Rat Brain Microvessel Endothelial Cells Research Articles

Study on the reversal of epileptic drug resistance by tetramethylpyrazine in combination with carbamazepine through modulation of P-Glycoprotein expression in rat brain microvessel endothelial cell.

The purpose of this study was to detect the changes of P-Glycoprotein (P-GP) expression in rat brain microvessel endothelial cell line RBE4 after the action of Tetramethylpyrazine (TMP) on Carbamazepine (CBZ), so as to clarify the potential mechanism of TMP combined with CBZ against intractable epilepsy drug resistance. The RBE4 cell line was utilized for in vitro analysis. Cells were divided into control, CBZ, and CBZ-TMP group. The expression of P-GP was assessed using Western blot and reverse transcription polymerase chain reaction (RT-PCR). Intracellular concentration of CBZ was measured through high-performance liquid chromatography (HPLC). The differential expression of mRNA was evaluated by RNA sequencing. The intracellular concentration of CBZ in the CBZ-TMP group was significantly higher than that in other groups. The expression of P-GP in the CBZ group was significantly higher than that in the control group, while in the CBZ&TMP group, it was significantly lower than that in the other groups. Comparative analysis also revealed some differentially expressed genes. Compared with the CBZ group, FAM106A, SLC3A2, TENM2, etc. were upregulated most significantly in the CBZ&TMP group. ZBTB10, WDR7, STARD13, etc. were downregulated most significantly. Results suggest that TMP increases the intracellular concentration of CBZ, downregulates the expression of P-GP increased by CBZ, and modulates related cellular metabolism and signaling pathways, thus reversing the drug resistance mechanism of intractable epilepsy, providing a theoretical basis for the combination of traditional Chinese medicine and antiepileptic drugs.

Ginkgolide C attenuates cerebral ischemia/reperfusion-induced inflammatory impairments by suppressing CD40/NF-κB pathway

Ethnopharmacological relevanceGinkgo biloba L. (Ginkgoaceae), a traditional Chinese medicine, has been applied for thousands of years for the treatment of cardio-cerebral vascular diseases in China. It is written in Compendium of Materia Medica that Ginkgo has the property of “dispersing poison”, which is now referred to as anti-inflammatory and antioxidant. Ginkgolides are important active ingredients in Ginkgo biloba leaves and ginkgolide injection has been frequently applied in clinical practice for the treatment of ischemic stroke. However, few studies have explored the effect and mechanism of ginkgolide C (GC) with anti-inflammatory activity in cerebral ischemia/reperfusion injury (CI/RI). Aim of the studyThe present study aimed to demonstrate whether GC was capable of attenuating CI/RI. Furthermore, the anti-inflammatory effect of GC in CI/RI was explored around the CD40/NF-κB pathway. Materials and methodsIn vivo, middle cerebral artery occlusion/reperfusion (MCAO/R) model was established in rats. The neuroprotective effect of GC was assessed by neurological scores, cerebral infarct rate, microvessel ultrastructure, blood-brain barrier (BBB) integrity, brain edema, neutrophil infiltration, and levels of TNF-α, IL-1β, IL-6, ICAM-1, VCAM-1, and iNOS. In vitro, rat brain microvessel endothelial cells (rBMECs) were preincubated in GC before hypoxia/reoxygenation (H/R) culture. The cell viability, levels of CD40, ICAM-1, MMP-9, TNF-α, IL-1β, and IL-6, and activation of NF-κB pathway were examined. In addition, the anti-inflammatory effect of GC was also investigated by silencing CD40 gene in rBMECs. ResultsGC attenuated CI/RI as demonstrated by decreasing neurological scores, reducing cerebral infarct rate, improving microvessel ultrastructural features, ameliorating BBB disruption, attenuating brain edema, inhibiting MPO activity, and downregulating levels of TNF-α, IL-1β, IL-6, ICAM-1, VCAM-1, and iNOS. Coherently, in rBMECs exposed to H/R GC enhanced cell viability and downregulated levels of ICAM-1, MMP-9, TNF-α, IL-1β, and IL-6. Furthermore, GC suppressed CD40 overexpression and hindered translocation of NF-κB p65 from the cytosol to the nucleus, phosphorylation of IκB-α, and activation of IKK-β in H/R rBMECs. However, GC failed to protect rBMECs from H/R-induced inflammatory impairments and suppress activation of NF-κB pathway when CD40 gene was silenced. ConclusionsGC attenuates cerebral ischemia/reperfusion-induced inflammatory impairments by suppressing CD40/NF-κB pathway, which may provide an available therapeutic drug for CI/RI.

