Use of human indigenous strain lactobacillus rhamnosus tg032 in combination with allopurinol for the preparation of a medicament for the treatment of type 2 diabetes

Abstract

This invention discloses the application of a combination of human-derived native strain *Lactobacillus rhamnosus* TG032 and allopurinol in the preparation of a drug for treating type 2 diabetes, belonging to the field of biomedical technology. The drug contains an effective amount of human-derived native strain *Lactobacillus rhamnosus* TG032 and an effective amount of allopurinol. Experiments show that, compared with the single components, the combination can synergistically reduce serum uric acid, synergistically reduce the levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β, and synergistically increase the level of interleukin-10, synergistically increase the level of glucagon-like peptide-1, and synergistically improve insulin resistance, thereby synergistically improving abnormal glucose metabolism and abnormal uric acid metabolism in type 2 diabetes, with good safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a human-derived native strain of Lactobacillus rhamnosus TG032 in combination with allopurinol in the preparation of a drug for treating type 2 diabetes. Background Technology

[0002] Metabolic diseases are a group of chronic diseases caused by disorders in the metabolism of substances such as carbohydrates, lipids, and purines. They mainly include diabetes mellitus, hyperuricemia, obesity, and metabolic syndrome. In recent years, with population aging and changes in lifestyle and dietary structure, the incidence of metabolic diseases has continued to rise, becoming a significant public health problem seriously affecting human health. Type 2 diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia, and its development is often accompanied by abnormalities in lipid and purine metabolism. Numerous studies have shown a close link between abnormal glucose metabolism and abnormal uric acid metabolism, with insulin resistance, chronic inflammation, and oxidative stress all contributing to their development. Patients with type 2 diabetes often have elevated serum uric acid levels, and abnormal uric acid metabolism can further exacerbate glucose metabolism disorders. Therefore, simultaneously improving abnormal glucose and uric acid metabolism has become an important direction for the comprehensive intervention of metabolic diseases.

[0003] Allopurinol is a classic clinical uric acid-lowering drug, belonging to the xanthine oxidase (XOD) inhibitor class. It reduces uric acid synthesis in the body by competitively inhibiting xanthine oxidase. However, in actual clinical application and long-term intervention for patients with metabolic syndrome, the use of chemical drugs alone has revealed significant limitations of "single-target intervention" and the risk of cumulative toxic side effects. On the one hand, allopurinol has a single mechanism of action, focusing only on the uric acid synthesis link in the purine metabolism pathway, and cannot substantially intervene in the pathological links of abnormal glucose metabolism such as concurrent insulin resistance and hyperglycemia. In clinical practice, abnormal glucose metabolism and abnormal uric acid metabolism are often mutually causal. For example, hyperinsulinemia inhibits the renal tubular excretion of uric acid. If uric acid is lowered solely by allopurinol, the overall metabolic microenvironment in the patient's body is not fundamentally reversed. On the other hand, long-term high-dose use of allopurinol carries a clear risk of liver and kidney toxicity and is prone to inducing severe allergic reactions such as drug rashes. If multiple chemical hypoglycemic drugs are used in combination to achieve blood sugar control, it will further increase the metabolic burden on the liver and kidneys and the risk of adverse drug reactions.

[0004] In recent years, the application of probiotics in metabolic diseases has received widespread attention. Studies have shown that probiotics can play a positive role in abnormal glucose metabolism by regulating gut microbiota composition, improving intestinal barrier function, and modulating inflammatory responses and host metabolism. *Lactobacillus rhamnosus* (… Lactobacillus rhamnosusAs a type of lactic acid bacteria with good safety and probiotic properties, some strains have been reported to improve glucose metabolism or degrade purine nucleosides and lower blood uric acid. However, existing technologies have not provided any technical insights into combining specific human Lactobacillus rhamnosus strains with allopurinol to achieve a "synergistic" improvement in abnormal glucose and uric acid metabolism: First, existing Lactobacillus rhamnosus strains have a narrow degradation spectrum and limited degradation efficiency for purine nucleosides, making it difficult to fully block the absorption of exogenous purines at the source; Second, the compatibility rules, dosage ratios, and synergistic effects of combining chemical uric acid-lowering drugs with probiotics are still unclear, and those skilled in the art cannot predict whether the combination will produce a synergistic effect rather than a simple additive effect; Third, existing technologies do not involve technical solutions for using specific strains in combination with allopurinol to simultaneously and synergistically lower blood uric acid, synergistically inhibit systemic inflammation, synergistically promote glucagon-like peptide-1 (GLP-1) secretion, and synergistically improve insulin resistance.

