Molecular mechanisms and biomarkers underlying the protective roles of the nutraceutical laminarin against ischaemic strokes

ABSTRACT

Stroke is the second leading cause of death and a major cause of disability worldwide. Ischemic stroke accounts for approximately >60% of all stroke, and >80% of strokes can be prevented. Middle cerebral artery occlusion (MCAO) is a common cause of stroke in humans. MCAO is associated with blood vessel-related problems, such as reduced vascular plasticity and hypertension. These problems can be attenuated by the use of nutraceuticals such as laminarin. Laminarin is a storage glucan commonly found in brown algae. Accumulating evidence and studies have revealed that laminarin is a promising candidate drug for treating ischaemic stroke. However, details on pharmaceutical targets and molecular mechanisms underlying laminarin’s beneficial effects for treating ischaemic stroke remains largely unknown. Herein, we applied network pharmacology, bioinformatic analysis, and middle cerebral artery occlusion model to delineate the protective role of laminarin. By comparing the laminarin- and MCAO-associated genes, we identified 23 potential targets including tumour necrosis factor (TNF), vascular endothelial growth factor A, selectin P, presenilin 1, fibroblast growth factor 2, microtubule-associated protein tau, caspase 3, matrix metallopeptidase 1, 5-hydroxytryptamine receptor 2A, telomerase reverse transcriptase, interleukin 2, signal transducer and activator of transcription 3 (STAT3), ATP binding cassette subfamily B member 1, catalase, superoxide dismutase 2, adenosine A1 receptor, adenosine A2a receptor, 5-hydroxytryptamine receptor 1B, heat shock protein 90 alpha family class A member 1, matrix metallopeptidase 8, BCL2-like 1, galectin 3, and epoxide hydrolase 2 of laminarin against ischaemic stroke. The gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses highlighted the importance of this gene cluster in the development of blood vessels, neuronal cell death, brain functions, and neuroinflammation. Furthermore, molecular docking analysis suggested a direct binding of laminarin to its target proteins STAT3 and TNF. Our results provide the pharmaceutical targets and delineate the details regarding the molecular mechanisms underlying the beneficial effects of laminarin against ischaemic stroke. Moreover, our findings support those of previous studies suggesting laminarin as a promising drug for treating ischaemic stroke.

KEYWORDS:

• Laminarin
• anti-neurodegeneration
• network pharmacology
• ischaemic stroke
• molecular mechanisms
• pharmaceutical targets

Introduction

According to the data from World Stroke Organization (https://www.world-stroke.org), ischaemic stroke accounts for approximately 62% of overall stroke incidence. Globally, approximately 7.6 million new ischaemic stroke cases are recorded, leading to 3.3 million healthy lives lost yearly. Ischaemic stroke is one of the leading causes of death and disability worldwide (Duloquin et al., 2021), although 80% of strokes are generally preventable (Katan & Luft, 2018). The major causes of ischaemic stroke is middle cerebral artery occlusion (MCAO), which is associated with metabolic risks including hypertension and an unhealthy diet that poses the greatest risk for stroke disability-adjusted life years (Avan & Hachinski, 2021). Thus, a healthy diet that can improve the quality of blood vessels could help to avoid ischaemic stroke.

In the recent years, the use of seaweeds as nutraceuticals for preventing cardiovascular associated diseases has been reported; seaweeds contain unique bioactive metabolites that are not present in terrestrial plants (Ryu et al., 2021). For example, a polysaccharide isolated from the red seaweed Bangia fusco-purpurea was discovered to have pro-angiogenic properties (Jiang et al., 2021). Durvillaea antarctica is the seaweed that promoted the decrease in the glycemic levels and lowered cholesterol levels and cardiovascular risk (Guerrero-Wyss et al., 2023). Lectin from red algae Amansia multifida Lamouroux was also discovered to have an anti-inflammatory activity by reducing the formation of edema via the modulation of vascular mediators (Mesquita et al., 2021). Therefore, the isolated active compounds from seaweeds could be promising candidates for the development of therapeutic drugs for treating ischaemic stroke.

