Silencing of circular RNA ANRIL attenuates oxygen-glucose deprivation and reoxygenation-induced injury in human brain microvascular endothelial cells by sponging miR-622

BACKGROUND: Circular RNA (circRNA) is highly expressed in the brain tissue, but its molecular mechanism in cerebral ischemia-reperfusion remains unclear. Here, we explored the role and underlying mechanisms of circRNA antisense non-coding RNA in the INK4 locus (circ_ANRIL) in oxygen-glucose deprivation and reoxygenation (OGD/R)-induced cell injury. RESULTS: The expression of circ_ANRIL in OGD/R-induced human brain microvascular endothelial cells (HBMECs) was significantly up-regulated, while that of miR-622 was significantly down-regulated. Overexpression of circ_ANRIL significantly inhibited the proliferation of OGD/R-induced HBMECs and aggravated OGD/R-induced cell apoptosis. Moreover, circ_ANRIL overexpression further increased the secretion of interleukin (IL)-1ß, IL-6, tumor necrosis factor-a, and monocyte chemoattractant protein-1 in OGD/R-treated HBMECs. The results of bioinformatics analysis and luciferase reporter assay indicated that circ_ANRIL served as an miR-622 sponge to negatively regulate the expression of miR-622 in OGD/R-treated HBMECs. Additionally, circ_ANRIL silencing exerted anti-apoptotic and anti-inflammatory effects by positively regulating the expression of miR-622. Furthermore, inhibition of OGD/R-induced activation of the nuclear factor (NF)-kB pathway by circ_ANRIL silencing was significantly reversed by treatment with miR-622 inhibitor. CONCLUSIONS: Knockdown of circ_ANRIL improved OGD/R-induced cell damage, apoptosis, and inflammatory responses by inhibiting the NF-κB pathway through sponging miR-622.

Effect of Sijunzi Decoction on regulation of Aβ-related proteins across blood-brain barrier / 中国中药杂志

According to traditional Chinese medicine, "spleen transport" is closely related to the metabolism of substance and energy. Studies have shown that Alzheimer's disease(AD) is a disease related to glucose and lipid metabolism and energy metabolism. The traditional Chinese medicine Jiangpi Recipe can improve the learning ability and memory of AD animal model. Sijunzi Decoction originated from Taiping Huimin Hefang Prescription is the basic prescription for strengthening and nourishing the spleen, with the effects of nourishing Qi and strengthening the spleen. In this experiment, human brain microvascular endothelial cells(HBMEC) and Sijunzi Decoction water extract(0.25, 0.5, 1 mg·L~(-1)) were pre-incubated for 2 h, and then Aβ_(25-35) oligomers(final concentration 40 μmol·L~(-1)) was added for co-culture for 22 hours. The effect of Sijunzi Decoction on the activity of Aβ_(25-35) oligomer injured cells and the expression of related proteins were investigated. Q-TOF-LC-MS was used first for principal component analysis of Sijunzi Decoction water extract. Then MTT assay was used to investigate the effect of Sijunzi Decoction water extract on the proliferation of HBMEC cells. Real-time fluorescence quantitative PCR(RT-qPCR) was employed to detect the mRNA expression of GLUT1, RAGE, and LRP1. The expression of Aβ-related proteins across blood-brain barrier(RAGE, LRP1) was detected by Western blot. The results showed that 40 μmol·L~(-1) Aβ_(25-35) oligomers could induce endothelial cell damage, reduce cell survival, increase expression of RAGE mRNA and RAGE protein, and reduce expression of GLUT1 mRNA, LRP1 mRNA, and LRP1 protein. Sijunzi Decoction water extract could reduce the Aβ_(25-35) oligomer-induced cytotoxicity of HBMEC, decrease the expression of RAGE mRNA and RAGE protein, and increase the expression of GLUT1 mRNA, LRP1 mRNA and LRP1 protein. The results indicated that Sijunzi Decoction could reduce the injury of HBMEC cells induced by Aβ_(25-35) oligomer, and regulate the transport-related proteins GLUT1, RAGE and LRP1, which might be the mechanism of regulating Aβ_(25-35) transport across the blood-brain barrier.

