Use of bifidobacterium longum subsp. longum bl21 in the preparation of a preparation for improving hematopoietic dysfunction

Abstract

The present application relates to the application of Bifidobacterium longum subsp.longum BL21 in the preparation of a preparation for improving hematopoietic dysfunction, wherein the Bifidobacterium longum subsp.longum BL21 is a Bifidobacterium longum subsp.longum BL21 strain with a preservation number of CGMCC No.10452.The present application develops a new microbial strategy for improving hematopoietic dysfunction, and uses the Bifidobacterium longum subsp.longum BL21 in the preparation of a preparation for improving hematopoietic dysfunction.It is found that the Bifidobacterium longum subsp.longum BL21 can increase the number of peripheral blood cells (red blood cells and white blood cells), the content of platelets or the content of hemoglobin, reverse the weight loss caused by hematopoietic dysfunction, and improve the serum inflammatory response and the spleen inflammatory response caused by hematopoietic dysfunction.The present application also provides a new idea for the wide application of the Bifidobacterium longum subsp.longum BL21.

Description

Technical Field

[0001] This invention belongs to the field of probiotic technology and relates to a novel use of Bifidobacterium longum subsp. BL21, specifically the application of Bifidobacterium longum subsp. BL21 in the preparation of formulations that improve hematopoietic dysfunction. Background Technology

[0002] In recent years, with the rapid development of organ transplantation technology, the recovery of hematopoietic function after transplantation has become a key factor affecting patient prognosis. During the hematopoietic reconstitution process after hematopoietic stem cell transplantation (HSCT) and solid organ transplantation, recipients often face multiple challenges such as hematopoietic suppression, increased risk of infection, and delayed graft function.

[0003] A close "gut-bone marrow axis" regulatory mechanism exists between the gut microbiota and bone marrow hematopoietic function: gut commensal bacteria, through the production of signaling molecules such as short-chain fatty acids (SCFA), tryptophan metabolites, and indole-3-lactic acid, can remotely activate the AhR / IL-22 pathway in bone marrow endothelial cells via blood circulation, enhancing VE-cadherin expression and thereby improving the adhesion, transendothelial migration, and engraftment efficiency of hematopoietic stem / progenitor cells (HSPCs). However, the use of broad-spectrum antibiotics and proton pump inhibitors during the perioperative period of transplantation, as well as stress-induced gastrointestinal mucosal ischemia, leads to a sharp decrease in the recipient's gut commensal bacteria, becoming a neglected but reversible hematopoietic inhibitory factor.

[0004] In existing technologies, although some scholars have attempted to rebuild the gut microbiota through oral fecal microbiota capsules or single probiotic strains, the survival rate of live bacteria reaching the colon is very low due to the influence of gastric acid, bile acids, and immunosuppressants, resulting in limited clinical benefits. Furthermore, traditional intervention methods share the following common drawbacks: they target only a single point of intervention, failing to simultaneously address the triple obstacles of stem cell quantity, quality, and microenvironment damage; the short half-life of bioactive molecules in vivo necessitates repeated administration of high doses, increasing toxic side effects; and there is a lack of delivery platforms that can precisely control the release rhythm and match the timing of transplantation surgery.

[0005] Therefore, developing a probiotic strain that can colonize simultaneously with organ transplantation, is resistant to gastric acid and bile, and efficiently synthesizes SCFA and specific extracellular polysaccharides, and corrects the recipient's bone marrow microenvironment damage in situ through the "gut-bone marrow" remote regulatory axis, activating endogenous HSPC amplification and homing, is expected to break through the bottleneck of traditional cytokine therapy, significantly reduce transfusion dependence, infection, and graft function delay, and has important clinical application value. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a new use for Bifidobacterium longum subsp. BL21, specifically the application of Bifidobacterium longum subsp. BL21 in the preparation of preparations to improve hematopoietic dysfunction.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides the application of Bifidobacterium longum subsp. BL21 in the preparation of formulations that improve hematopoietic dysfunction;

[0009] The Bifidobacterium longum subsp. longum strain mentioned is the Bifidobacterium longum subsp. longum strain BL21 with the accession number CGMCC No.10452.

