CYP2E1 Expression Research Articles

Hepatocyte-Targeted Lipid Nanoparticle Delivery of HERC2 Plasmid Controls Drug-Induced Hepatotoxicity by Limiting β-Catenin-Regulated CYP2E1 Expression.

Understanding the molecular mechanisms that bridge hepatic inflammation and liver injury is crucial for developing effective therapeutic strategies for drug-induced liver injury (DILI) management. HECT domain and RCC1-like domain 2(HERC2) belongsto the large Herc family of ubiquitin E3 ligases, which are implicated in tissue development and inflammation. The observation reveals a pronounced HERC2 expression in specific hepatocyte subsets that proliferate in response to DILI in humans, prompting an investigation into the role of HERC2 in distinct DILI progression. Under the APAP challenge, liver-specific HERC2-deficient mice suffer more severe liver damage. Integrated single-cell RNA sequencing analysis unveils a negative correlation between HERC2 and CYP2E1, a vital metabolic enzyme for xenobiotics, in hepatocytes from APAP-challenged mice. Mechanistically, HERC2 interacts with β-catenin to promote its ubiquitination, thereby governing CYP2E1 transcriptional regulation. Targeted hepatic delivery of lipid nanoparticle-encapsulated HERC2-overexpressing plasmid markedly reduces liver damage caused by APAP overdose. Collectively, these findings elucidate a previously unrecognized protective role of HERC2 in protecting against acute liver injury associated with drug metabolism disorders, highlighting its potential as a therapeutic target in treating DILI.

Mixed active metabolites of the SNP-6 series of novel compounds mitigate metabolic dysfunction-associated steatohepatitis and fibrosis: promising results from pre-clinical and clinical trials

BackgroundMetabolic dysfunction-associated steatohepatitis (MASH) is a growing global health concern with no effective pharmacological treatments. SNP-630, a newly developed synthetic molecule with multiple mechanisms of action, and a mixture of two of its active metabolites (SNP-630-MS) inhibit CYP2E1 expression to prevent reactive oxygen species generation, thereby reducing the accumulation of hepatic triglycerides and lowering chemokine levels. This study investigated the SNP-630’s potential to alleviate the liver injury in MASH and its efficacy in both a mouse model and patients with MASH to identify a drug candidate that targets multiple pathways implicated in MASH.MethodsSNP-630 and SNP-630-MS were separately administered to the MASH mouse model. The tolerability, safety, and efficacy of SNP-630-MS were also evaluated in 35 patients with MASH. The primary endpoint of the study was assessment of the changes in serum alanine aminotransferase (ALT) levels from baseline to week 12, while the secondary endpoints included the evaluation of liver inflammation, steatosis, and fibrosis parameters and markers.ResultsSNP-630 treatment in mice improved inflammation, liver steatosis, and fibrosis compared with that in the MASH control group. Both SNP-630 and SNP-630-MS treatments markedly reduced ALT levels, hepatic triglyceride content, and the expression of inflammatory cytokines monocyte chemoattractant protein 1 and fibrotic collagen (i.e., Col1a1, Col3a1, and Timp1) in mice. In the clinical trial, patients treated with SNP-630-MS exhibited significant improvement in ALT levels at week 12 compared with baseline levels, with no reports of severe adverse events. This improvement in ALT levels surpassed that achieved with most other MASH candidates. SNP-630-MS demonstrated potential antifibrotic effects, as evidenced by a significant decrease in the levels of fibrogenesis-related biomarkers such as CCL4, CCL5, and caspase 3. Subgroup analysis using FibroScan measurements further indicated the efficacy of SNP-630-MS in ameliorating liver fibrosis.ConclusionsSNP-630 and SNP-630-MS demonstrated favorable results in mice. SNP-630-MS showed excellent tolerability in mice and patients with MASH. Efficacy analyses indicated that SNP-630-MS improved liver steatosis and injury in patients with MASH, suggesting that SNP-630 and 630-MS are promising therapeutic options for MASH. Larger scale clinical trials remain warranted to assess the efficacy and safety of SNP-630 in MASH.Trial registration: ClinicalTrials.gov NCT03868566. Registered 06 March 2019-Retrospectively registered, https://clinicaltrials.gov/study/NCT03868566

Polygala fallax Hemsl polysaccharides alleviated alcoholic fatty liver disease by modifying lipid metabolism via AMPK

