Composition for alleviating symptoms caused by vascular hyperpermeability

Abstract

[Problem] To provide a composition for alleviating various symptoms caused by vascular hyperpermeability. [Solution] The present invention is achieved by a composition that is for controlling the expression of Robo4 and that contains rhamnan sulfate. The composition is preferably for alleviating symptoms caused by vascular hyperpermeability, and is preferably for oral administration. In addition, it is possible to provide a food or drink that is for alleviating symptoms caused by vascular hyperpermeability, and that contains the composition.

Description

Composition for alleviating symptoms caused by increased vascular permeability

[0001] The present invention relates to a composition for alleviating symptoms caused by increased vascular permeability.

[0002] The present inventors have previously reported that the expression of the procoagulant tissue factor (TF) and the platelet aggregation factor von Willebrand factor (VWF) produced by cultured vascular endothelial cells stimulated with inflammatory substances (thrombin, TNFα, and lipopolysaccharide (LPS)) is significantly suppressed in the presence of rhamnan sulfate (RS) derived from horseradish, demonstrating its potent anti-inflammatory effects (Non-Patent Document 1). Furthermore, we have reported that oral administration of RS dose-dependently suppresses inflammatory lesions in lungs and livers and increased vascular permeability in lung tissues in mice intraperitoneally administered with the bacterial endotoxin LPS (Non-Patent Documents 2 and 3).

[0003] Okamoto T, Akita N, Terasawa M, Hayashi T, Suzuki K. Rhamnan sulfateextracted from Monostroma nitidum attenuates blood coagulation and inflammationof vascular endothelial cells. J. Nat. Med. 2019, 73, 614-619.Terasawa M, Hiramoto K, Uchida R, Suzuki K. Anti-inflammatory activityof orally administered Monostroma nitidum rhamnan sulfate against lipopolysaccharide-induced damage to mouse organs and vascular endothelium.Mar. Drugs 2022, 20.Suzuki K, Terasawa M. Biological activities of rhamnan sulfateextract from the green algae Monostroma nitidum (Hitoegusa). Mar. Drugs 2020,18.

[0004] Research into RS has a short history, and there is still room for further research into the physiological effects of RS. Meanwhile, the Roundabout 4 (Robo4) gene is known to be specifically expressed in vascular endothelial cells. Robo4 functions in vascular permeability of substances and cells, and suppressing Robo4 expression is known to suppress the production of inflammatory cytokines such as IL-6 from vascular endothelial cells and inhibit inflammation in sepsis. However, the relationship between RS and Robo4 has not been clarified. The present invention was made in light of the above-mentioned circumstances, and its purpose is to provide a composition for alleviating various symptoms caused by increased vascular permeability by regulating Robo4 expression.

[0005] In this study, we analyzed the expression of the vascular permeability inhibitor Roundabout 4 (Robo4) in the lung tissue of ApoE-deficient (ApoE- / -) mice (atherosclerosis model mice) fed a high-fat diet. Robo4 expression was significantly reduced in the lung tissue of ApoE-deficient mice fed an RS diet. Furthermore, Robo4 expression was found to be equal to or greater than that in the lung tissue of ApoE-deficient mice fed a normal diet. These results suggest that oral RS intake suppresses the reduction in Robo4 expression in vascular endothelial cells of atherosclerosis-induced mice, thereby strongly suppressing vascular permeability. Thus, we demonstrated that RS is a novel substance that enhances the expression of Robo4, a molecule that inhibits vascular permeability (inflammatory damage of the vascular endothelium) during inflammation.

[0006] Inflammation of endothelial cells lining the vascular endothelium leads to increased blood coagulation, increased platelet aggregation leading to the formation of thrombi, and the activation of various white blood cells (increased migration and adhesion) leading to the expansion of inflammation, leading to a pathological condition in which blood leaks out of the blood vessels (increased vascular permeability). Increased vascular permeability occurs due to a breakdown in the adhesion mechanism between endothelial cells lining the vascular lumen. Specifically, this is due to a decrease in the binding strength between vascular endothelial cadherin (VE-cadherin), a vascular endothelial adhesion molecule involved in the adhesion between endothelial cells. This breakdown in the adhesion mechanism between endothelial cells allows blood components within the blood vessels, such as red blood cells and white blood cells (neutrophils, monocytes, macrophages, etc.), as well as fluid and many proteins (functional molecules) dissolved in the fluid, to easily pass through the gaps between vascular endothelial cells and leak into extravascular tissues.

