Synbiotics Alleviate Hepatic Damage, Intestinal Injury and Muscular Beclin-1 Elevation in Rats after Chronic Ethanol Administration

The purpose of this study was to investigate the beneficial effects of synbiotics on liver damage, intestinal health, and muscle loss, and their relevance in rats with chronic ethanol feeding. Thirty Wistar rats fed with a control liquid diet were divided into control and synbiotics groups, which were respectively provided with water or synbiotics solution (1.5 g/kg body weight/day) for 2 weeks. From the 3rd to 8th week, the control group was divided into a C group (control liquid diet + water) and an E group (ethanol liquid diet + water). The synbiotics group was separated in to three groups, SC, ASE, and PSE. The SC group was given a control liquid diet with synbiotics solution; the ASE group was given ethanol liquid diet with synbiotics solution, and the PSE group was given ethanol liquid diet and water. As the results, the E group exhibited liver damage, including increased AST and ALT activities, hepatic fatty changes, and higher CYP2E1 expression. Intestinal mRNA expressions of occludin and claudin-1 were significantly decreased and the plasma endotoxin level was significantly higher in the E group. In muscles, beclin-1 was significantly increased in the E group. Compared to the E group, the PSE and ASE groups had lower plasma ALT activities, hepatic fatty changes, and CYP2E1 expression. The PSE and ASE groups had significantly higher intestinal occludin and claudin-1 mRNA expressions and lower muscular beclin-1 expression when compared to the E group. In conclusion, synbiotics supplementation might reduce protein expression of muscle protein degradation biomarkers such as beclin-1 in rats with chronic ethanol feeding, which is speculated to be linked to the improvement of intestinal tight junction and the reduction of liver damage.

Drug Repurposing in Alternative Medicine: Sochehwan, a Polyherbal Traditional Korean Digestant, Protects against Alcoholic Steatohepatitis by Regulating Cytochrome P450 2E1 Expression

Sochehwan (SCH) is an herbal prescription from traditional oriental medicine and is currently used to treat digestive ailments. In a previous study, SCH was found to have the potential to attenuate metabolic syndrome (MetS) by activating AMPK and downstream signaling. From the view of drug repurposing, the efficacy of SCH on alcoholic liver injury is implied in classic medical texts but is yet to be proven. C57BL/6J mice were pre-treated with SCH orally for 5 days and challenged by providing a pair-fed Lieber DeCarli diet containing alcohol for 20 days. Hepatic enzyme and triglyceride levels and endoplasmic reticulum (ER) stress-related markers were analyzed. Moreover, mitogen-activated protein kinases (MAPKs) and cytochrome P450 2E1 (CYP2E1) levels were determined. CYP2E1-transfected HepG2 cells were used to test the cytoprotective efficacy of SCH against the adverse effects of alcohol in vitro. In mice, SCH administration notably reduced hepatic enzyme activity and neural lipid levels. Furthermore, ER-stress markers and MAPK phosphorylation were reduced due to ROS suppression, which was attributed to decreased CYP2E1 expression in liver tissue. In addition, SCH successfully protected CYP2E1-transfected HepG2 cells against ethanol. Our findings suggest SCH attenuated alcohol-induced liver injury by inhibiting CYP2E1 expression and indicate drug repurposing should be considered as a valuable option for drug development in traditional herbal medicines.

Inhibition of androgen/AR signaling inhibits diethylnitrosamine (DEN) induced tumour initiation and remodels liver immune cell networks

A promotional role for androgen receptor (AR) signaling in hepatocellular carcinogenesis is emerging. In pre-clinical models, including diethylnitrosamine- (DEN-) induced hepatocellular carcinoma (HCC), anti-androgen therapies delay hepatocarcinogenesis. However, pharmacologic anti-androgen therapy in advanced HCC patients fails, suggesting that AR plays a role in HCC onset. This study aims to characterize AR expression and function throughout DEN-induced liver inflammation and carcinogenesis and evaluate the efficacy of prophylactic AR antagonism to prevent hepatocarcinogenesis. We demonstrate that pharmacologic AR antagonism with enzalutamide inhibits hepatocellular carcinogenesis. With enzalutamide treatment, we observe decreased CYP2E1 expression, reducing DEN-induced hepatocyte death and DNA ethyl-adducts. AR protein expression analyses show that DEN causes an initial upregulation of AR in portal fibroblasts and leukocytes, but not hepatocytes, suggesting that hepatocyte-autonomous AR signaling is not essential for DEN-induced carcinogenesis. Ablating androgen signaling by surgical castration reduced pre-carcinogen Kupffer cell populations but did not alter DEN-mediated immune cell recruitment nor AR expression. In this study, we identified that anti-androgen interventions modulate mutagenic DNA adducts, tumour initiation, and immune cell composition. Additionally, we find that AR expression in hepatocytes is not present during nor required for early DEN-mediated carcinogenesis.

