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Table of Contents
Year : 2021  |  Volume : 64  |  Issue : 2  |  Page : 88-96

Protection of the neurovascular unit from calcium-related ischemic injury by linalyl acetate

1 Department of Basic Nursing Science, School of Nursing, Korea University, Seoul, Republic of Korea; Department of Nursing, School of Nursing, National Taipei University of Nursing and Health Sciences, Taipei, Taiwan
2 Department of Basic Nursing Science, School of Nursing, Korea University, Seoul, Republic of Korea
3 Department of Basic Nursing Science, School of Nursing; BK21 FOUR Program of Transdisciplinary Major in Learning Health Systems, Graduate School, Korea University, Seoul, Republic of Korea

Date of Submission20-Nov-2020
Date of Decision17-Mar-2021
Date of Acceptance23-Mar-2021
Date of Web Publication28-Apr-2021

Correspondence Address:
Prof. Geun Hee Seol
Department of Basic Nursing Science, College of Nursing, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841
Republic of Korea
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cjp.cjp_94_20

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Calcium-related ischemic injury (CRII) can damage cells of the neurovascular unit (NVU). Here, we investigate the protective effects of linalyl acetate (LA) against CRII-induced NVU damage and evaluate the underlying mechanisms. The protective effects of LA in cell lines representative of NVU components (BEND, SH-SY5Y, BV2, and U373 cells) were evaluated following exposure to oxygen-glucose deprivation/reoxygenation alone (OGD/R-only) or OGD/R in the presence of 5 mM extracellular calcium ([Ca2+]o) to mimic CRII. LA reversed damage under OGD/R-only conditions by blocking p47phox/NADPH oxidase (NOX) 2 expression, reactive oxygen species (ROS) production, nitric oxide (NO) abnormality, and lactate dehydrogenase (LDH) release only in the BEND cells. However, under CRII-mimicking conditions, LA reversed NO abnormality and matrix metalloproteinase (MMP)-9 activation in the BEND murine brain endothelial cells; inhibited p47phox expression in the human SH-SY5Y neural-like cells; decreased NOX2 expression and ROS generation in the BV2 murine microglial cells; and reduced p47phox expression in the U373 human astrocyte-like cells. Importantly, LA protected against impairment of the neural cells, astrocytes, and microglia, all of which are cellular components of the NVU induced by exposure to CRII-mimicking conditions, by reducing LDH release. We found that LA exerted a protective effect in the BEND cells that may differ from its protective effects in other NVU cell types, following OGD/R-induced damage in the context of elevated [Ca2+]o.

Keywords: Calcium-related ischemic injury, linalyl acetate, neurovascular unit

How to cite this article:
Hsieh YS, Shin YK, Seol GH. Protection of the neurovascular unit from calcium-related ischemic injury by linalyl acetate. Chin J Physiol 2021;64:88-96

How to cite this URL:
Hsieh YS, Shin YK, Seol GH. Protection of the neurovascular unit from calcium-related ischemic injury by linalyl acetate. Chin J Physiol [serial online] 2021 [cited 2022 Aug 17];64:88-96. Available from: https://www.cjphysiology.org/text.asp?2021/64/2/88/315093

  Introduction Top

Calcium-related ischemic stroke continues to be a clinical challenge, and its underlying mechanisms remain elusive. It is associated with hypertension, which can lead to greater localized increases in blood–brain barrier (BBB) permeability[1] or disruption in patients who suffer an ischemic stroke.[2] Destruction of the BBB is a severe complication that promotes the development of neurological dysfunction 6–48 h after the onset of an ischemia stroke.[3] Even in the United States, ~33% of patients experience this complication.[4]

Recent investigations of mechanisms involved in ischemia have shown that the neurovascular unit (NVU), consisting of neural cells, astrocytes, microglia, and vascular endothelial cells, is a target of ischemia-induced cell damage, warranting studies of possible protective mechanisms.[5],[6] It has been reported that the NVU is involved in the pathogenesis of BBB damage, neuronal cell degeneration, glial reactions, and immune cell responses in ischemic stroke.[7] Neuroprotective effects on the NVU are thought to be involved in preventing BBB disruption and endothelial damage caused by ischemic stroke.[8] However, to date, therapeutic options for ischemic stroke patients are limited to tissue plasminogen activator, antiplatelet drugs, and catheter devices – all of which have side effects and complex constraints.[9] Given these less-than-ideal options, it is important to identify safer and more effective strategies.

