Application of combination of glibenclamide and bifidobacterium B960 in preparation of medicine for treating type 2 diabetes mellitus

Abstract

The invention provides a combined application of glibenclamide and bifidobacterium B960 in preparation of a medicine for treating type 2 diabetes mellitus. According to the invention, the influence of the synergistic effect of glibenclamide and bifidobacterium B960 on the treatment effect of type 2 diabetes mellitus is systematically researched. Experimental results show that the bifidobacterium B960 and glibenclamide are combined for treatment, so that the treatment effect on type 2 diabetes mellitus is remarkably improved. Glucose tolerance tests show that the blood glucose change of a combined treatment group is more ideal, and the side effects of glibenclamide, including reduction of transaminase activity, regulation of lipid metabolism, reduction of inflammatory response and the like, are effectively relieved. In addition, the effect of combined treatment is similar to that of single use of the bifidobacterium B960 in the aspect of increasing the content of short-chain fatty acid, and the intestinal microenvironment can be improved. The invention provides a theoretical basis and a new strategy for comprehensive treatment of type 2 diabetes mellitus, and is expected to improve the living quality of patients and reduce the occurrence rate of complications.

Description

Technical Field

[0001] The invention belongs to the technical field of treatment of type 2 diabetes and relates to application of glibenclamide combined with bifidobacterium B960 in preparing a medicine for treating type 2 diabetes. Background Art

[0002] With the changes in modern lifestyles and the acceleration of the aging of the population, type 2 diabetes (T2DM) has gradually become a global public health problem, and its incidence continues to rise. According to data released by the International Diabetes Federation in 2021, approximately 537 million adults worldwide suffer from diabetes, of which approximately 90% suffer from type 2 diabetes. Long-term hyperglycemia can expose patients with type 2 diabetes to the threat of multiple complications such as cardiovascular disease, nephropathy, and retinopathy, which seriously affect the patient's quality of life and shorten their life expectancy. Therefore, exploring safe and effective treatments is crucial to improving the health of patients with type 2 diabetes.

[0003] In the traditional treatment of type 2 diabetes, sulfonylurea drugs, such as glibenclamide, lower blood sugar by stimulating pancreatic beta cells to secrete insulin. However, long-term use of such drugs is not only prone to cause adverse reactions such as hypoglycemia and weight gain, but may also cause the body to develop tolerance to the drug, making it difficult for a single drug to achieve good blood sugar control. Although combined medication can improve the treatment effect to a certain extent, it also increases the cost of treatment and the risk of side effects. Therefore, exploring new treatment strategies to enhance the treatment effect while reducing side effects has become an important direction of current research on the treatment of type 2 diabetes. Summary of the invention

[0004] The present invention aims to provide the use of glibenclamide in combination with Bifidobacterium B960 in the preparation of a medicament for treating type 2 diabetes, which provides a theoretical basis and a new strategy for the comprehensive treatment of type 2 diabetes, and is expected to improve the therapeutic effect, reduce side effects, and achieve individualized precision treatment.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] The use of glibenclamide in combination with Bifidobacterium B960 in the preparation of a drug for the treatment of type 2 diabetes. The Bifidobacterium B960 was deposited in the Guangdong Provincial Microbiological Culture Collection Center GDMCC on October 23, 2024. The storage address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The storage number is GDMCC No: 65330, and the classification name is: Bifidobacterium animalis.

[0007] Furthermore, the combination of glibenclamide and Bifidobacterium B960 can enhance the effect of lowering blood sugar, and the blood sugar fluctuation is smaller and the stability is better.

[0008] Furthermore, the combination of glibenclamide and Bifidobacterium B960 can better promote insulin secretion or improve pancreatic β-cell function, thereby enhancing the ability to regulate blood sugar.

[0009] Furthermore, the combination of glibenclamide and Bifidobacterium B960 can have a positive effect on the secretion or metabolism of glucagon-like peptide-1, thereby regulating blood sugar.

[0010] Furthermore, the combination of glibenclamide and Bifidobacterium B960 can significantly reduce the content of glycated serum protein, thereby more effectively controlling the long-term stability of blood sugar and reducing the damage to the body caused by blood sugar fluctuations.