[0007] As a result, the migration and accumulation of monocytes and macrophages in the arterial media induces arteriosclerosis. The extravasation of water and proteins into blood vessels causes edema in various tissues. Bacterial leakage leads to severe systemic infections and inflammatory conditions, such as sepsis and disseminated intravascular coagulation (DIC). Furthermore, viral leakage facilitates infection of systemic tissues by influenza and coronavirus, resulting in severe clinical symptoms. Furthermore, it enables cancer cells to infiltrate systemic tissues, leading to metastasis of cancer lesions and worsening of the disease. Thus, the inventors discovered that orally administered RS strongly suppressed the decrease in Robo4 expression, which increases vascular permeability during inflammation. This suggests that RS has therapeutic and / or preventive effects that alleviate and improve various inflammatory symptoms and pathologies caused by increased vascular permeability, and essentially led to the completion of this invention.

[0008] Thus, the composition of the present invention is characterized by regulating the expression of Robo4, which contains rhamnan sulfate. Preferably, the composition is intended for alleviating symptoms caused by increased vascular permeability. The following pathologies (diseases) may be effectively alleviated by alleviating symptoms caused by increased vascular permeability: 1. Acute respiratory distress syndrome (ARDS). ARDS is a disease with a poor prognosis and high mortality rate caused by non-cardiogenic pulmonary edema, in which vascular permeability is increased due to damage to alveolar vascular endothelial cells, accompanied by the production of inflammatory cytokines, leading to leakage and accumulation of blood components into the pulmonary arteries. It can be caused by various diseases, such as severe pneumonia, sepsis, or trauma. Furthermore, many patients who become seriously ill with COVID-19, a viral infection currently wreaking havoc worldwide, also develop ARDS, which can lead to death if the condition worsens. 2. Inflammatory lung diseases, including idiopathic pulmonary fibrosis and bleomycin-induced lung injury. These diseases are pathologically associated with increased permeability of pulmonary microvessels. 3. 3. Alleviation of cancer symptoms: It has been found that vascular endothelial growth factor (VEGF) forms fragile and highly permeable tumor blood vessels in cancer tissues, contributing to the progression of cancer. Therefore, RS intake may alleviate cancer symptoms. 4. Alleviation of edema caused by inflammatory diseases such as bacterial infections and nephritis.

[0009] In this case, the composition is preferably for oral administration. Furthermore, the food and beverage product of the present invention contains the composition and is intended for alleviating symptoms caused by increased vascular permeability. The daily dose of rhamnan sulfate is preferably 10 mg to 1,000 mg, more preferably 30 mg to 500 mg, and even more preferably 50 mg to 300 mg. The composition of the present invention is provided by combining an amount of rhamnan sulfate effective for alleviating symptoms caused by increased vascular permeability with pharmaceutically acceptable carriers and additives. This composition can be provided as a pharmaceutical or quasi-drug. The pharmaceutical composition is used internally or externally. This pharmaceutical composition can be used in the form of oral preparations, injections such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and / or intraperitoneal injections, transmucosal preparations, transdermal preparations, and the like. Rhamnan sulfate is particularly effective when administered orally or transdermally, and is therefore preferably used internally, transmucosally, or transdermally.

[0010] The dosage form of the pharmaceutical composition can be appropriately determined, and examples thereof include solid preparations such as tablets, granules, capsules, powders, and dusts; liquid preparations such as solutions and suspensions; and semi-solid preparations such as ointments and gels. As for food and beverage products, rhamnan sulfate can be incorporated into various foods as a food ingredient to produce food compositions. Examples of food compositions include general foods, as well as foods for specified health uses, foods with nutrient functions, foods with functional claims, foods for hospital patients, and supplements. It can also be used as a food additive. Examples of food compositions include beverages (soft drinks, alcoholic beverages, carbonated beverages, dairy beverages, fruit juice beverages, tea, coffee, energy drinks, concentrated beverages, etc.), powdered beverages (powdered juice, powdered soup, etc.), confectioneries (candy (throat lozenges), cookies, biscuits, gum, gummy candies, chocolate, etc.), bread, cereal, seasonings, etc.