The concentration of intracellular calcium ([Ca2+]i) plays a key role in the regulation of blood vessels.[10] Abnormal increases in [Ca2+]i caused by oxidative stress are capable of disrupting both vasoconstriction and vasorelaxation,[11] leading to perturbation of BBB function and apoptosis of human brain endothelial cells.[12] Linalyl acetate (LA), the major constituent of the extracts of Citrus bergamia Risso, Lavandula angustifolia, and Salvia sclarea, has been reported to exert therapeutic effects by inhibiting Ca2+ influx in the vascular endothelial cells[13] and inducing relaxation in the mouse aorta;[14] it has also been shown to possess excellent anti-inflammatory properties in diabetic rats by virtue of its ability to decrease the expression of inflammatory nuclear factors.[15] Our previous work demonstrated that LA exerts excellent preventive effects against calcium-related ischemic injury (CRII) by inhibiting expression of NADPH oxidase (NOX) subunit p47phox and production of reactive oxygen species (ROS), by modulating expression of endothelial nitric oxide (NO) synthase, and by virtue of its ability to prevent cytotoxicity in the rat aorta endothelial cells.[16] These findings show that LA possesses a broad range of beneficial functions; however, the underlying mechanisms may be diverse and are largely unexplored.

Accordingly, in this study, we investigated the protective effects of LA on four cell lines representative of cells of the NVU following exposure to oxygen–glucose deprivation/reoxygenation (OGD/R) in the presence of high extracellular calcium concentration ([Ca2+]o), to mimic CRII, or under OGD/R-only conditions, and provided insight into the underlying mechanism.

  Materials and Methods Top

Cell culture

Cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). BEND murine brain endothelial cells, SH-SY5Y human neural-like (neuroblastoma) cells, BV2 murine brain microglia, and U373 human astrocyte-like cells were incubated in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum and penicillin/streptomycin (100 U/ml each).

Oxygen-glucose deprivation/reoxygenation model

Cells seeded onto 6-well plates (2 × 105 cells/well) were divided randomly into five groups: NOGD control group (no OGD/R, 1.5 mM [Ca2+]o), OGD/R-only group (OGD/R + vehicle control (dimethyl sulfoxide [DMSO]), 1.5 mM [Ca2+]o), OGD/R-LA500 group (OGD/R + 500 μM LA, 1.5 mM [Ca2+]o), CRII group (OGD/R + DMSO, 5 mM [Ca2+]o), and CRII-LA500 group (OGD/R + 500 μM LA, 5 mM [Ca2+]o). At the beginning of the OGD treatment period, the culture medium was changed to DMEM without glucose (Sigma, Burlington, MA, USA), and cells were exposed to hypoxic conditions (ischemia) for 3 h using an Airlock Anaerobic Chamber (Coy Laboratory Products, Grass Lake, MI, USA). After this 3-h OGD treatment, 1 M glucose was added to a final concentration of 5%, and cells were incubated at 37°C for 20 h (reoxygenation step) in the presence of 1.5 or 5 mM [Ca2+]o; thereafter, cells were treated with LA (Sigma) and different concentrations of CaCl2 for 30 min.[17]

Cell viability assay

The effects of LA on the BEND, SH-SY5Y, BV2, and U373 cell viability were determined using 3- [4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays. Briefly, cells were plated in 96-well plates (2 × 104 cells/well) and incubated at 37°C for 24 h, after which 50, 500, or 5000 μM LA or DMSO was added to each well. After incubating for 24 h, cells were washed with phosphate-buffered saline (PBS) and treated with 10 μl MTT (5 mg/ml) in cell culture medium for 3 h. DMSO (100 μl) was then added to dissolve formazan crystals, and optical density was measured at a wavelength of 540 nm using a microplate enzyme-linked immunoassay (ELISA) reader (BMG Labtech, Ortenberg, Germany).[17]

Western blotting

Cells were homogenized and lysed using a protein extraction kit (InTron, Korea). Proteins in the samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels and then transferred to nitrocellulose membranes. Membranes were incubated overnight at 4°C with rabbit polyclonal anti-NOX subunit p47phox, anti-NOX2, and anti-glyceraldehyde 3-phosphate dehydrogenase (GADPH) primary antibodies and then incubated with anti-rabbit immunoglobulin G horseradish peroxidase (HRP)-linked secondary antibody for 2 h at room temperature. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Signals were visualized using an ECL Plus Western blot detection kit (Bio-Rad, Hercules, CA, USA) and analyzed densitometrically using ImageJ software (National Institutes of Health [NIH], Bethesda, MD, USA).

