Application of probiotics in reducing acute gout morbidity and improving acute gout inflammation under continuous hyperuricemia

Abstract

The invention belongs to the technical field of probiotics, and relates to application of probiotics to reduction of acute gout morbidity and improvement of acute gout inflammation under continuous hyperuricemia. Furthermore, the invention relates to the application of the probiotics (Lactobacillus gasseri BKR-007) in reducing the incidence rate of acute gout and improving the inflammation of acute gout under the condition of continuous hyperuricemia. The invention finds that the strain BKR-007 can reduce the risk of gout induced by an organism under hyperuricemia by inhibiting the activity of inflammasomes NLRP3 and NF-kappa B; the inflammation level in vivo in the acute gout attack period can also be inhibited, and the swelling and pain symptom is remarkably improved; and the traditional Chinese medicine composition has a protective effect on renal function impairment caused by hyperuricemia.

Description

Technical Field

[0001] This invention belongs to the field of probiotic technology and relates to the application of a probiotic in reducing the incidence of acute gout and improving acute gout inflammation in patients with persistent hyperuricemia. Background Technology

[0002] Inflammatory bodies are intracellular polyprotein complexes with a molecular weight of at least 700 kDa, containing caspase-1, cysteine ​​aspartate protease 1 apoptosis-associated speck-like protein (ASC), and a pyrazine-domain-containing nucleotide-binding oligomerization domain-like receptor (NLRP). This complex is present in the cytoplasm of activated immune cells in the human body, including monocytes, macrophages, and dendritic cells. [1] .

[0003] Gout is an inflammatory disease characterized by redness, swelling, and pain in the joints. Hyperuricemia is generally considered the underlying cause of gout, where supersaturated uric acid deposits in the joint cavity to form monosodium urate (MSU), causing local redness, swelling, and pain. This joint redness and pain is considered a non-infectious inflammatory arthritis caused by the deposition of MSU crystals in the joints and surrounding tissues. [2] .

[0004] Although hyperuricemia is an important biochemical basis for the development of gout, only a portion of patients with hyperuricemia eventually develop gout. Gout is essentially a crystal-related arthropathy caused by the deposition of monosodium urate (MSU) crystals in the joints and surrounding tissues. The acute onset of this disease begins with the recognition and phagocytosis of MSU crystals by macrophages in the joint. This process activates the intracellular NLRP3 inflammasome, which then releases pro-inflammatory factors such as interleukin-1β (IL-1β) and IL-18 through caspase-1 cleavage. [3,4,5] Simultaneously, the activation of the NF-κB pathway drives a strong acute inflammatory response and recruits neutrophils to aggregate, ultimately leading to the characteristic clinical manifestations of gout: redness, swelling, heat, and pain. [6,7,8] Generally speaking, gout attacks in humans are self-limiting, usually resolving spontaneously within 7-10 days. [9] In the model mice, remission occurred within 24-36 hours because uricase in the body can degrade MSU crystals. [10,11] However, in severe cases, persistent MSU crystal deposition can cause irreversible joint damage.

[12] This has a huge impact on the patient's quality of life.

[0005] Probiotics can improve gout, mainly through two mechanisms: First, certain strains can directly degrade purines, precursors of uric acid, in the intestines, thereby reducing the production of uric acid at its source; second, these probiotics can focus on regulating the body's immune inflammatory response, effectively relieving clinical symptoms such as joint redness, swelling, heat, and pain during acute gout attacks. Previous studies have shown that Lactobacillus gasseri BKR-007 (CN114908006B) can reduce the risk of elevated serum uric acid by reducing the intake of dietary uric acid precursor purines; the combination of Bifidobacterium longum BL21 and Lactobacillus salivarius LS97 (CN118147021B) has been reported to reduce pain sensitivity in mice with gouty arthritis and simultaneously reduce the number of white blood cells and neutrophil aggregation in the joints, manifested by: reducing the activity of myeloperoxidase in periarticular tissues, reducing the production of inflammatory factors CXCL1 and IL-1β, and increasing the expression level of the anti-inflammatory cytokine IL-10 in joint tissues.

