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. Author manuscript; available in PMC: 2020 May 1.
Published in final edited form as: Alcohol. 2018 Aug 21;76:81–89. doi: 10.1016/j.alcohol.2018.08.005

Influence of Low-Dose Alcohol Consumption on Post-Ischemic Inflammation: Role of Cystathionine γ-Lyase

Kimberly D McCarter 1, Chun Li 1, Jiyu Li 1,2, Guodong Xu 1, Hong Sun 1
PMCID: PMC6416088  NIHMSID: NIHMS1510940  PMID: 30597416

Abstract

Low-dose alcohol consumption (LAC) has been shown to suppress post-ischemic inflammation and alleviate cerebral ischemia/reperfusion (I/R) injury. Cystathionine γ-Lyase (CSE) is one of the enzymes that endogenously produce hydrogen sulfide (H2S), which has an anti-inflammatory property at low concentration. We determined the potential role of CSE in the protective effect of LAC. Male C57BL/6J mice were divided into two groups, an ethanol group and a control group, and gavage fed with 0.7 g/kg/day ethanol or volume-matched water once a day for 8 weeks. Transient focal cerebral ischemia was induced by unilateral middle cerebral artery occlusion (MCAO) for 90 minutes. CSE inhibitors were intraperitoneally given 30 minutes prior to the ischemia. Cerebral I/R injury, H2S production, adhesion molecules, IL-1 receptor accessory protein (IL-1RAcP), IL-1β, microglial activation, and neutrophil infiltration were evaluated at 24 hours of reperfusion. Eight-week ethanol feeding upregulated CSE in the cerebral cortex and reduced cerebral I/R injury. Moreover, ethanol increased post-ischemic H2S production and alleviated the post-ischemic inflammatory response (expression of adhesion molecules, IL-1RAcP, IL-1β, microglial activation, and neutrophil infiltration) in the peri-infarct cerebral cortex. Both inhibitors of CSE, DL-Proparglyglycine (PAG) and β-cyano-L-alanine (BCA), abolished the protective effect of ethanol on cerebral I/R injury. In addition, PAG attenuated the inhibitory effect of ethanol on the post-ischemic inflammation. Thus, LAC may protect against cerebral I/R injury by suppressing post-ischemic inflammation via an upregulated CSE.

Keywords: Ethanol, brain, ischemia/reperfusion, inflammation, cystathionine γ-lyase

INTRODUCTION

Stroke continues to be one of the leading causes of death and permanent disability in adults worldwide. Ischemic stroke accounts for 87% of all diagnosed strokes (Benjamin et al., 2017; Favate and Younger, 2016). Intravenous recombinant tissue plasminogen activator (tPA) and intra-arterial therapy (IAP) are currently used to treat acute ischemic stroke. Both treatments result in a recanalization/reperfusion. Thus, transient focal cerebral ischemia has become one of the most common types of ischemic stroke. Although recanalization/reperfusion is critical for restoring normal function, it can paradoxically result in secondary damage, called cerebral ischemia/reperfusion (I/R) injury (Jean et al., 1998). The mechanisms underlying cerebral I/R injury are complex and involve several interacting elements, including oxidative/nitrosative stress, activation of apoptotic and autophagic pathways, and increased inflammatory response (Chen et al., 2014; Jean et al., 1998; Kalogeris et al., 2012). After transient focal cerebral ischemia, an acute inflammatory response, which is characterized by expression of adhesion molecules, elaboration of cytokines/chemokines, activation of microglia, and infiltration of leukocytes, subsequently worsens the injury in the penumbra area. Alcohol is one of the most commonly and regularly used chemical substances. The brain is one of the major target organs of the action of alcohol (Alfonso-Loeches S, and Guerri, 2011). Epidemiological studies suggest that alcohol consumption has dual effects on both the incidence and prognosis of ischemic stroke. This dual effect generates a J-shaped pattern in the relationship between chronic alcohol intake and stroke incidence and prognosis. Heavy alcohol consumption has been defined as 4 or more American standard drinks per day, while low consumption has been defined as 1 to 2 American standard drinks per day (Hansagi et al., 1995; Ikehara et al., 2008; Ronksley et al., 2011). In a recent study, we found that low-dose alcohol consumption (LAC) was neuroprotective against cerebral I/R injury via a suppression of post-ischemic inflammation in rats (McCarter et al., 2017). Thus, the first goal of the present study was to corroborate the neuroprotective effect of LAC in a mouse model of transient focal cerebral ischemia.

