that GAE contains crucial types of phenolic acids, which are associated with its antiradical activities. GAE improved CCl4-induced Vero cell damage and restored renal architecture damage, total antioxidant capacity, ROS, TBARS, NO, GSH, GPX, SOD, and MPO in rats. GAE downregulated the gene expression of renal NF-κB, TNF-α, iNOS, and COX-2, as well as kidney injury molecule-1 (KIM-1) in rats. The GAE improved blood urea, creatinine, cholesterol, and reducing power. The computational analysis revealed the competitive inhibitory mechanism of selected phenolic composites of GAE on COX-2 and iNOS activities. The GAE exhibited higher potency than Ks in most of the studied parameters, as observed by the heatmap plots. Thus, GAE is a promising extract for the treatment of kidney toxicity.

[Adeno-associated virus-mediated hepatocyte-specific NDUFA13 overexpression protects against CCl4-induced liver fibrosis in mice by inhibiting hepatic NLRP3 activation].

OBJECTIVE: To investigate the protective effect of NDUFA13 protein against acute liver injury and liver fibrosis in mice and explore the possible mechanisms. METHODS: BALB/C mice (7 to 8 weeks old) were divided into normal group, CCl4 group, CCl4+AAV-NC group and CCl4+AAV-NDU13 group (n=18). Mouse models of liver fibrosis were established by intraperitoneal injection of CCl4 twice a week for 3, 5 or 7 weeks, and the recombinant virus AAV8-TBG-NC or AAV8-TBG-NDUFA13 was injected via the tail vein 7-10 days prior to CCl4 injection. After the treatments, pathological changes in the liver of the mice were observed using HE and Masson staining. Hepatic expression levels of NDUFA13 and α-SMA were detected with Western blotting, and the coexpression of NDUFA13 and NLRP3, TNF-α and IL-1ß, and α-SMA and collagen Ⅲ was analyzed with immunofluorescence assay. RESULTS: HE and Masson staining showed deranged liver architecture, necrotic hepatocytes and obvious inflammatory infiltration and collagen fiber deposition in mice with CCl4 injection (P < 0.001). NDUFA13 expression markedly decreased in CCl4-treated mice (P < 0.001), while a significant reduction in inflammatory aggregation and fibrosis was observed in mice with AAV-mediated NDUFA13 overexpression (P < 0.001). In CCl4+AAV-NDU13 group, immunofluorescence assay revealed markedly weakened activation of NLRP3 inflammasomes (P < 0.001), significantly decreased TNF-α and IL-1ß secretion (P < 0.001), and inhibited hepatic stellate cell activation (P < 0.05) and collagen formation in the liver (P < 0.001). CONCLUSION: Mitochondrial NDUFA13 overexpression in hepatocytes protects against CCl4- induced liver fibrosis in mice by inhibiting activation of NLRP3 signaling.

Fe(II) coordination transition regulates reductive dechlorination: The overlooked abiotic role of lactate.

The coordination environment of Fe(II) significantly affect the reductive reactivity of Fe(II). Lactate is a common substrate for enhancing microbial dechlorination, but its effect on abiotic Fe(II)-driven reductive dechlorination is largely ignored. In this study, the structure-reactivity relationship of Fe(II) is investigated by regulating the ratio of lactate:Fe(II). This work shows that lactate-Fe(II) complexing enhances the abiotic Fe(II)-driven reductive dechlorination with the optimum lactate:Fe(II) ratio of 10:20. The formed hydrogen bond (Fe-OH∙∙∙∙∙∙O = C-) and Fe-O-C metal-ligand bond result in a reduced Fe(II) coordination number from six to four, which lead to the transition of Fe(II) coordination geometry from octahedron to tetrahedron/square planar. Coordinatively unsaturated Fe(II) results in the highest reductive dechlorination reactivity towards carbon tetrachloride (k1 = 0.26254 min-1). Excessive lactate concentration (> 10 mM) leads to an increased Fe(II) coordination number from four to six with a decreased reductive reactivity. Electrochemical characterization and XPS results show that lactate-Fe(II)-I (C3H5O3-:Fe(II) = 10:20) has the highest electron-donating capacity. This study reveals the abiotic effect of lactate on reductive dechlorination in a subsurface-reducing environment where Fe(II) is usually abundant.

