Use of a bifidobacterium and extracellular vesicles thereof in the preparation of a preparation against diseases caused by a high-fat diet

Abstract

The application belongs to the technical field of Bifidobacterium transformation, and particularly relates to application of Bifidobacterium and extracellular vesicles thereof in preparation of a preparation for resisting diseases caused by high-fat diet. The Bifidobacterium is isolated from rabbit feces, and is Bifido mag and extracellular vesicles secreted by the Bifido mag. Intervention is performed on ApoE knockout mice fed with high-fat and New Zealand rabbits fed with high-fat and subjected to balloon injury, and it is observed that the Bifido mag and the extracellular vesicles thereof can significantly inhibit hyperlipidemia, formation of atherosclerotic plaques and pathological lipid deposition in the liver. It is found for the first time that the Bifido mag and the extracellular vesicles thereof can significantly improve lipid metabolism disorder caused by high-fat diet, reduce the area of atherosclerotic plaques and pathological lipid deposition in the liver, and have considerable application and industrialization prospects in the field of prevention and treatment of diseases caused by high-fat diet.

Description

Technical Field

[0001] This invention relates to the field of Bifidobacterium transformation and application technology, specifically to the application of Bifidobacterium and its extracellular vesicles in the preparation of formulations to combat diseases caused by a high-fat diet. Background Technology

[0002] With the advent of the industrial age, human lifestyles have undergone drastic changes, primarily reflected in increased dietary intake of high-fat, high-calorie foods. Lipid metabolism disorders caused by a high-fat diet can lead to a series of diseases, such as atherosclerosis, hypertension, fatty liver, obesity, diabetes, malignant tumors (colon cancer, rectal cancer, etc.), and osteoporosis, with atherosclerosis being one of the most prevalent. Atherosclerotic lesions often affect large and medium-sized muscular arteries; once they progress to the point of obstructing the arterial lumen, the tissues or organs supplied by that artery will experience ischemia or necrosis. Therefore, preventing and treating diseases such as atherosclerosis caused by excessive high-fat diets is urgent and necessary. However, current medications for preventing and treating atherosclerosis all have certain side effects. For example, statins, which lower blood lipids, can affect male sexual function; and antiplatelet and anticoagulant drugs such as aspirin and warfarin can cause coagulation disorders, impacting long-term drug use and patient confidence in treatment. Therefore, providing a safe, efficient, and long-term usable food or medicine to prevent and treat lipid metabolism disorders such as atherosclerosis will contribute to people's health and economic and social development.

[0003] Bifidobacteria are an important component of the gut microbiota in humans and animals, and some strains of Bifidobacteria can be used as probiotics in food and medicine. Bifidobacterium magnum (Bifidomag) is a Bifidobacterium isolated from rabbit feces. Since its discovery in 2001, its function has been poorly understood. Our previous research found a positive correlation between the amount of Bifidobacterium magnum in rabbit feces and its blood lipid concentration and the severity of atherosclerosis, suggesting that Bifidobacterium magnum may be a substance that lowers blood lipids and improves atherosclerosis.

[0004] Extracellular vesicles are a widely studied and important medium for biological communication. Studies have reported that gut microbiota can interact with organisms and generate corresponding biological functions through extracellular vesicles. Currently, there are no reports of directly using Bifidobacterium macrocarpa extracellular vesicles to prevent or treat diseases caused by excessive intake of high-fat diets. Summary of the Invention

[0005] In view of the above-mentioned shortcomings, this invention provides the application of Bifidobacterium and its extracellular vesicles in the preparation of formulations to combat diseases caused by a high-fat diet. This invention isolates Bifidobacterium macrocarpa (Bifidobacterium magnum, Bifido mag) and its secreted extracellular vesicles from rabbit feces. Through intervention in ApoE knockout mice fed a high-fat diet and New Zealand rabbits with sac-injured bacteria fed a high-fat diet, it was observed that both Bifidobacterium macrocarpa and its extracellular vesicles can significantly inhibit hyperlipidemia, the formation of atherosclerotic plaques, and pathological lipid deposition in the liver. This invention is the first to discover that both Bifidobacterium macrocarpa and its extracellular vesicles can significantly improve lipid metabolism disorders caused by a high-fat diet, reduce the area of ​​atherosclerotic plaques and pathological lipid deposition in the liver, showing promising application and industrialization prospects in the prevention and treatment of diseases related to a high-fat diet.

