Application of Bacteroides xylanacinium DSM 18836 in the preparation of drugs for treating diabetic nephropathy

Abstract

The application belongs to the technical field of microorganisms, and discloses application of Bacteroides xylanisolvens DSM 18836 in preparation of a medicine for treating diabetic nephropathy. After the diabetic nephropathy mice are treated by gavage with Bacteroides xylanisolvens DSM 18836 live bacterial suspension or fermentation supernatant, the fasting blood glucose of the mice is significantly reduced, the urine protein and microalbumin positive rate are obviously decreased, the renal tubular vacuolar degeneration is reduced, and the kidney histological damage score is reduced. It is found for the first time that Bacteroides xylanisolvens DSM 18836 and the fermentation supernatant have obvious therapeutic effects on type 2 diabetic nephropathy, and Bacteroides xylanisolvens DSM 18836 and the fermentation supernatant have potential application prospects in preparation of a medicine for treating type 2 diabetic nephropathy.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and more specifically, relates to the application of Bacteroides xylana DSM 18836 in the preparation of a drug for treating diabetic nephropathy. Background Technology

[0002] With the development of multi-omics technologies, the remote regulation of kidney function by the gut microbiota through metabolic and immune pathways has attracted widespread attention. Studies have shown that patients with diabetic nephropathy (DKD) exhibit significant gut microbiota dysbiosis; a decrease in beneficial bacteria and an increase in pathogenic bacteria can exacerbate inflammatory responses and drive DKD progression. Clinical reports indicate that dapagliflozin combined with probiotics (Bifidobacterium longum, Lactobacillus bulgaricus, and Streptococcus thermophilus) can improve blood glucose and kidney function in DKD patients; probiotics combined with irbesartan can also reduce kidney damage in DKD rats. Therefore, correcting gut microbiota imbalance through probiotic supplementation holds promise as a new strategy for the prevention and treatment of DKD, but the currently applicable strains remain very limited.

[0003] Digestive kidney disease (DKD) is the most common and serious microvascular complication of diabetes mellitus, characterized by persistent proteinuria and a progressive decline in glomerular filtration rate, ultimately leading to renal failure. DKD is not only a leading cause of end-stage renal disease but also an independent risk factor for cardiovascular disease, seriously endangering human health. Current clinical measures to delay DKD include lifestyle interventions and strict control of blood glucose and blood pressure, but their efficacy is limited. In cases of moderate to severe renal impairment, medication dosages need to be reduced or discontinued, and late-stage patients can only rely on dialysis or transplantation. Therefore, there is an urgent need to develop novel treatment strategies.

[0004] *Bacteroides xylanisolvens*, a symbiotic anaerobic bacterium isolated from the gut of healthy humans, exhibits good safety profiles and is a candidate strain for next-generation probiotics. Previous studies have shown that this bacterium can improve non-alcoholic fatty liver disease by synthesizing folic acid and can synergistically alleviate metabolic disorders in obese mice with *Clostridium butyricum*. Currently, there are reports of using *Bacteroides* strains to prepare drugs for treating diabetes. However, there are no reports of using *Bacteroides xylanisolvens* and its fermentation supernatant for the treatment of diabetic nephropathy, and the specific applications of existing *Bacteroides* strains in the treatment of DKD still require further development. Summary of the Invention

[0005] Based on the aforementioned deficiencies in the existing technology, the present invention aims to provide a new use for Bacteroides xylanase DSM 18836 in the treatment of DKD, and to provide a safe and effective probiotic preparation for clinical use.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] This invention first provides the use of Bacteroides xylanisolvens DSM 18836 in the preparation of a medicament for treating diabetic nephropathy.

[0008] Specifically, the aforementioned diabetic nephropathy refers to type 2 diabetic nephropathy.

[0009] This invention also provides the use of Bacteroides xylanisolvens DSM 18836 in the preparation of a medicament for diabetic nephropathy in mice.