Therapeutic Potential of Diosgenin in Amelioration of Carbon Tetrachloride-Induced Murine Liver Injury.

Diosgenin is a sapogenin with antidiabetic, antioxidant, and anti-inflammatory properties. The current study investigated whether diosgenin could ameliorate carbon tetrachloride (CCL4)-induced liver injury. To cause liver injury, CCL4 was injected intraperitoneally twice a week for 8 weeks. Daily oral administration of diosgenin at doses of 20, 40, and 80 mg/kg was started one day before CCL4 injection and continued for 8 weeks. Finally, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and also albumin were assessed. Catalase and superoxide dismutase (SOD) activities in addition to glutathione (GSH) and malondialdehyde (MDA) levels were also quantified in the liver homogenate and routine histological evaluation was also conducted. Elevated serum levels of liver enzymes and decreased serum level of albumin caused by CCL4 were significantly restored following diosgenin administration at doses of 40 and 80 mg/kg. Long-term administration of CCL4 increased inflammatory and apoptotic factors such as IL-1ß, caspase 3, TNF-α, and IL-6 and decreased SOD and catalase activities as well as GSH level in liver homogenates; while MDA level was increased. Treatment with diosgenin increased SOD and catalase activities and GSH levels in the liver of injured animals. In addition, liver MDA, IL-1ß, caspase 3, TNF-α, and IL-6 level or activity decreased by diosgenin treatment. Additionally, diosgenin aptly prevented aberrant liver histological changes. According to obtained results, diosgenin can dose-dependently diminish CCl4-induced liver functional deficits and histological changes in a dose-dependent manner, possibly due to its antioxidant and anti-inflammation properties, and its beneficial effect is comparable to known hepatoprotective agent silymarin.

Yinchen gongying decoction mitigates CCl4-induced chronic liver injury and fibrosis in mice implicated in inhibition of the FoxO1/TGF-ß1/ Smad2/3 and YAP signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver fibrosis (LF) is a common reversible consequence of chronic liver damage with limited therapeutic options. Yinchen Gongying decoction (YGD) composed of two homologous plants: (Artemisia capillaris Thunb, Taraxacum monochlamydeum Hand.-Mazz.), has a traditionally application as a medicinal diet for acute icteric hepatitis. However, its impact on LF and underlying mechanisms remain unclear. AIM OF THE STUDY: This study aims to assess the impact of YGD on a carbon tetrachloride (CCl4) induced liver fibrosis and elucidate its possible mechanisms. The study seeks to establish an experimental foundation for YGD as a candidate drug for hepatic fibrosis. MATERIALS AND METHODS: LC-MS/MS identified 11 blood-entry components in YGD, and network pharmacology predicted their involvement in the FoxO signaling pathway, insulin resistance, and PI3K-AKT signaling pathway. Using a CCl4-induced LF mouse model, YGD's protective effects were evaluated in comparison to a positive control and a normal group. The underlying mechanisms were explored through the assessments of hepatic stellate cells (HSCs) activation, fibrotic signaling, and inflammation. RESULTS: YGD treatment significantly improved liver function, enhanced liver morphology, and reduced liver collagen deposition in CCl4-induced LF mice. Mechanistically, YGD inhibited HSC activation, elevated MMPs/TIMP1 ratios, suppressed the FoxO1/TGF-ß1/Smad2/3 and YAP pathways, and exhibited anti-inflammatory and antioxidant effects. Notably, YGD improved the insulin signaling pathway. CONCLUSION: YGD mitigates LF in mice by modulating fibrotic and inflammatory pathways, enhancing antioxidant responses, and specifically inhibiting FoxO1/TGF-ß1/Smad2/3 and YAP signal pathways.

