Strain composition for alleviating nephritis and use thereof

Abstract

The application discloses a strain composition for relieving nephritis and application thereof, and belongs to the technical field of biological medicine, wherein the strain composition comprises the following strains: Bifidobacterium longum HH-BL18, Lactobacillus acidophilus HH-LA26, Lactobacillus plantarum HH-LP56 and Lactobacillus helveticus HH-LPH17. The strain composition provided by the application can effectively improve biochemical indexes of nephritis, improve immunity, reduce inflammatory reaction and the like, and can be used for improving kidney function and relieving nephritis disease, thereby providing a new treatment method and thought for nephritis disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to bacterial strain compositions for alleviating nephritis and their applications. Background Technology

[0002] The primary physiological functions of the kidneys are to excrete metabolic waste products, regulate water, electrolyte, and acid-base balance, secrete various active substances, and maintain homeostasis to ensure normal physiological function. Nephritis is a group of kidney diseases mediated by the immune system and involving inflammatory mediators (such as complement, cytokines, and reactive oxygen species), ultimately leading to inflammatory changes in the intrinsic renal tissues and causing varying degrees of renal function decline. It can be caused by a variety of etiologies. Non-immune and non-inflammatory mechanisms are also involved in the chronic process.

[0003] Current technologies commonly use high doses of antibiotics, such as levofloxacin hydrochloride, to treat nephritis-related diseases. However, this method can lead to drug resistance and damage to the human body as the dosage of antibiotics increases.

[0004] The applicant previously disclosed the use of Lactobacillus helveticus HH-LPH17 in the prevention and treatment of cholestatic liver disease in Chinese patent CN114262671A, but there are no reports on the application of the above strain in the relief of nephritis. Summary of the Invention

[0005] The purpose of this invention is to provide a bacterial strain composition for alleviating nephritis and its application. This bacterial strain composition can effectively improve biochemical indicators of nephritis, enhance immunity, reduce inflammatory response, etc., and can be used to improve kidney function and alleviate nephritis, providing new treatment methods and ideas for nephritis.

[0006] In a first aspect, this application provides a strain of *Bifidobacterium longum*, named *Bifidobacterium longum* HH-BL18, which was deposited on March 24, 2022, at the China Center for Type Culture Collection (CCTCC), located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2022306.

[0007] Secondly, this application provides a strain of *Lactobacillus acidophilus*, named *Lactobacillus acidophilus* HH-LA26, which was deposited on March 24, 2022, at the China Center for Type Culture Collection (CCTCC), located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2022305.

[0008] Thirdly, this application provides a strain of *Lactobacillus plantarum*, named *Lactobacillus plantarum* HH-LP56, which was deposited on June 26, 2019, at the China Center for Type Culture Collection (CCTCC), located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2019497.

[0009] Fourthly, embodiments of this application provide a bacterial strain composition comprising the following strains:

[0010] The strains described in the first aspect, the strains described in the second aspect, the strains described in the third aspect, and Lactobacillus helveticus HH-LPH17;

[0011] The Lactobacillus helveticus HH-LPH17 is deposited at the China Center for Type Culture Collection, with accession number CCTCCNO: M2021725.

[0012] Fifthly, embodiments of this application provide the use of the strain composition described in the fourth aspect in the preparation of a drug for relieving nephritis.

[0013] Sixthly, embodiments of this application provide a formulation for relieving nephritis, the formulation comprising:

[0014] (a) the bacterial composition described in the fourth aspect or the bacterial powder of the bacterial composition described in the fourth aspect; and

[0015] (b) Pharmaceutical excipients.

[0016] Further, by weight, the preparation comprises: 20-60 parts of the bacterial powder of the strain described in the first aspect, 20-60 parts of the bacterial powder of the strain described in the second aspect, 20-60 parts of the bacterial powder of the strain described in the third aspect, and 10-50 parts of the bacterial powder of Lactobacillus helveticus HH-LPH17.

[0017] Further, by weight, the preparation comprises: 35-50 parts of the bacterial powder of the strain described in the first aspect, 35-50 parts of the bacterial powder of the strain described in the second aspect, 35-50 parts of the bacterial powder of the strain described in the third aspect, and 20-30 parts of the bacterial powder of Lactobacillus helveticus HH-LPH17.