[0005] Therefore, developing a safe and effective approach that can simultaneously and synergistically improve abnormal glucose metabolism and abnormal uric acid metabolism in type 2 diabetes, and synergistically improve the shared inflammatory pathological basis of both, has significant clinical application value. Summary of the Invention

[0006] Technical problems to be solved To address the problems of existing technologies, such as allopurinol's single target and inability to effectively improve glucose metabolism abnormalities in type 2 diabetes, the risk of liver and kidney toxicity with long-term high-dose use, and the cumulative adverse reactions of combined drug use in comprehensive interventions for glucose and uric acid metabolism abnormalities, this invention provides a combined application scheme of human-derived native strain Lactobacillus rhamnosus and allopurinol. Allopurinol inhibits xanthine oxidase to reduce uric acid synthesis, while human-derived native strain Lactobacillus rhamnosus regulates intestinal flora to improve glucose metabolism abnormalities, achieving a comprehensive and synergistic improvement of glucose and uric acid metabolism abnormalities in type 2 diabetes, and reducing the risk of toxic side effects from single-drug use.

[0007] Technical solution This invention provides the application of a human-derived native strain of *Lactobacillus rhamnosus* TG032 combined with allopurinol in the preparation of a medicament for treating type 2 diabetes. The human-derived native strain of *Lactobacillus rhamnosus* is a human-derived native strain of *Lactobacillus rhamnosus* (… Lactobacillus rhamnosus TG032, the drug comprises an effective amount of the human-derived native strain Lactobacillus rhamnosus TG032 and an effective amount of allopurinol. Its preservation number is CCTCC NO: M 20251083. The human-derived native strain refers to a strain isolated from the intestinal flora of a healthy human and belonging to the host's own native flora.

[0008] In some embodiments, the human-derived native strain of Lactobacillus rhamnosus TG032 is a live bacterial cell or a freeze-dried bacterial cell powder.

[0009] In some embodiments, the viable count of the human-derived native strain Lactobacillus rhamnosus TG032 in the drug is 1 × 10⁻⁶. 6 ~1×10 12 CFU / g or 1×10 6 ~1×10 12 The CFU / mL concentration of allopurinol is 0.01–100 mg / g or 0.01–100 mg / mL. Preferably, the viable count of the human-derived native strain *Lactobacillus rhamnosus* TG032 is 1 × 10⁻⁶. 8 ~1×10 10 CFU / g or 1×10 8 ~1×10 10 CFU / mL, with allopurinol content ranging from 0.1 to 50 mg / g or 0.1 to 50 mg / mL.

[0010] In some embodiments, the drug is an oral preparation, and the dosage form is tablet, capsule, granule, powder, oral liquid or lyophilized powder.

[0011] In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier, diluent, excipient, or excipient.

[0012] In some embodiments, the human-derived native strain of Lactobacillus rhamnosus TG032 and the allopurinol are each present in their respective independent formulation units.

[0013] Beneficial effects Compared with the prior art, the present invention has the following beneficial effects: I. Synergistic reduction of blood uric acid: It acts on the dual sources of uric acid production, resulting in a significant synergistic effect.

[0014] Allopurinol blocks endogenous uric acid synthesis by inhibiting xanthine oxidase (XOD); the strain TG032 of this invention exhibits highly efficient degradation activity against adenine, adenosine, guanosine, and inosine (in vitro degradation rates of 100%, 100%, 94.60%, and 89.29%, respectively, significantly stronger than other strains of the same species), thus blocking the absorption of exogenous purine nucleosides at the intestinal source. The two form a dual blocking mechanism of "intestinal purine precursor degradation + inhibition of endogenous uric acid synthesis." Animal experiments showed that the serum uric acid level in the type 2 diabetes model group was 377.3±17.86 μmol / L; the TG032 monotherapy group decreased to 273.0±58.67 μmol / L (improvement rate 49.2%); the allopurinol monotherapy group decreased to 166.2±17.51 ​​μmol / L (improvement rate 99.6%); and the combination group further decreased to 95.13±20.04 μmol / L (improvement rate 133.2%), which was not only significantly better than the two monotherapy groups, but also lower than the level of the normal control group. Calculated using the King's Law, the synergistic index Q for the combination group in reducing serum uric acid was 1.334 (>1.15), indicating that the two drugs have a clear synergistic effect in reducing serum uric acid in type 2 diabetes, an effect that cannot be predicted by the simple summation of the effects of the two monotherapy drugs.