[0006] Another probiotic and prebiotic composition (CN113181365A) has been reported to have the function of degrading uric acid, dissolving uric acid crystals and tophi, so this composition also has the ability to improve joint redness, swelling and recurrent acute gout attacks.

[0007] Therefore, current probiotics primarily reduce serum uric acid levels by decreasing dietary purine intake, or may promote the dissolution of uric acid crystals through some unclear mechanisms, thereby preventing acute gout attacks to some extent, or alleviating local inflammation by directly dissolving existing uric acid crystals. However, when uric acid crystals have already formed and have not been effectively cleared, current probiotics have not shown the ability to inhibit inflammatory pathways at their source, and cannot block the activation and cascade amplification of inflammatory signals. Furthermore, there is currently insufficient research evidence to suggest that existing probiotics can effectively prevent gout or reduce the incidence of acute gout in a state of persistent hyperuricemia. Therefore, in preventing acute gout attacks or reducing the severity of inflammation after an acute attack and shortening the course of the disease, there is still an urgent need to develop new strategies that can fundamentally intervene in uric acid metabolism and inflammatory pathways.

[0008] References:

[0009] [1]Groslambert M,Py B F.Spotlight on the NLRP3inflammasome pathway[J].Journal ofinflammation research,2018:359-374.

[0010] [2]Dalbeth N,Merriman TR,Stamp LK.Gout[J].The Lancet,2016,388(10055):2039-2052.DOI:10.1016 / S0140-6736(16)00346-9.

[0011] [3]Martinon F,Pétrilli V,Mayor A,et al.Gout-associated uric acidcrystals activate the NALP3inflammasome[J].Nature,2006,440(7081):237-241.

[0012] [4]Ramirez M L G,Poreba M,Snipas S J,et al.Extensive peptide andnatural protein substrate screens reveal that mouse caspase-11has muchnarrower substrate specificity than caspase-1[J].Journal of BiologicalChemistry,2018,293(18):7058-7067.;

[0013] [5]Xi H,Zhang Y,Xu Y,et al.Caspase-1inflammasome activation mediateshomocysteine-induced pyrop-apoptosis in endothelial cells[J].Circulationresearch,2016,118(10):1525-1539.;

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[0015] [7]Slaats J,Ten Oever J,van de Veerdonk F L,et al.IL-1β / IL-6 / CRP andIL-18 / ferritin:distinct inflammatory programs in infections[J].PLoSPathogens,2016,12(12):e1005973.

[0016] [8]Campden R I,Zhang Y.The role of lysosomal cysteine cathepsins inNLRP3inflammasome activation[J].Archives of Biochemistry and Biophysics,2019,670:32-42.

[0017] [9]Martin W J,Harper J L.Innate inflammation and resolution in acutegout[J].Immunology and Cell Biology,2010,88(1):15-19.

[0018]

[10] Amaral F A,Costa V V,Tavares L D,et al.NLRP3inflammasome-mediatedneutrophil recruitment and hypernociception depend on leukotriene B4in amurine model of gout[J].Arthritis&Rheumatism,2012,64(2):474-484.

[0019]

[11] I,Vago J P,Barroso L C,et al.Annexin A1 promotes timelyresolution of inflammation in murine gout[J].European journal of immunology,2017,47(3):585-596.

[0020]

[12] Rees F, Hui M, Doherty M. Optimizing current treatment of gout[J]. Nature Reviews Rheumatology, 2014, 10(5): 271-283. Summary of the Invention

[0021] In view of the problem that existing technologies can only reduce the risk of gout by lowering serum uric acid levels and intra-articular uric acid crystals, the present invention aims to provide an application of probiotics in reducing the incidence of acute gout and improving acute gout inflammation in patients with persistent hyperuricemia.

[0022] To achieve the above objectives, the present invention adopts the following technical solution:

[0023] Application of a probiotic in reducing the incidence of acute gout and improving acute gout inflammation in patients with persistent hyperuricemia.

