Application of betaine in preparation of products for losing weight and treating metabolic disorder

Abstract

The invention discloses application of betaine in preparation of a product for losing weight and treating metabolic disorder. The betaine is applied to improvement and treatment of obesity and related diseases caused by high-fat diet for the first time. Experiments prove that the betaine can significantly inhibit adipogenesis of 3T3-L1 preadipocytes and reduce fat accumulation; animal experiments prove that the betaine can improve glucose and lipid metabolism disorder caused by high fat diet and can relieve liver and kidney injury, inflammation and the like caused by high fat diet, so that the betaine has a prospect of being applied to preparation of medicines for preventing, relieving and treating obesity induced by high fat and related diseases thereof. And a new direction is provided for the development of anti-obesity preparations.

Description

Technical Field

[0001] The present invention relates to the technical field of development and application of betaine, and in particular to the application of betaine in the preparation of anti-obesity preparations. Background Art

[0002] The global prevalence of obesity, driven by sedentary lifestyles and unhealthy eating habits, has skyrocketed, becoming a major public health challenge and drawing widespread global attention to human health. Obesity is not only a disease in itself but can also lead to multiple complications, including cardiovascular disease and diabetes. Natural weight loss supplements are attracting increasing research attention due to their potential to prevent and treat obesity and metabolic syndrome. Compared to synthetic weight loss drugs, these supplements derived from natural foods and herbs have demonstrated improved safety, offering new perspectives for obesity treatment.

[0003] Alkaloids are a class of alkaline nitrogen-containing organic compounds found widely in natural plants and exhibit a variety of physiological activities, including antimicrobial, antitumor, anti-obesity, anti-inflammatory, and anti-diabetic properties. In particular, betaine (N,N,N-trimethylglycine), a water-soluble quaternary ammonium alkaloid found in wheat germ, sugar beets, and some marine organisms, possesses antioxidant and anti-inflammatory properties and plays a key role in regulating various diseases, including non-alcoholic fatty liver disease and metabolic syndrome. However, the role of betaine in the prevention and treatment of obesity has not been comprehensively and systematically investigated.

[0004] The development of obesity is multifactorial and complex, regulated by multiple signaling pathways. Therefore, combining experimental cell and animal models can improve understanding of the effects of substance interventions to fully understand their mechanisms of action related to obesity. 3T3-L1, a well-established preadipocyte cell line, serves as a standard model system for identifying regulators of adipogenesis and adipocyte function and is a well-established and suitable model for studying adipocyte lipid metabolism and obesity in vitro. Importantly, the gut microbiome is a recognized factor in the pathophysiology of obesity and plays an important role in obesity and lipid metabolism. Therefore, modulating the gut microbiome through diet may become an effective strategy for treating obesity and its related metabolic diseases. Summary of the Invention

[0005] The present invention provides an anti-obesity formulation prepared using betaine for use in preventing, alleviating, or treating obesity and related diseases caused by a high-fat diet. The betaine of the present invention can be used to prepare anti-obesity formulations, as well as formulations for preventing and / or alleviating disorders of glucose and lipid metabolism, fatty liver, liver and kidney damage, inflammatory reactions, intestinal barrier function impairment, and intestinal flora imbalance caused by a high-fat diet.

[0006] The technical solution provided by the present invention is:

[0007] Application of betaine in the preparation of anti-obesity preparations.

[0008] Furthermore, the anti-obesity refers to preventing or alleviating obesity.

[0009] Furthermore, the obesity is caused by a high-fat diet.

[0010] Based on the same inventive concept, the present invention also provides the use of betaine in preparing a preparation for preventing and / or alleviating diseases related to a high-fat diet.

[0011] Furthermore, the high-fat diet-induced related diseases include at least one of the following:

[0012] ① Disorders of glucose and lipid metabolism caused by a high-fat diet;

[0013] ②Fatty liver caused by high-fat diet;

[0014] ③ Liver and kidney damage caused by a high-fat diet;

[0015] ④ Liver and kidney damage caused by a high-fat diet;

[0016] ⑤Inflammatory response caused by high-fat diet;

[0017] ⑥ Damage to intestinal barrier function caused by a high-fat diet.

[0018] Based on the same inventive concept, the present invention also provides the use of betaine in preparing a preparation for regulating intestinal flora imbalance caused by a high-fat diet.

[0019] Furthermore, the preparation is a food, a medicine, or a health product.

[0020] Based on the same inventive concept, the present invention also provides a preparation for preventing and / or alleviating obesity and related diseases caused by a high-fat diet, which comprises an effective amount of betaine.

[0021] Furthermore, the related diseases include at least one of the following: glucose and lipid metabolism disorders caused by a high-fat diet, fatty liver caused by a high-fat diet, liver and kidney damage caused by a high-fat diet, inflammatory response caused by a high-fat diet, and intestinal barrier function damage caused by a high-fat diet.

[0022] Furthermore, the related diseases also include intestinal flora imbalance caused by a high-fat diet.

[0023] Furthermore, the preparation is a food, an oral medicine, or an oral health product.

[0024] Furthermore, the betaine is the only active ingredient.