Curcumin Derivative Cur20 Attenuated Cerebral Ischemic Injury by Antioxidant Effect and HIF-1α/VEGF/TFEB-Activated Angiogenesis.

In this paper, a curcumin derivative Cur20 was synthesized for better hydrolytic stability, which showed a higher angiogenic effect on zebrafish model than curcumin. In order to reveal the potential effects on neuroprotection, a mouse model of vascular dementia (VaD) induced by permanent right common carotid artery occlusion (rUCCAO) was established. After two weeks of curcumin administration, the cognitive function of mice was detected by Morris water maze and Y maze. The alteration on oxidative injuries and morphological damage were also analyzed by reactive oxygen species, superoxide dismutase, GSH, malondialdehyde tests, and Nissl stain on cortex/hippocampus. The angiogenesis and related signal factors were evaluated as well. The results showed that Cur20 significantly attenuated the cognitive dysfunction and histopathological changes of the VaD mice with enhanced antioxidant system and angiogenesis. In addition, primary rat brain microvessel endothelial cells (rBMECs) with oxygen glucose deprivation (OGD) were applied to further verify the possible mechanisms of Cur20-induced angiogenesis. The results demonstrated that the proliferation effect and the activation of pro-angiogenesis factors such as HIF-1α, VEGF, and TFEB might contribute to the protection of ischemic injury. Based on the above, our conclusion is that Cur20 can be considered as a promising therapeutic strategy for VaD.

Molecular and Functional Study of Transient Receptor Potential Vanilloid 1-4 at the Rat and Human Blood-Brain Barrier Reveals Interspecies Differences.

Transient receptor potential vanilloid 1-4 (TRPV1-4) expression and functionality were investigated in brain microvessel endothelial cells (BMEC) forming the blood–brain barrier (BBB) from rat and human origins. In rat, Trpv1-4 were detected by qRT-PCR in the brain cortex, brain microvessels, and in primary cultures of brain microvessel endothelial cells [rat brain microvessel endothelial cells (rPBMEC)]. A similar Trpv1-4 expression profile in isolated brain microvessels and rPBMEC was found with the following order: Trpv4 > Trpv2 > Trpv3 > Trpv1. In human, TRPV1-4 were detected in the BBB cell line human cerebral microvessel endothelial cells D3 cells (hCMEC/D3) and in primary cultures of BMEC isolated from human adult and children brain resections [human brain microvascular endothelial cells (hPBMEC)], showing a similar TRPV1-4 expression profile in both hCMEC/D3 cells and hPBMECs as follow: TRPV2 > > TRPV4 > TRPV1 > TRPV3. Western blotting and immunofluorescence experiments confirmed that TRPV2 and TRPV4 are the most expressed TRPV isoforms in hCMEC/D3 cells with a clear staining at the plasma membrane. A fluorescent dye Fluo-4 AM ester was applied to record intracellular Ca2+ levels. TRPV4 functional activity was demonstrated in mediating Ca2+ influx under stimulation with the specific agonist GSK1016790A (ranging from 3 to 1000 nM, EC50 of 16.2 ± 4.5 nM), which was inhibited by the specific TRPV4 antagonist, RN1734 (30 μM). In contrast, TRPV1 was slightly activated in hCMEC/D3 cells as shown by the weak Ca2+ influx induced by capsaicin at a high concentration (3 μM), a highly potent and specific TRPV1 agonist. Heat-induced Ca2+ influx was not altered by co-treatment with a selective potent TRPV1 antagonist capsazepine (20 μM), in agreement with the low expression of TRPV1 as assessed by qRT-PCR. Our present study reveals an interspecies difference between Rat and Human. Functional contributions of TRPV1-4 subtype expression were not identical in rat and human tissues reflective of BBB integrity. TRPV2 was predominant in the human whereas TRPV4 had a larger role in the rat. This interspecies difference from a gene expression point of view should be taken into consideration when modulators of TRPV2 or TRPV4 are investigated in rat models of brain disorders.