[0010] The BL21 strain involved in this invention was deposited in 2015 at the China General Microbiological Culture Collection Center (CGMCC No. 10452). It is a probiotic with excellent acid and bile salt tolerance. Currently, there are no reports on the application of BL21 in promoting hematopoietic function, especially after organ transplantation.

[0011] This invention develops a novel microbial strategy for improving hematopoietic dysfunction. It utilizes *Bifidobacterium longum* subsp. BL21 to prepare a formulation that improves hematopoietic dysfunction. The invention creatively discovers that *Bifidobacterium longum* subsp. BL21 can increase the number of peripheral blood cells (red blood cells and white blood cells), platelet count, or hemoglobin level; it can reverse weight loss caused by hematopoietic dysfunction; and it can improve serum inflammatory responses and splenic inflammatory responses caused by hematopoietic dysfunction. This invention also provides new ideas for the widespread application of *Bifidobacterium longum* subsp. BL21.

[0012] Preferably, the hematopoietic dysfunction includes a decrease in the number of peripheral blood cells, a decrease in platelets, and a decrease in hemoglobin; the peripheral blood cells include red blood cells and white blood cells.

[0013] Preferably, the hematopoietic dysfunction includes serum inflammatory response, spleen inflammatory response, and weight loss caused by hematopoietic dysfunction.

[0014] Preferably, the dosage form of the formulation includes lyophilized powder, tablets, capsules, microcapsules, granules, or solutions. The formulation of the present invention can be formulated into various dosage forms to meet the needs of different usage scenarios.

[0015] Preferably, the formulation further contains excipients; the excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

[0016] Preferably, the number of *Bifidobacterium longum* subsp. BL21 in the preparation is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×109 CFU / g, for example 1×10 9 CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), 5×10 10 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 3×10 11 CFU / g (CFU / mL), 5×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), 1×10 13 CFU / g (CFU / mL), etc., and other specific point values ​​within this range can be selected, which will not be elaborated here.

[0017] Preferably, the hematopoietic dysfunction is a hematopoietic dysfunction following organ transplantation.

[0018] Bioinformatics analysis of *Bifidobacterium longum* subsp. *longum* BL21 in this invention revealed that BL21 exhibits systemic advantages in multiple pathways related to immune metabolic regulation, bacterial-host interactions, glycolipid structural modification, and anti-inflammatory signaling. These pathways work synergistically, enabling BL21 to reshape the immune microenvironment at the host level and reduce the risk of organ transplant-related immune rejection by restricting effector T cell metabolism, promoting the export of immune-tolerant metabolites, weakening antigen stimulation, and buffering inflammatory responses. Furthermore, this mechanism is not a single immunosuppressive effect but rather achieved through the synergistic effect of multiple pathways. Therefore, BL21 can reduce the risk of organ transplant immune rejection, and its application in improving post-organ transplant hematopoietic dysfunction shows broad promise, synergistically restoring the body's hematopoietic function and alleviating the body's organ transplant immune rejection response.

[0019] In a second aspect, the present invention provides the application of Bifidobacterium longum subsp. BL21 in the preparation of formulations that increase the number of peripheral blood cells, platelet count, or hemoglobin content.

[0020] The Bifidobacterium longum subsp. longum strain mentioned is the Bifidobacterium longum subsp. longum strain BL21 with the accession number CGMCC No.10452.

[0021] Preferably, the dosage form of the formulation includes lyophilized powder, tablets, capsules, microcapsules, granules, or solutions. The formulation of the present invention can be formulated into various dosage forms to meet the needs of different usage scenarios.