The hepatoprotective potentials of Olea europaea L. leaves against carbon tetrachloride-induced hepatic injury in rats.

OBJECTIVE: To examine the therapeutic effects of Olea europaea L. leaves extract on carbon tetrachloride-induced liver injury in rats. Methods: The experimental study was conducted at the Department of Physiology, University of Karachi, Karachi, in July 2021, and comprised Albino Wistar male rats weighing 180-220gm. The animals were divided into control group I, carbon tetrachloride group II, Olea europaea L. + carbon tetrachloride group III and Olea europaea L. group IV. In Vitro model of hepatic toxicity was developed by carbon tetrachloride. A daily dose of 50mg/kg of aqueous extract of olive leaves was administered orally and 0.8ml/kg of carbon tetrachloride was administered twice a week subcutaneously for 28 days. On the 29th day, the animals were sacrificed, and tested for hepatic enzymes, lipid peroxidation markers and histopathology. Data was analysed using SPSS 20. RESULTS: Of the 24 rats, 6(25%) were in each of the 4 groups. Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and total bilirubin levels were significantly reduced (p<0.05) in group II whereas, 4- hydroxynonenal, isoprostane and malondialdehyde levels were significantly increased (p<0.05). However, total antioxidant level increased significantly (p<0.05) in group III compared to group II. Histopathology showed severe liver damage in group II and mild damage in group III. Conclusion: Olea europaea L. leaves extract was found to have profound hepatoprotective effects.

A Mouse Model of Non-Alcoholic Steatohepatitis and Hepatocellular Carcinoma Induced by Western Diet and Carbon Tetrachloride.

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD). Obesity is a known risk factor of NASH, which, in turn, increases the risk of developing cirrhosis (liver scarring) and hepatocellular carcinoma (HCC). In addition to being a potentially life-threatening condition, public health concerns surrounding NASH are amplified by the lack of FDA-approved treatments. Although various preclinical models reflecting both the histopathology and the pathophysiological progression of human NASH exist, most of these models are diet-based and require 6-13 months for NASH symptom manifestation. Here, we describe a simple and rapid-progression model of NASH and NASH-driven HCC in mice. Mice received a western diet equivalent (WD; i.e., a high-fat, high-fructose, and high-cholesterol diet), high-sugar water (23.1 g/L fructose and 18.9 g/L glucose), and weekly intraperitoneal injections of carbon tetrachloride (CCl4) at a dose of 0.2 µL/g of body weight. The resulting phenotype, consisting in liver fibrosis and HCC, appeared within 24 weeks of diet/treatment initiation and presented similar histological and transcriptomic features as human NASH and NASH-driven HCC, thereby supporting the adequacy of this preclinical model for the development and evaluation of drugs that can prevent or reverse these diseases.

On a deterministic mathematical model which efficiently predicts the protective effect of a plant extract mixture in cirrhotic rats.

In this work, we propose a mathematical model that describes liver evolution and concentrations of alanine aminotransferase and aspartate aminotransferase in a group of rats damaged with carbon tetrachloride. Carbon tetrachloride was employed to induce cirrhosis. A second groups damaged with carbon tetrachloride was exposed simultaneously a plant extract as hepatoprotective agent. The model reproduces the data obtained in the experiment reported in [Rev. Cub. Plant. Med. 22(1), 2017], and predicts that using the plants extract helps to get a better natural recovery after the treatment. Computer simulations show that the extract reduces the damage velocity but does not avoid it entirely. The present paper is the first report in the literature in which a mathematical model reliably predicts the protective effect of a plant extract mixture in rats with cirrhosis disease. The results reported in this manuscript could be used in the future to help in fighting cirrhotic conditions in humans, though more experimental and mathematical work is required in that case.

Oral delivery of aescin-loaded gelatin nanoparticles ameliorates carbon tetrachloride-induced hepatotoxicity in Wistar rats.