[0025] The beneficial effects achieved by the present invention are:

[0026] Betaine has a significant preventive and alleviating effect on high-fat-induced obesity and its related diseases and intestinal flora imbalance. Therefore, the application of betaine in the preparation of prevention and relief of high-fat-induced obesity and its related diseases and their treatment is promising, and also provides a new direction for the development of anti-obesity preparations.

[0027] Experimental findings revealed that: ① Betaine significantly inhibited lipogenesis in 3T3-L1 preadipocytes and reduced fat accumulation; ② Betaine significantly inhibited weight gain and reduced tissue weight in mice fed a high-fat diet; and ③ Betaine improved the glucose and lipid metabolism disorders induced by a high-fat diet, lowering serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C).

[0028] ④ Betaine can reduce liver and kidney damage caused by a high-fat diet, and reduce the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRE) and blood urea nitrogen (BUN).

[0029] ⑤ Betaine inhibited the mRNA expression of the inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in the liver, which was induced by a high-fat diet. ⑥ Betaine upregulated the mRNA expression of zonula occludens-1 (ZO-1), occludin, claudin-1 protein (Claudin-1), mucin 2 (MUC2), and mucin 3 (MUC3) in the colon, which was induced by a high-fat diet. ⑦ Betaine modulated the composition of the intestinal microbiota, increasing the relative abundance of beneficial bacteria (Muribaculaceae, Allobaculum, and Parabacteroides) and decreasing the relative abundance of harmful bacteria (Colidextribacter and Desulfovibrionaceae). BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Effects of betaine on adipogenesis in 3T3-L1 preadipocytes:

[0031] (A) Chemical structure of betaine;

[0032] (B) Cell viability assay of betaine in 3T3-L1 preadipocytes;

[0033] (C) Oil red O staining of lipid droplet accumulation in differentiated adipocytes;

[0034] (D) Intracellular TG content was measured using a triglyceride assay kit at 550 nm;

[0035] (E) mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT / enhancer binding protein α (CEBP / α), CD36, stearoyl-CoA desaturase 1 (SCD1), PR domain zinc finger protein 16 (Prdm16), peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), and uncoupling protein 1 (UCP1) in 3T3-L1 preadipocytes;

[0036] (F) Protein expression of PPARγ, CEBP / α, CD36, and UCP1 in 3T3-L1 preadipocytes.

[0037] Figure 2 Effects of betaine on body weight and glucose metabolism in mice fed a high-fat diet:

[0038] (A) Food intake;

[0039] (B) Energy intake;

[0040] (C) weight gain;

[0041] (D) tissue weight;

[0042] (E) Glucose tolerance test;

[0043] (F) Insulin tolerance test.

[0044] Figure 3 Effects of betaine on obesity and related complications in mice fed a high-fat diet:

[0045] (A) Photomicrographs of sections of mouse epididymal fat, liver, and colon stained with hematoxylin-eosin;

[0046] (B) Relative mRNA expression of adipocyte-specific transcription factors PPARγ, CEBP / α, CD36, and SCD1 in mouse epididymal fat;

[0047] (C) mRNA expression of inflammatory factors IL-1β, IL-6, and TNF-α in mouse liver;

[0048] (D) mRNA expression of ZO-1, Occludin, Claudin-1, MUC2, and MUC3 in mouse colon.

[0049] Figure 4 Effects of betaine on intestinal microbial diversity in mice fed a high-fat diet:

[0050] (A) Number of OUTs;

[0051] (B) α diversity;

[0052] (C) β diversity;

[0053] (D) Venn diagram;

[0054] (E) Heat map.

[0055] Figure 5 Effects of betaine on the composition of intestinal flora in mice fed a high-fat diet:

[0056] (A) Relative abundance at the phylum level;

[0057] (B) Relative abundance at the genus level;

[0058] (C) Linear discriminant analysis effect size (LEfSe) analysis;

[0059] (D) Bacterial function prediction based on FAPROTAX.

[0060] Figure 6 Heat map analysis of Spearman correlation between gut microbiota and obesity factors at the genus level.

[0061] Figure 7 Effects of betaine on obesity in pseudo-germ-free mice fed a high-fat diet:

[0062] (A) Weight gain;

[0063] (B) tissue weight;

[0064] (C) Glucose tolerance test;

[0065] (D) Insulin tolerance test;

[0066] (E) Photomicrographs of sections of mouse epididymal fat, liver, and colon stained with hematoxylin-eosin. DETAILED DESCRIPTION

[0067] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0068] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0069] Example 1: Betaine improves high-fat diet-induced obesity and its related diseases and intestinal flora imbalance in mice

[0070] 1. Experimental Plan

[0071] The betaine used in the following experiments was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with the CAS number of 107-43-7 ( Figure 1 A).

[0072] (1) Cell testing and treatment

[0073] Mouse 3T3-L1 adipocytes were purchased from the National Cell Line Resource Bank and cultured in an incubator maintained at 37°C, 5% CO2, and sufficient humidity. The medium was changed daily. When the cells reached 70-80% confluence, they were digested with trypsin and passaged at a ratio of 1:2-1:3. The cell culture method is as follows:

[0074] ① Early stage of cell differentiation

[0075] 3T3-L1 adipocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum until confluence and incubated for 2 days.

