Application of reuterin in preparation of medicine for relieving or treating atherosclerosis

Abstract

The invention discloses application of intestinal flora metabolite reuterin in preparation of a medicine for relieving or treating atherosclerosis, and relates to the technical field of medicine. The reuterin can reduce the blood sugar level, the total cholesterol level and the low-density lipoprotein cholesterol level, reduce liver lipid deposition, improve liver fatty degeneration, promote growth of beneficial bacteria, inhibit proliferation of potential harmful bacteria and inhibit cell penetration and uptake of low-density lipoprotein in endothelial cells. According to the application, the fact that the reuterin can be used for relieving or treating the atherosclerosis is disclosed for the first time, a new direction is provided for treating the atherosclerosis, and experiments show that the reuterin does not cause adverse effects on liver and kidney functions of mice after being used for a long time, and shows that the reuterin has good biological safety.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of reuterin in the preparation of a drug for alleviating or treating atherosclerosis. Background Art

[0002] Atherosclerosis is a chronic disease affecting the arteries, characterized by lesions that begin in the intima of the affected arteries, typically manifesting as the accumulation of cholesterol-rich plaques on the inner walls of the arteries. Over time, this can cause the arteries to narrow, harden, or become blocked, leading to reduced blood flow to vital organs and tissues, thereby increasing the risk of heart attack, stroke, and peripheral cardiovascular disease. Globally, cardiovascular disease based on atherosclerotic lesions is the leading cause of death. The retention of atherogenic lipoproteins in the subendothelial layer of arteries is a key initial step in the development and progression of atherosclerosis. The development and progression of atherosclerosis is closely related to disorders in the body's metabolic system. Identifying endogenous small molecule metabolites associated with the disease may provide new directions for the discovery of safe and effective therapeutic drugs for atherosclerosis. These endogenous molecules have significant clinical application potential due to their inherent biocompatibility and safety.

[0003] Disturbances in the gut microbiome and its derived metabolites can influence the development of atherosclerosis. Resident microorganisms may influence host physiology in various ways. Dietary components can alter the composition of the gut microbiome or be processed into metabolites that can delay or accelerate the development of atherosclerosis. Lactobacillus reuteri (L. reuteri) is a widely studied lactic acid-producing probiotic with known cholesterol-lowering and anti-inflammatory properties, and has been shown to significantly reduce cardiac injury after ischemia-reperfusion events. Prophylactic supplementation with L. reuteri in individuals with elevated cardiovascular risk factors is a feasible approach to reduce heart disease risk. L. reuteri modulates the gut microbiome through multiple metabolic mechanisms, improving gut microbial profiles and counteracting the colonization of harmful bacteria, exerting beneficial effects on health. Apolipoprotein E knockout mice fed a high-fat diet have lower lactic acid bacteria abundance than those fed a control diet. L. reuteri produces a non-protein, broad-spectrum antimicrobial substance called reuterin during glycerol metabolism. Reuterin plays a crucial role in altering redox homeostasis. However, few studies have explored the role of reuterin in the cardiovascular field.

[0004] As a classic and clinically used probiotic, L. reuteri's primary metabolite, reuterin, in the human body, is worthy of investigation. Whether and how its metabolite, reuterin, affects the progression of cardiovascular disease is a topic of considerable interest. While statins are well-established for their ability to treat atherosclerosis, improved living standards and unhealthy dietary habits are disrupting the intestinal microbiome, a major endocrine organ. Disruption of microbial homeostasis poses a challenge to intestinal barrier function, and as harmful substances enter the circulatory system, the harm they pose to human health cannot be ignored.

[0005] Therefore, determining the role of reuterin in the development of atherosclerosis is an urgent problem to be solved in the present invention. Summary of the Invention

[0006] The present invention provides a use of reuterin, a metabolite of intestinal flora, in the preparation of a drug for alleviating or treating atherosclerosis, aiming to solve the problems existing in the above-mentioned background technology.

[0007] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:

[0008] The invention discloses application of reuterin in preparing medicine for alleviating or treating atherosclerosis.

[0009] The invention also discloses the use of reuterin in preparing medicines for alleviating or treating the reduction of blood sugar levels, and reducing total cholesterol and low-density lipoprotein cholesterol levels.

[0010] The invention also discloses the use of reuterin in preparing a medicine for reducing liver lipid deposition and improving liver fatty degeneration.

[0011] The invention also discloses the use of reuterin in preparing a medicine for promoting the growth of beneficial bacteria and inhibiting the proliferation of potential harmful bacteria.

[0012] Furthermore, the beneficial bacteria are Lactobacillus and Akkermansia, and the potentially harmful bacteria are Lachnoclostridium and Staphylococcus.