Alcoholic fatty liver disease (AFLD) is characterized by excessive lipid accumulation in the liver. This study aimed to investigate the protective effects and mechanisms of Polygala fallax Hemsl polysaccharides (PFPs) on AFLD. PFPs were purified and structurally characterized. An AFLD model was established in mice using alcohol and a high-fat diet. A significant reduction in hepatic steatosis was observed following PFPs treatment, evidenced by decreased fat deposition in liver tissues. Additionally, PFPs reduced various liver injury markers, increased levels of antioxidant enzymes, and improved significantly liver function. RNA sequencing revealed that PFPs improved lipid and CYP450 metabolic pathway abnormalities in AFLD mice. Furthermore, PFPs activated the AMPK pathway, reducing lipid accumulation and enhancing lipid metabolism. A HepG2 cell model treated with ethanol and oleic acid showed significant biochemical improvements with PFPs pretreatment, including reduced lipid accumulation and lower reactive oxygen species (ROS) levels. To further elucidate the AMPK and PFPs correlation in AFLD, an AMPK inhibitor (compound C) was used. In vitro and in vivo qRT-PCR and Western blot results confirmed that PFPs protected against AFLD by activating AMPK phosphorylation, regulating lipid synthesis, and inhibiting lipid accumulation. PFPs also modulated CYP2E1 and oxidative stress-related gene expression, affecting liver metabolism.

Ameliorative effects of Monascus-fermented hawthorn extract on a high-fat diet-induced rat model of non-alcoholic fatty liver disease

ObjectiveThe aim of this study was to elucidate the effects of fermented hawthorn extract on high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in rats, and explore the possible underlying mechanisms. MethodsA total of 42 male adult Sprague-Dawley rats were randomly divided into five groups: normal control group (given a normal feed diet and distilled water by gavage), NAFLD model (given HFD and distilled water by gavage), low-, medium-, and high-dose fermented hawthorn extract treatment groups (given HFD and different doses of fermented hawthorn extract by gavage). After 12 weeks of gavage administration, changes in body weight, liver/body weight ratio, serum liver enzymes, as well as triglyceride (TG) content and oxidative stress levels in rat liver tissueswere detected. Histological evaluation was performed to observe the degree of fat accumulation (steatosis). qRT-PCR and western blotting were performed to detect the mRNA and protein expression of cytochrome P4502E1 (CYP2E1, a key enzyme associated with lipid peroxidation), and lipogenic factors (sterol regulatory element-binding protein 1c (SREBP-1c) and fatty acid synthase (FAS)) in rat liver tissues. ResultsFermented hawthorn extract significantly reduced the body weight, decreased the levels of liver enzymes, improved hepatic steatosis, and exhibited obvious antioxidant effects. Fermented hawthorn extract also significantly down-regulated the mRNA and protein expression levels of CYP2E1, SREBP-1c and FAS. ConclusionOur findings suggested that fermented hawthorn extract can markedly reduce body weight, ameliorate HFD-induced NAFLD in rats, and exhibits significant antioxidant effects. Its underlying mechanism may depend on the inhibition of CYP2E1, SREBP-1c, and FAS expression.

Laurus nobilis L. leaves Suppress Alcohol-Related Liver Disease by Exhibiting Antioxidant and Anti-Inflammatory Effects in Alcohol-Treated Hepatocytes and Mice.

Excessive and prolonged alcohol consumption can lead to a serious health condition known as alcohol-related liver disease (ARLD). This ailment represents a significant worldwide health challenge, affecting populations across various demographics. ARLD has a multifactorial pathogenesis involving oxidative stress, inflammation, dysregulated lipid metabolism, and apoptosis. In this study, we investigated the hepatoprotective effects of Laurus nobilis L. leaf water extract (LLE) against ARLD in alcohol-treated hepatocytes and mice. LLE exhibited antioxidant and anti-inflammatory properties by enhancing antioxidant enzyme activities and suppressing proinflammatory cytokines and CYP2E1 expression in ethanol-treated hepatocytes. Moreover, LLE mitigated lipogenesis by modulating the expression of lipogenic factors in ethanol-treated hepatocytes. In vivo, LLE administration attenuated liver injury, oxidative stress, inflammation, and lipid accumulation induced by alcohol consumption in mice. Additionally, LLE suppressed apoptosis signaling pathways implicated in alcohol-induced hepatocyte apoptosis. These findings suggest that LLE functions as a multifaceted therapeutic agent for ARLD by modulating multiple cellular mechanisms, including the reduction of oxidative damage, mitigation of inflammatory responses, alleviation of lipid-mediated toxicity, and regulation of programmed cell death pathways.

