Part 1:Human Milk Oligosaccharide 2/-Fucosyllactose Induces Neuroprotection From Intracerebral Hemorrhage Stroke
Mar 22, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Tsai-Wei Hung 1, Kuo-Jen Wu 1, Yu-Syuan Wang 1, Eun-Kyung Bae 1, YoungHa Song 2 , JongWon Yoon 2 and Seong-Jin Yu1,*
1Center for Neuropsychiatric Research, National Health Research Institutes, Zhunan 35053, Taiwan;
pilypily@nhri.edu.tw (T.-W.H.); kjwu@nhri.edu.tw (K.-J.W.); yswang@nhri.edu.tw (Y.-S.W.);
baee@nhri.edu.tw (E.-K.B.)
2Advanced Protein Technologies Corporation, Suwon-si 16229, Korea; yhsong@aptech.biz (Y.S.);
jongwon.yoon91@gmail.com (J.Y.)
Correspondence: b7508@nhri.edu.tw; Tel.: +886-37206166 (ext. 36710)
Abstract:
Intracerebral hemorrhage (ICH) occurs when brain blood vessels rupture, causing inflammation and cell death. 2-Fucosyllactose (2FL), a human milk oligosaccharide, has potent antiapoptotic and anti-inflammatory effects. The purpose of this study was to examine the protective effect of 2FL in cellular and rodent models of ICH. Hemin was added to a primary rat cortical neuronal and BV2 microglia coculture to simulate ICH in vitro. IBA1 and MAP2 immunoreactivities were used to determine inflammation and neuronal survival. Hemin significantly increased IBA1, while it reduced MAP2 immunoreactivity. 2FL significantly antagonized both responses. The protective effect of 2FL was next examined in a rat ICH model. Intracerebral administration of type VII collagenase reduced open-field locomotor activity. Early post-treatment with 2FL significantly improved locomotor activity. Brain tissues were collected for immunohistochemistry and qRT-PCR analysis. 2FL reduced IBA1 and CD4 immunoreactivity in the lesioned striatum. 2FL downregulated the expression of ER stress markers (PERK and CHOP), while it upregulated M2 macrophage markers (CD206 and TGFβ) in the lesioned brain. Taken together, our data support that 2FL has a neuroprotective effect against ICH through the inhibition of neuroinflammation and ER stress. 2FL may have clinical implications for the treatment of ICH.
Keywords: 2-fucosyllactose; intracerebral hemorrhage stroke; inflammation; endoplasmic reticulum
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1. Introduction
Intracerebral hemorrhage (ICH) is a major neurological disorder associated with a high risk of death in adults [1,2]. ICH constitutes 10–15% of all acute stroke cases [3]. A few epidemiological studies have supported gender disparities in patients with ICH [4] or cerebrovascular diseases [5]. A small portion of ICH patients develops hemorrhagic lacunar syndrome [6]. Current treatment for ICH focuses on controlling the bleeding, removing the blood clot, and relieving intracranial pressure caused by bleeding. There is no effective pharmacological therapy for ICH.
Inflammation plays a critical role in ICH-induced neurodegeneration. After the onset of ICH, blood cells enter the brain parenchyma, activate the microglia, and initiate the production of proinflammatory cytokines [7,8]. Hemin, a byproduct of hemoglobin, causes neuronal death and induces apoptosis and inflammation [9,10]. Several studies have supported the effectiveness of anti-inflammatory treatment in animal models of ICH [11,12]. We recently demonstrated that early post-treatment with a CXCR4 antagonist CX807 inhibited inflammation and improved behavioral function in experimental ICH rats [13]. These data suggest that anti-inflammatory therapy may reduce brain damage
after ICH.
Human breastmilk contains nutrition and several critical bioactive ingredients [14], e.g., human milk oligosaccharides (HMOs) [15]. 2′-Fucosyllactose (2FL) is a major oligosaccharide in human milk. Oral delivery of 2FL improved memory and learning in rats [16,17]. In addition, 2FL suppressed lipopolysaccharide-induced CD14 expression and inflammation in human enterocytes [18]. We recently reported that 2FL mitigated neuroinflammation in a brain suffering ischemic stroke [19]. These data suggest that 2FL is a potent anti-inflammatory agent and may, thus, be useful for the treatment of ICH.
