Long-term administration of CU06-1004 ameliorates cerebrovascular aging and BBB injury in aging mouse model

CU06-1004 was synthesized as described previously [23]. Briefly, CU06-1004 was synthesized via tetrahydropyran deprotection and subsequent glycosidation with 4,6-di-O-acetyl-2,3-didieoxyhex-2-enopyran in the presence of an acid. A working solution of CU06-1004 (10 µg/µl) was prepared in dimethyl sulfoxide (DMSO, Sigma, #D2650) for in vitro experiments. For in vivo experiments, CU06-1004 was dissolved in olive oil (Sigma, #O1514) for oral administration. Mice (72 weeks old) were divided into two groups. The old-vehicle group was administered vehicle only (n = 15), and the old-1004 group was administered CU06-1004 (10 mg/kg, n = 15). Both vehicle and CU06-1004 treatments were orally administered 6 days per week using a Zonde needle (100 µl, Jeung Do Bio & Plant Co, #JD-S124) for 6 months (age 18–24 months). No symptoms, such as diarrhea, were observed. However, natural weight loss was observed with increased age in both old-vehicle and old-1004 mice, with no significant difference in body weights between the two aged groups.

Male C57BL/6 J mice (72 weeks old) were purchased from Charles River Laboratories Japan (Yokohama, Kanagawa, Japan). Additionally, 6-week-old male C57BL/6 J mice (DBL, Korea) were used as young mice, and 24-month-old male C57BL/6 J mice were used as aged mice. All mice were housed under controlled conditions (24 °C ± 1 °C, 12-h light/dark cycles, 55% humidity, and specific pathogen–free) and provided with free access to food and water. All animal facilities and experiments were performed in accordance with the Korean Food and Drug Administration guidelines. All procedures were approved by the Institutional Animal Care and Use Committee at Yonsei University (permit number: IACUC-A-202010-1154-01).

Human BMECs (HBMECs) were purchased from ScienCell (Cat. No. 1000) and cultured in endothelial cell growth medium 2 (EGM-2; Lonza, CC-3156) supplemented with the EGM-2 SingleQuots™ kit (Lonza, CC-4176), 20% fetal bovine serum, and 1% penicillin/streptomycin (Cat. No. 0503). Cells were routinely passaged at 80–90% confluency, and cells between passages 3 and 6 were used for experiments. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂.

Colorimetric 3-(4,5-dimetylthialzol-2-yl)-2,5-diphenyltertrazolium bromide (MTT; Thermo Fisher Scientific, #M6494) assay was used to measure cell viability. MTT is reduced to formazan by mitochondrial dehydrogenases, and the absorbance (570 nm) is directly proportional to the viable cell count. HBMECs were seeded into a gelatin-coated 24-well plate at 1 × 10⁵ cells/well and incubated at 37 °C in EGM-2 medium overnight. The following day, the cells were treated with either CU06-1004 or H₂O₂. The cells were then washed with 1 × phosphate-buffered saline (PBS) and incubated for 4 h at 37 °C with MTT solution (0.1 mg/ml) to evaluate cell viability. After the 4-h incubation, the MTT solution was removed, and a 50:50 solution of DMSO and ethanol was added (200 µl/well) to solubilize formazan crystals. Absorbance was detected at a wavelength of 570 nm, and cell viability was calculated as a percentage of the absorbance detected from the control cells.

To perform reverse transcription–polymerase chain reaction (RT-PCR), total RNA was extracted from HBMECs using easy-BLUE™ (iNtRON, #17061). Total RNA was reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Promega Corporation, #M1701) in the presence of oligo(dT) primers and dNTP. The following temperature protocol was used for reverse transcription: Denaturation at 70 °C for 5 min, annealing at 25 °C for 10 min, and extension at 42 °C for 50 min. The following primers were used for PCR: p21, 5′-GCTTCATGCCAGCTACTTCC-3′ (forward), 5′-CCCTTCAAAGTGCCATCTGT-3′ (reverse); p16, 5′-CCTCGTGCTGATGCTACTGA-3′ (forward), 5′-CATCATCATGACCTGGTCTTCT-3′ (reverse); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-CCACCCATGGCAAATTCC-3′ (forward), 5′-TCGCTCCTGGAAGATGGTG-3′ (reverse). All results were normalized to GAPDH expression levels.

