A method of lactic acid-enhanced mdsc immunosuppression for treating acute liver injury

Abstract

The application relates to the fields of biological medicine and immunotherapy, and discloses a method for treating acute liver injury by using lactic acid to enhance MDSC immunosuppression, which comprises the following steps: first, an acute liver injury model is established by injecting ConA into the intraorbital venous vein of a C57 / B6 wild type mouse; experiment grouping comprises a wild type group and a lactic acid pretreatment group; the dynamic change of serum ALT level is detected through orbital blood sampling; ELISA is used to detect cytokine secretion; liver tissue injury is evaluated through HE staining; liver lymphocytes are extracted by using a Percoll separation method; the proportion of immune cells in the liver, the spleen and the bone marrow and the intracellular cytokine expression are analyzed by using a flow cytometry; meanwhile, a lactic acid detection kit is used to detect the lactic acid production in the body; in addition, mouse bone marrow precursor cells are separated; the bone marrow precursor cells are induced to differentiate into MDSC by using GM-CSF and IL-6; the proportion of G-MDSC and the expression change of CXCR2 are detected by using a flow cytometry; and the method is verified by in-vivo and in-vitro experiments of mice, so that the lactic acid provides a new treatment strategy for acute liver injury by regulating MDSC.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and immunotherapy, specifically to a method for treating acute liver injury by using lactate-enhanced MDSC immunosuppression. Background Technology

[0002] Acute liver injury (AII) is a severe clinical syndrome with rapid onset and progression, often secondary to viral hepatitis, drug-induced liver injury, and autoimmune hepatitis (AIH). Its core pathology involves excessive inflammatory response, immune cell infiltration, and massive hepatocyte death. Currently, there is a severe lack of specific treatments for AII, necessitating the development of new intervention strategies. Autoimmune hepatitis is an acute liver injury mediated by an abnormal autoimmune response, with a cytokine storm triggered by excessive T cell activation being its main pathogenic mechanism. The concanavalin A (ConA)-induced AII model is widely recognized as an ideal experimental research model because it specifically activates T cells and NKT cells, mimicking the immunopathological process of human AIH.

[0003] The role of immunomodulation in liver injury is widely recognized, but precisely regulating the immune microenvironment to suppress excessive inflammation while avoiding immune paralysis remains a key challenge in current research. Myeloid-derived suppressor cells (MDSCs) are a group of immature myeloid cells with potent immunosuppressive functions. They can proliferate in large numbers under pathological conditions such as cancer and inflammation, and limit tissue damage by secreting molecules such as arginase-1 (Arg-1), inducible nitric oxide synthase (iNOS), and IL-10 to inhibit the activity of effector T cells. Previous studies have shown that in acute liver injury, MDSCs can participate in the liver repair process by regulating the immune response. Therefore, targeting MDSCs to restore hepatic immune homeostasis is a potentially effective strategy for treating acute liver injury.

[0004] Lactic acid, long considered a waste product of glucose metabolism, has recently been revealed to play a crucial role in immune regulation. Lactic acid can enter cells via its receptor GPR81 or monocarboxylic acid transporters, influencing immune cell function, such as inducing macrophage polarization towards the M2 type and inhibiting the release of inflammatory factors. In the tumor microenvironment, lactic acid has been shown to promote the expansion and functional activation of MDSCs, forming an immunosuppressive barrier. However, in acute inflammation models, the lactate-MDSC axis may play a drastically different protective role. For example, in studies of hypoxic-ischemic brain injury in newborns, lactate has been found to be a key effector molecule in the neuroprotective role of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). However, in acute liver injury, particularly ConA-induced immune liver injury, it remains unclear whether exogenous lactate exerts a protective effect by regulating MDSC function, and the specific cellular and molecular mechanisms involved are also unknown. Currently, there is a lack of effective methods to combine lactate with MDSC enhancement for the treatment of acute liver injury. Summary of the Invention

[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for treating acute liver injury by using lactate-enhanced MDSC immunosuppression. This method offers advantages such as highly effective protection of the liver from ConA-induced immune liver injury, precise regulation of the liver's immune microenvironment, high safety, and specific targeting to enhance MDSC proliferation and immunosuppressive function. It solves the problems in existing technologies, such as the lack of effective strategies for treating acute liver injury by using lactate to regulate MDSCs, the difficulty in suppressing excessive inflammation while avoiding immune paralysis, and the unclear mechanism of action of lactate in immune liver injury.

[0006] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a method for treating acute liver injury by using lactate-enhanced MDSC immunosuppression, comprising the following steps: Step 1: Establishment of ConA-induced acute liver injury model: Dissolve concanavalin A powder in phosphate buffer, randomly group experimental animals according to the principle of balanced body weight and mark individual numbers, and inject ConA into C57 / B6 wild-type mice through the inner canthal vein to establish an acute liver injury model. Step 2, Dynamic ALT Level Measurement: Blood was collected from the orbital region of mice at different time points according to experimental requirements, and the serum ALT level was detected using an ALT kit. Step 3: Cytokine and Liver Injury Detection: Collect mouse blood according to experimental requirements, separate serum, and use ELISA to detect the secretion of various cytokines; collect mouse liver tissue, perform HE staining on paraffin sections, and detect liver damage in wild-type mice and mice supplemented with lactate. Step 4: Extraction of liver lymphocytes from mice: Wild-type mice were harvested, the eyeballs were removed to expel blood, and the liver tissue was taken out and placed in pre-prepared ice-cold PBS for later use. Liver lymphocytes were extracted using the Percoll separation method. Step 5, Flow cytometry: Lymphocytes are isolated from designated organs of mouse liver, spleen and bone marrow, and the cell surface and intracellular cytokine staining is performed. The proportion of these cytokines is then analyzed by flow cytometry. Step 6, Lactic acid content determination: The amount of lactic acid produced in the WT-LA group was determined by lactic acid detection kit II, and the wild-type mouse group without in vitro lactic acid injection was used as a blank control. Step 7: Bone marrow MDSC induction differentiation experiment: Mouse bone marrow progenitor cells were isolated and induced to differentiate into MDSCs using GM-CSF and IL-6. The proportion of G-MDSCs and changes in CXCR2 expression were detected by flow cytometry.

[0007] Preferably, the ConA-induced mouse acute liver injury model is established in step one: S1.1. Open the biosafety cabinet in advance and irradiate it with ultraviolet light for 25-30 minutes; S1.2 Dissolve ConA in sterile PBS buffer to prepare a ConA solution with a concentration of 10 mg / mL. Mix well by pipetting to ensure complete dissolution. S1.3 Place the prepared ConA solution in a refrigerator or ice bath at 1-4℃ and let it stand for 0.4-0.5 hours. During this period, mix it with a pipette every 10-12 minutes. S1.4 After 25-30 minutes, once the solution is confirmed to be completely dissolved, the same batch of mice are anesthetized using inhalation anesthesia at the same time. S1.5 After anesthesia, liver injury was induced by intravenous injection of ConA solution at a dose of 10 mg / kg based on the mouse's body weight. S1.6 After 10-12 hours, the mice were euthanized using the cervical dislocation method, and their livers were removed for further analysis.

[0008] Preferably, the dosage and treatment method of the experimental animals—mice in each group—in step one are as follows: (1) WT group: ConA with a content of 10 mg / kg was injected into the inner canthus, and the body was dissected 12 hours later; (2) WT-LA group: 24 hours in advance, lactate with a content of 160 mg / kg was injected intraperitoneally, followed by ConA with a content of 10 mg / kg at the inner canthus, and the body was dissected 12 hours later; (3) WT-LA-Inhibitor group: 24 hours in advance, lactic acid with a content of 160 mg / kg and SB225002 with a content of 5 mg / kg were injected intraperitoneally; 2 hours in advance, ConA with a content of 10 mg / kg was injected into the inner canthus, followed by another intraperitoneal injection of SB225002 with a content of 5 mg / kg; and the body was dissected 12 hours after the ConA injection.