AIM: The liver plays a crucial role in biotransformation but it is susceptible to chemical-induced damage, known as hepatotoxicity. Traditional therapies for protecting the liver face significant challenges, including poor bioavailability, off-target effects, adverse reactions, drug breakdown, and inadequate uptake. These issues emphasize the need for precise, targeted therapeutic approaches against hepatotoxicity. MATERIALS AND METHODS: The objective of our research was to develop a customized, biocompatible, and biodegradable nanodrug delivery system for hepatoprotection. We chose collagen hydrolyzed protein, or gelatin, as the base material and utilized solvent evaporation and nanoprecipitation methods to create nanoparticles with size ranging from 130 to 155 nm. The resulting nanoparticles exhibited a spherical and smooth surface, as confirmed by scanning and transmission electron microscopy. KEY FINDINGS: Bioactive aescin (AES), into these gelatin nanoparticles (AES-loaded gel NPs), we tested these nanoparticles using a hepatotoxicity model. The results were indicating a significant reduction in the levels of key biomolecules, including NF-κB, iNOS, BAX, and COX-2 and decreased serum levels of enzymes ALT and AST. This reduction correlated with a notable alleviation in the severity of hepatotoxicity. Furthermore, the treatment with AES-loaded gel NPs resulted in the downregulation of several inflammatory and liver-specific biomarkers, including nitrite, MPO, TNF-α, and IL-6. SIGNIFICANCE: In summary, our study demonstrates that the AES-loaded gel NPs were markedly more effective in mitigating experimental hepatotoxicity when compared to the free aescin. The nanoparticles exhibited a propensity for suppressing liver damage, showcasing the potential of this targeted therapeutic approach for safeguarding the liver from harmful chemical insults.

Single-Cell Transcriptome Analysis Identified Core Genes and Transcription Factors in Mesenchymal Cell Differentiation during Liver Cirrhosis.

BACKGROUND: Mesenchymal cells, including hepatic stellate cells (HSCs), fibroblasts (FBs), myofibroblasts (MFBs), and vascular smooth muscle cells (VSMCs), are the main cells that affect liver fibrosis and play crucial roles in maintaining tissue homeostasis. The dynamic evolution of mesenchymal cells is very important but remains to be explored for researching the reversible mechanism of hepatic fibrosis and its evolution mechanism of hepatic fibrosis to cirrhosis. METHODS: Here, we analysed the transcriptomes of more than 50,000 human single cells from three cirrhotic and three healthy liver tissue samples and the mouse hepatic mesenchymal cells of two healthy and two fibrotic livers to reconstruct the evolutionary trajectory of hepatic mesenchymal cells from a healthy to a cirrhotic state, and a subsequent integrative analysis of bulk RNA sequencing (RNA-seq) data of HSCs from quiescent to active (using transforming growth factor ß1 (TGF-ß1) to stimulate LX-2) to inactive states. RESULTS: We identified core genes and transcription factors (TFs) involved in mesenchymal cell differentiation. In healthy human and mouse livers, the expression of NR1H4 and members of the ZEB families (ZEB1 and ZEB2) changed significantly with the differentiation of FB into HSC and VSMC. In cirrhotic human livers, VSMCs transformed into HSCs with downregulation of MYH11, ACTA2, and JUNB and upregulation of PDGFRB, RGS5, IGFBP5, CD36, A2M, SOX5, and MEF2C. Following HSCs differentiation into MFBs with the upregulation of COL1A1, TIMP1, and NR1H4, a small number of MFBs reverted to inactivated HSCs (iHSCs). The differentiation trajectory of mouse hepatic mesenchymal cells was similar to that in humans; however, the evolution trajectory and proportion of cell subpopulations that reverted from MFBs to iHSCs suggest that the mouse model may not accurately reflect disease progression and outcome in humans. CONCLUSIONS: Our analysis elucidates primary genes and TFs involved in mesenchymal cell differentiation during liver fibrosis using scRNA-seq data, and demonstrated the core genes and TFs in process of HSC activation to MFB and MFB reversal to iHSC using bulk RNA-seq data of human fibrosis induced by TGF-ß1. Furthermore, our findings suggest promising targets for the treatment of liver fibrosis and provide valuable insights into the molecular mechanisms underlying its onset and progression.