Rutaecarpine Protects against Acetaminophen-Induced Acute Liver Injury in Mice by Activating Antioxidant Enzymes

Rutaecarpine, an indolopyridoquinazolinone alkaloid isolated from the unripe fruit of Evodia rutaecarpa, is used to treat hypertension, postpartum hemorrhage, dysentery, and amenorrhea as a traditional medicine in Asia. We investigated the effect of rutaecarpine on acetaminophen-induced hepatotoxicity in mice. Rutaecarpine was administered orally daily for seven consecutive days, followed by intraperitoneal injection of acetaminophen in mice on day seven to induce hepatotoxicity. Rutaecarpine pretreatment significantly decreased acetaminophen-induced serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) activities and hepatic malondialdehyde content and prevented acetaminophen-induced hepatic glutathione depletion. Furthermore, CYP2E1 expression was decreased by rutaecarpine pretreatment in a dose-dependent manner. Rutaecarpine pretreatment inhibited acetaminophen-induced expression of inflammatory cytokines by inhibiting NF-κB activation by JNK1/2. Also, rutaecarpine pretreatment promoted Nrf2-mediated activation of the antioxidant enzymes GCLC, HO-1, and NQO1. This indicates that the protective effect of rutaecarpine during acetaminophen-induced acute liver injury is mediated by the activation of antioxidant enzymes. Therefore, rutaecarpine has a protective effect of APAP-induced liver damage.

Scutellarein Aggravated Carbon Tetrachloride-Induced Chronic Liver Injury in Gut Microbiota-Dysbiosis Mice

Scutellarein (SCU) is an herbal flavonoid, showing hepatoprotective potentials. The study was aimed to investigate whether the hepatoprotective effect of SCU is dependent on the integrity of gut microbiota. Mice received repeated intraperitoneal injections of CCl4, followed with or without SCU treatment (15, 30, and 60 mg/kg). Gut microbial community of mice was disrupted by administrating a cocktail of antibiotics (ampicillin, neomycin sulfate, metronidazole, and vancomycin) in drinking water. The results showed SCU plus antibiotics aggravated CCl4-induced chronic liver injury, as demonstrated by liver function analysis, histological analysis, and TUNEL assay. SCU activated CYP2E1 expression and worsened CYP2E1-mediated lipid peroxidation and oxidative stress as coadministered with antibiotics. Moreover, when gut microbiota was disrupted by antibiotics, SCU activated IκBα/NF-κB pathway and promoted the release of subsequent proinflammatory cytokines including interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). Remarkably, the 16 S rRNA sequencing demonstrated that SCU greatly decreased the relative abundance of Bifidobacterium and Lactobacillus and increased the relative abundance of Enterococcus in gut microbiota-dysbiosis mice. Spearman correlation analysis showed that Lactobacillus was positively correlated with SOD and negatively correlated with AST. Collectively, the hepatoprotective effect of SCU is reversed under antibiotics intervention, which may partly involve the activation of CYP2E1 and IκBα/NF-κB pathway and diminishment of Lactobacillus.