[0009] Preferably, the ALT dynamic level measurement process in step two is as follows: S2.1. Take the mice to be tested and use capillary glass tubes to collect blood from the retro-orbital vein at different time points. Collect 0.2-0.3 mL each time and place it in an EP tube. The specific time interval is every 4 h or 6 h. Continuously monitor for 72 h to obtain the dynamic changes in ALT levels. S2.2 After blood samples are collected, each group of samples is marked with time and group. S2.3. Place the whole blood sample in a room temperature environment for 1 hour to allow for coagulation. S2.4. After the blood has been coagulated, the blood sample is basically separated into layers and placed in a constant temperature centrifuge for the first centrifugation process. The speed is set to 3500-3800 rpm × 15-16 min. S2.5. Use a 200µL pipette to transfer the pale yellow supernatant to a new sterile 1.5mL centrifuge tube; S2.6 Under the same temperature control conditions, the transferred supernatant is centrifuged a second time, with the speed set at 3000-3500 rpm and the centrifugation time at 5-6 min. S2.7 Finally, use a micropipette to aspirate the clear serum; do not aspirate red blood cells. S2.8. After diluting the collected serum samples 5 times with physiological saline, the changes in ALT and AST levels of the two groups in the serum at different time points were detected using ALT and AST kits. Finally, the OD values ​​were detected using an ELISA reader to plot the changes in the curve.

[0010] Preferably, in step three, mouse liver RNA extraction is performed as follows: (1) After the mice were euthanized in step one, the abdomen was opened, and a piece of liver tissue the size of a mung bean was cut off with tweezers and placed into an enzyme-free EP tube. 200 μL of Trizol was added to the tissue and the mixture was ground completely with a mixer until no tissue was visible to the naked eye. Then, 800 μL of Trizol was added to the EP tube. (2) Add 200 μL of chloroform along the wall, shake vigorously up and down to mix evenly, let stand on ice for 8-10 min, then transfer to a centrifuge at 1-4℃ and centrifuge at 12000 rpm for 15 min; (3) After centrifugation, carefully aspirate 500 μL of the clear supernatant and transfer it into a new enzyme-free EP tube; (4) Add 500 μL of isopropanol to the EP tube, mix until there are no filaments, and let stand in an ice bath for 8-10 minutes; (5) Transfer to a centrifuge at 1-4℃ and centrifuge at 10000-12000rpm for 15-16min; (6) Remove the EP tube, discard the supernatant and add 1 mL of 75% ethanol. Use a vortex mixer to shake for 4-5 min at 1-4℃ and 7000-7500 rpm. (7) Remove the EP tube and use a vacuum pump to remove the supernatant; (8) Add 10-20 μL of RNase-free water according to the amount of precipitate, mix well by pipetting to dissolve RNA, and transfer to a refrigerator at 1-4℃ for 30 min-1 h; (9) Measure the RNA concentration using the instrument, take 1-2 μL of RNA and judge the RNA purity according to the A260 / 280 ratio, and store the rest at -80℃ for short-term use; (10) Estimate the required RNA concentration for reverse transcription based on the measured RNA concentration.

[0011] Preferably, the preparation and staining of the paraffin sections in step three are as follows: S3.1 Preparation of paraffin sections: (1) Sample processing: After the experimental animals were sacrificed, the liver tissue was completely removed and fixed in 4% paraformaldehyde for 24 hours; (2) Gradient dehydration: After the sample is removed from the fixative, it is first rinsed with running water for 3-5 minutes, and then subjected to gradient dehydration treatment according to the standard procedure; (3) Paraffin embedding: Place the tissue block impregnated with paraffin into the embedding box, pour in liquid paraffin, and after cooling and solidification, form a regular paraffin block containing the tissue; (4) Serial sectioning: Fix the embedded sample on the microtome, set the section thickness to 4-8 μm, collect the tissue sections using an anti-roll plate, and quickly transfer them to a constant temperature water bath at 41-42℃ for development; (5) Slide attachment: The flattened tissue sections are attached to pre-labeled glass slides and dried in a slide dryer at 55-60℃; S3.2 HE staining procedure for paraffin sections: (1) Pretreatment of glass slides: Place the glass slides in a constant temperature oven at 60-65℃ and bake for ≥12h to enhance tissue adhesion; (2) Tissue transparency and hydration treatment: Tissue transparency is achieved by soaking the tissue in xylene to completely dissolve the paraffin and make the tissue transparent. Then, hydration treatment is performed by immersing the sections in a gradient of alcohols to gradually absorb water. Finally, the sections are rinsed with distilled water to remove any alcohol residue, thus completing the transition from an organic environment to an aqueous environment. (3) Immerse in hematoxylin stain for 5-8 minutes, rinse with running deionized water, and then absorb the residual liquid with absorbent paper. (4) Counterstain the cytoplasm with eosin solution for 1-2 min, and stop the staining reaction with distilled water; (5) Gradient dehydration and wax impregnation treatment: Immersion in molten paraffin to allow the wax to fully penetrate the interstitial spaces; (6) Add neutral resin to the tissue area, cover with a coverslip, cure at room temperature in the dark for 24 hours, and then seal and store.

[0012] Preferably, in step four, mouse lymphocyte extraction is performed as follows: S4.1, Liver lymphocyte extraction: (1) Enucleation and bloodletting of the eyes of WT mice; (2) Transfer the extracted liver tissue sample to a sterile culture dish containing pre-cooled sterile phosphate buffer for temporary storage; (3) In a sterile culture dish, assemble a 200-mesh stainless steel cell sieve and spread the liver tissue evenly on the surface of the sieve; (4) Use the piston end of a 5mL syringe to perform mechanical homogenization, apply vertical pressure and grind repeatedly until the tissue is completely dissociated; (5) During homogenization, pre-cooled PBS was continuously added dropwise; S4.2, Cell suspension treatment: (1) Rinse the cell sieve with pre-cooled PBS; (2) Use a pipette to transfer the homogenized mixture to a 50mL centrifuge tube and centrifuge at 1-4℃, 500rpm×1min; (3) Filter the supernatant into a new 50mL centrifuge tube using a stainless steel mesh, centrifuge at 2200rpm×10min, discard the supernatant and keep the precipitate; S4.3, Perform Percoll separation: (1) Prepare a separation solution of 40% Percoll and 70% Percoll; (2) Resuspend the precipitate after centrifugation in 3 mL of 40% Percoll, dispense 70% Percoll into 15 mL centrifuge tubes, 3 mL / tube, and centrifuge briefly and quickly until no solution remains on the wall. (3) Use a sterile Pasteur pipette to add the resuspended 40% Percoll dropwise to the upper layer of 70% Percoll to form a clear boundary layer; (4) Perform density gradient centrifugation in a centrifuge at 1-4℃; S4.4 Cell Collection: (1) After 30 minutes, use a sterile Pasteur pipette to slowly aspirate 3-4 mL of the middle milky white mononuclear cell layer and transfer it to a new 15 mL centrifuge tube; (2) Add sterile PBS pre-cooled at 1-4℃, bring the volume to 14mL, and vortex the cells; (3) Centrifuge again at 300g×10min and discard the supernatant to retain the cell pellet. The obtained cell pellet is mainly enriched with lymphocyte populations derived from liver tissue. S4.5, Extraction of mouse bone marrow lymphocytes: (1) Place the mouse on a dissecting tray and use dissecting scissors to cut open the skin to expose the femur; (2) Use pliers to fix the femur, then use another pair of pliers to break the femur at the joint, remove the skin and tissue to expose the bone, and cut off both ends to expose the bone marrow; (3) Using a 5mL syringe plunger with a 1mL syringe pillow, draw up PBS buffer and gently rinse the bone marrow; (4) Centrifuge at 1-4℃, 1400rpm×7min, discard the supernatant and keep the precipitate; (5) Add 2 mL of red blood cell lysis buffer, mix by pipetting, let stand for 6-7 min, then stop with 10 mL of PBS, and then filter through a 0.22 µm steel mesh into a new 15 mL centrifuge tube. (6) Centrifuge again at 1400 rpm for 7 min, and perform subsequent MDSC flow cytometry staining on the resulting cell pellet; S4.6 Extraction of single lymphocytes from the spleen: (1) Pour PBS into a sterile culture dish, place the spleen on the ground surface of a glass slide, rub the upper and lower ground surfaces together until no clumps of particles are visible to the naked eye, and rinse with PBS. (2) Take the mixture and filter it through a 0.22µm steel mesh into a 15mL centrifuge tube. Centrifuge at 1-4℃ and 1300-1400rpm for 7-9min. (3) Discard the supernatant, add 2 mL of red blood cell lysis buffer, pipette mix well and let stand for 5-7 min; (4) Stop the red lysis with 10 mL PBS and filter it again into a new 15 mL centrifuge tube. Repeat centrifugation at 1400 rpm for 7 min. (5) Discard the supernatant to obtain cell precipitate and perform flow cytometry single-staining.