[0011] Furthermore, the combined use of glibenclamide and Bifidobacterium B960 can significantly reduce the activities of alanine aminotransferase and aspartate aminotransferase.

[0012] Furthermore, the combination of glibenclamide and Bifidobacterium B960 can significantly reduce the levels of serum cholesterol and low-density lipoprotein cholesterol, and significantly increase the level of high-density lipoprotein cholesterol.

[0013] Furthermore, the combination of glibenclamide and Bifidobacterium B960 can reduce the levels of interleukin IL-6 and tumor necrosis factor α.

[0014] Furthermore, the combination of glibenclamide and Bifidobacterium B960 is effective in improving liver pathology and protecting pancreatic islet function.

[0015] Furthermore, the combination of glibenclamide and Bifidobacterium B960 can significantly increase the contents of acetic acid, propionic acid, butyric acid, isobutyric acid and isovaleric acid.

[0016] The beneficial effects of the present invention are:

[0017] The present invention combines in vitro and in vivo experiments, uses high performance liquid chromatography to screen out Bifidobacterium B960 with a lower degradation of glibenclamide, and systematically studies the effect of the synergistic effect of glibenclamide and Bifidobacterium B960 on the therapeutic effect of type 2 diabetes. The experimental results show that the combined treatment of Bifidobacterium B960 and glibenclamide significantly improves the therapeutic effect of type 2 diabetes. The glucose tolerance test showed that the blood sugar changes in the combined treatment group were more ideal, and the side effects of glibenclamide were effectively alleviated, including reducing transaminase activity, regulating lipid metabolism, and reducing inflammatory response. In addition, the combined treatment and the use of Bifidobacterium B960 alone are similar in terms of improving the content of short-chain fatty acids, which helps to improve the intestinal microenvironment. The present invention provides a theoretical basis and new strategies for the comprehensive treatment of type 2 diabetes, which is expected to improve the quality of life of patients and reduce the incidence of complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The degradation rate of glibenclamide after 6 h of co-culture with Bifidobacterium is shown.

[0019] Figure 2 Shows the monitoring results of fasting blood glucose of mice in different groups.

[0020] Figure 3 Shows the test results of blood glucose, insulin, glucagon-like peptide-1, and glycated serum protein in the serum of mice in different groups.

[0021] Figure 4 Shows the results of glucose tolerance test of mice in different groups.

[0022] Figure 5 The results of the tests on alanine aminotransferase activity, aspartate aminotransferase activity, triglyceride content, serum cholesterol content, high-density lipoprotein cholesterol content, low-density lipoprotein cholesterol content, interleukin IL-6 content and tumor necrosis factor α content of mice in different groups are shown.

[0023] Figure 6 Shown are liver and pancreas sections from mice in different groups.

[0024] Figure 7 The results of the tests on the contents of acetate, propionate, butyrate, caproate, isobutyrate and isovalerate in mice in different groups are shown. DETAILED DESCRIPTION

[0025] The present invention is described in detail below in conjunction with specific implementation methods. The following specific embodiments are helpful for those skilled in the art to further understand the present invention, but do not limit the present invention in any form.

[0026] This application revolves around the following core scientific issues: First, during the metabolism of glibenclamide in the body, probiotics may degrade the drug and weaken its hypoglycemic effect, so it is necessary to screen strains with lower decomposition rates to ensure the efficacy of glibenclamide. Secondly, the synergistic effect of probiotic bifidobacteria and glibenclamide may enhance the therapeutic effect by improving the intestinal microecological environment and regulating blood sugar metabolism, but its specific synergistic mechanism is still unclear, so its mechanism of action needs to be further explored. Finally, this application will also explore whether the synergistic treatment effectively reduces the side effects of glibenclamide, as well as the effects on metabolic indicators such as blood lipids and inflammatory factors.

[0027] Example

[0028] 1. Materials and Methods

[0029] 1. In vitro experiments

[0030] 1.1 Experimental Materials

[0031] The bifidobacteria used in the experiment were provided by the tropical probiotic lactic acid bacteria resource bank of Hainan University. The experimental drugs glibenclamide and streptozotocin were purchased from Sigma-Aldrich.

[0032] Bifidobacterium B960 (also known as Bifidobacterium animalis H22B960) was deposited in the Guangdong Provincial Microbial Culture Collection Center GDMCC on October 23, 2024. The storage address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The storage number is GDMCC No: 65330, and the classification name is: Bifidobacteriumanimalis.

