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. 2020 Feb 25;36(6):625–638. doi: 10.1007/s12264-020-00471-0

Aloin Protects Against Blood–Brain Barrier Damage After Traumatic Brain Injury in Mice

Yao Jing 1,#, Dian-Xu Yang 1,#, Wei Wang 1, Fang Yuan 1, Hao Chen 1, Jun Ding 1,✉, Zhi Geng 2,✉, Heng-Li Tian 1,✉
PMCID: PMC7270422  PMID: 32100248

Abstract

Aloin is a small-molecule drug well known for its protective actions in various models of damage. Traumatic brain injury (TBI)-induced cerebral edema from secondary damage caused by disruption of the blood–brain barrier (BBB) often leads to an adverse prognosis. Since the role of aloin in maintaining the integrity of the BBB after TBI remains unclear, we explored the protective effects of aloin on the BBB using in vivo and in vitro TBI models. Adult male C57BL/6 mice underwent controlled cortical impact injury, and mouse brain capillary endothelial bEnd.3 cells underwent biaxial stretch injury, then both received aloin treatment. In the animal experiments, we found 20 mg/kg aloin to be the optimum concentration to decrease cerebral edema, decrease disruption of the BBB, and improve neurobehavioral performance after cortical impact injury. In the cellular studies, the optimum concentration of 40 μg/mL aloin reduced apoptosis and reversed the loss of tight junctions by reducing the reactive oxygen species levels and changes in mitochondrial membrane potential after stretch injury. The mechanisms may be that aloin downregulates the phosphorylation of p38 mitogen-activated protein kinase, the activation of p65 nuclear factor-kappa B, and the ratios of B cell lymphoma (Bcl)-2-associated X protein/Bcl-2 and cleaved caspase-3/caspase-3. We conclude that aloin exhibits these protective effects on the BBB after TBI through its anti-oxidative stress and anti-apoptotic properties in mouse brain capillary endothelial cells. Aloin may thus be a promising therapeutic drug for TBI.

Keywords: Aloin, Blood–brain barrier, Traumatic brain injury, Oxidative stress, Apoptosis

Introduction

Traumatic brain injury (TBI) is a crucial factor in disability and even death in young adults world-wide [1]. About 5.3 million people in the USA and ~ 7.7 million people in Europe are living with TBI-induced death and disabilities [2]. The population-based mortality from TBI in China is estimated to be ~ 13 per 100,000 [3]. Whether these patients die or survive with disability, the economic consequences of TBI are enormous [4].

TBI involves primary injury and secondary damage. The initial primary injury is caused by direct mechanical damage to brain tissue and tends to be irreversible. As a consequence of the injury, a cascade of effects is initiated, involving disruption of the blood–brain barrier (BBB), apoptosis, oxidative stress, formation of brain edema, and so on, leading to secondary damage [1, 2]. Some treatments are currently available to reduce the severity of secondary damage, but few are effective [5, 6].

The BBB is the major functional barrier in the central nervous system. It inhibits the extravasation of intravascular contents such as toxic substances, pathogens, and blood cells into the brain parenchyma, and pumps out cerebral waste materials so that the balance of the biochemical environment is maintained to ensure basic neural functions [7, 8]. Cerebrovascular endothelial cells (ECs) are the main components of the BBB and are connected by the continuous intercellular tight junctions (TJs), mainly consisting of zonula occludens (ZO) and occludin [9, 10]. After TBI, the function of the BBB is disrupted as a result of breakage of the continuous intercellular TJs, triggering secondary damage [7]. Therefore, ensuring the protection of the BBB is considered a promising therapeutic strategy for reducing secondary brain damage.

Aloin, an anthraquinone glycoside, is one of the major active ingredients extracted from Aloe species [11]. According to previous studies, aloin has shown anti-aging activity in a D-galactose-induced mouse model [12], anti-oxidative stress and anti-apoptotic activity in oxygen- and glucose-deprivation-induced PC12 cell injury [13], an anti-tumor growth effect in human colorectal cancer [14], and immunomodulatory and anti-inflammatory responses in both a lipopolysaccharide-activated human umbilical vein EC model and a model of ultraviolet B-induced paw sunburn in rats [11, 15]. However, there is little evidence that aloin has a protective action on the BBB after TBI, and if so, what the possible mechanism may be.

In this study, we aimed to explore the protective effects of aloin on the BBB in TBI models in vivo and in vitro and to investigate the possible underlying mechanisms.