Reactive oxygen species assay

Cells were plated in 6-well plates (2 × 105 cells/well) and incubated at 37°C for 24 h. After washing twice with PBS, cells were incubated for 45 min at 37°C with 5 μM 2,7-dichlorofluorescin diacetate (DCFH-DA) (Abcam, Cambridge, UK) to probe for ROS, followed by incubation for 5 min at 37°C with Hoechst 33342 (Sigma) to counterstain nuclei. DCFH-DA fluorescence was detected using a Nikon DS-Ri2 fluorescence microscope (Nikon, Tokyo, Japan) at a magnification of 400×. Fluorescence intensity was quantified using NIS Elements image analysis software (Nikon, Tokyo, Japan) and ImageJ software (NIH); the results are presented as mean relative fluorescence intensity.[16]

Measurement of intracellular calcium concentration

After pretreatment, 1 × 106 cells were incubated with 2.5 μg of Fura-2 acetoxymethyl ester (Fura-2AM) for 30 min at room temperature. Cells were placed in a quartz cuvette, and emitted fluorescence was measured at alternating wavelengths of 340 nm and 380 nm (Photon Technology Instruments, Birmingham, NJ, USA). Maximum fluorescence (340/380 nm; Rmax) was measured after adding 5 μM ionomycin (positive control), and minimum fluorescence (340/380 nm; Rmin) was added after adding 10 mM ethylene glycol-bis(β-aminoethyl ether)-N, N, N', N'-tetraacetic acid. [Ca2+]i was calculated according to the formula, [Ca2+]c = Kd × b × (R – Rmin)/(Rmax – R), where Kd is the dissociation constant of Fura-2AM (224 nM) and b is luminescence intensity at a wavelength of 380 nm.[13] Mean values were calculated for 10 samples treated with each concentration of LA or DMSO.

Nitrite assay

Culture medium conditioned by cells was collected, and the level of nitrite was measured by adding 100 μl Griess reagent (0.1% naphthylethylenediamine and 1% sulfanilamide) and 50 μl cell culture medium and then incubating for 10 min at room temperature. Nitrite production was determined by measuring absorbance at 540 nm using a microplate ELISA reader (BMG Labtech).[15]

Lactate dehydrogenase assay

The level of lactate dehydrogenase (LDH) released into the cell culture medium was assayed using a CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Fitchburg, WI, USA) according to the manufacturer's instructions. Changes in absorbance at 340 nm were measured using a microplate ELISA reader (BMG Labtech).[14]


The level of matrix metalloproteinase (MMP)-9 protein in the BEND cells was measured by zymography using 8% SDS-PAGE gels containing 1 mg/ml gelatin (Sigma, Burlington, MA, USA). After determining protein content, samples were prepared so as to contain the same amount of total protein and then were analyzed by gelatin zymography. After electrophoresis, proteins in gels were renatured with 2.5% Triton X-100 for 4 h at room temperature, after which gels were incubated at 37°C for 20 h in 50 mM Tris-Cl buffer (pH 7.4) containing 10 mM CaCl2 and 0.02% NaN3. Gels were stained for 2 h with 0.5% Coomassie Brilliant Blue (Sigma, Burlington, MA, USA) in 7.5% acetic acid and 10% isopropyl alcohol, followed by destaining to visualize MMP-9 bands. Absolute densities were analyzed using ImageJ software (NIH).[18]

Statistical analysis

Results of tests are presented as mean ± standard error of the mean (SEM). Differences in mean among groups were analyzed by one-way ANOVA with Fisher's LSD post hoc using SPSS Statistics 22 version (IBM, USA). P < 0.05 was considered statistically significant.[19]

  Results Top

Tolerance of neurovascular unit cellular components to different concentrations of linalyl acetate

