Xing-Pi-Qing-Gan decoction alleviates alcoholic liver disease by down-regulating DDIT3 and restoring Nrf2/HO-1 antioxidant signaling: Multi-omics and experimental evidence.

BACKGROUND: Alcoholic liver disease (ALD) is driven by oxidative stress, lipid metabolism, inflammation, and apoptosis. Current therapies lack efficacy in targeting multi-pathway mechanisms. Xing-Pi-Qing-Gan decoction (XPQG) is an improved traditional Chinese medicine designed to alleviate ALD, but its molecular mechanism remains unknown.

AIM: To illustrate the therapeutic targets and molecular pathways of XPQG for the treatment of ALD by integrating chemical profiling, network pharmacology, transcriptomics, and experimental verification in vivo and in vitro.

METHODS: The components of XPQG were analyzed using ultra-high performance liquid chromatography quadrupole-time-of-flight mass spectrometry. Then, the protective effect of XPQG on ethanol-induced liver injury, especially its regulatory effect on DDIT3 expression and associated Nrf2/HO-1 antioxidant signaling, was investigated through in vivo animal experiments and in vitro cell experiments. A mouse model of ALD was developed, and the mechanism of XPQG was validated through hematoxylin and eosin (H&E) staining, Western blot, and quantitative RT-PCR. In addition, the key role of DDIT3 in XPQG-mediated protection was further verified by siDDIT3 cell transfection technology. Animal experiments with the reactive oxygen species inhibitor N-acetylcysteine (NAC) further validated the mechanism of XPQG to alleviate liver injury by regulating oxidative stress.

RESULTS: In ethanol-treated HepG2 cells, XPQG dose-dependently reduced the formation of lipid droplets, inhibited the expression of tumor necrosis factor-α, interleukin-6, interleukin-1β, and alleviated oxidative stress. In mice, XPQG (15.2 g/kg) lowered the liver/body weight ratio, alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase; H&E and Oil Red O demonstrated a reduction in steatosis. Network pharmacology and RNA-seq converged on MAPK signaling, suggesting DDIT3 as a likely key effector in XPQG-mediated protection. DDIT3 knockdown in HepG2 cells attenuated the benefits of XPQG, supporting DDIT3 as a critical effector mechanism in XPQG-mediated protection. The use of NAC further illustrates the correlation of drugs to oxidative stress in disease effects.

CONCLUSION: In summary, the results of the study suggest that XPQG is effective in improving ethanol-induced acute liver injury (ALD). Its mechanism involves the suppression of DDIT3 and the enhancement of Nrf2/HO-1 pathway activity.