[0018] Furthermore, the viable cell count in the bacterial powder of the strains described in the first aspect, the second aspect, the third aspect, and the *Lactobacillus helveticus* HH-LPH17 is all 1.0 × 10⁻⁶. 9 -1.0×10 12 CFU / g.

[0019] Furthermore, the dosage form of the preparation includes injectable preparations, oral preparations, spray preparations, or rinse preparations.

[0020] Compared with the prior art, the above-mentioned solution provided in the embodiments of this application has at least the following beneficial effects:

[0021] This application provides, for the first time, a bacterial strain composition for alleviating nephritis. This bacterial strain composition can effectively improve biochemical indicators of nephritis, enhance immunity, reduce inflammatory response, etc., and can be used to improve kidney function and alleviate nephritis, providing a new treatment method and approach for nephritis. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The flowchart for the identification of Bifidobacterium longum HH-BL18, Lactobacillus acidophilus HH-LA26, and Lactobacillus plantarum HH-LP56 strains provided in the embodiments of the present invention;

[0024] Figure 2 The image shows the electrophoretic detection results of the Bifidobacterium longum HH-BL18 strain provided in the embodiments of the present invention.

[0025] Figure 3 The image shows the electrophoretic detection results of Lactobacillus acidophilus HH-LA26 strain provided in the embodiments of the present invention;

[0026] Figure 4 The image shows the electrophoretic detection results of the Lactobacillus plantarum HH-LP56 strain provided in this embodiment of the invention. Detailed Implementation

[0027] 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.

[0028] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0029] The overall concept of the technical solution provided by the embodiments of the present invention is as follows:

[0030] In a first aspect, this application provides a strain of *Bifidobacterium longum*, named *Bifidobacterium longum* HH-BL18, which was deposited on March 24, 2022, at the China Center for Type Culture Collection (CCTCC), located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2022306.

[0031] The *Bifidobacterium longum* HH-BL18 strain provided in this application was isolated from the adult intestine. The fermentation temperature was 36–38°C, and the fermentation time was 6–36 h. The culture medium used was MRS medium or a modified medium containing MRS medium. After isolation and purification, the 16S rRNA gene was used as a marker fragment, and the 16S rRNA sequence was amplified using universal primers. Electrophoresis was then performed, and the sequence was compared with the NT database to obtain species information with similar sequences. Homology comparison was used to further determine the species information, and the strain was classified and named *Bifidobacterium longum*, with the strain number HH-BL18. Its 16S rRNA characteristic sequence is shown in SEQ ID NO. 1.

[0032] Secondly, this application provides a strain of *Lactobacillus acidophilus*, named *Lactobacillus acidophilus* HH-LA26, which was deposited on March 24, 2022, at the China Center for Type Culture Collection (CCTCC), located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2022305.

[0033] The *Lactobacillus acidophilus* HH-LA26 strain provided in this application was isolated from the adult intestine. The fermentation temperature was 36–38°C, and the fermentation time was 6–36 h. The culture medium used was MRS medium or a modified medium containing MRS medium. After isolation and purification, the 16S rRNA gene was used as a marker fragment, and the 16S rRNA sequence was amplified using universal primers. Electrophoresis was then performed, and the sequence was compared with the NT database to obtain species information with similar sequences. Homology comparison was used to further determine the species information, and the strain was classified and named *Lactobacillus acidophilus*, with strain number HH-LA26. Its 16S rRNA characteristic sequence is shown in SEQ ID NO.2.

[0034] Thirdly, this application provides a strain of *Lactobacillus plantarum*, named *Lactobacillus plantarum* HH-LP56, which was deposited on June 26, 2019, at the China Center for Type Culture Collection (CCTCC), located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 2019497.

[0035] The *Lactobacillus plantarum* HH-LP56 strain provided in this application was isolated from traditional Sichuan fermented pickled vegetables. The fermentation temperature was 36–38°C, and the fermentation time was 6–36 h. The culture medium used was MRS medium or a modified medium containing MRS medium. After isolation and purification, the 16S rRNA gene was used as a marker fragment, and the 16S rRNA sequence was amplified using universal primers. Electrophoresis was then performed, and the sequence was compared with the NT database to obtain species information with similar sequences. Homology comparison was used to assist in determining the species information, and the strain was classified and named *Lactobacillus plantarum*, with the strain number HH-LP56. Its 16S rRNA characteristic sequence is shown in SEQ ID NO.3.