Hydrogen Sulfide (H2S) is a well-known toxic gas. Recent experimental studies have revealed that H2S is produced enzymatically in all mammalian species and serves as a gaseous signaling molecule involved in numerous biological processes. There is emerging evidence to indicate that H2S is cytoprotective at a low concentration in various organ systems including the heart, liver, kidney, and brain (Wu et al., 2015). In a rat model of global brain ischemia followed by reperfusion, Yin et al. reported that the H2S donor, sodium hydrosulfide (NaHS), reduced brain infarct size and improved neurological function (Yin et al., 2013). In a mouse model of transient focal cerebral ischemia, Wang et al. recently found that the H2S donor, 5-(4-methoxyphenyl) −3H-1, 2dithiole-3-thione (ADT), protected the blood-brain barrier (BBB) integrity and reduced cerebral I/R injury (Wang et a., 2014). Interestingly, both studies suggest that the neuroprotective effect of this H2S donor may be related to its anti-inflammatory property. They found that the H2S donor suppresses pro-inflammatory cytokines (TNFα, MCP-1, and IL-1β) and increases anti-inflammatory cytokines (IL-10) (Wang et a., 2014; Yin et al., 2013). Most recently, the H2S donor was shown to promote a shift in microglial polarization from ischemia-induced pro-inflammatory phenotypes toward anti-inflammatory phenotypes (Zhang et al., 2017). Moreover, an early study found that H2S inhibits leukocyte adhesion and infiltration in mesenteric venules (Zanardo et al., 2006). Thus, a low concentration of H2S may inhibit inflammation in several ways. H2S is endogenously produced in mammalian cells by three different enzymes: cystathionine β-synthase (CBS), 3-mercaptopyruvate sulfurtransferase (3-MST), and cystathionine γ-lyase (CSE). Evidence indicates that CSE may be the key inflammatory-modulating H2S-synthesizing enzyme in animal models of cardiovascular disease (Pan et al., 2012). However, we are not aware of any studies that have examined CSE in the brain during LAC. Thus, the second goal of the present study was to determine the potential role of CSE in the neuroprotective effect of LAC against post-ischemic inflammation and cerebral I/R injury. We hypothesized that LAC protects against cerebral I/R injury by suppressing post-ischemic inflammation via an upregulated CSE.

METHODS

Animal models of Ethanol Preconditioning

All of the procedures and protocols were approved by the Institutional Animal Care and Use Committee at the Louisiana State University Health Science Center-Shreveport and performed in accordance with the National Institutes of Health Guide for the Care and Use Laboratory Animals. Adult (postnatal age: 60 days) male C57BL/6J mice (25–30 g) were divided into two groups, ethanol (n=42) and control (n=42). The ethanol group was gavage fed with 0.7 g/kg/day ethanol (13.5% v/v) once a day for eight weeks. The control group was fed with volume-matched water. Plasma ethanol concentration was measured using an Ethanol Assay Kit (ab65343, Abcam). Blood pressure was measured using a CODA mouse tail-cuff system (Kent Scientific, Torrington, CT, USA). At the end of 8 weeks of feeding, mice were subjected to transient focal cerebral ischemia.

Pharmacological Inhibition of CSE

We examined the influences of two CSE inhibitors, PAG and BCA, on the neuroprotective effect of low-dose alcohol. PAG (10 mg/kg) and BCA (50 mg/kg) were administered intraperitoneally 30 minutes prior to the ischemia.

