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Research ArticleArticle

Insights into the Authentic Active Ingredients and Action Sites of Oral Exogenous Glutathione in the Treatment of Ischemic Brain Injury Based on Pharmacokinetic-Pharmacodynamic Studies

Chong Chen, Qingqing Ding, Boyu Shen, Tengjie Yu, He Wang, Yangfan Xu, Huimin Guo, Kangrui Hu, Lin Xie, Guangji Wang and Yan Liang
Drug Metabolism and Disposition January 2020, 48 (1) 52-62; DOI: https://doi.org/10.1124/dmd.119.089458

Abstract

Glutathione (GSH) has been reported to be closely related to various diseases of the central nervous system, yet its authentic active ingredients and action sites remain unclear. In the present study, oral exogenous GSH significantly alleviated ischemic brain injury, but this result was inconsistent with its low bioavailability and blood-brain barrier (BBB) permeability. To ascertain the exposure of GSH-derived ingredients, including GSH, cysteine (CYS), glutamate (Glu), glycine (GLY), CYS-GLY, and γ-glutamylcysteine (γ-GC) were systematically studied both in vitro and in vivo. The outcomes demonstrated that oral GSH not only increases the GSH and CYS levels in rat striatum and cortex, but it also can decrease the rise of intracerebral Glu concentration caused by ischemia/reperfusion surgery. Then the influence of GSH on the BBB was investigated via measuring IgG leakage, intracerebral endotoxin, and tight-junction proteins. All indicators showed that GSH dosing can repair the destroyed BBB. Oral GSH greatly enhances the exposure of GSH, CYS, CYS-GLY, and γ-GC in rat duodenum, jejunum, ileum, and colon. Accumulating evidence reveals a close link between brain injury and gastrointestinal dysfunction. Our findings further suggest that oral GSH significantly improves intestinal inflammatory damage and barrier disruptions. In conclusion, oral GSH can have a direct therapeutic role in brain injury by stabilizing intracerebral levels of GSH, CYS, and Glu. It can also play an indirect therapeutic role by enhancing the intestinal exposure of GSH, CYS, CYS-GLY, and γ-GC and improving intestinal barrier disruptions.

SIGNIFICANCE STATEMENT The authentic active ingredients and action sites of exogenous glutathione (GSH) in the treatment of ischemic brain injury are unclear. We have shown that oral exogenous GSH not only stabilizes intracerebral levels of GSH, cysteine (CYS), and glutamate (Glu) to act directly on brain injury, but it can also exert an indirect therapeutic role by improving intestinal barrier disruptions. These findings have great significance for revealing the therapeutic effect of GSH on ischemic brain injury and for promoting its further development and clinical application.

Introduction

Ischemic brain injury remains a leading cause of morbidity and mortality worldwide, and a therapeutic regimen to prevent cell death and regenerate damaged cells is lacking (Kuroda et al., 1996; Siesjö et al., 1999; Kharrazian, 2015; Kahl et al., 2018). Oxidative stress and mitochondrial impairment are early events in ischemic brain injury (Lin et al., 1993; Balkaya et al., 2013). The blood-brain barrier (BBB) is also considered a major impediment to treatment of central nervous system (CNS) diseases (Moskowitz et al., 2010; Obermeier et al., 2013; Jamieson et al., 2017). An increasing number of studies have indicated a bidirectional route of communication between the gastrointestinal tract and the CNS, termed the gut-brain axis (Filpa et al., 2016). The degree of intestinal barrier disruption, such as increased intestinal permeability, proinflammatory factors, and endotoxemia, positively correlates with the severity of cerebral ischemia/reperfusion (I/R) injury (Faries et al., 1998). Therefore, the causes and consequences of cerebral I/R injury injuries are complex and serious, and effective therapeutic strategies for cerebral I/R injury, via reducing oxidative damage and/or protecting the integrity of blood-brain and intestinal barriers, are seriously needed.

L-c-glutamyl-l-cysteinylglycine (GSH), an intracellular thiol tripeptide present in all mammalian tissues, plays a crucial role in antioxidation and some other metabolic functions, but it is always depleted during inflammatory responses (Pallardó et al., 2009; Nosareva et al., 2017; Shi et al., 2017; Yang et al., 2019). Some clinical studies have demonstrated that certain neurologic disorders, like Alzheimer disease (AD), Parkinson disease (PD), and stroke, are associated with decreased GSH levels (Merad-Boudia et al., 1998; van Leyen et al., 2006; Ansari and Scheff, 2010; Cojocaru et al., 2013). In addition, the GSH’s γ-peptide linkage can be hydrolyzed to cysteine (CYS)-glutathione (GLY) and glutamate (Glu) by the intestinal γ-glutamyltransferase (γ-GC), and the CYS-GLY can be further cleaved to generate CYS and GLY (Hanigan and Ricketts, 1993; Meister, 1994). Interestingly, most degradation products of GSH have been associated with oxidative stress and brain damage. For instance, depletion of CYS may cause oxidative stress, which can be associated with neurodegenerative disorders such as Huntington disease, amyotrophic lateral sclerosis, AD, and PD (Vandiver et al., 2013; Paul et al., 2014, 2018; Scheltens et al., 2016). The agents that stimulate synthesis of GSH from CYS can inhibit apoptosis induced by oxidative stress (Ratan et al., 1994; Paul et al., 2018). The amino acid GLY, another degradation product of GSH, is a major inhibitory neurotransmitter that binds to GLY receptors to inhibit postsynaptic neurons (Lynch, 2009). Liu et al. (2019) found that injection of GLY could indirectly reduce ischemia-induced neuronal death, brain damage, and functional recovery. As another degradation product of GSH, Glu is reported to be a principal excitatory neurotransmitter in the nervous system. Excessive release of Glu into extracellular spaces may lead to excitotoxic neuronal damages; thus, it is important that Glu concentrations be strictly controlled in the brain (Nishizawa, 2001; Ji et al., 2019). Song et al., 2015 have reported that exogenous intravenous injection of GSH attenuated cerebral infarct volume after ischemic stroke by promoting the PI3K/Akt pathway and inhibiting the translocation of FOXO3 into the nucleus; however, GSH cannot pass the BBB easily. The poor ability to permeate the BBB and the unstable structural characteristics of GSH make its therapeutic effects in ischemic brain injuries disputed. The numerous doubts can be clarified as follows: 1) Does oral GSH also play therapeutic roles in brain injury? 2) What are GSH-related active ingredients? 3) Does GSH act directly on the brain or indirectly on the periphery?