To determine the appropriate concentrations of LA that are nontoxic to cells of the NVU, we tested cell viability by MTT assay following incubation with LA. Concentrations of LA greater than 5000 μM significantly reduced cell viability, whereas little or no cytotoxicity was observed following treatment with 500 μM LA. On the basis of these results, we selected 500 μM as the appropriate concentration for use in further experiments [Figure 1].
Figure 1: Neurovascular unit cell types after exposure to different concentrations of LA. Data are shown as means ± standard error of the mean (n = 5 per group). #P < 0.05, ##P < 0.01 and ###P < 0.001 versus the control group. LA50, 50 μM LA; LA500, 500 μM LA; LA5000, 5000 μM LA; LA, linalyl acetate.

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Effect of linalyl acetate on NADPH oxidase subunit p47phox and NOX2

Among important cellular sources of ROS is the plasma membrane enzyme complex, NOX. Here, we evaluated the level of NOX subunits NOX2 (also known as gp91phox) and p47phox in cells of the NVU following exposure to OGD/R in the presence of 1.5 mM [Ca2+]o (OGD/R-only conditions) or 5 mM [Ca2+]o (CRII conditions), with or without 500 μM LA (see Materials and Methods for details). The cell lines, BEND, SH-SY5Y, BV2, and U373, corresponding to the four NVU cell types, were each divided into five groups: NOGD control group (no OGD/R, 1.5 mM [Ca2+]o), OGD/R-only group (OGD/R + DMSO, 1.5 mM [Ca2+]o), OGD/R-LA500 group (OGD/R + 500 μM LA, 1.5 mM [Ca2+]o), CRII group (OGD/R + DMSO, 5 mM [Ca2+]o), and CRII-LA500 group (OGD/R + 500 μM LA, 5 mM [Ca2+]o). Compared with the NOGD control group, exposure to OGD/R at 1.5 mM [Ca2+]o (OGD/R-only group) induced overexpression of p47phox in BEND, SH-SY5Y, and U373 cells [Figure 2]a and overexpression of NOX2 in BEND, SH-SY5Y, and BV2 cells [Figure 2]b; these effects were significantly reversed by LA (OGD/R-LA500 group; [Figure 2]a and [Figure 2]b). Exposure to OGD/R at 5 mM [Ca2+]o (CRII group) induced p47phox overexpression in the SH-SY5Y and U373 cells, an effect that was significantly reversed by LA (CRII-LA500 group) [Figure 2]a; NOX2 expression was not significantly different between CRII and CRII-LA500 groups [Figure 2]b.
Figure 2: Effects of linalyl acetate on NOX2 and p47phox subunit expression induced by exposure to OGD/R-only or calcium-related ischemic injury (CRII) conditions. (a) Relative expression of p47phox. (b) Relative expression of NOX2 protein, detected by western blotting. Glyceraldehyde 3-phosphate dehydrogenase protein was used as an internal control. Data are shown as means ± standard error of the mean (n = 3 per group). ##P < 0.01 and ###P < 0.001 versus the NOGD control group; *P < 0.05 and ***P < 0.001 versus the OGD/R-only group; +P < 0.05, ++P < 0.01 and +++P < 0.001 versus the CRII group. NOGD control group, no OGD/R in 1.5 mM [Ca2+]o; OGD/R-only group, oxygen-glucose deprivation/reoxygenation + dimethyl sulfoxide in 1.5 mM [Ca2+]o; OGD/R-LA500 group, OGD/R + 500 μM linalyl acetate in 1.5 mM [Ca2+]o; CRII group, OGD/R + dimethyl sulfoxide in 5 mM [Ca2+]o; CRII-LA500 group, OGD/R +500 μM linalyl acetate in 5 mM [Ca2+]o.

