Application of euglena in preparation of product for prevention and / or adjuvant therapy of cerebral arterial thrombosis

Abstract

The invention relates to the technical field of medicines, in particular to application of euglena in preparation of a product for prevention and / or adjuvant therapy of cerebral arterial thrombosis. Experiments prove that by means of supplementation of the euglena, the infarct volume is remarkably reduced, the neurological function defect is relieved, the synaptic plasticity of mouse hippocampus is enhanced, and the neuroprotective effect of the euglena is related to improvement of the integrity of intestinal barriers, reduction of systemic and neuroinflammation and regulation of intracerebral insulin signal channels; and the euglena can favorably change the composition of intestinal microbiota and promote the production of short-chain fatty acids. In conclusion, the euglena can relieve the cerebral arterial thrombosis through the enterocerebral axis, so that the euglena can be well used for developing related products for prevention and / or adjuvant therapy of the cerebral arterial thrombosis.

Description

Technical Field

[0001] The present application belongs to the field of medical technology, and in particular relates to the use of euglena in preparing a product for preventing and / or assisting in the treatment of ischemic stroke. Background Art

[0002] Stroke (commonly known as stroke) is divided into two types: ischemic stroke and hemorrhagic stroke. Ischemic stroke accounts for more than 70% of all stroke cases and is usually caused by local vascular blockage or arterial stenosis in the brain. Ischemic stroke can lead to reduced blood supply to specific brain areas, resulting in brain damage and neurological dysfunction, and poses a serious risk to survival. The pathological mechanisms of ischemic stroke include oxidative stress, insulin resistance, blood-brain barrier disruption, microglial activation, neuroinflammation, and endothelial damage. In addition to age, metabolic disorders have also been identified as common risk factors for ischemic stroke, and people with metabolic disorders (such as abnormal blood pressure, glucose lipid metabolism disorders, overweight or obesity) have a significantly higher incidence rate. Western diets are usually rich in saturated fats and simple carbohydrates, while low in fiber and micronutrients, which are believed to be related to the development and progression of metabolic dysfunction, which is associated with stroke by releasing inflammatory mediators and damaging cerebral blood vessels. Recent studies have shown that lifestyle adjustments, including dietary changes, can help reduce the occurrence of stroke events.

[0003] Ischemic stroke is mainly caused by cerebral artery obstruction, leading to significant neurological dysfunction and is still the leading cause of disability and death worldwide. Therefore, it is of great significance to develop products for the prevention and / or auxiliary treatment of ischemic stroke. Summary of the invention

[0004] The purpose of the present application is to provide an application of euglena in the preparation of a product for preventing and / or assisting in the treatment of ischemic stroke, aiming to solve the problem of how to better prevent and / or assist in the treatment of ischemic stroke.

[0005] In order to achieve the above application purpose, the technical solution adopted in this application is as follows: The present application provides an application, namely, the application of Euglena in preparing a product for preventing and / or assisting in the treatment of ischemic stroke.

[0006] This application experimentally studies the role of Euglena in the ischemic stroke model of middle-aged C57BL / 6J mice. The data show that the supplementation of Euglena significantly reduces the infarct volume, alleviates neurological deficits, and enhances the synaptic plasticity of the mouse hippocampus. The neuroprotective effect of Euglena is related to improving the integrity of the intestinal barrier, reducing systemic and neuroinflammation, and regulating the insulin signaling pathway in the brain; and the supplementation of Euglena favorably changes the composition of the intestinal microbiota and promotes the production of short-chain fatty acids (SCFAs). Therefore, Euglena can be well used for the prevention and / or adjuvant treatment of ischemic stroke and the development of corresponding products. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0008] Figure 1 The results are as follows: Euglena powder reduces infarct volume and neuronal loss, alleviates microglial activation and synaptic changes in the middle cerebral artery occlusion mouse model under a high-fat, high-fructose diet; A is the Nissl staining image and the Nissl + Relative quantitative analysis of cell density (n=4); B is the assessment of neural damage based on a 48-point scoring system after 24 hours (n=10); C is the relative protein level of Iba-1, a marker of microglial activation (n=3); D is the immunofluorescence staining of Iba-1, a marker of microglial activation, in the CA1, CA3 and DG regions of the hippocampus (scale bar: 50 μm, inset 20 μm, n=3); E is the relative protein level of SYN, a presynaptic membrane protein, in the hippocampus (n=3); F is the relative protein level of PSD95, a postsynaptic density protein, in the hippocampus (n=3); G is an electron microscopy image of the CA1 region of the hippocampus showing the ultrastructure of synapses (n=3); HJ are the image analysis data results of the length of PSD, the thickness of PSD, and the width of SC (n=3), respectively, PSD represents the postsynaptic density area, SC represents the synaptic cleft, and SV represents the synaptic vesicle; all values ​​are expressed as mean ± standard deviation, and statistical significance is marked with **** P <0.0001,*** P <0.001,** P <0.01,* P <0.05.