[0036] Fourthly, embodiments of this application provide a bacterial strain composition comprising the following strains:

[0037] The strains described in the first aspect, the strains described in the second aspect, the strains described in the third aspect, and Lactobacillus helveticus HH-LPH17;

[0038] The Lactobacillus helveticus HH-LPH17 is deposited at the China Center for Type Culture Collection, with accession number CCTCCNO: M2021725.

[0039] The Lactobacillus helveticus HH-LPH17 is a publicly available biological material, which has been published by the applicant in Chinese patent CN114262671A.

[0040] Fifthly, embodiments of this application provide the use of the strain composition described in the fourth aspect in the preparation of a drug for relieving nephritis.

[0041] The bacterial composition provided in this application can effectively improve biochemical indicators of nephritis, enhance immunity, reduce inflammatory response, and can also be used to improve kidney function and alleviate nephritis, providing new treatment methods and ideas for nephritis.

[0042] Sixthly, embodiments of this application provide a formulation for relieving nephritis, the formulation comprising:

[0043] (a) the bacterial composition described in the fourth aspect or the bacterial powder of the bacterial composition described in the fourth aspect; and

[0044] (b) Pharmaceutical excipients.

[0045] This application provides a preparation for relieving nephritis. The preparation is based on the bacterial composition or bacterial powder of the bacterial composition described in the fourth aspect above. Since the preparation adopts some or all of the technical solutions of the bacterial composition described in the fourth aspect above, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be repeated here.

[0046] The bacterial composition described in the fourth aspect, or the bacterial powder of the bacterial composition described in the fourth aspect, serves as the active ingredient (also known as the therapeutic ingredient) of the formulation. The amount added can be selected according to the technical content disclosed in the prior art and actual needs, reaching a "therapeutic effective amount." A "therapeutic effective amount" refers to an amount of reagent sufficient to provide the desired biological outcome. This outcome can be a reduction and / or alleviation of signs, symptoms, or causes of disease, or any other desired change in a biological system. For example, a "therapeutic effective amount" can specifically refer to the amount of bacterial composition containing the formulation as the active ingredient of the present invention that clinically significantly reduces nephritis. In any case, an appropriate "effective" amount can be determined by a person skilled in the art using conventional experiments.

[0047] In some specific embodiments, all the content related to the preparation of bacterial powder in Chinese Patent CN111500483A is incorporated into this application. The preparation of the bacterial powder can be carried out with reference to the content disclosed in the applicant's previous Chinese Patent CN111500483A, and will not be described in detail here.

[0048] Pharmaceutical excipients refer to pharmaceutically acceptable additives in pharmaceutical preparations, excluding the active pharmaceutical ingredient. Their types and dosages can be selected based on publicly available technical information and actual needs. Specifically, examples include binders, fillers, disintegrants, and lubricants in tablets; the base portion in semi-solid preparations such as ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solvents, osmotic pressure regulators, and colorants in liquid preparations.

[0049] As one embodiment of this application, the preparation comprises, by weight, 20-60 parts of the bacterial powder of the strain described in the first aspect, 20-60 parts of the bacterial powder of the strain described in the second aspect, 20-60 parts of the bacterial powder of the strain described in the third aspect, and 10-50 parts of the bacterial powder of Lactobacillus helveticus HH-LPH17.

[0050] This application has found through research that when Bifidobacterium longum HH-BL18 strain, Lactobacillus acidophilus HH-LA26 strain, and Lactobacillus plantarum HH-LP56 strain are used in combination in the above ratio, the therapeutic effect on nephritis is good.

[0051] Preferably, the preparation comprises, by weight, 35-50 parts of the bacterial powder of the strain described in the first aspect, 35-50 parts of the bacterial powder of the strain described in the second aspect, 35-50 parts of the bacterial powder of the strain described in the third aspect, and 20-30 parts of the bacterial powder of Lactobacillus helveticus HH-LPH17.

[0052] In one embodiment of this application, the viable count of the bacterial powder of the strain described in the first aspect, the strain described in the second aspect, the strain described in the third aspect, and the *Lactobacillus helveticus* HH-LPH17 is all 1.0 × 10⁻⁶. 9 -1.0×10 12 CFU / g.

[0053] In some specific embodiments, the strains described in the first aspect, the second aspect, the third aspect, and the *Lactobacillus helveticus* HH-LPH17 can all be 1.0 × 10⁻⁶. 9 CFU / g, 1.0×10 10 CFU / g, 1.0×10 11 CFU / g, 1.0×10 12 CFU / g, etc.