[0024] The application of the probiotics in the preparation of drugs that inhibit the in vivo inflammasome NLRP3 and NF-κB during the acute onset of gout.

[0025] The probiotic is Lactobacillus gasseri SYP-B4432 (BKR-007).

[0026] The Lactobacillus gasseri SYP-B4432(BKR-007) is deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCCNo. 22950 and deposit date of July 26, 2021.

[0027] Advantages of this invention:

[0028] The Lactobacillus gasseri BKR-007 of this invention can directly inhibit the activity of NLRP3 inflammasome and NF-κB in a state of persistent hyperuricemia, making it difficult for MSU crystals to activate the immune response, thereby reducing the risk of acute gout.

[0029] Although a few probiotics and compositions have the ability to reduce some inflammatory factors during acute gout attacks, the strain BKR-007 of this invention can inhibit the activity of NLRP3 and NF-κB at the source, and therefore has a good inhibitory effect on various inflammatory factors such as IL-1β and TNF-α produced downstream, thereby reducing inflammation and shortening the course of the disease. Attached Figure Description

[0030] Figure 1The inhibitory effect of strain BKR-007 on joint swelling and pain in mice after acute gout modeling provided in Example 1 of the present invention.

[0031] Figure 2 The inhibitory effect of strain BKR-007 on macrophages, neutrophils and MPO in mouse joints after acute gout modeling provided in Example 1 of the present invention.

[0032] Figure 3 The inhibitory effect of strain BKR-007 on the expression levels of various genes of the inflammasome NLRP3 in mouse joints after acute gout modeling provided in Example 1 of the present invention.

[0033] Figure 4 The acute gout model provided in Example 1 of this invention demonstrates the inhibitory effect of strain BKR-007 on some inflammatory factors and NF-κB in the joints of mice.

[0034] Figure 5 The inhibitory effect of strain BKR-007 on joint swelling and pain in mice during the acute gout recovery period provided in Example 2 of the present invention.

[0035] Figure 6 The inhibitory effect of strain BKR-007 on macrophages, neutrophils and MPO in mouse joints during the acute gout recovery period provided in Example 2 of the present invention.

[0036] Figure 7 The BKR-007 strain showed an inhibitory effect on some inflammatory factors in the joints of mice during the acute gout recovery period, as provided in Example 2 of this invention.

[0037] Figure 8 The serum uric acid level of the hyperuricemic mice provided in Example 3 of the present invention.

[0038] Figure 9 The BKR-007 strain was used to inhibit the incidence of acute gout in hyperuricemic mice provided in Example 3 of this invention.

[0039] Figure 10 The BKR-007 strain inhibited some inflammatory factors in the serum of hyperuricemic mice provided in Example 3 of this invention.

[0040] Figure 11 The protective effect of strain BKR-007 on renal function in hyperuricemic mice provided in Example 3 of the present invention. Detailed Implementation

[0041] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0042] This invention utilizes Lactobacillus gasseri BKR-007 to inhibit NLRP3 inflammasomes and NF-κB activated by MSU crystals in the body, thereby reducing the risk of acute gout induced by persistent hyperuricemia; comprehensively improving the body during the acute gout phase, thereby reducing the level of inflammatory factors in the body, and comprehensively relieving joint swelling and pain; achieving a reduction in the incidence of gout and protecting the kidneys; and shortening the course of the disease and improving the inflammatory response after the occurrence of acute gout.

[0043] The strain BKR-007 involved in the following examples has been described in patent document application number 2022104408921, and a corresponding deposit certificate has been provided. Its classification name is Lactobacillus gasseri, and its deposit number is CGMCC No. 22950.

[0044] The culture media involved in the following examples are as follows:

[0045] MRS medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 15g / L, lactose 15g / L, yeast extract 5g / L, diammonium hydrogen citrate 2g / L, K2HPO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4 0.05g / L, Tween 80 1mL / L, cysteine ​​0.5g / L.