[0013] The invention also discloses the use of reuterin in preparing medicine for inhibiting the permeation and uptake of low-density lipoprotein in endothelial cells.

[0014] Furthermore, the reuterin specifically downregulates the expression of caveolin-1 to inhibit the transcytosis of low-density lipoprotein, thereby reducing the deposition of lipids in endothelial cells.

[0015] The present invention also discloses a medicine for alleviating or treating atherosclerosis, characterized in that the active ingredient includes reuterin.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention discloses for the first time that reuterin can be used to alleviate or treat atherosclerosis, providing a new direction for the treatment of atherosclerosis. Experiments show that reuterin has no adverse effects on the liver and kidney functions of mice during long-term use, indicating that it has good biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A flowchart of the animal experiment on the effect of reuterin on atherosclerosis provided by the present invention;

[0019] Figure 2 ApoE after reuteri therapy - / - Figure 3. Changes in aortic plaques in mice; (A) Microscopic photographs of the aortic arches of mice in the control (PBS) and reuteriin (Reu) groups, with arrows pointing to atherosclerotic plaques located in the brachiocephalic artery, left subclavian artery, and lesser curvature of the aortic arch; (B) Representative images of the gross Oil Red O staining results of the aortas of mice in the PBS and Reu groups and statistical diagrams of the quantitative evaluation of the percentage of plaque areas (n=11), **P<0.01; t-test.

[0020] Figure 3 ApoE after reuteri therapy - / - Figure 4: Changes in mouse aortic valve plaques; (A) Representative images of Oil Red O staining of aortic valves in mice in the PBS and Reu groups; (B) Quantitative evaluation of plaque area percentage (n = 11); (C) HE staining of a transverse section of the mouse aorta. Scale bar: 500 μm (A), 200 μm (C). **P < 0.01; t-test.

[0021] Figure 4 ApoE in the reuteri intervention group - / - Mouse weight change curve;

[0022] Figure 5 ApoE after reuteri therapy - / - Changes in circulating reuterin, blood glucose, and blood lipid levels in mice;

[0023] Figure 6 Effect of reuterin on Western diet-induced ApoE - / - Figure 1: Effects of fatty degeneration on mouse livers. (A) Representative images of HE staining of liver sections of mice in the PBS and Reu groups; (B) Representative images of Oil Red O staining of liver sections of mice in the PBS and Reu groups. Scale bar: 100 μm.

[0024] Figure 7Figures show the results of the biosafety assessment of reuterin in mice; (A) HE staining results of the heart, liver, spleen, lung, and kidney tissues of mice in the Reu and PBS groups; (B) Quantitative analysis of liver and kidney function indicators of mice in the Reu and PBS groups (n=3); ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CR, creatinine. Scale bar 200 μm (heart, liver, spleen, lung); scale bar 100 μm (kidney).

[0025] Figure 8 Figure 2 is a graph showing the changes in intestinal microbial diversity of mice after treatment with reuteri. (A) PCoA results of intestinal microbial communities of mice in the Reu and PBS groups (n=10); (B-C) Quantitative analysis results of Shannon and Chao1 indices of α-diversity of intestinal microbial communities of mice in the Reu and PBS groups (n=10);

[0026] Figure 9 Figure 2 shows the results of LEfSe analysis of the intestinal microbiota of mice in the reuteri-treated group and the PBS control group. The vertical axis represents the microorganisms with significant differences between the groups, and the horizontal axis represents the logarithmic score of the LDA analysis of the microorganisms. The microorganisms are sorted according to the size of the score. The longer the bar, the more significant the difference between the groups of the microorganism. The color of the bar indicates the sample group with higher abundance of the corresponding microorganism.

[0027] Figure 10 The activity of reuterin at different concentrations and treatment times on HUVECs; (A) Cytotoxicity of HUVECs at different reuterin treatment concentrations (n=4); (B) Cytotoxicity of HUVECs at different treatment times at a specific reuterin concentration (n=4); *P<0.05; t test (A); two-way ANOVA test (B);

[0028] Figure 11 Flowchart for establishing an in vitro LDL translocation model; (A) Preparation of FITC-labeled LDL (FITC-LDL); (B) In vitro LDL translocation assessment process;

[0029] Figure 12 Figure 3. Reuteri intervention inhibits LDL transcytosis in a dose-dependent manner in vitro. In the presence of different concentrations of reuteri, 50 μg / mL FITC-LDL was added to the upper transcytosis chamber and transcytosis was continued for 24 hours. The fluorescence intensity in the lower chamber was measured to reflect the transcytosis amount (n=3). *P<0.05, **P<0.01, ***P<0.001; one-way ANOVA test.

