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Purpose To clarify the effect of adenosine on brain metastasis of lung cancer and the possible mechanism of adenosine promoting brain metastasis of lung cancer. Methods Western blot was used to dynamically detect the expression level of hypoxia inducible factor-1 (HIF-1) in lung cancer cells and tight junction protein ZO-1 in brain microvascular endothelial cells on blood-brain barrier. The content of adenosine in lung cancer cell culture was determined by ELISA. Fluorescence analysis was used to detect the changes of permeability of the blood-brain barrier model in vitro. Hemocytometer counts the number of A549 lung cancer cells in Transwell's lower chamber. Results The expression level of HIF-1 in lung cancer cells and the content of adenosine in lung cancer cell culture reached the highest level when lung cancer cells were deprived of oxygen for 12 hours. At the same time, the expression level of ZO-1 protein in the blood-brain barrier was the lowest, the blood-brain barrier permeability was the highest (7.11), and the number of lung cancer cells passing through the blood-brain barrier model was the highest (84.6). The permeability of the blood-brain barrier model increased after the action of adenosine, and its change trend was consistent with the effect of hypoxic lung cancer cell culture solution. Conclusion Hypoxia can induce the lung cancer cell to release adenosine, the increased adenosine can reduce the expression of tight junction protein ZO-1 in blood brain barrier, which leads to the increase of permeability of blood-brain barrier and eventually lead to brain metastasis of lung cancer.
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Objective To investigate the effects of downregulated pregnane X receptor (PXR)on expression and activity of P-glycoprotein(P-gp)in mouse brain microvascular endothelial cells (mBMEC)exposed to glutamate (GLU)to mimic conditions during seizures. Methods The bEnd.3 cells,were cultured in vitro and treated with culture medium containing 0 μmol,10 μmol,50 μmol,100 μmol GLU for 30 min and exposed to 100 μM GLU for different durations (0 min,15 min,30 min). With PXR knockdown using siRNA,the cells were divided into NC siRNA plus GLU group and siRNA-PXR plus GLU group. Western blotting was used to detect the protein expressions of P-gp and PXR in each group. Immunofluorescence assay was used to detect localization of PXR in cells. The expression of P-gp mRNA was detected by RT-qPCR. Rhodamine123 (Rh123)accumulation assay was used to study the activity of the P-gp in cells.Results PXR and P-gp protein expressions in the 50 μmol and 100 μmol GLU group were significantly higher than that in the blank group(P＜0.05),especially maximal expressions occurred in the 100 μmol GLU group. GLU exposures as short as 15 min and 30 min significantly increased PXR expressions(all P＜0.05);P-gp expression in the 30 min groups was higher than that in the blank group (P＜0.05 ). The data of immunofluorescence analysis suggested that the PXR nuclear accumulation increased in the 100 μM GLU group, compared with the blank group(P＜0.05). Compared with NC siRNA plus GLU group,the protein level of PXR was decreased by approximately 37%[(1.00 ± 0.00)vs(0.63 ± 0.18);t＝3.41,P＝0.02]and the levels of P-gp protein and mRNA were respectively decreased by 43%[(1.00 ± 0.00)vs (0.57 ± 0.09);t＝7.94,P＝0.00] and 52%[(1.00 ± 0.04)vs (0.48 ± 0.08);t ＝10.98,P＝0.00]in the siRNA-PXR plus GLU group. The fluorescence intensity of intracellular Rh123 in the GLU group (0.72 ± 0.01)was lower than that in the blank group [(1.00 ± 0.03);t ＝9.66,P＝0.00]. The fluorescence intensity of Rh123 in the GLU plus verapamil (P-gp inhibitor)group (1.07 ± 0.04)was higher than that in the GLU group (t＝ －11.93,P＝0.00). The fluorescence intensity of Rh123 in the siRNA-PXR plus GLU group (0.91 ± 0.03)was higher than that in the NC siRNA plus GLU group[(0.69 ± 0.05);t＝ －7.52,P＝0.00]. Conclusions Downregulation of PXR expression results in the inhibition of P-gp expression and activity in the mBMEC exposed to GLU to mimic seizures. PXR may play an important role in the regulation of seizure-induced expression and activity of P-gp in the blood-brain barrier.