[0015] Second, it synergistically inhibits inflammation, with the most prominent synergistic effect.

[0016] The type 2 diabetes model group exhibited typical systemic low-grade inflammation: TNF-α 527.4±16.91 pg / mL, IL-1β 100.3±3.71 pg / mL, IL-6 89.69±3.23 pg / mL, and the anti-inflammatory factor IL-10 decreased to 169.0±8.10 pg / mL. After intervention: Regarding TNF-α, the level in the TG032 monotherapy group was 448.7±22.91 (improvement rate 33.1%), the level in the allopurinol monotherapy group was 412.7±22.06 (48.2%), and the level in the combination group decreased to 279.2±15.21 (104.4%), which was lower than that in the normal control group (289.6). The synergistic index Q = 1.596, which was the highest among all indicators. Regarding IL-1β, the level in the combination group decreased to 68.22±3.13 (81.7%), Q = 1.315. Regarding IL-6, the level in the combination group decreased to 71.12±4.35 (53.0%), Q = 1.161. At the same time, the level of the anti-inflammatory factor IL-10 in the combination group increased to 283.7±14.55 (80.1%), Q = 1.151. The synergistic inhibition of pro-inflammatory factors and the synergistic enhancement of anti-inflammatory factors indicate that this combination can synergistically reverse the systemic low-grade inflammatory state in type 2 diabetes, an effect that cannot be achieved by using either component alone.

[0017] Third, it synergistically promotes GLP-1 secretion and synergistically improves insulin resistance.

[0018] In the type 2 diabetes model group, serum GLP-1 decreased to 1.632±0.062 pg / mL, and fasting insulin increased to 29.43±0.931 pg / mL. In the TG032 monotherapy group, GLP-1 was 1.998±0.103 (improvement rate 32.0%), and in the allopurinol monotherapy group, it was 2.234±0.126 (52.7%). The combination group increased to 2.623±0.119 (86.7%), approaching the level of the normal control group (2.775±0.149), with a synergistic index Q=1.278. Regarding fasting insulin, the combination group decreased to 19.39±1.274 pg / mL (75.9%), with Q=1.179, showing a significantly better effect in improving insulin resistance than the monotherapy groups. The synergistic increase in GLP-1 levels and the synergistic improvement in insulin resistance corroborate each other, explaining the mechanism by which this combination synergistically improves glucose metabolism abnormalities in type 2 diabetes from the perspective of the incretin pathway.

[0019] Fourth, it improves liver function indicators, reduces the risk of adverse reactions from single-drug use, and has good safety profile.

[0020] In the type 2 diabetes model group, ALT was 182.0±25.00 U / L, while in the allopurinol monotherapy group it was 100.70±11.41 U / L, and in the combination group it further decreased to 60.40±12.40 U / L, significantly better than the allopurinol monotherapy group. AST levels in the combination group (242.8±17.88 U / L) were also the lowest among all groups. This indicates that the combination therapy not only did not increase the burden on the liver but also improved liver enzyme levels, helping to reduce the potential liver toxicity risk of long-term allopurinol use. Simultaneously, multiple inflammatory and metabolic indicators in the combination group decreased to near or even better than normal levels, and no safety abnormalities were observed, suggesting that this combination therapy has a good safety profile. Attached Figure Description

[0021] Figure 1 This figure shows the effects of human-derived Lactobacillus rhamnosus TG032 (internal code TH02368) combined with allopurinol on glucose metabolism parameters in type 2 diabetic mice. The figure shows the changes in fasting blood glucose levels in each group of mice, with the combined group showing the best hypoglycemic effect.