[0046] The following examples illustrate the method for preparing probiotic suspensions:

[0047] The strain BKR-007, preserved in glycerol tubes, was inoculated into MRS medium and cultured at 37°C until OD500. 600 The concentration of the culture medium was >1.2. The fermentation broth was transferred to centrifuge tubes and centrifuged at 4000 rpm for 10 min. The supernatant was discarded, and the bacterial cells were recovered. The bacterial cells were resuspended in physiological saline to a concentration of 1.0 × 10⁻⁶. 10 CFU / ml available for use.

[0048] The experimental animals involved in the following examples are:

[0049] Healthy male 8-week-old SPF-grade C57BL / 6J mice weighing 20–25g were used. Mice were housed in standard transparent plastic cages at 22–25°C with free access to food and drink. All experiments were conducted according to the animal care and handling procedures of the International Association for the Study of Pain (IASP).

[0050] The experimental results were statistically analyzed using GraphPad. # indicates a significant difference compared to the positive control group (p < 0.01), while * indicates no significant difference compared to the negative control group (p > 0.05).

[0051] Example 1: Inhibitory effect of Lactobacillus gasseri BKR-007 on acute gout attacks.

[0052] Test method:

[0053] Thirty mice were divided into three groups: Group 1 was the negative control group (n=10), Group 2 was the positive control group (n=10), and Group 3 was the probiotic intervention group (n=10). One week prior to the establishment of the gouty arthritis model in the mouse knee joint, all three groups were allowed free access to food, but Group 3 was simultaneously administered 0.1 ml (1×10⁻⁶) of the probiotic suspension obtained above via gavage daily. 9 After 7 days of intervention (CFU / mouse / day), mice were used to establish the knee model. The first negative control group received an intra-articular injection of sterile saline (10 μL) under isoflurane anesthesia. The second and third groups were induced by intra-articular injection of 10 μL of MSU suspension (10 μg / μL) under short-acting isoflurane anesthesia. The swelling diameter of the mouse knee joint was measured with calipers at 1, 3, 5, 7 and 15 hours after modeling, and the swelling value was expressed as Δmm / joint.

[0054] Because mice with gout have a low pain threshold to external pressure, an electronic pressure gauge was used to assess their pain perception. The withdrawal threshold induced by flexion was used to infer pain-related behavioral responses. Pain responses were induced by applying progressive pressure to the animal's hind limbs. Once the animal exhibited a clear pain response, the force of the applied pressure was recorded. Tests were conducted at 1, 3, 5, 7, and 15 hours. Results are expressed as mean measurements (in grams).

[0055] Knee joint lavage fluid was collected 15 h after MSU crystal injection for leukocyte recruitment assay. The joint cavity was flushed three times with 3.3 μL of physiological saline containing 1 mM EDTA, and then diluted to a final volume of 50 μL PBS / EDTA to assess leukocyte migration. Total leukocyte count was determined in a Neubauer chamber (erythrocytes lysed with Turk solution, diluted 1:2). Cell classification and counting were performed under an optical microscope using Rosenfeld-stained sections, and results are expressed as the number of monocytes or neutrophils per cell.

[0056] MPO activity in the knee joint (assessing the extent of neutrophil recruitment to the knee joint) was measured 15 h after MSU crystal injection. Mice were deeply anesthetized, and knee joint tissue was homogenized in 200 μL of buffer (pH 6.0, 50 mM K₂HPO₄ + 0.5% HTAB). The homogenate was then centrifuged (16000 g, 2 min, 4 °C), and the supernatant was collected. 30 μL of the supernatant was placed in a 96-well plate and mixed with 200 μL of 50 mM K₂HPO₄ buffer (pH 6.0, 0.0167% ortho-dianisidinedihydrochloride + 0.05% H₂O₂) for 15 min, and the absorbance was measured at 450 nm. The MPO activity of the samples was compared with a standard curve of neutrophils and expressed as MPO activity / mg of protein.