[0033] 1.2 Screening of experimental strains using high performance liquid chromatography

[0034] High performance liquid chromatography was used to screen the probiotics suitable for this experimental study. First, a 0.68 mg / mL glibenclamide stock solution was accurately prepared. At the same time, the corresponding culture medium and bacterial solution were prepared. In a clean workbench, the experimental solution was prepared according to the ratio of culture medium: bacterial solution: glibenclamide stock solution = 12 mL: 400 μL: 200 μL, and mixed thoroughly.

[0035] The chromatographic analysis conditions were set as follows: a TIANHE Kromasil C18 chromatographic column was selected, and the column temperature was controlled at 30°C. Degassed methanol-0.1% sodium dihydrogen phosphate solution (volume ratio of 70:30, pH value of 3.0±0.05) was used as the mobile phase, the flow rate was set to 1 mL / min, the detection wavelength was 233 nm, and the injection volume was 10 μL. After the experiment, qualitative analysis was performed by chromatographic peak retention time and ultraviolet absorption spectrum, and quantitative analysis was performed by peak area using the external standard method, that is, a standard curve was first prepared, and then the sample peak area was substituted to calculate the content.

[0036] 2. In vivo experiments

[0037] 2.1 Experimental animals

[0038] This application was carried out after obtaining the approval of the Animal Ethics Committee of Hainan University, and all animal experimental operations were carried out in strict accordance with the relevant provisions of the "Guidelines for the Care and Use of Laboratory Animals" promulgated by Hainan University. The experiment selected 30 4-week-old male SPF-grade C57BL / 6 mice, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The mice were housed in a standard laboratory environment, which maintained a 12-hour light cycle day and night, the humidity was controlled at about 60%, and the temperature was maintained at about 23°C to ensure that the C57BL / 6 mice were in the best living condition. During the experiment, the mice had free access to standard food and drinking water provided by Beijing Keao Xieli Feed Co., Ltd. to meet their daily nutritional needs. Before the formal experiment began, C57BL / 6 mice were given a one-week adaptation period to reduce the impact of environmental factors on the experimental results.

[0039] 2.2 Construction of type 2 diabetes mouse model

[0040] After the adaptation period of C57BL / 6 mice, the normal control group was fed with 10% low-fat feed (protein content of 20%, carbohydrate content of 70%, and fat content of 10%), and the remaining mice were fed with 60% high-fat feed (protein content of 20%, carbohydrate content of 20%, and fat content of 60%). During the feeding period, all mice had free access to water.

[0041] After 8 weeks of diet feeding, the experimental mice were intraperitoneally injected with freshly prepared streptozotocin solution for 5 consecutive days, with an injection dose of 40 mg / kg / mouse per day. Streptozotocin was dissolved in cold citrate buffer at pH 4.5 and 0.01 M. At the same time, the control group was only given the same concentration of citrate buffer.

[0042] On the seventh day after the last injection, the mice were fasted for 12 hours and their fasting blood glucose was measured. If the fasting blood glucose value of the mice was greater than 11.1mmol / L, it was determined that the type 2 diabetes model was successfully established, and the mice were then divided into groups and subsequent experiments were carried out.

[0043] 2.3 In vivo treatment

[0044] The mice were randomly divided into 5 groups (n=6), namely:

[0045] Normal control group (CON): Normal control mice were intragastrically administered with 200 μL of 0.5% sodium carboxymethyl cellulose every day.

[0046] Type 2 diabetes group (T2DM): diabetic mice were intragastrically administered with 200 μL 0.5% sodium carboxymethyl cellulose every day.

[0047] Glibenclamide group (G): diabetic mice were intragastrically administered with 200 μL of 100 mg / kg glibenclamide dissolved in 0.5% sodium carboxymethylcellulose every day.

[0048] Glibenclamide + probiotics group (G+probiotics): diabetic mice were intragastrically administered with 200 μL, 100 mg / kg glibenclamide + 10 9 cfu probiotics, dissolved in 0.5% sodium carboxymethylcellulose.

[0049] Probiotic group (probiotics): diabetic mice were intragastrically administered 200 μL of 10 9 cfu probiotics, dissolved in 0.5% sodium carboxymethylcellulose.

