Protective effect and mechanism of Qishiwei Zhenzhu pills on cerebral ischemia-reperfusion injury via blood-brain barrier and metabonomics.

Stroke is an acute cerebrovascular disease caused by the sudden rupture of cerebral blood vessels or vascular obstruction from brain tissue damage or dysfunction, thereby preventing blood flow into the brain. Cerebral ischemia-reperfusion injury (CI/RI), a common syndrome of ischemic stroke, is a complex pathological process whose physiological mechanism is still unclear. Qishiwei Zhenzhu pills (QSW), a famous Tibetan medicine preparation, has the effect of tranquilizing by heavy settling, dredging channels and activating collaterals, harmonizing Qi and blood, restoring consciousness, and inducing resuscitation. Here, we investigated the protective effect of QSW on CI/RI in rats and its potential mechanism. First, the volatile and liposoluble components in QSW were determined using gas chromatography-mass spectrometry (GCMS). After 24 h of CI/RI, the neuroprotective effect was determined by evaluating the neurological function, cerebral infarction, histopathology, and blood-brain barrier (BBB) function. Immunofluorescence, real-time quantitative PCR (RT-qPCR), and western blot (WB) were used to detect the expression of matrix metalloproteinase 9 (MMP-9), claudin-5, and occludin. Finally, GCMS metabonomics was used to identify different metabolites and analyze metabolic pathways. The results showed that 88 volatile components and 63 liposoluble components were detected in QSW. Following the experimental stroke operation, it was observed that rats administered QSW pretreatment had improved neurological function, reduced infarct volume (P < 0.01), increased Nissl bodies (P < 0.05), improved histopathology, and reduced BBB disruption. Immunofluorescence, RT-qPCR, and WB results showed that MMP-9 level in the brain tissue of the QSW pretreatment group had a decreasing trend and the expression of claudin-5 and occludin had a tendency to increase. Eleven metabolites related to lipid metabolism, fatty acid metabolism, and energy metabolism, were identified via GC-MS metabonomics. Our study shows that QSW preconditioning has a neuroprotective effect on CI/RI; however, its mechanism requires further study.

Inducers of the endothelial cell barrier identified through chemogenomic screening in genome-edited hPSC-endothelial cells.

The blood-retina barrier and blood-brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-ß pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-ß, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibiting multiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.

Plant-derived triterpene celastrol ameliorates oxygen glucose deprivation-induced disruption of endothelial barrier assembly via inducing tight junction proteins.

BACKGROUND: The integrity and functions of blood-brain barrier (BBB) are regulated by the expression and organization of tight junction proteins. OBJECTIVE: The present study was designed to explore whether plant-derived triterpenoid celastrol could regulate tight junction integrity in murine brain endothelial bEnd3 cells. METHODS: We disrupted the tight junctions between endothelial bEnd3 cells by oxygen glucose deprivation (OGD). We investigated the effects of celastrol on the permeability of endothelial monolayers by measuring transepithelial electrical resistance (TEER). To clarify the tight junction composition, we analyzed the expression of tight junction proteins by RT-PCR and Western blotting techniques. RESULTS: We found that celastrol recovered OGD-induced TEER loss in a concentration-dependent manner. Celastrol induced occludin, claudin-5 and zonula occludens-1 (ZO-1) in endothelial cells. As a result, celastrol effectively maintained tight junction integrity and inhibited macrophage migration through endothelial monolayers against OGD challenge. Further mechanistic studies revealed that celastrol induced the expression of occludin and ZO-1) via activating MAPKs and PI3K/Akt/mTOR pathway. We also observed that celastrol regulated claudin-5 expression through different mechanisms. CONCLUSION: The present study demonstrated that celastrol effectively protected tight junction integrity against OGD-induced damage. Thus, celastrol could be a drug candidate for the treatment of BBB dysfunction in various diseases.