[0054] As one embodiment of this application, the dosage form of the preparation includes injectable preparations, oral preparations, spray preparations, or rinse preparations.

[0055] The dosage form of the formulation in this application can be selected and prepared according to the technical content disclosed in the prior art and actual needs. Injectable formulations include lyophilized powder injections, etc.; oral formulations include tablets, capsules, or granules, etc.; spray formulations include atomizing agents, sprays, suspensions, etc.; and rinsing formulations include rinsing solutions, etc.

[0056] In some specific embodiments, solid dosage forms for oral administration include tablets, pills, powders, granules, or capsules, etc., and such solid dosage forms may be incorporated with at least one excipient during preparation, such as starch, calcium carbonate, sucrose, lactose, or gelatin. Furthermore, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used, but are not limited thereto.

[0057] In some specific embodiments, the liquid dosage form for oral administration corresponds to suspensions, oral solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used as conventional diluents, it may also include various excipients, such as wetting agents, sweeteners, flavoring agents, preservatives, etc., but is not limited thereto.

[0058] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0059] Example 1

[0060] The purpose of this embodiment is to provide the Bifidobacterium longum strain HH-BL18.

[0061] 1.1 Source of strains

[0062] It was isolated from the adult intestine, fermented at a temperature of 36–38°C for 6–36 hours, and the culture medium used was MRS medium or a modified culture medium containing MRS medium.

[0063] 1.2 Strain Identification

[0064] The bacterial strain was isolated and purified. Using the 16S rRNA gene as a marker fragment, the 16S rRNA sequence was amplified with universal primers, followed by electrophoresis detection and comparison with the NT database to obtain species information with similar sequences. Homology alignment was used to further determine the species information. The strain was classified and named *Bifidobacterium longum*, with strain number HH-BL18. Its 16S rRNA characteristic sequence is shown in SEQ ID NO.1. The overall identification process is as follows: Figure 1 As shown.

[0065] (1) Sequencing primers and PCR procedures

[0066] Primer sequences:

[0067] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; denoted as SEQ ID NO.4.

[0068] 1492R: 5'-GGTTACCTTGTTACGACTT-3'; denoted as SEQ ID NO.5.

[0069] The PCR reaction system is shown in Table 1.

[0070] Table 1

[0071]

[0072] The PCR reaction conditions are shown in Table 2.

[0073] Table 2

[0074]

[0075] (2) The characteristic 16S rRNA sequence of Bifidobacterium longum HH-BL18 is shown in SEQ ID NO.1:

[0076]

[0077] After amplification using universal 16S rRNA primers, electrophoresis was performed for detection. The electrophoresis conditions were 1% agarose gel, 120V for 30 min. The electrophoresis results are shown below. Figure 2 As shown, the DL2000 band distribution is: 2000, 1000, 750, 500, 250, 100bp.

[0078] 1.3 Preservation of Strains

[0079] The strain of Bifidobacterium longum HH-BL18 was deposited at the China Center for Type Culture Collection (CCTCC) on March 24, 2022, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. The accession number is CCTCC NO:M 2022306, and the classification name is Bifidobacterium longum.

[0080] Example 2

[0081] The purpose of this embodiment is to provide the Lactobacillus acidophilus HH-LA26 strain.

[0082] 2.1 Source of strains

[0083] It was isolated from the adult intestine, fermented at a temperature of 36–38°C for 6–36 hours, and the culture medium used was MRS medium or a modified culture medium containing MRS medium.

[0084] 2.2 Strain Identification

[0085] After isolating and purifying the bacterial strain, the 16S rRNA gene was used as a marker fragment, and the 16S rRNA sequence was amplified using universal primers. Electrophoresis was then performed, and the sequence was compared with the NT database to obtain species information with similar sequences. Homology alignment was used to further determine the species information, and the strain was classified and named *Lactobacillus acidophilus*, with strain number HH-LA26. Its 16S rRNA characteristic sequence is shown in SEQ ID NO.2. The overall identification process is as follows: Figure 1 As shown.

[0086] (1) Sequencing primers and PCR procedures are the same as in Example 1.