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OBJECTIVE@#To investigate the role of human host heme-oxygenase-1 (HO-1) in pathogenesis of cerebral malaria in the in vitro model.@*METHODS@#The effect of human host HO-1 [human brain microvascular endothelial cell (HBMEC)] on hemoglobin degradation in the co-culture model of HBMEC and ITG Plasmodium falciparum-infected red cells (iRBC) through measurement of the enzymatic products iron and bilirubin.@*RESULTS@#Following exposure to the HO-1 inducer CoPPIX at all concentrations, the HBMEC cells apoptosis occurred, which could be prominently observed at 15 μM of 3 h exposure. In contrast, there was no significant change in the morphology in the non-exposed iRBC at all concentrations and exposure time. This observation was in agreement with the levels of the enzymatic degradation products iron and bilirubin, of which the highest levels (106.03 and 1753.54% of baseline level, respectively) were observed at 15 μM vs. 20 μM at 3 h vs. 24 h exposure. For the effect of the HO-1 inhibitor ZnPPIX, HBMEC cell morphology was mostly unchanged, but significant inhibitory effect on cell apoptosis was seen at 10 μM for the exposure period of 3 h (37.17% of baseline level). The degree of the inhibitory effect as reflected by the level of iron produced was not clearly observed (highest effect at 10 μM and 3 h exposure).@*CONCLUSIONS@#Results provide at least in part, insight into the contribution of HO-1 on CM pathogenesis and need to be confirmed in animal model.
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Objective To investigate the role of human host heme-oxygenase-1 (HO-1) in pathogenesis of cerebral malaria in the in vitro model. Methods The effect of human host HO-1 [human brain microvascular endothelial cell (HBMEC)] on hemoglobin degradation in the co-culture model of HBMEC and ITG Plasmodium falciparum-infected red cells (iRBC) through measurement of the enzymatic products iron and bilirubin. Results Following exposure to the HO-1 inducer CoPPIX at all concentrations, the HBMEC cells apoptosis occurred, which could be prominently observed at 15 μM of 3 h exposure. In contrast, there was no significant change in the morphology in the non-exposed iRBC at all concentrations and exposure time. This observation was in agreement with the levels of the enzymatic degradation products iron and bilirubin, of which the highest levels (106.03 and 1753.54% of baseline level, respectively) were observed at 15 μM vs. 20 μM at 3 h vs. 24 h exposure. For the effect of the HO-1 inhibitor ZnPPIX, HBMEC cell morphology was mostly unchanged, but significant inhibitory effect on cell apoptosis was seen at 10 μM for the exposure period of 3 h (37.17% of baseline level). The degree of the inhibitory effect as reflected by the level of iron produced was not clearly observed (highest effect at 10 μM and 3 h exposure). Conclusions Results provide at least in part, insight into the contribution of HO-1 on CM pathogenesis and need to be confirmed in animal model.
