Sphingomonas sp. DS-28 and its probiotics in the treatment of endometritis

Abstract

This invention provides the application of a uterine-derived Sphingomonas DS-28 strain and its metabolites in the treatment of endometritis, relating to the field of microbial technology. The Sphingomonas DS-28 strain provided by this invention possesses uterine colonization ability, good niche adaptability, and significant efficacy in treating Escherichia coli-induced endometritis. Animal experiments show that the preparation of this strain can effectively alleviate uterine redness and swelling, reduce pathological damage, and significantly reduce [the condition / progression]. TNF-alpha , IL-6 , IL-8 It inhibits gene expression and suppresses the NF-κB signaling pathway, exhibiting superior anti-inflammatory and tissue repair effects compared to other Sphingomonas strains C3-25.1, C3-41.1, and C3-42.1. Furthermore, the metabiotic preparation of this strain can also alleviate Escherichia coli-induced endometritis in mice, improve uterine redness and swelling, and reduce pathological damage.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Sphingosine monocytogenes DS-28 isolated from the uterus ( Sphingomonas insulae The application of strain DS-28 and its derived post-genes in the treatment of endometritis. Background Technology

[0002] Endometritis is a common inflammatory disease of the female reproductive system, closely associated with recurrent miscarriage, infertility, and adverse pregnancy outcomes. In severe cases, it can lead to maternal sepsis and bacteremia. Currently, antibiotics remain the primary means of preventing and treating postpartum infections, but their application has significant limitations: First, the use of antibiotics among pregnant and postpartum women is highly controversial, with vastly different rates of antibiotic use for postpartum infections across different countries and regions (17.3%-92.8%), primarily due to potential risks to infant development. Second, antibiotics indiscriminately disrupt the host microbiota, exacerbating bacterial resistance.

[0003] Probiotics, as a novel antibiotic alternative based on microecological regulation, have shown great potential in disease prevention and treatment. They can not only regulate the microecological balance of specific niches through intermicrobial interactions, but also synthesize beneficial metabolites through their unique genes, becoming a biosynthetic pathway for various traditional Chinese medicine monomers. It is noteworthy that bacterial metabolic profiles are niche-specific; the colonization ability and functional expression of the same strain vary significantly in different microenvironments. Therefore, developing probiotics with highly efficient colonization and regulatory functions targeting specific host niches is crucial for maintaining host health.

[0004] In existing technologies, probiotics, as a novel alternative to antibiotics, are receiving increasing attention for their role in disease regulation. However, current probiotic research suffers from significant technical limitations: most studies focus on common ecological niches such as the gut, while resources for probiotics developed specifically for the unique microenvironment of the uterus are extremely scarce. Due to the unique structure and load of the uterine flora, most existing probiotic strains or preparations lack the ability to adapt specifically to uterine tissue, making it difficult to establish a stable flora at the lesion site and exert a sustained regulatory effect. This lack of niche adaptability limits the effectiveness of traditional probiotics in the prevention and treatment of endometritis.

[0005] Most other known sphingosine monocytogenes strains are isolated from environments such as soil and water, and their morphology and properties vary. Some may cause disease. While some strains have been reported to have in vitro anti-inflammatory effects, they are not derived from the uterus and lack the ability to colonize uterine tissue and the necessary niche adaptation. They cannot stably colonize the endometrial microenvironment and exert a sustained effect. When used for intrauterine administration, they are easily cleared by the body, making it difficult to achieve an effective concentration at the lesion site. Furthermore, due to the incompatibility of the strain's ecological niche, they may cause local microecological dysbiosis and stimulate non-specific inflammatory responses in the uterine mucosa, posing risks to safety and efficacy.

[0006] Although some sphingosine monocytogenes have shown certain anti-inflammatory activities in vitro or in other inflammatory models, their targets and mechanisms of action are significantly different from the pathophysiological process of endometritis, and they cannot specifically improve endometrial damage, inhibit local inflammatory pathways in the uterus, or promote tissue repair.

[0007] Therefore, developing specialized probiotics that can specifically adapt to the uterine microenvironment and regulate local immune function is of great strategic significance in breaking through the current treatment bottleneck of endometritis. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a strain of Sphingosine monocytogenes DS-28 isolated from the uterus of healthy mice, which has the function of regulating the body's immune system. Sphingomonas insulae (strain DS-28), and provides information on the metagener it produces and its use in the preparation of drugs for treating infectious endometritis.