Activation of NADPH/ROS pathway contributes to angiogenesis through JNK signaling in brain endothelial cells

Recent evidences have shown that reactive oxygen species (ROS) are involved in regulating angiogenesis and preventing tissue injury. However, the precise molecular mechanisms behind ROS-induced angiogenesis are still unknown. The aim of the present study was to investigate the effects of ROS-induced angiogenesis in rat brain microvessel endothelial cells (rBMECs) and identify involving the signal pathways. For initial experiments, the rBMECs were incubated with different concentrations of hydrogen peroxide (H2O2). For the second experiments, the rBMECs were respectively treated with ROS scavenger dimethylthiourea (DMTU), NADPH oxidase (Nox) inhibitor apocynin, small interfering RNAs-mediated knock down Nox2 or Nox4, or pretreated with c-Jun N-terminal kinase (JNK) inhibitor SP600125. The cell proliferation, migration, tube formation, and the expressions of several important neuroangiogenic factors including vascular endothelial growth factor (VEGF), brain derived neurotrophic factor (BDNF), matrix metalloproteinase (MMP) -9 and phos-JNK were measured. Low level of H2O2 significantly promoted endothelial cell (EC) proliferation, migration and tube formation and upregulated levels of VEGF, BDNF, MMP-9 and phos-JNK. DMTU and apocynin significantly inhibited endothelial angiogenesis and downregulated these protein levels. As expected, knockdown of Nox2 or Nox4 expression blocked endothelial angiogenesis and downregulated the expressions of pro-neuroangiogenic factors. Furthermore, H2O2-induced endothelial angiogenesis and high expressions of pro-neuroangiogenic factors were decreased by SP600125. In conclusion, Nox-derived ROS were required for endothelial angiogenesis. Low level of ROS may activate JNK signaling pathway and upregulate pro-neuroangiogenic factors, ultimately mediating endothelial angiogenesis.

HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway

P-glycoprotein (P-gp) is located on the luminal surface of brain vascular endothelium and its status may determine the delivery of the agents into the brain tissues. Previous study showed that upregulation of P-gp at the blood-brain barrier (BBB) after ischemic stroke were mediated by nuclear factor-B (NF-kB) and tumour necrosis factor-α (TNF-α). Based on middle cerebral artery occlusion (MCAO) rats and oxygen-glucose deprivation (OGD) in co-culture of rat brain microvessel endothelial cells (rBMECs) and astrocytes system, the present data indicated that potentiated P-gp expression and activity in brain microvessels or rBMECs were associated with the increase in high-mobility group box 1 (HMGB1), Toll-like receptor 4 (TLR4) and activation of NF-kB and that HMGB1 can release from nucleus to the cytoplasm in activated astrocytes, then into the medium. Moreover, changes in TLR4, TIR domain-containing adaptor protein (TIRAP), NF-kB and P-gp in rBMECs were attenuated by addition of 1 mM ethyl pyruvate (EP), 10 μM TAK-242 and 10 μM pyrrolidine dithiocarbamate (PDTC), respectively. These results demonstrated that HMGB1 promoted P-gp at the BBB after cerebral ischemia via TLR4/NF-κB signaling pathway.

Protective Effect of Buyang Huanwu Decoction on Neurovascular Unit in Alzheimer's Disease Cell Model via Inflammation and RAGE/LRP1 Pathway.