In the present study, the therapeutic effects of oral GSH on ischemic brain injuries were first investigated in I/R model rats (in vivo) and oxygen-glucose deprivation/reperfusion model cells (in vitro). The results suggested that oral GSH could improve the neurologic deficit score, infarct size, histologic lesions, proinflammatory cytokines, and BBB disruption caused by I/R surgeries. The intracerebral distribution of GSH-derived ingredients has shown that I/R surgeries could lead to a decrease in GSH and CYS in the striatum and cortex of injured cerebral hemisphere, where oral GSH could significantly enhance the GSH and CYS levels. The increased intracerebral Glu caused by I/R surgeries could also be reduced by oral GSH. More importantly, oral GSH could significantly enhance the intestinal exposure of CYS, CYS-GLY, and γ-GC, which can improve intestinal inflammatory damages and barrier disruptions by decreasing proinflammatory cytokines and upregulating intestinal tight-junction proteins.

Materials and Methods

Materials

GSH (lot no. B141015) was kindly supplied by Kunming Jida Pharmaceutical Co., LTD (Kunming, Yunnan, China). Cell Counting Kit 8 reagents were purchased from Beyotime Institute of Biotechnology (Shanghai, China). [3-13C]-L-GSH, CYS, CYS-GLY, γ-GC, Glu, GLY, captopril (CAP), N-ethylmaleimide (NEM), and 2′7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO). Buthionine sulfoximine (BSO) were purchased from Aladdin (Shanghai, China).

Animals and Treatments

Animals.

Male healthy Sprague-Dawley rats (aged 8 to 9 weeks, weighing 220–250 g) were purchased from the Sipper-BK Laboratory Animal Co., Ltd (Shanghai, China). The rats were housed under controlled condition (25°C, 55%–60% humidity and 12-hour light/dark cycle) with free access to laboratory food and water. All studies were strictly in compliance with animal care laws and guidelines and approved by the China Pharmaceutical University Animal Care and Use Committee.

Preparation of I/R Model Rats.

The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 ml/100 g). A silicone rubber-coated filament was used to occlude the left middle cerebral artery (MCA) via the external carotid artery into the internal carotid artery and MCA. The correct position was confirmed by meeting a mild resistance, approximately 21 mm beyond the common carotid artery bifurcation. After MCA occlusion (MCAO) for 2 hours, the occluding filament was withdrawn to allow reperfusion. Then the rats were intragastrically adminisered GSH (250 mg/kg) or saline (vehicle).

Neurologic Assessments and Measurement of Infarct Sizes.

Neurologic function was performed by behavioral test according to previous reports (Longa et al., 1989). The modified scoring system is divided into five scales (0–4), with higher scores indicating more severe neurologic injury. Rats were sacrificed 24 hours after I/R surgeries. The collected brains were sectioned into consecutive 2-mm-thick coronal slices with a cryomicrotome (CM1950; Leica, Nussloch, Germany) and then immersed in 1% triphenyltetrazolium chloride (TTC) medium for 10 minutes at 37°C. NIH Image-J 1.8.0 software was used to calculate infarct sizes.

Measurement of Proinflammatory Cytokines in Rat Brain and Ileum.

To evaluate the inflammatory injury caused by I/R surgeries and the protective functions of GSH, the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in the brain and ileum of rats were determined using the corresponding commercial enzyme-linked immunosorbent assay (ELISA) kits (Excellbio Technology, Shanghai, China) according to the manufacturer’s instructions. The results were calibrated by protein concentration.

Measurement of Tight-Junction Proteins in Rat Brain and Ileum.

To assess the expression of tight-junction proteins, rats’ brain and ileum were homogenized in ice-cold phosphate-buffered saline (PBS) buffer and then centrifuged at 12,000g for 10 minutes at 4°C. The protein expressions of ZO-1, Claudin-5, and Occludin in supernatants were measured by using commercial ELISA kits (Shanghai Enzyme-Linked Biotechnology, Shanghai, China).

Histopathological Analysis of Rat Brain and Intestine.

Fresh brains and intestines of rats were fixed in 4% paraformaldehyde for 2 days. Then the fixed tissues were sectioned and stained using a picrosirius red solution for 1 hour according to the protocols of histopathological technique. After washing three times with acidified water, tissue sections were counterstained with Carazzi hematoxylin.

Measurement of IgG Leakage in Rat Brain.

BBB disruption was examined by determining IgG leakage in rat brains. In this process, brain tissues were cut into 50-μm-thick sections and then attached to glass slides after incubation with 3% H2O2. Then the brain sections were incubated with antibody against IgG (1:500; Acam) for 1 hour at room temperature and labeled with diaminobenzidine.

Measurement of Endotoxin.

At 24 hours after the I/R surgeries, venous blood was collected into heparinized pyrogen-free tubes. Brain tissues were homogenized in 10-fold volume of pyrogen-free homogenization buffer. Samples were centrifuged at 12,000g for 5 minutes and then frozen at −80°C. Endotoxin concentration was determined by commercial kits (Horseshoe Reagent Biotechnology, Xiamen, China).

Cell Culture and Treatments

Cell Culture.

The human cerebral microvascular endothelial cell (hCMEC/D3) lines were purchased from Biotechnology Co., Ltd., Shanghai Enzyme Research (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s media (Gibco, Grand Island, New York) containing 1% penicillin/streptomycin, 2 mM of l-glutamine and 10% fetal bovine serum and maintained in a humidified incubator (95% air and 5% CO2) at 37°C.