[0009] This invention systematically evaluates the therapeutic effects of Sphingomonas DS-28 and its produced metabiotics on Escherichia coli-induced endometritis by detecting uterine morphology, the degree of pathological damage to uterine tissue, the expression level of key inflammatory factor genes, and the detection of inflammatory pathway proteins through animal experiments.

[0010] The Sphingomonas DS-28 provided by this invention is classified and named as follows: Sphingomonas insulae The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35151, on July 9, 2025. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0011] On the other hand, the present invention also provides three other sphingomonas strains, also isolated from the uterus of healthy mice, including Sphingomonas C3-25.1, Sphingomonas C3-41.1, and Sphingomonas C3-42.1. Their preservation information is as follows: Sphingosomalidone C3-25.1 (Sphingomonas kyungheensis It was deposited at the China General Microbiological Culture Collection Center on December 15, 2025, and classified and named as follows: Sphingomonas kyungheensis The accession number is CGMCC NO.37081. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0012] Sphingosomalidone C3-41.1 ( Sphingomonas hankookensis It was deposited at the China General Microbiological Culture Collection Center on December 15, 2025, and classified and named as follows: Sphingomonas hankookensis The accession number is CGMCC NO.37082. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0013] Sphingosomalidone C3-42.1 ( Sphingomonas yunnanensis It was deposited at the China General Microbiological Culture Collection Center on December 15, 2025, and classified and named as follows: Sphingomonas yunnanensis The accession number is CGMCC NO.37083. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0014] On the other hand, the present invention provides a metabiotic preparation obtained by fermentation of any of the aforementioned sphingomonas bacteria. It can also be obtained by co-fermentation of one or more of the aforementioned sphingomonas bacteria.

[0015] Preferably, the postbiotic preparation is prepared by culturing the bacteria and then filtering to remove the bacteria, thereby obtaining a cell-free filtrate containing bacterial metabolites.

[0016] On the other hand, the present invention provides a method for preparing a metabiotic preparation, which includes culturing bacterial cells in the logarithmic growth phase of Sphingomonas, removing the bacterial cells by filtration through a sterile filter membrane, and obtaining a cell-free filtrate containing bacterial metabolites, which is the metabiotic preparation.

[0017] Preferably, the bacterial culture includes a bacterial activation step, through which the bacterial strain reaches the logarithmic growth phase and maintains high activity of the bacterial strain.

[0018] On the other hand, the present invention provides a product prepared from one or more of the aforementioned Sphingomonas bacteria, or prepared from the fermentation product of one or more of the aforementioned Sphingomonas bacteria, wherein the product has the effect of inhibiting or treating endometritis. Preferably, the product is a tablet, powder, capsule, or liquid reagent.

[0019] On the other hand, the present invention provides a microbial preparation, characterized in that it is prepared from one or more of the aforementioned Sphingomonas bacteria, or from the fermentation product of one or more of the aforementioned Sphingomonas bacteria. Preferably, the fermentation product can be a metabiotic preparation; preferably, the metabiotic preparation is a cell-free filtrate containing bacterial metabolites obtained by filtration to remove bacterial cells after bacterial culture.

[0020] Preferably, the viable bacterial concentration of each dose of the above-mentioned strains, inoculants, ferments, compositions, products, microbial preparations, and biological agents is 1×10⁻⁶. 3 ~1×10 5 Between CFU; preferably, the viable bacteria concentration is 1×10⁻⁶. 3 ~1×10 4 Between CFUs, the volume of each dose is 10-200ul / dose, preferably 20-180ul, 20-150ul, 20-100ul, or 30-80ul per dose.

[0021] No less than 1×10 in microbial preparations 3 CFU / dose, not exceeding 1×10 4 CFU / dose, preferably 20-100 μL, preferably 60 μL. The dosage per dose refers to administration to the mouse uterus; for different animal species, the recommended concentration of the microbial preparation is 1 × 10⁻⁶. 4 CFU / dose, with the volume of each dose adjustable according to the size of the animal's uterus. The volume is appropriately increased to enhance the contact area between the drug and the uterine cavity, while maintaining a microbial concentration of 1×10⁻⁶ per dose. 3 ~10 4 CFU is preferred.

[0022] On the other hand, the present invention provides the use of the above-mentioned Sphingomonas, products or microbial preparations in the preparation of drugs for treating endometritis.