Discovery of CYP2E1 as a novel target in rheumatoid arthritis and validation by a new specific CYP2E1 inhibitor

Considerable evidence indicates that CYP2E1 is associated with a variety of inflammatory diseases. Here we evaluated CYP2E1 as a potential therapeutic target for rheumatoid arthritis (RA) and established the protective effect of a new CYP2E1 inhibitor. Gene-expression datasets were used to analyze the change in expression of CYP2E1 in RA patients; CYP2E1 activity in collagen-induced arthritis (CIA) rats was determined by HPLC. We further evaluated the protective effects of Cyp2e1 knockout and a CYP2E1-specific inhibitor, Q11, synthesized by our group, in CIA and adjuvant-induced arthritis (AIA) rats. The expression of CYP2E1 in synovial tissue was elevated in RA patients and in CIA rats and the activity of CYP2E1 in vivo and in vitro in CIA rats was greater than that of controls. Cyp2e1 knockout significantly reduced the incidence of CIA and alleviated the severity of symptoms. Treatment with different doses of Q11 decreased paw thickness, volume and arthritis scores and reduced the serum levels of IL‐6, TNF‐α, IL‐1β and MDA, and increased the level of GSH in CIA rats. A similar inhibitory effect was exhibited for Q11 in the AIA rats. Moreover, Q11 significantly impeded proliferation, migration, and invasion of human rheumatoid arthritis synovial fibroblasts cells. Q11 decreased the release of ROS and enhanced Nrf2 nuclear translocation and HO-1 expression in the cell nucleus. Overall, our results indicated that CYP2E1 may be a new target for RA and Q11 has potential protective effects against RA by reducing oxidative stress and opposing the inflammatory response via the ROS/Nrf2/HO-1 signaling pathway.

CYP2E1 mediated deoxynivalenol-induced hepatocyte toxicity by regulating ferroptosis

Deoxynivalenol (DON), one of the most common mycotoxins in food and feed, can cause acute and chronic liver injury, posing a serious health risk to humans and animals. One of the important manifestations of DON-induced hepatotoxicity is ferroptosis. It has been reported that CYP2E1 can mediated ferroptosis, but the role of DON-induced CYP2E1 in DON-induced ferroptosis in hepatocytes is unknown. In the present study, we observed that DON significantly increased the expression of CYP2E1 and decreased the expression of the ferroptosis inhibitory proteins GPX4 and SLC7A11, as well as GCLC and NQO1. This resulted in an increase in the levels of cell lipid ROS and FeII, 4-HNE, which ultimately led to cell ferroptosis. Notably, knockdown of CYP2E1 resulted in an increase in DON-induced low levels of GPX4 and SLC7A11, a decrease in DON-induced high levels of lipid ROS, FeII and cell secreted 4-HNE, thus ameliorating cell ferroptosis. Moreover, the ferroptosis inhibitor ferrostatin-1 was observed to antagonise the cell growth inhibitory toxicity induced by DON exposure. This was achieved by blocking the increase in lipid ROS and FeII overload, which in turn reduced the extent of ferroptosis and increased IGF-1 protein expression. In conclusion, the present study demonstrated that CYP2E1 played a regulatory role in DON-induced ferroptosis in hepatocytes. Targeting ferroptosis may prove an effective strategy for alleviating DON-induced cell growth retardation toxicity. These findings provided a potential target and strategies to mitigate DON hepatotoxicity in the future.

Mitigating Acetaminophen-Induced Kidney Injury: The Protective Role of Grape Seed and Peanut Skin Extracts through the iNOS/CYP2E1 Pathway.

