The BBB is a specialised neurovascular interface that guards the central nervous system, formed primarily by brain capillary endothelial cells welded together by tight junctions and buttressed by basement membranes, pericytes and astrocytic end-feet. Its restriction operates through distinct phenotypes. The physical barrier, built from tight junctions, limits the paracellular diffusion of ions and small molecules. The selective barrier, comprising influx transporters such as GLUT1, efflux pumps such as P-glycoprotein and the transcytosis machinery, governs transcellular traffic and actively expels many therapeutic compounds. A metabolic barrier can further degrade compounds in transit, although the review notes this aspect has not been systematically examined in the borneol literature and was left outside its scope. Because the desired direction of barrier modulation differs radically by disease, any compound that pushes permeability both ways demands careful scrutiny.

That scrutiny is exactly what the conflicting literature seemed to require. In ischaemic stroke, excessive barrier disruption fuels secondary injury, oedema and inflammatory cell infiltration, so restoring integrity is beneficial. In glioma, by contrast, the problem is the opposite: the intact barrier and blood-tumour barrier (BTB) block drug access, and transient permeability enhancement could improve delivery of chemotherapy. Borneol, remarkably, has been associated with both outcomes. It remains an essential component of classical anti-stroke formulas such as the Angong Niuhuang Pill, a Qing-dynasty preparation still used for acute cerebrovascular disease, and is classified in traditional frameworks as an ‘ushering drug’ that guides co-administered herbs toward the brain. The key question, the review argues, is not whether borneol affects the BBB but how its net effect becomes context-dependent.

The evidence from stroke models points consistently toward restoration. Ischaemia dismantles the barrier through multiple pathways: ATP depletion destabilises ion pumps and tight junctions; calcium overload activates proteases such as calpain that cleave junctional proteins; reactive oxygen species damage junctional components and activate matrix metalloproteinases that degrade the basal lamina; inflammatory cytokines alter permeability; and disruption of the MFSD2A-caveolin-1 axis increases endothelial endocytosis. A review of fifteen preclinical studies found that borneol intervenes at several of these points, restoring ATPase activity, reducing calcium load, boosting antioxidant defences and suppressing inflammatory mediators. In a rat model of middle cerebral artery occlusion, borneol treatment reduced albumin extravasation, and in an oxygen-glucose deprivation co-culture model of human cerebral microvascular endothelial cells with pericytes it reduced horseradish peroxidase transfer, two readouts of macromolecular leakage.

The most mechanistically defined pathway in the stroke context links borneol to the suppression of caveolae-mediated transcytosis via the MFSD2A and caveolin-1 (Cav-1) signalling axis. MFSD2A is a transporter for docosahexaenoic acid that maintains the distinctive lipid composition of brain endothelial membranes and thereby suppresses caveolae formation, while Cav-1 is the structural coat protein required for assembling caveolar vesicles. Under ischaemic conditions, MFSD2A is downregulated and Cav-1 upregulated, driving vesicular transcytosis and macromolecular leakage. Borneol treatment reversed both expression changes and markedly reduced endothelial vesicle counts on transmission electron microscopy. Complementary work in a permanent occlusion model showed reduced serum MMP-9, pointing to parallel protection of the extracellular matrix. The authors caution, however, that the MFSD2A/Cav-1 pathway was first identified through network pharmacology prediction and still awaits direct molecular-level confirmation.

In drug-delivery models, the picture flips. A preclinical pharmacokinetic review found that borneol co-administration increased maximum brain concentrations of twelve co-delivered compounds by 26 to 155 percent and area-under-the-curve values by 38 to 197 percent across intragastric, intranasal, intraperitoneal and intravenous routes. Critically, the effect is dose-dependent, transient and reversible within four to twenty-four hours. In rats given synthetic borneol, electron microscopy showed widening and discontinuity of endothelial tight junctions at four hours, peaking at eight, with junctional structure returning to control appearance by forty-eight hours. Junctional changes in cell monolayers resolved within twenty-four hours of removing the agent, indicating that the loosening depends on continued exposure rather than structural injury.

The clearest molecular insight into this opening mechanism comes from tight-junction dynamics. When mouse brain endothelial cells were treated with borneol at 10 micrograms per millilitre for six hours, transendothelial electrical resistance (TEER) dropped rapidly, most markedly at one hour, then began recovering by four hours. Crucially, total claudin-5 protein levels remained unchanged, but cytoplasmic claudin-5 increased significantly, and immunofluorescence confirmed redistribution of the protein from cell-cell junctions into the cytoplasm. Borneol, in other words, does not destroy a key junctional component; it displaces it from its functional position, weakening the barrier through reorganisation rather than degradation. In blood-tumour barrier studies, borneol lowered TEER, enhanced cisplatin delivery through reversible ZO-1 downregulation, and did not inhibit P-glycoprotein activity when used alone, though one study associated its barrier-opening effect with increased ICAM-1 expression, a marker that promotes leukocyte adhesion and endothelial damage.