[0030] Figure 13Figures show the results of laser confocal imaging of the inhibition of LDL uptake by reuteri. (A) Representative images of DiI-LDL uptake by HUVECs in the control and Reu groups; (B) Quantitative statistical analysis of DiI-LDL uptake by HUVECs in the control and Reu groups (n=3); Scale bar: 20 μm. **P<0.01; t-test.

[0031] Figure 14 Figures show the flow cytometry results of the inhibition of LDL uptake by reuteri. (A) Representative results of DiI-LDL uptake by HUVECs in the control group and the Reu group; (B) Quantitative statistical results of DiI-LDL uptake by HUVECs in the control group and the Reu group (n=3); *P<0.05; t-test;

[0032] Figure 15 Figure 2: Changes in expression levels of key genes during LDL transcytosis after intervention with different reuteri concentrations; (A) Changes in mRNA levels of each molecule (n=3); (B-C) Changes in SR-B1 and CAVIN1 protein levels (n=3); (D) Representative Western blot bands; (E) Statistical chart of changes in CAV1 protein levels (n=4); **P<0.01, ****P<0.0001; one-way ANOVA test;

[0033] Figure 16 The immunohistochemical results of CAV1 protein expression in mouse aorta after 12 weeks of reuteri therapy; scale bar 100 μm (left); scale bar 50 μm (right);

[0034] Figure 17 Figure 2 shows the CAV1 protein levels and quantitative analysis results at different time points after reuteri therapy; (A) Representative Western blot bands; (B) Statistical chart of CAV1 protein level changes (n=3); *P<0.05, **P<0.01; one-way ANOVA test;

[0035] Figure 18 Figure 2 shows that reuteri therapy reversed the increase in CAV1 expression caused by CAV1 overexpression; (A) Representative Western blot bands; (B) Statistical graph of CAV1 protein expression results (n=4); **P<0.01, ****P<0.0001; one-way ANOVA test;

[0036] Figure 19Figure 1 shows that reuteri therapy reversed the increased LDL transcytosis and uptake caused by CAV1 overexpression. (A-B) Representative images and quantitative analysis of confocal laser imaging (n = 3); (C-D) Representative images and quantitative analysis of flow cytometry (n = 3); (E) Quantitative analysis of LDL transcytosis (n = 3); Scale bar 20 μm. *P < 0.05, **P < 0.01, ****P < 0.0001; one-way ANOVA test. DETAILED DESCRIPTION

[0037] The following examples further illustrate the present invention but are not to be construed as limiting the present invention. Modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are intended to fall within the scope of the present invention. Unless otherwise specified, the techniques used in the examples are conventional means well known to those skilled in the art.

[0038] Example 1 Reuterin delays ApoE - / - Atherosclerosis progression in mice

[0039] To further explore the direct effects of reuterin on the progression of atherosclerosis and its potential mechanism, the present invention used a widely recognized animal model of atherosclerosis - male ApoE fed a Western diet. - / - Mice. This model is widely used in atherosclerosis research because it is prone to the formation of atherosclerotic plaques. - / - After one week of adaptive feeding with a normal diet, the mice were randomly assigned to a Western diet-fed (PBS) group or a Western diet-fed plus reuteri gavage (Reu) group, which received 50 mg / kg of reuteri or an equal amount of PBS daily by gavage as a control, and continued to be fed for 12 weeks.

[0040] The experimental design and process are detailed in Figure 1 The results showed that reuteriin had a significant inhibitory effect on the progression of atherosclerosis. Specifically, compared with the PBS-treated group, the aorta of mice in the reuteriin-treated group was ( Figure 2 ) and aortic valve ( Figure 3 The area of atherosclerotic plaques in the AB group was significantly reduced. This result directly confirmed the positive effect of reuterin in inhibiting the formation of atherosclerotic plaques. Further histological analysis (HE staining) revealed that the lipid deposition in the plaque area of mice treated with reuterin was significantly reduced ( Figure 3 C), and weight gain was also slower than that of the control group ( Figure 4 These findings suggest that reuterin may exert its anti-atherosclerotic effects by regulating lipid metabolism and energy balance.

[0041] In terms of pharmacokinetics, after 12 weeks of oral administration of reuterin, the level of reuterin in the mouse circulation increased significantly ( Figure 5 A). In addition, reuterin treatment significantly reduced the blood glucose levels in mice ( Figure 5 B) and improved lipid profiles, as evidenced by significant decreases in total cholesterol and low-density lipoprotein cholesterol levels ( Figure 5 C). However, reuterin had no significant effect on triglyceride and high-density lipoprotein cholesterol levels. These results suggest that reuterin may exert its anti-atherosclerotic effects by regulating blood glucose and lipid metabolism, particularly by lowering total cholesterol and low-density lipoprotein cholesterol levels.