Transient Focal Cerebral Ischemia

To avoid a direct effect of ethanol, ethanol was not given on the day prior to and after the ischemia. Transient focal cerebral ischemia was induced by unilateral MCAO for 90 minutes (Sun et al., 2012). Prior to the procedure, mice were anesthetized with isoflurane (induction at 5% and maintenance at 1.5%) in a gas mixture containing 30% O2/70% N2 via a facemask. A temperature-controlled heating pad (Harvard Apparatus, March, Germany) was used to maintain the rectal temperature at 37˚C. A laser-Doppler flow probe was attached to the right side of the dorsal surface of the skull to monitor regional cerebral blood flow (rCBF). The right common and external carotid arteries were exposed and ligated. The MCA was occluded by inserting a silicon rubber-coated monofilament (Doccol Corporation, MA, USA) from the basal part of the external carotid artery and advanced cranially into the internal carotid artery to the point where the middle cerebral artery branched off from the internal artery. The onset of the MCAO was indicated by an immediate drop in rCBF. After the occlusion of the right MCA for 90 minutes, reperfusion was initiated by withdrawing the suture and reopening the right common carotid artery. Animals were allowed to recover for 24 hours. A 24-point scoring system was used to evaluate sensory/motor deficits at 24 hours of reperfusion in all of the mice studied (Zhao et al., 2011). After neurological evaluations, the mice were euthanized with Inactin (150 mg/kg) and the brains were quickly removed. Brain samples from 36 mice (control: n=18; ethanol: n=18) were placed in ice-cold saline for 5 min and cut into six 2 mm-thick coronal sections. Sections were stained with 2% 2,3,5triphenyltetrazolium chloride (TTC; Sigma) for 15 min at 37˚C. Sliced images were digitalized, and the infarct lesion was evaluated using Image J. The infarct lesion was characterized as an area completely void of staining. Total lesion was expressed as a percentage of the total ipsilateral hemisphere.

Western Blot analysis.

After neurological evaluations, 24 mice (control: n=6; ethanol: n=6; control + PAG: n=6; ethanol + PAG: n=6) were euthanized with Inactin (150 mg/kg) and exsanguination. Cortical tissues were isolated from the peri-infarct area and contralateral corresponding area to measure protein expression of CSE, CBS, 3-MST, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin as well as IL-1RAcP and IL-1β. The samples were homogenized in ice-cold lysis buffer containing 150 mmol/l NaCl, 50 mmol/l Tris-HCl, 10 mmol/l EDTA, 0.1% Tween-20, 1% Triton, 0.1% mercaptoethanol, 0.1 mmol/l phenylmethyl sulfonylfluoride, 5 μg/ml leupeptin, and 5 μg/ml aprotinin, pH 7.4. Homogenates were centrifuged at 4˚C for 20 min at 12,000g and the supernatants were collected. Protein concentration was determined by the Bradford method (Bio-Rad) with BSA as the standard. SDS-PAGE was performed on a 10% gel on which 20 μg of total protein per well was loaded. After SDS-PAGE, the proteins were transferred to a polyvinylidene difluoride membrane. Immunoblotting was performed using mouse anti-CSE (Santa Cruz Biotechnology), rabbit anti-CBS (Santa Cruz Biotechnology), rabbit anti-3-MST (Santa Cruz, TX, USA), mouse anti-ICAM-1 (Santa Cruz Biotechnology), rabbit anti-VCAM-1 (AbCam), mouse anti-E-selectin (Cell signaling), mouse anti-P-selectin (R&D systems), and goat anti-IL1RAcP/IL-1β as primary antibodies and peroxidase conjugated goat anti-mouse, mouse anti-rabbit, and donkey anti-goat IgG as the secondary antibody. The bound antibody was detected using enhanced chemiluminescence (ECL) detection (Pierce Chemical), and the bands were analyzed using ChemiDoc MP Imaging System (Bio-Rad). To quantify, protein expression of CSE, CBS, 3-MST, ICAM-1, VCAM-1, E-selectin, P-selectin, IL-1RAcP, and IL-1β was normalized to GAPDH and expressed as percentage changes to the control.

L-cysteine-induced H2S Production Assay

L-cysteine-induced H2S production was measured according to Bucci et al. with modifications (Bucci et al., 2009). Sixteen mice (control: n=8; ethanol: n=8) were euthanized with Inactin (150 mg/kg) and exsanguinated at 24 hours of reperfusion. Cortical tissues were isolated from the peri-infarct area and contralateral corresponding area and homogenized in a lysis buffer (potassium phosphate buffer 100 mM pH = 7.4, sodium orthovanadate 10 mM and protease inhibitor cocktail (1:100 (v/v))).