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[Abstract ] Objective High-mobility group box-1 (HMGB1) is abundantly released in the epileptogenic brain tissue , but few reports are seen about the effect of HMGB 1 on the expression of P-glycoprotein ( P-gp) in the vascular endothelial cells of the epi-leptogenic tissue .This study is to explore whether HMGB 1 can regulate P-gp expression in the brain microvascular endothelial cells of the mouse in vitro . Methods Immortalized brain microvascular endothelial bEnd .3 cells of the mouse were cultured in vitro and al-located to different concentration groups ( treated with culture medium containing 10 , 100 , 500 , and 1000 ng/mL HMGB1 for 8 hours), treatment duration groups (treated with culture medium containing 100 ng/mL HMGB1 for 4, 8, 16, 24, and 32 hours), and a control group ( treated with culture medium without HMGB 1 ) .The mRNA expression of P-gp-encoding gene-multidrug resistance gene 1a (mdr1a) was detected by real-time qPCR, and its protein expression determined by Western blot and immunocytochemistry . Results The results of qPCR manifested that the expressions of mdr 1a mRNA were 1.646 ±0.176, 1.777 ±0.135, 1.617 ±0.043, and 1.398 ±0.182 in the 10, 100, 500, and 1000 ng/mL HMGB1 groups, respectively, significantly higher than 1.030 ±0.284 in the control group (P<0.05), and so were those in the 4, 8, 16, 24 h, and 32 h groups (2.655 ±0.112, 2.168 ±0.212, 1.823 ± 0.232, 1.418 ±0.376, and 1.445 ±0.123) than in the control (1.010 ±0.164) (P <0.05).Western blot showed a significant increase in the P-gp protein expression in all the concentration groups (P<0.05) as well as in the 8 h and 16 h treatment duration groups as compared with the control group (P<0.05).Immunocytochemis-try also revealed a higher P-gp expression in the HMGB1-treated than in the control cells (P<0.01). Conclusion HMGB1 can upregu-late the expressions of mdr1a mRNA and P-gp protein in the brain microvascular endothelial cells of the mouse , which may associated with drug resistance of central nervous system diseases , especially that of epilepsy .
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OBJECTIVE: To develop a kind of multifunctional targeting epirubicin liposomes for the treatment of brain tumor, to characterize their physicochemical properties, and to observe their targeting effects on the brain microvascular endothelial cells (BM-VECs) and on the brain glioma cells. METHODS: The 2-amino-2-deoxy-β-Z)-glucopyranose(NH2-Glu) was used as a targeting molecule and conjugated with a cholesterol-polyethylene glycol derivative (Chol-PEG2000-NHS) for obtaining the targeting functional material aimed at targeting to glucose transporter-1 (Glut-1) on the BMVECs of blood-brain barrier and further targeting to the glioma cells. To prepare the multifunctional targeting epirubicin liposomes, the targeting functional material was modified onto the surface of liposomes, and epirubicin was loaded into the core of liposomes as the anticancer drug. The encapsulation efficiency, particle size, polydispersity indexes and Zeta potential of the liposomes were measured, their cellular uptakes were performed on the BMVECs and the glioma cells. The inhibitory effect was performed on the glioma cells. RESULTS: The analysis by MALDI-TOF-MS demonstrated that the targeting functional material, Chol-PEG2000-Glu, was successfully synthesized. The multifunctional targeting epirubicin liposomes were prepared, and had an average particle size of approximately 125 nm, and were negatively charged. The encapsulation efficiency of epirubicin in the liposomes was about 93%. Results from flow cytometry indicated that the multifunctional targeting epirubicin liposomes had the highest cellular uptakes by BMVECs and by two kinds of brain glioma cells as compared with no-targeting epirubicin liposomes. The cytotoxic study showed that the multifunctional targeting epirubicin liposomes had the strongest inhibitory effect to brain glioma cells as compared with free epirubicin or no-targeting epirubicin liposomes. CONCLUSION: A new targeting material (Chol-PEG2000-Glu) and the multifunctional targeting epirubicin liposomes are developed, and the multifunctional targeting epirubicin liposomes exhibit the potential lortransporting across the blood-brain barrier (BBB), and selectively inhibiting the brain glioma cells.