[0022] Figure 2 This figure shows the effects of the human-derived native strain *Lactobacillus rhamnosus* TG032 (internal code TH02368) combined with allopurinol on blood lipids, serum aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in type 2 diabetic mice. The figure shows that the combined group significantly reversed the abnormal elevations in serum triglycerides, total cholesterol, and low-density lipoprotein cholesterol, and reduced the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Among these, " "This indicates a significant difference compared to the Model group." P < 0.05 indicates a significant difference. P < 0.01 indicates a highly significant difference. P<0.001 indicates a highly significant difference; "#" indicates a significant difference compared to the MIX group, #P<0.05 indicates a significant difference, ##P<0.01 indicates an extremely significant difference, and ###P<0.001 indicates a highly significant difference.

[0023] Figure 3 This figure shows the effects of human-derived Lactobacillus rhamnosus TG032 (internal code TH02368) combined with allopurinol on serum inflammatory factors and hormone levels in type 2 diabetic mice. The figure shows that the combined group significantly reduced the levels of pro-inflammatory factors TNF-α, IL-6, IL-1β, and NLRP3 inflammasomes, increased the level of the anti-inflammatory factor IL-10, decreased fasting insulin levels, and significantly reduced serum uric acid levels. Among these, " "This indicates a significant difference compared to the Model group." P < 0.05 indicates a significant difference. P < 0.01 indicates a highly significant difference. P<0.001 indicates a highly significant difference; "#" indicates a significant difference compared to the MIX group, #P<0.05 indicates a significant difference, ##P<0.01 indicates an extremely significant difference, and ###P<0.001 indicates a highly significant difference.

[0024] Figure 4 The image shows the effect of the human-derived native strain *Lactobacillus rhamnosus* TG032 (internal code TH02368) combined with allopurinol on serum glucagon-like peptide-1 (GLP-1) levels in type 2 diabetic mice. Among them, " "This indicates a significant difference compared to the Model group." P < 0.05 indicates a significant difference. P < 0.01 indicates a highly significant difference. P<0.001 indicates a highly significant difference; "#" indicates a significant difference compared to the MIX group, #P<0.05 indicates a significant difference, ##P<0.01 indicates an extremely significant difference, and ###P<0.001 indicates a highly significant difference. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to the embodiments. However, the present invention is not limited to the following embodiments, and any equivalent substitutions or modifications made based on the technical solutions of the present invention shall fall within the protection scope of the present invention.

[0026] Example 1: Isolation, screening and identification of Lactobacillus rhamnosus TG032 (internal code TH02368) The Lactobacillus rhamnosus used in this invention ( Lactobacillus rhamnosus TG032 (internal designation TH02368) is a superior strain obtained through targeted screening from feces of healthy donors. The specific isolation and identification methods are as follows: First, fresh fecal samples were taken from healthy donors and anaerobically enriched for 6 days using a modified acid-resistant BHIS liquid culture medium with a pH of 3 at 37°C to screen for potential probiotics with strong acid resistance.

[0027] Next, the enrichment solution was serially diluted and spread on TPY solid medium. It was then anaerobically cultured at 37°C. After a large number of single colonies formed, colonies with obvious morphological differences were picked and purified by three rounds of streak plating to obtain a stable pure culture.

[0028] Next, the purified single colonies were transferred to BHIS liquid medium and cultured anaerobically at 37°C for 72 hours, after which the bacterial cells were collected. Ten purified strains were selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rDNA gene sequencing. The sequencing results were compared and identified in the EzBioCloud database, and the results showed that all 10 strains were identified as *Lactobacillus rhamnosus* (Lactobacillus). Lactobacillus rhamnosus One of the strains with excellent performance was named TG032 and preserved long-term. The 16S rDNA sequence of TG032 (internal number TH02368) is shown in SEQ ID NO:1.

[0029] SEQ ID NO:1 Two other human-derived *Lactobacillus rhamnosus* strains were also isolated, internally designated TH02627 and TH02370. Their 16S rDNA sequences are as follows: The TH0237016S rDNA sequence is as follows: SEQ ID NO:2: The TH0262716S rDNA sequence is as follows: SEQ ID NO:3: Finally, the bacterial suspension of strain TG032 (internal number TH02368) was mixed with 10% (V / V) glycerol and then frozen at -80℃ to serve as the master cell bank.

[0030] It should be noted that the human-derived native strain Lactobacillus rhamnosus TG032 (internal number TH02368) is a preserved strain, which is deposited at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251083 and deposit date of May 16, 2025.