Materials and Methods

Experimental Animals and Protocols

We used adult male C57BL/6 mice weighing 20–25 g and aged 8–10 weeks (Shanghai SLAC Laboratory Animal Corp., Shanghai). The use of animals was approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University, Shanghai, China, and the animal experiments were performed according to the Guidelines for Laboratory Animal Research of Shanghai Jiao Tong University. Aloin (> 97% pure) was from Sigma (St. Louis, MO) and diluted in a mixture of dimethyl sulfoxide and normal saline. First, the animals were randomly divided into sham-operated, TBI, TBI+vehicle, and TBI+aloin (10, 20, and 30 mg/kg) groups to determine the optimum concentration of aloin by measuring brain water content. Subsequently, based on the results, we randomly divided the remaining mice into three groups: sham-operated, TBI+vehicle, and TBI+aloin (optimum concentration). Each group contained 6 or 12 mice (12 for the neurobehavioral tests only) in each experiment. The vehicle or aloin was injected intraperitoneally 30 min before TBI was induced and then injected once daily until the experiments were finished. Except for the mice used in behavioral tests, mice were euthanized 3 days after TBI model completion, and brain tissue was collected for further analyses.

Controlled cortical impact (CCI) was adopted as the TBI model. After each C57BL/6 mouse was anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg), we fixed its head in a stereotaxic frame, and a heating pad was placed under its body to maintain a temperature of ~ 37°C. Then, a midline incision ~ 10 mm long was made on the scalp under aseptic conditions. The skin and fascia were retracted and a bone window 4 mm in diameter was made by a trephine at the center of the right parietal bone, 1 mm lateral to the sagittal suture. Extreme care was required during this operation. If the dural integrity was compromised, the mouse was excluded from the study. At that point, the treatment of the sham-operated group was complete. Then, the TBI model was created using a CCI device (PinPoint Precision Cortical Impactor PCI3000; Hatteras Instruments Inc., Cary, NC), which has been used in previous studies [16, 17]. A rounded steel impactor tip 3 mm in diameter was placed precisely on the exposed intact dura, and the cortical surface was struck vertically at an impact velocity of 1.5 m/s, a deformation depth of 1.5 mm, and a dwell time of 100 ms. The injured cortical surface was compressed with sterile cotton until the bleeding was controlled. The cranial defects in these mice, including the sham-operated group, were sealed with sterile bone wax, and the incisions were closed with interrupted 6–0 silk sutures under aseptic conditions. All animals were placed in heated cages to regain full consciousness, and then returned to their home cages.

Measurement of Brain Water Content and Brain Edema

The brain water content of mice was measured using the wet-dry method. In brief, 3-mm coronal sections of the ipsilateral cortex, centered on the impact site, were used to evaluate the water content. The sections were immediately weighed to obtain the wet weight, and then dried for 24 h in an oven (100°C) to determine the dry weight. Brain water content was calculated as follows: (wet weight–dry weight)/wet weight × 100%.

Magnetic resonance imaging (MRI) of mouse heads was performed with a 3.0 T scanner (Excite; Siemens Signa, Buffalo Grove, IL) 3 days after TBI. The parameters of the T2-weighted MRI were as follows: repetition time, 3670 ms; echo time, 97 ms; slice thickness, 0.9 mm; field of view, 70 × 70 mm2; and number of excitations, 1.5. The volume of the brain edema lesion, including the contusion area, was assessed from coronal T2-weighted scans using ImageJ software (National Institutes of Health, Bethesda, MD) by a physician proficient in neuroimaging.

Analysis of Evans Blue (EB) Extravasation

The extravasation of EB was used to assess the degree of BBB damage 3 days after TBI. EB dye (2%) was injected intravenously at 4 mL/kg, and then 2 h later the mice were perfused through the heart with normal saline to completely wash out the intravascular dye. After the brain was removed, it was divided into two hemispheres and weighed immediately. Subsequently, each hemisphere was homogenized in 50% trichloroacetic acid and centrifuged for 20 min at 12,000×g. The supernatant was transferred into 3 volumes of ethanol and the EB content of the cerebral hemispheres was assessed by a spectrophotometer (BioTek, Winooski, VT) at 610 nm, and then quantified as micrograms per gram of brain tissue.

Neurological Severity Score Determination

The modified neurological severity score (mNSS) was adopted to assess the neurological status of mice before TBI and at 1, 3, 7, and 14 days after TBI. As described in a previous report [18], the mNSS includes motor, sensory, balance, and reflex tests, for which a normal score is 0 and the maximal deficit score is 14 points. One point is awarded for deficiencies in each of the categories listed above; the higher the score, the more severe the neurological injury.

The rotarod test was used to assess motor coordination as previously described [19]. Briefly, each mouse was trained on the rod at speeds that accelerated from 0 to 40 rounds/min within 5 min for 3 days. Each training day included 3 trials. Before TBI, the mean value for 3 trials was calculated as the baseline value for each mouse. After TBI, the rotarod test time data were collected at 1, 3, 7, and 14 days.

