Use of pediococcus pentosaceus a21243 in reducing blood uric acid and improving kidney function

Abstract

The application belongs to the technical field of functional microorganisms, and discloses application of Pediococcus pentosaceus A21243 in lowering blood uric acid and improving kidney function, and provides application of the Pediococcus pentosaceus A21243 in preparing products for preventing or treating nephropathy and hyperuricemia, wherein the Pediococcus pentosaceus is Pediococcus pentosaceus A21243, and the preservation number is GDMCC NO:63749. The Pediococcus pentosaceus A21243 has the effects of improving and delaying kidney dysfunction caused by hyperuricemia, hyperglycemia and cisplatin, meanwhile, the strain can inhibit the activity of purine metabolism related enzymes in the uric acid generation pathway, such as inhibiting the activity of xanthine oxidase (XOD), purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA), promote the excretion of uric acid through the intestinal feces, regulate the oxidative stress disorder caused by hyperuricemia, and achieve the beneficial effect of lowering blood uric acid.

Description

Technical Field

[0001] This invention belongs to the field of functional microbial technology, specifically relating to the application of Pediococcus pentosaceus A21243 in lowering blood uric acid and improving renal function. Background Technology

[0002] Hyperuricemia (HUA) is a chronic metabolic disease characterized by elevated serum uric acid levels due to disordered purine metabolism or impaired uric acid excretion. It can easily lead to acute gouty arthritis attacks, tophi deposition, chronic tophaceous arthritis, and joint deformities. It frequently affects the kidneys, causing chronic interstitial nephritis and uric acid kidney stones. The causes of hyperuricemia are generally divided into two categories: increased uric acid production and decreased uric acid excretion. Factors leading to increased uric acid production include high levels of purines in the diet and increased purine metabolism; decreased uric acid excretion is mostly related to kidney disease, and also includes some medications and competition between uric acid and other molecules for excretion.

[0003] Current treatments for hyperuricemia (HUA) primarily focus on reducing excessive urate production, increasing renal and / or gastrointestinal excretion, or both. Methods include pharmacological and non-pharmacological therapies, but all have adverse effects. Clinically used medications mainly include uricosuric agents, urate-inhibiting agents, and alkalizing agents, which alleviate hyperuricemia by increasing urate excretion, inhibiting urate production, or alkalizing urine. Insufficient urate excretion is the main cause of gout-related hyperuricemia. Dietary factors also significantly influence urate production. Furthermore, the burden of gout is exacerbated by the additional effects of common complications in gout patients, including hypertension (75%), chronic kidney disease (CKD) (70%), obesity (53%), and cardiovascular disease (CVD) (10% to 14%), which are associated with increased morbidity and mortality risks. Gout patients also have a higher incidence of metabolic syndrome, and asymptomatic hyperuricemia is more common in patients with metabolic syndrome, seriously endangering public health. Given the shortcomings and deficiencies of existing treatment methods, there is an urgent need to find a treatment strategy for HUA that is effective, stable, safe, and convenient.

[0004] With the improvement of living standards, changes in lifestyle, and population aging in my country, the incidence of metabolic diseases such as diabetes, hypertension, obesity, hyperuricemia, and gout is gradually increasing, leading to a year-on-year rise in the incidence of secondary kidney diseases. Sustained hyperglycemia can cause glomerulosclerosis, renal arteriolar and glomerular capillary filtration membrane lesions, which are the pathological basis for proteinuria and renal insufficiency. Sodium urate crystal deposition in the renal medulla can lead to chronic tubulointerstitial nephritis, causing renal fibrosis, and can also cause urinary tract stones and obstruction.

[0005] Pediococcus pentosaceus, a type of lactobacillus, plays an important role in food production, storage, and the promotion of human health.

[0006] To date, issues related to the practical application of *Pediococcus pentosaceus* as a probiotic remain unresolved, such as a lack of knowledge regarding mechanisms, side effects, usage, and dosage. Increasing evidence suggests that *Pediococcus pentosaceus* and its metabolites perform well in the food industry and for gut health. Currently, screening indicators for *Lactobacillus* strains, their in vivo effects on regulating serum uric acid, and their mechanisms are unclear, and there are few reports on *Pediococcus pentosaceus* improving hyperuricemia. Therefore, screening for *Pediococcus pentosaceus* strains that can lower uric acid and improve metabolic kidney diseases such as hyperuricemia, hyperglycemia, and hyperlipidemia is of great significance for the treatment of hyperuricemia and for research on drugs targeting hyperuricemia. Summary of the Invention

[0007] This invention aims to overcome the shortcomings of the prior art and provide a strain of Pediococcus pentosaceus derived from the fecal sample of a centenarian in Guangxi, which has the effects of lowering uric acid or protecting the kidneys and improving kidney function, as well as its post-biotic application. It was also found that this strain can degrade uric acid in vitro and regulate oxidative stress disorder in a mouse model of intestinal hyperuricemia, and can effectively reduce uric acid. It can be used to treat hyperuricemia and renal insufficiency.