[0006] To achieve the above objectives, the present invention provides the application of Bifidobacterium and its extracellular vesicles in the preparation of a formulation to combat diseases caused by a high-fat diet, wherein the Bifidobacterium is Bifidobacterium macrocarpa.

[0007] According to one aspect of the invention, the diseases caused by the high-fat diet include: atherosclerosis, hypertension, fatty liver, obesity, diabetes, malignant tumors, and osteoporosis.

[0008] Based on the same inventive concept, this invention also discloses a formulation for combating diseases caused by a high-fat diet, the formulation comprising any one or a combination of Bifidobacterium and Bifidobacterium extracellular vesicles.

[0009] According to one aspect of the invention, the formulation comprises at least one pharmaceutically, foodologically, or health-product-acceptable carrier.

[0010] According to one aspect of the invention, the carrier is one or both of excipients and adhesives.

[0011] According to one aspect of the invention, the dosage form of the preparation is any one of an injection, an oral liquid, a capsule, a tablet, a spray, or an ointment.

[0012] The beneficial effects of this invention are:

[0013] This invention provides the application of *Bifidobacterium macrocarpa* and its extracellular vesicles in the preparation of drugs, foods, or health products for the prevention and treatment of hyperlipidemia, atherosclerosis, fatty liver, and other lipid-induced diseases. By orally feeding *Bifidobacterium macrocarpa* and its extracellular vesicles, it is possible to reduce blood lipids, decrease the area of ​​atherosclerotic plaques, and alleviate pathological lipid deposition in the liver, thereby achieving the purpose of preventing and treating hyperlipidemia, atherosclerosis, and fatty liver, and providing a new strategy for the safe and effective prevention and treatment of hyperlipidemia, atherosclerosis, and fatty liver. Attached Figure Description

[0014] Figure 1 Transmission electron microscopy image of extracellular vesicle exosomes of *Bifidobacterium macrocarpa* according to an embodiment of this application;

[0015] Figure 2 The results of the detection of blood lipid levels in each group of mice in Example 1 of this application;

[0016] Figure 3 A is the result of Oil Red O staining of the gross aorta of mice in each group of mice after three months of intervention in Example 1 of this application; Figure 3 B is a comparison chart of the gross Oil Red O staining results of the aorta of mice in each group of mice after three months of intervention in Example 1 of this application;

[0017] Figure 4 This is a comparison of HE staining results of the livers of mice in different groups after three months of intervention in Example 1 of this application;

[0018] Figure 5 The results of the detection of blood lipid levels in each group of New Zealand rabbits in Example 2 of this application;

[0019] Figure 6 A shows the results of HE staining, Oil Red O staining, Masson staining, α-SMA staining, and RAM11 staining of the vascular cross sections of the New Zealand rabbits in Example 2 of this application after three months of intervention in Example 2. Figure 6 B is a comparative diagram of the results of HE staining, Oil Red O staining, Masson staining, α-SMA staining and RAM11 staining of the vascular cross sections of the New Zealand rabbits in Example 2 of this application after three months of intervention in Example 2.

[0020] Figure 7 This is a comparison of the liver HE staining results of the New Zealand rabbits in each group in Example 2 of this application after three months of intervention. Detailed Implementation

[0021] To make the present invention easier to understand, specific embodiments are described below to further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art; unless otherwise specified, the raw materials and reagents involved herein can be purchased commercially or obtained by known methods.

[0022] The Bifidobacterium macrocarpa strain used in this application is DSM 20222.

[0023] The preparation process of the extracellular vesicles of Bifidobacterium macrocarpa used in this application is as follows:

[0024] After being activated by subculture of Bifidobacterium macrocarpa, it was added to the culture medium and cultured under anaerobic conditions at 37°C with shaking at 60 rpm for 4 days. After centrifugation, the supernatant was collected and filtered. The filtrate was concentrated and extracted with exosome extraction reagent. After removing the supernatant, the precipitate was the outer membrane vesicle.

[0025] It should be noted that the culture medium is Bifidobacterium culture medium; the centrifugation conditions are 12000×g, 4℃ for 15 minutes; the filtration conditions are: filtering the supernatant through a filter with a pore size of 0.22μm; the concentration conditions are: concentrating the filtrate 300-500 times using an ultrafiltration tube with a pore size of 100KD; the exosome extraction reagent is ExoQuick-TC exosome extraction reagent, and the amount used is 1 / 5 of the concentrated solution; the extraction conditions are: reacting at 4℃ for 12 hours, centrifuging at 1500×g, 4℃ for 30 minutes, and taking the precipitate as the outer membrane vesicle.