The spatial learning and memory of mice were evaluated by a Morris water maze test [20]. The apparatus was a circular tank with warm water which contained white lime to make the water opaque. The pool was divided into 4 quadrants with visual cues, and a platform 10 cm in diameter was submerged 1 cm below the surface of the water in one quadrant. On training days (14–18 days after TBI), each mouse received 4 training trials per day; the starting positions were randomly selected from among the 4 quadrants. Mice were allowed 60 s to find the platform. If they failed to locate the platform within 60 s, they were manually guided to it and allowed to stay there for 15 s; this result was recorded as 60 s. The mean time per training day were recorded for comparison. On the test day (19 days after TBI), the probe trial was conducted in the absence of the platform. Each mouse was put into the quadrant opposite to the normal location of the platform and allowed to swim freely for 60 s. The latency to the platform location, the number of crossings, and the time spent in the target quadrant were recorded. All the neurobehavioral tests were assessed by an investigator who was blinded to the experimental design.

Experimental Cell Design

The bEnd.3 mouse brain capillary ECs were from the American Type Culture Collection (Manassas, VA). They were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Laboratories, Grand Island, NY) containing 10% fetal bovine serum and 1% penicillin/streptomycin in a 37°C humidified incubator infused with a mixture of 5% CO2 and 95% air [21]. First, we explored the effects on cell viability of different concentrations of aloin using a Cell Counting Kit-8 (CCK-8) assay (Dojindo, Tokyo). Next, we randomly divided the cells into control, stretch injury (SI), SI+vehicle, and SI+aloin (10, 20, 40, 60, and 80 μg/mL) groups to determine the optimum concentration of aloin using a lactate dehydrogenase (LDH) release assay. Finally, according to the results, the bEnd.3 cells were randomly divided into three groups: control, SI+vehicle, and SI+aloin (optimum concentration). The vehicle and aloin solutions were added 30 min before SI of cells, which were then cultured for an appropriate time after SI for most of the subsequent biological tests. Each experiment was repeated 6 times.

Mechanical SI of bEnd.3 cells was used to simulate TBI in vitro. The cells were seeded at 0.5 × 105 cells/cm2 onto BioFlex® 6-well culture plates (Flexcell International Corp., Burlington, NC) with collagen-coated Silastic membranes. After the cells were cultured overnight, a biaxial SI was induced in cells using the Cell Injury Controller II system (Virginia Commonwealth University, Richmond, VA). This instrument released a 50-ms burst of nitrogen gas to cause a 7.5-mm downward deformation of the Silastic membrane and the adherent cells, analogous to the mechanical stress exerted on brain tissue by rotational acceleration and deceleration injury [2, 22, 23].

Cell Viability Assay

The bEnd.3 cells were seeded into 96-well plates at 1 × 104 per well and cultured overnight. The different concentrations of aloin (10, 20, 40, 60, and 80 μg/mL) were administered to the cells, which were cultured for 4.5 h. After 10 μL of the CCK-8 reaction solution was added into each well, the cells were conventionally incubated for 2 h. The absorbance of each well was then measured at 450 nm using a spectrophotometer.

LDH Release Assay

LDH release from bEnd.3 cells was determined using a cytotoxicity detection kit (Roche, Manheim). The 100 μL supernatant from each group was transferred into 96-well plates. After adding 100 μL reaction solution to each well, the mixed samples were incubated for 30 min in the dark at 25°C. The LDH release was determined as the absorbance of each well at 490 nm measured using a spectrophotometer.

Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-end Labeling (TUNEL) Assay

Samples of bEnd.3 cells from the control, SI+vehicle, and SI+aloin groups were collected to count the apoptotic cells by TUNEL assay using the in-situ cell death detection kit, TMR red (Roche, Diagnostics, Basel). Briefly, after immersion in 4% paraformaldehyde for 30 min and 0.3% TritonX-100 for 10 min, the samples were allowed to react with the TUNEL mixture solution for 1 h at 37°C, and then stained with 4’,6-diamidino-2-phenylindole (DAPI; 1:1000; Beyotime Biotechnology, Nantong, Jiangsu) for 5 min in the dark. Apoptotic cells were observed and recorded under a confocal fluorescence microscope (Leica TCS SP5 II; Zeiss, Jena, Germany). The apoptosis rate was calculated as follows: apoptotic cells/all cells in a field × 100%.