BackgroundThe aim of this study was to investigate the protective mechanism of neurovascular unit of Buyang Huanwu decoction (BYHWD) in an Alzheimer’s disease (AD) cell model via RAGE/LRP1 pathway and find a reliable target for Alzheimer’s disease treatment.Material/MethodsRat brain microvessel endothelial cells (BMECs) were cultured in 10% FBS and 1% penicillin/streptomycin. The AD model was established by administration of 24 μmol/L amyloid-β peptides 25~35. Different concentrations of BYHWD (0.1 mg/mL, 1 mg/mL, and 10 mg/mL) were added as the drug intervention. The morphology of the cells was observed by light microscopy and the ultrastructure of the cells was observed by microscopy. The inflammatory factors IL-1β, IL-6, TNF-α, and Aβ25–35 were detected by ELISA. Flow cytometry was used to assess the apoptosis rate. The expressions of RAGE, LRP1, ICAM-1, VCAM-1, Apo J, Apo E, and NF-κBp65 were detected by Western blotting.ResultsThe structure of cells in BYHWDM and BYHWDH gradually recovered with increasing dose. BYHWD decreased the apoptotic rate of BMECs induced by Aβ25–35. The cells treated with different concentrations of BYHWD had significant difference in terms of anti-apoptotic effect. The therapeutic effect of BYHWD on AD was via the RAGE/LRP1 and NF-κBp65 pathways.ConclusionsBYHWD regulates Aβ metabolism via the RAGE/LRP1 pathway, inhibits vascular endothelial inflammation induced by ICAM-1 and VCAM-1 via the NF-κBP65 pathway, and promotes morphological changes induced by Aβ-induced brain microvascular endothelial cell damage.

An Alternative In Vitro Method for Examining Nanoparticle-Induced Cytotoxicity.

Conventional in vitro assays are often used as initial screens to identify potential toxic effects of nanoparticles (NPs). However, many NPs have shown interference with conventional in vitro assays, resulting in either false-positive or -negative outcomes. Here, we report an alternative method for the in vitro assessment of NP-induced cytotoxicity utilizing Fluoro-Jade C (FJ-C). To provide proof of concept and initial validation data, Ag-NPs and Au-NPs were tested in 3 different cell cultures including rat brain microvessel endothelial cells, mouse neural stem cells, and the human SH-SY5Y cell line. Conventional 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and lactate dehydrogenase (LDH) assays were run in parallel with the new method and served as references. The results demonstrate for the first time that FJ-C labeling can be a useful tool for assessing NP-induced cytotoxicity in vitro. Using these approaches, it was also demonstrated that removal of Ag-NPs-while keeping the Ag-ions that were released from the Ag-NPs in culture media-abolished the measured cytotoxicity, indicating that Ag-NPs rather than Ag-ions in solution contributed to the observed cytotoxic effects. Further, co-treatment of Ag-NPs with N-acetyl cysteine (NAC) prevented the observed cytotoxicity, suggesting a protective role of NAC in Ag-NP-induced cytotoxicity. Thus, this alternative in vitro assay is well suited for identify potential cytotoxicity associated with exposure to NPs.

Acute liver failure impairs function and expression of breast cancer‐resistant protein (BCRP) at rat blood–brain barrier partly via ammonia‐ROS‐ERK1/2 activation

We once reported that P-glycoprotein (P-GP) and multidrug resistance-associated protein 2 (MRP2) were oppositely regulated at the blood-brain barrier (BBB) of thioacetamide-induced acute liver failure (ALF) rats. This study aimed to investigate whether ALF affected function and expression of breast cancer-resistant protein (BCRP) at the BBB of rats and the role of ammonia in the regulation. ALF rats were developed by intraperitoneal (i.p.) injection of thioacetamide (300mg/kg) for 2days. Hyperammonemic rats were developed by NH4 Ac (i.p. 4.5mmol/kg). BCRP function and expression were measured by brain distribution of specific substrates (prazosin and methotrexate) and western blot, respectively. MDCK-BCRP cells and primarily cultured rat brain microvessel endothelial cells (rBMECs) were employed to investigate possible mechanisms through which ammonia regulated BCRP function and expression. The results showed that both ALF and hyperammonemia significantly weakened function and expression of BCRP in the brain of rats. The function and expression of BCRP in MDCK-BCRP cells and rBMECs were strikingly decreased after exposure to NH4 Cl and H2 O2 , accompanied by remarkable increases in the levels of phosphorylated ERK1/2 and reactive oxygen species (ROS). The altered BCRP expression and function by ammonia and H2 O2 were restored by ROS scavenger N-acetylcysteine and ERK1/2 inhibitor U0126. Markedly increased levels of ERK1/2 phosphorylation and ROS were found in the brains of ALF rats and hyperammonemic rats. All above results indicated ALF down-regulated expression and function of BCRP at BBB of rats partly via hyperammonemia. Activation of ROS-mediatedERK1/2 phosphorylation may be one of the reasons that ammonia impaired BCRP expression and function at the BBB. The present study showed that the expression and function of breast cancer resistant protein (BCRP) at blood-brain barrier (BBB) of thioacetamide-induced ALF rats were down-regulated which partly attribute to hyperammonemia. Activation of ROS-mediated ERK1/2 phosphorylation may be one of the reasons that ammonia suppressed BCRP expression and function. Impaired BCRP at BBB might enhanced pharmacological/toxic effects of corresponding substrates on CNS.