[0009] Figure 2The results are the effects of Euglena powder on the hippocampal insulin signaling pathway in the middle cerebral artery occlusion mouse model under a high-fat and high-fructose diet; AE represents the relative protein levels of PTP1B, p-IRS / IRS (Ser 307), p-Akt / Akt (Ser473), p-GSK / GSK (Ser 9) and p-Tau / Tau (S202T205) in the hippocampus (n=3); protein levels were standardized with β-Actin, all values ​​were expressed as mean ± standard deviation, and statistical significance was marked with **** P <0.0001,*** P <0.001,** P <0.01,* P <0.05.

[0010] Figure 3 The results show that Euglena powder can prevent intestinal barrier damage and alleviate systemic inflammation in the middle cerebral artery occlusion mouse model under high-fat and high-fructose diet; AB is the actual measurement and data statistics of colon length (n=5); CE is the relative protein levels of ZO-1, occludin and claudin in the colon (n=3); FH is the immunofluorescence staining of ZO-1, claudin and occludin in the colon (scale bar: 50 μm, inset 20 μm, n=3); IK is the concentration level of LPS, IL-1β and IL-6 in serum (n=4); all values ​​are expressed as mean ± standard deviation, and statistical significance is marked with **** P <0.0001,*** P <0.001,** P <0.01,* P <0.05.

[0011] Figure 4 The results are as follows: The results of the regulatory effect of Euglena powder on the intestinal flora of the middle cerebral artery occlusion mouse model under a high-fat and high-fructose diet; AD represents the alpha diversity evaluated by Chao1 index, ACE index, Shannon index and the number of observed OTUs (n=5); E represents the beta diversity evaluated by principal coordinate analysis (PCoA); F represents the Bray distance of the cecal microbiome based on the weighted UniFrac distance matrix (n=5); G represents the most significantly enriched taxa with different abundances in the microbiome by linear discriminant analysis (LDA) (LDA>3, P<0.05); All values ​​are expressed as mean ± standard deviation, and statistical significance is marked with * P <0.05.

[0012] Figure 5The results of the Spearman correlation analysis were used to evaluate the correlation between the main genera and synaptic plasticity, intestinal barrier integrity and systemic inflammation (n=5), including Synaptophysin (SYN) and Postsynaptic Density Protein 95 (PSD95) for synaptic plasticity, ZO-1, claudin, occludin and LPS for intestinal barrier integrity, and IL-1β and IL-6 for systemic inflammation; all values ​​are expressed as mean ± SD, and statistical significance is marked with * P <0.05.

[0013] Figure 6 This is a graph showing the effect of Euglena powder on the production of short-chain fatty acids (SCFAs) in a mouse model of middle cerebral artery occlusion under a high-fat, high-fructose diet; AG represents the levels of acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, valeric acid and total short-chain fatty acids (Total SCFAs) in cecal content samples (n=5); all values ​​are expressed as mean ± standard deviation, and statistical significance is marked as *** P <0.001,** P <0.01,* P <0.05.

[0014] Figure 7 This is a scanning electron micrograph of the amyloid extract from the cells of the algae (10,000 times).

[0015] Figure 8 It is the level of short-chain fatty acids at different time points in the anaerobic fermentation of Euglena cells and Euglena paramylon in vitro; the figure shows the levels of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and total short-chain fatty acids (Total SCFAs) at 0, 24 and 48 hours in the anaerobic fermentation samples of the blank group (Blank), Euglena group (Euglena) and Euglena paramylon group (Paramylon) respectively (n=3); different letters represent statistical differences P <0.05. DETAILED DESCRIPTION

[0016] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0017] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0018] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0019] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0020] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0021] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also indicate the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as µg, mg, g, kg, etc.

[0022] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0023] The gut-brain axis is a bidirectional channel between the gut and the brain through the immune system, nervous system, and intestinal microenvironment, which plays a role in ischemic stroke. Cerebral ischemia can rapidly induce intestinal ischemia and ultimately lead to intestinal dysbiosis, which in turn may aggravate cerebral infarction by enhancing systemic inflammation. In addition to negative changes in microbial composition, intestinal dysbiosis in stroke models may also reduce the relative abundance of short-chain fatty acid (SCFAs) producing bacteria such as Clostridium butyricum, which may be associated with post-stroke damage caused by the gut-brain axis. Notably, a high-fat diet (HFD) or a high-fructose diet can induce changes in the gut microbiota, impair insulin resistance, lead to synaptic dysfunction and neuroinflammation, and further aggravate cerebral ischemic injury.