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Effect of linalyl acetate on changes in reactive oxygen species production and intracellular calcium concentration

ROS include superoxide anion (O2−), hydrogen peroxide (H2O2), and peroxynitrite (ONOO), which lead to NO abnormality. We found that ischemic injury alone (OGD/R-only) induced ROS production in all four cell types of the NVU. Under these conditions, ROS production was significantly reduced by LA treatment (OGD/R-LA500 group) in the BEND, SH-SY5Y, and BV2 cells, but not in U373 cells. In contrast, ROS production induced under CRII conditions (OGD/R, 5 mM [Ca2+]o) was significantly ameliorated by LA (CRII-LA500 group) only in BV2 cells [Figure 3]a.
Figure 3: Antioxidative effects of linalyl acetate. (a) The antioxidative effects on increases in intracellular reactive oxygen species. (b) The antioxidative effects on the changes in [Ca2+]i induced by exposure to OGD/R alone or calcium-related ischemic injury (CRII) conditions. Data are shown as means ± standard error of the mean (n = 3–8 per group). #P < 0.05, ##P < 0.01 and ###P < 0.001 versus the NOGD control group; *P < 0.05, **P < 0.01 and ***P < 0.001 versus the OGD/R-only group; +P < 0.05 and +++P < 0.001 versus the CRII group. NOGD control group, no OGD/R in 1.5 mM [Ca2+]o; OGD/R-only group, oxygen-glucose deprivation/reoxygenation + dimethyl sulfoxide in 1.5 mM [Ca2+]o; OGD/R-LA500 group, OGD/R + 500 μM linalyl acetate in 1.5 mM [Ca2+]o; CRII group, OGD/R + dimethyl sulfoxide in 5 mM [Ca2+]o; CRII-LA500 group, OGD/R +500 μM linalyl acetate in 5 mM [Ca2+]o.

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[Ca2+]i, which is involved in a variety of intracellular signaling pathways, might be affected by OGD treatment and different levels of [Ca2+]o. Ischemic injury under both 1.5 mM [Ca2+]o (OGD/R-only) and 5 mM [Ca2+]o (CRII) conditions induced an increase in [Ca2+]i in all cell types, except SH-SY5Y cells. In BEND and SH-SY5Y cells under these conditions, LA significantly increased [Ca2+]i [Figure 3]b.

Effect of linalyl acetate on nitric oxide or peroxynitrite production and lactate dehydrogenase release

NO possesses biphasic characteristics that can result in cell protection or oxidative damage. In the OGD/R-only group, ischemic injury induced a decrease in NO production in BEND cells and an increase in ONOO in SH-SY5Y and BV2 cells. LA exerted a protective effect on NO production in the BEND cells and attenuated the increase in ONOO in the SH-SY5Y cells. In the presence of high [Ca2+]o concentrations (CRII group), ischemic injury reduced NO production in the BEND cells and enhanced the production of NO in the BV2 cells, with both of these changes significantly reversed by LA (CRII-LA500 group). Ischemic injury also reduced NO production in the CRII group of U373 cells, but this effect was only partially reversed by LA [Figure 4]a.
Figure 4: Effects of linalyl acetate on changes in the levels of nitrite and lactate dehydrogenase. (a) Effects of linalyl acetate on changes in the levels of nitrite. (b) Effects of linalyl acetate on changes in the levels of lactate dehydrogenase. Data are shown as means ± standard error of the mean (n = 5 per group). #P < 0.05, ##P < 0.01 and ###P < 0.001 versus the NOGD control group; **P < 0.01 and ***P < 0.001 versus the OGD/R-only group; +P < 0.05, ++P < 0.01 and +++P < 0.001 versus the calcium-related ischemic injury group. NOGD control group, no OGD/R in 1.5 mM [Ca2+]o; OGD/R-only group, oxygen-glucose deprivation/reoxygenation + dimethyl sulfoxide in 1.5 mM [Ca2+]o; OGD/R-LA500 group, OGD/R + 500 μM linalyl acetate in 1.5 mM [Ca2+]o; calcium-related ischemic injury group, OGD/R + dimethyl sulfoxide in 5 mM [Ca2+]o; CRII-LA500 group, OGD/R + 500 μM linalyl acetate in 5 mM [Ca2+]o.

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Ischemic injury under 1.5 mM [Ca2+]o conditions (OGD/R-only group) induced greater release of LDH, a biomarker of cytotoxicity, in the BEND and BV2 cells than in the SH-SY5Y or U373, indicating increased sensitivity of BEND and BV2 cells to cell damage. Notably, however, LA only reduced LDH secretion in the BEND cells under these conditions. In contrast, ischemic injury-induced increases in LDH release under 5 mM [Ca2+]o conditions (CRII group) were reversed by LA (CRII-LA500 group) in all cell types, except BEND cells, which only showed a decreasing trend without statistical significance [Figure 4]b.