Anti-inflammatory activity of Barleria lupulina: Identification of active compounds that activate the Nrf2 cell defense pathway, organize cortical actin, reduce stress fibers, and improve cell junctions in microvascular endothelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Hot aqueous extracts of the plant Barleria lupulina (BL) are used for treating inflammatory conditions and diabetic vascular complications. AIM OF THE STUDY: The goal was to identify active compounds in hot aqueous extracts of BL (HAE-BL) that are consistent with a role in reducing inflammation and reducing the vascular pathology associated with diabetes. In particular, we examined activation of the Nrf2 cell defense pathway because our initial findings indicated that HAE-BL activates Nrf2, and because Nrf2 is known to suppress inflammation. Activation of Nrf2 by HAE-BL has not been described previously. MATERIALS AND METHODS: Human endothelial cells, real-time PCR, western blotting, cytoskeletal analyses, and assay-guided fractionation with HPLC were used to identify specific compounds in HAE-BL that activate the Nrf2 cell defense pathway and reduce markers of inflammation in vitro. RESULTS: HAE-BL potently activated the Nrf2 cell defense pathway in endothelial cells consistent with its traditional use and reported success in reducing inflammation. Assay guided fractionation with HPLC identified three alkyl catechols: 4-ethylcatechol, 4-vinylcatechol, and 4-methylcatechol, that are each potent Nrf2 activators. In addition to activating Nrf2, HAE-BL and akyl catechols each profoundly improved organization of the endothelial cell actin cytoskeleton, reduced actin stress fibers, organized cell-cell junctions, and induced expression of mRNA encoding claudin-5 that is important for formation of endothelial tight junctions and reducing vascular leak. CONCLUSIONS: HAE-BL contains important alkyl catechols that potently activate the Nrf2 cell defense pathway, improve organization of the endothelial cell cytoskeleton, and organize tight cell junctions. All of these properties are consistent with a role in reducing inflammation and reducing vascular leak. Because activation of the Nrf2 cell defense pathway also prevents cancers, neuro-degeneration, age-related macular degeneration, and also reduces the severity of chronic obstructive pulmonary disorder and multiple sclerosis, HAE-BL warrants additional consideration for these other serious disorders.

EGb761 provides a protective effect against Aß1-42 oligomer-induced cell damage and blood-brain barrier disruption in an in vitro bEnd.3 endothelial model.

Alzheimer's disease (AD) is the most common form of senile dementia which is characterized by abnormal amyloid beta (Aß) accumulation and deposition in brain parenchyma and cerebral capillaries, and leads to blood-brain barrier (BBB) disruption. Despite great progress in understanding the etiology of AD, the underlying pathogenic mechanism of BBB damage is still unclear, and no effective treatment has been devised. The standard Ginkgo biloba extract EGb761 has been widely used as a potential cognitive enhancer for the treatment of AD. However, the cellular mechanism underlying the effect remain to be clarified. In this study, we employed an immortalized endothelial cell line (bEnd.3) and incubation of Aß(1-42) oligomer, to mimic a monolayer BBB model under conditions found in the AD brain. We investigated the effect of EGb761 on BBB and found that Aß1-42 oligomer-induced cell injury, apoptosis, and generation of intracellular reactive oxygen species (ROS), were attenuated by treatment with EGb761. Moreover, treatment of the cells with EGb761 decreased BBB permeability and increased tight junction scaffold protein levels including ZO-1, Claudin-5 and Occludin. We also found that the Aß(1-42) oligomer-induced upregulation of the receptor for advanced glycation end-products (RAGE), which mediates Aß cytotoxicity and plays an essential role in AD progression, was significantly decreased by treatment with EGb761. To our knowledge, we provide the first direct in vitro evidence of an effect of EGb761 on the brain endothelium exposed to Aß(1-42) oligomer, and on the expression of tight junction (TJ) scaffold proteins and RAGE. Our results provide a new insight into a possible mechanism of action of EGb761. This study provides a rational basis for the therapeutic application of EGb761 in the treatment of AD.