Uterine-derived sphingomonas DS-28 and application of postbiotics thereof in treatment of endometritis

Abstract

The invention provides application of uterus-derived sphingomonas DS-28 and a metagen thereof in treatment of endometritis, and relates to the technical field of microorganisms. The sphingomonas DS-28 provided by the invention has uterus colonization ability and good ecological niche adaptability, and also has a remarkable curative effect in treatment of Escherichia coli induced endometritis. Animal experiments show that the strain preparation can effectively relieve red and swollen uterus, relieve pathological damage, remarkably reduce gene expression of TNF-alpha, IL-6 and IL-8 and inhibit an NF-kappa B signal channel, and the anti-inflammatory and tissue repair effects of the strain preparation are superior to those of other sphingomonas strains C3-25.1, C3-41.1 and C3-42.1. Meanwhile, the metastatic preparation of the strain can also relieve mouse escherichia coli endometritis, improve red and swollen uterus and relieve pathological injury.

Description

Application of a uterine-derived Sphingomonas DS-28 and its postbiotic in the treatment of endometritis Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of Sphingomonas insulaestrain DS-28 isolated from the uterus and the metabiotics produced therefrom 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 Sphingomonas insulaestrain DS-28 isolated from the uterus of healthy mice, which has the function of regulating the body's immune system, and provides the metabiotic produced by it and its application 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 strain provided by this invention, classified as Sphingomonas insulae, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35151, deposited on July 9, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0011] On the other hand, this invention also provides three other sphingomonas strains, similarly 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: Sphingomonas C3-25.1 (Sphingomonas kyungheensis) was deposited on December 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), classified as Sphingomonaskyungheensis, with accession number CGMCC NO.37081. The depositary address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

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

[0013] Sphingomonas yunnanensis C3-42.1 was deposited on December 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), classified as *Sphingomonas yunnanensis*, with accession number CGMCC NO.37083. The depositary address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 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 filtration to remove bacterial cells after bacterial culture, resulting in 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, and the bacterial strain is cultured twice to reach the logarithmic growth phase and maintain the 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, the volume of each dose can be adjusted according to the size of the uterus of different animals. The volume is appropriately increased to increase the contact area between the drug and the uterine cavity, while the microbial concentration is maintained at 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: relieving Escherichia coli-induced endometrial inflammation in mice, significantly improving uterine redness and swelling, reducing uterine histopathological damage, and / or significantly downregulating the levels of TNF-α, IL-1β, and IL-8 inflammatory factors in the uterus.

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

[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 of the present invention, the present invention provides a method for preparing a sphingomonas postbiotic preparation, comprising the following steps: (1) rapidly thawing the frozen sphingomonas strain in a 37°C water bath, transferring it to R2A liquid culture medium at an inoculation rate of 1-10%, and culturing it at 28°C and 180 rpm for 48-96 h to obtain a seed culture; (2) taking out the seed culture obtained in (1), transferring it to fresh R2A liquid culture medium at an inoculation rate of 1%, and culturing it at 28°C and 180 rpm for 48-96 h to obtain a yellow and turbid bacterial culture.

[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 beneficial effects of this invention compared to the prior art are as follows: 1. This invention provides and verifies the significant efficacy of Sphingomonas insulaestrain DS-28 (accession number CGMCC No. 35151) in treating Escherichia coli-induced endometritis. Animal experimental results show that the administration of the Sphingomonas insulaestrain DS-28 biological agent described in this application can effectively alleviate uterine redness and swelling caused by Escherichia coli infection, significantly reduce pathological damage caused by infection, significantly reduce the expression levels of key inflammatory genes (TNF-α, IL-6, and IL-8) in uterine tissue, and effectively reduce 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 has more significant anti-inflammatory and tissue repair capabilities.

[0030] 2. This invention also provides a method for preparing a post-biotic formulation obtained by culturing *Sphingomonas DS-28* in vitro on R2A medium. The main components of this formulation are various bioactive terpenoid compounds. The preparation process of this post-biotic formulation is simple, allows for large-scale production, and exhibits good stability and safety, facilitating long-term storage and subsequent dosage form development. Experiments have confirmed that this post-biotic formulation can effectively alleviate *Escherichia coli*-induced endometrial inflammation in mice, significantly improve uterine redness and swelling, reduce histopathological damage, and significantly downregulate the levels of multiple inflammatory genes (TNF-α, IL-1β, and IL-8) in the uterus, providing a new non-live bacterial formulation option 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. 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 Sphingomonas C3-25.1, Sphingomonas DS-28, Sphingomonas C3-41.1, and Sphingomonas C3-42.1 treatments on the expression of inflammatory cytokine genes in the uterus. Figure 6A shows the effect of the strains on TNF-α gene expression in the uterus; Figure 6B shows the effect of the strains on IL-6 gene expression in the uterus; and Figure 6C shows the effect of the strains on IL-8 gene expression in the uterus.

[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. p 0.05, p 0.01, p 0.001. Figure 7A shows the protein imprinting images of p-P65 and p-IκB detected by Western blotting; Figure 7B shows the difference in p-P65 expression level; Figure 7C shows the difference in p-IκB expression level.