[0013] Preferably, in step five, flow cytometry is used to sort MDSC cells using magnetic beads. (1) Select WT and WT-LA group mice and extract liver and bone marrow lymphocytes; (2) Add 1×10 cells labeled with CD11b-Biotin and Gr-1-Biotin antibodies. 7 cells / 10μL, gently pipette to mix; (3) Place the mixture in a refrigerator at 1-4℃ and incubate in the dark for 15-30 minutes, stirring and mixing once every 4-5 minutes; (4) After incubation, add 10 mL of pre-cooled PBS to terminate the binding, centrifuge at 1400-1500 rpm for 5-6 min, discard the supernatant and keep the precipitate; (5) Add 1×10 7 / 30μL of Anti-Biotin magnetic beads were used to prepare 1mL of magnetic bead suspension. The magnetic beads were resuspended on ice and bound for 18-20min. (6) Place the magnetic sorting rack in the clean bench, slowly add the labeled cell suspension into the sorting column, let it stand for 8-10 minutes, collect the suspension after the MDSC cells bind to the magnetic beads, and detect them by flow cytometry. The proportion of double-positive cells.

[0014] Preferably, the lactic acid treatment method in step six is ​​as follows: S5.1. Select male C57BL / 6N mice aged 6 to 8 weeks as experimental subjects. Weigh lactic acid and dissolve it in high-pressure deionized water to prepare a lactic acid solution with a concentration of 16 mg / mL, and dispense it for use. S5.2. 24 hours in advance, calculate the required lactate injection dose based on the weight of the experimental animals, use a 1mL syringe to draw the corresponding volume of lactate solution, inject it into the peritoneum, and continue to observe the condition of the mice. S5.3 24 hours after anesthesia, ConA was injected into the inner canthus to induce liver injury. 12 hours later, blood and tissue samples were collected to observe the performance of lactated mice in ConA-induced liver injury. ALT and liver lymphocytes were analyzed, and the expression of MDSC-related chemokines was detected by RT-PCR.

[0015] Preferably, in step seven, the in vitro bone marrow cell MDSC differentiation induction experiment is as follows: after in vitro injection of lactate and ConA, the extracted WT and WT-LA mouse bone marrow lymphocyte suspensions are seeded into 24-well cell culture plates, with 1 mL of cell suspension added to each well; RPMI-1640 containing GM-CSF and IL-6 is added and the cells are cultured completely at 37°C and 5% CO2 for 96 h; finally, the cells are collected for surface staining, and the proportion of MDSC cells and the expression level of CXCR2 are detected by flow cytometry to evaluate the ability of MDSCs to differentiate into G-MDSCs and M-MDSCs.

[0016] Compared with the prior art, the present invention provides a method for treating acute liver injury by using lactate-enhanced MDSC immunosuppression, which has the following beneficial effects: 1. This invention demonstrates through in vitro and in vivo experiments in mice that exogenous lactic acid alone does not induce an inflammatory response and can significantly reduce ConA-induced serum transaminase (ALT) levels, while reducing hepatocyte necrosis and the release of inflammatory factors (IFN-γ, TNF-α, IL-12), thereby achieving a safe and efficient protective effect against acute immune liver injury in wild-type mice.

[0017] 2. This invention verifies through flow cytometry and bone marrow MDSC induction differentiation experiments that lactate can significantly increase the proportion of MDSCs (especially the G-MDSC subset) in mouse liver, thereby promoting MDSC proliferation and differentiation into G-MDSCs. At the same time, it upregulates the expression of immunosuppressive molecules Arg-1, TGF-β, iNOS and anti-inflammatory factor IL-10 and inhibits the secretion of IFN-γ by CD4+ T cells, NK cells and NKT cells, ultimately achieving the beneficial effect of precisely regulating the liver immune microenvironment by enhancing the immunosuppressive function of MDSCs.

[0018] 3. This invention, through Western blotting, RT-PCR, and specific inhibitor blocking experiments, reveals that lactate significantly upregulates the expression of chemokine receptor CXCR2 and its ligands CXCL2, CXCL5, and CXCL10 by inhibiting the activation of the hepatic STAT1 / STAT3 signaling pathway, and specifically recruits G-MDSCs to the liver via the "CXCR2-ligand" axis. Simultaneously, it demonstrates that inhibiting CXCR2 reverses the protective effect of lactate (manifested as a decrease in MDSC ratio, an increase in IFN-γ, and a recovery and increase in ALT), achieving the beneficial effect of elucidating the hepatoprotective mechanism mediated by the lactate-MDSC-CXCR2 signaling axis at the molecular level and providing new targets for drug development. Attached Figure Description