[0008] The first aspect of the present invention is to provide the use of Pediococcus pentosaceus A21243 or its products in the preparation of products for the prevention or treatment of kidney disease.

[0009] A second aspect of the present invention aims to provide the use of Pediococcus pentosaceus A21243 or its products in the preparation of products for the prevention or treatment of hyperuricemia.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] The first aspect of the present invention provides the use of Pediococcus pentosaceus A21243 or its products in the preparation of products for the prevention or treatment of kidney disease, wherein Pediococcus pentosaceus A21243 has the accession number GDMCC NO:63749.

[0012] Pediococcus pentosaceus A21243 is taxonomically named Pediococcus pentosaceus It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 24, 2023.

[0013] In some embodiments of the present invention, the kidney disease includes renal insufficiency caused by diabetes, hyperuricemia, or cisplatin.

[0014] In some embodiments of the present invention, the treatment includes relief or improvement.

[0015] In some embodiments of the present invention, the diabetes includes type 2 diabetes.

[0016] In some embodiments of the present invention, the product includes pharmaceuticals.

[0017] In some embodiments of the present invention, the dosage form of the medicine is selected from tablets (such as ordinary tablets, bilayer tablets, multilayer tablets, sustained-release tablets, single-compartment controlled-release tablets, dual-compartment controlled-release tablets, microporous controlled-release tablets, dispersible tablets, enteric-coated tablets), pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules (such as ordinary capsules, sustained-release capsules, controlled-release capsules, capsules containing microcapsules or small pieces, pH-dependent capsules containing microcapsules or small pieces, gastrointestinal compound capsules), suppositories, injections, sprays, and injections.

[0018] In some embodiments of the present invention, the product further includes pharmaceutically acceptable excipients.

[0019] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one selected from fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, humectants, flavoring agents, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, fragrances, and solvents.

[0020] In some embodiments of the present invention, the filler is selected from starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, or glucose, etc.; the binder is selected from cellulose derivatives, alginate, starch, water, dextrin, gelatin, or polyvinylpyrrolidone, etc.; the disintegrant is selected from microcrystalline cellulose, sodium methyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, or croscarmellose sodium; the lubricant is selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, microcrystalline silica, talc, or magnesium stearate; and the suspending agent is selected from microcrystalline silica, beeswax, cellulose, or solid polyethylene glycol. The wetting agent is selected from glycerin, Tween-80, hydrogenated castor oil, or lecithin; the solvent is selected from ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerin, propylene glycol, or vegetable oil, wherein the vegetable oil is selected from soybean oil, castor oil, peanut oil, blended oil, etc.; the surfactant is selected from sodium dodecylbenzenesulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan, or polysorbate (Tween), etc.; the flavoring agent is selected from aspartame, sucralose, flavoring, stevia, acesulfame potassium, citric acid, or sodium saccharin; the preservative is selected from at least one of methylparaben or propylparaben.

[0021] In some embodiments of the present invention, the number of *Pediococcus pentosaceus* A21243 in the product is >10. 7 CFU.

[0022] A second aspect of the present invention provides the use of Pediococcus pentosaceus A21243 or its products in the preparation of products for the prevention or treatment of hyperuricemia, wherein the Pediococcus pentosaceus A21243 has the accession number GDMCC NO:63749.

[0023] In some embodiments of the present invention, the drug achieves the purpose of preventing or treating hyperuricemia by reducing the uric acid content in the subject's serum, inhibiting the activity of xanthine oxidase, purine nucleoside phosphorylase and / or adenosine deaminase in the subject's liver, regulating the subject's oxidative stress disorder, and promoting the subject's excretion of uric acid.

[0024] In some embodiments of the present invention, the regulation of oxidative stress disorder in subjects includes regulating the levels of SOD, CAT, GSH and MDA in the liver of subjects.

[0025] In some embodiments of the present invention, the product includes pharmaceuticals.

[0026] In some embodiments of the present invention, the dosage form of the medicine is selected from tablets (such as ordinary tablets, bilayer tablets, multilayer tablets, sustained-release tablets, single-compartment controlled-release tablets, dual-compartment controlled-release tablets, microporous controlled-release tablets, dispersible tablets, enteric-coated tablets), pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules (such as ordinary capsules, sustained-release capsules, controlled-release capsules, capsules containing microcapsules or small pieces, pH-dependent capsules containing microcapsules or small pieces, gastrointestinal compound capsules), suppositories, injections, sprays, and injections.

[0027] In some embodiments of the present invention, the product further includes pharmaceutically acceptable excipients.

[0028] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one selected from fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, humectants, flavoring agents, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, fragrances, and solvents.