[0011] In the case of foods for specified health uses, foods with nutrient functions, and foods with functional claims, they can also be provided in the form of capsules, lozenges, syrups, granules, powders, etc. A food for specified health uses is a food containing functional health ingredients that affect physiological functions, etc., and can be labeled as suitable for specific health uses with the permission of the Commissioner of the Consumer Affairs Agency. In this invention, the food is sold with a label indicating a specific use, such as the effect of improving various symptoms caused by increased vascular permeability. A food with nutrient functions is a food used to supplement nutritional components (vitamins, minerals) and displays the function of the nutritional components. To be sold as a food with nutrient functions, the amount of nutritional components contained in the recommended daily intake must be within the specified upper and lower limits, and not only nutritional function labeling but also warning labeling is required.

[0012] A functional food is a food that displays functionality based on scientific evidence at the responsibility of the business operator. Information regarding the basis for safety and functionality is submitted to the Commissioner of the Consumer Affairs Agency before sale. The present invention contains rhamnan sulfate as an active ingredient and is used as a food for specified health uses, a food with nutrient functions, or a food with functional claims for healthy individuals, frail individuals, frail individuals who are not clearly ill but are not considered healthy, the elderly, etc. The present invention contains rhamnan sulfate as an active ingredient and is used as a food with functional claims for alleviating symptoms caused by increased vascular permeability in humans.

[0013] According to the present invention, a novel composition for alleviating symptoms caused by increased vascular permeability can be provided.

[0014] These graphs show the effects of RS on body weight and food intake in ApoE-deficient mice. a. Line graph showing body weight change over the study period; b. Bar graph showing body weight change before and after the study; c. Bar graph showing food intake change before and after the study. a. Open circles represent the normal diet (ND) group, filled circles represent the ND+RS group, open squares represent the high-fat diet (HFD) group, and filled squares represent the HFD+RS group. b. The two leftmost bars show the weight gain rates (compared to the weight of ApoE-deficient mice fed for 12 weeks) for the ND group and the ND+RS group, respectively. The two rightmost bars show the weight gain rates (compared to the weight of ApoE-deficient mice fed for 12 weeks) for the HFD group and the HFD+RS group, respectively. c. The two leftmost bars show the weight gain rates (compared to the weight of ApoE-deficient mice fed for 12 weeks) for the ND group and the ND+RS group, respectively. The two rightmost bars show the weight gain rates (compared to the weight of ApoE-deficient mice fed for 12 weeks) for the ND group and the ND+RS group, respectively. Data are presented as mean ± standard deviation. In the figure, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001, indicating significant differences between groups by two-way analysis of variance (n=6). Bar graphs show the effects of RS on plasma total cholesterol (TCHO), sterol regulatory element-binding protein 1 (Srebp1) mRNA expression, and plasma triglycerides (TG). a. TCHO concentration, b. Srebp1 mRNA levels in the aorta as determined by quantitative real-time PCR (qPCR), and c. TG concentration. Data are presented as mean ± standard deviation. In the figure, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001, indicating significant differences between groups by two-way analysis of variance (n=5 or 6 for TCHO or TG, n=3 for qPCR). 1 shows photomicrographs and a bar graph showing the results of examining the effect of RS on vascular cell adhesion molecule-1 (VCAM-1) expression in the liver and aorta of ApoE-deficient mice fed a ND or HFD.a) Micrographs showing the results of immunohistochemical analysis of VCAM-1 in the liver and aorta of mice from the ND, ND+RS, HFD, and HFD+RS groups (scale bar: 100 μm). b) Bar graphs showing the fluorescence intensity (FI) of VCAM-1 in the liver of mice from the ND and HFD groups. c) Bar graphs showing the FI of VCAM-1 in the aorta of mice from the ND and HFD groups. d) Bar graphs showing the amount of VCAM-1 mRNA in the aorta of mice from the ND and HFD groups quantified by qPCR. In b–d, white and gray columns represent the RS-untreated and RS-treated groups, respectively. Data are shown as mean ± standard deviation. In the figures, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. Significant differences were observed between groups by two-way analysis of variance (n=4 for immunohistochemistry, n=3 for qPCR). These are photomicrographs and bar graphs showing the effects of RS on macrophage infiltration in organ tissues of ApoE-deficient mice fed either ND or HFD. a. Photomicrographs showing the results of immunohistochemical analysis of F4 / 80 in the liver and aorta of mice from the ND, ND + RS, HFD, and HFD + RS groups (scale bar: 100 μm). b. Bar graphs showing the FI of F4 / 80 in the liver of mice from the ND and HFD groups. c. Bar graphs showing the FI of F4 / 80 in the aorta of mice from the ND and HFD groups. In b and c, white and gray columns represent the RS-untreated and RS-treated groups, respectively. Data are presented as mean ± standard deviation. "***" in the figure indicates a significant difference between groups by two-way analysis of variance (p < 0.001) (n = 4). Figure 1 shows photomicrographs and a bar graph showing the effects of RS on PDGFRβ expression in the liver and aorta of ApoE-deficient mice fed an ND or HFD. a) Photomicrographs showing the results of immunohistochemical analysis of PDGFRβ in the liver and aorta of mice from the ND, ND+RS, HFD, and HFD+RS groups (scale bar: 100 μm). b) Bar graphs showing the FI of PDGFRβ in the liver of mice from the ND and HFD groups. c) Bar graphs showing the FI of PDGFRβ in the aorta of mice from the ND and HFD groups.In b and c, the white and gray columns represent the RS-untreated and RS-treated groups, respectively. Data are shown as mean ± standard deviation. "***" in the figure indicates a significant difference between groups (p < 0.001) based on two-way analysis of variance (n = 4).