[0023] The endometritis was caused by Escherichia coli.

[0024] The drug has the following effects: alleviating E. coli-induced endometrial inflammation in mice, significantly improving uterine redness and swelling, reducing uterine histopathological damage, and / or significantly downregulating uterine endometrial function. TNF-α , IL-1β and IL-8 The level of inflammatory factors.

[0025] The above applications include any of the following effects: alleviating E. coli-induced endometrial inflammation in mice, significantly improving uterine redness and swelling, reducing uterine histopathological damage, and / or significantly downregulating uterine endometrial function. TNF-α , IL-1β and IL-8The level of inflammatory factors.

[0026] The present invention provides a method for culturing the above-mentioned Sphingomonas C3-25.1, Sphingomonas DS-28, Sphingomonas C3-41.1 and Sphingomonas C3-42.1, comprising: inoculating one or more of the above-mentioned strains into R2A liquid medium and culturing them at 28°C and 180 rpm for 24-48 h with shaking.

[0027] In another aspect, the present invention provides a method for preparing a Sphingomonas postbiotic preparation, comprising the following steps: (1) The frozen Sphingomonas strain was quickly thawed in a 37°C water bath and transferred to R2A liquid medium at an inoculation rate of 1-10%. The culture was then incubated at 28°C and 180 rpm for 48-96 h to obtain the seed culture. (2) Take out the seed liquid obtained in (1) and transfer it to fresh R2A liquid culture medium at an inoculation rate of 1%. Culture it at 28℃ and 180rpm for 48-96h to obtain a yellow and turbid bacterial liquid.

[0028] (3) Centrifuge the bacterial solution obtained in (2) at 4℃ and 1000×g for 30 min, collect the supernatant, and filter it with a 0.22μm sterile filter membrane to obtain the metabiotic preparation derived from Sphingomonas.

[0029] The advantages of this invention compared to the prior art are as follows: The beneficial effects of this invention are as follows: 1. This invention provides and verifies Sphingomonas DS-28 (accession number CGMCC No. 35151). Sphingomonas insulae The significant efficacy of strain DS-28 in treating Escherichia coli-induced endometritis. Animal experiments showed that administration of the Sphingomonas DS-28 biological agent described in this application effectively alleviated uterine redness and swelling caused by E. coli infection, significantly reduced pathological damage caused by infection, and significantly decreased the levels of key inflammatory genes in uterine tissue. TNF-α , IL-6 and IL-8 It effectively reduced the expression level of NF-κB and decreased the NF-κB signaling pathway. Compared with the aforementioned uterine symbiotic Sphingomonas C3-25.1, Sphingomonas C3-41.1, and Sphingomonas C3-42.1, it exhibited more significant anti-inflammatory and tissue repair capabilities.

[0030] 2. This invention also provides the preparation of a post-biotic formulation obtained by culturing *Sphingomonas DS-28* in vitro in R2A medium, the main components of which are various bioactive terpenoid compounds. The preparation process of this post-biotic formulation is simple, enabling large-scale production, and it exhibits good stability and safety, facilitating long-term storage and subsequent dosage form development. Experiments have confirmed that this post-biotic formulation can also effectively alleviate *Escherichia coli*-induced endometrial inflammation in mice, significantly improve uterine redness and swelling, reduce histopathological damage, and significantly downregulate multiple inflammatory genes in the uterus (…). TNF-α , IL-1β and IL-8 The level of [unclear] provides a new option of non-live bacterial preparations for the prevention and treatment of endometritis. Attached Figure Description

[0031] Figure 1 Colony morphology of Sphingomonas C3-25.1, Sphingomonas DS-28, Sphingomonas C3-41.1 and Sphingomonas C3-42.1.

[0032] Figure 2 Phylogenetic analysis of Sphingomonas C3-25.1, Sphingomonas DS-28, Sphingomonas C3-41.1 and Sphingomonas C3-42.1.

[0033] Figure 3 Morphology of Sphingosine Monoclonalella DS-28.

[0034] Figure 4 Effects of treatment with Sphingomonas C3-25.1, Sphingomonas DS-28, Sphingomonas C3-41.1 and Sphingomonas C3-42.1 on uterine morphology.

[0035] Figure 5 The effects of treatment with Sphingomonas C3-25.1, Sphingomonas DS-28, Sphingomonas C3-41.1 and Sphingomonas C3-42.1 on uterine histopathology.

