lncRNAs Transcriptomics Elucidates the Potential Mechanism of Naoshuantong Capsule in Alleviating Synaptic Dysfunction in a murine model of Cerebral Ischemia/Reperfusion Injury

Abstract

Background: Naoshuantong capsule (NST), a Traditional Chinese Medicine formulation, is used for ischemic stroke treatment; however, its molecular mechanisms are unclear. This study aimed to investigate the mechanistic basis of NST using lncRNA and mRNA transcriptomics. Methods: The metabolites of NST were analyzed. Additionally, its systemically absorbed metabolites (in plasma) and brain-distributed metabolites were identified using UHPLC-MS/MS. The therapeutic effects of NST were evaluated in a mouse model of MCAO using neurological scoring, behavioral testing, cerebral blood flow, and brain tissue staining. LncRNA and mRNA expression profiles were analyzed using the Agilent Mouse competing endogenous RNA microarray, followed by GO and KEGG enrichment analyses. Differentially expressed transcripts were validated using qRT-PCR. Results: UHPLC-MS/MS analysis detected 129 metabolites in NST; 33 metabolites in plasma; and 17 metabolites in brain tissue of rats administered with NST. NST treatment significantly reduced neurological deficit scores (Longa score), decreased beam-crossing latency, and increased forelimb grip strength in middle MCAO mice, indicating improved neurological function. Additionally, NST treatment enhanced cerebral blood flow recovery, ameliorated pathological damage, restored neuronal architecture, and increased Nissl-stained neuron density in peri-infarct brain tissue. NST also attenuated cellular apoptosis by upregulating Bcl-2 expression and downregulating Bax protein levels, exerting neuroprotective effects. Notably, NST treatment reversed 177 out of 5,378 differentially expressed lncRNAs and 52 out of 5,540 differentially expressed mRNAs that were dysregulated between the model and sham groups. These NST-modulated lncRNAs participate in key biological processes, including synaptic modulation, apoptosis regulation, and neuronal function. A synaptic plasticity-associated lncRNA-mRNA coexpression network was developed using NST-reversed transcripts. Validation using qRT-PCR confirmed the upregulation of NONMMUT050688.2 and NONMMUT044667.2, and the downregulation of NONMMUT092269.1 and NONMMUT101071.1, the downregulation of Nrn1, the upregulation of Grn, and the downward trend in Rasd2 expression in MCAO mice. All these alterations were reversed through NST treatment. In vivo experiments confirmed the efficacy of NST in ameliorating memory deficits, mitigating synaptic structural damage, and upregulating key synaptic protein expression (SYN and PSD95) in mice. Conclusion: NST may protect against cerebral ischemia/reperfusion injury by modulating lncRNA and mRNA expressions to enhance synaptic plasticity, thereby preserving neuronal structure and function.