VP1-binding protein glucose-regulated protein 78 as an important mediator for enterovirus 71 infecting human brain microvascular endothelial cells

Purpose: Enterovirus 71 (EV71) is one of the main pathogens causing hand, foot and mouth disease, which could even induce severe brain damage in some patients. As the underlying mechanism of the invasion and replication process still remains largely unknown, we investigated the role of candidate proteins expressed during EV71 invasion in human brain microvascular endothelial cells (HBMECs) to delineate the pathophysiological mechanism of EV-71 infection. Materials and Methods: Ninety-one candidate EV71-associated proteins which could bind the major capsid protein (viral protein 1 [VP1]) of EV71 on the HBMEC were identified by applying an analysis of glutathione-S-transferase pull-down coupling with liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). Seventy-eight kDa glucose-regulated protein 78 (GRP78) binding to the VP1 protein was further validated by co-immunoprecipitation, immunofluorescence and western blot analysis. To explore the role of GRP78 in EV71 infection, GRP78 was knocked down and overexpressed in HBMEC and was verified by TCID50 assay. Results: LC-ESI-MS/MS-identified 91 proteins were subjected to gene ontology analysis, and on molecular and biological function analysis revealed GRP78 act as an important binding protein in mediating EV71 infection. In addition, immunofluorescence demonstrated the co-localisation of GRP78 and VP1 in cytoplasm of the infected HBMEC. The TCID50 assay showed that knockdown of GRP78 could attenuate the replication capacity of EV71 in HBMEC, and the overexpression could increase the virus titre in HBEMC at 24 h post-infection suggesting that GRP78 was associated with the replication capacity of EV71 in HBMEC. Conclusion: These findings provided evidence that GRP78 plays an important role during the progression of EV71 infection as a mediator in HBMEC.

Effects of Treponema pallidum on the expression of chemokine ligand 6, 8, 10 in human brain microvascular endothelial cells / 中华皮肤科杂志

Objective To evaluate the effects of Treponema pallidum (T.pallidum) on the expression of chemokine ligands (CXCL) in human brain microvascular endothelial cells (HBMECs).Methods HBMECs were randomly divided into 4 groups,which were treated with viable T.pallidum suspension (T.pallidum group),heat-inactivated T.pallidum suspension (inactivated T.pallidum group),200 μg/L lipopolysaccharide (LPS group) and cell culture medium (blank control group),respectively,for 6,12 and 24 hours.Fluorescence-based quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were performed to determine the mRNA and protein expression of CXCL6,CXCL8 and CXCL10 in HBMECs in the above groups respectively.Transwell migration assay was conducted to evaluate the effects of T.pallidum-stimulated HBMECs on the chemotaxis of human promyelocytic HL-60 leukemia cells (HL-60 cells).Results At 6,12 and 24 hours,the T.pallidum group showed significantly higher mRNA expression of CXCL6,CXCL8 and CXCL10 in HBMECs compared with the blank control group and inactivated T.pallidum group (all P ＜ 0.05),while there were no significant differences between the blank control group and inactivated T.pallidum group (all P ＞ 0.05).Compared with the LPS group,the T.pallidum group showed significantly decreased mRNA expression of CXCL6 and CXCL8 (P ＜ 0.05),but similar mRNA expression of CXCL10(P ＞ 0.05)at 6,12 and 24 hours.At these time points,the levels of CXCL6 and CXCL8 in the culture supernatant of HBMECs were significantly higher in the T.pallidum group than in the blank control group and the inactivated T.pallidum group (all P ＜ 0.05),but no significant differences were observed between the blank control group and the inactivated T.pallidum group (both P ＞ 0.05).Moreover,there were no significant differences in the level of CXCL10 in the culture supernatant of HBMECs among the T.pallidum group,the inactivated T.pallidum group and the blank control group (all P ＞ 0.05).The number of migratory HL-60 cells in the lower Transwell chambers was significantly higher in the T.pallidum group than in the inactivated T.pallidum group and the blank control group (both P ＜ 0.05).Conclusion Viable T.pallidum can up-regulate the gene expression of CXCL6,CXCL8 and CXCL10 in HBMECs,promote the secretion of CXCL6 and CXCL8,and enhance the chemotactic effect of HBMECs on HL-60 cells,which may play a certain role in the occurrence of neurosyphilis.