[0031] Example 2: Evaluation of TG032's in vitro gastrointestinal environment tolerance This study used an in vitro simulated gastrointestinal fluid method to evaluate the survival ability of live bacteria TG032 (internal number TH02368) in the extreme environment of the gastrointestinal tract.

[0032] Take 1 mL of bacteria with a viable count of 5 × 10⁶ 8 Second-generation bacterial culture with CFU / mL was added to 9.0 mL of simulated gastric fluid (Regen Biosciences, catalog number CZ0213), vortexed to mix, and incubated statically in a 37°C anaerobic incubator. The culture was then incubated at pH 3.0 for 2 hours. Bacterial culture was aspirated before treatment (0 hours) and after treatment (2 hours) and serially diluted 10-fold with sterile PBS buffer to 10⁻⁶. -8 Select 50 μL of a suitable gradient dilution and spread it on MRS solid medium plates. After anaerobic incubation at 37℃ for 48 hours, colony counting is performed. Simulated gastric fluid survival rate (%) = (total number of viable bacteria in the 2h treatment group / total number of viable bacteria in the 0h control group) × 100%.

[0033] Take another 1 mL of bacterial suspension of the same concentration and add it to 9.0 mL of simulated intestinal fluid. Incubate in an anaerobic incubator at 37℃ and pH 6.75 for 3 hours. Samples were taken, diluted, spread, and counted before treatment (0 hours) and after treatment (3 hours). The survival rate of simulated intestinal fluid (%) = (total number of viable bacteria in the 3h treatment group / total number of viable bacteria in the 0h control group) × 100%.

[0034] The specific survival rate results are shown in Table 1.

[0035] Table 1 Survival rate of TG032 in simulated gastrointestinal fluid

[0036] Experimental results showed that strain TG032 (internal code TH02368) exhibited good tolerance in both simulated gastric and intestinal fluids. After 2 hours of treatment in simulated gastric fluid at pH 3.0, the survival rate remained at 31.65%, and after 3 hours of treatment in simulated intestinal fluid, the survival rate reached 104.14% (the survival rate of over 100% was due to the continued proliferation of bacteria in the intestinal fluid environment during treatment). This indicates that the strain can resist the erosion of digestive enzymes and has the potential to survive stably and successfully colonize in the intestine.

[0037] Example 3: Determination of the in vitro purine nucleoside degradation ability of TG032 (internal number TH02368) 1. Preparation of bacterial culture and preparation of standard products The second-generation bacterial suspension of TG032 (internal number TH02368) was prepared according to the method in Example 2. The bacterial suspension was centrifuged at 4°C and 10,000 r / min for 10 minutes, the supernatant was discarded, the bacterial cells were collected and washed twice with sterile PBS buffer, and finally resuspended in PBS to obtain a bacterial suspension.

[0038] Accurately weigh 20 mg of adenine and 20 mg of guanosine standards, dissolve them with a small amount of 0.1 mol / L NaOH, and then bring the volume to 50 mL with PBS to obtain a mixed standard solution containing 400 μg / mL each of adenine and guanosine. Prepare a mixed standard solution containing 400 μg / mL each of inosine and adenosine using the same method.

[0039] 2. Reaction System and Detection Take 0.75 mL of the above bacterial suspension and mix it with 0.75 mL of one of the two purine nucleoside mixed standard solutions mentioned above. Each reaction system should be set up in triplicate. A PBS system containing only the standard solution should be used as a control. Incubate the reaction system in a shaker at 37°C for 6 hours. Immediately after the reaction is complete, heat the reaction solution in a 90°C water bath for 10 minutes to terminate the reaction. After cooling, centrifuge at 4000 r / min for 10 minutes, collect the supernatant, filter it through a 0.22 μm filter membrane, and analyze it using high-performance liquid chromatography (HPLC).

[0040] HPLC conditions: mobile phase: 10 mM potassium dihydrogen phosphate (containing 1% methanol), pH=3.5; isocratic elution; flow rate: 1 mL / min; column temperature: 40℃; injection volume: 10 μL; detection wavelength: 254 nm; column: Waters C18 column (4.6 mm × 250 mm). The degradation rate was calculated by comparing the peak areas of the standards before and after the reaction. Degradation rate (%) = [(C0 - C1) / C0] × 100%, where C0 is the concentration of the standard without bacterial control, and C1 is the concentration of the standard after bacterial reaction.