Immunostaining

Brain cryosections 20 µm thick and Silastic membranes with bEnd.3 cells were fixed in cold anhydrous methanol. After penetration with 0.3% TritonX-100 and blocking with 10% bovine serum albumin, the samples were incubated overnight with the following primary antibodies at 4°C: rabbit anti-ZO-1 (1:200; Life Technologies, Carlsbad, CA)/goat anti-CD31 (1:200; R&D Systems, Minneapolis, MN) and mouse anti-occludin (1:200; Invitrogen, Carlsbad, CA)/goat anti-CD31 (1:200) double-staining for tissue; rabbit anti-ZO-1 (1:200) and mouse anti-occludin (1:200) single-staining for cells. Subsequently, the samples were incubated with the mixed corresponding secondary antibodies (1:400) for 1 h, and the cells were stained with DAPI (1:1000) for 10 min in the dark at room temperature. Immunofluorescence images were captured using the fluorescence microscope.

Western Blot Analysis

The brain tissue samples and bEnd.3 cells were lysed in a mixed lysis buffer at the same protein concentration. After denaturation, equal volumes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skimmed milk powder for 1 h and then incubated at 4°C overnight with antibodies against the following: ZO-1 and occludin (1:1000); p38, phospho-p38 (p-p38), p65, phospho-p65 (p-p65), B cell lymphoma (Bcl)-2, Bcl-2-associated X protein (Bax), cleaved caspase-3, and caspase-3 (1:1000; Cell Signaling Technology, Beverly, MA); β-actin, β-tubulin, and GAPDH (1:1000; Abcam, Cambridge). After washing 3 times, the membrane was incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (1:5000) for 1 h at room temperature. Protein signals were measured using a gel imaging system (Millipore, Billerica, MA) with the enhanced chemiluminescence reagent (Pierce, Rockford, IL), and then the results were analyzed with Quantity One software (BioRad, Hercules, CA).

Measurement of Intracellular Reactive Oxygen Species (ROS)

The intracellular ROS generation of bEnd.3 cells was assessed using a DCFH-DA Assay Kit (Beyotime Biotechnology, Nantong, China). Briefly, 1 mL of 10 µmol/L DCFH-DA diluent was added to a BioFlex® 6-well culture plate, and then the cells were incubated for 20 min in the dark at 37°C. After washing the wells 3 times with DMEM, the fluorescence intensity was measured with a fluorescence spectrophotometer (Horiba Scientific, Piscataway, NJ), a fluorescence microscope, and a flow cytometer (BD Biosciences, San Jose, CA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Determination of Mitochondrial Membrane Potential (ΔΨm)

The ΔΨm of bEnd.3 cells was assessed using a JC-1 assay kit (Beyotime Biotechnology). Briefly, 1 mL DMEM and 1 mL JC-1 reaction solution were added to a BioFlex® 6-well culture plate, and then the cells were incubated for 20 min in the dark at 37°C. After washing the wells twice with JC-1 buffer, ΔΨm fluorescence images were captured on the fluorescence microscope, and the ΔΨm fluorescence intensity was determined by flow cytometry. Red fluorescence indicated healthy cells with a normal ΔΨm, while green fluorescence indicated potentially apoptotic cells with a low ΔΨm. The ratio of red to green fluorescence was used to quantify the differences in ΔΨm in the different groups of bEnd.3 cells.

Statistical Analysis

All data are expressed as the mean ± standard deviation (SD). Comparisons among multiple groups were assessed by one-way analysis of variance followed by Tukey’s post-hoc test, and differences between two groups were determined by the unpaired Student’s t test. P < 0.05 was considered to be statistically significant. Statistical analyses were demonstrated on IBM SPSS version 20 (SPSS Inc., Chicago, IL). Quantified bar graphs of the data were created in GraphPad Prism version 6 (GraphPad Software, San Diego, CA).

Results

Aloin Reduces Brain Edema by Protecting the BBB after TBI in Mice

Brain water content is one of the most important indicators of the severity of edema after TBI, and we found that the water content markedly increased after TBI in mice. To determine the optimum concentration of aloin for the mouse TBI model, we measured the brain water content using the wet-dry method for each of the different concentrations of aloin (10, 20, and 30 mg/kg) at 3 days after TBI. There were no differences between the TBI and vehicle groups (82.14% ± 0.29% vs 82.17% ± 0.22%), or the 20 mg/kg and 30 mg/kg aloin groups (79.77% ± 0.38% vs 79.87% ± 0.38%) (both P > 0.05). There was a significant reduction in the 10 mg/kg aloin group compared with the vehicle group (80.92% ± 0.25% vs 82.17% ± 0.22%), as well as in the 20 mg/kg aloin group compared with the 10 mg/kg aloin group (79.77% ± 0.38% vs 80.92% ± 0.25%) (both P < 0.01; Fig. 1A). Therefore, 20 mg/kg was used as the optimum concentration for further experiments.

Fig. 1.