Oxygen-Glucose Deprivation/Reperfusion Model.

Numerous steps were involved in establishing the oxygen-glucose deprivation/reperfusion (OGD/R) model: 1) We washed the hCMEC/D3 cells seeded in plates using fresh glucose-free medium three times; 2) replaced fresh glucose-free medium with glucose-free Dulbecco’s modified Eagle’s medium (DMEM); 3) put the cell plates into a hypoxic chamber, which is continuously fed with 95% (volume fraction) N2, 5% CO2, and less than 1% O2; and 4) after 12 hours, all the cell plates were removed and replaced with DMEM medium containing glucose with 95% air and 5% CO2 for reoxygenation of cells for 1 hour. In addition, cells in the control group were cultured in DMEM containing glucose and placed under normal culture conditions for the same periods.

Cell Treatments.

For the GSH-dosed group, 2 mM of GSH was previously dissolved in the culture medium at pH 6.8. The hCMEC/D3 cells were treated with 2 mM GSH during OGD/R. For BSO-dosed group, 2 mM BSO (the inhibitor of GSH synthetase) was previously dissolved in the culture medium with 0.02% DMSO. Then the cells were treated with 2 mM BSO during OGD/R.

Cell Viability and Scratch Assays.

Cell viability was measured by adding Cell Couting Kit-8 reagent strictly according to the manufacturer’s instructions. The absorbance intensity was measured at 450 nm using a microplate reader. The results were expressed by the fold change relative to control cell viability. For scratch assays, the hCMEC/D3 cells were seeded in six-well plates in DMEM medium for 24 hours. After 90% confluence, cells were scratched along the diameter of the well. Then the cells were washed twice with serum-free medium and cultured in normal or OGD/R condition. Images were acquired at 12 and 24 hours using a microscope system.

Measurement of Intracellular Reactive Oxygen Species.

Oxidative stress was characterized by intracellularreactive oxygen species (ROS) levels using a DCFH-DA staining assay. Briefly, hCMEC/D3 cells were seeded in 96-well plates at a density of 1 × 105/well. Cells were washed gently by prewarmed sterilized PBS and then incubated in serum-free DMEM containing 10 μM DCFH-DA for 1 hour at 37°C. After washing the cells three times with PBS, images were acquired using laser scanning confocal microscopy. The fluorescence intensity of ROS was determined by a multifunctional fluorescence microplate reader; wavelengths were set at 488 and 525 nm for excitation and emission, respectively.

Quantitative Analysis of GSH, CYS, CYS-GLY, γ-GC, [3-13C]-L-GSH, and [3-13C]CYS-GLY in Biologic Matrices Using Liquid Chromatography-Tandem Mass Spectrometry

Pretreatment of Tissue Specimens.

Fresh rat tissues were homogenized in 10-fold volume of homogenization buffer containing 6 mg/ml of Tris, 0.2 mg/ml of serine, 1.24 mg/ml of boric acid, 4 μg/ml of acivicin, and 7.76 mg/ml of NEM. After adding 10 μl of internal standard solution (5 μg/ml of CAP-NEM) into 50 μl of tissue homogenization, derivation lasted for 1 hour under the light-proof environment. Then 200 μl of methanol was added to precipitate the proteins. After vortex mixing for 5 minutes and centrifuging at 30,000g for 10 minutes at 4°C, 5 μl of supernatant was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) 4000 system (SCIEX, Framingham, MA).

Pretreatment of Cell Specimens.

The cells were washed gently with serum-free medium three times. After adding 100 μl of H2O, cell suspension was collected after repeated freezing and thawing three times. In 50 μl of cell suspension, 10 μl of derivatization internal standard (5 μg/ml of CAP-NEM), and 50 μl of derivative reagent were added, and derivation lasted 1 hour under the light-proof environment. Methanol (200 μl) was added to remove the proteins, and 5 μl of supernatant was analyzed using LC-MS/MS.

LC-MS/MS Conditions.

Chromatographic separation was carried out on a Sepax Bio-ODS SP column (4.6 × 150 mm, 5 μm; Sepax Technologies, Newark, DE) under a gradient program. Mobile phase A consisted of H2O containing 0.1% formic acid and 2 mM ammonium formate; mobile phase B was methanol. The gradient program was as follows: 0 minute, 10% B →0.2 minute, 10% B → 0.4 minute, 50% B → 4 minutes, 50% B → 4.5 minutes, 10% B → 6 minutes, 10% B. The MS parameters were optimized as follows: ion spray voltage, 4500 V; ion source gas 1, 60 psi; curtain gas, 10 psi; ion source gas, 50 psi; collision gas, 6 psi; source temperature, 550°C. The optimized multiple reaction monitoring parameters are listed in Supplemental Table S1.

Quantitative Analysis of GLY and Glu in Biologic Matrices Using LC-Q-TOF MS

Pretreatment of Tissue Specimens.

The fresh rat tissues (50 mg) were homogenized in 500 μl of H2O. After centrifuging at 30,000g for 5 minutes, 400 μl of methanol containing 13C-glutamine (15 μg/ml) was added to 100 μl of the supernatant, followed by vortex mixing for 5 minutes and centrifuging at 30,000g for 10 minutes at 4°C. The supernatant (300 μl) was evaporated to dryness in a rotary evaporator, and the residue was reconstituted in 150 μl of H2O.

Pretreatment of Cell Specimens.

A total of 800 μl of methanol containing 1.5 μg/ml of 13C-glutamine was added to 200 μl of cell suspension. After vortex mixing for 5 minutes and centrifuging at 30,000g for 10 minutes, 300 μl of supernatant was evaporated to dryness in a rotary evaporator, and the residue was reconstituted in 100 μl of H2O.

LC-Q-TOF MS Conditions.

