ABSTRACT
The extent of immune-mediated hepatic damage (such as in viral hepatitis) is characterised by the downregulation of cytochrome P450s (CYPs), a class of drug-metabolising enzymes. However, whether this downregulation aids liver cells in maintaining their homeostasis or whether the damage is aggravated remains largely unexplored. Herein, we evaluated the effects of phosphorylation mediated by the protein kinase C (PKC)/cAMP-response element binding protein (CREB) and nitration mediated by inducible nitric oxide synthase (iNOS) on the downregulation of CYP2E1 during immune-mediated liver injury. Additionally, we investigated the regulatory mechanism mediated by the nuclear factor κB (NF-κB). The rat model of immune-mediated liver injury was replicated by administering a single i.v. injection of Bacillus Calmette-Guerin (BCG, 125 mg/kg) vaccine and three i.p. injections of ammonium pyrrolidine dithiocarbamate (25, 50, 100 mg/kg/d, days 11, 12, and 13); blood was then collected on day 14. Subsequently, the livers were extracted to identify the different pharmacokinetic and biochemical indicators involved in the process. Our study reports new findings on the dependence between PKC-mediated CREB phosphorylation in the anti-inflammatory pathway and nitration emergency induced by iNOS in pro-inflammatory pathways in the NF-κB pathway. The interaction of these two pathways leads to the downregulation and recovery of CYP2E1, thus alleviating inflammation and nitration stress. Our results confirm that BCG-mediated downregulation of CYP2E1 is linked to iNOS-induced nitration and PKC/NF-κB-mediated CREB phosphorylation, and that NF-κB is an important molecular target in this process. These findings suggest that the downregulation of CYP2E1 may be an autonomous process characteristic of liver cells, helping them adapt to environmental changes, alleviate further hypoxia in inflamed tissues, and minimise exposure to toxic and harmful metabolites.
ABSTRACT
In this study, we investigated the effect of Hippophae rhamnoides L. (HRP) on the activity of CYP2D6 via the CAMP/PKA/NF-κB pathway in rats with Bacille Calmette-Guerin (BCG)-induced immunological liver injury. BCG (125 mg/kg) was injected to establish the rat model of liver injury. HRP was administered intragastrically for one week as the intervention drug. Proteomics techniques were used to analyze protein expression levels, obtaining a comprehensive understanding of the liver injury process. ELISA or western blotting was used to detect specific protein levels. Dextromethorphan was detected using high-performance liquid chromatography to reflect the metabolic activity of CYP2D6. BCG downregulated the expression of CYP2D6, cAMP, PKA, IκB, and P-CREB and upregulated that of NF-κB, IL-1ß, TNF-α, and CREB in the liver; HRP administration reversed these effects. Therefore, HRP may restore the metabolic function of the liver by reversing the downregulation of CYP2D6 through inhibition of NF-κB signal transduction and regulation of the cAMP/PKA/CREB/CYP2D6 pathway. These findings highlight the role of HRP as an alternative clinical drug for treating hepatitis B and other immune-related liver diseases.
Subject(s)
Chemical and Drug Induced Liver Injury, Chronic , Hippophae , Rats , Animals , NF-kappa B/metabolism , Hippophae/metabolism , Cytochrome P-450 CYP2D6ABSTRACT
Background: The use of police breath alcohol detectors in rat breath alcohol detection experiments has always been a challenge because of the small lung capacity and inability of rats to actively inhale. However, the method of using gas chromatography to detect blood alcohol concentration is time-consuming, complex, relatively expensive, and cannot achieve on-site detection and multi-point unlimited non-invasive detection. Objectives: In this study, a laboratory method was validated for rat breath ethanol concentration (BrAC) measurement to estimate blood ethanol concentration (BAC) in rats. Methods: The rats were placed in a gas collection bottle, the breath sample was drawn out with a syringe, and injected into the mouthpiece of the breath alcohol detector through a rubber tube. The results were immediately detected and automatically converted to BAC. Male rats were randomly divided into three groups. The control group received an intraperitoneal injection of normal saline, the liver injury group received an intraperitoneal injection of 50% Carbon tetrachloride (CCL4 1 mL.kg-1), and the induction group received an intraperitoneal injection of phenobarbital sodium (75 mg.kg-1). Western blot analysis was used to detect the protein expression of CYP2E1. Similar grouping and experimental methods were used for female rats. Results: This method was reproducible. The metabolic activity of CYP2E1 was downregulated in the injury group and upregulated in the induction group, which was consistent with the results obtained for CYP2E1 protein expression. Conclusions: Our results confirmed that the rat gas cylinder breath alcohol assay can be used for multiple detections with immediate and non-invasive determination of alcohol metabolizing capacity. This is important for studies that require repeated assessment of blood alcohol levels.
ABSTRACT
BACKGROUND: Accumulating evidences indicate that periodontitis is closely associated with endothelial dysfunction. Trimethylamine-N-oxide (TMAO), a harmful microbiota generated metabolite, has been implicated as a nontraditional risk factor for impaired endothelial function. However, whether increased circulating levels of TMAO in periodontitis patients induces endothelial dysfunction remains unknown. METHODS: Patients with periodontitis and periodontally healthy controls were enrolled. Periodontal inflamed surface area (PISA) was calculated to assess the inflammatory burden posed by periodontitis. The circulating TMAO was measured by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Vascular endothelial function including peripheral endothelial progenitor cells (EPCs), brachial arterial flow-mediated vasodilation (FMD), and brachial-ankle pulse wave velocity (baPWV) were assessed. We also isolated and cultured EPCs from participants' peripheral blood to investigate the effect of TMAO on EPC functions in vitro. RESULTS: One hundred and twenty two patients with Stage III-IV periodontitis and 81 healthy controls were included. Patients with periodontitis presented elevated TMAO (P = 0.002), lower EPCs (P = 0.025), and declined FMD levels (P = 0.005). The TMAO concentrations were correlated with reduced circulating EPCs and FMD levels. Moreover, TMAO can injury EPCs function in vitro, and may induce cell pyroptosis via Bax/caspase-3/GSDME pathway. CONCLUSIONS: The present study demonstrates for the first time that circulating TMAO levels are increased in patients with Stage III-IV periodontitis, and correlated with vascular endothelial dysfunction. These findings may provide a novel insight into the mechanism of vascular endothelial dysfunction in patient with periodontitis via TMAO-downregulated EPC functions.