Ethanol-induced CYP2E1 Expression is Reduced by Lauric Acid via PI3K Pathway in HepG2 Cells

The metabolism of alcohol involves cytochrome P450 2E1 (CYP2E1)-induced oxidative stress, with the association of phosphatidylinositol-3-kinases (PI3K) and nuclear factor kappa B (NFκB) signalling pathways. CYP2E1 is primarily involved in the microsomal ethanol oxidising system, which generates massive reactive oxygen species (ROS) and ultimately leads to oxidative stress and tissue damage. Lauric acid, a major fatty acid in palm kernel oil, has been shown as a potential antioxidant. Here, we aimed to evaluate the use of lauric acid as a potential antioxidant against ethanol-mediated oxidative stress by investigating its effect on CYP2E1 mRNA expression and the signalling pathway in ethanol-induced HepG2 cells. HepG2 cells were firstly treated with different concentrations of ethanol, and subsequently co-treated with different concentrations of lauric acid for 24 h. Total cellular RNA and total protein were extracted, and qPCR and Western blot was carried out. Ethanol induced the mRNA expression of CYP2E1 significantly, but lauric acid was able to downregulate the induced CYP2E1 expression in a dose-dependent manner. Similarly, Western blot analysis and densitometry analysis showed that the phosphorylated PI3K p85 (Tyr458) protein was significantly elevated in ethanol-treated HepG2 cells, but co-treatment with lauric acid repressed the activation of PI3K. However, there was no significant difference in NFκB pathway, in which the normalised NFκB p105 (Ser933) phosphorylation remained constant in any treatment conditions in this study. This suggests that ethanol induced CYP2E1 expression by activating PI3K p85 (Tyr458) pathway, but not the NFκB p105 (Ser933) pathway in HepG2 cells.

Investigation on Intestinal Proteins and Drug Metabolizing Enzymes in Simulated Microgravity Rats by a Proteomics Method

The present study aimed to investigate the change of intestinal mucosa proteins, especially the alteration of intestinal drug metabolizing enzymes (IDMEs) following 14-day simulated microgravity. Morey–Holton tail-suspension analog was used to simulate microgravity. Intestinal mucosa proteins of rats were determined by label-free quantitative proteomic strategy. A total of 335 differentially expressed proteins (DEPs) were identified, 190 DEPs were upregulated, and 145 DEPs were downregulated. According to bioinformatic analysis, most of DEPs exhibited hydrolase, oxidoreductase, transferase, ligase, or lyase catalytic activity. DEPs were mainly enriched in metabolic pathways, including metabolism of amino acid, glucose, and carbon. Moreover, 11 of DEPs were involved in exogenous drug and xenobiotics metabolism. Owing to the importance of IDMEs for the efficacy and safety of oral drugs, the expression of cytochrome P450 1A2 (CYP1A2), CYP2D1, CYP3A2, CYP2E1, alcohol dehydrogenase 1 (ADH1), and glutathione S-transferase mu 5 (GSTM5) in rat intestine mucosa was determined by Western-blot. The activity of ADH, aldehyde dehydrogenase (ALDH) and GST was evaluated. Compared with control rats, the expression of CYP1A2, CYP2D1, CYP3A2, and ADH1 in the simulated microgravity (SMG) group of rats were dramatically decreased by 33.16%, 21.93%, 48.49%, and 22.83%, respectively. GSTM5 was significantly upregulated by 53.14% and CYP2E1 expression did not show a dramatical change in SMG group rats. Moreover, 14-day SMG reduced ADH activity, while ALDH and GST activities was not altered remarkably. It could be concluded that SMG dramatically affected the expression and activity of some IDMEs, which might alter the efficacy or safety of their substrate drugs under microgravity. The present study provided some preliminary information on IDMEs under microgravity. It revealed the potential effect of SMG on intestinal metabolism, which may be helpful to understand the intestinal health of astronauts and medication use.

Synonymous mutation rs2515641 affects CYP2E1 mRNA and protein expression and susceptibility to drug-induced liver injury

Aim: To evaluate whether the synonymous mutant rs2515641 could affect cytochrome P450 2E1 ( CYP2E1) expression and the response to acetaminophen (APAP) or triptolide (TP) treatment. Materials & methods: HepG2 cells were transfected with lentiviral vector containing either CYP2E1-1263C or CYP2E1-1263T. Some of these recombinant cells were then treated with APAP or TP. CYP2E1 gene expression was detected by PCR and western blot. Results: CYP2E1 gene expression decreased significantly both in mRNA and protein level after rs2515641 mutation, indicating that this polymorphism can affect both transcription and translation. Furthermore, rs2515641 mutation dramatically changes the response of CYP2E1 expression to APAP or TP treatment. Conclusion: Rs2515641 significantly changes CYP2E1 expression and function, which would be expected to affect drug disposition and response.