Chromatographic separation was carried out on a Waters bridge amide (3.5 μm, 4.6 × 100 mm) column under a gradient program. The flow rate was 0.4 ml/min. Mobile phase A consisted of H2O containing 5 mM ammonium acetate and 5% acetonitrile, and pH was adjusted to 9.0 with aqueous ammonia; mobile phase B was acetonitrile. The gradient program was set as follows: 0 minute, 15% B → 3 minutes, 15% B → 6 minutes, 70% B → 15 minutes, 98% B → 18 minutes, 98% B → 19 minutes, 15% B → 26 minutes, 15% B. MS analysis was performed using an AB Sciex 5600 Triple TOF MS, which operated in negative ionization mode with a DuoSpray ion source (Concord, ON, Canada). The source conditions were set as follows: ion-spray voltage floating −4.5 kV, declustering potential 70 V, turbo spray temperature 500°C, nebulizer gas (gas 1) 50 psi, heater gas (gas 2) 60 psi, curtain gas 30 psi. The accumulation time for MS1 full scan was 100 milliseconds for scanning a mass range from 100 to 1000 Da. The accumulation time for each information-dependent acquisition experiment was 50 milliseconds, and the Collision Energy(CE) was set to 35 V with a CE spread of 15 V in high-sensitivity mode. All parameters were controlled by Analyst TF 1.7 software, and data were processed by MultiQuant 2.0 Software (Sciex).

Statistical Analysis

All data were analyzed by one-way ANOVA using Graph Pad Prism (version 6.0; San Diego, CA). Differences among means were analyzed by post hoc analysis or Dunnett’s multiple comparisons test. Statistically significant difference was considered P < 0.05.

Results

Investigation of the Therapeutic Effect of Oral GSH on I/R in Rats.

The rats were intragastrically administered GSH (250 mg/kg) or saline (vehicle) after I/R surgeries, and brain infarct volume was estimated using TTC staining assay. As shown in Fig. 1A, cerebral infarcts were pronounced after I/R surgeries, and the degree of ischemia of GSH-dosed rats was significantly lower than that of I/R model rats. Infarct size could be decreased from 31.21% to 9.62% by oral GSH (Fig. 1B). Meanwhile, intragastric administration of GSH greatly decreased the neurologic deficit score of I/R-model rats (Fig. 1C). The results of H&E staining of coronal sections showed that the brains of I/R-model rats, mainly the striatal and cortical regions, had serious histologic lesions, characterized by incomplete cortical structure, necrotic neurons, and broken cerebrovascular tissues. The histologic lesions of I/R-model rats were effectively alleviated by intragastric administration of GSH (Fig. 1D). Proinflammatory cytokine (TNF-α, IL-1β, and IL-6) levels in rat brains were determined to investigate further the therapeutic effect of oral GSH on ischemic brain injury. As shown in Fig. 1, E–G, levels of TNF-α, IL-1β, and IL-6 in the I/R rat brain were significantly higher than those of the sham-operated group (one-way ANOVA, P < 0.01). GSH dosing could significantly reverse the upregulation of proinflammatory cytokine induced by I/R surgeries.

Fig. 1.
Fig. 1.

Therapeutic effect of oral GSH on ischemic brain injury in rats. (A) Coronal sections of TTC-stained brains, (B) cerebral infarct volume, (C) neurology deficit score, (D) H&E staining of coronal section, (E) levels of TNF-α, (F) levels of IL-1β, and (G) levels of IL-6. Data are mean ± SD. *P < 0.05 and **P < 0.01 by unpaired two-tailed Student’s t-test.

Investigation of the Therapeutic Effect of Exogenous GSH in hCMEC/D3 Cells.

The OGD/R hCMEC/D3 cell model was built to confirm the therapeutic effects of exogenous GSH on ischemic brain injury. As shown in Fig. 2A, the viability of hCMEC/D3 cells could be significantly enhanced by exogenous GSH (P < 0.01), whereas BSO reduced cellular viability significantly (P < 0.05). The following experiment was performed to investigate the effect of GSH on cell migration using a scratch assay. As shown in Fig. 2B and Supplemental Fig. S1, exogenous GSH significantly increased the migration rate of hCMEC/D3 cells cultured in normal conditions, whereas BSO dosing reduced the migration of cells only to some extent. In addition, exogenous GSH could significantly increase the migration rate of the OGD/R-model hCMEC/D3 cells at 12 hours (P < 0.01), whereas BSO dosing further inhibited cell migration. Furthermore, the influence of GSH on the oxidative stress levels was assessed, and the intracellular ROS was labeled with DCFH-DA probe. Clearly, the fluorescence intensity of OGD/R-model cells was much greater than that of the conventional cells, and BSO-treated cells had greater fluorescence intensity than that of model-group cells. GSH could dose dependently reduce the fluorescence intensity of OGD/R-model cells within the dose range of 0.5 to 5 mM (Fig. 2, C and D). Thus, exogenous GSH could effectively repair hypoxia-induced cell damage.

Fig. 2.
Fig. 2.

Therapeutic effect of oral GSH on ischemic brain injury in hCMEC/D3 cells. (A) The viability (%) of control, OGD/R-model, GSH-dosed, and BSO-dosed cells. (B) The hCMEC/D3 cell migration, (C) fluorescence image of intracellular ROS, and (D) fluorescence intensity of intracellular ROS. Data are mean ± SD.*P < 0.05 and **P < 0.01 by unpaired two-tailed Student’s t-test.

Distribution of GSH-Derived Ingredients in hCMEC/D3 Cells.