[0050] During the treatment period, the fasting blood glucose of the mice was measured once a week and the body weight was recorded once a week. Before the end of the treatment, the mice were subjected to a glucose tolerance test.

[0051] 2.4 Glucose tolerance test in mice

[0052] Before the glucose tolerance test, the mice were fasted for 12 hours and clean bedding was replaced. Prepare an appropriate amount of 20% reagent-grade glucose solution dissolved in sterile saline. According to the common dose of 2g / kg, the amount of glucose solution required was calculated according to the weight of the mouse, and the gavage volume for each mouse was about 200μL. During the gavage process, if resistance is encountered, it should not be forcibly perfused to prevent the solution from entering the trachea by mistake. After the first mouse was successfully gavaged, the timer was immediately started and counted down for 15 minutes. During this period, the gavage operation of all mice was completed, and the number of experimental mice in the same batch did not exceed 15. Start the timer from the first gavage mouse, and monitor the blood glucose levels of the mice at 0, 15, 30, 60, 90, and 120 minutes. After the experiment, the mice were returned to the cage and provided with sufficient food and drinking water.

[0053] 2.5 Sample collection and measurement

[0054] Fecal samples of mice were collected once before and after the treatment, and stored at -80°C in time. After 5 weeks of treatment, blood, tissue samples and colon contents of mice were collected under sterile conditions. The blood samples were centrifuged to obtain serum, and both the serum and colon contents were stored at -80°C for later use. Serum was used to measure blood indicators (using the ELISA kit provided by Shanghai Xinyu Biotechnology Co., Ltd.), colon contents were used to measure short-chain fatty acids, and fecal samples were used for metagenomic determination (completed by Beijing Novogene Technology Co., Ltd.). The dissected liver, kidney, and pancreatic samples were divided into two parts, one was placed in a sterile centrifuge tube, quickly frozen in liquid nitrogen, and then stored at -80°C; the other was rinsed with 0.85% saline, fixed in a paraformaldehyde solution, and used for subsequent tissue sectioning (by Wuhan Sevier Biotechnology Co., Ltd.). The determination of short-chain fatty acids was carried out by gas chromatography-mass spectrometry of Agilent Technologies Co., Ltd. The specific conditions were as follows: the column was Agilent DB-WAX (0.25 mm × 0.25 μm × 50 cm); the injection port temperature was set to 250 °C; the gas interface temperature was 250 °C; the carrier gas flow rate was 1.5 mL / min; the split ratio was 3:1; and the injection volume was 1 μL.

[0055] 3. Statistical Analysis

[0056] All statistical analyses were performed with the help of RStudio software, and the graphics were performed using "Graphpad Pism9.5" software. The p value was used as the basis for judgment, and the p value threshold was set to 0.05. When p < 0.05, it was considered that there was a statistically significant difference; when p < 0.01, the difference was considered to be extremely significant.

[0057] 2. Results

[0058] 2.1 Selection of Bifidobacterium B960 based on HPLC

[0059] The degradation rate of glibenclamide after 6 hours of co-culture with bifidobacteria was determined by high performance liquid chromatography. In the intestinal environment, glibenclamide is expected to maintain high stability and bioavailability. From the degradation rate results, Bifidobacterium B960 performed relatively well, and its degradation rate of glibenclamide was relatively low. Figure 1As shown. This means that when glibenclamide is co-cultured with Bifidobacterium B960, glibenclamide is less affected by degradation, and more drugs can play a hypoglycemic effect, thereby improving the efficacy and stability of the drug. Therefore, the selection of Bifidobacterium B960 and glibenclamide to further explore the synergistic effect between the two will help to gain a deeper understanding of the interaction mechanism between this specific strain and the drug, and provide valuable basis and reference for drug development, clinical medication, and the study of the relationship between intestinal flora and drug metabolism. It also opens up new ideas for optimizing the treatment strategy for related diseases such as diabetes.