[0042] Example 2 Reuterin reduces ApoE - / - Hepatic steatosis in mice

[0043] Hepatic steatosis has been widely shown to be closely associated with poor prognosis in cardiovascular disease. In this study, mice fed a Western diet showed significant microvesicular and macrovesicular steatosis in liver sections, characterized by the abnormal accumulation of large amounts of lipids within hepatocytes. However, compared with the control group, the size and number of lipid droplets in the livers of mice treated with reuteri were significantly reduced ( Figure 6 A), indicating that reuteri has a significant inhibitory effect on Western diet-induced hepatic steatosis. This result was further confirmed by Oil Red O staining, which showed that the hepatic lipid deposition in the reuteri treatment group was significantly reduced ( Figure 6 B). The above results suggest that the application of reuteri may have a positive impact on metabolic health by improving hepatic steatosis.

[0044] Example 3 Biosafety Assessment of Reuterin in Mice

[0045] In order to evaluate the safety of long-term supplementation of reuterin, the present invention used male C57BL / 6 mice fed a normal diet as a model and administered reuterin (50 mg / kg / day) or control (PBS) by gavage for 12 weeks. After the experiment, histopathological examinations were performed on the main organs of the mice, such as the heart, liver, spleen, lungs, and kidneys, and biochemical indicators related to liver and kidney function were tested to comprehensively evaluate the biosafety of reuterin. HE staining results showed that long-term gavage treatment with reuterin did not cause obvious pathological changes in the heart, liver, spleen, lungs, kidneys and other organs of the mice ( Figure 7 A). In addition, there were no significant differences in biochemical indicators related to liver and kidney function (such as ALT, AST, BUN, CREA, etc.) between the reuteri-treated group and the control group ( Figure 7B), further indicating that long-term use of reuterin did not cause adverse effects on the liver and kidney functions of mice.

[0046] Example 4: Reuterin improves the composition of the intestinal microbiota in ApoE- / - mice

[0047] Principal coordinate analysis (PCoA) revealed that supplementation with reuteri significantly improved the overall structure of the intestinal microbiota in mice ( Figure 8 A). However, the results of α-diversity analysis (Shannon index and Chao1 index) showed that there was no significant difference in microbial diversity between the PBS control group and the reuteriin-treated group ( Figure 8 BC). Further linear discriminant analysis effect size (LEfSe) analysis revealed significant differences in the abundance of specific bacterial groups between the two groups. Specifically, the abundance of Lachnoclostridium and Staphylococcus was higher in the PBS control group, while the abundance of Lactobacillus and Akkermansia was significantly increased in the reuteri intervention group ( Figure 9 These results suggest that reuteri can selectively promote the growth of beneficial bacteria while inhibiting the proliferation of potentially harmful bacteria, thereby improving the composition and function of the intestinal microbiota and exerting a potential anti-AS effect.

[0048] Example 5 Reuterin inhibits LDL transcytosis and uptake in HUVECs

[0049] (1) Effect of reuterin on HUVECs cell activity

[0050] The results showed that reuterin below 200 μM did not show significant cytotoxicity to HUVECs ( Figure 10 A), indicating that reuterin has good biocompatibility at lower concentrations. To further evaluate the safety of reuterin on HUVECs, this study tested the cytotoxicity of 25μM, 50μM, and 100μM reuterin, which are commonly used in the literature, at different time points. The results showed that within 24 hours, reuterin treatment had no significant effect on the cell viability of HUVECs ( Figure 10 B), further confirming its safety in a short period of time. Considering that the effect of reuterin in AS may rely on sustained stimulation at low concentrations in vivo, in vitro cell experiments are difficult to fully simulate such long-term, low-concentration physiological conditions. Therefore, the present invention uses a relatively high reuterin concentration (25-100 μM) and a short intervention time (24 hours) to simulate possible physiological effects in vivo.

[0051] (2) Reuterin inhibits LDL transcellular uptake and uptake in HUVECs

[0052] One of the initial steps of AS is the infiltration of excessive circulating LDL particles into the arterial endothelium and accumulation under the endothelium, a process that is considered to be a key early event in plaque formation. In order to evaluate the effect of reuterin on LDL transendothelial transport, the present invention established an in vitro LDL transcellular model ( Figure 11 The experimental results showed that reuterin significantly inhibited the transcellular process of LDL in HUVECs in a dose-dependent manner ( Figure 12 ), suggesting that reuterin may delay the progression of AS by reducing LDL transport in endothelial cells.