OGD/R-model hCMEC/D3 cells were treated with 2 mM of GSH or BSO for 1, 6, and 12 hours, respectively. Then the intracellular concentrations of GSH-derived ingredients, including GSH, CYS, Glu, GLY, CYS-GLY, and γ-GC, were measured using LC-MS/MS and LC-Q-TOF MS. As shown in Fig. 3A, GSH levels in the cells modeled by OGD/R for 1 hour were significantly higher than those in conventional cells. No significant change was found in GSH concentration after treatment with 2 mM GSH for 1 hour, whereas BSO treatment reduced intracellular GSH concentration significantly (P < 0.05). By contrast, GSH levels in the cells modeled by OGD/R for 6 and 12 hours were significantly lower than those in conventional cells. After treatment with 2 mM GSH for 6 or 12 hours, the intracellular GSH concentrations were much greater than those in OGD/R model cells. Exposure to BSO for 6 or 12 hours significantly reduced the intracellular GSH concentrations (P < 0.01). Then the intracellular concentrations of the degradation products of GSH were measured. As shown in Fig. 3B, the concentrations of CYS in control, OGD/R-model, OGD/R-model + GSH, and OGD/R-model + BSO groups had no significant difference at 1 and 6 hours; however, CYS levels in the OGD/R-model cells collected at 12 hours were significantly greater than those in the control group, indicating the reduced ability of CYS to synthesize GSH in OGD/R-model cells. In contrast, the decreased CYS levels in GSH-treated cells demonstrated that GSH could restore the synthesis of CYS by alleviating hypoxia-induced cell damage. Exposure of Glu is shown in Fig. 3C. The intracellular Glu concentrations in hCMEC/D3 cells modeled by OGD/R for 1, 6, and 12 hours were all significantly lower than those in control cells. GSH treatment could greatly increase the Glu levels, whereas BSO dosing reduced the intracellular Glu exposure at 12 hours. The other degradation products, including GLY, CYS-GLY, and γ-GC, were also quantitatively analyzed, and the results showed that GSH treatment had no significant effect on intracellular concentrations of GLY and CYS-GLY (Fig. 3, D and E). The concentration of γ-GC in hCMEC/D3 cells was lower than the minimum quantitative limit. In conclusion, exogenous GSH mainly affected the intracellular concentrations of GSH, CYS, and Glu.

Fig. 3.
Fig. 3.

Distribution of GSH-derived ingredients in hCMEC/D3 cells after treatment with 2 mM GSH or 2 mM BSO for 1, 6, and 12 hours. (A) GSH, (B) CYS, (C) Glu, (D) GLY, (E) CYS-GLY, (F) concentrations of [3-13C]-L-GSH in hCMEC/D3 cells, and (G) concentrations of GSH in hCMEC/D3 cells. Data are mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001 by unpaired two-tailed Student’s t-test.

To investigate further the influence of exogenous GSH on intracellular GSH levels, OGD/R model hCMEC/D3 cells were incubated with 200 μM [3-13C]-L-GSH, and the intracellular and [3-13C]-L-GSH and GSH concentrations were both measured using LC-MS/MS. As shown in Fig. 3, F and G, the intracellular concentration of [3-13C]-L-GSH was about 1/50 of that of GSH and was much lower than 200 μM. Thus, GSH had low uptake capability in hCMEC/D3 cells, and the endogenous GSH level in GSH-dosed hCMEC/D3 was much higher than that of exogenous GSH.

Distribution of GSH-Derived Ingredients in I/R Model Rat Brains.

The intracerebral distribution of GSH-derived ingredients in sham-operated, I/R-model, and I/R + GSH rats was measured to search the active ingredients and action sites of oral GSH. Clearly, the intracerebral GSH levels in the I/R-model rats were significantly lower than those of sham-operated group, and GSH dosing could significantly reverse the reduction of GSH concentration caused by I/R surgeries (Fig. 4A). The changing pattern of CYS was almost the same as that of GSH, and GSH dosing could enhance the intracerebral CYS levels in I/R-model rats (Fig. 4B); however, the changing pattern of Glu was in contrast to that of GSH. I/R surgeries significantly increased the intracerebral Glu exposure, and GSH dosing could decrease the Glu concentrations in the I/R-model rat brain (Fig. 4C). The intracerebral concentrations of CYS-GLY and GLY in sham-operated, I/R-model, and I/R + GSH groups had no significant difference (Fig. 4, D and E). Besides, the concentration of γ-GC was too low to be detected.

Fig. 4.
Fig. 4.

Intracerebral distribution of GSH-derived ingredients in sham-operated, I/R-model, and I/R + GSH rats. (A) GSH, (B) CYS, (C) Glu, (D) GLY, and (E) CYS-GLY. (F) Concentrations of GSH in striatum, (G) concentrations of GSH in cortex, (H) concentrations of GSH in hippocampus, (I) concentrations of GSH in hypothalamus, (J) concentrations of CYS in striatum, (K) concentrations of CYS in cortex, (L) concentrations of CYS in hippocampus, and (M) concentrations of CYS in hypothalamus. Data are mean ± SD.*P < 0.05, **P < 0.01 and ***P < 0.001 by unpaired two-tailed Student’s t-test.

The concentrations of GSH and CYS in different brain regions, including the striatum, cortex, hippocampus, and hypothalamus, were further quantitatively analyzed to seek the specific action sites of GSH. As shown in Fig. 4, F and G, the GSH concentrations in the striatum and cortex of left injured (ipsilateral) hemisphere were significantly lower than those of sham-operated group, and intragastric administration of GSH could greatly increase the GSH concentrations in ipsilateral striatum and cortex. By contrast, the GSH concentrations in ipsilateral hippocampus were significantly greater than those of the sham-operated group, and intragastric administration of GSH could greatly reverse the rise of GSH concentration caused by I/R surgeries (Fig. 4H). In addition, exogenous GSH could significantly enhance the GSH concentration in hippocampus of I/R rats (Fig. 4I). In the right noninjured (contralateral) hemisphere, the GSH concentrations had no significant difference among the sham-operated I/R model and I/R + GSH groups. Like GSH, the CYS concentrations in the ipsilateral striatum and cortex of I/R rats were significantly lower than those of sham-operated group rats, and intragastric administration of GSH greatly increased the GSH concentrations in these two regions. In the contralateral cerebral hemisphere, GSH increased the striatal CYS level, but it had no effect on cortical CYS concentrations (Fig. 4, J and K). In addition, the CYS concentrations in the ipsilateral hippocampus were decreased by I/R surgeries, and GSH dosing could increase the CYS to some extent, although the difference between I/R and I/R + GSH groups was not significant. Curiously, GSH dosing decreased the CYS levels in the contralateral hippocampus (Fig. 4L). Besides, the CYS levels in hypothalamus of I/R group were much higher than those of sham-operated rats, and GSH dosing could further increase the CYS concentration in hypothalamus. In conclusion, the therapeutic effect of oral GSH on ischemic brain injury might rely primarily on the concentration increase of GSH and CYS in the ipsilateral striatum and cortex, and reduction in intracerebral Glu exposure might also be one of the mechanisms of GSH to treat brain injury. Thus, oral GSH could play a direct role in the treatment of brain injury by stabilizing intracerebral levels of GSH, CYS, and Glu.