[0019] Figure 1 This is a diagram of liver damage in wild-type WT mice induced by lactate resistance to ConA, as described in this invention. Figure 2 The present invention demonstrates that lactic acid can reduce the level of IL-12 in the liver of mice stimulated by ConA and upregulate the expression of Arg-1, TGF-β and iNOS. Figure 3 This diagram serves as verification that lactic acid does not induce an inflammatory response in mice, as per the present invention. Figure 4 This is a graph illustrating how lactic acid can increase the proportion of MDSCs in mouse livers, as described in this invention. Figure 5 This is a graph showing the proportion of immune cells producing IFN-γ in the liver of WT mice under ConA stimulation with lactate reduction, as described in this invention. Figure 6 This is a diagram showing how lactic acid can increase the proportion of MDSCs and the expression of IL-10 in the liver of mice stimulated by ConA. Figure 7 This is a diagram illustrating how lactic acid enhances the differentiation of mouse bone marrow MDSCs into G-MDSCs, as described in this invention. Figure 8 This is a transcriptomics diagram of WT and WT-LA from this invention; Figure 9 This is an analysis diagram of the activation status of STAT1 and STAT3 after lactic acid treatment according to the present invention; Figure 10 This diagram illustrates the upregulation of CXCR2 expression in mouse liver by lactate, as described in this invention. Figure 11 This is a diagram of liver damage in mice caused by the inhibition of CXCR2 to restore lactate protection, as described in this invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Please see Figures 1-11 A method for treating acute liver injury by using lactate-enhanced MDSC immunosuppression includes the following steps: Step 1: Establishment of ConA (concanavalin A)-induced acute liver injury model: Dissolve concanavalin A powder in phosphate buffer, randomly group experimental animals according to the principle of balanced body weight and mark individual numbers, and inject ConA (concanavalin A) (10 mg / kg) into C57 / B6 wild-type mice through the inner canthal vein to establish an acute liver injury model. Step 2: Dynamic determination of ALT (alanine aminotransferase) levels: Blood was collected from the orbital sinus of mice at different time points according to experimental requirements, and the serum ALT level was detected using an ALT kit. Step 3: Cytokine and Liver Injury Detection: Collect mouse blood according to experimental requirements, separate serum, and use ELISA (enzyme-linked immunosorbent assay) to detect the secretion of various cytokines; collect mouse liver tissue, perform HE (hematoxylin-eosin staining) on ​​paraffin sections, and detect liver damage in WT (wild-type) mice and mice supplemented with lactate. Step 4: Extraction of liver lymphocytes from mice: Wild-type mice were harvested, the eyeballs were removed to expel blood, and the liver tissue was taken out and placed in pre-prepared ice-cold PBS (phosphate buffer) for later use. Liver lymphocytes were extracted using the Percoll separation method. Step 5, Flow cytometry: Lymphocytes are isolated from designated organs of mouse liver, spleen and bone marrow, and the cell surface and intracellular cytokine staining is performed. The proportion of these cytokines is then analyzed by flow cytometry. Step 6, Lactic acid content determination: The amount of lactic acid produced in the WT-LA group was determined by lactic acid test kit II, and the WT (wild type) group, which did not receive in vitro lactic acid injection, was used as a blank control. Step 7: Bone marrow MDSC (myeloid-derived suppressor cells) induction differentiation experiment: Mouse bone marrow progenitor cells were isolated and induced to differentiate into MDSCs using GM-CSF (granulocyte-macrophage colony-stimulating factor) and IL-6. The proportion of G-MDSCs and changes in CXCR2 (CXC chemokine receptor type 2) expression were detected by flow cytometry.

[0022] Example The present invention relates to a method for using lactate-enhanced MDSC immunosuppression to treat acute liver injury, and the specific verification process is as follows: I. Experimental Animals and Feeding: The mice used in the experiment were all wild-type (WT) male C57BL / 6N strain mice. All mice were age-matched (6-8 weeks old) SPF-grade C57BL / 6 male mice, weighing between 20-22g. The mice used in this invention were fed conventional feed and high-pressure drinking water, and were kept in an environment with 12 hours of light and 12 hours of darkness. All experimental operations were strictly carried out in accordance with the 3R principle.

[0023] II. Experimental Reagents: The main reagents used in this invention include isopropanol, ionomycin Ion, and protein transport inhibitor. Information on these reagents is shown in Table 1. Table 1. Experimental reagents used in this invention ; Specifically, the ConA injection solution is prepared as follows: Dissolve ConA powder in sterile PBS buffer to prepare a 10 mg / mL ConA solution. Gently pipette to mix thoroughly, then dispense into sterile EP tubes and store at -80°C. When ready for use, remove the tubes and dilute to a concentration of 1 mg / mL. See Table 2 for details. Table 2. Preparation system of ConA injection solution ; Specifically, in step one, the ConA-induced mouse model of acute liver injury is established: S1.1 Open the biosafety cabinet in advance and irradiate it with ultraviolet light for 25-30 minutes to ensure the sterility of the working environment; S1.2 Dissolve ConA in sterile PBS buffer to prepare a 10 mg / mL ConA solution. Gently pipette and mix until fully dissolved. This solution should be prepared immediately. S1.3 Place the prepared ConA solution in a refrigerator or ice bath at 1-4℃ and let it stand for 0.4-0.5 hours. During this period, gently mix it with a pipette every 10-12 minutes to ensure homogeneity. S1.4 After 25-30 minutes, check whether the solution is completely dissolved. If it is confirmed that there are no flocculent substances in the solution, it means that the solution is completely dissolved. At the same time, use inhalation anesthesia to anesthetize the same batch of mice. S1.5 After anesthesia, liver injury was induced by intravenous injection of ConA solution at a dose of 10 mg / kg based on the mouse's body weight. S1.6 After 10-12 hours, the mice were euthanized using the cervical dislocation method, and their livers were quickly removed for further analysis.

[0024] Specifically, the dosage and treatment methods for each group of mice in step one are as follows: (1) WT group (ConA): Dissection was performed 12 hours after ConA (10mg / kg) was injected into the inner canthus; (2) WT-LA group (ConA+LA): 24 hours before the procedure, lactic acid (160mg / kg) was injected intraperitoneally, followed by ConA (10mg / kg) injected into the inner canthus 12 hours later, and then the body was dissected. (3) WT-LA-Inhibitor group (ConA+LA+CXCR2 inhibitor): 24 hours in advance, lactate (160mg / kg) and SB225002 (5mg / kg) were injected intraperitoneally. ConA (10mg / kg) was injected into the inner canthus. SB225002 (5mg / kg) was injected again 2 hours before the injection. Dissection was performed 12 hours later.