Regulation of P-glycoprotein efflux activity by Z-guggulsterone of Commiphora mukul at the blood-brain barrier

The present study was to investigate whether Z-guggulsterone had the regulatory effect on the activity and expression of P-glycoprotein in rat brain microvessel endothelial cells (rBMECs) and in rat brain. Inorganic phosphate liberation assay, high performance liquid chromatography, and western blot analysis were performed to assess the P-glycoprotein ATPase activity, the accumulation of NaF and rhodamine 123, and P-glycoprotein and MRP1 expression. The results showed that Z-guggulsterone (0–100μM) significantly enhanced basal P-glycoprotein ATPase activity in a concentration-dependent manner. Tetrandrine (0.1, 0.3, 1μM) or cyclosporine A (0.1, 0.3, 1μM) had non-competitively inhibitory manner on Z-guggulsterone-stimulated P-glycoprotein ATPase activity, suggesting that Z-guggulsterone might have unique binding site or regulating site on P-glycoprotein. However, Z-guggulsterone (30, 100μM) had almost no influence on MRP1 expression in rBMECs. Further results revealed that Z-guggulsterone (50mg/kg) significantly increased the accumulation of rhodamine 123 by down-regulating P-glycoprotein expression in rat brain, as compared with control (P<0.05). Our studies suggested that Z-guggulsterone potentially inhibited the activity and expression of P-glycoprotein in rBMECs and in rat brain.

Stereoselective Regulation of P-gp Activity by Clausenamide Enantiomers in Caco-2, KB/KBv and Brain Microvessel Endothelial Cells.

The (−)- and (+)-clausenamide (CLA) enantiomers have different pharmacokinetic effects in animals, but their association with putative stereoselective regulation of P-glycoprotein (P-gp) remains unclear. Using three cells expressing P-gp—Caco-2, KBv and rat brain microvessel endothelial cells(RBMEC), this study investigated the association of CLA enantiomers with P-gp. The results showed that the rhodamine 123 (Rh123) accumulation, an indicator of P-gp activity, in Caco-2, KBv and RBMECs was increased by (−)CLA (1 or 5 μmol/L) at 8.2%–28.5%, but reduced by (+)CLA at 11.7%–25.9%, showing stereoselectivity in their regulation of P-gp activity. Following co-treatment of these cells with each CLA enantiomer and verapamil as a P-gp inhibitor, the (+)-isomer clearly antagonized the inhibitory effects of verapamil on P-gp efflux, whereas the (−)-isomer had slightly synergistic or additive effects. When higher concentrations (5 or 10 μmol/L) of CLA enantiomers were added, the stimulatory effects of the (+)-isomer were converted into inhibitory ones, leading to an enhanced intracellular uptake of Rh123 by 24.5%–58.2%; but (−)-isomer kept its inhibition to P-gp activity, causing 30.0%–63.0% increase in the Rh123 uptake. The biphasic effects of (+)CLA were confirmed by CLA uptake in the Caco-2 cells. (+)CLA at 1 μmol/L had significantly lower intracellular uptake than (−)CLA with a ratio[(−)/(+)] of 2.593, which was decreased to 2.167 and 1.893 after CLA concentrations increased to 2.5 and 5 μmol/L. Besides, in the non-induced KB cells, (+)CLA(5 μmol/L) upregulated P-gp expression at 54.5% relative to vehicle control, and decreased Rh123 accumulation by 28.2%, while (−)CLA(5 μmol/L) downregulated P-gp expression at 15.9% and increased Rh123 accumulation by 18.0%. These results suggested that (−)CLA could be a P-gp inhibitor and (+)CLA could be a modulator with concentration-dependent biphasic effects on P-gp activity, which may result in drug—drug interactions when combined with other P-gp substrate drugs.

EEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells.

ObjectiveWe investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K)/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs).MethodsCortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.Results Mdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.ConclusionseEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.

Identification of P-glycoprotein co-fractionating proteins and specific binding partners in rat brain microvessels.

Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. P-glycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood-brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP-containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post-translationally regulated at the BBB. The goal of the current study was to identify proteins that co-localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co-localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co-fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post-translational regulation of PgP activity at the BBB.

Hyperammonemia enhances the function and expression of P-glycoprotein and Mrp2 at the blood-brain barrier through NF-κB.

Ammonia is considered to be the main neurotoxin responsible for hepatic encephalopathy resulting from liver failure. Liver failure has been reported to alter expression and activity of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (Mrp2) at the blood-brain barrier (BBB). The aim of this study was to investigate whether ammonia is involved in abnormalities of expression and activity of P-gp and Mrp2 at the BBB. Hyperammonemic rats were developed by an intraperitoneal injection of ammonium acetate (NH4 Ac, 4.5 mmol/kg). Results showed that Mrp2 function markedly increased in cortex and hippocampus of rats at 6 h following NH4 Ac administration. Significant increase in function of P-gp was observed in hippocampus of rats. Meanwhile, such alterations were in line with the increase in mRNA and protein levels of P-gp and Mrp2. Significant increase in levels of nuclear amount of nuclear factor-κB (NF-κB) p65 was also observed. Primarily cultured rat brain microvessel endothelial cells (rBMECs) were used for in vitro study. Data indicated that 24 h exposure to ammonia significantly increased function and expression of P-gp and Mrp2 in rBMECs, accompanied with activation of NF-κB. Furthermore, such alterations induced by ammonia were reversed by NF-κB inhibitor. In conclusion, this study demonstrates that hyperammonemia increases the function and expression of P-gp and Mrp2 at the BBB via activating NF-κB pathway. Hyperammonemia, a proverbial main factor responsible for neurocognitive disorder and blood-brain barrier (BBB) dysfunction resulting from liver failure, could increase the expression and activity of P-glycoprotein and multidrug resistance-associated protein 2 (Mrp2) at the BBB both in vivo and in vitro. Furthermore, the NF-κB activation stimulated by hyperammonemia may be the potential mechanism underlying such abnormalities induced by hyperammonemia.

Attenuated Blood-Brain Barrier Dysfunction by XQ-1H Following Ischemic Stroke in Hyperlipidemic Rats.

Following ischemic stroke, blood-brain barrier (BBB) is disrupted and is further aggravated with the corresponding incidence of hyperlipidemia. BBB breakdown promotes inflammation infiltration into the brain, which exacerbates cerebral ischemic injury as a result. Here, we report that 10-O-(N,N-dimethylaminoethyl)-ginkgolide B methanesulfonate (XQ-1H), a novel analog of ginkgolide B, alleviates BBB breakdown in hyperlipidemic rats and protects endothelial cells against inflammatory response. Middle cerebral artery occlusion (MCAO) modeled ischemic stroke in rats. Before surgery, these rats were fed a cholesterol-rich diet to induce an experimental hyperlipidemic condition. Additionally, lipopolysaccharide (LPS) incubation with rat brain microvessel endothelial cells (rBMECs) was applied to mimic hyperlipidemia-induced inflammatory injury of BBB. The results indicated more severe infarct size, increased BBB permeability, excessive secretion of pro-inflammatory cytokines, and exaggerated inflammation infiltration of the brain in hyperlipidemic rats following MCAO when compared to rats fed with normal diet. XQ-1H protected BBB integrity, lessoned brain edema and inflammation penetration, downregulated MMP-9 and VCMA-1 expressions, and extenuated ischemic infarction. XQ-1H alleviated LPS-induced inflammatory response in rBMECs, characterized by promoting cell viability, inhibiting TNF-α, IL-1β, and IL-6 releasing, and downregulating NF-κB inflammatory signal and downstream proteins, such as VCAM-1 and iNOS. In conclusion, the present study shows that XQ-1H stabilizes BBB function following ischemic stroke in hyperlipidemic rats, and the possible mechanisms may be related to inflammation inhibition.