Protective effect of linalyl acetate on matrix metalloproteinase-9 inhibition

MMP-9 is an essential marker of BBB disruption during ischemic stroke. To further demonstrate the protective effect of LA in BBB function, we measured MMP-9 in the BEND cells. Under both OGD/R-only (1.5 mM [Ca2+]o) and CRII (5 mM [Ca2+]o) conditions, ischemic injury induced an increase in MMP-9. However, LA reduced MMP-9 activity significantly only under CRII conditions. These results indicate that the protective effect of LA against MMP-9 elevation might be associated with increased [Ca2+]o [Figure 5].
Figure 5: BBB protective effects of linalyl acetate under OGD/R alone or calcium-related ischemic injury (CRII) conditions, measured as decreases in OGD/R- or CRII-induced increases in the level of matrix metalloproteinase-9 in brain endothelial cells. Data are shown as means ± standard error of the mean ##P < 0.01 and ###P < 0.001 versus the NOGD control group; **P < 0.01 versus the OGD-only group; ++P < 0.01 versus the CRII group. NOGD control group, no OGD/R in 1.5 mM [Ca2+]o; OGD/R-only group, oxygen-glucose deprivation/reoxygenation + dimethyl sulfoxide in 1.5 mM [Ca2+]o; OGD/R-LA500 group, OGD/R +500 μM linalyl acetate in 1.5 mM [Ca2+]o; CRII group, OGD/R + dimethyl sulfoxide in 5 mM [Ca2+]o; CRII-LA500 group, OGD/R +500 μM linalyl acetate in 5 mM [Ca2+]o.

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  Discussion Top

BBB dysfunction is a severe complication after acute ischemic stroke. Loss of integrity of the BBB, resulting from ischemic damage, is also a precursor to hemorrhagic transformation and poor outcome. Hypertension has been found to be an independent predictor of and risk factor for BBB disruption after stroke, with hypertension being involved in almost 25% of ischemic strokes.[17] Mechanisms of brain injury in ischemic stroke were shown to involve multicellular interactions within the NVU, including the evolution of damage within the BBB, neuronal cell death, and immune cell infiltration.[7] Thus, neuroprotection within the NVU was thought to be related to the prevention of BBB disruption and endothelial damage. Altered [Ca2+]o levels are associated with cardiovascular disease[19] and ischemic stroke.[20] To mimic CRII, we simultaneously exposed cells of the NVU to elevated [Ca2+]o and ischemia (OGD/R in 5 mM [Ca2+]o) and then evaluated mechanisms underlying the protective effect of LA.

NOX is an important generator of ROS in the cerebral vasculature under physiological and ischemic conditions.[21] Exposure to damaging stimuli stimulates the p47phox/NOX2 pathway, generating ROS, which induces cerebral endothelial dysfunction[22] and neural cell injury.[23] Lung endothelial cells from both NOX2-deficient[24] and p47phox-deficient[25] mice showed complete abolition of ischemia/reperfusion-induced changes in cellular shape, impaired barrier function, and production of ROS. Left ventricular cavity dilatation and dysfunction after myocardial infarction were significantly reduced in both NOX2- and p47phox-knockout mice compared to wild-type mice.[26] In ischemic microglia and astrocytes, ROS can also be generated by NOX1[27] or NOX4[28] isoforms. Regulation of p47phox by the protein kinase C pathway may also prevent overproduction of ROS.[29]

Similarly, both NOX2 content and NOX activity were higher in the cerebral arteries than in systemic arteries or tissues after stroke.[30] The results largely confirm that genetic deletion or dysfunction of the NOX2/p47phox pathway has a neuroprotective effect in the brain. Thus, this study evaluated the source of ROS by measuring the expression of NOX2/p47phox. The NOX2 inhibitor, apocynin, was shown to have the same effect as NOX2 deletion on the same signaling pathway in acute stroke.[31] Moreover, treatment with OGD/R or hypoxia was found to inhibit ROS generation in BEND and BV2 cells[32] and in SH-SY5Y cells,[33] but not in U373 astrocytes. These results are consistent with our findings, indicating that ROS in astrocytes are produced through a different enzyme pathway than in other cells of the NVU.