[0036] Figure 6 Effects of treatment with Sphingomonas C3-25.1, Sphingomonas DS-28, Sphingomonas C3-41.1 and Sphingomonas C3-42.1 on the expression of inflammatory factor genes in the uterus. Figure 6 A represents the effect of bacterial strain on the uterus. TNF-α Effects on gene expression Figure 6 B represents the bacterial strain affecting the uterus. IL-6 Effects on gene expression and Figure 6 C represents the effect of bacterial strain on the uterus. IL-8 The impact on gene expression.

[0037] Figure 7 Effects of treatment with *Sphingomonas* C3-25.1, *Sphingomonas* DS-28, *Sphingomonas* C3-41.1, and *Sphingomonas* C3-42.1 on the NF-κB signaling pathway. (Figure) express p 0.05, express p 0.01, express p 0.001. Figure 7 A shows a Western blot image of p-P65 and p-IκB protein imprints. Figure 7 B represents the difference in p-P65 expression levels; Figure 7 C represents the difference in p-IκB expression levels.

[0038] Figure 8 The effect of treatment with a post-biotic preparation derived from Sphingomonas DS-28 on uterine morphology.

[0039] Figure 9 The effects of treatment with a post-biotic preparation derived from Sphingomonas DS-28 on uterine histopathology. express p 0.05, express p 0.01, express p 0.001.

[0040] Figure 10 The effect of treatment with a postbiotic preparation derived from Sphingomonas DS-28 on the expression of uterine inflammatory factor genes. Figure 10 A is an afterbiotic preparation. TNF-α Effects on gene expression Figure 10 B is an epigenetic preparation. IL-6 Effects on gene expression and Figure 10 C represents an afterbiotic preparation. IL-8 The impact on gene expression. Detailed Implementation

[0041] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0042] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0043] The pregnant mare serum gonadotropin (PMSG) used in this embodiment was purchased from Ningbo Sansheng Biotechnology Co., Ltd. (veterinary drug registration number 110914564).

[0044] Human chorionic gonadotropin (HCG) was purchased from Ningbo 3SBio Co., Ltd. (veterinary drug registration number 110911282).

[0045] Example 1: Isolation and Identification of Microbial Strains

[0046] 1. Strains were isolated from uterine lavage fluid of healthy female KM mice.

[0047] 2. Isolation and purification of strains: (1) Sampling: The uterine horn flushing fluid of mice was collected under sterile conditions and used as a separation sample.

[0048] (2) Initial culture: The uterine flushing solution was evenly spread on the surface of R2A agar medium and placed in a constant temperature incubator at 28℃ for 3 days.

[0049] (3) Isolation and screening: Single colonies growing on R2A agar medium were picked and examined under a microscope after Gram staining. Colonies that were initially screened as Gram-negative bacilli were preliminarily screened. Specific primers were further designed (Forward primer: 5'-CAACCCTCGCCTTTAGTTACC-3', SEQ ID NO:1; Reverse primer: 5'-TTGCAGAGAACAATCCGAACT-3', SEQ ID NO:2). Single colonies that were Gram-negative bacilli were picked and verified by PCR. Strains with positive amplification results were selected.

[0050] (4) Molecular identification: 16S rRNA sequencing was performed on the above PCR positive strains, and the obtained sequences were compared and analyzed by phylogenetic tree analysis.

[0051] The four strains were ultimately identified as Sphingomonas C3-25.1 (…Sphingomonas kyungheensis ), Sphingosomalmonella DS-28 ( Sphingomonas insulae ), Sphingosomalidone C3-41.1 ( Sphingomonas hankookensis ) and Sphingomonas C3-42.1 ( Sphingomonas yunnanensis ),like Figure 1 , 2 As shown in Figure 3.

[0052] Example 2: Preparation of microbial preparations

[0053] Preserved *Sphingomonas* strains C3-25.1, DS-28, C3-41.1, and C3-42.1 were inoculated into R2A liquid medium at 1-10% inoculum and subjected to activation subculturing for two generations. Subsequently, the second-generation activated bacterial culture was transferred to fresh R2A liquid medium at 1% inoculum and cultured at 28℃ and 180 rpm for 4.1 h, 7 h, 6.3 h, and 7.7 h, respectively, with shaking. After incubation, the bacterial culture was serially diluted (10000-fold) to adjust the final concentration to 1×10⁻⁶. 4 CFU / 60ul. Subsequently, the diluted bacterial suspension was centrifuged at 4°C and 1000×g for 30 min, the supernatant was discarded, and the resulting bacterial pellet was resuspended in sterile PBS to finally prepare a solution containing 1×10⁶ viable cells. 4 A biological agent containing CFU / 60ul.

