Nao Tan Qing exerts neuroprotection against traumatic brain injury via multi-targeted immunomodulation and neurorestoration

BACKGROUND: Traumatic brain injury (TBI) is a devastating neurological disorder with long-term functional deficits and limited effective therapies, where secondary injury driven by dysregulated immunity and disrupted signaling is pathogenic; Nao Tan Qing (NTQ), a Chinese herbal formula guided by the traditional principle of "resolving phlegm and inducing resuscitation" for "brain collateral obstruction", shows neuroprotective potential, but its role and mechanism in TBI treatment remain unclear. AIM: This study aimed to systematically investigate the neuroprotective effects of NTQ against TBI model mice and elucidate its underlying molecular mechanisms. METHODS: A controlled cortical impact (CCI) mouse model of TBI was established, and animals received NTQ treatment for 28 consecutive days. NTQ's neuroprotective efficacy was comprehensively evaluated via behavioral tests (functional recovery), cerebral blood flow imaging (vascular integrity), and electromyography (neural activity). Post-treatment inflammatory levels in TBI mice were assessed by quantifying inflammatory cytokine expression using quantitative real-time PCR and detecting microglial activation via immunofluorescence. Mechanistic exploration integrated network pharmacology, transcriptomics and bioinformatics analyses to identify NTQ's active components, potential targets, and associated pathways in TBI. RESULTS: In vivo experiments demonstrated that NTQ significantly improved behavioral outcomes, restored cerebral blood flow, and enhanced neural activity in TBI mice. Concurrent with these functional benefits, NTQ robustly suppressed neuroinflammation, as evidenced by reduced pro-inflammatory cytokine expression and attenuated microglial activation. Integrated network pharmacology and transcriptomic analyses confirmed that NTQ acts primarily through immune regulation after TBI, modifying key immune-related molecules and pathways; Specifically, NTQ intervention elicited pronounced downregulation of immune-inflammatory mediators, including Cd3g, Cd5, Cd8a, Epcam, Slamf7, Il16, Il17r, Il18rap, Cxcl9, Cxcr6, Tnfsf11, and Tnfsf15. Further mechanistic dissection identified six putative bioactive constituents of NTQ, including nicotinamide, curcumin, baicalin, chrysin, daidzein, and apigenin, which may remodel the intracerebral immune microenvironment after TBI through three core pathways: amine ligand-binding receptors, nuclear receptor meta-pathways, and arachidonic acid metabolism. CONCLUSION: Taken together, our integrated analyses demonstrate that NTQ exerts neuroprotective effects in TBI by modulating immune responses and suppressing neuroinflammation, thereby establishing NTQ as a promising multi-target therapeutic agent for TBI.