[0010] Preferably, in the above applications, the Bacteroides xylanisolvens DSM18836 has at least one of the following effects:

[0011] (1) Reduces fasting blood glucose in mice;

[0012] (2) Reduced the positive rate of urinary protein in mice;

[0013] (3) Reduce the positive rate of microalbumin in mice;

[0014] (4) Alleviate pathological damage to kidney tissue;

[0015] More preferably, in the above applications, the bacterial cells or fermentation supernatant of Bacteroides xylanisolvens DSM18836 are used as the active ingredient of the drug.

[0016] Preferably, when Bacteroides xylanisolvens DSM 18836 is used as the bacterial cell, the dosage for mice is 1×10⁻⁶. 9 CFU / mouse / day, when using Bacteroides xylanisolvens DSM18836 as fermentation supernatant, the dosage for mice is 0.1 mL / mouse / day.

[0017] The *Bacteroides xylanoplasmosis* DSM 18836 described in this invention can be a strain obtained in any manner, including but not limited to natural strains isolated from the intestines of healthy individuals, recombinant strains modified through genetic engineering, and derivative strains obtained through passage culture. The fermentation supernatant is a cell-free supernatant obtained by centrifuging and filtration after anaerobic culture of *Bacteroides xylanoplasmosis* DSM 18836 in a suitable culture medium.

[0018] The Bacteroides xylanain-derived DSM 18836 described in this invention can be in live or inactivated form; it can be the bacterial cell itself, its metabolites, or its extracts.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention discloses the application of Bacteroides xylanis DSM 18836 in the preparation of drugs for treating diabetic nephropathy.

[0021] Live xylanobacterium and its fermentation supernatant can significantly reduce fasting blood glucose in diabetic nephropathy mice. The fermentation supernatant is more effective in reducing the positive rate of urinary protein and microalbumin, and can completely block the occurrence of proteinuria.

[0022] Live Bacteroides xylana can significantly reduce renal tubular vacuolar degeneration, decrease renal histological damage scores, and improve PAS-positive extracellular matrix deposition in diabetic nephropathy mice, demonstrating a significant protective effect against kidney damage under hyperglycemic conditions.

[0023] The fermentation supernatant of Bacteroides xylanase can also effectively alleviate pathological damage to kidney tissue, but the degree of improvement is slightly weaker than that of live bacteria.

[0024] Live Bacteroides xylanacinium DSM 18836 and its fermentation supernatant can both be used to effectively delay the onset and progression of diabetic nephropathy. This invention provides a safe and effective novel probiotic for the clinical treatment of diabetic nephropathy. Attached Figure Description

[0025] Figure 1 To investigate the effect of Bacteroides xylania on fasting blood glucose in DKD mice; where A represents before intervention and B represents after intervention;

[0026] Figure 2 To investigate the effect of Bacteroides xylania on renal function in DKD mice; where A represents the positive rate of urinary protein and B represents the positive rate of microalbumin.

[0027] Figure 3 To investigate the effects of Bacteroides xylanica on the pathological damage of renal tissue in DKD mice; A represents HE and PAS staining results, B represents quantitative analysis of renal tubular vacuolar area, and C represents renal tissue damage score. The scale bar for HE and PAS staining is 100 μm. Detailed Implementation

[0028] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0029] Bacteroides xylanisolvens was purchased from BNCC, and its corresponding German Microbial Culture Collection (DSMZ) accession number is DSM 18836.

[0030] Example 1: Therapeutic effect of Bacteroides xylanisolvens on diabetic nephropathy mice

[0031] I. Experimental Methods

[0032] (1) Animal experimental design

[0033] After one week of acclimatization, mice were randomly divided into a normal control group (STZ+ND+PBS), a model control group (STZ+HFD+PBS), a BHI medium control group (STZ+HFD+BHI), a live bacteria *Bacteroides xylanoplastin* group (STZ+HFD+BX), a fermentation supernatant group (STZ+HFD+BX-SUP), and a positive control group (STZ+HFD+PI). The normal control group was given a standard maintenance diet (ND), while the other groups were given a high-fat diet (HFD) for 6 weeks (weeks 1-6).