[0060] 2.2 Bifidobacterium B960 synergizes with glibenclamide to enhance the therapeutic effect

[0061] The therapeutic effects of glibenclamide and probiotic B960 were evaluated by monitoring fasting blood glucose in type 2 diabetic mice. Figure 2 As shown in the figure, at the beginning of the experiment, the fasting blood glucose of the T2DM group, G group, B960 group and G+B960 group was higher than that of the CON group. During the treatment from 1 to 5 weeks, the decline in fasting blood glucose in the G+B960 group was significantly greater than that in the G group, B960 group and T2DM group. By the 5th week, the blood glucose level of the G+B960 group dropped to the lowest, indicating that the combined use of drugs is more effective in lowering blood glucose, which may be due to the synergistic promotion of insulin secretion by the two. In addition, the blood glucose fluctuations in the G+B960 group were smaller and more stable, which may be due to the regulatory effects of probiotics on the intestine and inflammation, which synergized with glibenclamide to maintain blood glucose stability. It can be seen that combined use of drugs has obvious advantages in lowering blood glucose and maintaining blood glucose stability.

[0062] In addition, the blood glucose, insulin, glucagon-like peptide-1, and glycosylated serum protein in the serum of type 2 diabetic mice after using glibenclamide and probiotic B960 were tested. Figure 3As shown in the figure: The serum blood glucose content in the G+B960 group was significantly lower than that in the T2DM group (p<0.01), and there was a trend of further decrease compared with the G group (p<0.05). This shows that glibenclamide combined with probiotics B960 has a synergistic effect in lowering blood glucose, which may be achieved through multiple pathways such as promoting glucose metabolism and regulating intestinal flora to improve insulin resistance. The insulin content in the G+B960 group was significantly higher than that in the T2DM group (p<0.01), and the difference was significant compared with the G group (p<0.05), indicating that the combined use can better promote insulin secretion or improve pancreatic β-cell function, thereby enhancing the ability to regulate blood glucose. The glucagon-like peptide-1 content in the G+B960 group was significantly different from that in the G group (p<0.05), but there was no significant difference compared with the B960 group and the T2DM group, which suggests that the combined use may have a positive effect on the secretion or metabolism of glucagon-like peptide-1, thereby regulating blood glucose. Glycated serum protein can reflect the average blood sugar level in the past 2 to 3 weeks. The glycated serum protein content in the G+B960 group was significantly lower than that in the T2DM group (p<0.01), and significantly lower than that in the G group and B960 group (p<0.05), indicating that the G+B960 group can more effectively control the long-term stability of blood sugar and reduce the damage of blood sugar fluctuations to the body.

[0063] In summary, the combined use of glibenclamide and Bifidobacterium B960 in regulating blood glucose and serum-related indicators in type 2 diabetic mice is more effective than the single use of glibenclamide, Bifidobacterium B960 and the unintervention T2DM group.

[0064] 2.3 Glucose tolerance test in mice

[0065] The blood glucose concentration change curves of different groups intuitively present the dynamic changes of blood glucose in mice after oral glucose administration. Figure 4As shown in the figure, after oral administration of glucose, the G+B960 group performed better than the G group in terms of blood sugar regulation. Specifically, about 15 minutes after oral administration of glucose, the peak blood sugar level of the G+B960 group was significantly lower than that of the G group, indicating that the G+B960 group can more effectively inhibit the short-term sharp rise in blood sugar, which may be due to the synergistic effect of the two, promoting the initial uptake and metabolism of glucose, or regulating the secretion of hormones related to blood sugar in the intestine. In the period of 15 to 120 minutes, the blood sugar level of the G+B960 group decreased faster, and its blood sugar level was significantly lower than that of the G group at 120 minutes. This shows that the G+B960 group has a more lasting and efficient effect on regulating blood sugar, which may be due to the improvement of intestinal microecology by Bifidobacterium B960, enhancing insulin sensitivity, and cooperating with glibenclamide to better promote the uptake and utilization of glucose by peripheral tissues, accelerating the clearance of blood sugar. In addition, the blood sugar fluctuation range of the G+B960 group was significantly smaller than that of the G group, reflecting that the G+B960 group has a significant advantage in maintaining blood sugar stability. This may be because Bifidobacterium B960 improves the intestinal barrier function and reduces the inflammatory response caused by the entry of harmful substances such as endotoxins into the blood, thereby alleviating the interference with the blood sugar regulation system. Therefore, these results suggest that the combined use of glibenclamide and Bifidobacterium B960 may have a positive effect on blood sugar regulation in type 2 diabetic mice through multiple synergistic mechanisms.