[0024] Neuroinflammation serves as a mediator in ischemic stroke models, linking synaptic and cognitive dysfunction to gut dysbiosis. Microglial activation plays a key role in neuroinflammation and synaptic loss in ischemic stroke, especially in aging models. After ischemic injury, danger-associated molecular patterns (DAMPs) are released into the extracellular space, stimulating pattern recognition receptors (PRRs) on microglia and triggering an inflammatory response. Multiple microglial receptors, channel proteins, and enzymes promote microglial polarization, mediating pro-inflammatory and anti-inflammatory functions. This leads to a prolonged connection between microglia and synapses and the disappearance of damaged presynaptic bodies.

[0025] Insulin is essential for brain function, and insulin resistance or dysregulation may lead to neurological damage. Insulin resistance is an independent risk factor for ischemic stroke and promotes thrombosis and atherosclerosis. It exacerbates inflammation, oxidative stress, and neurological damage, leading to poor prognosis in stroke patients. Protein tyrosine phosphatase 1B (PTP1B) regulates neuroinflammation and insulin signaling pathways. Overexpression of PTP1B may lead to hyperphosphorylation of Tau protein, ultimately leading to cognitive impairment. Conversely, inhibition of PTP1B has been shown to improve neuroinflammation, insulin resistance, and neurological damage, and performs well in a brain insulin resistance stroke model.

[0026] Euglena Euglena gracilis EG is a unicellular microalgae commonly found in freshwater. It contains functional ingredients such as protein, vitamins, minerals, polyunsaturated fatty acids (PUFAs) and abundant paramylon. EG is widely used as a dietary supplement worldwide. The examples of this application further confirm its relationship with ischemic stroke. Therefore, this application proposes the following scheme.

[0027] The present application embodiment provides an application, namely, the application of Euglena in preparing a product for preventing and / or assisting in the treatment of ischemic stroke, wherein Euglena refers to the entire Euglena cell.

[0028] In the examples of the present application, the neuroprotective effect of Euglena supplementation in the ischemic stroke model of middle-aged C57BL / 6J mice was studied, focusing on gut-brain axis regulation and neuroinflammation. These 52-week-old mice were divided into three groups: a high-fat, high-fructose group, a high-fat, high-fructose plus Euglena supplementation group, and a high-fat, high-fructose sham operation group. After 8 weeks of dietary intervention, cerebral ischemia was induced in the high-fat, high-fructose group by the middle cerebral artery occlusion (MCAO) modeling method, marked as the HFHF_MCAO group, and cerebral ischemia was induced in the high-fat, high-fructose plus Euglena supplementation group, marked as the EG_MCAO group. The high-fat, high-fructose sham operation group was marked as the HFHF_SHAM group. The data showed that EG supplementation significantly reduced infarct volume, alleviated neurological deficits, and enhanced synaptic plasticity in the mouse hippocampus. The neuroprotective effect of EG is associated with improved intestinal barrier integrity, reduced systemic and neuroinflammation, and regulation of insulin signaling pathways in the brain, suggesting that it is achieved through the gut-brain axis. Furthermore, EG supplementation favorably altered the composition of the intestinal microbiota, particularly the increased abundance of beneficial bacteria and the increased production of short-chain fatty acids. Therefore, EG supplementation could be a promising dietary or pharmacological strategy to mitigate high-fat, high-fructose diet-induced ischemic stroke via the gut-brain axis.

[0029] In some embodiments, the above-mentioned ischemic stroke includes ischemic stroke caused by a high-fat, high-fructose diet. Specifically, taking a mouse animal model as an example, the high-fat, high-fructose diet includes: a high-fat diet (HFD, 60% fat: 26% carbohydrates: 14% protein) and drinking water containing 10% fructose.

[0030] In some embodiments, the algae reduce neurological damage and inflammation caused by ischemic stroke. Alternatively, the algae promote the production of short-chain fatty acids by regulating the abundance of beneficial bacteria.

[0031] In some embodiments, the above-mentioned product for preventing and / or assisting in the treatment of ischemic stroke includes a drug or a dietary supplement.

[0032] For example, the product is a drug, the drug includes euglena and pharmaceutically acceptable excipients, wherein the euglena is used as an active ingredient of the drug. The dosage form of the drug includes at least one of powder, granules, tablets, and capsules. Alternatively, the product is a dietary supplement, including euglena and other excipients, wherein the euglena is used as a functional supplement as a component of food or nutrition.