N2 ameliorates neural injury during experimental ischemic stroke via the regulation of thromboxane A2 production

Thromboxane A2 (TXA2) promotes ischemic stroke injury and has strong effects in vascular contraction and vascular endothelial cell dysfunction. Agents that reduce TXA2 production have potential for ameliorating neural injury in ischemic stroke. Thromboxane synthetase (TXS) is essential for TXA2 production, and TXS inhibitors have been developed as drugs for the prevention and treatment of stroke. However, ozagrel, a typical TXS inhibitor currently in clinical use, must be delivered via intravenous injection (I.V.). N2, 4-(2-(1H-imidazol-1-yl) ethoxy)-3-methoxybenzoate, is a potential thromboxane synthetase (TXS) inhibitor, which is being developed as an orally available formulation. The aim of this study was to investigate the effects of N2 on focal cerebral ischemia–reperfusion injury and related mechanisms. Neurological deficits, a Y-maze test and infarct volume were measured to evaluate the effects of N2 post-treatment on middle cerebral artery occlusion (MCAO)-induced ischemia/reperfusion (I/R) injury in rats. Furthermore, the influence of N2 on U46619-induced rat aorta contraction was investigated ex vivo. Moreover, we investigated the protective effects of N2 on rat brain microvessel endothelial cells (RBMECs) in hypoxia/deoxygenating (H/R) induced by Na2S2O4 in vitro. Cell viability and TXA2 biosynthesis were measured by 3-(4, 5-dimethylthiazol-2-yl)- 195 2, 5-diphenyltetrazolium bromide (MTT) and enzyme-linked immunosorbent assay (ELISA) assays, respectively. The results showed that N2 treatment effectively improves performance in neurological deficit and the Y-maze test and reduces the infarct volume in I/R rats. U46619-induced rat aorta contraction was inhibited by N2 ex vivo. Furthermore, N2 incubation improved the morphology of RBMECs, increased cell viability, and suppressed TXA2 production by inhibiting TXS during H/R damage.In summary, this study demonstrated that N2 was neural protective in focal cerebral I/R injury, which might be associated with the effects of N2 on endothelium protection and vascular contraction inhibition. In depth, the mechanisms underlying this phenomenon might be the influence of N2 on TXA2 production targeting TXS.

Bile acids permeabilize the blood brain barrier after bile duct ligation in rats via Rac1-dependent mechanisms

The blood brain barrier tightly regulates the passage of molecules into the brain and becomes leaky following obstructive cholestasis. The aim of this study was to determine if increased serum bile acids observed during cholestasis permeabilize the blood brain barrier. Rats underwent bile duct ligation or deoxycholic or chenodeoxycholic acid injections and blood brain barrier permeability assessed. In vitro, the permeability of rat brain microvessel endothelial cell monolayers, the expression and phosphorylation of occludin, ZO-1 and ZO-2 as well as the activity of Rac1 was assessed after treatment with plasma from cholestatic rats, or bile acid treatment, in the presence of a Rac1 inhibitor. Blood brain barrier permeability was increased in vivo and in vitro following bile duct ligation or treatment with bile acids. Associated with the bile acid-stimulated increase in endothelial cell monolayer permeability was elevated Rac1 activity and increased phosphorylation of occludin. Pretreatment of endothelial cell monolayers with a Rac1 inhibitor prevented the effects of bile acid treatment on occludin phosphorylation and monolayer permeability. These data suggest that increased circulating serum bile acids may contribute to the increased permeability of the blood brain barrier seen during obstructive cholestasis via disruption of tight junctions.