Fig. 1

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Aloin attenuates TBI-induced brain edema caused by damage to the BBB in mice. A Brain water content in different groups at 3 days post-TBI. B Representative coronal T2-weighted MRI scans and brain lesion volumes in sham, TBI+vehicle, and TBI+aloin groups at 3 days after TBI (white contours indicate areas of edema). C Representative images of EB extravasation and statistics for EB content in the three groups at 3 days after TBI (blue areas indicate EB extravasation). n = 6/group, data are presented as the mean ± SD, **P < 0.01, #P < 0.05.

The brain edema lesions were directly visualized as high intensity areas contoured by white lines on contiguous coronal T2-weighted scans (Fig. 1B). The lesion areas were evident in the vehicle group and were significantly smaller after treatment with aloin at 3 days post-TBI. These results were consistent with the statistical analyses (24.67% ± 3.93% vs 14.83% ± 3.13%, P < 0.01; Fig. 1B).

The extent of damage to the BBB influenced the severity of brain edema as previously described [2]. We assayed the images of EB extravasation to assess the permeability of the BBB. Statistical analyses revealed that aloin treatment significantly reduced EB leakage at 3 days after TBI, indicating that TBI-induced BBB damage is attenuated by aloin (5.69 ± 0.56 μg/g vs 8.06 ± 0.59 μg/g, P < 0.01; Fig. 1C).

Aloin Alleviates the Disruption of TJ Proteins of the BBB in the Pericontusional Areas after TBI in Mice

TJ proteins, including ZO-1 and occludin, play important roles in maintaining the functions of the BBB. TBI led to the loss of ZO-1 and occludin in the pericontusional areas. Western blot analyses revealed that the levels of ZO-1 and occludin were much higher in the aloin-treated group than in the vehicle-treated group at 3 days after TBI (both P < 0.01) (Fig. 2A). At the same time, double immunostaining for ZO-1/CD31 and occludin/CD31 revealed continuous ZO-1 and occludin staining along the EC margin of the cerebral capillaries in the sham group. Gaps and losses were present in the vehicle group, but they were fewer after aloin treatment (both P < 0.01; Fig. 2B).

Fig. 2.

Fig. 2

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Aloin alleviates the loss of TJ proteins in the BBB in the pericontusional area 3 days after experimental TBI in mice. A Representative western blots and levels of ZO-1 and occludin in the sham, TBI+vehicle, and TBI+aloin groups. B Representative co-stained immunofluorescence and levels of ZO-1/CD31 and occludin/CD31 in the three groups (scale bars, 75 μm). n = 6/group, data are presented as the mean ± SD. **P < 0.01, aloin vs vehicle group.

Aloin Improves Recovery from Neurological Deficits after TBI in Mice

Neurological deficits were present after TBI in mice, and we explored the effects of aloin on the recovery of neurological functions using the mNSS, rotarod test, and Morris water maze test. The results of the mNSS, which included motor, sensory, balance, and reflex tests, were lower in the aloin group than in the vehicle group at 3, 7, and 14 days after TBI (all P < 0.05; Fig. 3A). In the rotarod test, which focused on motor coordination, the durations that mice stayed on the rod in the aloin group were longer than those in the vehicle group at 3, 7, and 14 days post-TBI (all P < 0.05; Fig. 3B), consistent with the mNSS data.

Fig. 3.

Fig. 3

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Aloin improves neurological functions after experimental TBI in mice. A, B mNSS (A), and rotarod latency (B) before TBI and at 1, 3, 7, and 14 days after TBI in the TBI + vehicle and TBI + aloin groups. C Morris water maze training results during 14–18 days after TBI in the sham, TBI + vehicle, and TBI + aloin groups. D–F Time of first arrival at the platform (D), number of times crossing the platform (E), and percentage of time in the platform quadrant (F) in the Morris water maze 19 days after TBI in the three groups. n = 12/group, data are presented as the mean ± SD, *P < 0.05, **P < 0.01, aloin vs vehicle group.

The Morris water maze was used to assess spatial learning and memory. In the training trials, the aloin group exhibited a shorter latency than the vehicle group at 17 and 18 days after TBI (both P < 0.05; Fig. 3C). In the probe trials, the neurobehavioral outcomes of latency to the platform, number of crossings, and time spent in the target quadrant were better in the aloin group than in the vehicle group at 19 days after TBI (P < 0.05, P < 0.05, and P < 0.01, respectively; Fig. 3D–F).