Multi-omics reveals deoxycholic acid modulates bile acid metabolism via the gut microbiota to antagonize carbon tetrachloride-induced chronic liver injury.

Deoxycholic acid (DCA) serves essential functions in both physiological and pathological liver processes; nevertheless, the relationship among DCA, gut microbiota, and metabolism in chronic liver injury remain insufficiently understood. The primary objective of this study is to elucidate the potential of DCA in ameliorating chronic liver injury and evaluate its regulatory effect on gut microbiota and metabolism via a comprehensive multi-omics approach. Our study found that DCA supplementation caused significant changes in the composition of gut microbiota, which were essential for its antagonistic effect against CCl4-induced chronic liver injury. When gut microbiota was depleted with antibiotics, the observed protective efficacy of DCA against chronic liver injury became noticeably attenuated. Mechanistically, we discovered that DCA regulates the metabolism of bile acids (BAs), including 3-epi DCA, Apo-CA, and its isomers 12-KLCA and 7-KLCA, IHDCA, and DCA, by promoting the growth of A.muciniphila in gut microbiota. This might lead to the inhibition of the IL-17 and TNF inflammatory signaling pathway, thereby effectively countering CCl4-induced chronic liver injury. This study illustrates that the enrichment of A. muciniphila in the gut microbiota, mediated by DCA, enhances the production of secondary bile acids, thereby mitigating chronic liver injury induced by CCl4. The underlying mechanism may involve the inhibition of hepatic IL-17 and TNF signaling pathways. These findings propose a promising approach to alleviate chronic liver injury by modulating both the gut microbiota and bile acids metabolism.

RNA-seq combined network pharmacology reveals that Fu-Gan-Wan (FGW) inhibits liver fibrosis via NF-κB/CCL2/CCR2 and lipid peroxidation via Nrf2/HMOX1 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver fibrosis is a serious complication of liver disease characterized by excessive collagen deposition, without effective therapeutic agents in the clinic. Fu-Gan-Wan (FGW) is an empirical formula used for the clinical treatment of hepatitis and cirrhosis. It has been shown to reverse experimental liver fibrosis. However, its corresponding mechanisms remain unclear. AIM OF THE REVIEW: This study aimed to elucidate the key pathways and target genes of FGW in attenuating liver fibrosis. MATERIALS AND METHODS: The therapeutic effects of different doses of FGW on liver fibrosis were investigated using a 2 mL/kg 15% CCl4-induced mouse model. Then, RNA-seq combined with network pharmacology was used to analyze the key biological processes and signaling pathways underlying the anti-liver fibrosis exertion of FGW. These findings were validated in a TGF-ß1-induced model of activation and proliferation of mouse hepatic stellate cell line JS-1. Finally, the key signaling pathways and molecular targets were validated using animal tissues, and the effect of FGW on tissue lipid peroxidation was additionally observed. RESULTS: We found that 19.5 g/kg FGW significantly down-regulated CCl4-induced elevation of hepatic ALT and AST, decreased collagen deposition, and inhibited the expression of pro-fibrotic factors α-SMA, COL1α1, CTGF, TIMP-1, as well as pro-inflammatory factor TGF-ß1. Additionally, FGW at doses of 62.5, 125, and 250 µg/mL dose-dependently blocked JS-1 proliferation, migration, and activation. Furthermore, RNA-seq identified the NF-κB signaling pathway as a key target molecular pathway for FGW against liver fibrosis, and network pharmacology combined with RNA-seq focused on 11 key genes. Significant changes were identified in CCL2 and HMOX1 by tissue RT-PCR, Western blot, and immunohistochemistry. We further demonstrated that FGW significantly attenuated CCl4-induced increases in p-p65, CCL2, CCR2, and HMOX1, while significantly elevating Nrf2. Finally, FGW significantly suppressed the accumulation of lipid peroxidation products MDA and 4-HNE and reconfigured the oxidation-reduction balance, including promoting the increase of antioxidants GPx, GSH, and SOD, and the decrease of peroxidation products ROS and GSSG. CONCLUSIONS: This study demonstrated that FGW exhibits potential in mitigating CCl4-induced hepatic fibrosis, lipid peroxidation, and iron metabolism disorders in mice. This effect may be mediated through the NF-κB/CCL2/CCR2 and Nrf2/HMOX1 pathways.