[0033] It should be noted that the use of euglena mentioned in the examples of the present application refers to the use of euglena cells as a whole.

[0034] Accordingly, a product for preventing and / or assisting in the treatment of ischemic stroke. Specifically, the product of the embodiment of the present application contains euglena.

[0035] The examples of this application reveal the role of Euglena in the protective mechanism mediated by the gut-brain axis through experiments. First, Euglena regulates the imbalance of the intestinal microbiome, repairs mucosal barrier dysfunction, and promotes the production of SCFAs. Increased levels of SCFAs help reduce serum lipopolysaccharide (LPS), thereby improving systemic inflammation and reducing the activation of microglia in the brain. In addition, EG improves the insulin signaling pathway, which contributes to better synaptic function and reduced neuroinflammation. These effects together reduce cerebral infarction and improve neurological function in MCAO mice.

[0036] In some embodiments, the product can be a medicine or a dietary supplement. For example, the product is a medicine, and the medicine includes, in addition to the euglena, a pharmaceutically acceptable excipient. The excipient includes at least one of a pharmaceutically acceptable carrier, a diluent, a filler, a binder, a preservative, a lubricant, a dispersant, a flavoring agent, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, an antioxidant, a colorant, and a stabilizer.

[0037] In some embodiments, the dosage form of the drug includes at least one of powder, granules, tablets, and capsules.

[0038] In some embodiments, the present application also confirms that the euglena cells can better regulate the intestinal flora than the euglena paramylon, so the application of the embodiments of the present application is the application of the euglena cells as a whole.

[0039] In summary, in the examples of this application, Euglena can resist intestinal barrier damage, intestinal microbiome disorder, neuroinflammation and hippocampal insulin resistance caused by high-fat and high-sugar diet and cerebral ischemia injury. The experiments of this application support the potential of Euglena as a dietary supplement for the prevention and recovery of stroke, and emphasize its role in regulating the gut-brain axis and neuroinflammation.

[0040] The following describes the invention in conjunction with specific embodiments.

[0041] Example 1 1 Materials and Methods 1.1 Animal model establishment In this example, 52-week-old C57BL / 6J male mice were obtained from the Guangdong Medical Laboratory Animal Center (Guangzhou, China) and housed under environmentally controlled conditions (22 ± 2 °C, relative humidity 60 ± 10%). All experimental procedures followed the relevant regulations on experimental animal welfare ethics and were approved by the Experimental Animal Welfare Ethics Committee of Shenzhen Lingfu Top Biotechnology Co., Ltd. (IACUC approval number: TOP-IACUC-2022-0195).

[0042] The euglena powder used in this example was provided by the Shenzhen Key Laboratory of Marine Microbiome Engineering, China. Specifically, the euglena powder ( Euglena gracilis Klebs, CCAP 1224 / 5Z) was cultured heterotrophically in a 30-liter fermenter according to the optimized method (Xie et al., 2023); after the culture, the euglena cells were collected by centrifugation and freeze-dried. The obtained euglena powder was tested to contain 68.9% euglena paramylon, 20.7% protein, 4.2% fat, 2.4% water, and 3.8% others.

[0043] After a 1-week adaptation period, the male mice were randomly divided into two groups for feeding: HFHF group (n=30, HFHF diet) and EG group (n=15, HFHF diet supplemented with Euglena powder). The high-fat and high-fructose diet in the HFHF group included a high-fat diet (HFD, 60% fat: 26% carbohydrate: 14% protein) and drinking water containing 10% fructose. The EG group was further supplemented with Euglena powder, and the energy of the HFHF diet supplemented with Euglena powder was the same as that of the HFHF group. The body weight of the mice was recorded once a week throughout the study.

[0044] At the end of the above-mentioned feeding period (8 weeks), cerebral ischemia was induced in the above-mentioned HFHF group (n=15) and EG group (n=15) by a modified transient intraluminal suture middle cerebral artery occlusion (MCAO) modeling method, which were marked as HFHF_MCAO group and EG_MCAO group, respectively. The specific steps were as follows: the mice were anesthetized with 4% isoflurane (Abbott, USA), and then maintained in anesthesia with 2% isoflurane for MCAO surgery. The left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were then exposed. A stump was made on the ECA to insert a monofilament with a silicon-coated tip. The monofilament was advanced from the ECA to the ICA until the MCA was blocked to achieve cerebral ischemia. The monofilament was removed 30 minutes after the operation. The remaining mice (n=15) in the HFHF group underwent sham surgery without the insertion of a monofilament to occlude the MCA, which was marked as the HFHF_SHAM group.