[0026] The extracellular vesicle exosomes of the above-prepared *Bifidobacterium macrocarpa* were analyzed by transmission electron microscopy, and the transmission electron microscopy results are as follows: Figure 1 As shown.

[0027] This application conducted animal experiments on the aforementioned Bifidobacterium macrocarpa and its extracellular vesicles (see Examples 1 and 2). Through intervention on ApoE knockout mice fed a high-fat diet and New Zealand rabbits with sac-injured livers fed a high-fat diet, it was observed that Bifidobacterium macrocarpa and its extracellular vesicles could significantly inhibit hyperlipidemia, the formation of atherosclerotic plaques, and pathological lipid deposition in the liver.

[0028] It should be noted that this application Figure 2-7 In the figure, “*”, “**” and “***” represent P<0.05, P<0.01 and P<0.001 respectively.

[0029] Example 1

[0030] Bifidobacterium macrocarpa and its extracellular vesicles can prevent and treat hyperlipidemia, atherosclerotic plaque formation, and fatty liver in ApoE knockout mice fed a high-fat diet.

[0031] Mouse model: Six-week-old ApoE knockout male SPF-grade mice, weighing 18–20 g, were purchased from Cavens Laboratory Animal Co., Ltd., Changzhou, Jiangsu Province. Each group consisted of 7–10 mice, with free access to food and water, and a circadian rhythm was maintained.

[0032] Grouping experiment: ApoE knockout male SPF mice were randomly divided into 4 groups, including normal diet + solvent control group, high-fat diet + solvent control group, high-fat diet + Bifidobacterium macrocarpa group, and high-fat diet + Bifidobacterium macrocarpa extracellular vesicle group.

[0033] Modeling: Mice in each group were fed different diets while undergoing intervention.

[0034] Administration method:

[0035] Normal diet + solvent control group [Normal diet, ND(M)]: Mice were fed a normal diet and were given phosphate buffer by gavage at a volume of 200 μL once a week. Samples were collected after three months of intervention.

[0036] High fat diet + solvent control group [High fat diet, HFD(M)]: Mice were fed a high fat diet and were simultaneously gavaged with phosphate buffer solution at a volume of 200 μL once a week. Samples were collected after three months of intervention.

[0037] High-fat diet + Bifidobacterium macrocarpa group [Bifido mag + HFD(M)]: Mice were fed a high-fat diet and simultaneously administered Bifidobacterium macrocarpa (10) 8 ~10 9 / time) Gavage, with a volume of 200μL / time, once a week, and samples were collected after three months of intervention.

[0038] High-fat diet + Bifidobacterium macrocarpa extracellular vesicle group [Bifido mag OMV+HFD(M)]: Mice were fed a high-fat diet and were simultaneously gavaged with Bifidobacterium macrocarpa extracellular vesicles (0.5-1.5 ug / μL) at a volume of 200 μL once a week. Samples were collected after three months of intervention.

[0039] Three months after the above intervention, samples were collected from each group of mice, and their blood lipid levels were analyzed. The results are as follows: Figure 2 As shown; the results of Oil Red O staining of the aorta of mice in each group were compared after three months of the above intervention. Figure 3As shown; the HE staining results of the livers of mice in each group were compared after three months of the above intervention, and the results are as follows. Figure 4 As shown.

[0040] Example 2

[0041] Bifidobacterium macrocarpa and its extracellular vesicles can prevent and treat hyperlipidemia, atherosclerotic plaque formation, and fatty liver in New Zealand rabbits fed a high-fat diet.

[0042] Rabbit model: Ten-week-old New Zealand white rabbits, weighing 2.5–3.0 kg, were selected and purchased from Hunan Slack Jingda Animal Experiment Co., Ltd. Each group consisted of 5–7 New Zealand white rabbits, with free access to food and water, and their diurnal rhythm was maintained.

[0043] Grouping experiment: New Zealand white rabbits were randomly divided into 3 groups, including normal diet + solvent control group, high-fat diet + solvent control group, high-fat diet + Bifidobacterium macrocarpa group, and high-fat diet + Bifidobacterium macrocarpa extracellular vesicle group; each group consisted of 5 to 7 rabbits.