[0025] Specifically, in step two: The Reiter method was used to dynamically measure the levels of ALT and AST in serum. S2.1. Take the mice to be tested and use capillary glass tubes to collect blood from the retro-orbital vein at different time points. Collect 0.2-0.3 mL each time and place it in an EP tube (without anticoagulant). The specific time interval is every 4 h or 6 h. Continuously monitor for 72 h to obtain the dynamic changes in ALT levels. S2.2 After blood samples are collected, each group of samples should be immediately marked with time and group. S2.3. Place the whole blood sample in a room temperature environment (25℃) for 1 hour to complete the coagulation process; S2.4. After the blood has been coagulated, the blood sample is basically separated into layers and placed in a constant temperature centrifuge (25℃) for the first centrifugation process. The speed is set to 3800 rpm × 15 min. S2.5. Use a 200µL pipette to transfer the pale yellow supernatant to a new sterile 1.5mL centrifuge tube; S2.6 Under the same temperature control conditions, the transferred supernatant was centrifuged a second time, with the speed set at 3500 rpm and the centrifugation time at 5 min. S2.7 Finally, use a micropipette to aspirate the clear serum; do not aspirate red blood cells. S2.8. After diluting the collected serum samples 5 times with physiological saline, the changes in ALT and AST levels of the two groups in the serum at different time points were detected using ALT and AST kits. Finally, the OD values ​​were detected using an ELISA reader to plot the curve changes. Quantitative RT-PCR: Total RNA was extracted from liver tissue using TRIzol reagent (Invitrogen), followed by reverse transcription using the Quantscript RT kit (Takara). Amplification was performed on an Applied Biosystems 7500 real-time quantitative PCR platform using TB Green Premix Ex Taq II premixed reagent (Takara Bio) for the SYBR Green method. HPRT was used as a control gene, and a 2 ( The method for assessing its relative expression is as follows: 1. RNA extraction from mouse liver: (1) The pipette tips, EP tubes and other materials used in the experiment were autoclaved in advance. After the mice were euthanized, the abdomen was opened and a piece of liver tissue the size of a mung bean was cut off with tweezers and placed into an enzyme-free EP tube. 200 μL of Trizol was added to the tissue and the mixture was ground completely with a mixer until no tissue was visible to the naked eye. Then, 800 μL of Trizol was added to the EP tube. (2) Add 200 μL of chloroform along the wall, shake vigorously up and down to mix evenly, let stand on ice for 10 min, then transfer to a centrifuge at 4℃ and centrifuge at 12000 rpm for 15 min. (3) After centrifugation, carefully aspirate the clear liquid (500 μL) from the top layer into a new enzyme-free EP tube (do not aspirate the white protein in the middle). (4) Add 500 μL of isopropanol to the EP tube, mix gently until there are no filaments, and let stand in an ice bath for 10 min; (5) Transfer to a 4℃ centrifuge and centrifuge at 12000 rpm for 15 min; (6) Remove the EP tube, discard the supernatant and add 1 mL of 75% ethanol (prepared with sterile enzyme-free water). Use a vortex mixer to shake for 5 min at 4℃ and 7000-7500 rpm. (7) Remove the EP tube and use a vacuum pump to remove the supernatant and keep it dry so that the precipitate becomes transparent; (8) Add 10-20 μL of RNase-free water according to the amount of precipitate, mix well by pipetting to dissolve the RNA, and transfer to a 4℃ refrigerator for 30 min-1 h (at this time the RNA concentration is relatively stable). (9) Measure the RNA concentration using the instrument, take 1-2 μL of RNA and judge the RNA purity according to the A260 / 280 ratio (A260 / 280 is best at 1.8~2.0), and store the rest at -80℃ for short-term use; (10) Estimate the required RNA concentration for reverse transcription based on the measured RNA concentration. 1000 / RNA concentration = RNA volume μL (measured concentration μg / mL → ng / μL). 2. Reverse Transcription: Strictly follow the QuantScript RT Kit (Takara Bio) operating guidelines to convert the RNA sample into cDNA via reverse transcription. The specific operating procedure is as follows: (1) Prepare reverse transcription (10 μL system) under ice bath conditions, see Table 3: Table 3. Preparation of reverse transcription system ; Note: The reaction system can be scaled up proportionally according to experimental requirements; (2) The parameters for reverse transcription thermal cycling are shown in Table 4; Table 4 cDNA reverse transcription program ; (3) After the reaction is complete, collect the cDNA product: PCR is performed using the TB Green Premix ExTaq II kit (Takara Bio) based on the SYBR Green method, and the Applied Biosystems 7500 real-time quantitative PCR instrument is used for detection. The experimental procedure is as follows: (1) Prepare a 20 μL amplification mixture under ice bath conditions, as shown in Table 5: Table 5 PCR system preparation ; (2) PCR reaction conditions: The following are the detailed steps of the PCR procedure, including the temperature, duration and number of cycles for each step, as shown in Table 6: Table 6 PCR Procedure ; (3) During the data analysis, the HPRT gene was selected as the internal reference gene. The mRNA expression level of each target gene was represented by the 2-ΔΔCt value, as shown in Table 7: Table 7. PCR Primer Sequence List ; Flow cytometry staining of immune cells in liver and bone marrow 1. Surface antibody labeling and staining: After centrifuging the lymphocytes isolated from in vivo tissues (spleen, liver, spinal cord), wash them once with PBS. Prepare the required surface staining antibody in PBS in advance (antibody ratio of 1:200-1:400) and vortex to mix. Add 100μL of staining solution to each tube to resuspend the cell pellet, mix by pipetting, and incubate the EP tubes horizontally in a 4°C refrigerator in the dark for 20-30 min. G-MDSC is CD11b+Ly6G+Ly6Clow, and M-MDSC is CD11b+Ly6G-Ly6Chigh.

[0026] 2. Intracellular cytokine staining: (1) Add phorbol 12-myristate 13-acetate PMA, ionomycin Ion and BD Golgi-Plug protein transport inhibitor to 1640 medium at a ratio of 1:1000 and vortex mix. Dispense 1 mL / well and mix with cells.

[0027] (2) Stimulate cells in vitro for 4-6 hours in a CO2-free incubator at 37℃; (3) Use the eBioscience Fixation / Permeabilization Kit to fix and permeabilize the membrane. Use Fix / Perm to fix and punch the membrane, 100 μL / well. Mix well by pipetting and place in a 4℃ refrigerator to incubate in the dark for 25 min. (4) Prepare perm / wash according to the reagent instructions: (10mL perm / wash (1×) = 9mL ddH2O + 1mL perm / wash stock solution (10×); (5) After incubation, add 200 μL of 1×perm / wash to stop staining and wash twice with PBS. Centrifuge at 5000 rpm for 5 min and discard the supernatant. (6) Detect the proportions of CD4+ T cells, NK, NKT cells, Treg cells and their subsets, and the secretion of corresponding cytokines such as IFN-γ. (7) Prepare the required intracellular staining working solution in advance, and vortex mix it. Repeat the incubation, centrifugation and washing process with 100 μl / well staining solution. Finally, reselect the precipitate with 300 μl 1×perm / wash and transfer it to a flow cytometer for detection. (8) The data were analyzed by flow cytometer (BD Calibur) and data were analyzed by Flowjo.

[0028] 3. ELISA detection of cytokines (taking IFN-γ as an example): (1) Dilute 5×Coating Buffer with deionized water at a volume ratio of 1:5 to prepare 1× working solution, and add 100 μL of the diluted capture antibody solution to each well of a 96-well plate. Seal the plate with a membrane and incubate overnight at 4°C for 16-18 h. (2) After incubation, the 96-well plate was placed in a plate washer for washing. The washing program was set as follows: 300 μL 1×PBST solution / well, 45 s / time, repeated 4 times. (3) Prepare 1×Assay diluentA solution in advance. After washing the plate, add 200 μL of 1×Assay diluentA solution to each well using a pipette and incubate on a shaker at 140 rpm for 1 h. (4) Prepare diluents for standards and samples: Take 10 μL of 105 pg / mL stock solution of standard, add 990 μL of 1×Assay Diluent A and mix thoroughly to prepare the first standard with a concentration of 1000 pg / mL; then use the 2-fold serial dilution method, take 500 μL of the preceding solution and mix it with 500 μL of the diluent in each tube to obtain standards with concentrations of 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL and 15.625 pg / mL in sequence. In addition, a 0 pg / mL blank control tube is set up containing only 100 μL of 1×Assay Diluent A. (5) After sealing, repeat the above plate washing operation, then add 100 μL of diluted test sample and calibrator to the 96-well plate, and incubate with shaking at 140 rpm for 2 h. (6) After incubation, repeat the plate washing operation, add 100 μL of biotin-labeled detection antibody diluted with 1×Assaydiluent A to the 96-well plate, seal with sealing film, and incubate at room temperature at 140 rpm for 2 h. (7) After incubation, wash the plate, add 100 μL of Lavidin-HRP per well to the 96-well plate, seal with sealing film, and incubate at room temperature for 30 min at 140 rpm. (8) Wash the plate after incubation, wash 5 times, add 100 μL / well of TMB substrate mixture, wrap with tin foil to protect from light for 15 min, and then add 100 μL / well of 2N sulfuric acid solution to terminate the reaction. (9) On-machine detection: use an enzyme-linked immunosorbent assay (ELISA) reader to detect absorbance values ​​at wavelengths of 450 nm and 570 nm; Specifically, in step three, paraffin section preparation: (1) Sample processing: After the experimental animals were sacrificed, the liver tissue was completely removed and fixed in 4% paraformaldehyde for 24 hours; (2) Gradient dehydration: After the sample is removed from the fixative, it is first rinsed with running water for 3-5 minutes, and then subjected to gradient dehydration treatment according to the standard procedure; (3) Paraffin embedding: Place the tissue block impregnated with paraffin into the embedding box, pour in liquid paraffin, and after cooling and solidification, form a regular paraffin block containing the tissue; (4) Serial sectioning: Fix the embedded sample on the microtome, set the section thickness to 4-8 μm, collect the tissue sections using an anti-roll plate, and quickly transfer them to a 42℃ constant temperature water bath for development; (5) Slide attachment: The flattened tissue sections are attached to pre-labeled glass slides and dried in a 60°C slide dryer.