[0054] Example 3: Preparation of post-biotic formulations

[0055] (1) Take out the frozen sphingomonas DS-28 strain, thaw it quickly in a 37℃ water bath, transfer it to R2A liquid medium at an inoculation rate of 1-10%, and culture it at 28℃ and 180 rpm for 48-96 h with shaking until the medium shows a uniform yellow turbidity to obtain the seed liquid. (2) Take out the seed liquid obtained in (1) and transfer it to fresh R2A liquid culture medium with an inoculation amount of 1%. Continue to culture at 28℃ and 180rpm for 48-96h with shaking to obtain yellow turbid bacterial liquid.

[0056] (3) Centrifuge the bacterial solution obtained in (2) at 4℃ and 1000×g for 30 min, collect the supernatant, filter it through a 0.22μm sterile filter membrane to completely remove the bacterial cells, and obtain a cell-free filtrate containing bacterial metabolites, which is the sphingosine monocytogenes DS-28 postbiotic preparation of the present invention.

[0057] Example 4: Effects of Sphingomonas DS-28 biological agent on endometritis

[0058] To investigate the effects of the four uterine-derived Sphingosine monocytogenes biological agents prepared in Example 2 on Escherichia coli-induced endometritis, the following animal experiments were conducted in this example: The experimental animals were SPF-grade 7-8 week old female KM mice, purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0059] 1. Experimental Grouping

[0060] After a week of acclimatization feeding, the experimental animals were randomly divided into 10 groups: Healthy control group (CTRL); Biological agents group: Sphingomonas C3-25.1 single treatment group (C3-25.1), Sphingomonas DS-28 single treatment group (Sp. DS-28), Sphingomonas C3-41.1 single treatment group (C3-41.1), Sphingomonas C3-42.1 single treatment group (C3-42.1); Disease model group ( E. coli ); Biological agent treatment groups: Sphingomonas C3-25.1 treatment group (E_C3-25.1), Sphingomonas DS-28 treatment group (E_Sp.DS-28), Sphingomonas C3-41.1 treatment group (E_C3-41.1), and Sphingomonas C3-42.1 treatment group (E_C3-42.1).

[0061] 2. Handling of laboratory animals

[0062] All mice were given intraperitoneal injections of PMSG (5 IU) on day 1 and HCG (5 IU) on day 3 to synchronize estrus.

[0063] An endometritis model was established 24 hours later. All mice were first anesthetized. For the CTRL group and the biological agent group (C3-25.1, Sp. DS-28, C3-41.1, and C3-42.1 groups): 60 μL of sterile PBS was perfused into the uterus. For the disease model group (… E. coli The groups receiving biological agents (E_C3-25.1, E_Sp. DS-28, E_C3-41.1, and E_C3-42.1) were infused with 60 μL of [a specific drug / method] into the uterus. E. coli (1×10) 12 CFU).

[0064] The model was successfully established. Pathological sections revealed that, compared to the CTRL group, neutrophils infiltrated the uterine cavity and endometrium, endometrial epithelial cells were destroyed, and some uterine glandular structures were damaged and edematous. (See [reference needed]). Figure 5 CTRL group andE.coli Comparison of pathological sections from different groups.

[0065] Treatment began 24 hours later and continued for 5 days. Mice were first anesthetized preoperatively. The CTRL group and the disease model group (…) E. coli Mice: 60 μL of sterile PBS was perfused into the uterus. For mice in the biologics group (C3-25.1, Sp. DS-28, C3-41.1, and C3-42.1 groups) and mice in the biologics treatment group (E_C3-25.1, E_Sp. DS-28, E_C3-41.1, and E_C3-42.1 groups): 60 μL of the biologics from Example 2 was perfused into the uterus.

[0066] Five days after treatment with biological agents, the mice were euthanized the following day, and uterine samples were collected and photographed (e.g., Figure 4 The tissue samples were fixed in 4% paraformaldehyde, while the remaining tissues were placed in liquid nitrogen for 30 minutes. Finally, the samples were stored at -80°C for subsequent analysis. Animal experiments have been approved by the Ethics Committee of Jilin Agricultural University, China (Approval No.: 20230925001).