[0041] Meanwhile, strains TH02627 and TH02370 were set up as control experiments, and the method was the same as that for strain TG032 (internal number TH02368).

[0042] 3. Experimental Results The results are shown in Table 2.

[0043] Table 2. In vitro degradation rate of purine nucleosides by TG032

[0044] The results showed that strain TG032 (internal designation TH02368) exhibited broad-spectrum and highly efficient purine nucleoside degradation capabilities, demonstrating strong degradation activity against four common uric acid precursors. The degradation rates for adenine, adenosine, guanosine, and inosine were 100%, 100%, 94.60%, and 89.29%, respectively, significantly reducing intestinal absorption of purines from their source. Furthermore, strain TG032 (internal designation TH02368) showed significantly stronger degradation capabilities for adenine, guanosine, and inosine than its contemporaries TH02627 and TH02370.

[0045] Example 4: The effect of human-derived native strain Lactobacillus rhamnosus TG032 (internal code TH02368) combined with allopurinol on improving abnormal glucose and uric acid metabolism in type 2 diabetic mice. (1) Construction and grouping of diabetes models: SPF-grade male C57BL / 6J mice, weighing 18–22 g, were randomly divided into a normal control group (CON) and a model group after one week of acclimatization. The CON group was fed a standard maintenance diet throughout the acclimatization period. The model group was first fed a high-fat, high-sugar diet for 4 weeks to induce insulin resistance, followed by intraperitoneal injections of streptozotocin (STZ, 50 mg / kg) once daily for 5 consecutive days. After the last injection, the high-fat, high-sugar diet was resumed for another 7 days. Body weight, water intake, and general condition were monitored daily during this period. Fasting blood glucose was measured by tail blood samples taken on days 7 and 11 after fasting. A successful type 2 diabetes model was defined as two fasting blood glucose levels ≥11.1 mmol / L with an interval ≥72 hours between the two measurements.

[0046] After successful model establishment, mice were randomly divided into 5 groups: normal control group (CON group), model group (MOD group), TG032 single drug group (TH02368 group), allopurinol group (ALL group), and TG032 + allopurinol combination group (MIX group). Mice in each group were administered the corresponding dose of the intervention via gavage: the human-derived *Lactobacillus rhamnosus* group was given *Lactobacillus rhamnosus* TH02368 bacterial suspension (1×10⁻⁶ viable cells). 9The mice were administered CFU / mouse / day; the allopurinol group received allopurinol solution (10 mg / kg body weight / day); the combination group received a mixture of the same dose of human-derived Lactobacillus rhamnosus TH02368 bacterial suspension and allopurinol; the normal control group and the model control group received an equal volume of physiological saline by gavage. The gavage period lasted for 10 weeks, during which fasting blood glucose levels and related metabolic indicators were monitored regularly.

[0047] (2) Indicator detection: After the intervention period ended, patients fasted for 12 hours, and blood was drawn from the orbital sinus for serum separation. A fully automated biochemical analyzer was used to detect serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). An ELISA kit was used to detect levels of inflammatory factors interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), NLRP3 inflammasome, and the anti-inflammatory factor interleukin-10 (IL-10), as well as fasting insulin (INS), serum uric acid, and glucagon-like peptide-1 (GLP-1).

[0048] (3) Experimental results: Regarding fasting blood glucose (FBG): In the diabetes model group (MOD), blood glucose levels consistently remained at their highest and continued to rise, indicating successful model establishment. Among the intervention groups, the combined intervention group (MIX) showed the best hypoglycemic effect, significantly superior to the human-derived *Lactobacillus rhamnosus* monotherapy group and the allopurinol monotherapy group (see...). Figure 1 ).

[0049] Regarding serum liver enzyme activity: Compared with the normal control group, the ALT and AST activities in the diabetic model group mice were significantly increased, indicating that diabetes modeling induced clear liver damage. All intervention groups showed varying degrees of hepatoprotective effects, with the combined group showing the most significant reduction in ALT and AST (see...). Figure 2 ).

[0050] Regarding blood lipids: Compared with the normal control group, the serum levels of TG, TC, and LDL-C in the model group were significantly elevated. Intervention with the human-derived native strain of Lactobacillus rhamnosus, the allopurinol group, and the combined group all reversed these abnormalities to varying degrees. Among them, regarding TG, the combined group showed the best effect in improving blood lipids (see...). Figure 2 (and Table 3).