[0087] (2) The 16S rRNA characteristic sequence of Lactobacillus acidophilus strain HH-LA26 is shown in SEQ ID NO.2:

[0088]

[0089] After amplification using universal 16S rRNA primers, electrophoresis was performed for detection. The electrophoresis conditions were 1% agarose gel, 120V for 30 min. The electrophoresis results are shown below. Figure 3 As shown, the DL2000 band distribution is: 2000, 1000, 750, 500, 250, 100bp.

[0090] 2.3 Preservation of bacterial strains

[0091] Lactobacillus acidophilus strain HH-LA26 was deposited on March 24, 2022, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. The accession number is CCTCC NO:M 2022305, and the classification name is Lactobacillus acidophilus.

[0092] Example 3

[0093] The purpose of this embodiment is to provide the Lactobacillus plantarum HH-LP56 strain.

[0094] 3.1 Source of strains

[0095] It was isolated from traditional Sichuan fermented pickled vegetables. The fermentation temperature was 36-38℃ and the fermentation time was 6-36h. The culture medium used was MRS medium or a modified culture medium containing MRS medium.

[0096] 3.2 Strain Identification

[0097] After isolating and purifying the bacterial strain, the 16S rRNA gene was used as a marker fragment, and the 16S rRNA sequence was amplified using universal primers. Electrophoresis was then performed, and the sequence was compared with the NT database to obtain species information with similar sequences. Homology alignment was used to further determine the species information, and the strain was classified and named *Lactobacillus plantarum*, with strain number HH-LP56. Its 16S rRNA characteristic sequence is shown in SEQ ID NO.3. The overall identification process is as follows: Figure 1 As shown.

[0098] (1) Sequencing primers and PCR procedures are the same as in Example 1.

[0099] (2) The 16S rRNA characteristic sequence of Lactobacillus plantarum strain HH-LP56 is shown in SEQ ID NO.3:

[0100]

[0101] After amplification using universal 16S rRNA primers, electrophoresis was performed for detection. The electrophoresis conditions were 1% agarose gel, 120V for 30 min. The electrophoresis results are shown below. Figure 4 As shown, the DL2000 band distribution is: 2000, 1000, 750, 500, 250, 100bp.

[0102] 3.3 Preservation of bacterial strains

[0103] Lactobacillus plantarum HH-LP56 was deposited on June 26, 2019, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China. The accession number is CCTCCNO:M 2019497, and the classification name is Lactobacillus plantarum.

[0104] Example 4

[0105] The purpose of this embodiment is to provide the bacterial powder of the strain composition described in this invention according to the preparation process of bacterial powder in Chinese Patent CN111500483A, including the following steps:

[0106] 1) Bifidobacterium longum HH-BL18, Lactobacillus acidophilus HH-LA26, Lactobacillus plantarum HH-LP56 and Lactobacillus helveticus HH-LPH17 were inoculated into MRS liquid medium and fermented at 36-38℃ for 17.5h, 18h, 16h and 15h respectively to obtain fermentation broths of Bifidobacterium longum HH-BL18, Lactobacillus acidophilus HH-LA26, Lactobacillus plantarum HH-LP56 and Lactobacillus helveticus HH-LPH17.

[0107] 2) Centrifuge the fermentation broths of Bifidobacterium longum HH-BL18, Lactobacillus acidophilus HH-LA26, Lactobacillus plantarum HH-LP56, and Lactobacillus helveticus HH-LPH17 from step 1) above at 14,500 rpm, 14,000 rpm, 15,000 rpm, and 13,000 rpm respectively, collect the precipitates, and obtain Bifidobacterium longum HH-BL18 bacterial sludge, Lactobacillus acidophilus HH-LA26 bacterial sludge, Lactobacillus plantarum HH-LP56 bacterial sludge, and Lactobacillus helveticus HH-LPH17 bacterial sludge.