[0057] NF-κB activity in the knee joint was measured 15 hours after MSU crystal injection. The extracted joint tissue was lysed in pre-cooled lysis buffer (Cell Signaling Technology, Beverly, MA, United States). The levels of total NF-κB p65 and phosphorylated NF-κB p65 were measured by ELISA (450 nm) using PathScan kits #7836 and #7834 (Cell Signaling Technology, Beverly, MA, United States), respectively, to obtain the phospho-p65 / total-p65 ratio.

[0058] Another part of the knee joint tissue was Reagent processing was performed to isolate total RNA according to the manufacturer's instructions. The purity of the total RNA was measured using a spectrophotometer; the wavelength absorbance ratio (260 / 280 nm) of all preparations was between 1.8 and 2.0. 2-Step RT-qPCR System (Promega) and Specific Primers (Applied) Reverse transcription and qPCR were performed on total RNA to cDNA.

[0059] The primer sequences used are as follows:

[0060] Tnf-α:

[0061] Forward: 5′-TCTCATCAGTTCTATGGCCC-3′ Reverse: 5′-GGGAGTAGACAAGGTACAAC-3′

[0062] Pro-Il-1β (Il-1β):

[0063] Forward: 5′-GAAATGCCACCTTTTGACAGTG-3′ Reverse: 5′-TGGATGCTCTCATCAGGACAG-3′

[0064] Nlrp3:

[0065] Forward: 5′-CGTGGTTTCCTCCTTTTGTATT-3′ Reverse: 5′-CGACCTCCTCTCCTCTCTTCTT-3′

[0066] Asc (apoptosis-associated speck-like protein containing a CARD):

[0067] Forward: 5′-TCACAGAAGTGGACGGAGTG-3′ Reverse: 5′-TGTCTTGGCTGGTGGTCTCT-3′

[0068] Caspase-1:

[0069] Forward: 5′-CGTGGAGAGAAACAAGGAGTG-3′ Reverse: 5′-AATGAAAAGTGAGCCCCTGAC-3′

[0070] Experimental results:

[0071] 1. Effects of strain BKR-007 on joint swelling and pain during acute gout attacks

[0072] The degree of swelling and pain in the knee joint of mice 1–15 hours after injection of MSU crystals was determined by [the following factors]. Figure 1 As shown in the figure. Figure 1 As shown in Figure A, at all time points from 1 to 15 hours after modeling, the degree of joint swelling in the probiotic group (MSU+BKR-007) was significantly lower than that in the positive control group (MSU). Figure 1 As shown in Figure B, the probiotic group mice experienced a significant reduction in pain at each stage from 1 to 15 hours after modeling.

[0073] 2. Effect of strain BKR-007 on the number of intra-articular white blood cells in the acute phase of gout

[0074] Fifteen hours after injection of MSU crystals into the knee joint of mice, the number of total leukocytes, macrophages, and neutrophils recruited within the knee joint were as follows: Figure 2 As shown in the figure. Figure 2 As shown in Figure A, the total white blood cell count in the MSD group was significantly increased after modeling (6.2 ± 0.7 × 10⁻⁶). 4 (CFU / cavity), the probiotic group reduced the total white blood cell count to 4.2 ± 0.7 × 10⁻⁶.4 CFU / cavity; among which macrophages ranged from 1.6 ± 0.2 × 10⁻⁶. 4 CFU / cavity decreased to 1.2 ± 0.18 × 10⁻⁶ 4 CFU / cavity Figure 2 B), while the number of neutrophils decreased from 4.6 ± 0.5 × 10 4 CFU / cavity decreased to 3.0 ± 0.5 × 10⁻⁶ 4 CFU / cavity Figure 2 .C). By Figure 2 As can also be seen from D, myeloperoxidase (MPO) activity, which is used to assess the degree of neutrophil recruitment to the knee joint, was also significantly reduced (from 1800±300 to 1010±180). This result also indicates mechanistically that strain BKR-007 can reduce the recruitment of immune cells at the lesion site during the acute phase of gout.