Laminarin is a storage β-1,3-glucan that is commonly present in brown algae (Chen et al., 2021). It has been reported as a nutraceutical that plays important roles in anticoagulation, anti-inflammation, and immunoregulation (Lomartire et al., 2021). Accumulating studies have demonstrated the potential roles of laminarin for treating ischaemic stroke. For instance, laminarin demonstrated noticeable antioxidant properties and promoted wound healing (Sellimi et al., 2018). It has also been considered a dectin-1 antagonist that can prevent neuroinflammation (Ye et al., 2020). More importantly, a study on gerbils has revealed that pretreatment with laminarin protected hippocampal neurons and attenuated reactive gliosis after forebrain ischaemia (Lee et al., 2020). All these important features on neuroprotection make laminarin a promising nutraceutical for preventing ischaemic stroke and promoting recovery from ischaemic stroke. However, the core targets of laminarin and the molecular mechanisms underlying its beneficial effect on ischaemic stroke remain largely unknown. Herein, we applied network pharmacology, systematic bioinformatic analysis, and middle cerebral artery occlusion model to delineate the biological processes and pathways controlled by laminarin for blood vessel quality improvement and neuroprotection.

Materials and methods

Network pharmacology of laminarin’s targets against MCAO

The core targets of laminarin were identified by searching the Comparative toxicogenomics database (Davis et al., 2020), Swiss Target Prediction database (Daina et al., 2019), and PharmMapper database (Wang et al., 2017). The identified targets were subjected to conversion to human genes using the UniProt database (Swiss-Prot) (The UniProt Consortium, 2021). The MCAO-associated genes were obtained by searching the GeneCards database (Stelzer et al., 2016), Online Mendelian Inheritance in Man database (Hamosh et al., 2002), and National Center for Biotechnology Information database (National Center for Biotechnology Information (NCBI), n.d.). Then, the laminarin’s targets and MCAO-associated genes were overlapped. The STRING (Version 11.0) tool was used to determine protein–protein interaction (PPI) of laminarin’s targets against MCAO (Szklarczyk et al., 2019). The overlapped laminarin- and MCAO-associated targets were subjected to gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses using the ClusterProfiler and GOplot packages of the R-language software. The network of biological processes and signalling pathways involved in laminarin against MCAO was constructed using Cytoscape (Shannon et al., 2003).

Molecular docking analysis

Molecular docking analysis was performed to assess the binding of laminarin to its core targets. The core targets were prioritised using Cytoscape_v3.8.2 and set under median or maximum degree of freedom. The core targets were obtained under the upper limit of the screening range with the maximum degree value in the topology data, and the lower limit was twice the median degree of freedom (Shannon et al., 2003). The top two-ranked targets, tumour necrosis factor (TNF) and signal transducer and activator of transcription 3 (STAT3), were subjected to molecular docking analysis using the Chem Bio Office 2010 software and Autodock Tools 1.5.6 (Morris et al., 2009). The chemical structure of laminarin was obtained from the PubChem database (Wang et al., 2017). The protein structures of TNF and STAT3 were obtained from the Protein Data Bank (PDB) database (Berman et al., 2000). The PDB file was converted to pdbqt file that can be recognised by the Autodock programme. The docking parameter setting was assessed according to the root mean square deviation (RMSD) of the ligand molecule; the threshold for the conformation of the ligand molecule was set at RMSD ≤ 4 Å.

Middle cerebral artery occlusion (MCAO) model and laminarin treatment.