The formation of ROS was measured using a ROS-sensitive indicator dye: 2’,7’-dichlorodihydrofluorescein diacetate (H₂-DCFDA, Invitrogen, #D399). HBMECs were seeded at 1 × 10⁴ cells/well in a black, clear-bottom, 96-well plate containing 100 µl culture medium and incubated at 5% CO₂ and 37 °C overnight. The following day, HBMECs were starved of media for 2 h and then pretreated with CU06-1004 (10 µg/ml) for 1 h. The media were removed, and the cells were washed twice with PBS, followed by incubation with 100 µM H₂O₂ for 2 h to stimulate ROS development. The cells were then incubated with 10 µM H₂-DCFDA for 30 min at 37 °C. The fluorescent product formation was quantified with a spectrofluorometer at 485/520 nm. The fluorescent cells were then washed twice with PBS and observed using a fluorescence microscope (Microscope, Olympus DX51; Camera, Olympus DP72).

Samples were fixed with 3.7% formaldehyde for 10 min and washed with cold 1 × PBS for 15 min at room temperature (RT). Samples were washed twice more with PBS and then incubated with senescence-associated β-galactosidase (SA-β-Gal) staining solution [1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal, MERCK, #B4252), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl₂, and 0.01% Nonidet-P-40 (NP-40)] for 24 h at 37 °C without CO₂. After the 24-h incubation, samples were washed with PBS, and the degree of blue color development was used to indicate aging [26]. Staining and imaging were observed under a phase-contrast microscope (Nikon, Japan).

The integrity of the BBB was determined by the detection of cerebral perivascular extravasation of the plasma protein immunoglobulin G (IgG), a widely used and established method [27]. To clear blood and remove vascular IgG, 24-month-old mice were anesthetized using avertin (2, 2, 2-tribromoethanol, Sigma Aldrich, #T48402; 250 mg/kg of body weight) and perfused with 0.9% saline solution injected into the apex of the left ventricle. Brain tissues were immersion-fixed in 4% paraformaldehyde for 24 h and immersed in 15% and 30% sucrose each day. The tissues were then frozen in optimal cutting temperature (OCT) compound and stored at − 80 °C. Brain cryosections (25-µm-thick) were placed on Polysine™-coated microscope slides (Leica, #3800050CL). The sections were prefixed in acetone for 30 min at − 70 °C. Non-specific binding was blocked by incubation with 10% goat serum in PBS for 30 min. Sections were incubated with goat anti-mouse IgG conjugated with Alexa 488 (1 mg/ml, 1:50, Invitrogen, #A28175) at 4 °C for 20 h. After washing sections with 0.2% Tris-buffered saline containing Tween 20 (TBST) and 1 × PBS, the sections were mounted with mounting solution (DAKO, #S3023). Immunofluorescent images were acquired using a Zeiss LSM980 confocal microscope (Zeiss, Germany) at 20 × magnification. Images were analyzed by Zen blue software (Zeiss, Germany). Quantification of fluorescence intensity was performed using Photoshop version CS6 (Adobe Systems, San Jose, CA). For each cortical and hippocampal region, 5–6 images were randomly obtained from each brain section, and all images were used for subsequent quantitative analyses.

After 6 months of drug administration, 24-month-old mice were anesthetized using avertin (2, 2, 2-tribromoethanol, Sigma Aldrich, #T48402; 250 mg/kg of body weight) and perfused with 0.9% saline solution injected into the apex of the left ventricle. Brain tissue was removed and fixed in 4% paraformaldehyde in PBS (pH 7.4) overnight at 4 °C. Following overnight fixation, brain tissue was incubated in 15% sucrose overnight at 4 °C and then transferred to 30% sucrose at 4 °C until the tissue sank. Fixed tissue was encapsulated in Tissue-Tek OCT embedding medium for 30 min at RT, transferred to an embedding mold filled with OCT, frozen on dry ice, and stored at − 70 °C. Frozen section (25-µm-thick) were cut at − 20 °C, and slides were stored at − 80 °C until stained for immunofluorescence. Sections were prefixed in acetone for 30 min at − 70 °C and air dried. OCT was washed off with running tap water. Sections were incubated in blocking solution for 1 h at RT and then incubated overnight at 4 °C with the following primary antibodies: CD31 (1 µg/ml, 1:200; Abcam, #ab24590), glial fibrillary acidic protein (GFAP; 1:200; Millipore, #MAB360), claudin-5 (0.5 mg/ml, 1:200; Invitrogen, #35-2500), and occludin (0.25 mg/ml, 1:200; Invitrogen, #711500). After incubation, sections were washed three times with 0.2% Triton X-100 in PBS (10 min/wash) and further incubated separately with appropriate 488-conjugated secondary antibody (1 mg/ml, 1:400, Invitrogen, #A28175), 594-conjugated secondary antibody (2 mg/ml, 1:400; Invitrogen, #A21207), and 4′,6-diamidino-2-phenylindole (DAPI; 1 mg/ml, 1:1000, Duolink, #D9542). Stained sections were analyzed using a confocal microscope (LSM 880 META; Carl-Zeiss).