Aloin Decreases Apoptosis, and Protects the Integrity of TJ Proteins in bEnd.3 Cells after Stretch Injury

To explore the cytotoxicity of aloin, we used CCK-8 assays to evaluate the viability of bEnd.3 cells at different concentrations (10, 20, 40, 60, and 80 μg/mL) for 4.5 h. There were no differences between the control, 10 μg/mL, 20 μg/mL, and 40 μg/mL aloin (all P > 0.05). With increasing aloin concentrations, cell viability continuously declined (P < 0.05 for both 40 vs 60 μg/mL and 60 vs 80 μg/mL; Fig. 4A). In addition, the release of LDH, an apoptosis-associated index, increased significantly after SI and was used to determine the optimum concentration of aloin at 4 h post-SI. Consistent with the CCK-8 assays, 40 μg/mL was the optimum concentration and this was used in subsequent experiments (Fig. 4B).

Fig. 4.

Fig. 4

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Aloin reduces the damage to bEnd.3 cells by experimental SI. A Cell viability at different concentrations of aloin after 4.5 h assessed by CCK-8. B Effects of different concentrations of aloin on cells 4 h after SI assessed by LDH release. C Representative TUNEL staining of apoptotic cells and quantified apoptosis rate at 4 h after SI in control, SI+vehicle, and SI+aloin groups (scale bar, 75 μm). n = 6/group, data are presented as the mean ± SD, *P < 0.05, **P < 0.01, #P > 0.05.

TUNEL and DAPI double-positive cells verified as apoptotic cells were detected in the control and SI groups. The statistical analyses revealed that the apoptosis rates were significantly lower after aloin treatment than in the vehicle-treated group at 4 h post-SI (P < 0.01; Fig. 4C).

Immunostaining and western blot analyses showed that the ZO-1 and occludin expression levels increased after aloin treatment compared with the vehicle-treated group at 4 h post-SI (all P < 0.01; Fig. 5A, B).

Fig. 5.

Fig. 5

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Aloin alleviates the loss of TJ proteins after experimental SI in bEnd.3 cells. A Representative immunofluorescence images and quantification of ZO-1 and occludin proteins 4 h after SI in the three groups (scale bars, 30 μm). B Representative western blots and quantification of ZO-1 and occludin proteins 4 h after SI in the three groups. n = 6/group, data are presented as the mean ± SD, **P < 0.01, aloin vs vehicle group.

Aloin Attenuates Intracellular ROS Generation after Stretch Injury of bEnd.3 Cells

After SI, a great deal of ROS occurred in bEnd.3 cells, as assessed by DCFH-DA. The level of ROS reached its highest point 2 h after SI. Aloin (40 μg/mL) noticeably reduced the ROS production at 1 h, 2 h, and 4 h after SI (all P < 0.01). There were no differences among 20, 40, and 60 μg/mL aloin at 1 h after SI (both P > 0.05), and the 40 μg/mL aloin had the optimum effect compared with 20 μg/mL (both P < 0.01) and 60 μg/mL (P < 0.01, P < 0.05) at 2 h and 4 h after SI, so we used 40 μg/mL aloin in subsequent assays (Fig. 6A). The DCFH fluorescence analyses showed that the relative ROS level of each cell after aloin treatment was markedly lower than in the vehicle-treated group 2 h post-SI (P < 0.01; Fig. 6B), and similar results were found using flow cytometry (Fig. 6C). The DCFH fluorescence intensity of all cells in the aloin-treated group decreased significantly compared with the vehicle-treated group (P < 0.01).

Fig. 6.

Fig. 6

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Aloin decreases intracellular ROS generation after experimental SI in bEnd.3 cells, as detected by DCFH-DA. A ROS levels at different time points post-SI after treatment with different concentrations of aloin. B Representative intracellular ROS fluorescence images with bright field and quantification 2 h after SI in the control, SI+vehicle, and SI+aloin groups (scale bar, 75 μm). C Representative fluorescence intensity using flow cytometry and quantification 2 h after SI in the three groups. n = 6/group, data are presented as the mean ± SD, *P < 0.05, **P < 0.01, #P > 0.05.

Aloin Protects against the Changes in Mitochondrial Membrane Potential after Stretch Injury of bEnd.3 Cells

The changes in ΔΨm from red to green fluorescence after SI in bEnd.3 cells were detected by the JC-1 kit and showed that the relatively lower ΔΨm of each cell in the vehicle-treated group was remarkably reversed after aloin treatment (P < 0.01; Fig. 7A). Consistent with the above results, the flow cytometry-based analyses of the red/green fluorescence ratio revealed that aloin significantly increased the levels of ΔΨm at 2 h post-SI (P < 0.01; Fig. 7B).

Fig. 7.