Influence of Exogenous GSH on the Distribution of GSH in Rat Tissues.

The intratissue concentrations of GSH-derived ingredients in rat plasma, heart, liver, stomach, duodenum, jejunum, ileum, colon, and kidney were measured to investigate the influence of exogenous GSH on the distribution of GSH-derived ingredients. As shown in Fig. 5A, intragastric administration of GSH could significantly increase the concentrations of GSH in I/R-model rat plasma collected at 7 and 24 hours. Both I/R surgeries and oral exogenous GSH had no obvious effect on GSH level in the rat heart, liver, kidney, and stomach (Fig. 5, B–E); however, I/R surgeries and exogenous GSH had great effects on intestinal GSH level (Fig. 5, F–I). For instance, intragastric administration of GSH increased the duodenal GSH level of sham-operated rats by 10-fold. The GSH levels in the duodenum of I/R + GSH-group rats collected at 7 and 24 hours were about 5 and 18 times greater than those in the I/R model group, respectively. Besides, intragastric administration of GSH could greatly enhance the GSH levels in the rat jejunum, ileum, and colon of both sham-operated and I/R-model groups.

Fig. 5.
Fig. 5.

Distribution of GSH in rat tissues. (A) Plasma, (B) heart, (C) liver, (D) kidney, (E) stomach, (F) duodenum, (G) jejunum, (H) ileum, and (I) colon. Data are mean ± SD.*P < 0.05, **P < 0.01 and ***P < 0.001 by unpaired two-tailed Student’s t-test.

The intratissue concentrations of CYS, CYS-GLY, γ-GC, Glu, and GLY were also measured, and the results were shown in Supplemental Figs. S2–S6 . Intragastric administration of GSH could significantly increase the concentrations of CYS in I/R- model rat plasma and duodenum. GSH dosing could also greatly enhance the CYS levels in the I/R model rat jejunum and ileum collected at 7 hours, whereas the effect of exogenous GSH on the CYS level in other tissues was not significant (Supplemental Fig. S2). The analysis results of CYS-GLY showed that the CYS-GLY concentrations in rat plasma, liver, kidney, and heart almost could not be altered by GSH dosing. Nevertheless, intragastric administration of GSH significantly increased the concentrations of CYS-GLY in duodenum, jejunum, ileum, and colon of sham-operated and I/R model rats (Supplemental Fig. S3). The exposure of γ-GC was also quantitatively analyzed, although its concentrations were extremely low, and the γ-GC levels in the plasma and intestine could also be greatly enhanced by oral exogenous GSH (Supplemental Fig. S4). The Glu and GLY concentrations in rat tissues were much higher than those of CYS-GLY, γ-GC, and CYS, and the effect of oral exogenous GSH on the exposure of Glu and GLY was significantly lower than that of other ingredients (Supplemental Figs. S5 and S6). In conclusion, intragastric administration of GSH could greatly enhance the intestinal CYS, CYS-GLY, and γ-GC levels in both sham-operated and I/R-model rats.

Influence of Exogenous GSH on Intestinal Injury Caused by I/R Surgeries.

Accumulating evidence reveals a close link between brain injury and gastrointestinal dysfunction. Our findings suggested that oral exogenous GSH greatly enhanced the concentrations of GSH, CYS, CYS-GLY, and γ-GC in rat intestines. In our study, the effects of I/R surgeries and GSH dosing on gastrointestinal dysfunction were investigated by observing pathologic sections and measuring the levels of inflammatory factors and tight junction proteins. More precisely, the intestines of I/R-model rats were characterized by severe accumulation of inflammatory cells, incomplete structure, or even shedding partial villus, gland degeneration, and crypt structure change (Fig. 6A). Intragastric administration of GSH greatly improved the intestinal pathologic features. Besides, the levels of IL-6, TNF-α, and IL-1β in the I/R rat intestine were significantly higher than those of sham-operated group (one-way ANOVA, P < 0.01), and GSH-dosing could significantly reverse the upregulation of proinflammatory cytokines induced by I/R surgeries (Fig. 6, B–D). In addition, the tight-junction proteins in rat intestine were determined based on ELISA assay. As shown in Fig. 6, E–G, the intestinal levels of ZO-1, claudin-5, and occluding proteins in the I/R group were significantly lower than those of the sham-operated group. I/R surgeries might lead to the destruction of intestinal barrier, and GSH dosing could significantly reverse the downregulation of intestinal ZO-1 and claudin-5 proteins. Thus, oral exogenous GSH could alleviate intestinal damage caused by brain injury by reducing intestinal inflammation and intestinal barrier.

Fig. 6.
Fig. 6.

Influence of exogenous GSH on intestinal injury caused by I/R surgeries. (A) Pathologic sections, (B) levels of IL-6, (C) levels of TNF-α, (D) levels of IL-1β, (E) expression of ZO-1, (F) expression of claudin-5 proteins, and (G) expression of occludin proteins. Data are mean ± SD.*P < 0.05, **P < 0.01, ***P <0.001 and **** P < 0.0001 by unpaired two-tailed Student’s t-test.