[0015] These are photomicrographs and bar graphs showing the effects of RS on α-smooth muscle actin (αSMA) expression in the liver and aorta of ApoE-deficient mice fed either ND or HFD. a. Photomicrographs showing the results of immunohistochemical analysis of αSMA in the liver and aorta of mice from the ND, ND + RS, HFD, and HFD + RS groups (scale bar: 100 μm). b. Bar graphs showing the FI of αSMA in the liver of mice from the ND and HFD groups. c. Bar graphs showing the FI of αSMA in the aorta of mice from the ND and HFD groups. In b and c, white and gray columns represent the RS-untreated and RS-treated groups, respectively. Data are presented as mean ± standard deviation. "***" in the figure indicates a significant difference between groups by two-way analysis of variance (p < 0.001) (n = 4). Figure 1 shows a photomicrograph and bar graph showing the effect of RS on roundabout 4 (Robo4) expression in the livers of ApoE-deficient mice fed ND or HFD. a. Photomicrograph showing the results of immunohistochemical analysis of Robo4 in the livers of mice from the ND, ND+RS, HFD, and HFD+RS groups (scale bar: 100 μm). b. Bar graph showing the fluorescence intensity of Robo4 in the livers of mice from the ND and HFD groups. Data are shown as mean ± standard deviation. "**" in the figure indicates a significant difference between groups by two-way analysis of variance (p<0.01) (n=4). Figure 1 shows a photomicrograph and bar graph showing the effect of RS on Robo4 expression in the aortas of ApoE-deficient mice fed ND or HFD. a. Photomicrograph showing the results of immunohistochemical analysis of Robo4 in the aortas of mice from the ND, ND+RS, HFD, and HFD+RS groups (scale bar: 100 μm). b. This is a bar graph showing the fluorescence intensity of Robo4 in the aorta of mice from the ND and HFD groups. Data are shown as mean ± standard deviation. "**" in the figure indicates a significant difference between groups by two-way analysis of variance at p<0.01 (n=4). This is a bar graph showing the effect of RS on Mmp-2 and Mmp-9 mRNA expression in the aorta of ApoE-deficient mice fed ND or HFD.a) Bar graph showing Mmp-2 mRNA expression levels in the aortas of mice from the ND, ND+RS, HFD, and HFD+RS groups, as quantified by qPCR. b) Bar graph showing Mmp-9 mRNA expression levels in the aortas of mice from the ND, ND+RS, HFD, and HFD+RS groups, as quantified by qPCR. In a) and b), white and gray columns represent the RS-untreated and RS-treated groups, respectively. Data are shown as mean ± standard deviation. An * in the figure indicates a significant difference between groups by two-way analysis of variance (p < 0.05) (n = 3). These are photomicrographs and bar graphs showing the effects of RS on RAW264.7 cell migration. The top nine micrographs show representative cell appearances after adding RS to the upper or lower chamber, incubating for 4 or 24 hours, and then fixing and staining with Hoechst 33342 solution. (x200; scale bar, 100 μm) The left and center bars in the bottom panel show the number of cells that migrated 4 and 24 hours after adding RS to the upper chamber, respectively. The right bar in the bottom panel shows the number of cells that migrated 24 hours after placing RS in the lower chamber. Data are shown as mean ± standard deviation. "***" in the figure indicates a significant difference between groups (p < 0.001) based on one-way analysis of variance (n = 5).