Homogenates were centrifuged at 4°C for 30 minutes at 15000 g. The supernatant was collected and protein concentration was determined by the Bradford method (Bio-Rad, CA, USA) with BSA as the standard. The supernatants (50 μl) were added in a reaction mixture (total volume 50 μl) containing piridoxal-5′-phosphate (5 mM, 10 μl), L-cysteine (100 mM, 4 μl), and NaCl (1M, 2 μl). The reaction was performed in parafilmed eppendorf tubes and initiated by transferring tubes from ice into an incubator (Fisher Scientific, MA, USA) at 37 °C. After 30 minutes, zi nc acetate (0.85% in 3% NaOH, 100 μl) was added to trap evolved H2S. Subsequently, DPD (2 μM in 7.2 M HCl, 5 μl) and FeCl3 (30 μM in 1.2 M HCl, 6 μl) were added. After 20 min, absorbance values were read at a wavelength of 650 nm. H2S production was normalized with protein concentration and expressed as percentage change to control mice without I/R.

Immunohistochemistry staining

After neurological evaluations, 24 mice (control: n=6; ethanol: n=6; control + PAG: n=6; ethanol + PAG: n=6) were anesthetized with Inactin and perfused transcardially with 1X phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 mmol/L PBS. The brains were removed, fixed overnight in 4% paraformaldehyde in 0.1 M PBS, dehydrated in a graded series of sugar solutions over the course of 72 hours, and then embedded in O.C.T. compound (Fisher Scientific) and quick frozen for 5 minutes in liquid nitrogen. The frozen brains were then cut into 0.14 μm sections and placed on frost free slides. The sections were washed with 1X PBS, blocked with 10% Bovine Serum Albumin (BSA) for at least 1 hour, and then incubated overnight at 4˚C with 1:100 rabbit anti-MPO (Abcam) for visualization of neutrophils or 1:100 rabbit anti- ionized calcium-binding adapter molecule 1 (Iba1) (Wako Chemicals Inc.) for visualization of microglia as primary antibodies. Then the sections were incubated with 1:200 AlexaFluor 555 donkey anti-rabbit (Santa Cruz Technology) for one hour at room temperature. Sections were mounted with DAPI mounting medium with Vector shield and visualized using a fluorescence microscope with a mounted Nikon camera. Cells positive for MPO represented infiltrating neutrophils. For quantitative analysis, positive cells were counted in three separate areas per section surrounding the infarct area (penumbra) in at least three slides per mouse in each group. To quantify microglia, cells positive for Iba1 were observed and counted. Resting microglia present with long processes extending from their cell body. Upon activation, microglia become ramified and draw in their processes. Microglia with three processes or less were deemed to be activated.

Statistical Analysis

Data are reported as means ± SE. Differences between groups were evaluated for statistical significance by Student’s t-tests or ANOVA with Tukey’s test where applicable. P ≤ 0.05 was considered to be significant. Data analysis was conducted using GraphPAD prism 7.

RESULTS

Control conditions

Eight weeks of gavage feeding with 0.7 g/kg/day ethanol did not significantly alter body weight (Table 1). In addition, there was no significant difference in mean arterial blood pressure (MABP) between the ethanol group and the control group. The plasma ethanol concentration was measured at 15 minutes, 30 minutes, 1 hour and 2 hours after gavage feeding. The highest concentration was 9.4 ± 1.5 mM appeared at 15 minutes after gavage feeding (Table 1).

Table 1.

Effect of low-dose ethanol consumption on body weight, blood pressure and plasma alcohol concentration (15 min, 30 min, 1 hour and 2 hours after gavage feeding). Values are means ± SE for 5 mice in each group.

Control Ethanol
Body Weight 22.5±1.4 (25.7 ± 1.5) 23.3±1.2 (23.9 ± 1.0)
M.A.B.P. (mmHg) 98.3 ± 6.7 86.6 ± 5.3
Blood
Alcohol
Content
(mM) 		15’ 9.4 ± 1.5
30’ 8.5 ± 2.3
60’ 2.9 ± 1.5
120’ 0.1 ± 0.1
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Protein expression of H2S-producting enzymes and L-cysteine-induced H2S production

Protein expression of CSE was significantly upregulated in ethanol-fed mice when compared to control mice. In contrast, there was no significant change in protein expression of CBS and 3-MST between the ethanol group and the control group (Figures 1A and 1B). On the other hand, L-cysteine-induced H2S production was significantly increased in ischemic side cerebral cortex of ethanol-fed mice but not control mice (Figure 1C).

Figure 1.

Figure 1.