The biological effects of magnetic fields on microvascular endothelial cells in the human brain / 中华物理医学与康复杂志

Objective To observe the biological effects of magnetic fields of different intensities on microvascular endothelial cells in the human brain (HBMECs).Methods HBMECs were cultured in vitro under normal conditions and randomly divided into a control group and several magnetic induction groups——26 mT,62.5 mT,110.7 mT and 215.6 mT at the center pole.Any changes to the cytomembranes were observed 72 h after planking using the lactate dehydrogenase (LDH) method.Superoxide dismutase and malondialdehyde methods were used to detect cellular oxidation due to the magnetic field.An inverted microscope was used to observe any changes in cell morphology,and flow cytometry was employed to detect cell apoptosis.Results Compared with the control group,the LDH value of the 215.6 mT group was significantly higher,but there were no significant differences in oxidative damage,apoptosis or morphology observed.Moreover,there were no significant differences between the controls and the 26 mT,62.5 mT and 110.7 mT groups in any of the above measurements.Conclusion Magnetic fields of different intensity have different biological effects on HBMECs.A 215.6 mT magnetic field influences their cell membranes but causes no oxidative damage,cell apoptosis or morphological changes.These observations and the mechanism need further exploration.

The effects of Celecoxib on human brain microvascular endothelial cells release 6-keto-PGF1α and TXB2 and apoptosis after radiation / 中华放射肿瘤学杂志

Objective To investigate the effect of Celecoxib on human brain microvascular endothelial cells release6-keto-PGF1α,TXB2 and apotosis after irradiation.Methods The logarithmic growth phase cells were divided into control groups (Con),simple irradiation (IR) groups and combination groups (IR+C).CCK-8 and clone formation experiment were used to evaluate the effects of radiosensitivity and toxicity of celecoxib.The results were observed atthe time point of 6 h,12 h,24 h,48 h after irradiation.ELISA was used to test the contents of 6-keto-PGF1α and TXB2,which metabolized by PGI2 and TXA2 from culture medium after irradiation at different time points in different groups.TXB2/6-keto-PGF1αratios were calculated.Annexin V-FITC/PI double staining method was used to measure the apoptosis rates at different time points in different groups.Western blot was used to measure the protein expression.Paired t test difference.Results Compared with simple irradiation group,there were no significant radiosensitivity (SER=0.96) in combination groups incubated with30 μmol/L of celecoxib.Compared with the control group,the ratio of TXB2/6-keto-PGF1αincreased at each time point in IR and IR+C (P<0.05),and the apoptosis rates increased (P<0.05).Cox-2,P-JNK and Cleaved caspase-3 increased.Compared with IR,the ratio of TXB2/6-keto-PGF1αdecreased at each time point in IR+C (P<0.05),and the apoptosis rates decreased (t=3.34~6.38,P< 0.05).The protein expression of Cox-2,P-JNK and Cleaved caspase-3 decreased.Conclusions Celecoxib may help to protect HBMECs from releasing TXA2 and decreasing the ratio of TXB2/6-keto-PGF1α,and inhibitting apoptosis after irradiation.The mechanisms of apoptosis inhibition may be related to the inhibition of Cox-2 and P-JNK,caspase-3 Cleaved proteinexpressions.