[0016] Next, embodiments of the present invention will be described with reference to the accompanying drawings. However, the technical scope of the present invention is not limited to these embodiments, and various embodiments can be implemented without departing from the spirit of the invention. <Test Methods> <Preparation of Rhamnan Sulfate> Any naturally occurring rhamnan sulfate (RS) can be used. In this embodiment, rhamnan sulfate obtained by hot water extraction from Single-stranded algae was used. Dried seaweed was washed with water and extracted with hot water. The resulting hot water extract was filtered to obtain an extract containing RS as the main component. This was used as the sample in this embodiment. The molecular weight of RS was determined by gel permeation chromatography (GPC) using a Shodex RI-71 refractive index detector (Showa Denko K.K.) and a Shodex SB-806M HQ column (8.0 x 300 mm). Rhamnan sulfate can also be prepared by methods other than those described above. The raw material is not limited to Single-stranded algae, but other sources such as Ulva pertusa and Ulva ribbon can also be used.

[0017] <Subject Animals> ApoE- / - mice (B6.KOR / StmSlc-Apoeshl strain, hereinafter referred to as "ApoE-deficient mice"), a hyperlipidemic Japanese wild mouse (KOR) lacking the ApoE gene, were purchased from Japan SLC, Inc. and housed under a 12-hour light / dark cycle. Twelve-week-old female ApoE-deficient mice were randomly assigned to four groups of six mice each. (1) The normal diet (ND) group received a standard diet (CLEA Rodent Diet CE-7, CLEA Japan), (2) the ND+RS group received a standard diet supplemented with 0.1% RS (w / w), (3) the high-fat diet (HFD) group received a high-fat animal diet containing 1.25% cholesterol (Research Diet D12108C), and (4) the HFD+RS group received a high-fat animal diet supplemented with 0.1% RS. The study period was 12 weeks. During the study, body weight, fasting blood glucose levels, and food intake were measured weekly. After the study, mice were euthanized by isoflurane (Pfizer) hyperanesthesia and underwent laparotomy. Blood samples were collected, and plasma triglycerides (TG) and total cholesterol (TCHO) were measured using the Wako L-type TG and Wako L-type TCHO assay kits (Fujifilm Wako Pure Chemical Industries, Ltd.), respectively. Liver and abdominal aortic tissues were collected and used for histological and quantitative real-time PCR (qPCR) analysis. The animal study was approved by the Mie University Ethics Committee (approval number: 28-4). Animal experiments were conducted in accordance with the Act on the Welfare and Care of Animals and international guidelines.

[0018] Quantitative Real-Time PCR (qPCR) Total RNA from liver and abdominal aorta tissues was purified using the QIAGEN RNeasy Miniprep Kit (Qiagen). cDNA was synthesized from 500 ng of total RNA using the ReverTra Ace qPCR RT Kit (Toyobo). qPCR was performed using Power SYBR Green Master Mix and the ABI StepOnePlus Real-Time PCR System (both Applied Biosystems). Relative mRNA levels were determined using hypoxanthine guanine phosphoribosyltransferase 1 (Hprt1) as an endogenous control gene. Primer sequences used to determine mRNA expression levels were prepared from the following genes: matrix metallopeptidase 2 (Mmp2), matrix metallopeptidase 9 (Mmp9), sterol regulatory element-binding transcription factor 1 (Srebf1), intercellular adhesion molecule 1 (Icam1), and vascular cell adhesion molecule 1 (Vcam1). The primer sequences used for PCR amplification are shown in Table 1 (SEQ ID NO: 1 to SEQ ID NO: 12).