[0075] 3. Effects of strain BKR-007 on intra-articular inflammasomes during acute gout attacks

[0076] The NLRP3 inflammasome is composed of sensor protein (NLRP3, which senses external stimuli such as MSU crystals), adaptor protein (ASC, which is responsible for recruiting effector proteins) and effector protein (Caspase-1, which is responsible for activating IL-1β, etc.).

[0077] Fifteen hours after injection of MSU crystals into the knee joint of mice, strain BKR-007 inhibited the transcriptional levels of three proteins in the NLRP3 inflammasome as follows: Figure 3 As shown in the figure. Figure 3 It can be seen that strain BKR-007 can reduce the transcription level of NLRP3 from 3.2±0.5 to 1.5±0.2. Figure 3 .A) can reduce the transcriptional level of ACS from 3.0±0.4 to 1.6±0.3 ( ). Figure 3 .B), which can reduce the transcriptional level of Caspase-1 from 3.4±0.4 to 1.6±0.2 ( ). Figure 3 (C). This result indicates that strain BKR-007 can inhibit the expression of NLRP3 inflammasomes induced by MSU crystals in the knee joint, and thus may inhibit inflammatory factors that cause pain, swelling and inflammation by inhibiting the activity of inflammasomes.

[0078] 4. Effects of strain BKR-007 on intra-articular inflammatory factors and NF-κB during acute gout attacks.

[0079] The effects of strain BKR-007 on major inflammatory factors IL-1β and TNF-α 15 hours after injection of MSU crystals into the knee joint of mice are as follows: Figure 4 As shown in the figure. Figure 4 It was found that strain BKR-007 significantly inhibited the activity of the NLRP3 inflammasome, thereby significantly reducing the expression levels of downstream inflammatory factors in the inflammatory pathway, with IL-1β expression decreasing from 4.2±0.4 to 2.1±0.21. Figure 4 TNF-α expression levels decreased from 3.8 ± 0.43 to 1.6 ± 0.21 (A). Figure 4 .B).

[0080] Studies have shown that IL-1β and TNF-α can also induce the activation of the nuclear transcription factor NF-κB (https: / / pubmed.ncbi.nlm.nih.gov / 28780730 / ) and, upon activation, form a positive feedback loop that amplifies the inflammatory response. Figure 4 As shown in Figure C, strain BKR-007 can effectively inhibit the activation of NF-κB, and the ratio of phosphorylated p56 subunits to total p56 subunits can be reduced from 0.45±0.04 to 0.24±0.02.

[0081] in conclusion:

[0082] Based on the above results, strain BKR-007 has a significant inhibitory effect on acute gouty arthritis induced by MSU crystals. Phenotypically, it can reduce edema and pain; at the cellular level, it can inhibit the recruitment of immune cells to key targets; and at the molecular level, it can inhibit the activity of NLRP3 inflammasome, nuclear transcription factor NF-κB, and major inflammatory factors (IL-1β and TNF-α), thus showing a strong therapeutic effect on acute gout attacks.

[0083] Example 2: Recovery after an acute gout attack using Lactobacillus gasseri BKR-007

[0084] Test method:

[0085] Thirty mice were divided into three groups: Group 1 was the negative control group (n=10), Group 2 was the positive control group (n=10), and Group 3 was the probiotic intervention group (n=10). After one week of free-range feeding, mice in all three groups underwent probiotic modeling (as described in Example 1). Fifteen hours after modeling, all three groups were intraperitoneally injected with potassium oxonate (270 mg / kg / day) to inhibit the breakdown of MSU crystals. Subsequently, Groups 1 and 2 were allowed free-range feeding, while Group 3, in addition to free-range feeding, was also administered 0.1 ml (1 × 10⁻⁶) of probiotic suspension daily by gavage. 9 (CFU / animal / day). The three groups received continuous intervention for a total of 7 days.