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

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

[0040] Figure 10. Effects of post-biotic preparations derived from Sphingomonas DS-28 on the expression of uterine inflammatory factor genes. Figure 10A shows the effect of post-biotic preparations on TNF-α gene expression, Figure 10B shows the effect of post-biotic preparations on IL-6 gene expression, and Figure 10C shows the effect of post-biotic preparations on IL-8 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 as a sample for isolation.

[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 finally identified as Sphingomonas kyungheensis C3-25.1, Sphingomonas insulae DS-28, Sphingomonas shankookensis C3-41.1, and Sphingomonas yunnanensis C3-42.1, as shown in Figures 1, 2, and 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, and transfer it to R2A liquid medium with an inoculum of 1-10%. 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 culture; (2) Take out the seed culture obtained in (1), transfer it to fresh R2A liquid medium with an inoculum of 1%, and continue to culture it at 28℃ and 180 rpm for 48-96 h with shaking to obtain the yellow turbid bacterial culture.

[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 Monoclonal antibodies prepared in Example 2 on Escherichia coli-induced endometritis, the following animal experiments were conducted in this example: SPF-grade 7-8 week old female KM mice were selected as experimental animals and purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0059] 1. Experimental Grouping

[0060] After one week of acclimatization feeding, the experimental animals were randomly divided into 10 groups: a healthy control group (CTRL); biological agent groups: Sphingomonas C3-25.1 alone (C3-25.1), Sphingomonas DS-28 alone (Sp. DS-28), Sphingomonas C3-41.1 alone (C3-41.1), and Sphingomonas C3-42.1 alone (C3-42.1); a disease model group (E. coli); and 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) and the biological agent treatment group (E_C3-25.1, E_Sp. DS-28, E_C3-41.1, and E_C3-42.1 groups), 60 μL of E. coli (1×10⁻⁶) was perfused into the uterus. 12 CFU).

[0064] The model was successfully established. Pathological sections showed 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 Figure 5 for a comparison of pathological sections between the CTRL group and the E. coli group.

[0065] Treatment began 24 hours later and continued for 5 days, starting with preoperative anesthesia for the mice. For the CTRL group and the disease model group (E. coli): 60 μL of sterile PBS was perfused into the uterus. For the biologics group (C3-25.1, Sp. DS-28, C3-41.1, and C3-42.1 groups) and 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 the biological agent, the mice were euthanized the following day, and uterine samples were collected and photographed (Figure 4). Tissue from a fixed area was fixed in 4% paraformaldehyde, while the remaining tissue was placed in liquid nitrogen for 30 minutes. Finally, the samples were stored at -80°C for subsequent analysis. The animal experiments were approved by the Ethics Committee of Jilin Agricultural University, China (Approval No.: 20230925001).

[0067] 3. Efficacy and verification: Comparative observation of uterine morphological changes, as shown in Figure 4, compared with the CTRL group, the E. coli group mice had significantly red and swollen uterus, while after treatment with Sphingomonas DS-28, the redness and swelling of the uterus 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 histopathological sections revealed that, compared with the CTRL group, the E. coli group mice showed extensive neutrophil infiltration in the uterine cavity and endometrium, endometrial epithelial structure destruction, reduced number of uterine glands, edema, and structural damage. Treatment with Sphingomonas DS-28 significantly improved these corresponding pathological findings. In contrast to Sphingomonas DS-28 treatment, neutrophil infiltration persisted after treatment with the other three Sphingomonas strains (E_C3-25.1, E_C3-41.1, and E_C3-42.1) (Figure 5).

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

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

[0072] In summary, Sphingomonas DS-28 showed the best performance in treating E. coli-induced endometritis in mice, mainly due to its ability to reduce neutrophil infiltration, downregulate the expression of inflammatory genes TNF-α, IL-6, and IL-8, and inhibit 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 significant efficacy of Sphingomonas insulae DS-28 (accession number CGMCC No. 35151) in treating Escherichia coli-induced endometritis. Animal experiments showed that administration of the Sphingomonas insulae DS-28 biological agent described in this application effectively alleviated uterine redness and swelling caused by E. coli infection, significantly reduced neutrophil infiltration, and damage to the structure of uterine glands and endometrial epithelial cells caused by the infection. While the other three Sphingomonas strains played a protective role in the structure of uterine glands and endometrial epithelial cells, they still exhibited varying degrees of neutrophil infiltration. Meanwhile, Sphingomonas DS-28 significantly reduced the expression levels of key inflammatory factors (TNF-α, relative decrease of 58.9%; IL-6, relative decrease of 84.6% and IL-8, relative decrease of 92.2%) in uterine tissue and effectively reduced the NF-κB signaling pathway, p-P65 (p>0.9999, relative decrease of 38.2%) and p-IκB (p=0.2976, relative decrease of 50.2%). Sphingosomalidobacterium C3-42.1 failed to effectively inhibit the expression level of IL-8 (E. colivsE_C3-42.1, p= 0.7920, relative decrease of 20.5%), and its inhibitory effect on TNF-α (CTRLvsE_C3-42.1, p= 0.4759; CTRLvsE_Sp. DS-28, p= 0.9943; E. colivsE_C3-42.1, p= 0.8990, relative decrease of 31.3%; E. colivsE_Sp. DS-28, p= 0.2055, relative decrease of 58.9%) was lower than that of Sphingosomalidobacterium DS-28. Similarly, the inhibitory effects of Sphingomonas C3-25.1 and Sphingomonas C3-41.1 on TNF-α (CTRLvsE_C3-25.1, p= 0.3187; CTRLvsE_C3-41.1, p= 0.0935; E. colivsE_C3-25.1, p= 0.9700, relative decrease of 25.4%; E. colivsE_C3-41.1, p> 0.9999, relative decrease of 11.4%) were also lower than those of Sphingomonas DS-28. Furthermore, Sphingomonas C3-41.1 (p = 0.0152, relative decrease of 3.65%) and Sphingomonas C3-42.1 (p = 0.0109, 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 experiment was conducted in this example: SPF-grade 7-8 week old female KM mice were selected as experimental animals and 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: negative control group (NC), the post-biotic preparation treatment group in Example 3 (SPS), the disease model group (E. coli), and the post-biotic preparation treatment group in Example 3 (ESPS).