The rising number of acute kidney injury cases worldwide due to acetaminophen (APAP) emphasizes the critical need for effective prevention strategies to counteract APAP's detrimental effects. This study examined the kidney-protective capabilities of ethanolic extracts from grape seeds and peanut skins (GSEE and PSEE, respectively) in comparison with silymarin in rats that experienced an APAP overdose. The phenolic compounds in these extracts were measured by using high-performance liquid chromatography (HPLC). In the experiment, Sixty adult male albino rats were divided into five groups of 12. The Control group received 0.5 mL of saline via a gastric tube. Group II received acetaminophen (APAP, 640 mg/kg per day via a gastric tube) to induce renal injury, following Ucar et al. and Islam et al. Groups III, IV, and V received silymarin (50 mg/kg), grape seed extract (200 mg/kg), and peanut skin extract (200 mg/kg), respectively, along with 640 mg of APAP/kg per day for 21 days. Post APAP treatment, significant increases in serum urea and creatinine levels were noted, along with notable decreases in the percentage of body weight gain. Furthermore, there were increases in oxidative stress and inflammatory markers in the kidney tissues, including heightened mRNA expressions of renal iNOS and CYP2E1, which were confirmed through histological studies. The administration of GSEE, PSEE, and silymarin mitigated these adverse effects, likely due to their high phenolic content, which is recognized for its antioxidant and anti-inflammatory effects. GSEE, in particular, showed efficacy comparable to that of silymarin. Molecular docking studies revealed that APAP impeded critical enzymes essential for cellular antioxidant defense, whereas the bioactive compounds in the grape seed and peanut skin extracts effectively inhibited key enzymes and receptors involved in inflammation and oxidative stress. These findings suggest that GSEE and PSEE could serve as viable alternative treatments for kidney damage induced by APAP. Further research to isolate and identify these effective compounds is recommended.

N-acetyl Cysteine Overdose Induced Acute Toxicity and Hepatic Microvesicular Steatosis by Disrupting GSH and Interfering Lipid Metabolisms in Normal Mice.

N-acetyl cysteine (NAC) is a versatile drug used in various conditions, but the limitations and toxicities are not clear. The acute toxicity and toxicological mechanisms of an intraperitoneal injection of NAC in normal mice were deciphered. The LD50 for male and female BALB/cByJNarl mice were 800 mg/kg and 933 mg/kg. The toxicological mechanisms of 800 mg/kg NAC (N800) were investigated. The serum biomarkers of hepatic and renal indices dramatically increased, followed by hepatic microvesicular steatosis, renal tubular injury and necrosis, and splenic red pulp atrophy and loss. Thus, N800 resulted in mouse mortality mainly due to acute liver, kidney, and spleen damages. The safe dose (275 mg/kg) of NAC (N275) increased hepatic antioxidant capacity by increasing glutathione levels and catalase activity. N275 elevated the hepatic gene expressions of lipid transporter, lipid synthesis, β-oxidation, and ketogenesis, suggesting a balance between lipid production and consumption, and finally, increased ATP production. In contrast, N800 increased hepatic oxidative stress by decreasing glutathione levels through suppressing Gclc, and reducing catalase activity. N800 decreased the hepatic gene expressions of lipid transporter, lipid synthesis, and interferred β-oxidation, leading to lipid accumulation and increasing Cyp2E1 expression, and finally, decreased ATP production. Therefore, NAC doses are limited for normal individuals, especially via intraperitoneal injection or similar means.

CYP2E1 deficit mediates cholic acid-induced malignant growth in hepatocellular carcinoma cells

BackgroundIncreased level of serum cholic acid (CA) is often accompanied with decreased CYP2E1 expression in hepatocellular carcinoma (HCC) patients. However, the roles of CA and CYP2E1 in hepatocarcinogenesis have not been elucidated. This study aimed to investigate the roles and the underlying mechanisms of CYP2E1 and CA in HCC cell growth.MethodsThe proteomic analysis of liver tumors from DEN-induced male SD rats with CA administration was used to reveal the changes of protein expression in the CA treated group. The growth of CA-treated HCC cells was examined by colony formation assays. Autophagic flux was assessed with immunofluorescence and confocal microscopy. Western blot analysis was used to examine the expression of CYP2E1, mTOR, AKT, p62, and LC3II/I. A xenograft tumor model in nude mice was used to examine the role of CYP2E1 in CA-induced hepatocellular carcinogenesis. The samples from HCC patients were used to evaluate the clinical value of CYP2E1 expression.ResultsCA treatment significantly increased the growth of HCC cells and promoted xenograft tumors accompanied by a decrease of CYP2E1 expression. Further studies revealed that both in vitro and in vivo, upregulated CYP2E1 expression inhibited the growth of HCC cells, blocked autophagic flux, decreased AKT phosphorylation, and increased mTOR phosphorylation. CYP2E1 was involved in CA-activated autophagy through the AKT/mTOR signaling. Finally, decreased CYP2E1 expression was observed in the tumor tissues of HCC patients and its expression level in tumors was negatively correlated with the serum level of total bile acids (TBA) and gamma-glutamyltransferase (GGT).ConclusionsCYP2E1 downregulation contributes to CA-induced HCC development presumably through autophagy regulation. Thus, CYP2E1 may serve as a potential target for HCC drug development.

