N6-Methyladenosine Profiling Reveals Xuefu Zhuyu Decoction Upregulates METTL14 and BDNF in a Rat Model of Traumatic Brain Injury

Ethnopharmacological relevance: The traditional Chinese herbal formula Xuefu Zhuyu decoction (XFZYD) is a classic formula in the category of invigorating blood circulation and resolving blood stasis. It has been proven to improve the neurological and ethological prognosis of traumatic brain injury. XFZYD promotes synaptic and axonal regeneration after traumatic brain injury, which is functionally modulated by the N6-methyladenosine (m6A) modification of RNA. However, the epigenetic effects of XFZYD on m6A modification remain unknown. Aim of the study: To explore how XFZYD protected against traumatic brain injury induced by controlled cortical impact (CCI) injury by altering RNA m6A modification.Materials and methods: Different doses of XFZYD were administered to CCI model rats for 14 days. The modified neurological severity scoring and Morris water maze were performed to evaluate the neuroprotective effects of XFZYD and screen the dose. Next, Western blotting and methylated RNA immunoprecipitation sequencing (MeRIP-Seq) were used to explore changes in RNA m6A modification in the perilesional cortex. The Metascape platform is used to analyze the Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway of the differential m6A-tagged genes. Furthermore, MeRIP-qPCR was conducted to quantify differences in the hub differential m6A modification gene brain-derived neurotrophic factor (Bdnf).Results: XFZYD significantly ameliorated the neurological deficits, spatial learning and memory impairments in rats post-CCI on day 14. XFZYD enhanced the expression of METTL14 and YTHDC2 in the perilesional cortex of CCI rats. In all the three groups, the 3′-untranslated regions and coding sequence were primarily enriched for m6A peaks. XFZYD reversed the increased proportion of 3′-untranslated regions, and the decreased proportion of coding sequence and 5′-untranslated regions in CCI rats. Moreover, XFZYD markedly downregulated 41 elevated m6A-tagged transcripts and upregulated 119 decreased m6A-tagged transcripts induced by CCI. Gene ontology and KEGG pathway analysis revealed that XFZYD-regulated m6A-tagged transcripts are predominantly enriched in synapse assembly, synaptic plasticity, learning or memory and MAPK signaling pathway. Then, the hub regulated m6A-tagged gene BDNF was identified using bioinformatics analysis. Both the m6A methylation level and the protein level of BDNF were ascended by XFZYD treatment.Conclusion: XFZYD improves neurological deficits, spatial learning and memory impairments in rats post-TBI probably through increasing the expression of METTL14 and BDNF in the cortex. Our study highlights a novel post-transcriptional regulation mechanism mediated by herbal medicine for traumatic brain injury treatment.