[0045] 1.2 Neurological assessment A 48-point scoring system was used to evaluate the behavioral ability of mice 24 hours after MCAO modeling (reference: Y. Li, Y. Wang, W. Yang, Z. Wu, D. Ma, J. Sun, H. Tao, Q. Ye, J. Liu, Z. Ma, L. Qiu, W. Li, L. Li and M. Hu, ROS-responsive exogenous functional mitochondria can rescue neural cells post-ischemic stroke, Frontiers in Cell and Developmental Biology , 2023). The maximum total score is 48 and is used to assess motor, sensory and reflex abilities, with higher scores indicating more severe neurological damage.

[0046] 1.3 Sample Collection At the end of the study, five mice from each group were euthanized by CO2 asphyxiation. Blood, hippocampus, cecal contents, and colon samples were collected and stored at -80 °C for analysis. Another group of mice (n = 3 per group) were euthanized by PBS perfusion, and the hippocampus was subsequently collected for transmission electron microscopy (TEM) observation. The remaining mice (n = 7 per group) were euthanized by transcardial perfusion of PBS. The intact brain tissue (n = 4 per group) and hippocampus (n = 3 per group) were fixed in 4% paraformaldehyde. The intact brain tissue was used for Nissl staining, and the hippocampus was used for immunofluorescence staining.

[0047] 1.4 Serum cytokine and endotoxin detection Serum cytokine and lipopolysaccharide (LPS) levels were measured using commercial serum cytokine ELISA kits (Elabscience, Wuhan, China) and LPS ELISA kits (Signalway Antibody, MD, USA) according to the manufacturers’ instructions.

[0048] 1.5 Western blotting Western blotting analysis was used to determine the expression levels of tight junction proteins in the colon and proteins associated with the insulin resistance signaling pathway in the hippocampus. Total proteins were extracted from hippocampal and colonic tissues at 4 °C using RIPA buffer (ThermoFisher Scientific, 89900) containing protease and phosphatase inhibitors. Samples were homogenized at 4 °C for 30 min and then centrifuged at 1700 × g for 10 min at 4 °C. The protein concentration in the supernatant was determined by Bicinchoninic Acid (BCA) assay. After SDS-PAGE separation, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat dry milk for 1 h and then incubated with the corresponding primary antibodies overnight at 4 °C. After incubation with HRP-labeled anti-rabbit IgG secondary antibody (Cat#7074s, 1:2000, CST) at room temperature for 1 h, protein bands were visualized using ECL substrate and images were captured using a ChemiDoc Touch imaging system (Bio-Rad, CA, USA). Quantitative protein analysis was performed using Image J software.

[0049] 1.6 Nissl staining After dehydration, brain tissue sections were stained with Nissl reagent (G1036, Servicebio) for 2 minutes. After differentiation with 0.5% acetic acid, the sections were dried in an oven at 60 °C for 4 hours. Finally, the sections were mounted with neutral glue, and Nissl bodies were observed under an optical microscope (Nikon Eclipse E100, Japan), and images were captured using an imaging system (Nikon DS-U3, Japan). Relative quantitative analysis of Nissl cells was performed using Image J.

[0050] 1.7 Immunofluorescence staining Immunofluorescence staining was used to detect Iba-1 in the hippocampus and ZO-1, claudin, and occludin in colon tissue samples. Tissue samples were fixed in 4% paraformaldehyde for 24 hours and then immersed in 15% and 30% sucrose solutions until the tissue sank. Tissues were embedded in optimal cutting temperature compound (OTC) (Cat#4583, Sakura), placed in dry ice for 20 minutes, and then cut into 10 μm sections using a freezing microtome (Thermo Scientific, USA). Blocking was then performed. Tissue sections were incubated in TBS solution containing 10% goat serum (Cat#C0265, Beyotime) at 37 °C for 30 min and then incubated with primary antibodies including rabbit anti-Iba1 antibody (Cat#17198T, 1:200, CST), rabbit anti-ZO-1 antibody (Cat#21773-1-AP, 1:200, PTG), rabbit anti-claudin 1 antibody (Cat#PA5-20754, 1:200, PTG), and rabbit anti-occludin antibody (Cat#37-4900-AP, 1:200, PTG) at 4 °C overnight. The sections were then washed with PBS (pH = 7.4) and incubated with secondary antibodies (Cat#111-165-003, 1:600, Jackson, Cy™3 AffiniPure Goat Anti-Rabbit IgG (H+L)) at 37 °C for 50 min. Finally, they were stained with DAPI (Cat#D9542, Sigma) for 10 min in the dark at room temperature and treated with anti-fluorescence quencher blocking slides (Cat#0100-01, Southern Biotech). Images were captured using a Nikon EclipseC1 fluorescence microscope (Nikon, Japan).

