Xuefu Zhuyu decoction promotes synaptic plasticity by targeting miR-191a-5p/BDNF-TrkB axis in severe traumatic brain injury.

BACKGROUND: Xuefu Zhuyu decoction (XFZYD) is a traditional Chinese herbal formula known for its ability to eliminate blood stasis and improve blood circulation, providing neuroprotection against severe traumatic brain injury (sTBI). However, the underlying mechanism is still unclear. PURPOSE: We aim to investigate the neuroprotective effects of XFZYD in sTBI from a novel mechanistic perspective of miRNA-mRNA. Additionally, we sought to elucidate a potential specific mechanism by integrating transcriptomics, bioinformatics, and conducting both in vitro and in vivo experiments. METHODS: The sTBI rat model was established, and the rats were treated with XFZYD for 14 days. The neuroprotective effects of XFZYD were evaluated using a modified neurological severity score, hematoxylin and eosin staining, as well as Nissl staining. The anti-inflammatory effects of XFZYD were explored using quantitative real-time PCR (qRT-PCR), Western blot analysis, and immunofluorescence. Next, miRNA sequencing of the hippocampus was performed to determine which miRNAs were differentially expressed. Subsequently, qRT-PCR was used to validate the differentially expressed miRNAs. Target core mRNAs were determined using various methods, including miRNA prediction targets, mRNA sequencing, miRNA-mRNA network, and protein-protein interaction (PPI) analysis. The miRNA/mRNA regulatory axis were verified through qRT-PCR or Western blot analysis. Finally, morphological changes in the neural synapses were observed using transmission electron microscopy and immunofluorescence. RESULTS: XFZYD exhibited significant neuroprotective and anti-inflammatory effects on subacute sTBI rats' hippocampus. The analyses of miRNA/mRNA sequences combined with the PPI network revealed that the therapeutic effects of XFZYD on sTBI were associated with the regulation of the rno-miR-191a-5p/BDNF axis. Subsequently, qRT-PCR and Western blot analysis confirmed XFZYD reversed the decrease of BDNF and TrkB in the hippocampus caused by sTBI. Additionally, XFZYD treatment potentially increased the number of synaptic connections, and the expression of the synapse-related protein PSD95, axon-related protein GAP43 and neuron-specific protein TUBB3. CONCLUSIONS: XFZYD exerts neuroprotective effects by promoting hippocampal synaptic remodeling and improving cognition during the subacute phase of sTBI through downregulating of rno-miR-191a-5p/BDNF axis, further activating BDNF-TrkB signaling.

N6-methyladenosine profiling reveals that 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 protects against traumatic brain injury induced by controlled cortical impact (CCI) injury by altering RNA m6A modification. MATERIALS AND METHODS: The modified neurological severity scoring and Morris water maze were performed to evaluate the neuroprotective effects of XFZYD for 14 days and screen the dose. Then, dot blot, 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 was 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 m6A level, and the expression of METTL14 and YTHDC2 in the perilesional cortex of CCI rats. In all 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 post-CCI. Moreover, XFZYD markedly downregulated 41 elevated m6A-tagged transcripts and upregulated 119 decreased m6A-tagged transcripts following CCI. Gene ontology and KEGG pathway analysis revealed that XFZYD-regulated m6A-tagged transcripts were predominantly enriched in synapse assembly, synaptic plasticity, learning or memory, and MAPK signaling pathway. Then, the hub-regulated m6A-tagged gene BDNF was identified. 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.