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Objective To investigate the virulence role of ompT of Escherichia coli in the patho-genesis of neonatal meningitis .Methods Adhesive abilities of the parent strain E 44 and the isogenic ompT-deletion mutant strain ( E44 ∶ΔompT) to human brain microvascular endothelial cells were evaluated in in vitro model.Low-copy-number plasmid pST containing ompT locus and point mutant plasmid pST 85 were transferred into E44 ∶ΔompT to construct the complemented mutant strain , and its adhesive ability was ana-lyzed.Influences of ompT deletion on E44 strain in its ability of bacterial intestinal colonization and ability of penetrating the blood-brain barrier were determined . Results In comparison with the parent strain , E44 ∶ΔompT strain showed significantly impaired adhesive ability to human brain microvascular endothelial cells, which could be partly restored by inserting the complementary plasmids of pST and pST 85.Deletion of the ompT did not affect Escherichia coli K1 in normal intestinal colonization in in vivo model.E44 ∶ΔompT strain could induce bacteremia , which was similar to that induced by the parent strain , but its ability of crossing the blood-brain barrier was significantly declined .Conclusion The study demonstrate that ompT plays an important role as the virulence element of Escherichia coli in binding to brain microvascular endothe-lial cells and penetrating the blood-brain barrier .Further study should be performed to investigate the influ-ences of OmpT proteinase on the virulence of Escherichia coil.
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Objective Shunaoxin Dropping Pills (SDPs), a Chinese patent medicine, has been used widely in China for the treatment of headache, amnesia, and insomnia. The aim of the present study is to observe the effect of SDPs on inducing angiogenesis and neurogenesis in vitro. Methods The present testing system using the serum obtained from animals ig treated with SDPs and a co-culture system in vitro was used to investigate if SDPs promotes brain microvascular endothelial cells (BMECs) tube formation and neural differentiation of neural stem/progenitor cells (NSPCs), which plays important roles in angiogenesis and neurogenesis. Results The SDPs serum sampled from rats ig treated with SDPs for 3 d dose-dependently promoted the tube like structure formation of cultured BMECs, and enhanced the fraction of MAP-2 positive cells of NSPCs, which co-cultured with the BMECs and astrocyte. In addition, there was no significant change in the percentage of glial fibrillary acidic protein positive cells. Conclusion Our results show that SDPs serum can induce neural differentiation and BMECs tube formation in vitro.
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The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phos-pholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase A2 (PLA2) and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA2. Acanthamoeba exhibited optimal phospholipase activities at 37degrees C and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA2-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.
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Humans , Acanthamoeba/enzymology , Cell Adhesion , Cells, Cultured , Endothelial Cells/parasitology , Keratitis/parasitology , Phospholipase D/genetics , Phospholipases A2/genetics , Protozoan Proteins/genetics , Soil/parasitologyABSTRACT
Objective To investigate the molecular mechanism for change in permeability in brain microvascular endothelial cells (bEnd.3) induced by lipopolysaccharide (LPS). Methods Monolayers of bEnd.3 were exposed to LPS,in the presence or absence of exoenzyme C3 transferase. We monitored the monolayer barrier integrity by transendothelial electrical resistance assay (TEER),activity of RhoA by pull down assay,NF-κB by luciferase reporter assay,and F-actin dynamic structure by Rhodamine-phalloidin staining. Results Incubation of monolayers with LPS caused substantial barrier hyperpermeability. Under the had been treated for 3 and 12 h with LPS (P＜0.05). Such effects could be inhibited partly by pretreatment of RhoA inhibitor exoenzyme C3 transferase. LPS activated RhoA and NF-κB at 0.5 h. The C3 transferase could significantly reverse the NF-κB activation (P＜0.05). The F-actin rearrangments displayed in a time-dependent manner and occurred originally after the stimulation of LPS for 3 h,which could be diluted by the pretreatment of C3 transferase as well. Conclusion LPS induces the disruption of F-actin cytoskeleton and brain microvascular endothelial barrier integrity,in part,through RhoA and NF-κB activation. The mechanism underlying this pathophysiological effect of RhoA is to influence the disruption of the F-actin cytoskeleton by regulating NF-κB activites.