[0051] Regarding hormones and inflammatory factors: Compared with the normal control group, the fasting insulin (INS) level in the model group was significantly elevated. The human-derived *Lactobacillus rhamnosus* group, allopurinol group, and combination group all reduced INS levels to varying degrees, with the combination group showing significantly better improvement than the single-drug group. Serum levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β were significantly elevated in the model group, as was the NLRP3 inflammasome level, while the level of the anti-inflammatory factor IL-10 was decreased, indicating the successful establishment of a systemic low-grade inflammatory state. The human-derived *Lactobacillus rhamnosus* group, allopurinol group, and combination group all reversed the above-mentioned inflammatory factor abnormalities to varying degrees, with the combination group exhibiting the strongest comprehensive anti-inflammatory effect, maximally inhibiting pro-inflammatory factor levels while effectively increasing the level of the anti-inflammatory factor IL-10 (see...). Figure 3 (and Table 4).

[0052] Regarding uric acid regulation: the serum uric acid level in the model group was significantly increased, while the combined treatment group significantly reduced serum uric acid levels, showing better results than the allopurinol monotherapy group (see...). Figure 3 ).

[0053] Regarding glucagon-like peptide-1 (GLP-1): Compared with the normal control group, the model group showed a significant decrease in GLP-1, and the Lactobacillus rhamnosus monotherapy group was superior to the model group. The combination group (MIX) showed the best improvement, superior to the Lactobacillus rhamnosus monotherapy group and the allopurinol monotherapy group (see...). Figure 4 (and Table 5).

[0054] Table 3. Effects of each intervention group on blood lipids, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in type 2 diabetic model mice.

[0055] Table 4. Effects of each intervention group on inflammatory factors and uric acid in mice.

[0056] Table 5. Effects of each intervention group on GLP-1 secretion in mice.

[0057] Synergistic Effect Analysis: To objectively evaluate the synergistic effect of the combined use of human-derived Lactobacillus rhamnosus TG032 (internal code TH02368) and allopurinol, Jin's formula was used to quantitatively analyze the synergistic effect of the combined drug administration. The correction rate E = (mean of model group) / (mean of model group) is used as the formula. (Intervention group mean) / (Model group mean) The individual effects of human-derived native strain Lactobacillus rhamnosus TG032 and allopurinol (Eb and Ealtopurinol) were characterized by (mean value of normal control group) × 100%; Eexpected = Eb + Ealtopurinol. The expected additive effect of E bacteria × E allopurinol was calculated after the combined application of the two single drugs; the synergy index Q = E measured / E expected was used for judgment, where Q ≥ 1.15 was judged as synergistic, 0.85 ≤ Q < 1.15 was judged as simple additive, and Q < 0.85 was judged as antagonistic (the calculation results are shown in Table 6).

[0058] Table 6. Calculation results of synergistic effects of various indicators (Kin's formula)

[0059] The results showed that: in reducing serum uric acid, E. coli achieved an improvement rate of 49.2%, E. allopurinol achieved an improvement rate of 99.6%, and the expected improvement rate of E was 99.8%, while the combined group achieved an actual improvement rate of 133.2% (Q = 1.334); in reducing tumor necrosis factor-α, E. coli achieved an improvement rate of 33.1%, E. allopurinol achieved an improvement rate of 48.2%, and the expected improvement rate of E was 65.4%, while the actual improvement rate of E was 104.4% (Q = 1.596); in reducing interleukin-1β, Q = 1.315; in increasing glucagon-like peptide-1, Q = 1.278; in reducing fasting insulin (improving insulin resistance), Q = 1.179; in reducing interleukin-6, Q = 1.161; and in increasing the anti-inflammatory factor interleukin-10, Q = 1.151. The synergistic index Q values ​​of the above seven indicators were all greater than 1.15, indicating that the combined group was significantly more effective than the simple sum of the effects of the two single drugs in reducing serum uric acid, inhibiting pro-inflammatory factors, increasing anti-inflammatory factors, increasing glucagon-like peptide-1, and improving insulin resistance. This means that the human-derived native strain *Lactobacillus rhamnosus* TG032 and allopurinol produced a clear synergistic effect in improving abnormal glucose and uric acid metabolism, an effect that cannot be predicted by the simple superposition of the effects of the two single drugs. Furthermore, the combined group showed better improvement in liver enzyme indicators such as alanine aminotransferase (60.40±12.40 U / L) and aspartate aminotransferase (242.8±17.88 U / L) than the allopurinol monotherapy group, suggesting that the combined use of the two drugs did not increase the metabolic burden on the liver and was beneficial in reducing the potential liver toxicity risk of long-term allopurinol use.