[0108] 3) Add freeze-drying protectant (based on solid content percentage: 50% skim milk powder, 25% maltodextrin, 20% lactose, 5% glycerol) to the *Bifidobacterium longum* HH-BL18 sludge from step 2), add freeze-drying protectant (based on solid content percentage: 45% skim milk powder, 30% maltodextrin, 20% lactose, 5% glycerol) to the *Lactobacillus acidophilus* HH-LA26 sludge, add freeze-drying protectant (based on solid content percentage: 45% skim milk powder, 30% maltodextrin, 20% lactose, 5% glycerol) to the *Lactobacillus plantarum* HH-LP56 sludge, add freeze-drying protectant (based on solid content percentage: 60% skim milk powder, 20% maltodextrin, 10% sucrose, 10% Tween-80) to the *Lactobacillus helveticus* HH-L... PH17 bacterial sludge was mixed with a freeze-drying protectant (based on the following percentages of solid content: 40% skim milk powder, 20% maltodextrin, 20% trehalose, 10% glucose, and 10% Tween-80), stirred at 120 rpm for 10 minutes to emulsify, and then subjected to vacuum freeze-drying for 38 hours (freeze-drying conditions: pre-freezing temperature -45℃, cold trap temperature -60℃, vacuuming started, and desorption temperature controlled at 28℃~30℃) to obtain Bifidobacterium longum HH-BL18 bacterial powder, Lactobacillus acidophilus HH-LA26 bacterial powder, Lactobacillus plantarum HH-LP56 bacterial powder, and Lactobacillus helveticus HH-LPH17 bacterial powder.

[0109] Example 5

[0110] The purpose of this embodiment is to investigate the therapeutic effects of different ratios of Bifidobacterium longum HH-BL18 powder, Lactobacillus acidophilus HH-LA26 powder, Lactobacillus plantarum HH-LP56 powder, and Lactobacillus helveticus HH-LPH17 powder on nephritis.

[0111] 1. Preparation of experimental subjects and experimental models

[0112] Experimental subjects: 160 female SD rats were randomly divided into 16 groups of 10 rats each, weighing 185-230g. Their feeding conditions were: constant temperature of 25℃, relative humidity of 70-75%, and alternating light and dark for 12 hours.

[0113] Model preparation method: An acute pyelonephritis rat model was established by direct intravesical injection of *E. coli* solution via the urethra without abdominal incision. Specific procedures: One day before modeling, the rats were weighed and their rectal temperature was measured. After 24 hours of water deprivation, the rats were anesthetized with 20% urethane. An external hard catheter was inserted 4 cm into the urethra. Clear fluid was observed draining from the outer hole of the catheter, indicating successful catheterization. Subsequently, 1 ml of *E. coli* solution (10...) was drawn using a syringe. 7 0.6 ml of CFU was transferred to an adventitious rigid catheter, and the syringe was gently pushed to inject the bacterial solution into the bladder. The rat's urethral opening was clamped with an arterial clamp. After 8 hours, the arterial clamp was released, and hematuria and a significant increase in body temperature indicated that the model was successfully established.

[0114] 2. Experimental Groups

[0115] Blank control group: Healthy female SD rats, without drug administration;

[0116] Model control group: Female SD rats used in the experimental model, without drug administration;

[0117] Negative control group: Female SD rats in the experimental model were administered a lyophilized protectant without active ingredients by gavage;

[0118] Experimental groups: Examples 1-8, Comparative examples 1-5; the specific drug components of Examples 1-8 and Comparative examples 1-4 are shown in Table 3, by weight (in g); Comparative example 5 is levofloxacin hydrochloride.

[0119] Table 3

[0120]

[0121]

[0122] Note: The Bifidobacterium longum HH-BL18 powder, Lactobacillus acidophilus HH-LA26 powder, Lactobacillus plantarum HH-LP56 powder, and Lactobacillus helveticus HH-LPH17 powder in Table 3 were all prepared according to the method described in Example 4.

[0123] 3. Experimental Methods

[0124] Drug administration began on the first day after model establishment, with rats being administered the drug by gavage daily (twice, once at 10:00 AM and once at 10:00 PM). The blank control group, negative control group, and experimental group were administered the corresponding drug at 30 mg / kg via gavage for 15 consecutive days. Rectal temperature was measured and recorded daily, and body weight was measured every two days. On day 12 after drug administration, 24-hour urine was collected from the rats, centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected and stored at -80°C. On day 14 after drug administration, the rats were fasted for 24 hours, and their body temperature and body weight were recorded. On day 15, the rats were sacrificed, and their kidneys were harvested. One kidney was longitudinally cut; half was used for renal tissue bacterial culture, and the other half was fixed with 10% formaldehyde. Blood was collected from the abdominal aorta, centrifuged at 3000 rpm for 15 minutes, and serum was prepared.

[0125] Indicator determination: kidney tissue culture; determination of creatinine concentration (urine creatinine concentration was determined by taking urine stored on day 12 and using the sarcosine oxidase method, purchasing a commercially available test kit and determining the concentration according to the kit instructions); determination of serum IL-2 (IL-2 content was determined by taking rat serum and using the ELISA method, purchasing a commercially available test kit and determining the concentration according to the kit instructions).