[0076] ②Medium stage of cell differentiation

[0077] The DMEM medium containing 10% fetal bovine serum was discarded, and after washing with PBS, the cells were incubated for 2 days in Differentiation Medium I (DMEM medium containing 1 μM dexamethasone (Dex), 0.5 mM isobutylmethylxanthine (IBMX), 5 μg / mL insulin (INS), and 10% fetal bovine serum. Differentiation Medium I was discarded, and after washing with PBS, the cells were incubated for 2 days in Differentiation Medium II (DMEM medium containing 10 μg / mL INS and 10% fetal bovine serum).

[0078] ③Terminal stage of cell differentiation

[0079] The differentiation medium II was discarded, the cells were washed with PBS buffer, and the cells were incubated in DMEM medium containing 10% fetal bovine serum for 2 days to differentiate into mature adipocytes.

[0080] (2) Cell viability assay

[0081] 3T3-L1 adipocytes in the logarithmic growth phase were seeded in a 96-well cell culture plate (100 μL, approximately 10,000 cells). After the cells adhered, different concentrations of DL-norvaline were added for 24 h. The reaction solution was discarded, and 10 μL of CCK-8 reagent was added to each well. The culture plate was incubated in an incubator for 4 h. The absorbance was measured at 450 nm using a microplate reader, and the cell viability was calculated: cell viability (%) = absorbance of the experimental group / absorbance of the control group.

[0082] (3) Oil Red O staining

[0083] Induced 3T3-L1 adipocytes were fixed with 4% paraformaldehyde for 20 minutes, washed three times with PBS, and allowed to air dry. Hematoxylin O staining solution was added dropwise for 15 minutes, followed by three washes with PBS buffer and air dried. Hematoxylin staining solution was added dropwise for nuclear counterstaining for 1 minute. After air drying, the cells were observed and photographed under a microscope.

[0084] (4) Analysis of triglyceride (TG) content

[0085] Cell samples: After induced 3T3-L1 adipocytes, the culture medium was discarded and digested with 1 mL of trypsin for 1 minute. The cells were then incubated with 3 mL of DMEM supplemented with 10% fetal bovine serum to terminate the digestion and dispersed by pipetting. The pellet was centrifuged at 1000 rpm for 10 minutes at 4°C, and the supernatant discarded. The pellet was washed twice with PBS, mixed with 250 μL of PBS buffer, and ground (-30°C, 20 m / s) for 3 minutes. The pellet was centrifuged at 12,000 rpm at 4°C for 5 minutes and stored at -20°C until analysis. Tissue samples: Mouse tissue was ground in PBS buffer (1:9 w / v) using a frozen tissue grinder (-30°C, 20 m / s) for 3 minutes. The supernatant was then centrifuged at 12,000 rpm at 4°C for 10 minutes and stored at -20°C until analysis. TG content in the samples was determined according to the instructions of the commercial kit.

[0086] (5) Animal experiment grouping and treatment

[0087] C57BL / 6J mice (8 weeks old) were purchased from Spefox (Beijing) Biotechnology Co., Ltd. (Beijing, China). The experimental animals were housed in a standard animal room under strict conditions in accordance with the Chinese National Standard for Laboratory Animal Environment and Facilities (GB 14925-2010): temperature 22 ± 2°C, humidity 50% ± 15%, 12 h light / dark cycle, and free access to food and water. The animals were acclimated to the feeding regimen for one week before the start of the experiment. All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (8th edition, ISBN-10: 0-309-15401-4) and approved by the Animal Ethics Committee of Northwest Agriculture and Forestry University.

[0088] C57BL / 6J males were randomly divided into 3 groups (n=6 / group):

[0089] ① Low-Fat Diet (LFD) group: fed with low-fat feed (TP2330055AC, 10% fat, 75% carbohydrate, 15% protein, Nantong Trophy Feed Technology Co., Ltd., China) and water.

[0090] ② High-Fat Diet (HFD) group: fed with high-fat feed (TP2330055A, 60% fat, 25% carbohydrate, 15% protein, Nantong Trophy Feed Technology Co., Ltd., China) and water.

[0091] ③ Betaine intervention group: fed with high-fat feed and water containing 1% betaine.

[0092] The mice were orally gavaged once daily for 6 consecutive weeks, and their body weight and food intake were recorded every two days. After euthanasia, blood was collected from the eyeballs and centrifuged at 2000 rpm for 15 minutes at 4°C. The supernatant was collected, and the liver, spleen, kidney, and adipose tissue were collected and stored at -80°C for subsequent analysis.

[0093] (6) Biochemical index detection

[0094] Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine (CRE), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using biochemical kits purchased from Nanjing Jiancheng Bioengineering Institute.

[0095] (7) Glucose tolerance and insulin tolerance analysis

[0096] Glucose tolerance test: In the last week of betaine intervention, mice were gavaged with 2 g / kg glucose solution after fasting for 10 h. Blood glucose was tested by sampling from the tail vein at 0, 15, 30, 60, 90 and 120 min, and the curve was drawn. The area under the blood glucose curve (AUC) was calculated.