Fig. 7

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Aloin protects against the changes in ΔΨm after SI in bEnd.3 cells. A Representative ΔΨm fluorescence images and red/green fluorescence ratios 2 h after SI in the control, SI + vehicle, and SI + aloin groups (scale bar, 75 μm). B Representative fluorescence intensity using flow cytometry and red/green fluorescence ratios 2 h after SI in the three groups. n = 6/group, data are presented as the mean ± SD, **P < 0.01, aloin vs vehicle group.

Aloin Regulates Mitogen-Activated Protein Kinase (MAPK), Nuclear Factor-kappa B (NF-κB), and Apoptosis-Associated Pathways to Reduce the Damage in Mice Post-TBI and in bEnd.3 Cells after Stretch Injury

To determine the effects of aloin on the MAPK, NF-κB, and apoptosis-associated pathways in the in vivo and in vitro TBI models, we used western blot analyses. In the animal model, aloin remarkably decreased the high levels of p-p38/p38 in the MAPK pathway and p-p65/p65 in the NF-κB pathway induced by TBI after 3 days (both P < 0.01; Fig. 8A, B). Furthermore, in the mitochondrial apoptotic pathways, the ratios of Bax/Bcl-2 and cleaved caspase-3/caspase-3 were clearly lower in the aloin-treated group than in the vehicle group (both P < 0.01; Fig. 8C). In the cell-injury model, the levels of phosphorylation of p38 and p65 were elevated at 2 h after SI, while they were reversed by aloin treatment (both P < 0.05; Fig. 9A, B). Meanwhile, the ratios of Bax/Bcl-2 and cleaved caspase-3/caspase-3 increased remarkably at 2 h after SI, and aloin reduced these ratios (both P < 0.01; Fig. 9C, D).

Fig. 8.

Fig. 8

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Aloin regulates MAPK, NF-kB, and apoptosis-associated pathways in mouse TBI models. A–C Representative western blots of p-P38 and P38 (A), p-P65 and P65 (B), and Bax, Bcl-2, cleaved caspase-3, and caspase-3 proteins (C), along with their quantifications 3 days after TBI in the sham, TBI+vehicle, and TBI+aloin groups. n = 6/group, data are presented as the mean ± SD, **P < 0.01, aloin vs vehicle group.

Fig. 9.

Fig. 9

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Aloin regulates MAPK, NF-kB, and apoptosis-associated pathways after experimental stretch injury in bEnd.3 cells. A–D Representative western blots of p-P38 and P38 (A), p-P65 and P65 (B), and Bax, Bcl-2 (C), cleaved caspase-3, and caspase-3 proteins (D), along with quantifications at 2 h after SI in the control, SI+vehicle, and SI+aloin groups. n = 6/group, data are presented as the mean ± SD, *P < 0.05, **P < 0.01, aloin vs vehicle group.

Discussion

TBI is a public health problem that leads to high rates of disability and mortality in modern-day society [24]. Therefore, to explore the pathogenesis and possible therapies, several TBI models in mice have been developed over time to simulate human TBI, such as the fluid percussion injury model [25], the weight-drop-based injury model [26], the penetrating brain injury model [27], and the blast brain injury model [28]. In our study, the CCI model was used due to its strong operability and high accuracy compared with the above models. We were able to manually control the impact parameters to obtain different degrees of pathological brain injury. According to a previous report [29], moderate TBI was created in mice for our experiments.

After TBI, the reversible secondary damage is an important target for therapy [30]. Disruption of the BBB is a crucial part of this secondary damage, leading to the generation of vasogenic brain edema. The more severe the BBB damage, the more edema forms [31]. Aloin, a small molecule, has been used and explored in our studies of its anti-apoptosis [13] and anti-oxidative stress response [15] properties. At the optimum treatment concentration of 20 mg/kg, aloin significantly reduced the volume of edema after TBI, and attenuated the BBB damage as evidenced by the extravasation of EB. Furthermore, the integrity of the BBB is maintained mainly by the TJ proteins ZO-1 and occludin in ECs [32]. The loss of these proteins in the pericontusional area post-TBI was reversed by aloin, reducing the permeability of the BBB.

TBI-induced secondary damage inhibits the recovery of neurological deficits [33]. In this study, the mNSS, rotarod test, and Morris water maze test were used to comprehensively assess the recovery of neurological functions. The motor, sensory, balance, and reflex functions in mice are evaluated by the mNSS and the rotarod test [34], while spatial learning and memory are examined by the Morris water maze test [34, 35]. Clearly, aloin improved the neurobehavioral performance of mice after TBI.

In the animal TBI model, we found that aloin regulated the MAPK, NF-κB, and apoptosis-associated pathways. To further investigate the possible underlying mechanism of action of aloin in TBI therapy, we used an SI model in bEnd.3 cells similar to the moderate TBI, which is usually used to explore the properties of the BBB in vitro [36]. Based on the cell viability and LDH release tests, 40 μg/mL aloin had the best therapeutic effect. This concentration significantly reduced the apoptosis and minimized the loss of the TJ proteins ZO-1 and occludin in bEnd.3 cells after SI. Potentially, aloin protects the integrity of TJ proteins by alleviating the apoptosis of cerebrovascular ECs after TBI.

