Gan-Jiang-Ling-Zhu decoction improves steatohepatitis induced by choline-deficient-high-fat-diet through the METTL14/N6-methyladenosine-mediated Ugt2a3 expression.

Abstract

Ethnopharmacological relevance: Gan-Jiang-Ling-Zhu (GJLZ) decoction, a classical Chinese herbal prescription, can be applied for the treatment of metabolic diseases including liver steatosis. Although GJLZ decoction has been widely applied clinically for thousands of years, the mechanism of GJLZ decoction behind treatment of nonalcoholic steatohepatitis (NASH) remains relatively unelucidated.

Aim of the study: To elucidate the efficacy of GJLZ decoction in the treatment of NASH and to investigate its underlying mechanisms from an epigenetic perspective.

Materials and methods: The quality control of chemical components in GJLZ decoction was conducted. C57BL/6J mice with NASH were induced by feeding them a choline-deficient-high-fat-diet (CDHFD), along with GJLZ decoction intervention for 4 weeks. Then NASH phenotypes including histological steatosis, inflammation, hepatic apoptosis, fibrosis, serum liver enzyme and lipid level were measured. N6-methyladenosine (m⁶A) and transcriptome sequencing were performed. Levels and functions of methyltransferases and different genes were performed by quantitative polymerase chain reaction, immunofluorescence, gene knockdown, oil red O staining and western blotting.

Results: GJLZ decoction significantly reduced liver weight, liver index and improved hepatic steatosis, and inflammation, as well as inhibited hepatic apoptosis and fibrosis. Moreover, GJLZ decoction significantly reduced the levels of lactate dehydrogenase, aminotransferase, triglyceride, aspartate aminotransferase, and inhibited levels of interleukin 6 and tumor necrosis factor α. Transcriptome and m⁶A sequencing revealed the landscape of transcriptome and m⁶A modification influenced by NASH and the following GJLZ decoction intervention. Eleven differential genes were identified, and GJLZ markedly promoted m⁶A level of UDP glucuronosyltransferase family 2 member A3 (Ugt2a3), to promote its expression. Additionally, GJLZ significantly promoted methyltransferase 14 (METTL14) expression, whereas METTL14 knockdown aggravated hepatocellular steatosis. Finally, METTL14 knockdown significantly reduced the level of Ugt2a3 by promoting its degradation, whereas, Ugt2a3 overexpression could markedly inhibit hepatocellular steatosis.

Conclusions: GJLZ decoction demonstrates potential in alleviating CDHFD-induced NASH by modulating the METTL14-m⁶A-Ugt2a3 axis, offering a novel therapeutic approach for NASH treatment.