[0029] HE staining procedure for paraffin sections: (1) Pretreatment of glass slides: Place the glass slides in a constant temperature oven at 65℃ for ≥12h to enhance tissue adhesion; (2) Tissue clearing and hydration treatment: Tissue clearing is achieved by thoroughly dissolving paraffin in xylene to make the tissue transparent. Then, hydration treatment is performed by immersing the sections in a gradient of alcohols to gradually absorb water. Finally, the sections are rinsed with distilled water to remove any alcohol residue, completing the transition from an organic environment to an aqueous environment. See Table 8 for details. Table 8. Tissue Transparency and Hydration Steps ; (3) Immerse in hematoxylin stain for 5 minutes, rinse with running deionized water, and then absorb the residual liquid with absorbent paper. (4) Counterstain the cytoplasm with eosin solution for 1-2 min, and stop the staining reaction with distilled water; (5) Gradient dehydration and paraffin infiltration treatment: Immersion in molten paraffin allows the wax to fully penetrate the interstitial spaces, providing uniform and firm support for subsequent sectioning, ensuring section integrity and staining quality. See Table 9 for details. Table 9 Gradient Dehydration and Wax Impregnation Procedure ; (6) Add an appropriate amount of neutral resin to the tissue area, slowly cover with a coverslip to avoid air bubbles, cure at room temperature in the dark for 24 hours, and then seal and store.

[0030] The process of protein immunoblotting: T1. Sample preparation (conducted entirely on ice): (1) The mice were euthanized by dislocation of the neck, and part of the liver tissue was cut and placed into an enzyme-free EP tube. Then, RIPA protein lysis buffer and protease phosphatase inhibitor were added. The ratio of RIPA:protease inhibitor:phosphatase inhibitor was 100:1:1 (the specific ratio should be referred to the kit instructions). (2) Grind the tissue with a tissue grinder until no tissue clumps are visible to the naked eye, vortex thoroughly, and then place on ice for 30 min-1 h to lyse.

[0031] (3) Centrifuge at 12000 rpm for 20 min, transfer the supernatant to a new EP tube, and take 2-3 μL of BCA; T2. Measure protein concentration: (4) Based on the quantitative results, calculate the volume required for loading, add 5×LoadingBuffer at a ratio of 4:1, vortex to mix, and centrifuge briefly; (5) After boiling in a 100℃ metal bath for 10 minutes, immediately place it on ice to cool and precipitate it. Store it in portions at -20℃ and avoid repeated freeze-thaw cycles, otherwise the protein will easily degrade or aggregate. T3, SDS-PAGE electrophoresis: (1) Prepare a suitable separating gel concentration (10%) in advance according to the target protein molecular weight range (10-200kDa), as shown in Table 10: Table 10 Protein Gel Preparation ; (2) Remove the sample and protein marker from -20℃ and let them dissolve at room temperature; (3) Take out the electrophoresis tank, pour in the prepared 1× electrophoresis solution, load the denatured protein sample onto the gel (protein marker 2-3 μL), and perform the electrophoresis at a constant voltage of 90V. After the sample enters the separating gel, adjust the voltage to 130V until the bromophenol blue front reaches the bottom of the separating gel. T4, protein electroporation: (1) Soak SDS-PAGE gel, filter paper and other materials in 1× electroporation solution in advance. PVDF membrane needs to be activated by soaking in methanol for 3-5 seconds in advance. (2) Place the protein gel after electrophoresis on the activated PVDF membrane and assemble it into a "sandwich" structure according to the following order: cathode-sponge-filter paper-gel-membrane-filter paper-sponge-anode. Gently scrape off the air bubbles and then place it in the electrophoresis tank. (3) Place an ice box inside the electrostatic precipitator and fix the electrostatic precipitator on ice to assist in cooling. It is usually carried out under constant current conditions (300mA 1.5h). Blocking: After electroporation, remove the membrane with tweezers, wash with TBST, and then block in BSA blocking solution at room temperature on a shaker for 1-2 hours to reduce nonspecific binding; The incubation process for T5 primary and secondary antibodies is as follows: (1) After the membrane is blocked, prepare a primary antibody (specific antibody) and incubation solution according to the required dilution based on the protein content, and incubate overnight at 4°C; (2) Remove the membrane the next day and wash it with TBST buffer for 10 min × 3 times (60-70 r) to remove unbound antibodies; (3) Place the membrane in the incubation solution of secondary antibody (HRP-labeled rabbit anti-mouse 1:1000) and incubate at room temperature for 1.5 h; (4) Wash the membrane with TBST buffer for 10 min × 3 times (60-70 r); T6. Detection and Quantification: For HRP-labeled secondary antibodies, chemiluminescence detection was performed using ECL reagent (1:1). The data was exposed on X-ray film and the original data was saved. The bands were quantified using ImageJ image analysis software.

[0032] Specifically, in step four, mouse lymphocyte extraction: S4.1, Extraction of liver lymphocytes: (1) The eyes of WT mice were removed and blood was expelled to ensure the validity of the samples and experimental ethics; (2) The extracted liver tissue samples were transferred to sterile culture dishes containing pre-cooled sterile phosphate buffer (sterile PBS, pH 7.4) for temporary storage to maintain tissue viability; (3) In a sterile culture dish, assemble a 200-mesh stainless steel cell sieve and spread the liver tissue evenly on the surface of the sieve; (4) Use the piston end of a 5mL syringe to perform mechanical homogenization, apply vertical pressure and grind repeatedly until the tissue is completely dissociated, ensuring that there are no visible tissue blocks remaining; (5) During homogenization, pre-cooled PBS was continuously added to maintain tissue hydration and protect cell membrane integrity; S4.2, Cell suspension treatment: (1) Gently rinse the cell sieve with pre-cooled PBS (phosphate buffer) to remove residual tissue debris, ensuring a slow and uniform flow rate during rinsing to avoid cell loss; (2) Use a pipette to transfer the homogenized mixture to a 50mL centrifuge tube and centrifuge at 1-4℃, 500rpm×1min. Obvious precipitation will be visible. (3) Filter the supernatant into a new 50mL centrifuge tube using a stainless steel mesh, centrifuge at 2200rpm×10min, discard the supernatant and keep the precipitate; S4.3, Perform Percoll separation: (1) Prepare a separation solution of 40% Percoll and 70% Percoll; (2) Resuspend the precipitate after centrifugation in 3 mL of 40% Percoll, dispense 70% Percoll into 15 mL centrifuge tubes, 3 mL / tube, and centrifuge briefly and quickly until no solution remains on the wall. (3) Using a sterile Pasteur pipette, add the resuspended 40% Percoll dropwise to the upper layer of 70% Percoll to form a clear boundary layer; (4) Perform density gradient centrifugation in a centrifuge at 1-4℃, under the conditions of 1260g (increase 6, decrease 2) for 30min; S4.4, Cell Collection: (1) After 30 minutes, use a sterile Pasteur pipette to slowly aspirate the middle milky white mononuclear cell layer (3-4 mL) and transfer it to a new 15 mL centrifuge tube; (2) Add sterile PBS pre-cooled at 4°C, bring the volume to 14 mL, and vortex the cells; (3) Centrifuge again at 300g (corresponding to 2200rpm for an angular rotor) for 10min. After centrifugation, discard the supernatant and retain the cell pellet. The obtained cell pellet mainly enriches the lymphocyte population derived from liver tissue and is used for flow cytometry staining; S4.5, Extraction of mouse bone marrow lymphocytes: (1) Place the mouse on a dissecting tray and use dissecting scissors to cut open the skin to expose the femur; (2) Use pliers to fix the femur, then use another pair of pliers to break the femur at the joint, remove the skin and tissue to expose the bone, and cut off both ends to expose the bone marrow; (3) Using a 5mL syringe plunger with a 1mL syringe pillow, draw up PBS buffer and gently rinse the bone marrow; (4) Centrifuge at 4℃, 1400rpm×7min, discard the supernatant and keep the precipitate, a large number of red blood cells can be seen precipitated; (5) Add 2 mL of red blood cell lysis buffer, mix by pipetting, let stand for 7 min, then stop with 10 mL of PBS (at least 2 times the volume of PBS), and then filter through a 0.22 µm steel mesh into a new 15 mL centrifuge tube. (6) Centrifuge again at 1400 rpm for 7 min, and perform subsequent MDSC flow cytometry staining on the resulting cell pellet; S5.6 Extraction of single lymphocytes from the spleen (for single-staining tube adjustment and compensation). (1) Pour PBS into a sterile culture dish, place the spleen on the ground surface of a glass slide, rub the upper and lower ground surfaces together until no clumps of particles are visible to the naked eye, and rinse with PBS. (2) Take the mixture and filter it into a 15mL centrifuge tube using a 0.22µm steel mesh. Incubate at 4℃, 1400rpm for 7min. (3) Discard the supernatant to see a large amount of red blood cell precipitate. Add 2 mL of red blood cell lysis buffer, pipette to mix well and let stand for 5 min. (4) Stop the red lysis with 10 mL PBS and filter it again into a new 15 mL centrifuge tube. Repeat centrifugation at 1400 rpm for 7 min. (5) Discard the supernatant to obtain cell pellet, and perform flow cytometry single-staining (FITC, PE, Percp, APC).