Hydrogen Peroxide Induces Ethanol-inducible CYP2E1 via the NFkB-classical Pathway: CYP2E1 mRNA Levels are not High in Alcoholic Hepatitis.

The aim of the present study is to elucidate the mechanism of CYP2E1 induction as a causative factor of alcoholic hepatitis (AH) and its relationship with inflammation. Chronic alcohol consumption induces CYP2E1, which is involved in the development of alcoholic hepatitis (AH). However, the mechanisms underlying the induction of CYP2E1 by alcohol remain unclear. Therefore, we herein investigated the induction of drug-metabolizing enzymes, particularly CYP2E1, by hydrogen peroxide (H2O2), the concentration of which is elevated under inflammatory conditions. The mechanisms underlying the induction of CYP2E1 by H2O2 were examined with a focus on Keap1, a target factor of H2O2. We assessed changes in the expression of drug-metabolizing enzymes in the human hepatoma cell line, Hep3B, following treatment with H2O2, and evaluated changes in the expression of the NFkB-related factor RelA(p65) after the knockdown of Keap1, a regulator of Nrf2 expression by reactive oxygen species. We also performed a promoter analysis using the upstream region of the CYP2E1 gene. We herein used the GSE89632 series for non-alcoholic hepatitis (NASH) and the GSE28619 series for AH. The induction of CYP2E1 by H2O2 was significantly stronger than that of other drugmetabolizing enzymes. On the other hand, the knockdown of Keap1, a target of H2O2, markedly increased RelA(p65), an NFkB factor. Furthermore, the overexpression of RelA(p65) strongly induced the expression of CYP2E1. Four candidate p65-binding sequences were identified upstream of the CYP2E1 gene, and promoter activity assays showed that the third sequence was responsive to the overexpression of RelA(p65). We used the GSE89632 series for NASH and the GSE28619 series for AH in the present study. The expression of CYP2E1 mRNA in the liver was significantly lower in AH patients than in HC patients, but was similar in HC patients and NASH patients. We herein demonstrated that the expression of CYP2E1 was induced by H2O2. The overexpression of RelA(p65) also induced CYP2E1 mRNA expression, whereas H2O2 did not after the knockdown of RelA. These results suggest that H2O2 acts on Keap1 to upregulate RelA (p65) in the NFkB system. One of the mechanisms underlying the induction of CYP2E1 was dependent on the H2O2-Keap1-RelA axis. The results of the database analysis revealed that the expression of CYP2E1 in the liver was significantly lower in AHH patients than in NASH patients, suggesting that CYP2E1 is not the main cause of AH; however, CYP2E1 may exacerbate the pathogenesis of AH.

Phytic acid attenuates acetaminophen-induced hepatotoxicity via modulating iron-mediated oxidative stress and SIRT-1 expression in mice.

Introduction: Administration of high doses of acetaminophen (APAP) results in liver injury. Oxidative stress and iron overload play roles in the pathogenesis of APAP-induced hepatotoxicity. The present study assessed the potential hepatoprotective effects of phytic acid (PA), a natural antioxidant and iron chelator, on APAP-induced hepatotoxicity and the possible underlying mechanism through its effects on CYP2E1 gene expression, iron homeostasis, oxidative stress, and SIRT-1 expression levels. Methods: Twenty-four adult male albino mice were used in this study. Mice were divided into four groups (six mice in each group): control, APAP-treated, PA-treated and APAP + PA-treated groups. Liver function tests, serum and liver tissue iron load were evaluated in all the study groups. Hepatic tissue homogenates were used to detect oxidative stress markers, including malondialdehyde (MDA) and reduced glutathione (GSH). Histological hepatic evaluation and immunohistochemistry of SIRT-1 were performed. Quantitative real-time PCR was used for the assessment of CYP2E1 and SIRT-1 gene expressions. APAP-induced biochemical and structural hepatic changes were reported. Results: PA administration showed beneficial effects on APAP-induced hepatotoxicity through improvements in liver functions, decreased CYP2E1 gene expression, decreased serum and liver iron load, decreased MDA, increased GSH, increased SIRT-1 expression level and improvement in hepatic architecture. Conclusion: Conclusively, PA can be considered a potential compound that can attenuate acetaminophen-induced hepatotoxicity through its role as an iron chelator and antioxidant, as well as the up-regulation of SIRT-1 and down-regulation of CYP2E1.