Western blotting was performed as previously described [28]. Briefly, HBMECs were lysed using radioimmunoprecipitation assay (RIPA) buffer (100 mM Tris–Cl, 5 mM EDTA, 50 mM NaCl, 50 mM β-glycerophosphate, 50 mM NaF, 0.1 mM Na₃VO_4, 0.5% NP-40, 1% Triton X-100, and 0.5% sodium deoxycholate) at 4 °C. Sample protein concentrations were quantified using the SMART™ BCA Protein Assay Kit (iNtRON, #21071). Cell lysates (25 µg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with 3% bovine serum albumin in 0.1% TBST and probed with primary antibodies. Membranes were then incubated with horseradish peroxidase–conjugated goat anti-rabbit IgG (0.8 mg/ml, 1:1000, Thermo Scientific, #31460) or goat anti-mouse IgG (0.8 mg/ml, 1:1000, Thermo Scientific, #31430) secondary antibodies. β-Actin was used as the loading control. The following primary antibodies were obtained from Cell Signaling Technology and were used at a 1:1000 dilution: phospho–NF-κB inhibitor (IκB)-α (#9242), IκB-α (#9242). The other primary antibodies used were intracellular adhesion molecule 1 (ICAM-1; 200 µg/ml, 1:1000, Santa Cruz Biotechnology, #SC-8439), vascular cell adhesion molecule 1 (VCAM-1; 200 µg/ml, 1:1000, Santa Cruz Biotechnology, #SC-13160), cyclooxygenase 2 (COX-2; 200 µg/ml, 1:1000, Santa Cruz Biotechnology, #SC-376861), and β-actin (1 µg/ml, 1:2000, Thermo Fisher Scientific, #MA5-15739).

Spatial working memory was assessed using a simple T-maze test [30]. Each trial consisted of a sample run and a choice run. During the sample run, one of the goal arms was blocked, forcing the mouse to enter the other goal arm (e.g., the left arm). A 30-s interval separated the sample run from the choice run, and a 30-s interval was used between trials. During the choice run, both arms were open, and the mouse was able to choose either arm. Even without a reward, driven by curiosity, mice usually selected the previously unvisited arm (e.g., the right arm). The animal was considered to have made a correct choice (+) if it visited the previously unsampled arm and an incorrect (−) choice if it visited the previously sampled arm. A total of 10 free choices made by each mouse were measured, and the percentage of correct arm choices during the choice trials was calculated. Each arm of the T-maze was cleaned between sessions using ethanol to remove any olfactory cues, which may have affected the behavior of the next mouse tested.

Prior to investigating the protective properties of CU06-1004 (Fig. 1A) against H₂O₂ treatment, the cytotoxic potential of CU06-1004 was examined in HBMECs. The MTT assay indicated no cytotoxic effects for CU06-1004 treatment at concentrations ranging from 1 to 20 µg/ml (1, 2, 5, 10, and 20 µg/ml). Conversely, CU06-1004 treatment significantly increased HBMEC growth in a dose-dependent manner (Fig. 1B). Further MTT analyses revealed that treatment with 50 µM H₂O₂ did not inhibit HBMEC growth, whereas treatment with 100 µM H₂O₂ significantly inhibited HBMEC growth (Fig. 1C). However, CU06-1004 pretreatment effectively prevented the H₂O₂-induced inhibition of cell growth (Fig. 1D). These results demonstrate that CU06-1004 pretreatment is able to prevent the H₂O₂-induced inhibition of HBMEC growth.