However, NOX also stimulates the generation of ROS in the cerebral vasculature under physiological and ischemic conditions.[21] Exposure to damaging stimuli activates the p47phox/NOX2 pathway, which generates of ROS, causing cerebral endothelial dysfunction[22] and neural cell injury.[23] These findings should be considered when evaluating the ability of LA to inhibit other NOX isoforms in these cells.

ROS can be generated by NOX1[27] and NOX4[28] isoforms in ischemic neural cells, such as microglia and astrocytes. The protein kinase C pathway is associated with the regulation of p47phox, thus preventing ROS overproduction. The results of the present study indicate that ROS in the microglia and astrocytes of the NVU are derived from different enzymatic sources. Because this research focused on the effects of LA on NVU cells with ischemic injury, ROS generation by NOX2 and its p47phox subunit was evaluated in an NVU model. The molecular mechanisms underlying the cardiovascular effects of LA are still poorly understood, suggesting the need for additional studies, including evaluation of the inhibitory effects of LA on other NOX isoforms.

Excessive ROS can lead to cell damage and elevated [Ca2+]i and thereby trigger disease. Here, we found that LA exerted an antioxidative effect on ROS production induced by ischemia alone (OGD/R) in the brain endothelial cells, neural cells, and microglia, but not in astrocytes. Interestingly, the effect of LA in the brain endothelial cells and neural cells in this pathological setting might be related to increased [Ca2+]i, reflecting Ca2+ release from intracellular stores. A previous study suggested that LA could modulate Ca2+ homeostasis in human vascular endothelial cells by mobilizing Ca2+ from intracellular stores and blocking excessive Ca2+ channel-mediated Ca2+ influx that might otherwise generate excess NO.[13] Although large increases in neuronal [Ca2+]i can induce neuronal damage, moderate increases activate neural cell survival signals, including the p42/44 mitogen-activated protein kinase pathway.[34] Increasing [Ca2+]i to 50–200 nM also enhances cellular resistance to ischemia in the rat hippocampal neurons.[35] Our results suggest that increases in [Ca2+]i induced by LA might be related to NO generation and triggering of cell survival signals.

[Ca2+]o may play an important role in maintaining BBB function by preventing disruption of adherent junctions and tight junctions.[36] Previous studies in the rat mesenteric arteries,[37] arterial endothelial cells,[38] rabbit aorta,[39] and human aortic endothelial cells[40] have shown that increasing [Ca2+]o from 2.5 to 5 mM modulates endothelium-dependent relaxation. Our results indicate that the elevation of [Ca2+]o to 5 mM decreased ischemic injury-induced NOX2- or p47phox-derived ROS compared with 1.5 mM [Ca2+]o in the brain endothelial cells. These results indicate that, in CRII, elevated [Ca2+]o may induce a protective reflex in brain endothelial cells that serves to maintain cell survival.

Coordinate signaling resulting from interactions between Ca2+ and ROS signaling may be beneficial or detrimental.[41] Our previous results suggested that LA could regulate Ca2+ homeostasis in endothelial cells, mobilize Ca2+ from intracellular Ca2+ stores, and block excessive Ca2+ influx via Ca2+ channels in cell membranes, thereby preventing cell damage.[13] The present results showed that LA treatment induced marked increases of [Ca2+]i in the BEND and SH-SY5Y cells, suggesting that [Ca2+]i may play different roles in brain endothelial and neural cells.

NO in the endothelial cells has been reported to exert a cardiovascular protective effect.[42] Importantly, NO produced by eNOS regulates vascular resistance, which ultimately affects blood pressure in humans.[42] Consistent with previously results,[16] our findings suggest that LA reverses ischemic injury-induced NO abnormality in brain endothelial cells. However, NO exhibits a biphasic characteristic in the central neural system. In pathologic circumstances, metabolic pathways are regulated so as to promote NO generation in neural cells, but injury leads to neuronal death through synthesis of ONOO from NO.[43] The results presented here show that NO was decreased when [Ca2+]o was elevated to 5 mM in the microglia and astrocytes. In fact, it has been reported that NO exerts a protective effect in astrocytes by decreasing mitochondrial ROS[44] and that increasing [Ca2+]o attenuates cell death in microglia and astrocytes by mediating glutamate transport.[45] Although the relationship between NO and elevated [Ca2+]o in microglia and astrocytes remains unclear, [Ca2+]o seems to be important in maintaining normal function in cells of the NVU and appears to contribute to the effect of LA.