Pharmacogene expression during progression of metabolic dysfunction-associated steatotic liver disease: Studies on mRNA and protein levels and their relevance to drug treatment

Metabolic dysfunction-associated steatotic liver disease (MASLD) is common worldwide. Genes and proteins contributing to drug disposition may show altered expression as MASLD progresses. To assess this further, we undertook transcriptomic and proteomic analysis of 137 pharmacogenes in liver biopsies from a large MASLD cohort.We performed sequencing on RNA from 216 liver biopsies (206 MASLD and 10 controls). Untargeted mass spectrometry proteomics was performed on a 103 biopsy subgroup. Selected RNA sequencing signals were replicated with an additional 187 biopsies.Comparison of advanced MASLD (fibrosis score 3/4) with milder disease (fibrosis score 0–2) by RNA sequencing showed significant alterations in expression of certain phase I, phase II and ABC transporters. For cytochromes P450, CYP2C19 showed the most significant decreased expression (30 % of that in mild disease) but significant decreased expression of other CYPs (including CYP2C8 and CYP2E1) also occurred. CYP2C19 also showed a significant decrease comparing the inflammatory form of MASLD (MASH) with non-MASH biopsies. Findings for CYP2C19 were confirmed in the replication cohort. Proteomics on the original discovery cohort confirmed decreased levels of several CYPs as MASLD advanced but this decrease was greatest for CYP2C19 where levels fell to 40 % control. This decrease may result in decreased CYP2C19 activity that could be problematic for prescription of drugs activated or metabolized by CYP2C19 as MASLD advances. More limited decreases for other P450s suggest fewer issues with non-CYP2C19 drug substrates. Negative correlations at RNA level between CYP2C19 and several cytokine genes provided initial insights into the mechanism underlying decreased expression.

The Role of Oxidative Stress in Alcoholic Fatty Liver Disease: A Systematic Review and Meta-Analysis of Preclinical Studies.

Alcoholic Fatty Liver Disease (AFLD) is characterized by the accumulation of lipids in liver cells owing to the metabolism of ethanol. This process leads to a decrease in the NAD+/NADH ratio and the generation of reactive oxygen species. A systematic review and meta-analysis were conducted to investigate the role of oxidative stress in AFLD. A total of 201 eligible manuscripts were included, which revealed that animals with AFLD exhibited elevated expression of CYP2E1, decreased enzymatic activity of antioxidant enzymes, and reduced levels of the transcription factor Nrf2, which plays a pivotal role in the synthesis of antioxidant enzymes. Furthermore, animals with AFLD exhibited increased levels of lipid peroxidation markers and carbonylated proteins, collectively contributing to a weakened antioxidant defense and increased oxidative damage. The liver damage in AFLD was supported by significantly higher activity of alanine and aspartate aminotransferase enzymes. Moreover, animals with AFLD had increased levels of triacylglycerol in the serum and liver, likely due to reduced fatty acid metabolism caused by decreased PPAR-α expression, which is responsible for fatty acid oxidation, and increased expression of SREBP-1c, which is involved in fatty acid synthesis. With regard to inflammation, animals with AFLD exhibited elevated levels of pro-inflammatory cytokines, including TNF-a, IL-1β, and IL-6. The heightened oxidative stress, along with inflammation, led to an upregulation of cell death markers, such as caspase-3, and an increased Bax/Bcl-2 ratio. Overall, the findings of the review and meta-analysis indicate that ethanol metabolism reduces important markers of antioxidant defense while increasing inflammatory and apoptotic markers, thereby contributing to the development of AFLD.