[0022] Preferably, the formulation further contains excipients; the excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

[0023] Preferably, the number of *Bifidobacterium longum* subsp. BL21 in the preparation is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g, for example 1×10 9 CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), 5×10 10 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 3×10 11 CFU / g (CFU / mL), 5×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), 1×10 13 CFU / g (CFU / mL), etc., and other specific point values ​​within this range can be selected, which will not be elaborated here.

[0024] Thirdly, the present invention provides the application of Bifidobacterium longum subsp. BL21 in the preparation of a formulation to reverse weight loss caused by hematopoietic dysfunction;

[0025] The Bifidobacterium longum subsp. longum strain mentioned is the Bifidobacterium longum subsp. longum strain BL21 with the accession number CGMCC No.10452.

[0026] Preferably, the dosage form of the formulation includes lyophilized powder, tablets, capsules, microcapsules, granules, or solutions. The formulation of the present invention can be formulated into various dosage forms to meet the needs of different usage scenarios.

[0027] Preferably, the formulation further contains excipients; the excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

[0028] Preferably, the number of *Bifidobacterium longum* subsp. BL21 in the preparation is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g, for example 1×10 9CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), 5×10 10 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 3×10 11 CFU / g (CFU / mL), 5×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), 1×10 13 CFU / g (CFU / mL), etc., and other specific point values ​​within this range can be selected, which will not be elaborated here.

[0029] Fourthly, this invention provides the application of Bifidobacterium longum subsp. BL21 in the preparation of formulations that improve serum inflammatory responses caused by hematopoietic dysfunction;

[0030] The Bifidobacterium longum subsp. longum strain mentioned is the Bifidobacterium longum subsp. longum strain BL21 with the accession number CGMCC No.10452.

[0031] Preferably, the dosage form of the formulation includes lyophilized powder, tablets, capsules, microcapsules, granules, or solutions. The formulation of the present invention can be formulated into various dosage forms to meet the needs of different usage scenarios.

[0032] Preferably, the formulation further contains excipients; the excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

[0033] Preferably, the number of *Bifidobacterium longum* subsp. BL21 in the preparation is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g, for example 1×10 9 CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), 5×10 10 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 3×10 11 CFU / g (CFU / mL), 5×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), 1×10 13CFU / g (CFU / mL), etc., and other specific point values ​​within this range can be selected, which will not be elaborated here.

[0034] Fifthly, the present invention provides the use of Bifidobacterium longum subsp. BL21 in the preparation of a formulation that improves splenic inflammatory response caused by hematopoietic dysfunction;

[0035] The Bifidobacterium longum subsp. longum strain mentioned is the Bifidobacterium longum subsp. longum strain BL21 with the accession number CGMCC No.10452.

[0036] Preferably, the dosage form of the formulation includes lyophilized powder, tablets, capsules, microcapsules, granules, or solutions. The formulation of the present invention can be formulated into various dosage forms to meet the needs of different usage scenarios.

[0037] Preferably, the formulation further contains excipients; the excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

[0038] Preferably, the number of *Bifidobacterium longum* subsp. BL21 in the preparation is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g, for example 1×10 9 CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), 5×10 10 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 3×10 11 CFU / g (CFU / mL), 5×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), 1×10 13 CFU / g (CFU / mL), etc., and other specific point values ​​within this range can be selected, which will not be elaborated here.

[0039] Sixthly, the application of Bifidobacterium longum subsp. BL21 in the preparation of agents to improve immune rejection response in organ transplantation;

[0040] The Bifidobacterium longum subsp. longum strain mentioned is the Bifidobacterium longum subsp. longum strain BL21 with the accession number CGMCC No.10452.

[0041] Preferably, the dosage form of the formulation includes lyophilized powder, tablets, capsules, microcapsules, granules, or solutions. The formulation of the present invention can be formulated into various dosage forms to meet the needs of different usage scenarios.