Male Sprague–Dawley rats (240 ± 20 g, specific pathogen-free, 9 weeks old) were obtained from the Central Animal Facility of Guangxi Medical University (Nanning, China). The animals were housed in clean cages under standard conditions and light and dark cycles (12 h:12 h; temperature: 25°C) with free access to food and water. All animal studies were conducted according to the approved protocols and guidelines of the Institutional Animal Ethical Care Committee of the Guangxi Medical University Experimental Animal Center. For the cerebral ischaemia model, the rats were anesthetised with chloral hydrate (10%, 3 mL/kg), the inner and outer muscles of the sternocleidomastoid muscles were separated to expose and isolate the right common, external, and internal carotid arteries. The occlusion was performed by inserting a monofilament (approximately 2 cm) from the external carotid artery to the middle cerebral artery, avoiding the pterygopalatine artery. After the monofilament was inserted, the common carotid artery was ligated to complete the occlusion and induce ipsilateral ischaemia. After 2 h of ischaemia, the monofilament was gently pulled out, and ligation of the common carotid artery was relieved to cause reperfusion. The wound was disinfected with iodine and sutured. The rats were randomly divided into two groups as follows: (1) the MCAO group: the rats were subjected to 2 h ischaemia followed by 24 h reperfusion, and then were intraperitoneally injected with the same amount of normal saline as the laminarin treatment group daily for 7 days. (2) MCAO + laminarin treatment group: rats were intraperitoneally injected with laminarin (dissolved in normal saline) at 10 mg/kg per day for 7 days after MCAO.

Immunohistochemical staining

The brain sections were incubated in 0.3% H₂O₂ (in 10 mM PBS, pH 7.4) at room temperature for 20 min and followed by incubation in 5% normal horse serum (in 10 mM PBS, pH 7.4) at room temperature for 30 min. Then, the sections were incubated with primary antibody (mouse anti-VEGFA (1:800, Proteintech, Rosemont, IL, USA) or rabbit anti-SOD2 (1:200, Sabbiotech, LA, USA)) at 4°C overnight. The sections were incubated with biotinylated secondary antibody (Zhongshan Golden Bridge, Beijing, China) and avidin–biotin-peroxidase (Zhongshan Golden Bridge) before being exposed to diaminobenzidine (DAB; Zhongshan Golden Bridge) for 1 min and then counterstained with Haematoxylin.

Results

Identification and functional characterisation of laminarin targets against MCAO

By searching the databases, a total of 132 laminarin-associated genes and 625 MCAO-associated genes were identified (Figure 1A). When we overlapped the laminarin- and MCAO-associated genes, we identified 23 potential targets including TNF, vascular endothelial growth factor A (VEGFA), selectin P (SELP), presenilin 1 (PSEN1), fibroblast growth factor 2 (FGF2), microtubule-associated protein tau (MAPT), caspase 3 (CASP3), matrix metallopeptidase 1 (MMP1), 5-hydroxytryptamine receptor 2A (HTR2A), telomerase reverse transcriptase (TERT), interleukin 2 (IL2), STAT3, ATP binding cassette subfamily B member 1 (ABCB1), catalase (CAT), superoxide dismutase 2 (SOD2), adenosine A1 receptor (ADORA1), adenosine A2a receptor (ADORA2A), 5-hydroxytryptamine receptor 1B (HTR1B), heat shock protein 90 alpha family class A member 1 (HSP90AA1), matrix metallopeptidase 8 (MMP8), BCL2-like 1 (BCL2L1), galectin 3 (LGALS3), and epoxide hydrolase 2 (EPHX2) of laminarin against MCAO (Figure 1A & Table 1). Of these targets, 18 core targets demonstrated a PPI (Figure 1B). These targets were subjected to a GO enrichment analysis to determine the functional roles of laminarin against MCAO. Our results highlighted the biological processes related to blood vessel such as regulation of blood vessel diameter and blood pressure, leading to the regulation of oxidative stress (Figure 2A). These changes could result in the modulation of neuron cell death such as neuron apoptotic and glial cell apoptotic processes (Figure 2B). More importantly, the laminarin’s core targets contributed to brain functions including synaptic transmission and regulation of neurotransmitters (Figure 2C). In the molecular function analysis, we observed the highlight of neurofunctions, such as serotonin receptor, neurotransmitter receptor, and calcium channel activities (Figure 3A). In addition, the immune and inflammatory responses including cytokine receptor binding, cytokine activity, and chemoattractant activity were enriched (Figure 3A). All these biological processes and molecular functions were involved in brain compartments, such as the presynaptic membrane, neuronal cell body, neuron projection membrane, and axon terminus (Figure 3A). Moreover, the KEGG enrichment analysis revealed the involvement of laminarin’s targets in inflammatory responses, cell death, brain functions, neurological disorders, and signalling pathways (Figure 3B). Altogether, our data suggested that laminarin targeted the genes involved in the pathogenesis of MCAO.