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Effect of low-dose ethanol consumption on expression of H2S-producing enzymes and L-cysteine-induced H2S production. (A) Representative Western blots. (B) Values are means ± SE for 4 mice in each group. (C) Values are means ± SE for 8 mice in each group. *P < 0.05 vs Control. #P < 0.05 vs Without I/R. Analyzed using Student t-test.

Cerebral I/R Injury

There was a significant reduction in total infarct volume in the ethanol-fed mice compared to the control mice (Figures 2A and 2B). Consistent with the findings regarding the infarction, post-ischemic sensory-motor functions were significantly improved in the ethanol-fed mice (Figure 2C). Two CSE inhibitors, PAG and BCA, were used to determine whether the beneficial effect of ethanol on cerebral I/R injury is related to a CSE-mediated mechanism. Both PAG and BCA did not alter the infarct volume and post-ischemic neurological deficits in control mice, but significantly increased the infarct volume and worsened the neurological deficits in the ethanol-fed mice (Figure 2). PAG and BCA inhibition yielded similar results that were not statistically different. Since PAG does not inhibit CBS even if used at high dose (Asimakopoulou et al., 2013), PAG was used as an inhibitor in subsequent experiments.

Figure 2.

Figure 2.

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Effect of low-dose ethanol consumption on cerebral I/R injury in the presence and absence of PAG or BCA. (A) Representative brain sections stained with TTC. (B) Total infarct volume. (C) Neurological deficit score. Values are means ± SE for 6 mice in each group. *P < 0.001 vs. Control. #P < 0.001 vs. Ethanol. Analyzed using ANOVA with Tukey’s post-hoc.

Protein expression of Adhesion Molecules

Eight-week feeding of ethanol significantly reduced the baseline expression of ICAM-1 (Figure 3A) and E-selectin (Figure 3B). Transient focal cerebral ischemia induced an upregulation in expression of ICAM-1 and E-selectin in both the control and ethanol groups. However, the magnitude of the increase was significantly less in the ethanol group compared to the control. In contrast, expression of VCAM-1 and P-selectin was not altered at 24 hours of reperfusion in either control mice or ethanol-fed mice (data not shown). PAG did not alter baseline and post-ischemic expression of ICAM-1 and E-selectin in the control group, but increased baseline expression of E-selectin and post-ischemic expression of ICAM-1 and E-selectin in the ethanol group (Figure 3).

Figure 3.

Figure 3.

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Effect of low-dose ethanol consumption on expression of ICAM (A) and E-selectin (B) in the presence and absence of PAG. Values are means ± SE for 5 mice in each group. *P < 0.05 vs Control without I/R. **P < 0.001 vs Control with I/R. ***P < 0.05 vs Control + PAG without I/R. #P < 0.001 vs Ethanol without I/R. ##P < 0.001 vs Ethanol + PAG without I/R. ###P < 0.05 vs Ethanol with I/R. Analyzed using ANOVA with Tukey’s post-hoc.

Expression of IL-1RAcP and IL-1β

Ethanol significantly reduced the baseline expression of IL-1RAcP (Figures 4A and 4B). Transient focal ischemia produced an increase in IL-1RAcP in the control group but not in the ethanol group. On the other hand, although ethanol did not alter baseline IL-1β, it significantly attenuated the post-ischemic increase in IL-1β (Figures 4A and 4C). PAG increased baseline IL-1RAcP expression in the ethanol group. In addition, PAG abolished the inhibitory effect of ethanol on the post-ischemic increase of IL-1RAcP (Figures 4A and 4B) and IL-1β (Figures 4A and 4C).

Figure 4.

Figure 4.

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Effect of low-dose ethanol consumption on expression of IL-1RAcP and IL-1β in the presence and absence of PAG. (A) Representative Western Blots. (B & C). Values are means ± SE for 5 mice in each group. *P < 0.05 vs Control without I/R. **P < 0.05 vs Control with I/R. ***P < 0.05 vs Control + PAG without I/R. #P < 0.001 vs Ethanol without I/R. ##P < 0.05 vs Ethanol with I/R. ###P < 0.05 vs Ethanol + PAG without I/R. Analyzed using ANOVA with Tukey’s post-hoc.