[0042] Preferably, the formulation further contains excipients; the excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

[0043] Preferably, the number of *Bifidobacterium longum* subsp. BL21 in the preparation is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g, for example 1×10 9 CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), 5×10 10 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 3×10 11 CFU / g (CFU / mL), 5×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), 1×10 13 CFU / g (CFU / mL), etc., and other specific point values ​​within this range can be selected, which will not be elaborated here.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] This invention develops a novel microbial strategy for improving hematopoietic dysfunction. It utilizes *Bifidobacterium longum* subsp. BL21 to prepare a formulation that improves hematopoietic dysfunction. The invention creatively discovers that *Bifidobacterium longum* subsp. BL21 can increase the number of peripheral blood cells (red blood cells and white blood cells), platelet count, or hemoglobin level; it can reverse weight loss caused by hematopoietic dysfunction; and it can improve serum inflammatory responses and splenic inflammatory responses caused by hematopoietic dysfunction. This invention also provides new ideas for the widespread application of *Bifidobacterium longum* subsp. BL21.

[0046] The BL21 strain involved in this invention is classified as *Bifidobacterium longum* subsp. *longum*, deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 27, 2015, with accession number CGMCC No. 10452, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0047] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0048] The following method for preparing the suspension of Bifidobacterium longum subsp. BL21 involved in the experiment is as follows: The strain was inoculated into MRS liquid medium at an inoculum of 2% (v / v) and cultured statically at 37°C for 24 h to obtain the seed culture; then, it was inoculated into MRS liquid medium at an inoculum of 2% (v / v) and cultured statically at 37°C for 24 h to obtain the bacterial culture; the bacterial culture was centrifuged at 4000 rpm for 5 min at 4°C, filtered, and the bacterial cells were obtained; the bacterial cells were resuspended in pure water and diluted as needed to obtain the bacterial suspension.

[0049] MRS medium: Weigh 10 g peptone, 10 g beef extract, 20 g glucose, 2 g anhydrous sodium acetate, 5 g yeast extract, 2 g diammonium citrate, 2.6 g K2PO4·3H2O, 0.1 g MgSO4·7H2O, 0.05 g MnSO4, and 0.5 g L-cysteine ​​hydrochloride. Dissolve in deionized water, add 1 mL Tween 80, and bring the volume to 1 L. Sterilize and cool before use.

[0050] Example 1

[0051] This embodiment verifies the effect of Bifidobacterium longum subsp. BL21 on a hematopoietic suppression model mouse. The steps are as follows:

[0052] (1) Forty-eight male SPF-grade BALB / c mice, aged 7 weeks, were selected and acclimatized for 7 days in an animal room (room temperature: 22±2℃, humidity: 50±10%, 12-hour light-dark cycle) with ordinary feed (free access to food and water).

[0053] (2) Grouping: Mice were randomly divided into 4 groups: blank control group (Control), model group (Model), positive control group (rhG-CSF), and Bifidobacterium longum subsp. BL21 group (BL21), 12 mice / group.

[0054] (3) Feeding: All groups were fed regular feed, and the water and bedding were changed twice a week.

[0055] (4) Modeling method: The model group, positive control group and Bifidobacterium longum subsp. BL21 group were all induced to establish a hematopoietic suppression model by cyclophosphamide (CTX, prepared with sterile PBS, 10 mg / mL). The model group, positive control group and Bifidobacterium longum subsp. BL21 group were subjected to a single intraperitoneal injection (0.1 mL / 10g, 100 mg / kg) for 3 consecutive days. The blank control group was simultaneously injected intraperitoneally with an equal volume of physiological saline (0.2 mL / 20 g). The injection frequency was exactly the same as the experimental group to ensure the effectiveness of the control.

[0056] (5) Intervention method: After modeling, the drugs were administered continuously for 6 weeks. Mice in the model group and the positive control group were injected intraperitoneally with 0.2 mL / 20 g saline and 22.5 μg / kg rhG-CSF, respectively. The Bifidobacterium longum subsp. BL21 group and the blank control group were administered 1×10 g of rhG-CSF orally. 9 Mice were administered a CFU / mL suspension of Bifidobacterium longum subsp. BL21 and 0.2 mL / 20 g physiological saline via gavage at a volume of 20 mL / kg, and the survival rate and weight changes of the mice were monitored periodically.