Synergistic inhibition of angiogenesis and glioma cell-induced angiogenesis by the combination of temozolomide and enediyne antibiotic lidamycin

Present work mainly evaluated the inhibitory effects of lidamycin (LDM), an enediyne antibiotic, on angiogenesis or glioma-induced angiogenesis in vitro and in vivo, especially its synergistic anti-angiogenesis with temozolomide (TMZ). LDM alone efficiently inhibited proliferations and induced apoptosis of rat brain microvessel endothelial cells (rBMEC). LDM also interrupted the tube formation of rat brain microvessel endothelial cells (rBMEC) and rat aortic ring spreading. The blockade of rBMEC invasion and C6 cell-induced rBMEC migration by LDM was associated with decrease of VEGF secretion in a co-culture system. TMZ dramatically potentiated the effects of LDM on anti-proliferation, apoptosis induction, and synergistically inhibited angiogenesis events. As determined by western blot and ELISA, the interaction of tumor cells and the rBMEC was markedly interrupted by LDM plus TMZ with synergistic regulations of VEGF induced angiogenesis signal pathway, tumor cell invasion/migration, and apoptosis signal pathway. Immunofluorohistochemistry of CD31 and VEGF showed that LDM plus TMZ resulted in synergistic decrease of microvessel density (MVD) and VEGF expression in human glioma U87 cell subcutaneous xenograft. This study indicates that the high efficacy of LDM and the synergistic effects of LDM plus TMZ against glioma are mediated, at least in part, by the potentiated anti-angiogenesis.

Transport of active flavonoids, based on cytotoxicity and lipophilicity: An evaluation using the blood–brain barrier cell and Caco-2 cell models

This in vitro study aims to evaluate and compare transmembrane transport of eight cardio-cerebrovascular protection flavonoids including puerarin, rutin, hesperidin, quercetin, genistein, kaempferol, apigenin and isoliquiritigenin via the rat blood–brain barrier cell and Caco-2 cell monolayer models, based on the data of cytotoxicity and lipophilicity. The cytotoxicity of the flavonoids to rat brain microvessel endothelial cell was determined by the MTT assay. The apparent permeability coefficients (Papp) of the flavonoids were calculated from the unilateral transport assays in Transwell system with simultaneous determination using a high performance liquid chromatography. The results showed that the cytotoxicity and oil–water partition coefficient of the flavonoids modified by the number and position of the glycoside and hydroxyl group were the key determinant for the transmembrane transport. The Papp values of the flavonoids reduced adversely when the numbers of glycoside and hydroxyl groups of the flavonoids increased accordingly. The tested flavonoids exhibited time-dependent Papp values in these models. The efflux mechanism related with P-glycoprotein also existed with the polar flavonoids; verapamil could enhance the permeation of rutin and quercetin via inhibition of P-glycoprotein. We propose that genistein and isoliquiritigenin with the permeation priority in vitro Caco-2 and BBB cell model could be better as the drug candidates for cardio-cerebral vascular protection. These findings provided important information for establishing the transport relationship for the flavonoid compounds and evaluating the potential oral bioavailability and brain distribution of the flavonoids.

CJY, an isoflavone, interacts with ATPase of P-glycoprotein in the rat brain microvessel endothelial cells (RBMECs)

Our previous study reported CJY, an isoflavone, can reverse P-glycoprotein (P-gp) efflux activity in rat brain microvessel endothelial cells (RBMECs). In the present report, by assessment of ATPase activity of RBMECs, we gained further insight into the nature of the CJY interactions with P-gp. The results revealed that the basal P-gp ATPase activity was increased by CJY. Kinetic studies on ATPase activity showed the effects of Tetrandrine (Tet) on CJY-stimulated, CsA on CJY-stimulated, and CsA on Tet-stimulated P-gp ATPase activity were all non-competitive inhibition, indicating that these substrates can simultaneously but independently bind to diverse sites on P-gp. Furthermore, the combined effects of CJY with Tet, and CJY with CsA were also evaluated isobolographically. The results showed synergistic interactions in both combinations, implying that combined treatment of CJY with other modulators may exert synergistic interactions for the drug’s penetration into the brain and the treatment of neurological disorders.