[0019]

[0020] Immunohistochemistry: Liver and aorta specimens were fixed in 4% phosphate-buffered paraformaldehyde, embedded in a frozen tissue sectioning compound (Sakura Finetech Japan), and cut into 5-μm-thick sections. Sections for immunohistochemical analysis were stained with antibodies as follows. Liver and aorta specimens were incubated with primary antibodies, including rat monoclonal anti-F4 / 80 (macrophage marker) antibody (1:100 dilution, BioRad), rabbit monoclonal anti-alpha smooth muscle actin (αSMA) antibody (1:100 dilution, Aligo BioLaboratories), rabbit monoclonal anti-PDGF receptor β (PDGFRβ) antibody (1:100 dilution, Cell Signaling Technology), rabbit monoclonal anti-VCAM-1 antibody (1:100 dilution, Abcam), and mouse monoclonal anti-Roundabout 4 (Robo4, an endothelial-specific vascular permeability inhibitor) antibody (1:100 dilution, Santa Cruz). Subsequently, sections were incubated with fluorescein- or tetramethylrhodamine isothiocyanate-conjugated anti-rabbit, anti-rat, or anti-mouse secondary antibodies (1:30 dilution, Dako Cytomatin). The expression of F4 / 80, αSMA, PDGFRβ, VCAM-1, and Robo4 was evaluated by immunohistochemistry using a fluorescence microscope. Fluorescence intensity was measured using Image J software version 1.53 (National Institutes of Health, USA).

[0021] <Cell migration assay> Mouse macrophage-like RAW264.7 cells (RCB0535, RIKEN) were used to perform a cell migration assay using a transwell migration assay (Corning). Cell culture was performed in low-glucose Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% BSA (Sigma) and 1% penicillin-streptomycin (Fujifilm Wako Pure Chemical Industries, Ltd.). 1 x 10 5Cells were suspended in serum-free medium and seeded in the upper chamber. 500 μL of whole serum medium was poured into the lower chamber. RS (10 or 100 μg / mL) was added to the upper or lower chamber and incubated for 4 or 24 hours. The medium was then removed from the upper and lower chambers, and the chambers were washed twice with PBS. Cells migrated from the upper chamber to the lower chamber through the membrane filter. Untreated cells were used as a control. Non-migrating cells in the upper chamber were scraped off the top surface of the membrane filter using a cotton swab. Cells that migrated to the underside of the membrane filter were fixed with 4% formaldehyde solution, stained with Hoechst / DAPI (4',6-diamidino-2-phenylindole), and quantified by light microscopy.

[0022] Statistical Analysis: All data are presented as mean ± standard deviation. Multiple comparisons between groups were analyzed by two-way ANOVA followed by Tukey's post-hoc test using GraphPad Prism software version 10 (GraphPad Software). Results of the cell migration assay were analyzed by one-way ANOVA with Dunnett's post-test using Prism software. Statistical significance was set at 5% (p ≤ 0.05).

[0023] <Test Results> <Chemical Composition of RS> After RS ​​was extracted from the human liver, its chemical properties were analyzed. Analysis revealed a single major peak on the GPC column, with weight-average (Mw), number-average (Mn), and Z-average (Mz) molecular weights of 148, 28, and 844 kDa, respectively. RS content was defined as the sum of the constituent monosaccharides, sulfate groups, and cations bound to sulfate groups. Per 100 g of RS, the monosaccharide content was 55 g, sulfate groups 32 g, and cations 6.9 g. This resulted in an RS content of 94 g per 100 g (94% purity). The monosaccharide composition of RS included glucuronic acid, rhamnose, glucose, galactose, and xylose, with the respective contents per 100 g being 3.2, 49, 0.7, <0.4, and 2.3 g. The cation composition of RS consisted of sodium, potassium, magnesium, and calcium, with the contents per 100 g being 0.12, <0.05, 0.54, and 6.2 g, respectively.