[0057] (6) Clinical symptom observation: Clinical symptom observation was conducted once in the morning and once in the afternoon during the modeling period, and the number of surviving mice and clinical symptoms were recorded.

[0058] (7) Weight: The weight of the mice was measured once after the acclimatization period ended and before the modeling began; the weight was measured once after the modeling was completed 3 days later; the weight of the mice was measured weekly after the modeling was completed and the data were statistically analyzed; the weight of the experimental mice was measured a total of 8 times.

[0059] (8) Peripheral blood and serum collection: Two hours after the last administration, mouse blood samples were collected via the tail vein for the detection of peripheral blood indicators. Another portion of the whole blood sample was transferred to centrifuge tubes, allowed to stand at room temperature for 20 min, and centrifuged at 3500 rpm for 10 min to obtain the supernatant liquid. After centrifugation, the supernatant serum was collected, aliquoted into sterile centrifuge tubes, and immediately transferred to a -80℃ ultra-low temperature storage system for long-term preservation of biological samples.

[0060] (9) Organ collection: After blood collection, the experimental animals were euthanized by CO2 inhalation, and tissue samples of the spleen, lungs, liver, kidneys, heart, femur, and tibia, as well as the contents of the cecum, were collected quickly, and the wet weight of the organs was measured. The organ index was calculated as follows: Organ index (mg / g) = organ mass (mg) / mouse body weight (g).

[0061] (10) Indicator testing:

[0062] (10.1) Clinical symptom observation:

[0063] During the experiment, all mice in each group were in good condition with no obvious abnormalities and no animals died.

[0064] (10.2) Changes in mouse body weight:

[0065] As shown in Table 1 (data in the table are expressed as mean ± standard deviation, # and ## represent comparisons with the blank control group), the mice in the blank control group showed normal weight gain during the experiment; compared with the blank control group, the mice in the model group showed significant weight changes (P < 0.05, P < 0.01), indicating that the hematopoietic injury model was successfully established. Compared with the model group, the weight of mice in the positive control group and the BL21 group both showed varying degrees of recovery, indicating that rhG-CSF and BL21 have a mitigating effect on CTX-induced weight loss.

[0066] Table 1

[0067]

[0068] (10.3) Changes in organ indices in mice:

[0069] Organ index is an important indicator for evaluating drug toxicity and can reflect the physiological state of various organs in mice, as shown in Table 2 (the data in the table are expressed as mean ± standard deviation, # and ## represent the difference between the control group and the control group, respectively, P < 0.05 and P < 0.01). , (P < 0.05 and P < 0.01, respectively, compared with the model group). After treatment, the organ indices of the mice did not change significantly, indicating that BL21 did not cause any side effects during treatment, which preliminarily demonstrates the safety of BL21.

[0070] Table 2

[0071]

[0072] (10.4) Effects on mouse peripheral blood cells:

[0073] The number of white blood cells (WBC), red blood cells (RBC), platelets (PLT), and hemoglobin (Hb) in blood samples was analyzed using a fully automated cell analyzer. The test results are shown in Table 3.

[0074] Table 3

[0075]

[0076] As shown in Table 3, compared with the blank group, the number of peripheral blood WBC, RBC, PLT and Hb in the model group mice was significantly reduced (P<0.05, P<0.01); compared with the model group, the number of peripheral blood WBC, RBC, PLT and Hb in the positive control group and BL21 group mice was significantly increased (P<0.05, P<0.01). The drugs rhG-CSF and Bifidobacterium longum subsp. BL21 can improve the peripheral blood (WBC, RBC, PLT and Hb) content in hematopoietic suppression model mice (data in the table are expressed as mean ± standard deviation, # and ## indicate that compared with the blank control group, P<0.05 and P<0.01, respectively). , "P < 0.05" and "P < 0.01" respectively, compared to the model group.