[0067] 3. Efficacy and verification: Comparative observation of uterine morphological changes, such as Figure 4 Compared to the CTRL group, E. coli The uterus of mice in the group was significantly swollen and red, but after treatment with Sphingomonas DS-28, the uterine swelling and redness were significantly reduced.

[0068] No significant differences in uterine morphology were observed between treatment with Sphingomonas DS-28 and treatment with the other three Sphingomonas strains (E_C3-25.1, E_C3-41.1, and E_C3-42.1). Figure 4 ).

[0069] Hematoxylin-eosin stained pathological tissue sections showed that, compared with the CTRL group, E. coli In the group of mice, the uterine cavity and endometrium showed extensive neutrophil infiltration, endometrial epithelial structure destruction, reduced number of uterine glands, edema, and structural damage. Treatment with Sphingomonas DS-28 significantly improved these pathological findings. Compared to Sphingomonas DS-28 treatment, neutrophil infiltration remained even after treatment with the other three Sphingomonas strains (E_C3-25.1, E_C3-41.1, and E_C3-42.1). Figure 5 ).

[0070] Real-time quantitative PCR detection of inflammatory genes related to mouse uterine tissue TNF-α ( Figure 6 A) IL-6 (Figure 6 B) and IL-8 ( Figure 6 C) Expression level. Results showed that, compared to the CTRL group, E. coli group of mice TNF-α ( p 0.0001) IL-6 ( p = 0.0007) and IL-8 ( p The expression levels of the 0.0001 gene increased by 4.73, 2.61, and 7.2 times (372.6%, 160.9%, and 620.3%), respectively, and were significantly reduced after treatment with Sphingomonas DS-28. TNF-α ( p = 0.9943), IL-6 ( p = 0.6801) and IL-8 Gene expression levels (>0.9999) and... E. coli Compared with the previous group, the rates decreased by 58.9%, 84.6%, and 92.2%, respectively. (Compared with E_C3-25.1) TNF-α , p = 0.3187, a relative decrease of 25.4%; IL-6 , p = 0.9389, a relative decrease of 54.5%; IL-8 , p = 0.9600, a relative decrease of 71.1%), E_C3-41.1 ( TNF-α , p = 0.0935, a relative decrease of 11.4%; IL-6 , p = 0.8816, a relative decrease of 43.4%; IL-8 , p > 0.9999, a relative decrease of 89.8%) and E_C3-42.1 ( TNF-α , p = 0.4756, a relative decrease of 31.3%; IL-6 , p = 0.9214, a relative decrease of 44.8%; IL-8 , p =0.0003, a relative decrease of 20.5% (compared to Sphingomonas DS-28). TNF-α, IL-6 and IL-8 It exhibited a more stable regulatory effect.

[0071] like Figure 7As shown in Figure A, Western blotting was used to detect proteins related to the NF-κB pathway. The results showed that, compared with the CTRL group, E. coli Group p-P65 ( p = 0.0054, a relative increase of 53.9% ( Figure 7 B) and p-IκB ( p = 0.0008, a relative increase of 52.0% ( Figure 7 C) expression was significantly increased after treatment with Sphingomonas DS-28, p-P65 ( p >0.9999, a relative decrease of 38.2%) and p-IκB ( p = 0.2976, a relative decrease of 50.2%) significantly reduced, indicating inhibition of the NF-κB signaling pathway. Compared with sphingomyelin-monas DS-28 treatment, E_C3-25.1 significantly reduced p-P65 ( p = 0.9999, a relative decrease of 30.7% and p-IκB ( p No significant difference was observed in the regulation of >0.9999 (relative decrease of 36.9%), and it could also inhibit the NF-κB signaling pathway; E_C3-41.1 and E_C3-42.1 significantly reduced p-IκB ( p > 0.9999, a relative decrease of 34.1% and p =0.9238, a relative decrease of 42.8%) expression, but failed to reduce p-P65 ( p = 0.0152, a relative decrease of 3.65% and p =0.0109, a relative decrease of 2.47% (expression).