[0097] Insulin tolerance test: In the last week of betaine intervention, mice were fasted for 8 hours and intraperitoneally injected with 0.5 IU / kg insulin solution. Blood was collected from the tail vein at 0, 15, 30, 60, 90 and 120 minutes for blood glucose testing, and the curve was drawn. The blood glucose AUC was calculated at the same time.

[0098] (8) Hematoxylin-eosin staining

[0099] Mouse tissues were fixed in 4% paraformaldehyde for 24 hours. After washing away the fixative, the tissues were dehydrated in 70%, 80%, 95%, and 100% ethanol, then immersed in xylene twice to render them transparent and embedded in molten paraffin. The embedded paraffin blocks were cut into 4-μm-thick slices on a microtome, flattened in water at 42°C, removed with a glass slide, and dried in a 60°C incubator for 4 hours to prepare paraffin sections. The sections were rehydrated using xylene, anhydrous ethanol, 95% ethanol, 85% ethanol, 75% ethanol, 50% ethanol, purified water, and phosphate-buffered saline (PBS) buffer (pH 7.4). The sections were then stained with hematoxylin-eosin (H&E), mounted with neutral gum, and observed and photographed under a microscope.

[0100] (9) Real-time fluorescence quantitative PCR

[0101] Weigh an appropriate amount of tissue and 1 mL of Trizol reagent and grind them in a frozen tissue grinder (-30°C, 20 m / s) for 3 min, centrifuge at 12000 r / min for 10 min at 4°C, and take the supernatant. According to the RNA extraction operating instructions, total RNA was obtained, and the quality and concentration of total RNA were detected by 1% agarose gel electrophoresis and ultramicro spectrophotometer. The RNA was reverse transcribed into cDNA using a reverse transcription kit and diluted for real-time fluorescence quantitative PCR (RT-qPCR) detection and analysis. The temperature program was as follows: pre-denaturation: 94°C 30s, denaturation: 94°C 5s, annealing: 60°C 30s, 40 amplification cycles. The dissolution program was as follows: 60°C, 20s, 95°C, 20s. The GAPDH gene was used as an internal reference, and the expression of the target gene was analyzed according to the 2-ΔΔCt method. The primer sequences for RT-qPCR are as follows (Table 1):

[0102] Table 1. Primer sequences for RT-qPCR

[0103]

[0104] (10) Western blot detection

[0105] Cells were added to 1 mL of radioimmunoprecipitation assay (RIPA) lysis buffer containing 1% phenylmethanesulfonyl fluoride (PMSF) and 2% protein phosphatase inhibitors. The cells were mixed and ground in a freezer grinder (-30°C, 20 m / s) for 3 minutes. The supernatant was centrifuged at 12,000 rpm for 10 minutes at 4°C, and the total protein content in the homogenate supernatant was determined. The supernatant was diluted to a consistent concentration and then added with protein loading buffer. The cells were heat-denatured at 100°C for 5 minutes, cooled, and aliquoted and stored at -20°C until analysis. A lower separation gel of varying concentrations was prepared based on the molecular weight of the target protein. After the gel solidified, the comb was removed, and the protein sample was loaded and electrophoresed in Tris(hydroxymethyl)aminomethane (Tris)-glycine running buffer. Electrophoresis was performed at 80V until bromophenol blue dye entered the lower separating gel. The voltage was then increased to 120V and electrophoresis was continued until bromophenol blue reached the bottom of the gel. After activating the polyvinylidene fluoride (PVDF) membrane by soaking it in methanol for 5 minutes, the gel and PVDF membrane were sandwiched between filter paper, two transfer cotton pads, and a transfer clip. The membrane was transferred in an electrophoresis tank filled with electrophoresis transfer buffer at 250 mA for 150 minutes. After transfer, the membrane was removed and washed twice with PBS for 5 minutes each, then blocked in 5% nonfat milk blocking buffer for 2 hours. For primary antibody incubation, the blocking buffer was discarded and the membrane was washed once with Tris-buffered saline and Tween 20 (TBST) for 5 minutes each. The membrane was then incubated in the corresponding primary antibody solution at 4°C overnight. Incubation with secondary antibodies: Recover the primary antibody, wash the membrane three times with TBST buffer (5 minutes each), then incubate in the corresponding secondary antibody solution at room temperature for 2 hours. Recover the secondary antibody and wash the membrane three times with TBST buffer (5 minutes each). Immerse the membrane in enhanced chemiluminescence (ECL) for 1 minute, image the membrane using a chemiluminescence imaging system, and perform quantitative analysis using ImageJ software.

[0106] (11) 16S rRNA sequencing analysis of intestinal flora

[0107] use DNA was extracted from the microorganisms in the mouse feces using a DNA extraction kit, and degradation and contamination were monitored on a 1% agarose gel, and its purity and concentration were determined using a microspectrophotometer. Then, primers (upper primer 338F and lower primer 806R were 5′-ACTCCTACGGGAGGCAGCAG-3′ and

[0108] 5′-GGACTACHVGGGTWTCTAAT-3′) to amplify the variable region V3-V4 of the 16S rRNA gene.