[0051] 1.8 Transmission Electron Microscopy The ultrastructure of the CA1 region of the hippocampus was evaluated using transmission electron microscopy (TEM). First, the CA1 region was isolated from the hippocampus and fixed with 2.5% glutaraldehyde for 24 h at 4°C. After washing, the samples were postfixed with 1% osmium tetroxide in PBS for 2 h at room temperature. Subsequently, the tissues were dehydrated through a series of increasing concentrations of ethanol (from 50% to 100% (v / v)) for 15 min each and then dehydrated in 100% acetone for 30 min. Next, the tissues were embedded in 50% epoxy resin in acetone for 4 h and then in 33% epoxy resin (v / v) for 8 h. Finally, the tissues were embedded in pure epoxy resin at 37°C overnight and cured at 60°C for 48 h. The samples were then cut into 70 nm ultrathin sections using a Leica ultramicrotome (Leica, Germany) and mounted on Diatome copper grids. The sections were stained with 2% urethane acid and 6% lead citrate aqueous solutions. The ultrastructure of the CA1 region was visualized by transmission electron microscopy (Hitachi Limited, Japan), and image analysis was performed using Image-Pro Plus 6.0 software.

[0052] 1.9 16S rRNA analysis Total genomic DNA was extracted from cecal contents using the FastDNA SPIN kit (MP Biomedicals, Santa Ana, CA) following the manufacturer’s protocol. The integrity of the genomic DNA was assessed by agarose gel electrophoresis, and the concentration and purity of the DNA were measured using a Nanodrop 2000 and Qubit 3.0 spectrophotometer. Primers were used to amplify the V3-V4 hypervariable region of the 16S rRNA gene and then sequenced on the Illumina NovaSeq 6000 platform. The resulting amplification products were sequenced and analyzed using Quantitative Insights into Microbial Ecology 2 (QIIME2). High-quality reads were clustered into operational taxonomic units (OTUs) at a 97% sequence similarity threshold and deposited for further analysis.

[0053] 1.10 Determination of short-chain fatty acids (SCFAs) Six SCFAs (including acetate, propionate, isobutyrate, butyrate, isovaleric acid, and valeric acid) in cecal contents were quantified using a gas chromatography-mass spectrometry (GC-MS) system (7890B-5977B, Agilent) equipped with a DB-FFAP capillary column (30 m × 0.25 mm × 0.25 μm, Agilent). 100 mg of cecal contents were resuspended in 1 mL of methanol and centrifuged at 1000 × g for 10 min at 4 °C. The supernatant was mixed with 3.5 mM 2-methylbutyric acid (internal standard) solution in a 1:1 ratio and then centrifuged at 1000 × g for 10 min at 4 °C. The supernatant was filtered through a 0.22 μm filter membrane before GC-MS analysis. Calibration curves were constructed using authentic SCFA standards. Helium was used as the carrier gas at a flow rate of 1 mL / min. The column temperature was initially set to 80°C, held for 3 min, then increased to 170°C at a rate of 10°C / min and held at this temperature for 12 min. Total SCFAs were calculated as the sum of the six SCFAs.

[0054] 1.11 Statistical analysis All data were analyzed using SPSS 25.0 (USA) and GraphPad Prism 9.0 (GraphPad Software, SanDiego, CA, USA), and the results are presented as mean ± SD. The significance level was set at P < 0.05. The Brown-Forsythe test and Shapiro-Wilk test were used to assess homogeneity of variance and normal distribution. For normally distributed data, two-group comparisons were performed using the independent sample t test, while for multiple group comparisons, one-way analysis of variance (ANOVA) with Tukey HSD test was used. When the data did not conform to normal distribution, two-group comparisons were performed using the Mann-Whitney test, and multiple group comparisons were performed using the Kruskal-Wallis test and Dunn's test. For metagenomic data analyzed by 16S rRNA, the Adonis test (with 999 permutations) was used to determine the statistical significance of the separation between groups in PCoA. The linear discriminant analysis effect size (LEfSe) was used to identify differential taxa between groups. Spearman correlation analysis was performed to assess the association of major genera with synaptic plasticity (e.g., SYN and PSD95), intestinal barrier integrity (e.g., ZO-1, claudin, occludin, and LPS), and systemic inflammation (e.g., IL-1β and IL-6).