Neutrophil Infiltration

To examine neutrophil infiltration, positive signals from immunofluorescence staining with MPO were analyzed and counted. There was no neutrophil infiltration detected in the contralateral hemisphere of the ischemic brain. Neutrophil infiltration in the ipsilateral hemisphere of the ischemic brain was significantly attenuated in the ethanol group when compared to the control (Figure 5). PAG did not affect neutrophil infiltration in the control mice, but reversed the inhibitory effect of ethanol (Figure 5).

Figure 5.

Figure 5.

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Effect of low-dose ethanol consumption on neutrophil infiltration in the presence and absence of PAG. (A) Representative MPO staining. (B) Values are mean ± SE for 4 mice in each group. *P < 0.001 vs. Control with I/R. #P < 0.001 vs Ethanol with I/R. Analyzed using ANOVA with Tukey’s post-hoc.

Microglial Activation

To assess microglial activation, positive signals from immunofluorescence staining with Iba1 were analyzed and counted. There were no activated microglia detected in the contralateral hemisphere of the ischemic brain. Microglial activation in the ipsilateral hemisphere of the ischemic brain was significantly reduced in ethanol-fed mice when compared to control mice (Figure 6). PAG did not affect microglial activation in the control mice, but abolished the inhibitory effect of ethanol.

Figure 6.

Figure 6.

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Effect of low-dose ethanol consumption on microglia infiltration in the presence and absence of PAG. (A) Representative lba1 staining. (B) Values are mean ± SE for 4 mice in each group. *P < 0.05 vs. Control with I/R. #P < 0.05 vs Ethanol with I/R. Analyzed using ANOVA with Tukey’s post-hoc.

DISCUSSION

The present study investigated the role of CSE in the neuroprotective effect of LAC against transient focal cerebral ischemia. There are several new findings from this study. First, LAC significantly upregulated the baseline expression of CSE and increased post-ischemic H2S production in the cerebral cortex. Second, inhibition of CSE by PAG and BCA abrogated the protective effect of LAC on brain tissue damage and neurological deficits following transient focal cerebral ischemia. Third, inhibition of CSE reversed the inhibitory effect of LAC on the ischemia-induced expression of ICAM-1 and E-selectin. Fourth, CSE inhibition significantly abolished the LAC-induced suppression of IL-1RAcP and IL-1β expression following transient focal cerebral ischemia. Fifth, the LAC-induced attenuation of neutrophil infiltration and microglial activation was significantly abolished by inhibition of CSE. We speculate that the neuroprotective effect of LAC against post-ischemic inflammation and cerebral I/R injury may be related to an upregulation of CSE.

We previously have shown that two-month feeding with a liquid diet containing low-dose alcohol (1%) protected against cerebral I/R injury in rats and mice (Sun et al., 2012; Zhao et al., 2011). In a recent study, low-dose alcohol was given via an oral gavage once a day, which is an animal model of a drinking pattern similar to social drinking and regular light-moderate drinking in humans (McCarter et al., 2017). In that study, we found that chronic gavage feeding with low-dose red wine or ethanol protected the brain against its I/R injury in rats (McCarter et al., 2017). In the present study, the neuroprotective effect of low-dose ethanol was evaluated in mice. Consistently, chronic consumption of low-dose ethanol reduced infarct volume and improved neurological function following transient focal cerebral ischemia. Therefore, the present study further corroborated the neuroprotective effect of LAC. In the present study, the peak blood ethanol concentration of the dose associated with a neuroprotective effect observed in the ethanol group was 9.4 mM, which usually can be seen in a man with average body weight (70 kg or 154 Ibs) after ingestion of 2 American standard drinks. Thus, the results of the present study are also in agreement with a recent epidemiologic study (Patra et al., 2010).

