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 kappa-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.