[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 to synchronize estrus. An endometritis model was established 24 hours later. All mice were first anesthetized. For mice in the NC and SPS groups: 60 μL of sterile PBS was perfused into the uterus. For mice in the E. coli and ESPS groups: 60 μL of E. coli (1 × 10⁻⁶) was perfused into the uterus. 12 (CFU / 60μL). Treatment was initiated 24 hours later and continued for 5 days. Mice were first anesthetized preoperatively. For mice in the NC and E. coli groups, 80μL of sterile PBS was perfused into the uterus. For mice in the SPS and ESPS groups, 80μL of the metabiotic preparation from Example 3 was perfused into the uterus. After 5 days of treatment with the metabiotic 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 minutes and finally stored at -80℃ for subsequent analysis.

[0081] 3. Efficacy Verification: Morphological changes in the uterus were compared and observed. Hematoxylin-eosin stained pathological tissue sections were analyzed, and the expression levels of key inflammatory genes (IL-1β, TNF-α, and IL-8, Figure 9) were detected using real-time quantitative PCR to determine the level of uterine inflammation in each group of mice. Data are presented as mean ± standard deviation. Statistical significance was determined using one-way ANOVA and Tukey's test.

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

[0083] Hematoxylin-eosin stained histopathological sections showed that, compared with the NC group, the E. coli group mice had neutrophil infiltration in the endometrium, disrupted endometrial epithelial cell structure, and edematous and structurally damaged uterine glands. After treatment, the ESPS group maintained good endometrial structure and no neutrophil infiltration was observed (Figure 9).

[0084] Real-time quantitative PCR results showed that, compared with the NC group, the E. coli group mice had lower levels of TNF-α (p0.05). 0.0001), IL-1β (p= 0.0007) and IL-8 (p The expression levels of TNF-α in the ESPS group mice were significantly increased. After postbiotic treatment, the expression level of TNF-α in the ESPS group mice was significantly lower than that in the E. coli group (p = 0.0035) (Figure 10A), while the gene levels of IL-1β (p = 0.9900) (Figure 10B) and IL-8 (p > 0.9999) (Figure 10C) decreased significantly and returned to a level that was not significantly different from that in 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 effectively alleviate E. coli-induced endometrial inflammation in mice, significantly improve uterine redness and swelling, reduce histopathological damage, and significantly downregulate the levels of multiple inflammatory factors (TNF-α, IL-1β, and IL-8) in the uterus, providing a new non-live bacterial formulation option 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 mentioned is sphingomonas DS-28, classified as Sphingomonas insulae, and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35151 on July 9, 2025.

2. A type of sphingosine monocytogenes, characterized in that, The *Sphingomonas* strain is derived from the uterine flora of mammals, possesses uterine colonization ability, and also has a therapeutic effect on *Escherichia coli*-induced endometritis. *Sphingomonas* is one of a)-c): a) *Sphingomonas* is classified as *Sphingomonas kyungheensis*, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37081, deposited on December 15, 2025; b) *Sphingomonas* is classified as *Sphingomonas hankookensis*, deposited at the CGMCC with accession number CGMCC NO.37082, deposited on December 15, 2025; c) *Sphingomonas* is classified as *Sphingomonas yunnanensis*, deposited at the CGMCC with accession number CGMCC. NO.37083, deposited on December 15, 2025.

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

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

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

6. The microbial preparation according to claim 5, 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.

7. The microbial preparation according to claim 6, 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.

8. The use of Sphingomonas as described in claim 1 or 2, the product as described in claim 3 or 4, or the microbial preparation as described in any of claims 5-6 in the preparation of a medicament for treating endometritis.

9. The application as described in claim 8, characterized in that, The endometritis was caused by Escherichia coli.

10. 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) Reduces pathological damage to uterine tissue; d) Significantly downregulates the levels of TNF-α, IL-1β, and IL-8 inflammatory factors in the uterus.

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