[0033] Specifically, in step five, flow cytometry is used to sort MDSC cells using magnetic beads: (1) Select WT and WT-LA group mice and extract liver and bone marrow lymphocytes; (2) Add CD11b-Biotin and Gr-1-Biotin antibody-labeled cells (1×10⁻⁶) 7 (cells / 10μL), gently pipette to mix; (3) Place the mixture in a refrigerator at 4°C and incubate in the dark for 15-30 minutes, stirring and mixing once every 5 minutes; (4) After incubation, add 10 mL of pre-cooled PBS to terminate the binding, centrifuge at 1500 rpm for 5 min, discard the supernatant and keep the precipitate; (5) Add Anti-Biotin magnetic beads (1×10) 7 / 30μL), to prepare 1mL of magnetic bead suspension (1×10 7 Cells were bound with 30 μL magnetic beads + 970 μL buffer, and the magnetic beads were resuspended on ice for 20 min. (6) Place the magnetic sorting rack in the clean bench, slowly add the labeled cell suspension into the sorting column, and let it stand for 10 minutes. At this time, MDSC cells bind to the magnetic beads. Collect the suspension and detect it by flow cytometry. The proportion of double-positive cells.

[0034] Specifically, the lactic acid treatment method in step six: S5.1. Select male C57BL / 6N mice aged 6 to 8 weeks as experimental subjects. Weigh lactic acid and dissolve it in high-pressure deionized water to prepare a lactic acid solution with a concentration of 16 mg / mL, and dispense it for use. S5.2. 24 hours in advance, calculate the required lactate injection dose based on the weight of the experimental animals to ensure that the injection dose for each mouse reaches 160mg / kg. Use a 1mL syringe to draw the corresponding volume of lactate solution and inject it into the peritoneum. Continue to observe the condition of the mice. S5.3 24 hours after anesthesia, ConA was injected into the inner canthus to induce liver injury. 12 hours later, blood and tissue samples were collected to observe the performance of lactated mice in ConA-induced liver injury. ALT and liver lymphocytes were analyzed, and the expression of MDSC-related chemokines was detected by RT-PCR.

[0035] Specifically, mouse injection methods can be categorized as follows: 1. Intraperitoneal injection method for mice: (1) Wear lab coats, gloves and masks, prepare the required drug concentration in advance, weigh the mice and record the results, and administer the drugs according to their body weight; (2) Grab the mouse by the tail, take it out of the cage, place it on a clean experimental table, and use the "spine pinching method" to press the mouse's back to make its abdomen bulge and fix its body. (3) Locate the injection point, observe the mouse's abdomen, and find the area located on both sides of the lower abdomen; (4) Hold the mouse in place with one hand and hold the 1mL syringe in the other hand. Gently insert the needle into the mouse’s skin at a 45-degree angle and continue to push the needle into the abdominal cavity. Slowly inject the drug or solution and observe the mouse’s reaction. (5) After the injection is completed, gently pull out the needle, put the mouse back in the cage, and give it appropriate observation and care.

[0036] 2. Intracanthal injection method in mice: (1) Preparation: Wear lab coats, gloves and masks to ensure safety, and prepare inhaled anesthetic (30% isoflurane) in advance, according to the required drug concentration; (2) Grab the mouse by the tail, take it out of the cage, put it into a glass beaker filled with anesthetic gas, and tightly cover it. Observe the mouse's reaction carefully. When the breathing slows down and the breathing rate is about 1-3 seconds / time, take it out of the beaker and place it on the experimental table. (3) Use your left hand to hold the mouse’s head still and stabilize it. Press down on the mouse’s eyelids so that the inner canthus of the mouse’s eyeballs is exposed and protrudes towards the operator. (4) Hold a 1mL syringe in your right hand and gently insert it into the inner canthus vein. After confirming that the needle has entered the vein correctly, slowly push the injection drug smoothly and without any abnormal obstruction (avoid the needle slipping out or damaging the blood vessel). (5) After injection, remove the needle and apply pressure to the injection site with an alcohol swab to prevent bleeding; (6) Put the mouse back into the cage and observe its condition.

[0037] Lactic acid content analysis: When determining lactate content using the Solarbio lactate dehydrogenase (LDH) activity assay kit, the sample should first be centrifuged and the supernatant collected. Then, the sample, buffer, coenzyme (NAD⁺), and chromogenic agent (2,4-dinitrophenylhydrazine) should be added sequentially to the reaction system. LDH catalyzes the oxidation of lactate to pyruvate. Pyruvate reacts with the chromogenic agent under alkaline conditions to form a brownish-red pyruvate-dinitrophenylhydrazone. After incubation in a 37°C water bath for 15 min, NaOH is added to terminate the reaction. The absorbance is measured at a wavelength of 450 nm. Finally, the amount of pyruvate generated is calculated based on the standard curve plotted using sodium pyruvate standard. Combined with the reaction time, sample volume, and dilution factor, the lactate content in the sample is calculated.

[0038] Specifically, in step seven, the in vitro bone marrow cell MDSC differentiation induction experiment was performed as follows: After in vitro injection of lactate and ConA, bone marrow lymphocytes from WT and WT-LA mice were extracted according to the above method. The cell suspension was seeded into 24-well cell culture plates, with 1 mL of cell suspension added to each well (1.5 × 10⁶ cells / well). RPMI-1640 complete medium (containing 10% FBS) containing GM-CSF (20 ng / mL) and IL-6 (5 ng / mL) was added and cultured at 37°C and 5% CO₂ for 96 h. Finally, the cells were collected and surface stained (Ly-6G-FITC, CD11b-Percp, Gr-1-APC, ly-6C PE). Flow cytometry was used to detect the proportion of MDSC cells and the expression level of CXCR2 to evaluate the ability of MDSCs to differentiate into G-MDSCs (Ly-6G+) and M-MDSCs (Ly-6C+).