[0055] 2 Experimental Results EG supplementation improves neural injury, microglial activation, and synaptic ultrastructure Nissl staining is often used to evaluate the functional status of neurons. The morphology and number of Nissl bodies are directly related to neuronal activity. Nissl staining is used to assess infarct volume and neuronal loss to determine the severity of cerebral ischemia. + The infarct volume was determined by relative quantitative analysis of the density of cells. The results of the examples of the present application showed that fewer Nissl bodies were observed in mice with ischemic stroke, and Nissl bodies were found to be in a destroyed or dissolved state in damaged neurons. Figure 1 As shown in Figure A, fewer Nissl bodies were observed on the left side of the brain in the HFHF_MCAO group compared with the EG_MCAO group, and the neurons in the EG_MCAO group were more densely arranged. In addition, the Nissl bodies in the HFHF_MCAO group had blurred outlines and weaker staining, indicating that the degree of neurological damage in this group was higher than that in the EG_MCAO group, which was also supported by the overall smaller infarct size in the EG_MCAO group. These data indicate that EG supplementation has a protective effect on neurological damage caused by ischemic stroke. The neurological scoring scale based on the 48-point scoring system also showed that the neurological deficits in the EG_MCAO group were reduced compared with those in the HFHF_MCAO group ( Figure 1 The expression of Iba-1, a marker of microglial activation, was further examined by immunofluorescence staining, and the ultrastructure of hippocampal synapses was evaluated by TEM. Compared with the HFHF_MCAO group, EG supplementation reduced the expression of Iba-1, indicating that microglial activation in the CA1, CA3, and DG regions was attenuated in MCAO mice fed a HFHF diet ( Figure 1 Western blotting showed that SYN ( Figure 1 E) and PSD95 ( Figure 1 F) significantly increased the expression of EG, suggesting that EG supplementation has neuroprotective potential. It also improved the synaptic ultrastructure of ischemic stroke mice ( Figure 1 (shown in GJ).

[0056] EG supplementation restores altered hippocampal insulin signaling The role of hippocampal insulin signaling pathway in the neuroprotective effect of EG on MCAO mice was explored by immunoblotting analysis of key signaling molecules, including PTP1B, p-IRS-1 / IRS-1 (Ser307), p-Akt / Akt (Ser473), p-GSK-3β / GSK-3β (Ser9), and p-tau / tau (S202 / T205). Figure 2As shown in AE, EG supplementation downregulated the expression of PTP1B, p-IRS, and p-Tau, and significantly upregulated the expression of p-AKT and p-GSK3β. These results suggest that the hippocampal insulin signaling pathway is a potential target for EG to exert neuroprotective effects in disease models.

[0057] 2.3 Protective effects of EG supplementation on intestinal barrier function and systemic inflammation Colon length is associated with inflammation. Figure 3 As shown in AB in the present invention, it was observed that the colon length of mice in the HFHF_MCAO group was shorter than that in the HFHF_SHAM group, and EG supplementation could effectively prevent the shortening of the colon in MCAO mice. EG supplementation also had a protective effect on the intestinal barrier function of mice, which was supported by the increased expression of tight junction proteins (such as ZO-1, Claudin and Occludin) in the EG_MCAO group compared with the HFHF_MCAO group, see Figure 3 In addition, EG supplementation reduced serum LPS levels ( Figure 3 Furthermore, EG supplementation alleviated systemic inflammation in ischemic stroke mice fed a HFHF diet, as evidenced by a reduction in the expression of proinflammatory cytokines IL-1β and IL-6 ( Figure 3 ).

[0058] EG supplementation restored intestinal flora dysbiosis and promoted SCFA production Intestinal flora imbalance is closely related to cerebral ischemic stroke and high-fat diet. To investigate whether EG plays a protective role in ischemic stroke mice by regulating intestinal microorganisms, the α-diversity and β-diversity of mouse cecal contents were measured. Figure 4 As shown in Figures AD, the EG_MCAO group showed higher α-diversity than the MCAO_HFHF group. The β-diversity was evaluated by PCoA analysis of weighted UniFrac distance, and the results showed that the microbiota of mice in the MCAO model group was significantly separated from that in the control group (see Figure 4 The MCAO_HFHF group had significantly altered β-diversity, whereas EG supplementation appeared to counteract this adverse effect (see Figure 4 F in.

