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

Abstract

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.

Keywords: Alcoholic liver disease; Xing-Pi-Qing-Gan decoction; DDIT3; Nrf2/HO-1; Oxidative stress

Core Tip: Xing-Pi-Qing-Gan decoction (XPQG) is effective in improving ethanol-induced acute liver injury. XPQG alleviates alcoholic liver disease by suppressing DDIT3 and enhancing Nrf2/HO-1-mediated antioxidant responses, reduce lipid accumulation, inflammation, and apoptosis, as validated through multi-omics and experimental studies.