Protective effects of three phenylallyl compounds from Guizhi decoction ox-LDL-induced oxidative stress injury of human brain microvascular endothelial cells / 中国中药杂志

The main objective of this research is to observe protective effects of three phenylallyl compounds(cinnamyl alcohol,cinnamaldehyde and cinnamic acid)from Guizhi decoction against ox-LDL-induced oxidative stress injury on human brain microvascular endothelial cells(HBMEC).In this study,the toxicity and optimal protective concentration of three phenylallyl compounds from Guizhi decoction were determined by MTT assay.The HBMEC were divided into control group(DMSO),model group(ox-LDL),tert-butylhydroquinone (t-BHQ) group,cinnamyl alcohol group, cinnamaldehyde group and cinnamic acid group.The model group were treated with ox-LDL (50 mg•L⁻¹)for 24 h,other groups were separately treated with t-BHQ, cinnamyl alcohol, cinnamaldehyde and cinnamic acid of 20 μmol•L⁻¹, and exposed to ox-LDL (50 mg•L⁻¹) for 24 h at the same time.The survival rate of HBMEC was detected by MTT assay,reactive oxygen species(ROS) production of injured cells were detected using laser scanning confocal microscope (LSCM),the content of SOD, MDA, eNOS and NO in HBMEC was determined by ELISA, and the expressions of Nrf2 mRNA were detected by quantitative Real-time PCR(qRT-PCR).The results shows that oxidative stress injury of HBMEC could be induced by ox-LDL, the three phenylallyl compounds from Guizhi decoction did not affect morphology and viability of normal HBMEC.Compared with model group, the three phenylallyl compounds from Guizhi decoction could improve the above oxidative stress status and up-regulate Nrf2 mRNA expressions in injured HBMEC(P<0.05, P<0.01) .These findings suggested that the three phenylallyl compounds from Guizhi decoction have certain protective effects against ox-LDL-induced oxidative stress injury on HBMEC(cinnamaldehyde> t-BHQ> cinnamic acid>cinnamyl alcohol),the protective mechanism maybe related to regulation of antioxidant enzymes gene expression in HBMEC by Nrf2.

High concentrations of urea induce human brain microvascular endothelial cell line to produce inflammatory cytokines / 基础医学与临床

Objective To explore high concentrations of urea-induced human brain microvascular endothelial cell line( HBMECs) to produce inflammatory cytokines and possible mechanism .Methods HBMECs were incubated in high concentrations of urea or mannitol ( as osmotic control ) for 3,6,12 and 24 hours.Expression of TNF-αand iNOS was observed by immunofluorescence .Western blot analysis was employed to assess the protein expressions of TNF-α, iNOS, COX-2, NF-κB/P65 and p-P65.NO concentration was determined by a commercial NO assay kit . Results Immunofluorescence showed high positive immunostaining of TNF-αand iNOS after incubation in high concentration of urea stimulued as compared with control group .The protein expressions of TNF-α, COX-2 and p-P65 were significantly increased at 3 and 6 hours after high urea treatment (P<0.01), and iNOS was continued to increase from 3 to 24 hours ( P<0.01 ) .Moreover , NO content was increased at 3 hours after high urea treatment ( P<0.05 ) .Conclusions High concentration of urea can induce HBMECs to produce inflammatory cytokines .

Attachment of Treponema pallidum to human brain microvascular endothelial cells in vitro:, an experimental study / 中华皮肤科杂志