The translational stakes are vividly illustrated by nanoparticle work. Borneol co-treatment increased the four-hour transport ratio of paclitaxel-loaded nanoparticles across an in vitro BBB model from 13.11 to 19.33 percent for PEG-coated particles and from 18.38 to 23.85 percent for particles modified with the CGKRK tumour-targeting peptide. In U87MG glioma-bearing mice, fluorescence imaging showed greater brain and tumour accumulation, and mean survival time rose from 17.4 days with saline to 38.9 days with the combination of CGKRK-modified nanoparticles and borneol. L-borneol has also been shown to transiently open the mouse BBB across doses of 100 to 900 milligrams per kilogram, enhance cisplatin brain penetration and extend survival in glioma-bearing animals, establishing the adjuvant concept in vivo.

The review’s decisive analytical move is to compare matched readouts across the two contexts. On TEER, borneol increased resistance in oxygen-glucose-deprived human endothelial monolayers but decreased it rapidly in intact mouse endothelial monolayers. On junctional structure, electron microscopy showed preservation of tight-junction ultrastructure in ischaemic models, whereas intact barriers showed claudin-5 redistribution into the cytoplasm. On macromolecular passage, albumin leakage fell in stroke models while Evans blue albumin entry rose in healthy rats, and endothelial vesicle numbers fell in stroke while nanoparticle transport ratios rose in delivery settings. Although these experiments were not conducted in a single directly comparable design, the directional contrast across matched measures is strikingly consistent: borneol shifts injured barriers toward restoration and intact restrictive barriers toward transient passage.

What remains unresolved, the authors stress, is the upstream regulatory basis of this bidirectionality. Whether the trajectory is governed by inflammatory state, endothelial metabolic stress, baseline barrier integrity or a combination of factors is not yet determined, and no single study has examined borneol under both injured and intact barrier conditions within one design. Substantial heterogeneity in model, species, dose, formulation and timing further complicates interpretation, and risks of transient BBB opening, including neuroinflammation, leukocyte trafficking and oedema, cannot be ignored. Yet this apparent flexibility may be borneol’s most interesting pharmacological property. A barrier-active compound that drives the endothelium in a therapeutically relevant direction depending on pathological context could, if its responsiveness can be brought under sufficient control, offer advantages over rigidly unidirectional agents. The next step is to identify the conditions under which its adaptability becomes a predictable and governable therapeutic property, transforming a two-thousand-year-old adjuvant into a pharmacologically controllable instrument of brain access.

Subject of Research: Context-dependent effects of the monoterpene borneol on blood-brain barrier integrity and permeability

Article Title: Comparative evaluation of borneol as a context‐dependent modulator of blood-brain barrier function: barrier-restorative or permeability-enhancing agent?

Article References: Yin, X., & Thomas, S. A. (2026). Comparative evaluation of borneol as a context‐dependent modulator of blood-brain barrier function: barrier-restorative or permeability-enhancing agent?. Frontiers in Drug Delivery, 6, Article 1934025. https://doi.org/10.3389/fddev.2026.1934025

Image Credits: AI Generated

DOI: 10.3389/fddev.2026.1934025

Keywords: blood-brain barrier, borneol, ischaemic stroke, drug delivery, tight junctions, claudin-5, MFSD2A, caveolin-1, transcytosis, TEER, glioma, traditional Chinese medicine

The intestinal epithelial barrier is the body’s frontline customs office, a single layer of cells that decides what passes from the gut lumen into the bloodstream and what stays out. Its defining feature is the tight junction, a belt of protein complexes that seals the gaps between neighboring epithelial cells. These junctions perform what biologists call a gate-and-fence function: they permit the selective passage of water, ions, and nutrients through the spaces between cells while blocking bacteria and their toxins. When tight junctions fail, the result is the condition popularly known as leaky gut, in which unwanted molecules slip through and provoke inflammatory responses. Disrupted barrier function is implicated in inflammatory bowel disease, metabolic disorders, and a growing list of chronic conditions.

The key players at these junctions are the tight junction proteins, including occludin and members of the claudin family. Occludin, the first of these proteins to be identified, stabilizes the structural framework of the junction. Claudin-2, one of several tissue-specific isoforms, is associated with permeability properties of the intestinal epithelium, and its behavior is subtle: depending on context, it can either loosen or remodel the barrier. Another important player is lipocalin-2, an antibacterial, iron-sequestering protein of innate immunity that participates in the barrier’s defensive repertoire and is often induced by inflammatory stimuli. The inflammatory cytokine tumor necrosis factor alpha, meanwhile, can undermine the barrier by triggering the internalization of occludin, raising permeability.

Probiotic lactobacilli have repeatedly been shown to bolster this system. Certain strains of Lactiplantibacillus plantarum increase the production and correct localization of tight junction proteins by upregulating the genes that encode them, thereby reducing permeability and guarding against inflammation. Researchers have also explored postbiotics, the cell-free supernatants left behind after bacterial growth, which contain the bioactive metabolites of probiotic cultures without the living cells themselves. What has remained murky is how the food matrix itself contributes when probiotics are delivered as part of a fermented food, rather than as a purified supplement. The new study set out to probe exactly that boundary.