[0077] (10.5) Effects on inflammatory factors in mouse serum:

[0078] Inflammatory signaling regulates the development and homeostasis of hematopoietic stem cells and progenitor cells, which is crucial for the hematopoietic system. Inflammatory factors can affect the generation, migration, and differentiation of hematopoietic stem cells, and can also inhibit hematopoiesis by damaging hematopoietic stem cells, ultimately leading to bone marrow damage. IL-4, as a Th2-type anti-inflammatory factor, can inhibit the secretion of pro-inflammatory mediators and regulate the activation state of macrophages / neutrophils, stabilizing the hematopoietic microenvironment. Meanwhile, pro-inflammatory factors such as IFN-γ, TNF-α, and IL-6 affect the hematopoietic microenvironment by interfering with intercellular communication and cytokine production, promoting inflammatory responses, and in some cases, potentially leading to abnormal proliferation or negative effects on hematopoietic stem cells.

[0079] Mouse serum was used to detect inflammatory factors (IL-4, IFN-γ, TNF-α, IL-6). The levels of these inflammatory factors were detected using an ELISA kit. The results are shown in Table 4.

[0080] Table 4

[0081]

[0082] As shown in Table 4, the anti-inflammatory factor (IL-4) in the serum of mice in the model group was significantly decreased (P < 0.01), while the pro-inflammatory factors (IFN-γ, TNF-α, IL-6) were significantly increased (P < 0.05), indicating that the model group mice were successfully modeled. Compared with the model group, the positive control group and BL21 group showed significantly increased IL-4 levels (P < 0.05) and significantly decreased IFN-γ levels (P < 0.05), with little difference in IFN-γ levels between the positive control group and BL21 group. Compared with the model group, the positive control group showed significantly decreased levels of TNF-α and IL-6 (P < 0.05), while the BL21 group showed no significant difference (P > 0.05), but the specific levels of both were reduced. All experimental results indicate that *Bifidobacterium longum* subsp. *longum* BL21 can alleviate serum inflammatory response in mice with hematopoietic dysfunction (data in the table are expressed as mean ± standard deviation, # and ## indicate P < 0.05 and P < 0.01 compared with the blank control group, respectively). , "P < 0.05" and "P < 0.01" respectively, compared to the model group.

[0083] (10.6) Effects on inflammatory factors in mouse spleen cells:

[0084] The spleen is a crucial extramedullary hematopoietic and immune organ. It works with the bone marrow to maintain the homeostasis of the human hematopoietic and immune system. Together, they maintain the normal function of the blood system and immune regulation. If the spleen becomes inflamed, it will directly affect its extramedullary hematopoietic function.

[0085] Mouse spleen tissue was used to detect inflammatory factors (IL-4, IFN-γ, TNF-α, IL-6). Spleen samples were mixed with physiological saline and then homogenized at 60 Hz for 3 × 30 s for mechanical lysis. The supernatant was enriched by differential centrifugation, and the resulting experimental samples were analyzed using an ELISA kit to detect inflammatory factors in spleen cells. The results are shown in Table 5.

[0086] Table 5

[0087]

[0088] As shown in Table 5, compared with the blank control group, the IL-4 content in the model group was significantly reduced (P < 0.01), while the contents of IFN-γ, TNF-α, and IL-6 were significantly increased (P < 0.01). Compared with the model group, the IL-4 content in the spleen cells of the positive control group and BL21 was increased, while the contents of pro-inflammatory factors (IFN-γ, TNF-α, and IL-6) were reduced; indicating that administering a certain amount of rhG-CSF or BL21 to mice can restore the spleen's anti-inflammatory factor function to a certain extent (all data in the table are expressed as mean ± standard deviation, # and ## indicate that compared with the blank control group, P < 0.05 and P < 0.01, respectively). , "P < 0.05" and "P < 0.01" respectively, compared to the model group.