[0029] In some embodiments of the present invention, the filler is selected from starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, or glucose, etc.; the binder is selected from cellulose derivatives, alginate, starch, water, dextrin, gelatin, or polyvinylpyrrolidone, etc.; the disintegrant is selected from microcrystalline cellulose, sodium methyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, or croscarmellose sodium; the lubricant is selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, microcrystalline silica, talc, or magnesium stearate; and the suspending agent is selected from microcrystalline silica, beeswax, cellulose, or solid polyethylene glycol. The wetting agent is selected from glycerin, Tween-80, hydrogenated castor oil, or lecithin; the solvent is selected from ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerin, propylene glycol, or vegetable oil, wherein the vegetable oil is selected from soybean oil, castor oil, peanut oil, blended oil, etc.; the surfactant is selected from sodium dodecylbenzenesulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan, or polysorbate (Tween), etc.; the flavoring agent is selected from aspartame, sucralose, flavoring, stevia, acesulfame potassium, citric acid, or sodium saccharin; the preservative is selected from at least one of methylparaben or propylparaben.

[0030] The beneficial effects of this invention are:

[0031] This invention provides the application of *Pediococcus pentosaceus* A21243 in the preparation of products for the prevention or treatment of kidney disease and hyperuricemia. The *Pediococcus pentosaceus* is *Pediococcus pentosaceus* A21243, with accession number GDMCC NO: 63749. *Pediococcus pentosaceus* A21243 in this invention has the effect of improving and delaying metabolic renal insufficiency caused by hyperuricemia and hyperglycemia, as well as renal insufficiency caused by cisplatin. Simultaneously, this strain can also reduce blood uric acid levels by inhibiting the activity of purine metabolism-related enzymes in the uric acid production pathway, such as xanthine oxidase (XOD), purine nucleoside phosphorylase (PNP), and adenosine deaminase (ADA), promoting uric acid excretion through feces, and regulating oxidative stress disorders caused by hyperuricemia. It has extremely high development and practical value, providing new ideas for the development and application of related products. Attached Figure Description

[0032] Figure 1 Serum uric acid concentrations in mice in each group before (day 0) and after (day 28) intervention with different doses of A21243 are shown in the figure. In the figure, ns represents no significant difference, and ** represents... P <0.01, *** represents P <0.001, **** represents P <0.0001.

[0033] Figure 2The changes in serum uric acid in HUA mice during intervention with different doses of A21243.

[0034] Figure 3 The figure shows the inhibitory effect of A21243 on the activities of xanthine oxidase (XOD), purine nucleoside phosphorylase (PNP), and adenosine deaminase (ADA) in the liver of HUA mice. In the figure, ns represents no significant difference, and * indicates... P <0.05, ** represents P <0.01, **** represents P <0.0001.

[0035] Figure 4 The figure shows the regulatory effect of A21243 on oxidative stress disorder in HUA mice. In the figure, ns represents no significant difference, and * represents... P <0.05, ** represents P <0.01, *** represents P <0.001, **** represents P <0.0001.

[0036] Figure 5 The figure shows the promoting effect of A21243 on intestinal uric acid excretion in HUA model mice. In the figure, ns represents no significant difference, and ** represents... P <0.01.

[0037] Figure 6 The figure shows the effect of A21243 on reducing renal function indicators creatinine (Cr) and blood urea nitrogen (BUN) and restoring renal coefficient in HUA mice. ** represents... P <0.01, *** represents P <0.001, **** represents P <0.0001.

[0038] Figure 7 The effect of A21243 on improving the pathological changes in the kidneys and liver of HUA mice.

[0039] Figure 8 The figure shows the effects of A21243 on fasting blood glucose, glycated serum protein (GSP), and renal function indicators creatinine (Cre) and blood urea nitrogen (BUN) in DKD mice. * indicates... P <0.05, ** represents P <0.01, *** represents P <0.001, **** represents P <0.0001.

[0040] Figure 9 This study aimed to improve the renal pathological changes in HUA mice by using A21243.

[0041] Figure 10 The figure shows the effect of A21243 on serum and urinary creatinine in CKD mice. ** represents... P <0.01, *** represents P <0.001.

[0042] Figure 11 This study aimed to improve the renal pathological changes in CKD mice using A21243. Detailed Implementation

[0043] The present invention will be further described in detail below through specific embodiments.

[0044] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0046] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0047] Example 1: In vitro uric acid-lowering effect of Pediococcus pentosaceus A21243

[0048] Pediococcus pentosaceus ( Pediococcus pentosaceus Pediococcus pentosaceus strain A21243 (preservation number GDMCCNO:63749, disclosed in the invention patent "A Pediococcus pentosaceus strain A21243 with therapeutic and / or fat-reducing functions and its application, publication number: CN117625478A") was inoculated at 1% in MRS liquid medium containing 10 mM uric acid. After anaerobic incubation at 37°C for 24 h, the uric acid content in the medium was determined by HPLC. The same medium without Pediococcus pentosaceus A21243 was set as a control. The HPLC determination was performed using an Agilent Zobax SB-Aq (4.6 mm × 250 mm, 5 μm) at 30°C with a detection wavelength of 293 nm. The mobile phase was 20 mM pH 7.4 K2HPO4-KH2PO4 buffer and methanol (volume ratio 90:10), the flow rate was 0.5 mL / min, and the analysis time was 15 min.