LDH is a dependable biomarker of cytotoxicity in the vascular endothelium that is related to NO[46] and ONOO bioavailability.[47] In the current study, although cytotoxicity was reversed by LA in cells of the NVU under CRII conditions, LDH release might not be completely dependent on NO or ONOO. In fact, LDH release is not only induced by ONOO, but also by H2O2,[48] O2,[49] or pro-inflammatory cytokines.[50] Although ischemic injury-induced cytotoxicity was mediated by different pathways in each cell type, LA had beneficial, protective effects on all cells of the NVU, except for brain endothelial cells, on which LA showed only a slight effect.

Degradation of tight junctions during ischemia is shown mainly by the predominant protease MMP-9, which is involved in BBB disruption after acute ischemic injury.[51] MMP-9 was also reported to be upregulated after ischemic stroke and to contribute to irreversible elevation of BBB permeability. In humans, MMP-9 is mainly derived from vessel endothelium after ischemic stroke.[52] Moreover, MMP-9 is elevated in OGD/R models of BEND cells.[53] To further investigate the mechanisms underlying the protective effect of LA in BEND cells, we measured the level of MMP-9, a key enzyme involved in BBB destruction.[54] MMP-9 inhibitors have been reported to significantly reduce ischemia-induced BBB damage,[55] suggesting that LA may indirectly protect the BBB against ischemic injury through inhibition of MMP-9. Although LA treatment reversed the elevated MMP-9 expression under CRII conditions in the BEND cells, LA had no effect under OGD/R-only conditions. These findings suggest that LA protection of the BBB may relate to increase in intracellular Ca2+ level and decreased NO dysfunction.

A summary of our results is illustrated schematically in [Figure 6]. Ischemia injury alone (OGD/R-only group) induced cell damage in brain endothelial cells and microglia, but LA only reversed damage in brain endothelial cells and did so by blocking p47phox/NOX2 expression, ROS production, NO abnormality, and LDH release [Figure 6]a. In contrast, ischemic injury under high [Ca2+]o conditions (CRII group) induced damage in all cells of the NVU. LA reversed CRII effects on NO abnormality and MMP-9 activation in brain endothelial cells; p47phox expression and LDH release in the neural cells; NOX2 expression, ROS production, and LDH release in microglia; and p47phox expression and LDH release in astrocytes [Figure 6]b. Although ischemic injury-induced cytotoxicity was mediated by different pathways in each cell type, the most prominent effects of LA were amelioration of cytotoxicity toward cells of the NVU. LA also maintained BBB function by inhibiting MMP-9 in the brain endothelial cells.
Figure 6: Up/downstream signals and the effects of linalyl acetate under oxygen-glucose deprivation/reoxygenation-only and calcium-related ischemic injury conditions. (a) In condition of oxygen–glucose deprivation/reoxygenation-only. (b) In condition of calcium-related ischemic injury. Arrows represent the protective effect of linalyl acetate and factors that were blocked.

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Two potential limitations of this study are important. The first was that NVU cell lines were incubated separately rather than being co-cultured, preventing the evaluation of cell–cell interactions in the NVU. Second, the molecular mechanisms underlying the cardiovascular effects of LA remain poorly understood. Thus, additional studies, including evaluations of the effect of LA on other NOX isoforms and subunits, are required.

The results of the present study indicate that LA might protect the NVU through multiple different pathways, but additional mechanistic research is necessary to resolve this issue.

  Conclusion Top

Collectively, our results suggest that LA could be a novel therapeutic agent for protection against ischemic injury. Our results provide novel insight into the action of LA in protecting against cytotoxicity in situations in which hypertension and ischemic stroke occur simultaneously and suggest the potential of LA treatment as a new strategy for protecting the NVU and may indirect maintaining BBB function in CRII. The results presented here further provide a descriptive basis for further research and suggest potential future benefits for patients in a clinical setting.

Financial support and sponsorship

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF-2018R1D1A1B07050048). This article is a revision of the first author's doctoral dissertation from Korea University.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]


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