Yin Yang 1 facilitates the activation, inflammation, and extracellular matrix deposition of hepatic stellate cells in hepatic fibrosis.

Chronic hepatic diseases often involve fibrosis as a pivotal factor in their progression. This study investigates the regulatory mechanisms of Yin Yang 1 (YY1) in hepatic fibrosis. Our data reveal that YY1 binds to the prolyl hydroxylase domain 1 (PHD1) promoter. Rats treated with carbon tetrachloride (CCl4) display heightened fibrosis in liver tissues, accompanied by increased levels of YY1, PHD1, and the fibrosis marker alpha-smooth muscle actin (α-SMA). Elevated levels of YY1, PHD1, and α-SMA are observed in the liver tissues of CCl4-treated rats, primary hepatic stellate cells (HSCs) isolated from fibrotic liver tissues, and transforming growth factor beta-1 (TGF-ß1)-induced HSCs. The human HSC cell line LX-2, upon YY1 overexpression, exhibits enhanced TGF-ß1-induced activation, leading to increased expression of extracellular matrix (ECM)-related proteins and inflammatory cytokines. YY1 silencing produces the opposite effect. YY1 exerts a positive regulatory effect on the activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and PHD1 expression. PHD1 silencing rescues the promotion of YY1 in cell activation, ECM-related protein expression, and inflammatory cytokine production in TGF-ß1-treated LX-2 cells. Overall, our findings propose a model wherein YY1 facilitates TGF-ß1-induced HSC activation, ECM-related protein expression, and inflammatory cytokine production by promoting PHD1 expression and activating the PI3K/AKT signaling pathway. This study positions YY1 as a promising therapeutic target for hepatic fibrosis.

Deficiency of purinergic P2X4 receptor alleviates experimental autoimmune hepatitis in mice.

Purinergic P2X4 receptor (P2X4R) has been shown to have immunomodulatory properties in infection, inflammation, and organ damage including liver regeneration and fibrosis. However, the mechanisms and pathophysiology associated with P2X4R during acute liver injury remain unknown. We used P2X4R-/- mice to explore the role of P2X4R in three different models of acute liver injury caused by concanavalin A (ConA), carbon tetrachloride, and acetaminophen. ConA treatment results in an increased expression of P2X4R in the liver of mice, which was positively correlated with higher levels of aspartate aminotransferase and alanine aminotransferase in the serum. However, P2X4R gene ablation significantly reduced the severity of acute hepatitis in mice caused by ConA, but not by carbon tetrachloride or acetaminophen. The protective benefits against immune-mediated acute hepatitis were achieved via modulating inflammation (Interleukin (IL)-1ß, IL-6, IL-17A, interferon-γ, tumor necrosis factor-α), oxidative stress (malondialdehyde, superoxide dismutase, glutathione peroxidase, and catalase), apoptosis markers (Bax, Bcl-2, and Caspase-3), autophagy biomarkers (LC3, Beclin-1, and p62), and nucleotide oligomerization domain-likereceptorprotein 3(NLRP3) inflammasome-activated pyroptosis markers (NLRP3, Gasdermin D, Caspase-1, ASC, IL-1ß). Additionally, administration of P2X4R antagonist (5-BDBD) or agonist (cytidine 5'-triphosphate) either improved or worsened ConA-induced autoimmune hepatitis, respectively. This study is the first to reveal that the absence of the P2X4 receptor may mitigate immune-mediated liver damage, potentially by restraining inflammation, oxidation, and programmed cell death mechanisms. And highlight P2X4 receptor is essential for ConA-induced acute hepatitis.