[0049] The in vitro degradation rate of uric acid by Pediococcus pentosaceus A21243 is shown in Table 1, suggesting that Pediococcus pentosaceus A21243 has an in vitro uric acid-lowering effect.

[0050] Table 1. Degradation of uric acid by A21243 under eutrophic conditions.

[0051]

[0052] Example 2: Preparation of a lyophilized powder with uric acid-lowering effect in a hyperuricemia model

[0053] The freeze-dried powder preparation method of this embodiment includes the following steps:

[0054] Preparation of the preservative: Sterilize 12% (W / V) skim milk and 12% (W / V) trehalose at 115℃ for 15 min, cool and set aside. When needed, mix 12% (W / V) skim milk and 12% (W / V) trehalose in a 1:1 volume ratio.

[0055] Strain activation and culture: *Pediococcus pentosaceus* A21243 was streaked onto an MRS plate using an inoculation loop and incubated anaerobicly at 37°C for 24 h. Activated single colonies were streaked again, and the resulting colonies were inoculated into MRS liquid medium and incubated anaerobicly at 37°C for 18 h. Single colonies were then transferred to 10 mL of MRS liquid medium and incubated anaerobicly at 37°C for 12 h to obtain seed culture 1. Seed culture 1 was inoculated into 150 mL of MRS liquid medium at a 3% inoculation rate and incubated anaerobicly at 37°C for 20–22 h to obtain seed culture 2. Seed culture 2 was inoculated into 300 mL of MRS liquid medium at a 3% inoculation rate and incubated anaerobicly at 37°C for 20–24 h to obtain the scaled-up bacterial culture. The bacterial culture was collected and centrifuged at 4°C, 5000 rpm for 5 min using a refrigerated centrifuge, and the supernatant was discarded. Wash the bacterial cells twice with physiological saline, centrifuge at 4℃, 5000 rpm for 5 min, and weigh the wet weight of the bacterial cells.

[0056] Vacuum freeze-drying: The freeze-drying protectant and wet bacterial cells were resuspended at a volume ratio of 1:1 (W:V), dispensed into EP tubes, and pre-cooled at -80℃ for 1 h. The dispensed EP tubes were placed into freeze-drying bottles, and the samples were freeze-dried using a four-ring Foring vacuum freeze dryer. The material bottle was inserted into the T-shaped rack of the vacuum freeze dryer, and the switch was turned on to connect the ampoule to the vacuum unit. The bacterial agent was dried continuously for 18 h to obtain a freeze-dried powder containing Pediococcus pentosaceus A21243. The finished product was stored in a dry place for later use, and a portion of the freeze-dried powder was randomly selected to prepare a bacterial suspension for specific amplification verification and viable cell counting.

[0057] Example 3: Uric acid-lowering effect of Pediococcus pentosaceus A21243 on hyperuricemia model mice.

[0058] This example was used to investigate the uric acid-lowering effect of Pediococcus pentosaceus A21243 on a mouse model of hyperuricemia (HUA). The specific animal experimental steps are as follows:

[0059] Forty-eight male SPF-grade C57BL / 6 mice, aged 6–8 weeks and weighing 18–20 g, were purchased. Before the experiment, the mice were acclimatized for 7 days under a 12-hour light cycle, at a temperature of 20–26°C and humidity of 40%–60%, with free access to food and water. After the acclimatization period, the mice were randomly divided into four groups: a normal control group (Control), a hyperuricemia model group (Model), a high-dose Pediococcus pentosaceus group (A21243 H), and a low-dose Pediococcus pentosaceus group (A21243 L), with 12 mice in each group. The specific grouping procedures are as follows:

[0060] Normal control group (Control): fed with basal feed, 0.2 mL of physiological saline was administered by gavage and 0.1 mL of physiological saline was administered by intraperitoneal injection for 14 days during the modeling period, and treatment was carried out after the modeling period, with 0.2 mL of physiological saline administered by gavage and 0.1 mL of physiological saline administered by intraperitoneal injection for 28 days during the treatment period;

[0061] Hyperuricemia model group (Model): During the modeling period, the rats were fed a high-purine diet containing 28.5% yeast powder, and were given 250 mg / kg ethambutol hydrochloride by gavage and 350 mg / kg potassium oxonate by intraperitoneal injection for 14 days. After the modeling period, the rats entered the treatment period, and during the treatment period, the hyperuricemia model was maintained by a high-purine diet and intraperitoneal injection of 350 mg / kg potassium oxonate working solution for 28 days.