[0044] Modeling: After feeding the animals with normal or high-fat diets for two weeks, sac damage was performed on each group. After sac damage was completed, each group was treated with phosphate buffer or Bifidobacterium macrocarpa and its extracellular vesicle solution.

[0045] Administration method:

[0046] Normal feed + solvent control group [ND(R)]: After balloon injury, New Zealand rabbits were fed a normal feed and were given phosphate buffer by gavage at a volume of 5 mL once a week. Samples were collected after three months of intervention.

[0047] High-fat diet + solvent control group [HFD(R)]: After balloon injury, New Zealand rabbits were fed a high-fat diet and were given phosphate buffer by gavage at a volume of 5 mL once a week. Samples were collected after three months of intervention.

[0048] High-fat diet + Bifidobacterium macrocarpa group [Bifido mag + HFD(R)]: New Zealand rabbits were fed a high-fat diet after sac injury, and were given Bifidobacterium macrocarpa (10) 9 ~10 10 / time) Gavage, with a volume of 5mL / time, once a week, and samples were collected after three months of intervention.

[0049] High-fat diet + Bifidobacterium macrocarpa extracellular vesicle group [Bifido mag OMV+HFD(R)]: After sac injury, New Zealand rabbits were fed a high-fat diet and were given Bifidobacterium macrocarpa extracellular vesicles (5 mg / time) by gavage once a week. Samples were collected after three months of intervention.

[0050] The blood lipid levels of New Zealand white rabbits collected three months after the above intervention were analyzed, and the results are as follows: Figure 5 As shown; the results of HE staining, Oil Red O staining, Masson staining, α-SMA staining, and RAM11 staining of vascular sections in each group of New Zealand white rabbit atherosclerosis models collected three months after the above intervention were compared, and the results are as follows. Figure 6 As shown; the HE staining results of livers from New Zealand white rabbits collected three months after the above intervention were compared, and the results are as follows. Figure 7 As shown.

[0051] Results analysis:

[0052] Depend on Figure 2-7 As shown, this invention demonstrates through animal experiments that *Bifidobacterium macrocarpa* and its extracellular vesicles are highly effective in improving hyperlipidemia, fatty liver, and atherosclerosis in apolipoprotein E (ApoE) knockout mice fed a high-fat diet and New Zealand white rabbits with balloon-induced aortic injury. In the ApoE mouse model fed a high-fat diet, after intervention with *Bifidobacterium macrocarpa* and its extracellular vesicles, gross Oil Red O staining results showed that the area of ​​atherosclerotic plaques in the arteries was significantly reduced compared to the solvent control group (P < 0.05); serological tests showed that the serum triglyceride content was significantly reduced compared to the solvent control group (P < 0.05); and liver HE staining results showed that pathological lipid deposition in the liver was significantly reduced compared to the solvent control group. In a New Zealand white rabbit model of aortic injury induced by balloon feeding with high-fat diet, intervention with Bifidobacterium macrocarpa and its extracellular vesicles showed that H&E staining, Oil Red O staining, Masson staining, α-SMA staining, and RAM11 staining significantly reduced the area of ​​atherosclerotic plaques in the arteries compared to the solvent control group (P < 0.05), while significantly increasing the collagen content and stability of the plaques compared to the solvent control group (P < 0.05). Serological tests showed that serum triglyceride levels were significantly lower than those in the solvent control group (P < 0.05). Liver HE staining showed a significant reduction in pathological lipid deposition in the liver compared to the solvent control group.

[0053] Therefore, this invention conducted animal experiments on Bifidobacterium macrocarpa and its extracellular vesicles. Through intervention on ApoE knockout mice fed a high-fat diet and New Zealand rabbits with sac-injured livers fed a high-fat diet, it was observed that Bifidobacterium macrocarpa and its extracellular vesicles could significantly inhibit hyperlipidemia, the formation of atherosclerotic plaques, and pathological lipid deposition in the liver. Its application and industrialization prospects in the prevention and treatment of diseases caused by a high-fat diet are promising.

[0054] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Extracellular vesicles of Bifidobacterium longum Bifidobacterium magnum Use of extracellular vesicles of Bifidobacterium longum The disease is atherosclerosis, hyperlipidemia or fatty liver.

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