[0086] Following the intervention, joint pain and swelling in mice were assessed on days 1, 3, 5, and 7 (experimental methods as described in Example 1). On days 5 and 7, five mice from each group were anesthetized and euthanized, and the number of macrophages and neutrophils, MPO content (experimental methods as described in Example 1), and IL-1β and TNF-α levels in the joints were measured. TNF-α was detected using a tumor necrosis factor-α kit (Nanjing Jiancheng Biotechnology Institute); IL-1β was detected using an interleukin-1β kit (Nanjing Jiancheng Biotechnology Institute).

[0087] Experimental results:

[0088] 1. During treatment, strain BKR-007 aids in the recovery of joint swelling and pain.

[0089] Depend on Figure 5 As shown, mice induced by MUS crystallization and treated with strain BKR-007 for 7 days showed significant efficacy by day 3. Compared with the positive control group, the swelling decreased from 0.27±0.035mm to 0.16±0.025mm (P<0.001), and was basically recovered by day 7 (no significant difference compared with the negative control group, P>0.05) (see...). Figure 5 A). For example Figure 5 As shown in Figure B, the degree of pain in mice was similar to the change in swelling, with significant improvement observed by day 3 (from 2.9±0.5g to 5.7±0.45g, P<0.001), and near-complete recovery by day 7 (no significant difference compared to the negative control group, P>0.05) (see Figure B). Figure 5 .B)

[0090] 2. Effects of strain BKR-007 on immune cells during treatment

[0091] Depend on Figure 6 As shown, by day 5 of treatment, the total white blood cell, macrophage, and neutrophil counts were already at low levels. Although slightly higher than the negative control group (P<0.05), they were significantly lower than the positive control group (P<0.001). By day 7 of treatment, the macrophage and neutrophil counts had returned to normal, while the positive control group remained at higher levels (see...). Figure 6 .AB C). For example Figure 6 As can be seen from D, the MPO content, which represents the degree of neutrophil recruitment, returned to normal levels on day 5 (no significant difference compared with the negative control group, P>0.05).

[0092] 3. Effects of strain BKR-007 on immune factors during treatment.

[0093] Depend on Figure 7As shown, by day 5 of treatment, IL-1β and TNF-α levels had decreased to a low level. Although the levels were slightly higher than those in the negative control group (P<0.05), they were significantly lower than those in the positive control group (P<0.001). By day 7 of treatment, IL-1β and TNF-α had completely returned to normal levels (no significant difference compared to the negative control group, P>0.05).

[0094] in conclusion:

[0095] In summary, after establishing a MUS crystallization model in the mouse knee joint, injection of potassium oxonate inhibited the hydrolysis of MUS, allowing the knee joint inflammation to persist for a longer period, with a high concentration of inflammatory factors remaining in the joint even on day 7. Furthermore, strain BKR-007 effectively suppressed joint inflammation, with complete recovery observed by day 7, thus demonstrating that strain BKR-007 can significantly shorten the course of acute gout.

[0096] Example 3: Prevention of acute gout attacks in hyperuricemic states using Lactobacillus gasseri BKR-007

[0097] Test method:

[0098] Thirty mice were divided into three groups: Group 1 was the negative control group (n=10), Group 2 was the positive control group (n=10), and Group 3 was the probiotic intervention group (n=10). After one week of free-range feeding, mice in each group were used to establish the probiotic model. Group 1 (negative control group) received free-range feeding and 0.1 ml of saline solution via tail vein injection every 3 days; Group 2 (positive control group) received free-range feeding and 0.1 ml of MUS crystal suspension (600 μg / mouse) via tail vein injection every 3 days; Group 3 (probiotic group) received free-range feeding and 0.1 ml of the probiotic suspension obtained above (1×10⁻⁶) via gavage. 9 MUS crystal suspension (CFU / mouse / day) was administered to mice with free access to food and water. Every 3 days, mice received a tail vein injection of 0.1 ml (100 μg / mouse) of MUS crystal suspension. Simultaneously, all three groups received an intraperitoneal injection of potassium oxonate (270 mg / kg / day) to inhibit the breakdown of MUS crystals in mice.