To elucidate the possible mechanisms through which CU06-1004 prevents the H₂O₂-induced inhibition of HBMEC growth and determine the effect of CU06-1004 on H₂O₂-induced oxidative stress, we used H₂-DCFDA, a cell-permeable dye that fluoresces upon oxidation by ROS. HBMECs treated with H₂O₂ for 2 h showed significantly enhanced ROS generation compared with untreated HBMECs. However, pretreatment with CU06-1004 for 1 h significantly inhibited the H₂O₂-induced increase in ROS generation in a dose-dependent manner (Fig. 2A, B). Many studies have determined that oxidative stress induces an inflammatory response, either directly or indirectly, and oxidative stress and inflammation are primary mechanisms related to the onset of age-related vascular endothelial dysfunction [31‐33]. Therefore, to investigate whether CU06-1004 pretreatment was able to alleviate the inflammatory response induced by oxidative stress, we examined the expression of inflammatory proteins in H₂O₂‑treated HBMECs. As shown in Fig. 2C–G, CU06-1004 pretreatment effectively inhibited the H₂O₂-induced expression of ICAM-1, VCAM-1, and COX-2, an inflammation-mediating enzyme. Furthermore, the transcription factor NF-κB is considered a major mediator of the inflammatory response, and the level of phosphorylated IκBα, an indicator of NF-κB activity, was significantly reduced in HBMECs pretreated with CU06-1004. Together, these results suggest that CU06-1004 pretreatment exerts a protective effect against ROS-induced damage by inhibiting the inflammatory response in HBMECs.

To investigate the effect of CU06-1004 treatment on HBMECs with a senescent phenotype, cells were treated with 100 µM H₂O₂ to induce cellular senescence (Fig. 3A). At 3 days post-H₂O₂ exposure, the presence of senescent HBMECs was confirmed by the presence of cells with an enlarged shape and cytoplasmic granularity. After 5 days, the SA-β-Gal staining assay was used to assess the level of senescence among HBMECs exposed to H₂O₂. A threefold increase in the percentage of SA-β-Gal⁺ cells was observed in H₂O₂-treated HBMECs compared with control cells. However, pretreatment with CU06-1004 significantly inhibited this effect and reduced the percentage of SA-β-Gal⁺ cells to twofold that of control cells (Fig. 3B). Senescence is a state of permanent cellular arrest that is established and maintained by the expression of cyclin-dependent kinase inhibitors. As p16^INK4a and p21 are known to mediate permanent cell cycle arrest [34, 35], we used RT-PCR to determine the mRNA levels associated with these genes. The expression levels of p16 ^INK4a and p21 were downregulated in the CU06-1004 treatment group, confirming the anti-senescence effect of CU06-1004 in HBMECs (Fig. 3C–E).

Based on results showing the anti-inflammatory and anti-senescence effects of CU06-1004 in HBMECs, we examined whether the long-term administration of CU06-1004 (10 mg/kg via oral gavage) to 18-month-old mice (late middle age) had protective effects against cerebrovascular aging. First, we measured the maximal cortical diameter and the brain-to-body weight ratio at the postmortem examination. The mean cortical diameter decreased by 8% in 24-month-old mice (late age) compared with 6-week-old mice (young age). However, among 24-month-old mice, no differences in cortical diameter were detected between those treated with vehicle (old-vehicle) and those treated with CU06-1004 (old-1004; Fig. 4A, B). We also measured the brain-to-body weight ratio because this measure serves as a rough estimate of intelligence and brain function in animals [36]. The relative brain-to-body weight ratio was significantly reduced in the old-vehicle group but was less reduced in the old-1004 group (Fig. 4C). The brains of CU06-1004-treated mice were heavier than the brains of vehicle-treated mice (Additional file 1: Figure S1). To examine age-related changes in the brain microvasculature, the patterning of the brain vasculature in the cortex and hippocampus was analyzed in the aged mouse brain through the immunofluorescent staining of endothelial markers. A significant reduction in capillary vessel density was observed for the microvasculature of old mice compared with the microvasculature of young mice. However, greater capillary vessel density and higher numbers of branch points were observed in the old-1004 group than in the old-vehicle group, suggesting that CU06-1004 improves vessel maintenance and inhibits cerebral microvascular rarefaction in aged mice (Fig. 4D, E). Additionally, SA-β-Gal⁺ cells were observed in vessels located in the cortex of aged mice, but the prevalence of SA-β-Gal⁺ cells decreased in the old-1004 group compared with the old-vehicle group (Fig. 4F). Interestingly, in the old-1004 group, SA-β-Gal⁺ cell prevalence was reduced in both vascular and non-vascular region, suggesting that CU06-1004 may alleviate cerebrovascular aging in both vascular and non-vascular cells (Fig. 4G). These results suggest that long-term administration of CU06-1004 prevents age-associated cerebral microvascular rarefaction and cerebrovascular aging.