[0024] Effect of RS on Lipid Profile in the HFD Group: We investigated the effects of orally administered RS on body weight and lipid metabolism in ApoE-deficient mice. The results are shown in Figure 1. Compared with the ND group, the HFD group showed a significant increase in body weight (p<0.001). Furthermore, when comparing the HFD+RS group with the HFD group, a significant increase in body weight was observed in the RS group (p<0.05). These results indicate that RS did not adversely affect growth in the HFD group. Furthermore, RS did not affect food intake during the feeding experiment. Figure 2 shows the results for plasma TCHO (Figure 2a), Srebp1 mRNA (Figure 2b), and plasma TG (Figure 2c). As shown in Figure 2a, plasma TCHO concentrations were significantly increased in the RS-untreated HFD group compared with the RS-untreated ND group (p<0.001). Furthermore, the RS-treated ND group had significantly lower cholesterol than the RS-untreated ND group (p<0.05), and the RS-treated HFD group had significantly lower cholesterol than the RS-untreated HFD group (p<0.01). This indicates that RS suppressed the increase in cholesterol production in the ND and HFD groups of ApoE-deficient mice.

[0025] To confirm the inhibitory effect of RS on TCHO production in mice, we examined the effect of RS on sterol regulatory element-binding protein 1 (Srebp1) mRNA expression in the aortas of ApoE-deficient mice fed either a normal diet or a high-fat diet (HFD) by qPCR. As shown in Figure 2b, Srebp1 mRNA levels were significantly higher in the aortas of the HFD group compared with the ND group (p < 0.05), and significantly lower in the aortas of the HFD + RS group compared with the HFD group (p < 0.05). These results suggest that RS suppresses Srebp1 mRNA expression in the aorta, which is associated with a decrease in plasma cholesterol levels. Figure 2c shows the results of plasma triglyceride concentrations. Plasma triglyceride concentrations were significantly higher in the HFD group compared with the ND group (p < 0.05). They were also significantly lower in the HFD + RS group compared with the HFD group (p < 0.001). This indicates that RS strongly suppresses the increase in triglyceride production induced by the HFD. These results suggest that RS improved the lipid profile of ApoE-deficient mice fed a HFD.

[0026] Effect of RS on VCAM-1 Expression in the Liver and Aorta of the HFD Group. Figure 3 shows the results of VCAM-1 expression in the liver and aorta. VCAM-1 protein expression was significantly higher in the HFD group than in the ND group (p<0.001), and significantly lower in the HFD+RS group than in the HFD group (p<0.001). Furthermore, as shown in Figure 3d, Vcam-1 mRNA expression was significantly higher in the HFD group than in the ND group (p<0.05), and significantly lower in the HFD+RS group than in the HFD group (p<0.01). These results indicate that RS significantly suppressed Vcam-1 mRNA expression in the aorta of the HFD group. Although not shown, ICAM-1 expression in the liver and aorta was low in both the ND and HGD groups, indicating that it was not affected by RS administration in either tissue. Furthermore, when we examined the expression level of Icam-1 mRNA in the aorta, RS did not affect Icam-1 mRNA expression in the ND or HFD groups.

[0027] Effect of RS on Macrophage Accumulation in the Liver and Aorta of HFD Groups. Macrophages accumulated at inflammatory sites differentiate into tissue-specific cell types, such as aortic foam cells and hepatic Kupffer cells, after ingesting oxidized LDL. Therefore, we investigated whether RS ​​administration inhibited macrophage infiltration into the liver and aorta using an antibody against the macrophage-specific marker, the 160 kDa cell surface glycoprotein F4 / 80. Figure 4 shows the results of F4 / 80 expression in the liver and aorta. F4 / 80 expression in both the liver and aorta was significantly higher in the HFD group compared with the ND group (p<0.001) and significantly lower in the HFD+RS group compared with the HFD group (p<0.001). These results indicate that RS administration inhibited macrophage accumulation and proliferation in both the liver and aorta of ApoE-deficient mice, regardless of whether they were on an ND or HFD.

[0028] Effect of RS on PDGFRβ Expression in the Aorta of the HFD Group Figure 5 shows the effect of RS on PDGFRβ expression in the liver and aorta of ApoE-deficient mice fed either ND or HFD. PDGFRβ expression was lower in the liver of the ND group, but tended to decrease in the aorta of the ND + RS group. PDGFRβ expression in the liver and aorta was significantly higher in the HFD group compared to the ND group (p<0.001). PDGFRβ expression did not decrease in the liver, but was significantly lower in the aorta of the HFD + RS group compared to the HFD group (p<0.001). These results indicate that RS administration suppresses PDGFRβ-mediated atherosclerosis promotion.