Figure 1. Identification of laminarin- and middle cerebral artery occlusion (MCAO)-associated genes. (A) The Venn diagram presents the number of common laminarin- and MCAO-associated genes. (B) The STRING analysis demonstrated the protein–protein interaction of laminarin’s targets.

Figure 2. Functional characterization of laminarin’s targets against ischaemic strokes. The gene ontology enrichment analysis demonstrated the involvement of laminarin’s targets in biological processes related to (A) blood vessels, (B) cell death, and (C) brain functions. The size of the bubble represents the number of genes, and the colour of the bubble represents the significance of the terms.

Figure 3. Molecular functions, cellular components, and pathways of laminarin’s targets against ischaemic strokes. (A) Gene ontology enrichment analysis demonstrated the involvement of laminarin’s targets in molecular function related to neurofunctions and inflammatory responses; and the cellular components. (B) The Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated the involvement of laminarin’s targets in inflammatory responses, cell death, brain functions, neurological disorders, and signaling pathways. The size of the bubble represents the number of genes, and the colour of the bubble represents the significance of the terms.

Table 1. Core target genes of laminarin against ischaemic stroke.

Gene name	Gene symbol
Tumour necrosis factor	TNF
Vascular endothelial growth factor A	VEGFA
Selectin P	SELP
Presenilin 1	PSEN1
Fibroblast growth factor 2	FGF2
Microtubule-associated protein tau	MAPT
Caspase 3	CASP3
Matrix metallopeptidase 1	MMP1
5-Hydroxytryptamine receptor 2A	HTR2A
Telomerase reverse transcriptase	TERT
Interleukin 2	IL2
Signal transducer and activator of transcription 3	STAT3
ATP binding cassette subfamily B member 1	ABCB1
Catalase	CAT
Superoxide dismutase 2	SOD2
Adenosine A1 receptor	ADORA1
Adenosine A2a receptor	ADORA2A
5-Hydroxytryptamine receptor 1B	HTR1B
Heat shock protein 90 alpha family class A member 1	HSP90AA1
Matrix metallopeptidase 8	MMP8
BCL2-like 1	BCL2L1
Galectin 3	LGALS3
Epoxide hydrolase 2	EPHX2

Binding of laminarin to its targets TNF and STAT3

The top two-ranked targets, TNF and STAT3, were prioritised for molecular docking analysis. The protein structures of TNF (ID: 3L0 V) (Yu et al., 2010) and STAT3 (ID: 6SM8) (Su et al., 2020) were obtained from the PDB database. Their binding affinity with laminarin was determined using the AutoDock Vina programme. The negative value of the binding affinity represented the possible direct binding of laminarin to its target proteins. We identified the formation of hydrogen bonds between laminarin with amino acid residues of VAL-434 (2.0 Å), HIS-405 (2.6 Å), GLU-406 (2.3 Å), THR-347 (2.1 Å), PRO-437 (2.2 Å), ALA-439 (1.8 Å), ALA-440 (2.3 Å), and TYR-436 (2.4 Å) of TNF (ID: 3L0 V) (Figure 4A). The binding affinity was −6.31 Kcal/mol. For the STAT3 (ID: 6SM8), its amino acid residues GLY-1020 (2.1 Å), ASP-1021 (1.9 Å), LSY-908 (2.7 Å), GLU-883 (2.2 Å), HIS-885 (2.7 Å), ASP-1003 (2.1 Å), ARG-1007 (1.8 Å), and ASN-1008 (2.4 Å) were observed to form hydrogen bonds with laminarin, and the binding affinity was −6.42 Kcal/mol (Figure 4B). Collectively, our results suggested possible direct binding of laminarin to TNF and STAT3 through the formation of hydrogen bonds.