[0060] The above results indicate that the combined use of human-derived native strain Lactobacillus rhamnosus TG032 (internal number TH02368) and allopurinol can synergistically improve abnormal glucose metabolism and abnormal uric acid metabolism in type 2 diabetic mice, and the overall effect is better than that of the single component.

[0061] Example 5: Compound tablets containing human-derived native strain Lactobacillus rhamnosus TG032 (internal code TH02368) and allopurinol and their preparation method (1) Formula composition:

[0062] (2) Preparation method: ① The human-derived native strain of Lactobacillus rhamnosus TG032 (internal number TH02368) lyophilized powder, allopurinol, microcrystalline cellulose, and lactose were passed through an 80-mesh sieve for later use; ② Take the lyophilized powder of human-derived Lactobacillus rhamnosus TG032 (internal number TH02368) and allopurinol, and mix them evenly using the equal-volume incremental addition method to obtain the active ingredient mixture; ③ Add the main drug mixture, microcrystalline cellulose, lactose, and pregelatinized starch into a three-dimensional mixer and mix at 20 r / min for 30 min to ensure that the materials are fully and evenly mixed. ④ Add magnesium stearate and continue mixing for 5 minutes; ⑤ Compress the mixed powder directly into tablets, controlling the tablet weight to approximately 300 mg and the hardness to be 5–8 kg / cm². 2 This yields compound tablets.

[0063] Each tablet contains approximately 20 mg of allopurinol and approximately 1 × 10⁻⁶ live bacteria of the human-derived native strain Lactobacillus rhamnosus TG032 (internal code TH02368). 9 CFU. This compound tablet can be taken orally. The recommended dose is 1-2 tablets twice daily, or adjusted according to the patient's condition and the doctor's guidance.

[0064] This tablet contains both probiotics and chemical drugs, enabling the combined use of human-derived native strain Lactobacillus rhamnosus and allopurinol to improve glucose metabolism and uric acid metabolism abnormalities in type 2 diabetes.

[0065] In addition to the above embodiments, the human-derived native strain of *Lactobacillus rhamnosus* TG032 (internal number TH02368) in this invention can also be used in the form of its cell lysate, fermentation supernatant, etc., and allopurinol can be used in the form of its pharmaceutically acceptable salt or solvate. Other conventional excipients, such as antioxidants, sweeteners, flavoring agents, stabilizers, disintegrants, binders, etc., can also be added to the product to meet the preparation requirements of different dosage forms.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. The application of a human-derived native strain of Lactobacillus rhamnosus TG032 in combination with allopurinol in the preparation of a drug for treating type 2 diabetes, characterized in that: The drug contains an effective amount of human-derived native strain Lactobacillus rhamnosus TG032 and an effective amount of allopurinol; the preservation number of the human-derived native strain Lactobacillus rhamnosus TG032 is CCTCC NO: M 20251083.

2. The application according to claim 1, characterized in that, The human-derived native strain of Lactobacillus rhamnosus TG032 is a live bacterial cell or a freeze-dried bacterial cell powder.

3. The application according to claim 1, characterized in that, The viable count of the human-derived native strain of Lactobacillus rhamnosus TG032 in the drug is 1×10⁻⁶. 6 ~1×10 12 CFU / g or 1×10 6 ~1×10 12 The concentration of allopurinol is CFU / mL, and the concentration is 0.01–100 mg / g or 0.01–100 mg / mL.

4. The application according to claim 1, characterized in that, The dosage form of the drug is tablets, capsules, granules, powders, or oral liquids.

5. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers, diluents, excipients, or excipients.

6. The application according to any one of claims 1 to 5, characterized in that, In the drug, the human-derived native strain of Lactobacillus rhamnosus TG032 and the allopurinol are each contained in their own independent formulation unit.

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