Design and expression of recombinant Xuanwei ham antioxidant peptide in Escherichia coli BL21

Xuanwei Ham Peptide (XHP) is a bioactive compound isolated and extracted from Xuanwei ham. It enhances the oxidative defense system by down-regulating CYP2E1 expression, reducing ROS production, and by activating the Nrf2/HO-1 pathway. In order to solve the problems of low yield, high cost and time-consuming separation process of active peptides from ham. In this study, we designed three peptide sequences derived from Xuanwei ham and expressed them recombinantly. The results showed that the peptides using flexible linker peptide ((GGGGS)2), rigid linker peptide ((EAAAK)2) linkage as well as the head-to-tail tandem linkage were successfully expressed in the E. coli expression system. Peptide monomers were released from the fusion proteins by formic acid cleavage and purified sequentially by Ni-IDA affinity chromatography, membrane filtration and RP-HPLC. The molecular weight of XHP was confirmed to be 717.35 by ESI-MS analysis. The results of the antioxidant activity of XHP showed that the hydroxyl radical scavenging rates of the three recombinant peptides were as high as 79.46%, 87.42%, and 53.60%, respectively. The hydroxyl radical activity of formic acid-cleaved XHP was reduced to 35.31%, but it showed good stability in heat, pH, metal ions and gastrointestinal digestion (GID). The molecular docking results showed that the docking of XHP to Keap1 protein was evaluated by five Molecular dynamics simulation with a maximum binding energy of -8.5 kcal/mol. This study provides references and ideas for the expression of active peptides, which can be widely used in the future can be used in the field of nutraceuticals, functional foods and pharmaceuticals.

Characterization of diseased primary human hepatocytes in an all-human cell-based triculture system

Liver diseases, including NAFLD, are a growing worldwide health concern. Currently, there is a lack of suitable in vitro models that sustain basic primary human hepatocyte (PHH) morphology and functionality while supporting presentation of disease-associated phenotypic characteristics such as lipid accumulation and inflammasome activation. In TruVivo, an all-human triculture system (hTCS), basic metabolic functions were characterized in PHHs isolated from normal or diseased livers during two-weeks of culture. Decreases in albumin and urea levels and CYP3A4 activity were seen in diseased-origin PHHs compared to normal PHHs along with higher CYP2E1 expression. Positive expression of the macrophage markers CD68 and CD163 were seen in the diseased PHH preparations. Elevated levels of the pro-inflammatory cytokines IL-6 and MCP-1 and the fibrotic markers CK-18 and TGF-β were also measured. Gene expression of FASN, PCK1, and G6PC in the diseased PHHs was decreased compared to the normal PHHs. Further characterization revealed differences in lipogenesis and accumulation of intracellular lipids in normal and diseased PHHs when cultured with oleic acid and high glucose. TruVivo represents a promising new platform to study lipogenic mechanisms in normal and diseased populations due to the preservation of phenotypic differences over a prolonged culture period.

ERRγ-inducible FGF23 promotes alcoholic liver injury through enhancing CYP2E1 mediated hepatic oxidative stress

Fibroblast growth factor 23 (FGF23) is a member of endocrine FGF family, along with FGF15/19 and FGF21. Recent reports showed that under pathological conditions, liver produces FGF23, although the role of hepatic FGF23 remains nebulous. Here, we investigated the role of hepatic FGF23 in alcoholic liver disease (ALD) and delineated the underlying molecular mechanism. FGF23 expression was compared in livers from alcoholic hepatitis patients and healthy controls. The role of FGF23 was examined in hepatocyte-specific knock-out (LKO) mice of cannabinoid receptor type 1 (CB1R), estrogen related receptor γ (ERRγ), or FGF23. Animals were fed with an alcohol-containing liquid diet alone or in combination with ERRγ inverse agonist. FGF23 is mainly expressed in hepatocytes in the human liver, and it is upregulated in ALD patients. In mice, chronic alcohol feeding leads to liver damage and induced FGF23 in liver, but not in other organs. FGF23 is transcriptionally regulated by ERRγ in response to alcohol-mediated activation of the CB1R. Alcohol induced upregulation of hepatic FGF23 and plasma FGF23 levels is lost in ERRγ-LKO mice, and an inverse agonist mediated inhibition of ERRγ transactivation significantly improved alcoholic liver damage. Moreover, hepatic CYP2E1 induction in response to alcohol is FGF23 dependent. In line, FGF23-LKO mice display decreased hepatic CYP2E1 expression and improved ALD through reduced hepatocyte apoptosis and oxidative stress. We recognized CBIR-ERRγ-FGF23 axis in facilitating ALD pathology through hepatic CYP2E1 induction. Thus, we propose FGF23 as a potential therapeutic target to treat ALD.

The Benzene-induced Hepatic Cytochrome P450 2E1 Expression and Activity are Reduced by Quercetin Administration in Mice.