Figure 4. Direct binding of laminarin to its targets TNF and STAT3. Molecular docking revealed the hydrogen bond formation between (A) laminarin and TNF (ID: 3L0 V); and between (B) laminarin and STAT3 (ID: 6SM8).

Laminarin treatment induced the expression of SOD2 and VEGFA in middle cerebral artery occlusion model

In order to validate the findings from network pharmacology and bioinformatic analysis, we used MCAO model to determine the effect of laminarin on the expression of the identified targets, SOD2 and VEGFA. The result of IHC staining showed the significant induction of SOD2 (Figure 5A) and VEFGA (Figure 5B) in the MCAO model.

Figure 5. Treatment of laminarin induced the expression of SOD2 and VEGFA in the middle cerebral artery occlusion (MCAO) model. Immunohistochemical (IHC) staining showed the significant induction of (A) SOD2 and (B) VEGFA in the MCAO model. Left panel: the representative picture of IHC staining. Right panel: the result of quantitation of positive stained cells. * Represented the statistically significant, p < 0.05. Number of replicates >20.

Discussion

In this report, we aimed to investigate the laminarin controlled biological processes and pathways involved in promoting quality of vessel and neuroprotection against ischaemic stroke. Using a network pharmacology approach, we compared the laminarin- and MCAO-associated genes to identify laminarin’s core targets against ischaemic stroke. Network pharmacology is a valuable evidence-based tool to understand a medicine’s putative actions, indications, and mechanisms (Hopkins, 2007). It is commonly used for screening drug targets involved in prevention and treatment of stroke (Casas et al., 2019; Wang et al., 2021). In our network pharmacology results, we identified 23 potential targets of laminarin against ischaemic stroke, including TNF, VEGFA, SELP, PSEN1, FGF2, MAPT, CASP3, MMP1, HTR2A, TERT, IL2, STAT3, ABCB1, CAT, SOD2, ADORA1, ADORA2A, HTR1B, HSP90AA1, MMP8, BCL2L1, LGALS3, and EPHX2. The gene enrichment and KEGG pathway analyses further highlighted their importance in the development of blood vessels, brain cell death, brain functions, and inflammatory responses.

We identified that laminarin targets VEGFA, HTR2A, HTR1B, SOD2, and EPHX2 play a role in vessel development. VEGFA is a well-documented glycoprotein that plays an important role in neurons and is a major inducer in the development of blood vessels both during physiological and pathological conditions (Melincovici et al., 2018; Setyopranoto et al., 2019). A comparative study including 35 matched pairs of patients with acute ischaemic stroke and non-ischaemic stroke has revealed that the VEGFA level was significantly higher in the non-stroke group than in the stroke group (Setyopranoto et al., 2019). More importantly, the serum level of VEGFA was associated with the promotion of brain repair and improvement of cognitive function in a cerebral small vascular disease model. Moreover, our results revealed that the 5-hydroxytryptamine receptor family was also targeted by laminarin, and limited studies have demonstrated the functional roles of this receptor family in the blood vessels. HTR2A was induced in the pulmonary artery of patients with the neurodevelopmental disease Williams–Beuren syndrome (Ma et al., 2021). Meanwhile, HTR1B was highly abundant in neuronal tissues and vascular smooth muscle cells, and has been reported to mediate vasocontraction (De Vries et al., 1999). SOD2 is an enzyme that plays an important role in apoptotic signalling and oxidative stress (Danial & Korsmeyer, 2004). SOD2 has been reported to ameliorate pulmonary hypertension in sleep apnea (Fu et al., 2020). Moreover, overexpression of SOD2 resulted in induction of in vivo blood vessel formation (Connor et al., 2005). Hence, SOD2 was critical to preventing cell death from ischaemia and reperfusion injury subsequent to a stroke (Flynn & Melov, 2013). Another enzyme, EPHX2, was also discovered to be targeted by laminarin. A genetic variation study of the EPHX2 gene in 601 patients with ischaemic stroke and 736 matched controls has suggested that nucleotide polymorphisms of EPHX2 demonstrated associations with an increased risk for ischaemic stroke (Gschwendtner et al., 2008). This might be due to the regulation of epoxyeicosatrienoic acids (EETs), the substrate of EPHX2, because EETs have been reported to play an important role in dilating blood vessels, lowering blood pressure, and preventing inflammation (Spector et al., 2004).