[0126] 4. Test Results

[0127] 4.1 Infected rat kidney tissue was collected and the experimental procedure was carried out in a sterile operating table. Tissue from the same location was cut and a 10% tissue bacterial solution was prepared. After homogenization of the kidney, 0.1 ml of the solution was spread on a solid culture medium and incubated in a constant temperature incubator at 37℃. After 24 h, single colony counts were performed, and the results are shown in Table 4.

[0128] Table 4

[0129]

[0130] Note: The test results in Table 4 are the average values ​​of each group of test results.

[0131] Table 4 shows that after 15 days of continuous administration to rats, Examples 1-8 all exhibited significant antibacterial effects on kidney tissue. Compared to levofloxacin (Comparative Example 5), the antibacterial effects of Examples 1-4 on kidney tissue were slightly less pronounced. The antibacterial effects of Examples 5 and 7 on kidney tissue were similar to those of levofloxacin. The antibacterial effect of Example 6 on kidney tissue was superior to that of levofloxacin. Example 8 showed the best antibacterial effect on kidney tissue and was significantly superior to that of levofloxacin.

[0132] 4.2 The results of the urine creatinine concentration measurement are shown in Table 5. Compared with the blank control group, the urine creatinine concentration in the model control group and the negative control group was significantly increased. Compared with the model control group, the urine creatinine concentration in Example groups 1-8 was significantly decreased, with Example group 8 showing the best effect.

[0133] Table 5

[0134]

[0135]

[0136] Note: The test results in Table 5 are the average values ​​of each group of test results.

[0137] 4.3 The results of serum IL-2 measurement are shown in Table 6. Compared with the blank control group, the serum IL-2 concentration in the model control group and the negative control group was significantly increased. Compared with the model control group, the serum IL-2 concentration in Examples 1-8 was significantly decreased, with Example 8 showing the best effect.

[0138] Table 6

[0139]

[0140]

[0141] Note: The test results in Table 6 are the average values ​​of each group of test results.

[0142] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A bacterial strain composition, characterized in that, The bacterial composition Including the following strains: A strain of Bifidobacterium longum, named Bifidobacterium longum HH_BL18, was deposited on March 24, 2022, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with accession number CCTCC NO: M 2022306. A strain of Lactobacillus acidophilus, named Lactobacillus acidophilus HH_LA26, was deposited on March 24, 2022, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with accession number CCTCC NO: M2022305. A strain of Lactobacillus plantarum, named Lactobacillus plantarumHH_LP56, was deposited on June 26, 2019, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO:M 2019497. And Lactobacillus helveticus HH_LPH17, wherein Lactobacillus helveticus HH_LPH17 is deposited at the China Center for Type Culture Collection, accession number CCTCCNO: M2021725.

2. The use of the bacterial composition of claim 1 in the preparation of a medicament for relieving acute pyelonephritis.

3. A preparation for relieving acute pyelonephritis, characterized in that, The formulation includes: (a) the bacterial composition of claim 1 or the bacterial powder of the bacterial composition of claim 1; and (b) Pharmaceutical excipients.

4. The formulation according to claim 3, characterized in that, The preparation comprises, by weight, 20-60 parts of Bifidobacterium longum bacterial powder, 20-60 parts of Lactobacillus acidophilus bacterial powder, 20-60 parts of Lactobacillus plantarum bacterial powder, and 10-50 parts of Lactobacillus helveticus HH_LPH17 bacterial powder.

5. The formulation according to claim 3, characterized in that, The preparation comprises, by weight, 35-50 parts of Bifidobacterium longum bacterial powder, 35-50 parts of Lactobacillus acidophilus bacterial powder, 35-50 parts of Lactobacillus plantarum bacterial powder, and 20-30 parts of Lactobacillus helveticus HH_LPH17 bacterial powder.

6. The formulation according to claim 4 or 5, characterized in that, The viable counts of *Bifidobacterium longum* strain, *Lactobacillus acidophilus* strain, *Lactobacillus plantarum* strain, and *Lactobacillus helveticus* HH_LPH17 bacterial powder were all 1.0 x 10⁻⁶. 9 -1.0 x10 12 CFU / g.

7. The formulation according to claim 3, characterized in that, The dosage forms of the preparations include injectable preparations, oral preparations, spray preparations, or rinse preparations.

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