Objective To observe the attachment of Treponema pallidum to human brain microvascular endothelial cells (HBMECs) in vitro.Methods Some primary cultured HBMECs were inoculated into in 24-well plates to be cocultured with the suspension of T.pallidum at a concentration of 1.6 × 107 treponemes/ml.After 0.5,2 and 4 hours of co-culture,scanning electron microscopy was conducted to observe the attachment of T.pallidum to HBMECs.Some HBMECs were cocultured with the presence of T.pallidum suspensions at different concentrations (4 × 106,8 × 106,1.6 × 107 treponemes/ml) for 2,4,6 and 16 hours,then,dark-field microscopy was performed to count the number of treponemes that attached to single HBMECs.Statistical analysis was carried out by using repeated-measures analysis of variance.Results As scanning electron microscopy showed,treponemes gathered at some regions on the surface of HBMECs when they attached to HBMECs.In addition,T.pallidum partly merged with the membrane of HBMECs at the site of attachment.After co-culture with T.pallidum suspensions,the number of treponemes that attached to single HBMECs was significantly different among different time points (F =387.72,P ＜ 0.001) and among different concentrations of T.pallidum suspensions (F =593.23,P ＜ 0.001),with an interaction effect between the concentration of T.pallidum suspensions and incubation period (F =98.74,P ＜ 0.001).Concretely speaking,the number of treponemes that attached to single HBMECs increased over time until 6 hours after the start of coculture,then showed a decreasing trend,and reached the nadir value at 16 hours.Conclusion T.pallidum can adhere to cultured HBMECs in vitro,likely by the merger of its end with the membrane of HBMECs at some regions.

Quercetin protects human brain microvascular endothelial cells from fibrillar β-amyloid1-40-induced toxicity

Amyloid beta-peptides (Aβ) are known to undergo active transport across the blood-brain barrier, and cerebral amyloid angiopathy has been shown to be a prominent feature in the majority of Alzheimer׳s disease. Quercetin is a natural flavonoid molecule and has been demonstrated to have potent neuroprotective effects, but its protective effect on endothelial cells under Aβ-damaged condition is unclear. In the present study, the protective effects of quercetin on brain microvascular endothelial cells injured by fibrillar Aβ 1-40 (fAβ 1-40) were observed. The results show that fAβ 1-40-induced cytotoxicity in human brain microvascular endothelial cells (hBMECs) can be relieved by quercetin treatment. Quercetin increases cell viability, reduces the release of lactate dehydrogenase, and relieves nuclear condensation. Quercetin also alleviates intracellular reactive oxygen species generation and increases superoxide dismutase activity. Moreover, it strengthens the barrier integrity through the preservation of the transendothelial electrical resistance value, the relief of aggravated permeability, and the increase of characteristic enzyme levels after being exposed to fAβ 1-40. In conclusion, quercetin protects hBMECs from fAβ 1-40-induced toxicity.

Regulating effect of Tongxinluo on HIF-1α,VEGF generated by the human brain microvascular endothelial cells induced by β-amyloid1-42 / 中国药理学通报

Aim To study the regulating effect of Tongxinluo on HIF-1α,VEGF that Generated by the Human brain microvascular endothelial cells induced by β-amyloid 1-42.Methods Human brain microvascular endothelial cells were pre-incubated for 4h by Tongxinluo, then injured by β-amyloid 1-42.VEGF protein and HIF-1α were detected by Western blot.Result In the Aβ treated Human brain microvascular endothelial cells,viable cells decreased.The expression of VEGF was Reduced, and HIF-1α elevated. After the cells were treated with Tongxinluo, all the above indexes were improved.Conclusion Tongxinluo can enhance the VEGF protein expressions by the HIF-1α and protect the Human brain microvascular endothelial cells.

Regulating effect of Tongxinluo on HIF-1?,VEGF generated by the human brain microvascular endothelial cells induced by ?-amyloid1-42 / 中国药理学通报

Aim To study the regulating effect of Tongxinluo on HIF-1?,VEGF that Generated by the Human brain microvascular endothelial cells induced by ?-amyloid 1-42. Methods Human brain microvascular endothelial cells were pre-incubated for 4h by Tongxinluo,then injured by ?-amyloid 1-42. VEGF protein and HIF-1? were detected by Western blot. Result In the A? treated Human brain microvascular endothelial cells,viable cells decreased. The expression of VEGF was Reduced,and HIF-1? elevated. After the cells were treated with Tongxinluo,all the above indexes were improved. Conclusion Tongxinluo can enhance the VEGF protein expressions by the HIF-1? and protect the Human brain microvascular endothelial cells.