[0072] In conclusion, Sphingomonas DS-28 is effective in the treatment of... E. coli It showed the best performance in induced mouse endometritis, mainly due to its ability to reduce neutrophil infiltration and downregulate inflammatory genes. TNF-α , IL-6 , IL-8 Regarding the expression and inhibition of the NF-κB signaling pathway. Data are presented as mean ± standard deviation. Statistical significance was determined by one-way ANOVA and Tukey's test.

[0073] This invention discloses and verifies for the first time the sphingomonas DS-28 strain with accession number CGMCC No. 35151 ( Sphingomonas insulaeThe application demonstrates significant efficacy in treating Escherichia coli-induced endometritis. Animal experiments showed that administration of the Sphingomonas DS-28 biological agent described in this application effectively alleviated uterine redness and swelling caused by E. coli infection, significantly reduced neutrophil infiltration, and decreased damage to the structure of uterine glands and endometrial epithelial cells. While the other three Sphingomonas strains protected the structure of uterine glands and endometrial epithelial cells, they still exhibited varying degrees of neutrophil infiltration. Simultaneously, Sphingomonas DS-28 significantly reduced key inflammatory factors in uterine tissue (…). TNF-α A relative decrease of 58.9%; IL-6 A relative decrease of 84.6% and IL-8 The expression level of p-P65 decreased by 92.2% (relatively) and effectively reduced the NF-κB signaling pathway. p > 0.9999, a relative decrease of 38.2%) and p-IκB ( p = 0.2976, a relative decrease of 50.2%. However, Sphingomonas C3-42.1 failed to effectively inhibit... IL-8 ( E. coli vs E_C3-42.1, p = 0.7920, a relative decrease of 20.5%) expression level, and on TNF-α (CTRL) vs E_C3-42.1, p = 0.4759; CTRL vs E_ Sp. DS-28, p = 0.9943; E. coli vs E_C3-42.1, p =0.8990, a relative decrease of 31.3%; E. coli vs E_ Sp. DS-28, p = 0.2055, a relative decrease of 58.9%) showed lower inhibitory effects than Sphingomonas DS-28. Similarly, Sphingomonas C3-25.1 and Sphingomonas C3-41.1 showed similar inhibitory effects on... TNF- α (CTRL) vs E_C3-25.1, p = 0.3187; CTRL vs E_C3-41.1, p = 0.0935; E. coli vs E_C3-25.1, p = 0.9700, a relative decrease of 25.4%; E. colivs E_C3-41.1, p The inhibitory effects of *Sphingomonas* C3-41.1 (>0.9999, a relative decrease of 11.4%) were also lower than those of *Sphingomonas* DS-28. Furthermore, *Sphingomonas* C3-41.1 ( p = 0.0152, a relative decrease of 3.65%) and Sphingomonas C3-42.1 ( p = 0.0109, a relative decrease of 2.47%) could not effectively inhibit the protein expression of p-P65 in the NF-κB signaling pathway.

[0074] In summary, the uterine symbiotic sphingomonas DS-28 of the present invention has more significant anti-inflammatory and tissue repair capabilities compared with the control strains sphingomonas C3-25.1, sphingomonas C3-41.1 and sphingomonas C3-42.1.

[0075] Example 5: Effects of Sphingomonas DS-28 Postbiotic Preparation on Endometritis

[0076] To investigate the effect of the sphingomonas DS-28 postbiotic preparation prepared in Example 3 on Escherichia coli-induced endometritis, the following animal experiments were conducted in this example: The experimental animals were SPF-grade 7-8 week old female KM mice, purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0077] 1. Experimental Grouping

[0078] After a week of acclimatization feeding, the experimental animals were randomly divided into four groups: the negative control group (NC), the post-biotic preparation treatment group (SPS) as described in Example 3, the disease model group (…), and the… E. coli (and the post-epigenetic preparation treatment group (ESPS) in Example 3).

[0079] 2. Handling of laboratory animals

[0080] All mice were administered PMSG (5 IU) intraperitoneally on day 1 and HCG (5 IU) intraperitoneally on day 3 for estrus synchronization. An endometritis model was established 24 hours later, and all mice were first anesthetized. For mice in the NC and SPS groups, 60 μL of sterile PBS was perfused into the uterus. E. col Group i and ESPS mice: 60 μL was infused into the uterus E. coli (1×10) 12 CFU / 60μL). Treatment was initiated 24 hours later and continued for 5 days. Mice were first anesthetized preoperatively. The NC group and... E. coliMice in the first group: 80 μL of sterile PBS was perfused into the uterus. Mice in the SPS and ESPS groups: 80 μL of the post-biotic preparation from Example 3 was perfused into the uterus. After 5 days of treatment with the post-biotic preparation from Example 3, the mice were euthanized the following day, uterine samples were collected, photographed, and tissue from a fixed area was fixed in 4% paraformaldehyde. The remaining tissue was placed in liquid nitrogen for 30 min and finally stored at -80℃ for subsequent analysis.