[0053] Endothelial cell uptake of LDL is not only an intermediate step in LDL transport, but also a key link in the formation of AS. To further study the effect of reuteri on the uptake of LDL by endothelial cells, the present invention used DiI-labeled LDL (DiI-LDL) for experiments. After DiI-LDL was taken up by HUVECs, small and dispersed red fluorescent spots could be observed under a fluorescence microscope. The results showed that reuteri intervention significantly reduced the uptake of LDL by HUVECs, as shown by a significant decrease in the red fluorescence intensity in the cells of the reuteri treatment group ( Figure 13 This result indicates that reuterin can effectively inhibit the uptake of LDL by endothelial cells, thereby possibly reducing the accumulation of LDL under the endothelium. In addition, consistent with the results of fluorescence microscopy observations, the results of flow cytometry further confirmed the inhibitory effect of reuterin on the uptake of LDL by endothelial cells ( Figure 14 ).

[0054] Example 6 CAV1 is a key molecule in the inhibition of LDL transcytosis by reuterin

[0055] To further explore the molecular mechanism by which reuterin inhibits LDL transcytosis, the present invention systematically detected the expression levels of key molecules closely related to the LDL transcytosis process to evaluate whether reuterin exerts its inhibitory effect by regulating these molecules. The experimental results showed that reuterin treatment did not significantly change the expression levels of SR-B1 and CAVIN1. However, reuterin significantly reduced the mRNA and protein expression levels of CAV1, a key gene that promotes LDL transcytosis ( Figure 15 This finding suggests that reuterin may inhibit the transcytosis of LDL by specifically downregulating the expression of CAV1, thereby reducing lipid deposition in endothelial cells.

[0056] To further verify this result, the present invention used immunohistochemical staining technology to detect ApoE - / -The expression of CAV1 protein in the mouse aortic endothelium was analyzed. The results showed that after 12 weeks of supplementation with reuteri, the expression of CAV1 protein in the mouse aortic endothelium was significantly decreased ( Figure 16 ), which is consistent with the in vitro experimental data, indicating that reuteri can also effectively inhibit the expression of CAV1 in vivo. In addition, to explore whether reuteri has a rapid effect on the regulation of CAV1 expression, this study conducted a time gradient experiment in HUVECs cultured in vitro. The results showed that after only 12 hours of reuteri intervention in HUVECs, the level of CAV1 protein was significantly reduced ( Figure 17 ), further confirming the rapid inhibitory effect of reuterin on CAV1 expression.

[0057] To further clarify the key role of CAV1 in the inhibition of LDL transcytosis mediated by reuterin, the present invention conducted a functional verification experiment by overexpressing CAV1. The experimental results showed that CAV1 overexpression significantly increased the protein level of CAV1 in HUVECs ( Figure 18 ), and further promoted the uptake of LDL ( Figure 19 AD) and transcytosis ( Figure 19 E). This result is consistent with the known function of CAV1 in LDL transport, confirming CAV1's central role in regulating LDL endocytosis and transcytosis. However, treatment with reuterin significantly reversed the elevated CAV1 protein levels and its promotion of LDL uptake and transcytosis caused by CAV1 overexpression. This result suggests that reuterin, by specifically downregulating CAV1 expression, effectively blocks the promotion of LDL transport by CAV1 overexpression, thereby exerting its inhibitory effect on LDL transcytosis.

[0058] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Application of reuterin in the preparation of drugs for alleviating or treating atherosclerosis.

2. The use of reuterin in the preparation of drugs for lowering blood sugar levels, total cholesterol and low-density lipoprotein cholesterol levels.

3. The application of reuterin in the preparation of drugs for reducing liver lipid deposition and improving liver fatty degeneration.

4. The application of reuterin in the preparation of drugs that promote the growth of beneficial bacteria and inhibit the proliferation of potentially harmful bacteria.

5. The use according to claim 4, characterized in that The beneficial bacteria are Lactobacillus and Akkermansia, and the potentially harmful bacteria are Lachnoclostridium and Staphylococcus.

6. Use of reuterin in the preparation of drugs that inhibit the transcellular uptake and translocation of low-density lipoprotein in endothelial cells.

7. The use according to claim 6, characterized in that The reuterin specifically downregulates the expression of caveolin-1 to inhibit the transcytosis of low-density lipoprotein, thereby reducing the deposition of lipids in endothelial cells.

8. A drug for alleviating or treating atherosclerosis, characterized in that: The active ingredient includes reuterin.

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