CSE is one of the key enzymes that endogenously produce H2S. CSE is expressed in the cardiovascular system and has been demonstrated to be vasculoprotective (Mani et al., 2013). Furthermore, CSE is also expressed in the central nervous system (CNS) (Diwakar and Ravindranath, 2007; Gonzalez-Carter et al., 2017). Inhibition of CSE leads to loss of glutathione and aggravation of mitochondrial dysfunction mediated by an excitatory amino acid in the CNS (Diwakar and Ravindranath, 2007). Moreover, upregulation of CSE in microglia is associated with a reduced microglial inflammation (Gonzalez-Carter et al., 2017). Thus, CSE may confer neuroprotection by suppressing oxidative stress and inflammation. A recent study reported that acute treatment with high-dose ethanol increased CSE expression in the gastric mucosa (Medeiros et al., 2013). While the brain is one of the organs most affected by ethanol actions, no studies that we are aware of have determined the influence of chronic ethanol consumption on CSE expression in the brain. In the present study, CSE was upregulated in the cerebral cortex after 8-week feeding with low-dose ethanol. In addition, the neuroprotective effect of low-dose ethanol was abolished by CSE inhibitors, PAG and BCA. Both PAG and BCA are specific inhibitors of CSE. Although PAG does not inhibit CBS even if used at high dose, BCA also inhibits CBS when used above 1 mM (Asimakopoulou et al., 2013). Because both inhibitors were used at low doses in the present study, we suggest that the neuroprotective effect of low-dose ethanol may be related to the upregulated CSE.

The present study is the first to investigate the influence of chronic consumption of low-dose ethanol on the post-ischemic inflammatory response in a mouse model of transient focal cerebral ischemia. Consistent with previous findings from a rat model of transient focal cerebral ischemia, we found that ethanol similarly reduced ischemia-induced upregulation of ICAM-1 and E-selection in mice. In addition, ethanol downregulated the baseline expression of ICAM-1 and E-selection. The inhibitory effect of ethanol on adhesion molecules was abolished by CSE inhibition. A few studies have examined the effect of CSE/H2S on adhesion molecules. Mani et al reported that a CSE knockout produced an increase in ICAM-1 in aorta and the increased ICAM-1 could be reversed by H2S donor treatment (Mani et al., 2013). Pan et al found that a H2S donor concentration-dependently suppressed TNFα-induced upregulation of ICAM-1, VCAM-1, P-selectin, and E-selectin in human umbilical vein endothelial cells (Pan et al., 2011). In addition, a H2S donor prevented ischemia-induced upregulation of ICAM-1 in kidney and liver (Kang et al., 2009; Tripatara et al., 2002). On the other hand, CSE inhibition further increased post-ischemic upregulation of ICAM-1 (Kang et al., 2009). Thus, it is possible that reduced baseline and post-ischemic expression of adhesion molecules may be related to increased CSE in ethanol-fed mice. Adhesion molecules contribute to cerebral I/R injury by recruiting leukocytes. The selectins facilitate the diapedesis of leukocytes on the endothelial surface while ICAM-1 mediates the firm adhesion and transendothelial migration of leukocytes. Animals lacking ICAM-1 or treated with strategies that block ICAM-1 had decreased ischemic damage and less brain neutrophil infiltration (Kitagawa et al., 1998). In addition, E-selectin inhibition has been shown to be associated with improved neurological outcome (Huang et al., 2000). Of the various types of leukocytes, neutrophils are among the first to infiltrate the ischemic brain (Gronberg et al., 2013). Neutrophils may worsen brain injury in the post-ischemic brain by obstructing capillaries resulting in reduced blood flow during reperfusion as well as by releasing cytotoxic products once they infiltrate into the brain parenchyma. Many studies have shown that inhibition of neutrophil infiltration can decrease cerebral I/R injury (Egashira et al., 2013; Herz et al., 2015). Studies found the appearance of neutrophils as early as 30 min to a few hours of focal cerebral ischemia with a peak at days 1–3, and then they disappear or decrease rapidly with time (Mantovani et al., 2011). In the present study, reduced neutrophil infiltration was abolished by CSE inhibition in ethanol-fed mice. Therefore, the neuroprotective effect of low-dose ethanol against cerebral I/R injury may be related to a CSE-mediated reduction in expression of adhesion molecules and subsequent neutrophil infiltration. We evaluated the neutrophil infiltration at the 24-hour time point which suggests that ethanol plays a role in the suppression of neutrophil infiltration during acute inflammation however further studies are warranted to determine the long-term effects of ethanol on post-ischemic inflammation.