[0099] The experiment lasted for 21 days, and the joint pain and swelling of mice were detected every 3 days (the experimental method is as described in Example 1). When the force of the stress response of the mouse is <8g and the swelling of any joint is >0.15mm, the mouse is considered to have an acute gout attack.

[0100] Blood samples were collected every 7 days to measure serum uric acid levels, inflammatory factors (IL-1β and TNF-α), and BUN and Scr, which reflect renal function. Blood was collected from rat tails, and the collected blood was centrifuged at 3000 rad / min for 10 min to obtain serum. Serum uric acid levels in each group of rats were measured using a uric acid assay kit (BioVision Inc., Milpitas, CA, USA); serum TNF-α and IL-1β levels were measured using a tumor necrosis factor-α assay kit (Nanjing Jiancheng Biotechnology Institute) and an interleukin-1β assay kit (Nanjing Jiancheng Biotechnology Institute); and creatinine (Scr) was measured using the sarcosine method and blood urea nitrogen (BUN) was measured using the urease method.

[0101] Test results

[0102] 1. Serum uric acid levels in mice over 21 days

[0103] The concentration of serum uric acid in mice from 0 to 21 days after modeling is as follows: Figure 8 As shown, during continuous injection of MUS crystals, the serum uric acid concentration in mice peaked at 650±50 μmol / L on day 14 and remained there for 21 days, demonstrating successful model establishment. Furthermore, it was found that strain BKR-007 did not degrade MUS injected directly via intravenous injection.

[0104] 2. Incidence of gout in mice over 21 days

[0105] The criteria for determining a gout attack are: when a mouse responds to stress with a force <8g and swelling of any joint >0.15mm, it is considered to have an acute gout attack. Figure 9 As shown, by day 3 of modeling, the positive control group had one case of gout, 50% of mice developed the disease by day 12, and 90% by day 15. In the probiotic group, the first case appeared on day 6, but the incidence rate did not exceed 50% until day 21. Therefore, it can be concluded that strain BKR-007 can effectively reduce the acute incidence of gout under the sustained pressure of hyperuricemia.

[0106] 3. Inflammatory factors in mouse serum during 21 days

[0107] The changes in serum IL-1β and TNF-α in mice from 0 to 21 days after modeling are as follows: Figure 10 As shown in the figure. In the positive control group, IL-1β showed a significant increase on days 14 and 21. Although the probiotic group also showed an increase on days 14 and 21, the increase level was significantly lower than that in the positive control group (P<0.01) (see...). Figure 10 A). By Figure 10As shown in Figure B, TNF-α in the positive control group showed a significant increase on day 7 and continued to rise from day 14 to day 21. In contrast, the probiotic group showed a significant increase from day 14 to day 21, with no significant difference from day 14 (P>0.05). Therefore, the probiotic group was significantly lower than the positive control group from day 7 to day 21 (P<0.001).

[0108] 4. Changes in renal function indicators in mice over 21 days

[0109] like Figure 11 As shown, in the positive control group, no significant changes were observed in Scr and BUN levels before day 14, but a significant increase in both Scr and BUN was observed on day 21. However, in the probiotic group, Scr did not show a significant increase throughout the 21 days (P>0.05), while BUN was significantly higher on day 21 compared to the negative control group (P<0.05), but still much lower than the positive control group. This indicates that long-term hyperuricemia can cause significant damage to renal function in mice, but strain BKR-007 can effectively alleviate this renal damage caused by hyperuricemia.

Claims

1. The application of a probiotic in reducing the incidence of acute gout and improving acute gout inflammation in patients with persistent hyperuricemia.

2. The application according to claim 1, characterized in that: The application of the probiotics in the preparation of drugs that inhibit the in vivo inflammasome NLRP3 and NF-κB during the acute onset of gout.

3. The application according to claim 1 or 2, characterized in that: The probiotic is Lactobacillus gasseri SYP-B4432 (BKR-007).

4. The application according to claim 3, characterized in that: The Lactobacillus gasseri SYP-B4432(BKR-007) is deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 22950 and deposit date of July 26, 2021.

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