[0109] PCR was performed under the following conditions: 5–50 ng of DNA template, 0.3 μL of the upper primer (10 μM), 0.3 μL of the lower primer (10 μM), 5 μL of KOD FX Neo buffer, 2 μL of 2 mM dNTPs, and 0.2 μL of KOD FX Neo reagent. The total volume was made up to 20 μL with double-deionized water. After an initial denaturation at 95°C for 5 min, 20 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 40 s were performed, followed by a final extension at 72°C for 7 min. Amplified products were purified using a DNA purification kit and quantified using a Qsep-400 high-throughput bio-fragment analyzer. Paired-end sequencing (2 × 250 bp) of the amplicon library was performed on an Illumina novaseq 6000.

[0110] The raw data of 16S rRNA gene sequencing reads were filtered, assembled, and screened to obtain high-quality reads. The remaining sequences were divided into operational taxonomic units (OTUs) according to different similarity levels. Bioinformatics analysis was performed at OTUs with 97% similarity. Alignment was performed using the Greengenes database, and sequencing data were processed on the BMKCloud platform (www.biocloud.net).

[0111] 2. Experimental Results

[0112] (1) According to the attached Figure 1 The effects of betaine on adipogenesis in 3T3-L1 preadipocytes are shown. It can be seen that betaine has significant effects on lipid synthesis and accumulation as well as the regulation of related signaling pathways at the cellular level.

[0113] like Figure 1 As shown in B, in 3T3-L1 preadipocytes, betaine at a concentration of up to 200 μg / mL showed no cytotoxicity compared to the induced group, indicating that betaine exhibited only low cytotoxicity in 3T3-L1 preadipocytes. Based on this result, subsequent experiments used 50 and 100 μg / mL of betaine. Figure 1Oil red O staining results showed that compared with the induced group, two different doses of betaine treatment resulted in 59.39% and 20.35% adipogenesis rates, respectively, which indicates that betaine has a strong inhibitory effect on adipogenesis in the 3T3-L1 preadipocyte model. In addition, betaine treatment significantly reduced intracellular triglyceride content ( Figure 1 D) These results indicate that betaine can inhibit fat accumulation and triglyceride synthesis in 3T3-L1 preadipocytes. Figure 1 As shown in E, betaine significantly inhibited the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT / enhancer-binding protein α (C / EBPα). PPARγ can be activated by sterol regulatory element-binding protein-1 (SREBP-1), thereby inducing the expression of lipogenic genes, such as fatty acid transporter-differentiation cluster 36 (CD36). Figure 1 As shown in Figure E, betaine treatment inhibited CD36 gene expression compared to the control group. In addition, brown fat cells consume calories in the form of heat through non-shivering thermogenesis, which is characterized by upregulation of uncoupling protein 1 (UCP1) protein, leading to an increase in the number of mitochondria, which provides new ideas for developing new strategies for treating obesity. Moreover, the "browning" of white adipose tissue is regulated by different transcription factors, such as PR domain-containing 16

[0114] (PR-Domain-Containing 16, PRDM16) and Peroxisome Proliferator-Activated Receptor Gamma Co-activator 1α (Peroxisome Proliferator-Activated Receptor Gamma Co-activator 1α, PGC-1α). Figure 1 As shown in Figure E, after betaine treatment, the expression of UCP1, PGC-1α, and PRDM16 increased, indicating that betaine can promote the development of 3T3-L1 preadipocytes into brown adipocyte-like phenotype. Figure 1F, This observation is consistent with the results of Western blot analysis, further indicating that betaine can regulate the adipogenesis and “browning” process of 3T3-L1 preadipocytes through the PPARγ / C / EBPα / CD36 signaling pathway and the UCP1 / PGC-1α / PRDM16 axis.

[0115] (2) As attached Figure 2 The effects of betaine on body weight and glucose metabolism in mice fed a high-fat diet are shown. It can be seen that betaine plays a key role in regulating the overall metabolic homeostasis of obese mice fed a high-fat diet, and shows a potential core regulatory role in combating high-fat diet-induced obesity and related metabolic disorders.

[0116] like Figure 2 As shown in A and 2B, the food intake of the high-fat diet (HFD) group and the betaine group was significantly reduced compared with the low-fat diet (LFD) group, while there was no significant difference in energy intake among the three groups. However, the weight gain rate of the mice in the HFD group was significantly higher than that of the LFD group. However, after betaine treatment of the HFD-fed mice, their weight gain rate was significantly reduced to a value between the HFD and LFD groups ( Figure 2 C). In addition, HFD feeding induced a significant increase in the weight of liver, kidney, spleen, and epididymal adipose tissue compared with the LFD group. However, after betaine intervention, the tissue weights were significantly reduced ( Figure 2 D) In ​​addition, obesity is always accompanied by glucose metabolism disorders and insulin resistance, which are the most typical complications of obesity. Figure 2 As shown in E, during the glucose tolerance test, the blood glucose levels and area under the curve (AUC) at 0, 15, 30, 60, 90, and 120 minutes in the HFD group were significantly higher than those in the LFD group, while these were significantly reduced after betaine intervention. In the insulin tolerance test, the glucose AUC in the HFD group was approximately 1.5 times higher than that in the LFD group, while the blood glucose levels and AUC in the betaine group were significantly lower than those in the HFD group ( Figure 2 F).

