The blood-brain barrier (BBB), primarily composed of cerebral microvessel endothelial cells (CMECs), maintains CNS physiological function by preventing neurotoxic substances and pathogens from entering the brain. BBB breakdown contributes to various neurological diseases. Semen Ziziphi Spinosae (SZS), a functional food and traditional Chinese medicine, has shown anti-depressant, antioxidant, and anxiolytic effects, and its components, like spinosin, may regulate immune function and prevent Alzheimer's disease. However, its effect on BBB integrity remained unexplored. This study investigated the protective effect of SZS on LPS-induced BBB damage using histomorphology, permeability assays, and biochemical analyses, exploring the global proteomic profile and underlying mechanism using LC-MS analysis, western blot validation, and molecular docking experiments. The aim was to elucidate SZS's mechanism in preventing BBB breakdown and explore its potential as a neuroprotective functional food.

Extensive research supports the neuroprotective properties of various compounds and approaches. Studies have highlighted the role of BBB integrity in preventing neurodegenerative diseases, emphasizing the importance of maintaining its function. The use of mass spectrometry-based proteomics has become crucial in understanding the biological processes at the protein level, providing valuable insights into disease mechanisms and potential treatments. Lipopolysaccharide (LPS) is frequently used in in vivo and in vitro models to induce BBB damage due to its ability to cause neuroinflammation and oxidative stress. Existing literature shows that oxidative stress and neuroinflammation can damage BBB integrity through mechanisms such as degrading tight junction proteins and modulating pathways like Nrf2 and NF-κB. Alterations in cellular matrix adhesion also contribute to BBB permeability. However, prior to this study, there was a lack of research on the global proteomic profile of rat brains following SZS treatment and its underlying mechanism in protecting against BBB breakdown after LPS exposure.

The study employed both in vivo and in vitro approaches. In vivo, forty Sprague-Dawley rats were divided into four groups: control (CON), LPS, SZS (administered orally for 30 days), and a positive control (Vc). LPS was injected intraperitoneally to induce BBB damage. Blood and brain samples were collected for various analyses, including H&E, Nissl, and TUNEL staining to assess tissue morphology and apoptosis. Transmission electron microscopy (TEM) examined ultrastructure. Evans blue (EB) extravasation measured BBB permeability. Biochemical assays determined oxidative stress (SOD, GSH, MDA, H2O2, ROS, PCB) and inflammatory levels (IL-6, TNF-α). Western blot analysis assessed the expression of tight junction proteins (ZO-1, occludin), adherens junction proteins (E-cadherin, β-catenin), and P-glycoprotein (P-gp). In vitro, the hCMEC/D3 cell line was used, subjected to similar treatments and analyses as the in vivo study, including sodium fluorescein permeability and TEER measurements. A label-free proteomic strategy (LC-MS/MS) was employed to identify differentially expressed proteins (DEPs) in rat brains. The chemical composition of SZS decoction and rat plasma was analyzed using UPLC-HR-MS. Molecular docking experiments evaluated the interaction between SZS components and signaling pathway proteins. Statistical analysis was performed using one-way ANOVA.

SZS significantly attenuated LPS-induced rat brain injury, reducing inflammatory infiltration, nuclear shrinkage, cell swelling, and apoptosis. SZS decreased BBB permeability in rats and hCMEC/D3 cells, as evidenced by reduced EB extravasation and sodium fluorescein penetration, and increased TEER values. SZS reduced oxidative stress and inflammatory levels in both rat brains and hCMEC/D3 cells. SZS increased the expression of tight junction and adherens junction proteins (ZO-1, occludin, E-cadherin, β-catenin) and P-gp in rat brains and hCMEC/D3 cells. Proteomic analysis revealed 135 DEPs in the SZS/LPS comparison group, predominantly related to cell-cell adhesion and cell junctions. SZS regulated the expression of proteins associated with dendritic shaft, postsynaptic density, cell junction, and postsynaptic membrane. SZS activated the FAK-DOCK180-Rac1-WAVE2-Arp3 signaling pathway, increasing F-actin expression and the F/G-actin ratio in rat brains and hCMEC/D3 cells. UPLC-HR-MS identified 33 constituents in SZS decoction and 13 in vivo compounds in rat plasma. Molecular docking showed that spinosin, swertisin, and 6''-feruloylspinosin strongly bound to FAK, DOCK180, Rac1, and Arp3.

This study provides the first evidence that SZS prevents LPS-induced BBB dysfunction by upregulating the FAK-DOCK180-Rac1-WAVE2-Arp3 pathway. The findings demonstrate SZS's neuroprotective effects on the BBB, contrasting with studies focusing on RhoA/ROCK, NF-κB, or Nrf2 pathways. The activation of the FAK-DOCK180-Rac1-WAVE2-Arp3 pathway by SZS is crucial in regulating actin cytoskeleton dynamics, which is essential for maintaining BBB integrity. The identification of specific SZS components (spinosin, swertisin, and 6''-feruloylspinosin) that interact with key proteins in this pathway strengthens the mechanistic understanding of SZS's neuroprotective effects. The in vivo and in vitro findings are consistent, indicating the reliability of the results. The study also provides comprehensive proteomic data, revealing a more holistic understanding of BBB regulation by SZS.

This research demonstrates that SZS protects against LPS-induced BBB dysfunction by activating the FAK-DOCK180-Rac1-WAVE2-Arp3 signaling pathway, leading to enhanced BBB integrity. The identification of key SZS components strengthens the mechanistic basis for its neuroprotective effects, supporting its use as a functional food and traditional medicine for BBB-related neurological diseases. Future studies could focus on investigating the long-term effects of SZS on BBB integrity and exploring its therapeutic potential in animal models of neurodegenerative diseases.

The study primarily focused on the acute effects of LPS and SZS on BBB integrity. Long-term effects and the potential for tolerance or cumulative effects of SZS need further investigation. The molecular docking study provided insights into potential interactions but does not definitively prove direct interactions in vivo. Further studies with in vivo assays would be required to verify this interaction. The study used a single concentration of LPS; future studies could investigate dose-response effects. While the study examined several key proteins, a more comprehensive analysis of the entire proteome could provide a more detailed picture of the biological effects of SZS.