[0034] Starting in week 5, except for the normal control group, mice in all other groups were administered an antibiotic mixture (ABX) via gavage daily for 7 consecutive days to clear the intestinal flora. In week 6, ABX was added to the drinking water for another 7 days. In week 7, mice in the model control group and all treatment groups were intraperitoneally injected with streptozotocin (STZ, 50 mg / kg / day, freshly prepared with pH 4.5 sodium citrate buffer solution), fasted for 12 hours before injection, for 5 consecutive days. 72 hours after the last STZ injection, fasting blood glucose (FBG) was measured via tail vein sampling. A FBG ≥ 16.7 mmol / L sustained for 3 days was considered a successful diabetes model. The normal control group received an equal volume of citrate buffer. Weeks 8 and 9 were the recovery period after STZ injection; mice in all groups continued to be fed the same diet without gavage intervention. From week 10 to week 13, mice in each group received the corresponding gavage intervention: the normal control group and the model control group were gavaged with an equal volume of PBS; the BHI group was gavaged with BHI culture medium; and the BX group was gavaged with a suspension of live xylan-lysed Bacteroides (1×10⁻⁶). 9The interventions included: CFU / mouse / day for the BX supernatant group (0.2 mL / mouse / day via gavage); and pioglitazone solution (10 mg / kg / day) for the positive control group (0.2 mL / mouse via gavage). All interventions were administered once daily. During the intervention period, all groups continued to be fed the corresponding diet, and body weight and fasting blood glucose were recorded weekly. At the end of week 13, 24-hour urine was collected from mice in metabolic cages, and the mice were sacrificed. Blood and kidney tissue samples were collected for subsequent testing.

[0035] (2) Fasting blood glucose test

[0036] Before intervention (7 days after STZ injection) and after intervention (after the last gavage), mice in each group were fasted for 12 hours (with no restriction on water), and blood was collected from the tail vein. Fasting blood glucose (FBG) was measured using a blood glucose meter and matching test strips. Each sample was measured twice, and the average value was taken.

[0037] (3) Urine index testing

[0038] Urine protein (PRO), urine glucose (GLU), and microalbumin (MA) in mice were detected using a kit. Urine specific gravity (SG) and pH were measured using a dry chemical test strip method. A positive result for urine protein was defined as PRO ≥ 0.15 g / L, and a positive result for microalbumin was defined as MA ≥ 100 mg / L. The positive rates for each group were recorded.

[0039] (4) HE staining of kidney tissue

[0040] The removed kidney tissue was fixed in 4% paraformaldehyde solution for 24 h and then embedded in paraffin. After embedding, the tissue was sectioned. The sections were dewaxed in xylene, then washed sequentially with anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 min each, and finally rinsed with distilled water. Afterward, the sections were stained with hematoxylin for 5-10 min, rinsed thoroughly with water for bluing, stained with eosin for 5 min, dehydrated in anhydrous ethanol three times in a gradient, and then immersed twice in xylene. Finally, the sections were mounted with neutral resin and air-dried in a fume hood.

[0041] (5) PAS staining of kidney tissue

[0042] After dewaxing and hydration, paraffin sections were rinsed with distilled water; the sections were then immersed in a 1% periodic acid solution for oxidation at room temperature for 10–15 min, followed by thorough rinsing with distilled water; Schiff's reagent was applied for staining at room temperature in the dark for 10 min; the sections were then rinsed with running water for 5 min; subsequently, nuclear counterstaining (blue) was performed with Harrison hematoxylin for 3 min; the sections were then rinsed with running water for 5 min, and appropriate blue re-staining could be performed as needed; finally, routine dehydration, clearing, and mounting were performed. The sections were then scanned and observed using a slide scanner.