[0117] (2) As shown in Table 2, betaine has an effect on the blood lipid levels and liver and kidney functions of obese mice induced by a high-fat diet. It can be seen that betaine has a potential key regulatory value in regulating blood lipid homeostasis and maintaining the normal physiological functions of the liver and kidneys in obese mice fed a high-fat diet.

[0118] Table 2 Effects of betaine on blood lipid levels and liver and kidney function in obese mice induced by high-fat diet

[0119]

[0120] Data are presented as mean ± standard deviation, n = 6. Significance is indicated as follows: # indicates comparison with the control group, $ indicates comparison with the model group; # and $ indicate P < 0.05; ## and $$ indicate P < 0.01; ### and $$$ indicate P < 0.001.

[0121] As shown in Table 1, compared with the high-fat diet group, mice fed betaine significantly reduced serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Furthermore, long-term high-fat diet feeding may cause liver damage, as evidenced by increased levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRE), and blood urea nitrogen (BUN). However, betaine supplementation significantly reduced the levels of liver function (AST and ALT) and renal function (CRE and BUN).

[0122] (3) As attached Figure 3 The effects of betaine on obesity and related complications in mice fed a high-fat diet are shown. It can be seen that betaine has multi-target and systemic intervention effects on high-fat diet-induced inflammatory response and intestinal barrier damage.

[0123] like Figure 3 As shown in Figure A, morphological analysis of liver and epididymal fat sections from mice fed a LFD diet revealed no pathological signs and no significant steatosis. However, hepatocytes in mice fed a HFD diet showed disordered arrangement and numerous lipid droplets. Furthermore, adipocyte size in the HFD group was significantly larger than in the LFD group. However, betaine-treated mice significantly prevented hepatic steatosis and reduced adipocyte size compared with the HFD group. Furthermore, the epithelial cell membrane structure of the colonic mucosa in the LFD group was intact, with abundant, uniformly lengthed, and regularly arranged microvilli, and clear and neat tight junctions at the top of the intercellular surfaces. However, intestinal damage was observed in HFD-fed mice, with microvilli of varying length and fragmentation, and widened and irregular tight junctions. Betaine-treated mice showed some improvement in inflammatory cell infiltration and mucosal and microvilli damage. To further explore the underlying mechanisms, real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the expression levels of related genes. Consistent with the results of cell experiments, HFD upregulated the expression of lipogenic genes in epididymal fat of obese mice. However, betaine reduced the expression of C / EBPα and PPARγ and affected the transcription levels of downstream functional genes such as CD36 and SCD1 ( Figure 3B). At the same time, the relative mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the liver of mice in the HFD group increased significantly, indicating that low-grade inflammation had occurred in the liver tissue of HFD-induced obese mice. However, betaine supplementation in HFD mice caused a significant decrease in the expression of these inflammatory factors, indicating that betaine successfully alleviated inflammation in HFD-fed mice, confirming the results of liver morphological analysis ( Figure 3 C).

[0124] The integrity of the intestinal mucosa is an important indicator of intestinal barrier function. The integrity of the intestinal epithelium is maintained by tight junction proteins including ZO-1, Occludin and Claudin-1. Mucin is also involved in the organization and construction of the intestinal mucus layer on the epithelial surface. Figure 3 As shown in Figure 3, the mRNA expression levels of tight junction proteins and mucins were lower in the HFD group compared with the LFD group. In contrast, betaine treatment significantly upregulated the expression levels of ZO-1, Occludin, Claudin-1, MUC2, and MUC3, further confirming the results of colonic morphological analysis.

[0125] (4) As attached Figure 4 and attached Figure 5 The effects of betaine on the intestinal microorganisms of mice fed a high-fat diet are shown. It can be seen that betaine plays a key role in reshaping the intestinal microecology. It effectively intervenes in the intestinal microbial imbalance caused by a high-fat diet by regulating the composition structure, abundance ratio and metabolic functional activity of the microbial community.

[0126] like Figure 4 As shown in A, HFD led to a decrease in the operational taxonomic units (OTUs) of the intestinal microbiota, while betaine intervention helped increase the OTUs of the intestinal microbiota. Compared with mice fed with LFD, the α diversity shown by the Shannon index, ACE richness, Chao1 richness, and Simpson index was significantly reduced under HFD feeding, and although betaine treatment showed an increasing trend, it was not significant ( Figure 4 B). Beta diversity analysis based on principal component analysis (PCA) of Bray-Curtis distance showed that the clusters of the intestinal microbiota in each group were separated from each other. There was a clear separation of intestinal microbial structures between the LFD and HFD groups, while samples from the LFD and betaine groups were in tighter clusters ( Figure 4 C). Venn diagram analysis showed that among the total 2667 OTUs, 207 were shared among all samples ( Figure 4 C). In particular, the OTUs between the LFD group and the betaine group were more common, while the HFD group and the betaine group only shared 43 ( Figure 4C). The cluster heat map revealed that the intestinal microbiota patterns were significantly different among the groups, with the LFD group and the betaine group being similar, indicating that betaine intervention led to changes in the intestinal microbiota ( Figure 4 D).