[0062] The high-dose group of *Pediococcus pentosaceus* (A21243 H): During the modeling period, the rats were fed a high-purine diet containing 28.5% yeast powder, and received oral administration of 250 mg / kg ethambutol hydrochloride and intraperitoneal injection of 350 mg / kg potassium oxonate for 14 consecutive days. After the modeling period, treatment was initiated. During the treatment period, the hyperuricemia model was maintained by a high-purine diet and intraperitoneal injection of 350 mg / kg potassium oxonate. High-dose probiotics (i.e., 2 × 10⁻⁶) were administered during the treatment period. 9 Intervention was performed by gavage with CFU (Pediococcus pentosaccharide A21243) for 28 days;

[0063] The low-dose group of *Pediococcus pentosaceus* (A21243 L): During the modeling period, the rats were fed a high-purine diet containing 28.5% yeast powder, and received oral administration of 250 mg / kg ethambutol hydrochloride and intraperitoneal injection of 350 mg / kg potassium oxonate for 14 consecutive days. After the modeling period, treatment was initiated. During the treatment period, the hyperuricemia model was maintained by a high-purine diet and intraperitoneal injection of 350 mg / kg potassium oxonate for 28 consecutive days. Low-dose probiotics (1×10⁻⁶) were administered during the treatment period. 8Intervention was performed by gavage with CFU (Pediococcus pentosaccharide A21243) for 28 days;

[0064] After the modeling period, blood was collected from mice in all groups via orbital blood sampling. Mice were fasted for 12 hours prior to blood collection. The collected blood was centrifuged at 3500 r / min for 15 min, and the supernatant (serum) was collected and stored at -80℃ for later use. The uric acid content in the supernatant (serum) was determined using a uric acid detection kit from Nanjing Jiancheng Biotechnology Institute.

[0065] During the modeling period (14 days), the serum uric acid levels of each mouse in each group were measured, and the mean and standard deviation of serum uric acid levels for each group were calculated. The results are as follows: Figure 1 As shown, there were significant differences between the model group and the intervention groups of each strain and the normal group, and the serum uric acid in each group was 2.0 times higher than that in the normal group. Compared with the model group, there were no significant differences in the Pediococcus pentosaceus A21243 H group and the Pediococcus pentosaceus A21243L group. P >0.05), indicating that the hyperuricemia model was successfully established.

[0066] Intervention Period (28 days) and Intergroup Comparisons: After successful model establishment, the intervention period began. At 2.5 weeks of treatment (day 18), blood was collected from the orbital fossa of mice in both the high-dose Pediococcus pentosaceus group (A21243 H) and the low-dose Pediococcus pentosaceus group (A21243 L). Mice were fasted for 12 hours prior to blood collection. The collected blood was centrifuged at 3500 rpm for 15 min, and the supernatant (serum) was used to determine the uric acid content using a uric acid detection kit from Nanjing Jiancheng Bioengineering Institute. At the end of the treatment period (4 weeks, day 28), blood was collected from all mice. The collected blood was centrifuged at 3500 rpm for 15 min, and the supernatant (serum) was stored at -80℃ for later use and analysis as soon as possible.

[0067] The results of the comparison of uric acid levels in each group are as follows: Figure 1 As shown. After 28 days of treatment, serum uric acid in the model group was significantly higher than that in the normal group (P < 0.001). In mice in the high-dose group (A21243 H) and low-dose group (A21243 L) of Pediococcus pentosaceus, serum uric acid was significantly lower than that in the model group. P The result was <0.001, indicating that Pediococcus pentosaceus A21243 has the ability to reduce serum uric acid in mice with hyperuricemia, and this effect is dose-dependent.

[0068] Comparison of uric acid levels in mice before and after intervention: The changes in uric acid levels in each mouse of each group before and after intervention were compared, and the results are as follows: Figure 2As shown. In the control group and model group, there was no significant difference in serum uric acid levels before and after treatment. However, in the high-dose group, after 28 days of intervention, serum uric acid levels were significantly reduced (Paired T test). P <0.001), low-dose intervention significantly reduced serum uric acid in mice at 18 and 28 days (Paired T test). P <0.05), and from the perspective of significance in both periods, the difference in uric acid between the Pediococcus pentosaceus group and the model group was more significant with the extension of intervention time.

[0069] Example 4: Inhibitory effect of Pediococcus pentosaceus A21243 on xanthine oxidase (XOD), purine nucleoside phosphorylase (PNP), and adenosine deaminase (ADA) in mouse liver.

[0070] This example investigates the inhibitory effects of Pediococcus pentosaceus A21243 on xanthine oxidase (XOD), purine nucleoside phosphorylase (PNP), and adenosine deaminase (ADA) in the liver of HUA model mice. The specific process is as follows:

[0071] Preparation of liver homogenate supernatant: Based on Example 3, mice in each group were euthanized after treatment and dissected. The mouse livers were added to 9 times their volume of 0.9% physiological saline at a weight (g):volume (mL) ratio of 1:9. The homogenate was mechanically homogenized under ice-water bath conditions to prepare a 10% homogenate solution at 3000 rpm for 10 min. The supernatant was aliquoted and frozen at -80℃. It was removed only when needed, avoiding repeated freeze-thaw cycles. XOD (catalog number: A002-1-1) and ADA (catalog number: A048-2-1) detection kits from Nanjing Jiancheng Bioengineering Institute and PNP detection kits from Shanghai Enzyme-Linked Biotechnology Co., Ltd. were tested according to their instructions.