Influence of Oral Exogenous GSH on BBB Destruction after I/R Surgeries

The integrity of the BBB was investigated by determining cerebral IgG leakage, tight-junction proteins, and the levels of endotoxin. The results illustrated that there was no IgG leakage in the sham-operated rat brain. After 24 hours of I/R surgeries, severe IgG leakage occurred on the left injured hemisphere, and GSH dosing significantly ameliorated IgG leakage (Fig. 7A). Besides, GSH dosing dramatically reversed the downregulation of intracerebral ZO-1 and claudin-5 proteins induced by I/R surgeries (Fig. 7, B–D). The improved intestinal and BBB disruption might alter the levels of endotoxin, which positively correlate with severity of brain injuries (Faries et al., 1998). Herein, the intracerebral endotoxin levels were determined to further elucidate the protective effect of GSH on intestinal and BBB. As shown in Fig. 7E, the intracerebral endotoxin level in I/R model rats was significantly greater than that in the sham-operated group (P < 0.01). Intragastric administration of GSH could greatly decrease the intracerebral endotoxin level of the I/R rats (P < 0.05). Similarly, I/R surgeries could lead to elevated plasma endomycin level, whereas GSH dosing dramatically reduced it in the I/R model rat plasma (Fig. 7F). Thus, oral exogenous GSH could significantly repair the damage of BBB caused by brain injury.

Fig. 7.
Fig. 7.

Influence of exogenous GSH on destruction of BBB integrity caused by I/R surgeries. (A) IgG expression in rat brain, (B) expression of ZO-1 proteins, (C) expression of claudin-5 proteins, (D) expression of occludin proteins, (E) levels of endotoxin in brain, and (F) levels of endotoxin in plasma. Data are mean ± SD.*P < 0.05, **P < 0.01 and ***P < 0.001 by unpaired two-tailed Student’s t-test.

Discussion

Oxidative stress is associated with various CNS diseases, such as stroke, AD, PD, and others (Merad-Boudia et al., 1998; van Leyen et al., 2006; Ansari and Scheff, 2010; Cojocaru et al., 2013). As an intracellular thiol tripeptide present in all mammalian tissues, GSH plays a crucial role in cellular protection against oxidant damage. Reduction of the GSH level in vivo may lead to the degeneration of dopaminergic neurons (Li et al., 1997; Cadenas, 2004; Bilgin et al., 2019). Song et al., 2015 reported that intravenous injection of exogenous GSH attenuated cerebral infarct volume after ischemic stroke by promoting the PI3K/Akt pathway. The therapeutic effects of oral GSH were investigated in the I/R-model rats and OGD/R-model hCMEC/D3 cells in the present study. The results demonstrated that intragastric administration of GSH could not only greatly improve the neurologic deficit score, infarct size, and histologic lesions of the I/R-model rat, but it could also significantly reverse the upregulation of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) induced by I/R surgeries. In vitro PD studies further suggested that exogenous GSH could dose dependently increase the survival of hCMEC/D3 during OGD/R by increasing the migration rate of the OGD/R-model cells and suppressing the generation of ROS. BSO is an inhibitor of the rate-limiting enzyme γ-glutamylcysteine synthetase in GSH biosynthesis (Li et al., 1997). In addition to increasing intracellular ROS, BSO treatment could lead to a decrease in the survival and migration rate of OGD/R-model hCMEC/D3 cells. Thus, exogenous GSH could be used to treat ischemic brain injury; however, the effectiveness of oral GSH as a therapeutic agent for brain injuries may be limited because of its low bioavailability and poor ability to permeate BBB (Homma and Fujii, 2015). According to previous reports, the intestinal γ-glutamyltransferase could hydrolyze GSH’s γ-peptide linkage to produce CYS-GLY and Glu, and the CYS-GLY could be further cleaved to generate CYS and GLY (Hanigan and Ricketts, 1993; Meister, 1994). Interestingly, most degradation products of GSH, including CYS, GLY, and Glu, have been reported to be associated with oxidative stress and brain damage. For instance, CYS participates in a wide variety of redox reactions because of its sulfur atom (Vandiver et al., 2013; Paul et al., 2014, 2018; Scheltens et al., 2016). Another degradation product, GLY, is a major inhibitory neurotransmitter that binds to glycine receptors to inhibit postsynaptic neurons (Lynch, 2009; Liu et al., 2019). Besides, excessive release of Glu into extracellular spaces is proved to cause excitotoxic neuronal damages, and the concentration of Glu in the brain should be strictly controlled (Nishizawa, 2001; Ji et al., 2019).

To elucidate the paradox between the pharmacokinetics and pharmacodynamics of GSH, first we carried out studies on the distribution and uptake of GSH-derived ingredients, including CYS, GLY, Glu, CYS-GLY, and γ-GC, in both in vitro and in vivo models. In vitro studies have suggested that exogenous GSH mainly enhanced the intracellular concentrations of GSH and Glu but had almost no significant effect on exposure of GLY, CYS-GLY, and γ-GC in hCMEC/D3 cells. The intracellular CYS level increased significantly after OGD/R injury, and GSH treatment could decrease CYS levels by alleviating OGD/R-induced cell damage and restoring the synthesizing ability of CYS. To investigate more fully the uptake capacity of GSH, the OGD/R-model hCMEC/D3 cells were incubated with 200 μM of [3-13C]-L-GSH. The results proved that the concentration of endogenous GSH was significantly higher than that of exogenous GSH. GSH had low uptake capability, which was consistent with its low bioavailability. The cerebral distribution of GSH-derived ingredients (GSH, CYS, GLY, GLY, CYS-GLY, and γ-GC) showed that I/R surgeries could lead to decreased levels of GSH and CYS in the rat striatum and cortex of injured cerebral hemisphere, and intragastric administration of GSH could significantly reverse this decline in GSH and CYS levels caused by I/R surgeries. We also found that I/R surgeries resulted in an increase in intracerebral Glu concentration. The main reason might lie in the increased permeability of the BBB, which always produced severe clinical consequences, such as vasogenic brain edema, hemorrhagic transformation, and poor neurologic outcomes (Xu et al., 2005; Moskowitz et al., 2010; Khatri et al., 2012). Our results also suggested that the increase in Glu exposure in the I/R rat brain could be significantly reversed by oral GSH, which implied that exogenous GSH may have the function of repairing BBB. In fact, the leakage of serum proteins into brain parenchyma could be used to examine the integrity of the BBB (Mann et al., 2016). Then we investigated the influence of GSH on the BBB by determining the IgG leakage, intracerebral tight-junction proteins, and the levels of endotoxin. All indicators showed that GSH dosing had the function of repairing the damaged BBB caused by I/R operation. Thus, oral exogenous GSH could play a direct role in the treatment of brain injury by stabilizing intracerebral GSH, CYS, and Glu, repairing damaged BBB and downregulating proinflammatory cytokines.