[0059] Differential enrichment of taxa between the study groups was identified by LEfSe analysis (LDA>3). Figure 4 As shown in G, in the MCAO_HFHF group, significant taxa were differentially enriched compared with the SHAM and MCAO_EG groups, especially in the Proteobacteria ( Proteobacteria )of Gammaproteobacteria Head, EnterobacterialesKehe Escherichia-Shigella genus, which is associated with pathogenic effects. In addition, it was observed Firmicutes In the door Anaerotruncus Genus, Clostridium_XVIII Genus and Clostridium cocleatum EG supplementation partially restored the intestinal flora, and the MCAO_EG group showed enrichment of specific taxa, including Bacillota In the door Allobaculum Genus; Pseudomonadota In the door Pasteurellales Mewa Pasteurellaceae division; Bacillota In the door Butyricicoccus Genus; Bacteroidetes In the door Prevotella Genus, uncultured Bacteroidales Bacteria and Bacteroides stercorirosoris species; and Firmicutes Uncultured Clostridiales Bacteria. The enrichment of these beneficial bacteria plays an important role in the health-promoting effects of EG supplementation, such as maintaining intestinal health and integrity, reducing inflammation, and enhancing metabolic function.

[0060] Spearman correlation analysis was used to assess the association of the main genera with synaptic plasticity, intestinal barrier integrity, and systemic inflammation. Figure 5 As shown, inflammatory biomarkers are Akkermansia , Pseudoflavonifractor , Anaerobacterium or Anaerotruncus is negatively correlated with Bacteroides and Escherichia LPS level is also positively correlated with Bacteroides and Escherichia is positively correlated, but Parabacteroides , Pseudoflavonifractor and Anaerobacterium Among the tight junction proteins of the intestinal barrier, occludin is negatively correlated with Bacteroides and Mucispirillum was negatively correlated with Alloprevotella and Anaerobacterium In terms of synaptic plasticity biomarkers, SYN and PSD95 were positively correlated with Bacteroides However, SYN is negatively correlated with Parabacteroide s and Pseudoflavonifractor There is a positive correlation between PSD95 and Akkermansia and Anaerobacterium There is a positive correlation.

[0061] SCFAs play a key role in regulating the gut-brain axis. The cecal SCFA levels of MCAO mice on the HFHF diet were significantly lower than those of the control group, while EG supplementation could partially restore these SCFA levels (see Figure 6 AG in the intestine), indicating that EG has an enrichment effect on intestinal SCFAs-producing bacteria.

[0062] 3 Comparative Experiments The embodiment of the present application further extracts the main energy storage material of naked algae: naked algae paramylon. The specific steps include: adding 100 grams of naked algae powder to 1 liter of water, and then using an ultrasonic homogenizer for 15 minutes to break the cells. The suspension is then centrifuged at 4°C with a centrifugal force of 5000g for 5 minutes. The precipitate is then resuspended in a 1% SDS aqueous solution, incubated in a 90°C water bath for 60 minutes, and centrifuged again at a centrifugal force of 5000g for 10 minutes. Subsequently, 0.1% SDS solution is added to the precipitate, the suspension is placed in a 50°C water bath for 60 minutes, and then centrifuged at 4°C with a centrifugal force of 5000g for 5 minutes. Finally, the precipitate is washed twice with anhydrous ethanol and dried at room temperature to obtain naked algae paramylon. Its surface structure under a scanning electron microscope (SEM) is as shown Figure 7 shown.

[0063] The euglena and the above-extracted euglena paramylon were subjected to in vitro anaerobic fermentation with healthy human feces, and the samples were collected at 0, 24, and 48 hours and then SCFAs were detected using GC-MS. Figure 8 As shown, the results indicate that the ability of the whole Euglena to promote SCFAs production is better than that of Euglena paramylon. It can be inferred that the effect of the whole Euglena cells in regulating intestinal flora is better than that of paramylon in Euglena cells. In vitro anaerobic fermentation reveals that the effect of Euglena cells as a whole in regulating intestinal flora is better than that of paramylon in Euglena.

[0064] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. Use of Euglena in preparing products for preventing and / or assisting in the treatment of ischemic stroke.

2. The use according to claim 1, characterized in that The ischemic stroke includes ischemic stroke caused by a high-fat and high-fructose diet.

3. The use according to claim 1, characterized in that The euglena reduces nerve damage and inflammation caused by ischemic stroke; and / or, the euglena promotes the production of short-chain fatty acids by regulating the abundance of beneficial bacteria.

4. The use according to any one of claims 1 to 3, characterized in that: The product includes a medicine or a dietary supplement.

5. The use according to claim 4, characterized in that The product is a medicine, which includes the euglena and pharmaceutically acceptable excipients, and the dosage form of the medicine includes at least one of powder, granules, tablets and capsules.

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