[0066] 2.4 Bifidobacterium synergistically treats glibenclamide to alleviate the side effects of glibenclamide and type 2 diabetes

[0067] Experimental studies have found that the use of glibenclamide alone can increase transaminase activity, induce lipid metabolism disorders, and increase the level of inflammatory factors. This series of changes indicates that taking glibenclamide may cause damage to the liver, lead to the accumulation of lipid substances in the body, and persist in inflammatory reactions, thereby increasing the risk of cardiovascular disease and the occurrence of other complications.

[0068] However, when glibenclamide was combined with Bifidobacterium B960 for treatment, the experimental results showed positive changes. Figure 5 As shown, this result strongly shows that the combined treatment has a protective or improving function on the liver. In terms of lipid metabolism, the content of α-glucose aminotransferase decreased significantly (p<0.05), while that of β-glucose aminotransferase increased significantly (p<0.05), which indicates that the combined treatment can effectively regulate lipid metabolism and reduce the risk of cardiovascular disease. In addition, the activity of alanine aminotransferase and aspartate aminotransferase, serum cholesterol and low-density lipoprotein cholesterol, high-density lipoprotein cholesterol content, interleukin IL-6 and tumor necrosis factor α content decreased (p<0.05), which means that the combined treatment has an anti-inflammatory effect and is beneficial to improve insulin resistance.

[0069] The results of serum index characterization of experimental mice showed that supplementing Bifidobacterium B960 with glibenclamide in the treatment of type 2 diabetes can not only reduce the side effects of glibenclamide, but also enhance its therapeutic effect. This potential synergistic or auxiliary therapeutic value is worthy of in-depth exploration of its mechanism of action, and is expected to open up new ideas for the comprehensive treatment of type 2 diabetes.

[0070] By observing the liver sections, we can clearly see the differences in liver tissue pathology in different groups, such as Figure 6 As shown. In the liver tissue of the CON group, the hepatocytes around the central vein were arranged in a regular radial pattern, with clear cell boundaries, abundant microvacuoles in the cytoplasm, normal liver sinusoidal structure, no fibrosis, necrosis, or inflammatory infiltration, and were in a normal physiological state. Due to the influence of type 2 diabetes, the livers of the T2DM group had disordered arrangement of hepatocytes around the central vein, fatty degeneration and hydropic degeneration, abnormal nuclei of some hepatocytes, and occasionally intranuclear inclusions were observed. The tissue structure and cell morphology of the liver were obviously abnormal. After treatment with glibenclamide in the G group, the liver tissue showed various pathological changes. The hepatocytes lost their normal order, fatty degeneration and hydropic degeneration were obvious, and ballooning and necrosis also occurred, suggesting that glibenclamide was not effective in improving liver conditions and may even have adverse effects. The liver morphology of the G+B960 group was outstanding. Although the arrangement of liver cells had not yet completely recovered, the fatty degeneration was less severe and there was no obvious fibrosis, necrosis, or inflammatory infiltration, which fully proved that the combined treatment had a good protective and improving effect on the liver. The liver morphology of the B960 group was also good, with a low degree of fatty degeneration, which shows that the use of Bifidobacterium B960 alone can also protect the liver and reduce the damage to the liver caused by type 2 diabetes.

[0071] In this experiment, if Figure 6As shown in the figure, after observing and analyzing the pancreatic sections of each group, it was found that the pancreatic sections of the CON group showed that the islet structure was clearly visible, the islet cells were evenly distributed, and the cell morphology was normal, which fully demonstrated that the pancreatic tissue function was normal and the islet function was good. The pancreatic sections of the T2DM group showed that the islet structure was destroyed, the number of islet cells was significantly reduced, and the cells were loosely arranged. This phenomenon is most likely caused by islet cell apoptosis, which is consistent with the actual situation of impaired islet function in patients with type 2 diabetes, and then caused a series of problems such as insufficient insulin secretion. Although the islet structure and cell morphology of the G group were restored compared with the T2DM group, they still did not reach the normal level of the CON group. It can be seen that glibenclamide treatment can improve islet function to a certain extent, but its effect has certain limitations. The islet structure and cell morphology of the G+B960 group are closer to those of the CON group, and the islet cells are relatively evenly distributed. This strongly proves that the combined treatment of Bifidobacterium B960 and glibenclamide can more effectively protect the islet structure and function and promote the recovery of islet cells. It is worth noting that the pancreatic islet structure and cell morphology in the B960 group also improved to a certain extent, suggesting that when Bifidobacterium B960 is used alone, it also has a certain protective effect on the pancreas. Its possible mechanism of action is to indirectly have a positive effect on pancreatic islet function by regulating the intestinal microenvironment and other pathways.