[0081] 3. Efficacy verification: Comparative observation of uterine morphological changes, hematoxylin-eosin stained pathological tissue sections, and detection of key inflammatory genes using real-time quantitative PCR. IL-1β , TNF-α and IL-8 , Figure 9 The expression levels of [insert expression level here] were used to determine the changes in uterine inflammation in each group of mice. Data are presented as mean ± standard deviation. Statistical significance was determined by one-way ANOVA and Tukey's test.

[0082] The results showed that, compared with the NC group, E. coli The uterus of mice in the ESPS group was significantly swollen and shortened, while after treatment with sphingomonas DS-28-derived postbiotics (ESPS group), the uterine swelling was significantly reduced and the uterine length was restored. Figure 8 ).

[0083] Hematoxylin-eosin stained pathological tissue sections showed that, compared with the NC group, E. coli In the ESPS group, neutrophil infiltration was observed in the endometrium, the endometrial epithelial cell structure was disrupted, and the uterine glands were edematous and structurally damaged. However, after treatment, the ESPS group maintained a healthy endometrial structure and no neutrophil infiltration was observed. Figure 9 ).

[0084] Real-time fluorescence quantitative PCR results showed that, compared with the NC group, E. coli group of mice TNF-α ( p 0.0001) IL-1β ( p = 0.0007) and IL-8 ( p The gene expression level of 0.0001 was significantly increased in the ESPS group mice after metabiotic treatment. TNF-α Expression level significantly compared to E. coli Group low ( p = 0.0035) Figure 10 A), IL-1β ( p = 0.9900) Figure 10 B) andIL-8 ( p > 0.9999) Figure 10 C) Gene levels decreased significantly, returning to a level that was not significantly different from the NC group.

[0085] This post-biotic formulation has a simple preparation process, enabling large-scale production, and exhibits good stability and safety, facilitating long-term storage and subsequent dosage form development. Experiments have demonstrated that this post-biotic formulation can also effectively alleviate E. coli-induced endometrial inflammation in mice, significantly improve uterine redness and swelling, reduce histopathological damage, and significantly downregulate multiple inflammatory factors in the uterus. TNF-α , IL-1β and IL-8 The level of [unclear] provides a new option of non-live bacterial preparations for the prevention and treatment of endometritis.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0087] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of sphingosine monocytogenes, characterized in that, The sphingomonas strain mentioned is Sphingomonas DS-28, classified and named as follows: Sphingomonas insulae It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35151 and deposit date of July 9, 2025.

2. The product prepared from *Sphingomonas* as described in claim 1, characterized in that, The product has the effect of inhibiting or treating endometritis.

3. The product as described in claim 2, characterized in that, The product is in the form of tablets, powders, capsules, or liquid reagents.

4. A microbial preparation, characterized in that, It is prepared by fermentation of Sphingosine Monoclonal bacteria as described in claim 1.

5. The microbial preparation according to claim 4, characterized in that, The microbial preparation is prepared from the bacterial cells obtained by fermentation of Sphingosine Monoclonalella as described in claim 1 or its postbiotic preparation.

6. The microbial preparation according to claim 5, characterized in that, The postbiotic preparation is prepared by culturing bacteria and then filtering to remove the bacteria, resulting in a cell-free filtrate containing bacterial metabolites.

7. The use of Sphingomonas as described in claim 1, the product as described in claim 2 or 3, or the microbial preparation as described in any one of claims 4-6 in the preparation of a drug for treating endometritis.

8. The application as described in claim 7, characterized in that, The endometritis mentioned is caused by Escherichia coli.

9. The application as described in claim 8, characterized in that, The application includes any of the following features: a) Relieves E. coli-induced endometritis in mice; b) Significantly improves uterine redness and swelling; c) Reduce pathological damage to uterine tissues; d) Significantly downregulates uterine fluid. TNF-α , IL-1β and IL-8 The level of inflammatory factors.

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