[0029] Effect of RS on αSMA Expression in the Liver and Aorta of the HFD Group. Vascular smooth muscle cells express αSMA and contribute to vascular motility and contraction. Figure 6 shows the effects of RS on αSMA expression in the liver and aorta of the ND and HFD groups. The expression levels of αSMA-positive cells were low in the liver and aorta of the ND group, regardless of RS administration. Furthermore, αSMA expression levels were significantly higher in both the liver and aorta of the HFD group (p<0.001), but significantly lower in the HFD+RS group compared to the HFD group (p<0.001). These results suggest that RS administration suppresses vascular smooth muscle cell proliferation in the liver and aorta, potentially ameliorating or preventing the development of atherosclerosis.

[0030] Effect of RS on Robo4 Expression in the Liver and Aorta of the HFD Group Figure 7 shows Robo4 expression in the liver, and Figure 8 shows Robo4 expression in the aorta. As shown in the figures, no significant changes were observed in Robo4 expression in the liver or aorta of the ND group, regardless of oral administration of RS. In contrast, Robo4 expression was reduced by more than half in the liver and was significantly lower in the aorta of the HFD group compared with the ND group (p<0.01). Robo4 expression was also significantly higher in both the liver and aorta of the RS+HFD group compared with the HFD group (p<0.01). These results indicate that Robo4 expression is significantly reduced in the blood vessels of the HFD group, resulting in increased infiltration of monocytes and macrophages into the arterial media. Furthermore, the RS+HFD group demonstrates that the downregulation of Robo4 significantly reduces monocyte / macrophage infiltration into the arterial media due to the suppression of monocyte / macrophage infiltration into the arterial media.

[0031] Effect of RS on Mmp-2 and Mmp-9 mRNA in the Aorta. Figure 9 shows the qPCR analysis results of the effect of RS on Mmp-2 and Mmp-9 mRNA expression in the aorta of ApoE-deficient mice fed either ND or HFD. As shown in Figure 9a, Mmp-2 mRNA expression in the aorta of the HFD group was slightly higher than that in the aorta of the ND group. However, this increase was not affected by RS administration. On the other hand, as shown in Figure 9b, Mmp-9 mRNA expression tended to be higher in the aorta of the HFD group compared with that of the ND group, and this increase was significantly suppressed by RS administration (p<0.05). These results indicate that RS prevents atherosclerosis by suppressing MMP-9 production in ApoE-deficient mice.

[0032] Effect of RS on Migration of Murine Macrophage-Like Cells The effect of RS on macrophage migration was examined by an in vitro cell migration assay using RAW264.7 cells. The results are shown in Figure 10. The results show that the number of migrated cells (i.e., the number of stained cells on the underside of the membrane filter) increased with increasing incubation time of RS-untreated cells from 4 to 24 hours. Migration of cells treated with RS (≥10 μg / mL) was significantly inhibited after 4 hours (p < 0.001), regardless of whether RS ​​was added to the upper or lower chamber. These results suggest that RS directly acts on macrophages to reduce their migration ability, leading to the suppression of atherosclerosis.

[0033] As described above, oral intake of RS suppressed the decrease in Robo4 expression in vascular endothelial cells of atherosclerosis-induced mice, and demonstrated strong control of increased vascular permeability. RS was found to be a novel substance that increases the expression of Robo4, a molecule that inhibits increased vascular permeability (inflammatory damage to the vascular endothelium) that occurs during inflammation in the body. Thus, this embodiment provides a composition for alleviating symptoms caused by increased vascular permeability.

Claims

1. A composition that contains rhamnan sulfate and regulates the expression of Robo4.

2. A composition as described in claim 1 for relieving symptoms caused by increased vascular permeability.

3. A composition for relieving symptoms caused by increased vascular permeability as described in claim 2, wherein the composition is for oral administration.

4. A food or beverage for relieving symptoms caused by increased vascular permeability, comprising the composition described in claim 3.

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