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Objective To investigate the function of chemokine receptor 5（CCR5）in human brain microvascular endothelial cells（HBMEC）during T lymphocytes transendothelial migration induced by amyloid beta（Aβ）.Methods The in vitro model of blood-brain barrier was established with cultured HBMEC monolayer.The ability of T lymphocytes transendothelial migration induced by Aβ was analyzed by Transwell and Millicell-ERS endothelia volt-ohmmeter.Results Aβ could promote T lymphocytes to migrate through the HBMEC monolayer.Over-expression of CCR5 in HBMEC promoted T lymphocytes to migrate through HBMEC monolayer.Expression of CCR5 mutant in HBMEC inhibited the transendothelial migration of T lymphocytes.Conclusion CCR5 of HBMEC is involved in T lymphocytes transendothelial migration induced by Aβ.
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Objective To investigate the effect of injectable Xuebijing on the proliferation of mouse brain microvascular endothelial cell in vitro.Methods Cultured mouse brain microvascular endothelM cell line bEnd. 3 was treated by injectable Xuebijing of different concentrations,0 (control),5,25 and 50 mg/ml.The regulatory. effect of Xuebijing on the proliferation of cell line bEnd.3 was observed and studied by means of MTT method and cell cycle analyzed with flow cytometry.Results Compared to the control group,MTT and proliferation index (PI) of 5 and 25 mg/ml groups were significantly increased at 12 and 24 h,and PI,but not MTT,of these 2 groups was decreased remarkably at 48 h.Meanwhile,50 mg/ml group showed significantly decreased MTT at 24 and 48 h,and PI of this group was increased obviously at 12 and 24 h,but decreased significantly at 48 h. Conclusions Injectable Xuebijing at certain concentrations might promote the proliferation of cultured mouse brain microvascular endothelial,cells within specific time frame.
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@#The adhesion molecules expressed by brain microvascular endothelial cell maintain the structure and function of brain, ensure the continuity of endothelium cell and penetrate blood-brain barrier in physiology. They also intervene inflammation cells that remove or orientate regular or inflammation organism in pathology. The adhesion molecules of Immunoglobulin supperfamily and Selectin family are expressed by brain microvascular endothelial cell and have affinity with the ischemic brain damage. The advance in research on relationship between the expression of adhesion molecules by brain microvascular endothelial cell and ischemic brain damage is reviewed in this article.
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Objective: To observe the expression of cell adhesion molecules ICAM-1 and VCAM-1of cultured rat brain microvascular endothelial cells(MVEC),expecting to explore the mechanisms of new QingKaiLing injection protecting brain from injury of inflammatory cascade in cerebral ischemia diseases.Methods: Rat cerebral MVEC were extracted by separating microvessel sections and collagenase enzymatic digesting,an in vitro ischemia reperfusion model was established(Kreb,95%N2+5%CO2),the protein and mRNA expression of ICAM-1 and VCAM-1 were detected by using immunocytochemical stain and RT-PCR method.Results:The expression of adhesion molecules of model group were significantly higher than those of noral group(P
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Objective: To study the influence of conditioned medium of rat brain microvascular endothelial cells on the activity of cortical neurons and the protective effect of Tong Luo Jiu Nao Injection.Methods: We collected the conditioned media from 4 different cultured endothelial cell groups,which were normal endothelial cells,the normal ones and treated with Tong Luo Jiu Nao Injection,the injured ones damaged by simulated cerebral ischemia,as well as the injured ones and treated with Tong Luo Jiu Nao Injection,respectively(N-CM,NT-CM,I-CM,IT-CM).Then,the conditioned medium was added into the cultures of the normal neurons and the damage ones which are injured by simulated cerebral ischemia as well,respectively.The effect of each type of conditioned medium on the activities of neurons was determined through the measurement of MTT and the transduction rate of LDH.Results:(1) N-CM has no obvious effect on the normal neurons,but does show some protective effect on the injured ones by increasing its activity significantly;(2) I-CM could decrease the activity of the normal neurons as well as aggravate the damage on the injured ones,while this injury effect can be reversed remarkably by IT-CM.Conclusion: The paracrine secretion of the brain microvascular endothelial cells might be one of the vital mechanisms in cerebral ischemic injury,indicating that the brain microvascular endothelial cells could be the therapeutic targets of Chinese medicine,which are not able to permeate through the Blood-Brain Barrier.