Dragon’s blood (DB) has shown a protective effect on neurological diseases. Microgravity (MG) or simulated MG (SMG) can induce blood–brain barrier (BBB) dysfunction, which is a characteristic feature of neurological disorders. This study’s purpose was to evaluate the effect of DB on SMG-induced BBB dysfunction and explore its signaling pathway. Both DB and vitamin C (Vc) were administered orally for tail-suspended rats within 3 weeks. DB and Vc solutions were added to human brain microvascular endothelial cells (HCMEC/D3) cells, which were then exposed to SMG for 24 h. The protective effect of DB was assessed by hematoxylin and eosin and Nissl staining, ultrastructure observation, and permeability in rats. Cell apoptosis and the distribution of tight junction (TJ) and adherens junction (AJ) proteins and filamentous actin (F-actin) were examined in HCMEC/D3. The oxidative stress and inflammation, and TJ and AJ protein expressions were determined in rat brain and HCMEC/D3. The focal adhesion kinase (FAK) signaling pathway proteins were determined. DB protected SMG-induced rat BBB disruption by improving neuronal apoptosis, repairing widened intercellular space, and decreasing BBB permeability. DB effectively relieved SMG-induced HCMEC/D3 damage by inhibiting cell apoptosis and restoring F-actin spindle distribution. High doses of DB upregulated TJ and AJ protein expressions and decreased oxidative stress and proinflammatory cytokine levels in rat brain and HCMEC/D3. DB enhanced the expressions of FAK signal transduction proteins and F-actin/globular actin (G-actin) ratio in rat brain and HCMEC/D3, suggesting that DB promotes actin cytoskeleton polymerization, benefits the endothelial cell–cell and cell–extracellular matrix adhesion, and, in consequence, contributes to BBB integrity.

P-glycoprotein (P-gp) could maintain stability of the nerve system by effluxing toxins out of the blood-brain barrier. Whether it plays a very important role in drug brain distribution during space travel is not yet known. The present study was aimed at investigating P-gp function, expression, and its interacting proteins in a rat brain under simulated microgravity (SMG) by comparative proteomics approach. Rats were tail-suspended to induce short- (7-day) and long-term (21-day) microgravity. P-gp function was assessed by measuring the P-gp ATPase activity and the brain-to-plasma concentration ratio of rhodamine 123. P-gp expression was evaluated by Western blot. 21d-SMG significantly enhanced P-gp efflux activity and expression in rats. Label-free proteomics strategy identified 26 common differentially expressed proteins (DEPs) interacting with P-gp in 7d- and 21d-SMG groups. Most of the DEPs mainly regulated ATP hydrolysis coupled transmembrane transport and so on. Interaction analysis showed that P-gp might potentially interact with heat shock proteins, sodium/potassium ATP enzyme, ATP synthase, microtubule-associated proteins, and vesicle fusion ATPase. The present study firstly reported P-gp function, expression, and its potentially interacting proteins exposed to simulated microgravity. These findings might be helpful not only for further study on nerve system stability but also for the safe and effective use of P-gp substrate drugs during space travel.