[0043] (6) Quantitative analysis of renal tubular vacuolar area and histological damage score

[0044] For each HE-stained slide, five non-overlapping renal tubular fields were randomly selected at 200x magnification. Image analysis software (ImageJ) was used to analyze the proportion of vacuolar area in renal tubular epithelial cells, calculating the percentage of vacuolar area to the total field area. The average of the five fields was taken as the vacuolar area proportion for that sample. The degree of renal tubular damage was assessed using a semi-quantitative scoring system from 0 to 4 points, based on literature standards: 0 points for no damage, 1 point for <25% damage area, 2 points for 25-50%, 3 points for 50-75%, and 4 points for >75%. All pathological scores were performed by two independent observers without prior knowledge of the group assignments, and the results were averaged.

[0045] (7) Statistical analysis

[0046] Statistical analysis was performed using GraphPad Prism 8.0 software. Quantitative data were expressed as mean ± standard deviation (x ± SD), and one-way ANOVA was used for comparisons among multiple groups. Fisher's exact test was used for categorical data (positive rate). A p-value < 0.05 was considered statistically significant; * indicated p < 0.05, ** indicated p < 0.01, and *** indicated p < 0.001.

[0047] II. Experimental Results

[0048] (1) Effects of Bacteroides xylanisolvens on blood glucose and renal function in DKD mice

[0049] Effects of Bacteroides xylania on fasting blood glucose in DKD mice Figure 1 As shown in A and B, after STZ injection combined with a high-fat diet, the fasting blood glucose level of mice in the model group (STZ+HFD+PBS) was significantly increased. Figure 1 As shown in Figure A, before intervention, compared with the normal control group (STZ+ND+PBS), fasting blood glucose levels in all model groups (STZ+HFD+PBS, STZ+HFD+BX, STZ+HFD+BHI, STZ+HFD+BX-sup) were significantly elevated (P<0.05), while there were no significant differences among the model groups (P>0.05). Figure 1 As shown in Figure B, after 4 weeks of intervention, compared with the model control group (STZ+HFD+PBS), the fasting blood glucose levels in the BX group (STZ+HFD+BX) and the BX supernatant group (STZ+HFD+BX-sup) were significantly lower (P<0.05), while there was no significant difference in the BHI group. These results indicate that live Bacteroides xylana and its fermentation supernatant can effectively reduce fasting blood glucose in DKD mice.

[0050] Effects of Bacteroides xylania on renal function in DKD mice Figure 2 As shown in A and B, after induction with STZ combined with a high-fat diet, mice in the model group showed significant renal function impairment: the positive rate of urinary protein was 40%, and the positive rate of microalbumin was 60%. After gavage administration of live Bacteroides xylanalyticum (STZ+HFD+BX), the positive rate of urinary protein decreased to 0%, and the positive rate of microalbumin decreased to 50%, representing decreases of 100% and 16.7% respectively compared to the model group. Mice administered gavage with Bacteroides xylanalyticum fermentation supernatant (STZ+HFD+BX-sup) showed both urinary protein and microalbumin positive rates to 0%, completely blocking the occurrence of proteinuria. However, mice administered gavage with BHI medium (STZ+HFD+BHI) showed urinary protein and microalbumin positive rates of 60%, with no significant improvement compared to the model group (P>0.05). In the positive drug group (STZ+HFD+PI), the positive rate of urinary protein was 0%, and the positive rate of microalbumin was 16.7%. The above experimental results indicate that both live Bacteroides xylana and its fermentation supernatant can effectively alleviate the pathological damage to the kidney tissue of DKD mice, with the fermentation supernatant showing a more significant effect in reducing proteinuria.