[0072] The liver XOD values ​​of mice in each group were measured, and the results are as follows: Figure 3 As shown, compared with the normal group, the XOD activity of the model mice increased ( P <0.05), after intervention with Pediococcus pentosaceus A21243 H, the XOD of HUA mice was significantly reduced ( P <0.001). This indicates that Pediococcus pentosaceus A21243 has an inhibitory effect on XOD activity in the liver of mice with hyperuricemia.

[0073] The PNP and ADA values ​​in the livers of mice in each group were measured, and the results are as follows: Figure 3 As shown, after intervention with Pediococcus pentosaceus group A21243, PNP and ADA activities decreased significantly. P <0.05, P <0.01).

[0074] XOD, PNP, and ADA are three key enzymes involved in the purine nucleoside catabolism pathway, and the metabolite produced by these three enzymes is uric acid (UA). Inhibition of XOD, PNP, and ADA activity leads to inhibition of uric acid biosynthesis. Intervention with Pediococcus pentosaceus A21243 inhibited the activity of XOD, PNP, and ADA in the liver, indicating that it can inhibit uric acid (UA) production.

[0075] Example 5: Regulatory effect of Pediococcus pentosaceus A21243 on oxidative stress disorder in HUA model mice.

[0076] This embodiment was used to investigate the regulatory effect of Pediococcus pentosaceus A21243 on oxidative stress disorder in HUA model mice, including the determination of SOD (superoxide dismutase), CAT (catalase), GSH (glutathione peroxidase), and MDA (malondialdehyde) in mouse liver. The specific procedure is as follows:

[0077] Liver samples were weighed from each group of mice (mice treated for 28 days in Example 3), and physiological saline was added at a weight ratio of 1:9. The livers were then ground using a tissue homogenizer at 60 Hz for 10 seconds on, 10 seconds off, and 120 seconds. Afterward, the mixture was centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was collected, aliquoted, and stored at -80°C. It was removed only when needed, avoiding repeated freeze-thaw cycles. SOD, CAT, GSH, and MDA were measured according to the instructions of the SOD (catalog number: A001-3-2), CAT (catalog number: A007-1-1), GSH (catalog number: A006-2-1), and MDA (catalog number: A003-1-2) detection kits from Nanjing Jiancheng Biotechnology Institute.

[0078] The results are as follows Figure 4 As shown. Compared to the normal group, the model group showed a significant increase in MDA (as shown). P <0.05), SOD increased significantly ( P <0.05), CAT was relatively significantly increased ( P <0.01), after intervention with Pediococcus pentosaceus A21243, the MDA level was significantly lower relative to the model group ( P <0.001), SOD and CAT were significantly reduced ( P <0.01, P <0.001), GSH increased significantly ( P <0.001). After intervention, MDA, SOD, and CAT levels approached normal, indicating that Pediococcus pentosaceus A21243 can regulate oxidative stress disorders caused by elevated uric acid.

[0079] Example 6: The promoting effect of Pediococcus pentosaceus A21243 on intestinal uric acid excretion in HUA model mice.

[0080] This embodiment was used to investigate the promoting effect of Pediococcus pentosaceus A21243 on intestinal uric acid excretion in HUA model mice. The specific process is as follows:

[0081] After the experiment in Example 3 was completed, feces from all mice were collected. All collected feces were added to 9 volumes of 20 mmol / L pH 7.4 phosphate buffer at a weight (g):volume (mL) ratio of 1:9, mechanically homogenized to prepare a 10% homogenate, centrifuged at 3000 rpm for 10 min, and the supernatant was used for HPLC analysis. HPLC analysis was performed using an Agilent Zobax SB-Aq (4.6 mm × 250 mm, 5 μm) at 30°C to analyze uric acid content. The detection wavelength was 285 nm, the mobile phase was 20 mM pH 7.4 K₂HPO₄-KH₂PO₄ buffer and methanol (volume ratio 90:10), the flow rate was 0.5 mL / min, and the analysis time was 15 min.

[0082] The quantitative analysis results of uric acid in mouse feces are as follows: Figure 5 As shown. Compared with the model group, there was no significant difference in the normal control group, but the fecal uric acid level after Pediococcus pentosaceus A21243 intervention was significantly higher than that in the model group ( P <0.01). This indicates that the intervention of Pediococcus pentosaceus A21243H promoted the excretion of uric acid via feces, suggesting that Pediococcus pentosaceus A21243 has a promoting effect on intestinal uric acid excretion in HUA model mice.