Microglia are the primary immune cells of the brain and readily become activated in response to injury, as characterized by an amoeboid shape with little to no processes extending out (Ziebell et al., 2015). The penumbra is particularly vulnerable to microglia activation. Activated microglia may contribute to cerebral I/R injury via phagocytosis and elaboration of neuroinflammatory mediators toxic to cells (Ceulemans et al., 2010). Consistent with previous findings from a rat model of transient focal cerebral ischemia, post-ischemic microglia activation in the penumbra was also significantly attenuated by ethanol in the present study. H2S appears to be an inhibitory modulator of microglia activation. A previous study reported that a H2S donor inhibited lipopolysaccharide-induced iNOS expression and TNFα production in cultured microglia (Hu et al., 2007). CSE may be the only H2S producing enzyme existing in microglia (Lee et al., 2006). Recently, Gonzalez-Carter et al found that upregulated CSE is involved in the anti-inflammatory effects of silver nanoparticles in microglia (Gonzalez-Carter et al., 2017). In the present study, CSE inhibition abolished the negative effect of ethanol on post-ischemic microglia activation. Thus, it is also possible that ethanol inhibits post-ischemic inflammation via an upregulated CSE in microglia.

Following ischemic stroke, pro-inflammatory cytokines are elaborated from endothelial cells, leukocytes and resident cells within the brain, including microglia, astrocytes and neurons. An increase in pro-inflammatory cytokines is correlated with a larger infarct size in animal models and a worsened clinical outcome (Vila et al., 2003). IL-1 is a key pro-inflammatory cytokine and has been strongly implicated in the pathogenesis of the ischemic brain as a neurotoxic mediator (Kim et al., 2014). There are two IL-1 isoforms,IL-1α and IL-1β. IL-1β appeared more engaged than IL-1α in the ischemic pathogenesis (Boutin et al., 2001). IL-1 exerts its actions by binding to its receptor. However, the signal transduction is elicited by interaction of the receptor with an accessory protein, IL1RAcP. After ischemic stroke, IL-1RAcP expression is upregulated (Touzani et al., 2002). In the present study, although ethanol did not alter baseline IL-1β, it reduced baseline expression of IL-1RAcP. In addition, ethanol inhibited the post-ischemic upregulation of both IL-1RAcP and IL-1β. Thus, the neuroprotective effect of ethanol may be related to decreased elaboration of pro-inflammatory cytokines. A few studies have investigated the effect of H2S/CSE on IL-1β secretion. In a lung I/R injury model, exogenous hydrogen sulfide inhibited the production of IL-1 β while the opposite was observed when CSE was inhibited via PAG (Wu et al., 2013). Recently, Castelblanco et al reported that a H2S donor reduced monosodium urate-induced IL-1β secretion in both murine and human macrophages (Castelblanco et al., 2017). Furthermore, IL-1β secretion was higher in CSE-deficient bone marrow-derived macrophages (BMDMs) than in wild-type BMDMs (Castelblanco et al., 2017). In the present study, PAG did not alter IL-1β secretion and IL-1RAcP expression in control mice, but abolished the inhibitory effect of ethanol on IL-1β and IL-1RAcP. Therefore, it is possible that the inhibitory effect of ethanol on post-ischemic IL-1β elaboration/signaling is related to an upregulated CSE.

In summary, the present study further demonstrated that LAC protects the brain against its I/R injury via suppression of post-ischemic inflammation. Therefore, anti-inflammatory drugs may be not able to significantly improve prognosis of ischemic stroke in patients who consume alcohol in mild-moderate amounts. In addition, the present study is the first to investigate a potential role of CSE in the neuroprotective effect of LAC. The proposed pathway by which LAC confers its neuroprotective effect is illustrated in Figure 7. Further studies are warranted to delineate the specific mechanism by which ethanol upregulates CSE.

Figure 7.

Figure 7.

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Schematic of the effects of low-dose ethanol consumption on post-ischemic inflammation.

HIGHLIGHTS.

  1. LAC suppressed post-ischemic inflammation and reduced cerebral I/R injury in mice.

  2. LAC upregulated CSE and increased post-ischemic H2S production in the cerebral cortex.

  3. CSE inhibitors, PAG and BCA, abolished the protective effect of LAC on cerebral I/R injury.

  4. PAG attenuated the inhibitory effect of LAC on post-ischemic inflammation.

ACKNOWLEDGMENTS

This study was supported by a National Institutes of Health Grant (AA023610) and funds from Louisiana State University Health Sciences Center-Shreveport to HS and WGM, a postdoctoral fellowship to CL and a predoctoral fellowship to KDM from the Center for Cardiovascular Diseases and Sciences, Louisiana State University Health Science Center-Shreveport.

Footnotes

CONFLICTS OF INTEREST

We declare no conflict of interest.

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