Excessive ROS accumulation in cells is associated with oxidative stress, which exacerbates secondary damage [8]. ROS include superoxide anion, hydroxyl radical, hydrogen peroxide, and hypochlorous acid. However, the most common intracellular free radical after TBI is the superoxide anion, which attacks DNA, protein, transcription factor, and membrane lipid, leading to cell damage and apoptosis [8, 37]. Aloin inhibited the generation of excessive ROS in bEnd.3 cells after SI as determined by DCFH-DA. Consistent with previous studies [12, 38], we found that the high levels of ROS directly activated the MAPK and NF-κB signaling pathways. MAPKs, which belong to the highly-conserved family of serine/threonine protein kinases, mainly include the ERK, JNK, and p38 subgroups [39]. The phosphorylation of MAPKs is induced by the stimulation of intracellular ROS and is associated with apoptosis [6, 8, 40]. NF-κB, a family of DNA-binding proteins, is widely known to play an important role in the pathology of neuroinflammation after TBI [5, 12]. NF-κB in the cytoplasm is activated and phosphorylated by ROS, then translocated into the nucleus, where it induces inflammatory factors and apoptosis [6, 41]. The results from our studies demonstrated that aloin blocked the phosphorylation of p38 MAPK and the activation of p65 NF-κB. Taken together, apoptosis was alleviated post-TBI based on the action of aloin to remarkably decrease the levels of ROS that regulate MAPK and NF-κB.

After TBI, the decreased normal mitochondrial populations lead to abundant ROS, which aggravate the mitochondrial damages [37, 42]. We also found that the low ΔΨm post-SI in bEnd.3 cells was significantly reversed by aloin to prevent apoptosis. In addition, the endogenous mitochondria-related apoptosis pathways that are activated after TBI include Bax, Bcl-2, and cleaved caspase-3 [2, 8]. Bax, a pro-apoptotic protein, and Bcl-2, an anti-apoptotic protein, were detected on the membrane of mitochondria. A high ratio of Bax/Bcl-2 induces the release of cytochrome c from the mitochondria into the cytoplasm, which promotes the activation of caspase-3 [43, 44] and cleaved caspase-3 is a key player in the final execution phase of apoptotic [45–47]. We found that aloin inhibited the apoptotic pathway via significant reductions of the ratios of Bax/Bcl-2 and cleaved caspase-3/caspase-3.

There were some limitations to this study. First, cerebral edema is mainly regarded as the vasogenic edema caused by disruption of the BBB. However, some investigators argue that cytotoxic edema is also an important part of cerebral edema, coexisting with vasogenic edema [48]. We found no evidence for a relationship between aloin and cytotoxic edema. Second, we created a model of moderate TBI only and used it to investigate the protective effects of aloin on the BBB. Other TBI models of different severity (both milder and more severe) need to be established for future investigations. Third, we only assessed some of the protein factors in the MAPK, NF-κB, and apoptosis-associated pathways, and the effects of aloin on the other signaling pathways need to be explored in further experiments. Last, the interactions between ROS, ΔΨm, and signal pathways are complex. More detailed analyses would be valuable in the future.

Conclusions

In summary, our results demonstrated that aloin protects against disruption of the BBB to reduce the vasogenic edema resulting from secondary injury in a TBI model in mice. Subsequently, we showed that aloin attenuates the loss of TJs in ECs, and this was consistent with the experimental evidence in vitro. A possible mechanism is that aloin influences intracellular ROS generations and changes the ΔΨm by regulating the MAPK, NF-kB, and apoptosis-associated pathways to reduce the apoptosis of ECs. Aloin improves the recovery of neurological deficits and might be a promising therapeutic drug for TBI.

Acknowledgements

We thank Prof. Guo-yuan Yang (School of Biomedical Engineering and Med-X Research Institute of Shanghai Jiao Tong University, Shanghai, China) for guidance in experiments. This work was supported by the National Natural Science Foundation of China (81671207, 81701895, and 81501048) and the Shanghai Jiao Tong University Medicine-Engineering Research Fund (YG2016QN20).

Conflict of interest

The authors declare no competing interests.

Footnotes

Yao Jing and Dian-Xu Yang have contributed equally to this work.

Contributor Information

Jun Ding, Email: djdjdoc@126.com.

Zhi Geng, Email: gengzhi1998@163.com.

Heng-Li Tian, Email: tianhlsh@126.com.

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