The purpose of this study was to examine the neural protective actions of 2FL in cellular and animal models of ICH. Hemin was used to simulate ICH in cell culture [20]. We demonstrated that 2FL antagonized hemin-mediated activation of microglia. Type VII collagenase was used to generate focal ICH in adult rats [13]. We found that 2FL significantly mitigated ICH-induced microglia activation, CD4+ lymphocyte infiltration, and ER stress in the lesioned brain and improved behavioral function. Our data support that 2FL has a neuroprotective effect against ICH.
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2. Results
2.1. 2FL Reduced Hemin-Mediated Neurodegeneration in Primary Cortical Neurons and BV2 Microglia Coculture
The protective effect of 2FL was first examined in a cellular model of a cerebral hemorrhage. Primary cortical neuronal and BV2 microglia cocultures were treated with hemin (10 uM) for 48 h (see timeline in Figure1E). Hemin attenuated neuronal marker MAP2 (Figure 1A), while it increased microglia marker IBA1 (Figure1B). These responses were both antagonized by 2FL (1 uM, Figure 1A,B). The immunoreactivities of MAP2 and IBA1 were further analyzed by measuring the pixel density of specific markers. As seen in Figure 1C,D, hemin significantly reduced MAP2 immunoreactivity (Map2-ir) and increased IBA1-ir (p < 0.001, one-way ANOVA, n = 6 in each group). These responses were significantly antagonized by 2FL (MAP2: p < 0.001, F2,14 = 115.863; IBA1: p < 0.001, F2,15 = 134.644, one-way ANOVA).
F
Figure 1. The neuroprotective effect of 2FL in a primary cortical neuron and BV2 microglia coculture. (A) Representative photomicrographs demonstrate that hemin reduced MAP2 immunoreactivity (-ir). Coadministration with 2FL antagonized the hemin-mediated loss of MAP-ir. (B) Hemin increased IBA1-ir. Coadministration with 2FL reduced IBA1-ir. (C,D) Pixel density of MAP2-ir or IBA1-ir was analyzed using NIS Elements AR 5.11 Software. (C) 2FL significantly antagonized hemin-mediated loss of MAP2-ir. (D) 2FL significantly attenuated hemin-induced IBA1 activity. (E) Timeline of experiment. * p < 0.05. Scale bar = 100 um.
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2.2. Post-Treatment with 2FL Improved Locomotor Activity in ICH Rats
The protective effect of 2FL was next examined in an animal model of ICH. A total of 14 adult male rats were stereotactically administered a low dose (0.5 units/uL × 1 uL) of type VII collagenase to induce cerebral hemorrhage in the right striatum. 2FL (400 mg/kg/d, i.p., n = 7) or vehicle (saline, i.p., n = 7) was given daily for 5 days, starting 1 day after stroke (please see the timeline in Figure 2G). Then, 2 h after the last 2FL or vehicle injection on day 5, animals were individually placed in infrared activity chambers for 30 min. Open-field locomotor behavior was examined for 30 min. We found that 2FL significantly increased vertical activity (VACTV, Figure 2A), total distance traveled (TOTDIST, Figure 2B), vertical time (VTIME, Figure 2C), horizontal activity (HACTV, Figure 2D), movement time (MOVTME, Figure 2E), and movement number (MOVNO, Figure 2F). Detailed statistics (two-way ANOVA + post hoc Newman–Keuls (NK) tests) are listed in Table 1.
Figure 2. Post-treatment with 2FL improves neural functions in ICH rats. Adult rats were treated with type VII collagenase on D0 (G, timeline). 2FL or vehicle was given systemically daily for 5 days. Locomotor behavior was examined on D5. 2FL significantly improved (A) vertical activity, (B) total distance traveled, (C) vertical time, (D) horizontal activity, (E) movement time, and (F) movement
number. n = 7 in each group. * p < 0.05, two-way ANOVA; # p < 0.05, post hoc NK test.
Table 1. Significant differences in locomotor behaviors between ICH +veh and ICH +2FL.
2.3. Early Post-Treatment with 2FL Reduced Microglial Activation in the Peri-Lesioned Region
Eight ICH rats (2FL, n = 4; Veh, n = 4) were euthanized and perfused for IBA1 immunohistochemistry after the behavioral test on day 5. Typical IBA1-ir is demonstrated in Figure 3. IBA1-ir was enhanced in the peri-lesioned area in the striatum (Figure 3B), as compared with the corresponding sites in the contralateral non-lesioned striatum (Figure3A) in stroke rats receiving veh. At a high magnification, de-ramified or ameboid microglial cells were found in the lesioned area (Figure 3B, insert), while resting microglia exhibiting a ramified morphology were found in the non-lesioned striatum (Figure3A, insert). 2FL treatment reduced IBA1-ir and partially restored morphological ramification of the microglia (Figure3C, insert). IBA1-ir was quantified and averaged across three consecutive brain sections with a visualized anterior commissure in all animals. 2FL significantly reduced IBA1-or in the ICH brain (Figure 3D, p < 0.001, F2,12 = 50.357, one-way ANOVA).
