ncRNA network pharmacology integrated with miRNA omics and experimental validation reveal the anti-autophagic effect of Xuefu Zhuyu Decoction in traumatic brain injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Traumatic brain injury (TBI) is a major cause of death and long-term disability in humans. Xuefu Zhuyu Decoction (XFZYD) has potential therapeutic effects on TBI, but its molecular mechanism involving non-coding RNAs (ncRNAs) remains unclear.

AIM OF THE STUDY: To elucidate the mechanism of XFZYD, this study proposed an integrated ncRNA-based network pharmacology (ncRNP) strategy, combined with multi-omics analysis, in vivo and in vitro experiments. Unlike traditional network pharmacology which primarily focuses on protein-coding targets, the ncRNP strategy shifts the regulatory perspective to the transcriptional level, specifically targeting the herb-compound-ncRNA axis to bridge the gap between Traditional Chinese medicine (TCM) multi-component synergy and epigenetic regulation.

MATERIALS AND

METHODS

The ncRNP framework was established through four integrated modules: 1) construction of herbal-ncRNA regulatory networks; 2) high-throughput miRNA profiling of TBI mice; 3) multi-omics integration to identify core "herb-miRNA-pathway" nodes; and 4) experimental validation. Male C57BL/6J TBI mice were treated with different doses of XFZYD, and their neurological functions were examined. The ncRNP integrative strategy was established and used in XFZYD. Electron microscopy, western blotting, qRT-PCR, and immunofluorescence were further performed to confirm the anti-autophagy effects of XFZYD by miR-323-3p.

RESULTS

XFZYD treatment significantly improved neurological deficits and reduced neuronal loss and apoptosis. By implementing the ncRNP strategy which filters pharmacological targets through the lens of differential ncRNA expression, we identified a unique profile of 5 potential miRNAs. Through bioinformatics analysis and validation, miR-323-3p was selected as the key miRNA. We further confirmed that XFZYD normalized the pathological over-activation of autophagy after TBI by upregulating miR-323-3p, and subsequently suppressed the excessive activation of the AMPK/ULK1/Beclin-1 signaling axis. Overexpression of miR-323-3p directly inhibited the AMPK-dependent autophagy and restored cellular homeostasis.

CONCLUSIONS

XFZYD alleviates TBI-induced neuronal injury by rebalancing the miR-323-3p/AMPK-mediated autophagic flux. The established ncRNP strategy represents a methodological advancement over traditional network pharmacology by incorporating ncRNA-mediated epigenetic signatures, providing a more comprehensive and high-resolution framework for deciphering the complex pharmacological basis of TCM.