[0072] The results of liver and pancreatic sections showed that the synergistic treatment of Bifidobacterium B960 and glibenclamide was significantly effective in improving liver pathology and protecting pancreatic islet function, which was better than the use of glibenclamide or Bifidobacterium B960 alone. This result echoes the previous research results that combined treatment can alleviate the side effects of glibenclamide, regulate lipid metabolism, reduce inflammation, and improve the intestinal microenvironment.

[0073] 2.5 Increase short acid content and improve intestinal microenvironment

[0074] like Figure 7 As shown in the study of type 2 diabetic mouse model, it was found that the effect of glibenclamide alone on the content of short-chain fatty acids was relatively limited. However, when glibenclamide was used in combination with Bifidobacterium B960 for treatment, or when Bifidobacterium B960 was used alone for treatment, the contents of acetic acid, propionic acid, butyric acid, isobutyric acid and isovaleric acid were significantly increased. Moreover, the combined treatment had a similar effect to that of Bifidobacterium B960 alone in increasing the content of these short-chain fatty acids. This indicates that Bifidobacterium B960 plays an important role in regulating the metabolism of short-chain fatty acids in type 2 diabetic mice, and combined treatment with glibenclamide may produce a synergistic effect. However, no significant difference in the content of caproic acid was observed between the groups in the experiment, indicating that the effect of this treatment on the content of caproic acid was small or not obvious. These research results provide certain experimental basis for further exploring the treatment strategies of diabetes and the relationship between intestinal flora and diabetes.

[0075] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. The use of glibenclamide in combination with Bifidobacterium B960 in the preparation of a drug for the treatment of type 2 diabetes. The Bifidobacterium B960 was deposited in the Guangdong Provincial Microbiological Culture Collection Center GDMCC on October 23, 2024. The storage address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The storage number is GDMCC No: 65330, and the classification name is: Bifidobacteriumanimalis.

2. The use according to claim 1, characterized in that: The combined use of glibenclamide and Bifidobacterium B960 can enhance the effect of lowering blood sugar, and the blood sugar fluctuation is smaller and the stability is better.

3. The use according to claim 1, characterized in that: The combination of glibenclamide and Bifidobacterium B960 can better promote insulin secretion or improve pancreatic β-cell function, thereby enhancing the ability to regulate blood sugar.

4. The use according to claim 1, characterized in that: The combination of glibenclamide and Bifidobacterium B960 can have a positive effect on the secretion or metabolism of glucagon-like peptide-1, thereby regulating blood sugar.

5. The use according to claim 1, characterized in that: The combined use of glibenclamide and Bifidobacterium B960 can significantly reduce the content of glycated serum protein, thereby more effectively controlling the long-term stability of blood sugar and reducing the damage to the body caused by blood sugar fluctuations.

6. The use according to claim 1, characterized in that: The combined use of glibenclamide and Bifidobacterium B960 can significantly reduce the activities of alanine aminotransferase and aspartate aminotransferase.

7. The use according to claim 1, characterized in that: The combined use of glibenclamide and Bifidobacterium B960 can significantly reduce the levels of serum cholesterol and low-density lipoprotein cholesterol, and significantly increase the level of high-density lipoprotein cholesterol.

8. The use according to claim 1, characterized in that: The combination of glibenclamide and Bifidobacterium B960 can reduce the levels of interleukin IL-6 and tumor necrosis factor α.

9. The use according to claim 1, characterized in that: The combination of glibenclamide and Bifidobacterium B960 is effective in improving liver pathology and protecting pancreatic islet function.

10. The use according to claim 1, characterized in that: The combination of glibenclamide and Bifidobacterium B960 can significantly increase the contents of acetic acid, propionic acid, butyric acid, isobutyric acid and isovaleric acid.

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