Figure 3. Early post-treatment 2FL reduced microglial activation in the lesioned striatum. Animals received vehicle or 2FL after collagenase injection from days 1 to 5. Animals were sacrificed on day 5. Enhanced IBA1 immunoreactivity with de-ramified morphology was found in the lesioned striatum (B vs. A). Treatment with 2FL reduced IBA immunoreactivity (C vs. B). (D) 2FL significantly reduced IBA1 immunoreactivity in the lesioned brain. * p < 0.05, one-way ANOVA + NK test.
2.4. 2FL Mitigated CD4+ Lymphocyte Infiltration into the Lesioned Brains
Administration of collagenase enhanced CD4+ lymphocyte infiltration into the lesioned striatum (Figure 4B). Almost no CD4 cells were found in the non-lesioned striatum (Figure 4A). CD4 cell infiltration into the lesioned striatum was mitigated by 2FL treatment (Figure 4C vs. Figure 4B; p < 0.001, F2,6 = 173.485, one-way ANOVA, Figure 4D).
Figure 4. Post-treatment with 2FL reduced CD4+ lymphocyte infiltration into the lesioned brain. Representative photomicrographs demonstrate the CD4 immunoreactivity in (A) the non-lesioned striatum and (B) the lesioned striatum from a rat receiving vehicle at low (calibration = 50 um) and high magnification (insert, calibration = 10 um), as well as (C) the lesioned striatum from a rat receiving 2FL. (D) 2FL significantly reduced CD4-ir in the lesioned brain (* p < 0.001, one-
way ANOVA).
2.5. 2FL Differentially Altered the Expression of M1 and M2 Inflammatory Markers in ICH Brains
Striatal tissues were collected from eight rats (veh, n = 4; 2FL, n = 4) on day 5. The expression of M1/M2 inflammatory markers was examined by qRT-PCR. ICH significantly increased the expression of M1 marker CD86 (Figure5A, p = 0.005) and M2 markers CD206 and TGFβ (Figure 5B, C, p = 0.005). 2FL did not alter the expression of CD86 (Figure 5A, p = 0.356, two-way ANOVA). However, it significantly upregulated the expression of CD206 (Figure 5B) and TGFβ in the lesioned side (p < 0.05, NK test).
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2.6. 2FL Altered the Expression of ER Stress and Apoptotic Markers in ICH Brains
The expression of ER stress and apoptotic markers was examined in eight rats (veh, n = 4; 2FL, n = 4). 2FL significantly reduced the expression of PERK (Figure6A, p = 0.016), IRE1 (Figure6B, p = 0.018), CHOP (Figure6C, p = 0.032), and SigmaR1 (Figure6D, p = 0.017) in the lesioned striatum. 2FL did not alter the expression of BIP (Figure6E, p = 0.841) and ATF6 (Figure6F, p = 0.403). In addition, the expression of caspase3 was significantly re- duced by 2FL (Figure 6G, p = 0.029, t-test).
Figure 5. 2FL upregulated the expression of M2 markers in ICH brains. ICH significantly increased the expression of (A) M1 marker CD86, and M2 markers (B) CD206 and (C) TGFβ (# p = 0.005, two-way ANOVA). (A) 2FL significantly upregulated the expression of (B) CD206 and (C) TGFβ, but not (A) CD86, in the lesioned striatum (* p < 0.05, NK test).
Figure 6. 2FL suppressed the expression of ER stress and apoptotic markers. The expression of (A) PERK, (B) IRE1, (C) CHOP, (D) SigmaR1, and (G) caspase3 was significantly inhibited by 2FL in hemorrhagic brains. The expression of (E) BIP and (F) ATF6 was not altered by 2FL. * p < 0.05. ER, endoplasmic reticulum; PERK, protein kinase R-like ER kinase; IRE1, inositol-requiring enzyme 1; CHOP, CCAAT enhancer-binding protein homologous protein; SigmaR1, Sigma 1 receptor; ATF6, activating transcription factor 6.