[0083] Example 7: Effect of Pediococcus pentosaceus A21243 on renal function indicators in HUA model mice

[0084] This example was used to investigate the effect of Pediococcus pentosaceus A21243 on renal function indicators in HUA model mice. The specific process is as follows:

[0085] As in Example 3, on days 18 and 28, serum samples were collected from mice and measured according to the instructions of the creatinine (Cre) (catalog number: C011-2-1) and blood urea nitrogen (BUN) (catalog number: C013-2-1) assay kits from Nanjing Jiancheng Bioengineering Institute. Simultaneously, when mice in each group were euthanized, their body weight and kidneys were weighed, and the kidney coefficient (the ratio of kidney to body weight) was calculated.

[0086] The measurement results are as follows Figure 6 As shown, compared with the normal group, the serum creatinine Cre in the HUA model group was significantly increased ( P <0.01), blood urea nitrogen (BUN) was significantly elevated ( P<0.001). Cre and BUN are important indicators for evaluating renal function, indicating renal insufficiency in HUA model mice. After intervention with Pediococcus pentosus A21243, serum Cre and BUN were significantly reduced ( P <0.01).

[0087] By calculating the kidney coefficient of each group of mice, the results showed that the kidney coefficient of the HUA model group was significantly lower than that of the normal group, indicating that the kidneys may have atrophy or other degenerative changes. However, after intervention with Pediococcus pentosus A21243, the kidney coefficient returned to normal. Figure 6 This indicates that A21243 can improve renal insufficiency caused by high uric acid.

[0088] Example 8: Improvement of kidney and liver pathological changes in HUA model mice by Pediococcus pentosaceus A21243

[0089] This embodiment was used to investigate the effect of Pediococcus pentosaceus A21243 on the improvement of kidney and liver pathological changes in HUA model mice. The specific process is as follows:

[0090] After the experiment in Example 3, mice in each group were euthanized and tissue samples were collected. Kidney and liver tissue samples from each group were fixed using 4% paraformaldehyde. After dehydration with 50%, 70%, 80%, 90%, 95% and anhydrous ethanol, the samples were embedded in paraffin and sectioned using a microtome. The paraffin sections were then sequentially placed in clearing agents and ethanol of different concentrations to remove paraffin from the tissue. The sections were stained with hematoxylin for 3–8 min, rinsed with tap water, and differentiated using 1% hydrochloric acid-ethanol to remove excess hematoxylin dye. A 0.6% ammonia solution was used for blueing to restore the blue color of the cell nuclei. The sections were then stained with eosin for 1–3 min, followed by dehydration and clearing with ethanol and xylene of different concentrations. Finally, the sections were mounted with neutral resin for microscopic observation.

[0091] The results are as follows Figure 7 As shown, compared to the normal group, when ethambutol hydrochloride was used in combination to construct hyperuricemia with renal excretion disorder, the model group showed interstitial cell infiltration (red arrow), renal tubular degeneration (green arrow), and glomerular hypertrophy in the kidneys, compared to the normal group. However, the intervention of Pediococcus pentosaceus A21243 reduced the area of ​​renal pathological changes and the degree of corresponding pathological changes. At the same time, the model group showed mild heparin disorder, hepatocyte swelling (orange arrow), and mild inflammatory cell infiltration (red arrow) in the liver. The intervention of Pediococcus pentosaceus A21243 reduced the pathological changes in the liver.

[0092] Example 9: The effect of Pediococcus pentosaceus A21243 on delaying and improving renal insufficiency in diabetic nephropathy (DKD)

[0093] This embodiment was used to investigate the effects of Pediococcus pentosaceus A21243 on delaying and improving renal insufficiency in diabetic nephropathy (DKD). The specific process is as follows:

[0094] Thirty 6-week-old male C57BL / 6 mice were purchased and given a 7-day acclimatization period, fed a basal diet daily. After the acclimatization period, they were randomly divided into a normal control group, a model group, and an A21243 group according to their body weight. The model group and the A21243 group were fed a high-fat, high-fructose diet (Synerbio: XTHF60-1) for 14 days, while the A21243 strain was administered as a preventative intervention by gavage at a dose of 0.2 mL of 1×10⁻⁶ mol / L daily. 9 CFU. Streptozotocin (STZ) was injected intraperitoneally on days 14 and 17, at a dose of 84 mg / kg for the first time and 65 mg / kg for the second time. The control group was injected intraperitoneally with citrate buffer to establish a type 2 diabetes mellitus (T2DM) model.

[0095] Fasting blood glucose and blood glucose changes 2 hours after glucose ingestion were measured in the tail veins of mice in each group using a Roche blood glucose meter and blood glucose test strips. Successful model establishment was defined as a fasting blood glucose concentration greater than 7.0 mmol / L and a blood glucose concentration greater than 11.0 mmol / L 2 hours after glucose ingestion. In terms of mouse condition, compared to the normal group, the model group mice had loose, wet, and sticky fur, and produced more urine. The Pediococcus pentosaceus A21243 intervention group showed relatively better A21243 status.