[0127] To further elucidate the differences between the groups, component intestinal microbiota composition analysis was performed. Figure 5 As shown in A, at the phylum level, the main bacterial communities in all groups were Firmicutes, Bacteroidota, and Campylobacterota. In addition, HFD feeding of mice resulted in an increase in the ratio of Firmicutes / Bacteroidetes (F / B) and Desulfobacterota compared to LFD-fed mice. Fortunately, these abnormal changes in phylum-level bacterial composition caused by HFD were significantly inhibited by betaine supplementation. Specifically, HFD feeding increased the relative abundance of Colidextribacter ( Figure 5 B), while Colidextribacter was significantly positively correlated with inflammatory factors (TNF-α, IL-1β, IL-6, IL-18). Consistent with the results at the phylum level, Desulfovibrionaceae was also significantly enriched in the HFD group ( Figure 5 B). Desulfovibrionaceae, as harmful bacteria, can act as endotoxin-producing pathogens to produce lipopolysaccharide (LPS) and is also positively correlated with epididymal and perirenal fat mass, as well as liver triglyceride and total cholesterol levels. However, betaine treatment statistically reduced the abundance of Colidextribacter and Desulfovibrionaceae, and significantly increased the relative abundance of Muribaculaceae, Allobaculum, and Parabacteroides in HFD-fed mice ( Figure 5 B). Muribaculaceae can produce short-chain fatty acids from endogenous and exogenous polysaccharides and is associated with the mitigating effects of a plant-based diet on inflammatory bowel disease, obesity, and type 2 diabetes. In addition, as beneficial bacteria, a negative correlation between Allobaculum and obesity has been reported (Baldwin et al., 2016). Studies have shown that due to the important role of intestinal Parabacteroides in human health and disease, Parabacteroides has received increasing attention. For example, Parabacteroides can alleviate obesity and metabolic dysfunction by producing succinate and secondary bile acids. In order to further identify specific bacterial taxa under different treatment conditions, such as Figure 5As shown in C, the microbiota composition of the three experimental groups was compared by the linear discriminant analysis effect size (LEfSe) method. Bacteroidia and Muribaculaceae were enriched in the LFD group, and most of the microbiota in the HFD group were Clostridia, Firmicutes and Lachnospiraceae, while Bacteroides and Verrucomicrobiota were more abundant in the betaine group. Consistently, the linear discriminant analysis (LDA) scores confirmed that the above bacteria were biomarkers of the three experimental groups, indicating that betaine treatment widely alleviated intestinal flora imbalance. Based on the relative abundance of the above microorganisms, FAPROTAX was used to predict microbial functions. The most important functions of the intestinal microbiota in the three treatment groups were mainly chemoheterotrophy and fermentation, with a total relative abundance of approximately 50%. Compared with the LFD group, HFD increased the bacterial functions of chemoheterotrophy, fermentation and animal parasites or symbionts, and reduced the bacterial functions of human pathogens, human pathogen pneumonia, human intestine and mammalian intestine. However, betaine supplementation reversed these changes ( Figure 5 D).

[0128] Spearman correlation was further used to analyze the correlation between intestinal microbiota and obesity-related indicators. Figure 6 As shown in the data, unclassified_Muribaculaceae, uncultured_Bacteroidales_bacterium, Allobaculum, and Alloprevotella were significantly positively correlated with HDL-C levels, and negatively correlated with weight gain, TC, TG, LDL-C, AST, ALT, CRE, and BUN levels. Alistipes, unclassified_Desulfovibrionaceae, Colidextribacter, Helicobacter, Coriobacteriaceae_UCG_002, and Faecalibaculum showed the opposite trend.

[0129] The above results indicate that betaine treatment can significantly improve intestinal flora imbalance in high-fat diet-induced obese mice.

[0130] Example 2: Effects of betaine on high-fat diet-induced obesity and related diseases and intestinal flora imbalance in the "pseudo-germ-free mouse" model

[0131] 1. Experimental Plan

[0132] (1) Animal experiment grouping and treatment

[0133] C57BL / 6J mice (8 weeks old) were purchased from Spefox (Beijing) Biotechnology Co., Ltd. (Beijing, China). The experimental animals were housed in a standard animal room under strict conditions in accordance with the Chinese National Standard for Laboratory Animal Environment and Facilities (GB 14925-2010): temperature 22 ± 2°C, humidity 50% ± 15%, 12 h light / dark cycle, and free access to food and water. The animals were acclimated to the feeding regimen for one week before the start of the experiment. All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (8th edition, ISBN-10: 0-309-15401-4) and approved by the Animal Ethics Committee of Northwest Agriculture and Forestry University.

[0134] Mice were treated with an antibiotic cocktail (0 μg / mL clindamycin, 50 μg / mL metronidazole, 50 μg / mL penicillin, and 25 μg / mL vancomycin) for 7 consecutive days and then randomly divided into 3 groups (n=6 / group):

[0135] ① Antibiotic-treated low-fat diet (ALFD) group: fed with low-fat feed and antibiotic water.

[0136] ② Antibiotic-treated high-fat diet (ALFD) group: fed with high-fat feed and antibiotic water.

[0137] ② Antibiotic-treated betaine intervention (AB) group: fed with high-fat feed, antibiotic water, and water containing 1% betaine.