We then investigated whether age-induced cerebral microvascular rarefaction affects BBB integrity. BBB integrity was determined by detecting the cerebral extravasation of the plasma protein IgG. As shown in Fig. 5A, B, IgG was almost absent from the cortical and hippocampal parenchyma of young mice but was highly abundant in the parenchyma of aged mice. Notably, the IgG abundance was significantly decreased in the old-1004 group compared with the old-vehicle group. As BBB permeability is highly dependent on cerebrovascular endothelial tight junctions, we next examined tight junction integrity in the brains of young and aged mice. Compared with young mice, the cerebral vessels of aged mice expressed lower levels of the tight junction proteins claudin-5 and occludin, but claudin-5 was upregulated in aged mice that received CU06-1004 treatment (old-1004). However, no difference in occludin expression levels was observed between the aged groups (Fig. 5C, D). In addition, the protein expression levels of claudin-5 and occludin in aged mice brain tissues following old-1004, which revealed significantly increased protein expressions in old-1004 mice compared with old-vehicle (Additional file 2: Figure S2). We used electron microscopy to further examine the changes in tight junction complexes that might be responsible for cerebrovascular leakage. In young mice, ultrastructural analysis showed seamless tight junctions within a smooth endothelial layer surrounded by astrocytic end-feet. In aged mice, the capillary wall appeared thicker, and the astrocytic end-feet were considerably swollen; however, the tight junctional complex was less damaged in the old-1004 group than in the old-vehicle group (Additional file 3: Figure S3). These results indicate that aging accelerates the onset of BBB dysfunction, and the long-term administration of CU06-1004 might prevent damage to BBB integrity associated with aging.

Studies have shown that BBB dysfunction amplifies and may act as a key process in the development of neuroinflammation [37]. Therefore, we evaluated astrocyte activation, a widely accepted neuroinflammatory hallmark, in the brain tissues of young and aged mice. Histopathological alterations were evaluated using immunohistochemistry and immunofluorescence staining. As shown in Fig. 6A, B, GFAP-positive (activated) astrocytes were significantly increased in hippocampal brain sections from aged mice compared with hippocampal brain sections from young mice. Double immunofluorescence staining showed increased GFAP-positive astrocytes in the hippocampal sections of aged mice, suggesting that aging causes the upregulation of activated astrocytes. Notably, the long-term administration of CU06-1004 reduced systemic aging-associated increases in TNF-α and IL-6 levels (Fig. 6C). Additionally, we analyzed expression levels of inflammatory proteins from brain tissue extracts. The expression levels of proteins, such as ICAM-1, VCAM-1, and COX-2, were lower in brain tissues from aged mice that received CU06-1004 (old-1004) than in untreated aged mice (old-vehicle; Fig. 6D–G). Collectively, these results indicate that the long-term administration of CU06-1004 exerts anti-inflammatory and neuroprotective effects in aged mice.

Next, we quantified neuronal nuclear protein A60-positive (NeuN⁺) cells in the brains of aged mice. The number of NeuN⁺ cells was significantly reduced, and these cells were less compact in the cortical and hippocampal CA1 regions of the aged mouse brain than in the same regions of the young mouse brain. However, NeuN⁺ cell numbers and compactness were restored by CU06-1004 treatment (old-1004), demonstrating the efficacy of CU06-1004 in protecting against neuronal damage in the aged brain (Fig. 7A, B). We then examined whether aged mice treated with CU06-1004 showed behavioral and cognitive recovery (Fig. 7C). Motor function and working memory tests were performed with old-vehicle and old-1004 mice at 23 months. The rotarod test and wire hang test are classic methods for evaluating motor coordination and balance in aged animals. As shown in Fig. 7D, both the old-vehicle and old-1004 groups exhibited shorter average times to fall from the accelerating rotating rod than the young group, but no significant difference was detected between the old-vehicle and old-1004 groups. In the wire hang test, the old-1004 group showed a marked increase in hanging time (approximately threefold) compared with the average hanging time of the old-vehicle group, indicating that CU06-1004 enhances motor coordination and forelimb muscle strength (Fig. 7E). The T-maze test is a spontaneous alternation task for assessing spatial working memory. Aged mice demonstrated a significantly lower percentage of correct spontaneous alternation choices, indicating an impaired working memory. However, the old-1004 group displayed an increased percentage of correct spontaneous alternation choices compared with the old-vehicle group, indicating a significant effect of CU06-1004 treatment (Fig. 7F). These results suggest that the long-term administration of CU06-1004 reduces aging-associated neuromuscular strength impairments and damage to spatial working memory caused by neuronal cell damage.