The team, led by Jihen Missaoui with colleagues at the University of Bari Aldo Moro in Italy and the University of Monastir in Tunisia, worked with two lactobacilli previously isolated from traditional zgougou: Lactiplantibacillus paraplantarum A1 and Lactiplantibacillus plantarum A2. They prepared these bacteria in three forms: living cell biomass from liquid culture, cell-free supernatant from the same cultures, and zgougou juice fermented with both strains together as a starter consortium. For comparison, they also prepared zgougou juice that had fermented spontaneously, relying on whatever microbes happened to be present. Each preparation was applied to monolayers of Caco-2 cells, a human colon cancer cell line that, when grown for 21 days on Transwell inserts, differentiates into a faithful in vitro model of the intestinal epithelium.

To gauge barrier integrity, the researchers measured transepithelial electrical resistance, or TEER, a standard technique in which a small current is passed across the cell layer; higher resistance means tighter junctions and a less permeable barrier. As a positive control, they challenged cells with lipopolysaccharide, a component of the outer membrane of E. coli that reliably disrupts tight junctions. The LPS treatment produced the expected time-dependent response, with resistance dropping significantly within 30 minutes before partially recovering. Against this baseline, the effects of the bacterial preparations emerged clearly, though not uniformly.

Exposure to the biomass of L. paraplantarum A1 raised TEER values significantly at early time points, an effect that depended on both the dose and the dilution used. The cell-free supernatant of the same strain increased resistance after six hours, and the biomass and supernatant of L. plantarum A2 produced significant boosts at 30 minutes. The most striking result, however, came from the fermented food itself. When the monolayers were treated with zgougou juice fermented by the two lactobacilli together, diluted tenfold, TEER values climbed to between roughly 690 and 963 ohms per square centimeter at every time point measured, dramatically higher than the untreated control. Spontaneously fermented juice produced a smaller but still significant effect at one time point. The combination of the two strains, embedded in the pine seed matrix, outperformed anything the bacteria achieved alone.

The molecular story behind these measurements proved equally intriguing. Using reverse transcription quantitative PCR, the team measured the expression of genes encoding occludin, claudin-2, lipocalin-2, and TNF alpha after 24 hours of exposure. Both bacterial biomasses and the driven-fermented juice significantly increased TNF alpha gene expression, yet an ELISA assay of the culture medium detected no corresponding increase in the protein itself. The authors attribute this discrepancy to post-transcriptional regulatory mechanisms that can block cytokine synthesis even when the gene is actively transcribed, a reminder that messenger RNA levels do not always translate into protein output.

More encouraging were the tight junction results. Biomass and cell-free supernatant of both lactobacilli significantly upregulated the genes for occludin and lipocalin-2, with the strongest effects seen at the tenfold dilution. The fermented zgougou juice, whether driven by the starter cultures or fermented spontaneously, significantly increased expression of both claudin-2 and occludin, with the spontaneous juice producing the strongest upregulation. The apparent tension between rising claudin-2 expression and rising TEER, which might seem contradictory given claudin-2’s association with permeability, is resolved by context: the functional impact of claudin-2 depends on the overall composition of the junction complex, and its overexpression here likely signals junction remodeling and epithelial homeostasis rather than a leaky barrier.

The authors hypothesize that the observed effects arise from a synergy between the bioactive compounds of Aleppo pine seeds, which are rich in linoleic and alpha-linolenic acids, flavonoids, and phenolics with antioxidant and anti-inflammatory activity, and the metabolic activity of the fermenting lactobacilli. Vegetable matrices may also protect probiotic bacteria during their passage through the stomach and small intestine, releasing them in the colon where they can interact directly with the host. The study is the first to show that fermented zgougou juice can upregulate genes encoding tight junction proteins in an intestinal cell model, and it underscores how traditional fermented foods, from kefir to kimchi, may modulate gut permeability through the combined action of microbes, their metabolites, and plant phytochemicals. The researchers caution that the effects are strain-dependent and shaped by the host and its resident microbiota, and they call for untargeted metabolomics to identify the specific compounds responsible, followed by animal studies to confirm whether the barrier-reinforcing effect survives the journey from the dish to the organism.

Subject of Research: Effects of Tunisian fermented zgougou and probiotic lactobacilli on intestinal epithelial barrier integrity in Caco-2 cell models

Article Title: Impact of Tunisian Fermented Zgougou and Related Lactobacilli on Caco‐2 Cell Barrier Integrity: Upregulation of Key Tight Junction Proteins

Article References: Missaoui, J., Liso, M., Lataoui, M., Limongelli, R., Minervini, F., Achour, L., & De Angelis, M. (2026). Impact of Tunisian Fermented Zgougou and Related Lactobacilli on Caco‐2 Cell Barrier Integrity: Upregulation of Key Tight Junction Proteins. Food Science & Nutrition, 14(10), Article e72387. https://doi.org/10.1002/fsn3.72387

Image Credits: AI Generated

DOI: 10.1002/fsn3.72387

Keywords: zgougou, Aleppo pine seeds, fermented foods, lactobacilli, probiotics, gut barrier, tight junction proteins, occludin, claudin-2, Caco-2 cells, TEER, postbiotics