[0138] The mice were orally gavaged once daily for 6 consecutive weeks, and their body weight and food intake were recorded every two days. After euthanasia, blood was collected from the eyeballs and centrifuged at 2000 rpm for 15 minutes at 4°C. The supernatant was collected, and the liver, spleen, kidney, and adipose tissue were collected and stored at -80°C for subsequent analysis.

[0139] (2) Other experimental schemes are the same as in Example 1

[0140] 2. Experimental Results

[0141] To evaluate the impact of metabolic interactions between the host and gut microbiota on the effects of betaine, a pseudo-germ-free mouse model was prepared using a cocktail of antibiotics to alter their intestinal conditions, which is a valuable tool for understanding the effects of betaine on gut microbiota. Figure 7 The effects of betaine on obesity in pseudo-germ-free mice fed a high-fat diet indicate that intestinal flora plays a unique and critical role in improving high-fat diet-induced obesity and related diseases.

[0142] like Figure 7As shown in A, in the absence of intestinal microorganisms, the weight gain of mice fed an HFD for 6 weeks was still significantly higher than that of the LFD group, but after betaine intervention, the weight gain of mice was significantly reduced. Consistently, tissue weights of mice in the AHFD group were significantly increased in pseudo-germ-free mice compared with the ALFD group. Interestingly, in the "pseudo-germ-free mouse" model, betaine intervention reduced liver, kidney, and epididymal fat, while spleen weight had no significant effect. Similarly, blood glucose and the AUC of the glucose tolerance test and insulin tolerance test were significantly reduced in the AHFD group compared with the ALFD group. However, betaine supplementation did not significantly improve the pseudo-germ-free mice treated with an HFD. Figure 7 E showed that HFD caused a significant increase in adipocyte size, histopathological damage, and intestinal damage in pseudo-germ-free mice, while the AB group significantly reduced these damages compared with the AHFD group. Consistent with studies in normal mice, serum levels of TC, TG, LDL-C, and HDL-C, as well as serum liver and kidney damage markers such as AST, ALT, CRE, and BUN, were significantly increased in HFD-fed mice. However, betaine intervention only significantly improved TC, TG, and ALT levels, but had no effect on other markers.

[0143] The above results show that when the intestinal microbiota disappears, the improvement effect of betaine on high-fat diet-induced obesity is inhibited, indicating that the beneficial effects of betaine in improving high-fat diet-induced obesity and related diseases depend to a certain extent on intestinal microorganisms.

[0144] The conclusion is as follows:

[0145] Betaine shows significant improvement effects on high-fat diet-induced obesity and its related diseases, and is able to regulate dysbiosis and maintain microbial dysbiosis. The potential molecular mechanisms of betaine treatment in improving obesity may be as follows: 1) correcting intestinal microbial dysbiosis by regulating the composition of the intestinal microbiota, especially selectively reducing the relative abundance of harmful intestinal bacteria (Colidextribacter and Desulfovibrionaceae) and increasing the relative abundance of beneficial intestinal bacteria (Muribaculaceae, Allobaculum and Parabacteroides); 2) further helping to improve intestinal barrier function, inflammatory response and lipid metabolism; 3) by normalizing obesity-related indicators, betaine becomes a promising candidate drug for the treatment of obesity and its related metabolic syndrome. These findings suggest that betaine has great potential as a useful alternative or nutritional health product to combat obesity.

[0146] The present invention and its embodiments are described above. Such description is not restrictive. The embodiment shown in the embodiment is only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by the above, and does not deviate from the purpose of the invention, without creatively designing a structure and embodiment similar to the technical solution, they shall fall within the scope of protection of the present invention.

Claims

1. Application of betaine in the preparation of anti-obesity preparations.

2. The use according to claim 1, characterized in that The anti-obesity refers to preventing or alleviating obesity.

3. The use according to claim 1, characterized in that The obesity is caused by a high-fat diet.

4. The use of betaine in the preparation of preparations for preventing and / or alleviating diseases related to a high-fat diet.

5. The use according to claim 4, characterized in that The diseases caused by the high-fat diet include at least one of the following: ① Disorders of glucose and lipid metabolism caused by a high-fat diet; ②Fatty liver caused by high-fat diet; ③ Liver and kidney damage caused by a high-fat diet; ④ Liver and kidney damage caused by a high-fat diet; ⑤Inflammatory response caused by high-fat diet; ⑥ Damage to intestinal barrier function caused by a high-fat diet.

6. The use of betaine in the preparation of preparations for regulating intestinal flora imbalance caused by high-fat diet.

7. The use according to any one of claims 1 to 6, characterized in that The preparation is food, medicine or health product.

8. A preparation for preventing and / or alleviating obesity and related diseases caused by a high-fat diet, comprising an effective amount of betaine.

9. The preparation according to claim 8, characterized in that The related diseases include at least one of the following: glucose and lipid metabolism disorders caused by a high-fat diet, fatty liver caused by a high-fat diet, liver and kidney damage caused by a high-fat diet, inflammatory response caused by a high-fat diet, and intestinal barrier function damage caused by a high-fat diet.

10. The preparation for preventing and / or alleviating obesity and related diseases caused by a high-fat diet according to claim 8, characterized in that: The related diseases also include intestinal flora imbalance caused by a high-fat diet.

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