Silybin regulates P450s activity by attenuating endoplasmic reticulum stress in mouse nonalcoholic fatty liver disease.

Cytochrome P450s are important phase I metabolic enzymes located on endoplasmic reticulum (ER) involved in the metabolism of endogenous and exogenous substances. Our previous study showed that a hepatoprotective agent silybin restored CYP3A expression in mouse nonalcoholic fatty liver disease (NAFLD). In this study we investigated how silybin regulated P450s activity during NAFLD. C57BL/6 mice were fed a high-fat-diet (HFD) for 8 weeks to induce NAFLD, and were administered silybin (50, 100 mg ·kg-1 ·d-1, i.g.) in the last 4 weeks. We showed that HFD intake induced hepatic steatosis and ER stress, leading to significant inhibition on the activity of five primary P450s including CYP1A2, CYP2B6, CYP2C19, CYP2D6, and CYP3A in liver microsomes. These changes were dose-dependently reversed by silybin administration. The beneficial effects of silybin were also observed in TG-stimulated HepG2 cells in vitro. To clarify the underlying mechanism, we examined the components involved in the P450 catalytic system, membrane phospholipids and ER membrane fluidity, and found that cytochrome b5 (cyt b5) was significantly downregulated during ER stress, and ER membrane fluidity was also reduced evidenced by DPH polarization and lower polyunsaturated phospholipids levels. The increased ratios of NADP+/NADPH and PC/PE implied Ca2+ release and disruption of cellular Ca2+ homeostasis resulted from mitochondria dysfunction and cytochrome c (cyt c) release. The interaction between cyt c and cyt b5 under ER stress was an important reason for P450s activity inhibition. The effect of silybin throughout the whole course suggested that it regulated P450s activity through its anti-ER stress effect in NAFLD. Our results suggest that ER stress may be crucial for the inhibition of P450s activity in mouse NAFLD and silybin regulates P450s activity by attenuating ER stress.

A quantitative metabolomics assay targeting 14 intracellular metabolites associated with the methionine transsulfuration pathway using LC-MS/MS in breast cancer cells.

The methionine transsulfuration pathway plays an important role in some fundamental biological processes, such as redox and methylation reactions. However, quantitative analysis of the majority of intracellular metabolites is rather challenging. In this study, we developed a simple, fast and reliable method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous detection of 14 methionine-related metabolites, including methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), homocysteine (HCY), cystathionine (Cysta), cysteine (CYS), glutathione (GSH), dimethylglycine (DMG), betaine, serine, folic acid (FA), dihydrofolic acid (DHF), tetrahydrofolic acid (THF) and 5-methyltetrahydrofolic acid (5-MTHF), in MCF-7 and MDA-MB-231 breast cancer cells. By taking advantage of a surrogate matrix, the linearity, sensitivity, precision, accuracy, stability, matrix effect, recovery, dilution integrity and carryover of the established method were evaluated and validated. This method enabled the precise measurement of methionine-related metabolites both in cells and in the medium and was successfully applied to profile these metabolites involved in the methionine transsulfuration pathway. The data showed that cystine deprivation or excessive supplementation with cystine had a marked impact on methionine metabolism, in addition to its effects on intracellular CYS and GSH levels, indicating that the methionine transsulfuration pathway was dependent on intracellular cystine levels. The established method provides a reliable way to target metabolomics for the quantitative determination of intracellular metabolites in the methionine transsulfuration pathway, which can greatly facilitate the understanding of the mechanisms involved in methylation and redox homeostasis in cellular metabolomic studies.

Silybin Restored CYP3A Expression through the Sirtuin 2/Nuclear Factor κ-B Pathway in Mouse Nonalcoholic Fatty Liver Disease.

Silybin is widely used as a hepatoprotective agent in various liver disease therapies and has been previously identified as a CYP3A inhibitor. However, little is known about the effect of silybin on CYP3A and the regulatory mechanism during high-fat-diet (HFD)-induced liver inflammation. In our study, we found that silybin restored CYP3A expression and activity that were decreased by HFD and conditioned medium (CM) from palmitate-treated Kupffer cells. Moreover, silybin suppressed liver inflammation in HFD-fed mice and inhibited nuclear factor κ-B translocation into the nucleus through elevation of SIRT2 expression and promotion of p65 deacetylation. This effect was confirmed by overexpression of SIRT2, which suppressed p65 nuclear translocation and restored CYP3A transcription affected by CM. The hepatic NAD+ concentration markedly decreased in HFD-fed mice and CM-treated hepatocytes/HepG2 cells but increased after silybin treatment. Supplementing nicotinamide mononucleotide as an NAD+ donor inhibited p65 acetylation, decreased p65 nuclear translocation, and restored cyp3a transcription in both HepG2 cells and mouse hepatocytes. These results suggest that silybin regulates metabolic enzymes during liver inflammation by a mechanism related to the increase in NAD+ and SIRT2 levels. In addition, silybin enhanced the intracellular NAD+ concentration by decreasing poly-ADP ribosyl polymerase-1 expression. In summary, silybin increased NAD+ concentration, promoted SIRT2 expression, and lowered p65 acetylation both in vivo and in vitro, which supported the recovery of CYP3A expression. These findings indicate that the NAD+/SIRT2 pathway plays an important role in CYP3A regulation during nonalcoholic fatty liver disease. SIGNIFICANCE STATEMENT: This research revealed the differential regulation of CYP3A by silybin under physiological and fatty liver pathological conditions. In the treatment of nonalcoholic fatty liver disease, silybin restored, not inhibited, CYP3A expression and activity through the NAD+/ sirtuin 2 pathway in accordance with its anti-inflammatory effect.