Benzene as an environmental and industrial agent induces adverse effects that are mainly metabolism-dependent. Effects of Quercetin (QCN) on Benzene (BNZ)-induced changes in the hepatic Cytochrome P450 2E1 expression and activity were investigated. Thirty-six adult male mice were divided into 6 groups (n = 6) and nominated as control, BNZ (exposed to BNZ: 30 ppm), QCN (received QCN: 50 mg/kg, orally), and the fourth, fifth and sixth groups were exposed to 30 ppm BNZ and received 10, 50 and 100 mg/kg QCN respectively, for 28 days. The microsomal subcellular fraction was isolated from the liver samples and the activity of CYP 2E1 was measured based on the hydroxylation rate of 4-nitrophenol. The hepatic activity of myeloperoxidase also was assessed. Total antioxidant capacity and nitric oxide contents of the liver were determined. Expression changes of CYP 2E1 at the mRNA level were examined by qPCR technique. QCN lowered significantly (p < 0.05) the BNZ-increased hepatic nitric oxide levels and restored the BNZ-reduced antioxidant capacity. The BNZ-elevated activity of myeloperoxidase was declined in QCN-received mice. QCN downregulated the expression and activity of hepatic CYP 2E1 in BNZ-exposed animals. Our results suggest that QCN could be a novel hepatoprotective compound for BNZ-induced hepatotoxicities, which is attributed to its capability in the down-regulation of CYP 2E1 expression and activity.

Hepatoprotective effects of Elaeagnus latifolia fruit extract against acetaminophen-induced hepatotoxicity in mice: Mechanistic insights

Elaeagnus latifolia fruit extract (EE) has been reported to contain antioxidant and anti-inflammatory phytochemicals. The present study aimed to examine the anti-hepatotoxic effects of EE in acetaminophen (APAP)-injected mice. Pretreatment with EE for 7 days in APAP mice showed significant reductions in the levels of AST and ALT and necrotic area, accompanied by decreases in liver oxidative stress, nitric oxide, and infiltrating inflammatory cells, compared to the APAP-only group. EE markedly down-regulated expression of CYP2E1, JNK, Bax, NOX subunit, and NF-κB target genes, and up-regulated expression of Bcl-2 and Nrf2 target genes in the livers. Furthermore, EE effectively lowered serum and hepatic lipids, and down-regulated expression of SREBP-1c/2 genes. EE was found to contain phenolic compounds, flavonoids, and proanthocyanidins. Therefore, mechanistic insights into EE's anti-hepatotoxic effects may be associated with preventing the abnormal expression of genes involved in toxic metabolite generation, hepatotoxic signaling, apoptosis, oxidative stress, inflammation, and lipid synthesis.

The “Sweat Soaking Method”-processed Radix Wikstroemia indica Extracts Attenuate Liver Toxicity and its Effect on Cytochrome P450 Protein

Background: Radix Wikstroemia indica (RWI) is one of the most commonly used drugs in Miao medicine. However, RWI is characterized by high toxicity. Objectives: In this study, we aimed to observe the effect of the “sweat soaking method” processed on reducing the hepatotoxicity of RWI and on cytochrome P450 (CYP) protein expression. Materials and Methods: The study focused on investigating the impact of both RWI raw products and RWI processed products on the proliferation of L-02 cells. To assess this, a cell counting kit-8 was employed. After the administration of RWI through oral gavage for a duration of 15 days in rats, the corresponding kits were utilized to determine the serum levels of the liver index factor. Furthermore, both hematoxylin and eosin (H&amp;E) staining and western blot analysis were conducted to analyze the liver tissues. Results: Both the raw RWI and the processed RWI at high doses inhibited the proliferation of L-02 cells, but the cell viability of the concoction group was higher than that of the raw group. The liver tissues of rats in the control and processed product groups were normal, while those in the raw product groups showed different degrees of liver damage. Additionally, compared with the control group, the activity of alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine transaminase (ALT) in the raw product groups was notably increased in a dose-dependent manner; the three enzyme activities of the processed group were lower than those of the raw product group. In addition, the expression levels of CYP3A4, 2C19, 2C9, 1A2, and 2E1 in the liver tissue of rats in the processed group were lower than those in the raw group, while the protein expression trend of CYP2D6 was irregular. Conclusion: The detoxication mechanism of RWI, after undergoing processing, appears to be associated with reduced protein expression of CYP1A2, CYP2E1, CYP2C9, CYP2C19, and CYP3A4, ultimately leading to decreased liver injury.