[0089] Example 2

[0090] In this embodiment, whole-genome sequencing and bioinformatics analysis were performed on *Bifidobacterium longum* subsp. *longum* to analyze at the gene level how *Bifidobacterium longum* subsp. *longum* BL21 reduces the possibility of immune rejection in organ transplantation. The analysis results are shown in Table 6 (wherein, the difference in pathway integrity or the difference in the number of pathway genes refers to the difference between the BL21 strain and the type strains of the same species in the database, and positive values ​​indicate that the BL21 strain has higher pathway integrity and more functional genes in the corresponding immune-related pathways).

[0091] Table 6

[0092]

[0093]

[0094] The analysis results in Table 6 show that both "differences in pathway integrity" and "differences in the number of pathway genes" are positive, and some are specific to BL21, indicating that BL21 possesses higher pathway integrity and more functional genes in these immune-related pathways. Regarding immune tolerance and anti-rejection, BL21 exhibits a systemic advantage in multiple pathways related to immune metabolic regulation, bacterial-host interactions, glycolipid structural modification, and anti-inflammatory signaling. These pathways work synergistically, enabling BL21 to reshape the immune microenvironment at the host level and reduce the risk of organ transplant-related immune rejection by restricting effector T cell metabolism, promoting the export of immune tolerance metabolites, weakening antigen stimulation, and buffering inflammatory responses. This mechanism is not a simple immunosuppression but rather achieved through the synergistic action of multiple pathways.

[0095] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0096] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0097] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. Application of Bifidobacterium longum subsp. BL21 in the preparation of agents to improve hematopoietic dysfunction; The Bifidobacterium longum subsp. longum strain mentioned is the Bifidobacterium longum subsp. longum strain BL21 with the accession number CGMCC No.10452.

2. The application according to claim 1, characterized in that, The hematopoietic dysfunction includes a decrease in the number of peripheral blood cells, a decrease in platelets, and a decrease in hemoglobin. The peripheral blood cells include red blood cells and white blood cells.

3. The application according to claim 1, characterized in that, The hematopoietic dysfunction includes serum inflammatory response, spleen inflammatory response, and weight loss caused by hematopoietic dysfunction.

4. The application according to claim 1, characterized in that, The dosage forms of the preparation include lyophilized powder, tablets, capsules, microcapsules, granules, or solutions; Preferably, the formulation further contains excipients; the excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

5. The application according to claim 1, characterized in that, The hematopoietic dysfunction mentioned refers to hematopoietic dysfunction after organ transplantation.

6. The application according to claim 1, characterized in that, The number of *Bifidobacterium longum* subsp. BL21 in the preparation is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

7. Application of Bifidobacterium longum subsp. BL21 in the preparation of formulations that increase peripheral blood cell count, platelet count, or hemoglobin count; The Bifidobacterium longum subsp. longum strain mentioned is the Bifidobacterium longum subsp. longum strain BL21 with the accession number CGMCC No.10452.

8. Application of Bifidobacterium longum subsp. BL21 in the preparation of agents to reverse weight loss caused by hematopoietic dysfunction; The Bifidobacterium longum subsp. longum strain mentioned is the Bifidobacterium longum subsp. longum strain BL21 with the accession number CGMCC No.10452.

9. Application of Bifidobacterium longum subsp. BL21 in the preparation of agents that improve serum inflammatory responses caused by hematopoietic dysfunction; The Bifidobacterium longum subsp. longum strain mentioned is the Bifidobacterium longum subsp. longum strain BL21 with the accession number CGMCC No.10452.

10. Application of Bifidobacterium longum subsp. BL21 in the preparation of formulations that improve splenic inflammatory response caused by hematopoietic dysfunction; The Bifidobacterium longum subsp. longum strain mentioned is the Bifidobacterium longum subsp. longum strain BL21 with the accession number CGMCC No.10452.

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