Although oral exogenous GSH could play a direct therapeutic role in brain injury, we question whether GSH can also play an indirect therapeutic role owing to its low bioavailability and poor ability to permeate the BBB. The concentrations of GSH-derived ingredients in rat plasma, heart, liver, kidney, stomach, duodenum, jejunum, ileum, and colon were measured to identify other possible action sites of oral GSH. Unexpectedly, intragastric administration of GSH could significantly enhance the intestinal exposure of GSH-derived ingredients, including GSH, CYS, CYS-GLY, and γ-GC. According to previous reports, GSH was formed from CYS by the enzymatic action of glutamate-cysteine ligase (GCL), which comprised the catalytic subunit (GCLc) and the modulating subunit (GCLm) (Meister and Anderson, 1983; Lu, 2013; Homma and Fujii, 2015). The GCLc expression in rat liver, the main organ for GSH synthesis, was determined to elucidate the effect of exogenous GSH on GSH synthesis in vivo. As shown in Supplemental Fig. S7, the expression of GCLc in the rat liver was significantly decreased by I/R surgeries (P < 0.001), and oral administration of GSH had no obvious effect on GCLc expression. Thus, the increase in GSH concentrations in rat brains and intestines was not achieved by increasing GSH synthesis. Accumulating evidence reveals a close linkage of brain injury and gastrointestinal dysfunction. The influence of the gastrointestinal tract on the brain of human has been noted since the 19th century, and the neuroinflammation hypothesis has been advocated since the 21st century (Evrensel et al., 2019). The structure and function of the brain can be modulated by the gut; conversely, the brain regulates the gut microenvironment and microbiota composition (Maqsood and Stone, 2016; Zhao et al., 2018). Changes in the intestinal flora easily caused small intestinal immune dysfunction, which might suppress the transporting of IL-17-positive γδT cells and/or Th17 cells from the small intestine to the peripheral blood and then reduce systemic inflammation after brain injury (Benakis et al., 2016; Honda and Littman, 2016). Gastrointestinal dysfunction, including mucosal injury, barrier disruption, dysmotility, and inflammation, caused by brain injury might be one of the causes of morbidity and mortality (Tan et al., 2011; Olsen et al., 2013). Resveratrol has been reported to improve cerebral ischemia by decreasing the ischemia-induced transfer of cytokines (IL-17A, IL-23, IL-10, interferon-γ, and IL-4) from the small intestine to the blood by attenuating the small intestinal epithelial permeability (Dou et al., 2019). In this study, the effects of I/R surgeries and GSH dosing on the gastrointestinal dysfunction were investigated by observing pathologic sections and measuring the levels of inflammatory factors and tight-junction proteins. Our findings suggested that intragastric administration of GSH could significantly reduce intestinal inflammatory damage and improve intestinal barrier disruption by decreasing proinflammatory cytokines and upregulating intestinal tight junction proteins ZO-1 and claudin-5.

In summary, oral exogenous GSH not only plays a direct therapeutic role in brain injury by stabilizing intracerebral levels of GSH, CYS, and Glu, but it can also have an indirect therapeutic role by enhancing the intestinal exposure of GSH, CYS, CYS-GLY, and γ-GC and improving intestinal barrier disruptions.

Acknowledgments

We thank Haofeng Li, Jiajia Shen, and Changjian Li for contributing to study design and execution.

Authorship Contributions

Participated in research design: Chen, Ding, Wang, Liang.

Conducted experiments: Chen, Ding, Shen, Yu, Wang.

Contributed new reagents or analytic tools: Xu, Guo, Xie.

Performed data analysis: Chen, Liang.

Wrote or contributed to the writing of the manuscript: Chen, Ding, Hu, Wang, Liang.

Footnotes

    • Received September 22, 2019.
    • Accepted October 25, 2019.

  • ↵1 C.C. and Q.D. contributed equally to this work.

  • This study was supported by the National Nature Science Foundation of China [Grants 81573559, 81530098] and the Nature Science Foundation of Jiangsu Province [Grant BK20171395].

  • https://doi.org/10.1124/dmd.119.089458.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

Abbreviations

γ-GC
γ-glutamylcysteine
AD
Alzheimer disease
BBB
blood-brain barrier
BSO
buthionine sulfoximine
CAP
captopril
CNS
central nervous system
CYS
cysteine
DCFH-DA
2′7 ′-dichlorofluorescin diacetate
DMEM
Dulbecco’s modified Eagle’s medium
ELISA
enzyme-linked immunosorbeny assay
GCL
glutamate-cysteine ligase
GSH
glutathione
Glu
glutamate
GLY
glycine
hCMEC/D3
human cerebral microvascular endothelial cell
IL-6
interleukin-6
IL-1β
interleukin-1β
I/R
ischemia/reperfusion
MCA
middle cerebral artery
MCAO
middle cerebral artery occlusion
NEM
N-ethylmaleimide
OGD/R
oxygen-glucose deprivation/reperfusion
PBS
phosphate-buffered saline
PD
Parkinson disease
ROS
reactive oxygen species
TNF-α
tumor necrosis factor-α
TTC
triphenyltetrazolium chloride

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Active Ingredients and Action Sites of Oral Exogenous GSH

Chong Chen, Qingqing Ding, Boyu Shen, Tengjie Yu, He Wang, Yangfan Xu, Huimin Guo, Kangrui Hu, Lin Xie, Guangji Wang and Yan Liang
Drug Metabolism and Disposition January 1, 2020, 48 (1) 52-62; DOI: https://doi.org/10.1124/dmd.119.089458
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