Other than blood vessel development, laminarin would target the genes involved in neuron cell death, including MAPT, CASP3, and BCL2L1. MAPT is differentially expressed in different stages of neuronal maturation and neuron type (Beevers et al., 2017). The alteration of the tau protein could lead to a neurodegenerative disorder called tauopathy, which is characterised by an accumulation of MAPT in the neurons and glia, leading to brain cell death (Kovacs, 2017). Moreover, two well-reported candidates of cell death pathway, including CASP3 and BCL2L1, were also targeted by laminarin. CASP3, a pro-apoptotic enzyme, is a major mediator of cell apoptosis (Zhou et al., 2018). CASP3 has been detected in both cerebrospinal fluid and serum of patients after aneurysmal subarachnoid haemorrhage (Kacira et al., 2007), and CASP3 was associated with the c-Jun N-terminal kinase pathway for mediating neuronal cell death (Khan et al., 2019). A human study of neuronal death in brain ischaemia has reported that CASP3 was upregulated in neurons within infarcts during the first 2 days after infarction (Love et al., 2000), suggesting the importance of CASP3 in the neuron-programmed cell death. Meanwhile, BCL2L1 belongs to the BCL-2 protein family, which is critical for anti-apoptotic regulation (Loo et al., 2020). A study using BCL2L1-knockout mice has revealed that deletion of BCL2L1 caused severe brain hypoplasia via the increased apoptosis of cerebellar granule neurons (Veleta et al., 2021). Thus, all these laminarin-associated genes might serve as therapeutic targets of laminarin-mediated neuron cell death for treating ischaemic stroke.

In the molecular docking analysis, our results demonstrated direct binding of laminarin to its target proteins STAT3 and TNF. Hence, laminarin could be used to improve the recovery outcomes after ischaemic stroke through the promotion of neurofunctions and inhibition of neuroinflammation because STAT3 is well-reported to regulate the brain’s immune system (Seton-Rogers, 2018). Additionally, Chen et al. have demonstrated that suppression of STAT3 inhibited astrogliogenesis and enhanced neurogenesis in humans, suggesting the importance of STAT3 in brain inflammation and neural progenitor cell differentiation (Chen et al., 2013). More importantly, a study using a rat model of MCAO has demonstrated the contribution of STAT3 signalling pathway in inflammatory reaction after cerebral ischaemia via the crosstalk with JAK2 pathway (Wu et al., 2018). Our result also highlighted that laminarin could target TNF, which is also called TNFα, a pleotrophic polypeptide that plays a significant role in the brain’s immune and inflammatory activities (Feuerstein et al., 1994). TNFα has been reported as a risk factor for ischaemic stroke (Bokhari et al., 2014), and both progression and outcomes of stroke were affected by the relationship between the blood–brain barrier and TNFα (Pan & Kastin, 2007). A comparative study in 41 patients with ischaemic stroke indicated the elevation of serum TNFα levels after stroke onset and the involvement of TNFα in brain damage following ischaemic injury (Intiso et al., 2004).

In conclusion, the current report identified the pharmaceutical targets and delineated in detail the molecular mechanisms underlying the beneficial effects of laminarin against ischaemic stroke through the regulation of blood vessel development, neuroinflammation, and neuroprotection. However, further pre-clinical studies are needed to confirm our findings. In addition, signalling pathways are not the same in the acute phase and the recovery phase of laminarin’s treatment, so further molecular studies are needed to delineate the detail involved pathways.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This research was supported by Joint Project on Regional High-Incidence Diseases Research of Guangxi Natural Science Foundation [Grant numbers 2023GXNSFAA026193 and 2023GXNSFAA026197], the Scientific Research Project of Guangxi Health Commission [Grant numbers S2022103, Z-A20220669 and Z-A20220666], Guangxi Medical and health key discipline construction project (Department of Neurology, The Second Affiliated Hospital of Guangxi Medical University).