[0096] After successful modeling, mice were continuously administered Pediococcus pentosaceus via gavage for 28 days. Fasting blood glucose levels were measured before the end of the experiment, and an oral glucose tolerance test (OGTT) was performed. After the intervention, blood and tissue samples were collected from the mice's hearts, including serum and kidney tissues. Glycated serum protein (GSP) (using a kit from Nanjing Jiancheng, catalog number: A037-2-1), serum creatinine (Cr), and blood urea nitrogen (BUN) were measured, with the methods for detecting Cr and BUN being the same as in Example 7.

[0097] The results are as follows Figure 8 As shown. The fasting blood glucose level in the model group was significantly higher than that in the normal group ( P <0.0001), and blood glucose levels greater than 11 mmol / L, and the glycated serum protein in the model group was significantly higher than that in the normal group ( P <0.0001 indicates that the type 2 diabetes model was successfully established. The creatinine and blood urea nitrogen levels in the model group were also significantly higher than those in the normal group ( P <0.01, P <0.05), after intervention with Pediococcus pentosaceus A21243, the fasting blood glucose and glycated serum protein in the model mice were significantly reduced ( P <0.001, P<0.05), glucose metabolism improved, and renal function indicators creatinine and blood urea nitrogen decreased significantly ( P <0.0001, P <0.01), indicating that Pediococcus pentosaceus A21243 can improve renal insufficiency caused by type 2 diabetes.

[0098] Mouse kidney tissue was fixed, dehydrated, embedded, and sectioned, and then stained with HE and Masson staining and observed. The specific experimental procedure was the same as in Example 8. Masson staining was performed according to the instructions of the Masson trichrome staining kit provided by Beyotime Biotechnology Co., Ltd.

[0099] The results are as follows Figure 9 As shown in the diagram, compared to the normal group, the diabetic nephropathy model showed interstitial cell infiltration (red arrow), glomerular hypertrophy (red arrow), granulocyte infiltration (green arrow), glomerular capillary basement membrane thickening, and mild mesangial proliferation (the collagen in the basement membrane and mesangium was stained bright green, with orange-yellow arrows). However, after intervention with Pediococcus pentosaceus A21243, the corresponding pathological changes in the kidneys could be reduced or improved.

[0100] Example 10: The effect of Pediococcus pentosaceus A21243 on delaying and improving cisplatin-induced renal insufficiency.

[0101] This embodiment was used to investigate the effects of Pediococcus pentosaceus A21243 on delaying and improving the progression of cisplatin-induced acute kidney injury to chronic kidney disease. The specific process is as follows:

[0102] Ten 8-week-old male C57BL / 6 mice were purchased and given a 7-day acclimatization period, fed a basal diet daily. After the acclimatization period, they were randomly divided into a normal group, a model group, and an A21243 group according to their body weight. The A21243-containing bacterial powder from Example 2 was administered to the model group for 14 days via gavage at a dose of 1×10⁻⁶. 9 CFU was used to colonize the strain first, and the normal and model groups were administered physiological saline by gavage. The model group and the A21243 group were intraperitoneally injected with cisplatin on days 14, 21, and 28, respectively, at a dose of 7.5 mg / kg each time. Simultaneously, the A21243 group was administered 1×10⁻⁶ cisplatin by gavage. 9 CFU A21243 was administered via gavage to both the normal and model groups, along with physiological saline. After intervention, euthanasia was performed, and blood samples were collected from the heart to measure serum uric acid (Cre) and serum BUN, following the methods described in the preceding examples.

[0103] The results are as follows Figure 10 As shown, compared with the normal group, the serum creatinine in the model group was significantly increased ( P <0.0001), after intervention with strain A21243, serum creatinine decreased significantly relative to the model group ( P<0.01). There was no significant difference in urinary protein, however, the urinary creatinine level in the model group was significantly increased. Combined with the serum creatinine results, more creatinine was accumulated in the model mice, indicating a decrease in the kidney's ability to excrete creatinine and renal function impairment in the model group. A21243 intervention can improve cisplatin-induced renal insufficiency.

[0104] Simultaneously, following the methods described in the preceding embodiments, the kidneys were fixed, dehydrated, embedded, sectioned, stained, and observed. The results are as follows: Figure 11 As shown, since cisplatin is mainly excreted through the renal tubules and may be reabsorbed and accumulated in the tubules, morphological changes in the renal tubules, such as tubular nephritis, are most common. Figure 11 As shown by the red arrow, there are a large number of inflammatory cells near the renal tubules, and the renal tubular epithelial cells undergo vacuolar degeneration (green arrow). A21243 intervention alleviated the above pathological changes.

[0105] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Pediococcus pentosaceus ( Pediococcus pentosaceus The application of Pediococcus pentosaceus A21243 in the preparation of drugs for treating kidney disease, wherein the preservation number of Pediococcus pentosaceus A21243 is GDMCC NO:63749; The kidney disease is renal insufficiency caused by type 2 diabetes, hyperuricemia, or cisplatin.

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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