[0051] (2) Effects of Bacteroides xylanisolvens on renal pathological damage in DKD mice

[0052] HE staining results are as follows Figure 3 As shown in Figure A, the kidney tissue of mice in the normal control group (STZ+ND+PBS) was structurally intact, with regular glomerular morphology and neatly arranged tubules, showing no obvious pathological changes. The kidney tissue of mice in the model control group (STZ+HFD+PBS) showed significant damage, manifested as widespread vacuolar degeneration of renal tubular epithelial cells, dilation of some renal tubules, structural disorder, and some degree of interstitial changes. Compared with the model group, gavage administration of live Bacteroides xylana (STZ+HFD+BX) significantly reduced vacuolar degeneration of renal tubules and restored the tissue structure; gavage administration of fermentation supernatant (STZ+HFD+BX-sup) also improved renal tubular damage, but to a slightly weaker degree than the live bacteria group; no significant improvement was observed in the BHI control group (STZ+HFD+BHI).

[0053] PAS staining results are as follows Figure 3 As shown in Figure A, the PAS-positive material in the glomerular and tubular basement membranes of mice in the model group was significantly increased, as evidenced by deeper staining, indicating increased deposition of glycoproteins and extracellular matrix. After intervention with Bacteroides xylana, the PAS staining intensity in both the BX and BX-sup groups decreased, and the deposition of mesangial matrix and basement membrane was reduced. The improvement was more significant in the BX group, while no significant change was observed in the BHI group.

[0054] Further quantitative analysis of renal tubular vacuolar area, such as... Figure 3As shown in Figure B, the proportion of vacuolar area in the model group mice was significantly higher than that in the normal control group (P<0.001); both the BX bacteria group and the BX-sup group significantly reduced the vacuolar area (P<0.01 and P<0.05, respectively), with the BX bacteria group showing a greater reduction. The results of the renal tissue damage score were consistent with these findings. Figure 3 The results showed that the BX group score was significantly lower than that of the model group (P<0.01), the BX-sup group also showed improvement (P<0.05), while there was no significant difference in the BHI group.

[0055] The above experimental results indicate that both live Bacteroides xylana and its fermentation supernatant can effectively alleviate the pathological damage to the kidney tissue in DKD mice, with the live bacteria playing a more significant role in improving the kidney tissue structure.

[0056] In summary, both live *Bacteroides xylana* and its fermentation supernatant significantly reduced fasting blood glucose in DKD mice. The fermentation supernatant was particularly effective in reducing the positive rates of urinary protein and microalbumin, completely blocking proteinuria; while the BHI medium control showed no such effect. Live *Bacteroides xylana* also significantly alleviated renal tubular vacuolar degeneration in DKD mice, reduced renal histological damage scores, and improved PAS-positive extracellular matrix deposition, demonstrating a significant protective effect against kidney damage under hyperglycemic conditions. Simultaneously, the fermentation supernatant also effectively alleviated renal tissue pathological damage, but the degree of improvement was slightly weaker than that of the live bacteria. These results indicate that live *Bacteroides xylana* DSM 18836 and its fermentation supernatant can effectively delay the onset and progression of diabetic nephropathy.

Claims

1. Application of Bacteroides xylanisolvens DSM 18836 in the preparation of drugs for treating diabetic nephropathy.

2. Application of Bacteroides xylanisolvens DSM 18836 in the preparation of drugs for diabetic nephropathy in mice.

3. The application of Bacteroides xylanisolvens DSM 18836 according to claim 2, characterized in that, The Bacteroides xylanisolvens DSM 18836 described herein possesses at least one of the following effects: (1) Reduces fasting blood glucose in mice; (2) Reduced the positive rate of urinary protein in mice; (3) Reduce the positive rate of microalbumin in mice; (4) Relieve pathological damage to kidney tissue.

4. The application of Bacteroides xylanisolvens DSM 18836 according to claim 3, characterized in that, The active ingredient of the drug is either the bacterial cells or fermentation supernatant of Bacteroides xylanisolvens DSM 18836.

5. The application of Bacteroides xylanisolvens DSM 18836 according to claim 4, characterized in that, When Bacteroides xylanisolvens DSM 18836 is in bacterial form, the dosage for mice is 1×10⁻⁶. 9 CFU / mouse / day, when using Bacteroides xylanisolvens DSM18836 as fermentation supernatant, the dosage for mice is 0.1 mL / mouse / day.

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