[0039] CFSE proliferation experiment: (1) Dilute the sorted MDSC cells with CFSE at a ratio of 1:1000 (final concentration 5 μM) and incubate at 37°C in the dark for 10 min; (2) Add 5 times the volume of 1640 complete culture medium and stop the reaction on ice for 5 min; (3) Wash cells with PBS at 1000 rpm for 5 min each time, repeat twice; (4) Transfer to a 96-well plate and incubate at 37°C and 5% CO2 for 3-5 days; (5) After the culture is completed, wash twice with PBS and centrifuge to remove the supernatant; (6) Transfer the cells from the 96-well plate to flow cytometry tubes for analysis; 17. RNA-Seq Analysis: Total RNA was isolated using the TRIzol method (Thermo Fisher Scientific). RNA integrity was assessed using the Agilent 2100 Bioanalyzer system (Agilent Technologies). Samples with an RNA integrity index (RIN) ≥ 7.0 were selected for library construction. mRNA enrichment and fragmentation were performed according to the Illumina TruSeq chain-specific mRNA library construction kit (San Diego, CA) manual. High-throughput sequencing was performed by OE Biotech Co, Ltd. on the Illumina NovaSeq 6000 sequencing platform, using a paired-end 150bp sequencing mode to acquire raw data. Bioinformatics analysis was performed using the DESeq2 analysis tool to screen for differentially expressed genes. The significance threshold was set at p < 0.05 and an expression fold change > 2-fold. The raw sequencing data were uploaded to the NCBI SRA database.

[0040] The following analysis results were obtained from the above examples: (1) Lactic acid can protect WT mice from ConA-induced liver injury: The same batch of male mice were selected, and WT mice were injected intraperitoneally with 160 mg / kg of lactic acid 24 h in advance, and then injected intravenously with ConA (10 mg / kg) to induce acute liver injury. Blood was collected at different time points to detect the dynamic changes in serum ALT levels. The results showed that the serum ALT level of WT mice supplemented with lactic acid was significantly lower than that of the control group. Figure 1 In Part A of the study, paraffin section HE staining was used to detect cell necrosis in mouse liver tissue, and serum IFN-γ, TNF-α, and IL-12 expression levels were detected using an ELISA kit. The results showed that, compared to the WT control group, almost no inflammatory factor infiltration or hepatocyte necrosis was observed in the livers of WT mice supplemented with lactate. Figure 1 (Part B) Furthermore, it was found that the serum levels of IL-12, IFN-γ, and TNF-α in the lactate group mice were significantly lower than those in the WT group mice. Figure 1 In the C-1E section, to rule out the possibility that lactate alleviated liver damage in mice due to its intrinsic resistance to apoptosis independent of inflammation, anti-Fas antibodies were injected intraperitoneally into both WT and lactate-stimulated mice, and the apoptotic response of hepatocytes was then examined. Figure 1 As shown in section F, after treatment with anti-Fas antibody, the serum ALT levels of WT and lactate mice were similar. Therefore, the hepatocytes of lactate and WT mice showed similar responsiveness to Fas-mediated apoptosis.

[0041] (2) Lactic acid can reduce the level of IL-12 in the liver of mice under ConA stimulation and upregulate the expression of Arg-1, TGF-β and iNOS: The expression levels of liver inflammatory factors in mice 2 h after ConA stimulation were detected by RT-PCR. The results showed that compared with the WT group mice, the mRNA level of IL-12 in the liver of the lactate group mice was significantly reduced ( Figure 2 As shown in Part A), the expression of FasL and Fas did not change significantly. Figure 2 (See section B-2C). The results above are consistent with the previous results that lactate can reduce serum IL-12 levels and has no significant effect on Fas antibody response. In addition, it was found that the expression of Arg-1, TGF-β and iNOS in the liver of mice in the lactate group was significantly upregulated (see section 2D-2F in the figure).

[0042] (3) Lactic acid does not induce inflammatory response in mice: Given the previous results showing that lactic acid can protect WT mice from ConA-induced liver damage and reduce the level of inflammatory factors in mouse serum, further verification was conducted to determine whether lactic acid itself would affect mice. Mice were injected with lactic acid 24 hours in advance, and the levels of lactic acid in the mice and the levels of ALT, AST, IL-6, and TNF-α in the serum were analyzed using a kit. The results showed that compared with the normal WT group mice, the levels of lactic acid in the serum and liver of the lactic acid group mice were significantly increased, but the lactic acid levels were within the normal physiological range. Figure 3 (As shown in section A-3B), it was also found that the serum levels of ALT, AST, IL-6, and TNF-α in the lactate group mice were not significantly different from those in the normal WT group mice. Figure 3 As shown in C-3F, the above results indicate that lactic acid itself has no effect on the mouse body.

[0043] (4) Lactic acid can increase the proportion of MDSCs in mouse liver: To further verify whether lactic acid itself has an effect on immune cells in mouse liver, flow cytometry was used to detect changes in immune cells in the liver of mice after lactic acid stimulation. The analysis results showed that the number of CD4+ T cells and NKT cells in the liver of lactic acid group mice was not significantly different from that of WT mice. Figure 4 (Part A-4D), and the proportion of MDSCs in the liver of lactate-treated mice was significantly higher than that in WT-treated mice, verifying that lactate affects immunosuppressive cells in mouse liver ( Figure 4 (As shown in section E-4F).

[0044] (5) Lactic acid can reduce the proportion of IFN-γ produced by immune cells in the liver of WT mice under ConA stimulation: To verify the effect of lactic acid on immune cells in the liver of WT mice under ConA stimulation, WT mice were injected intraperitoneally with 160 mg / kg lactic acid in advance, and then injected with 10 mg / kg ConA 24 h later. The changes in the number of immune cells in the liver of mice and the production of IFN-γ were detected by flow cytometry 2 h after ConA stimulation. The results showed that the proportions of NK cells, NKT cells and CD4+ T cells in the liver of lactic acid group mice did not change significantly compared with those of WT group mice after ConA stimulation. Figure 5(As shown in sections A-5E); Based on the previous analysis, it was found that the serum IFN-γ level in lactate-treated mice was significantly downregulated, and that CD4+ T lymphocytes, NK cells, and NKT cells played a core effector function in the ConA-mediated liver injury model. Therefore, flow cytometry was used to analyze the IFN-γ production of NK cells, NKT cells, and CD4+ T cells. The results showed that, compared with WT mice, the proportions of IFN-γ secreted by CD4+ T cells, NK cells, and NKT cells in the liver of lactate-treated mice were significantly reduced. Figure 5 (As shown in the F-5K section).

[0045] (6) Lactic acid can increase the proportion of MDSCs and the expression of IL-10 in the liver of mice stimulated by ConA: Since lactic acid causes a decrease in the proportion of IFN-γ secreted by CD4+ T cells, NK cells and NKT cells in the liver of mice, it is speculated that this phenomenon is caused by an increase in immunosuppressive cells in the liver. To verify this hypothesis, WT mice were first injected intraperitoneally with 160 mg / kg lactic acid, and then injected with 10 mg / kg ConA 24 h later. The level of MDSCs in the liver of mice 2 h after ConA stimulation was detected by flow cytometry. The results showed that the proportion of MDSCs in the liver of mice in the lactic acid group was significantly increased compared with that in the WT group. Figure 6 (As shown in section A-6B); further analysis of changes in Treg cells in the liver revealed that, compared to WT mice, the proportion of Treg cells in the liver of lactate mice did not change significantly ( Figure 6 (As shown in section C-6D of Figure 6), it was also found that the proportion of CD4+ T cells producing IL-10 in the liver of mice in the lactate group was significantly increased (as shown in section E-6F of Figure 6). The above results indicate that lactate can cause acute liver injury induced by ConA in mice due to the increase of MDSCs and immunosuppressive factors in the liver.

[0046] (7) Lactic acid can promote the proliferation and differentiation of mouse MDSCs into G-MDSCs: To verify the effect of lactic acid on the differentiation and proliferation of mouse MDSCs, the liver MDSCs of WT mice and mice treated with lactic acid were analyzed after ConA stimulation. The results showed that, compared with WT mice, the proportion of G-MDSCs in the liver and bone marrow MDSCs of lactic acid-treated mice was significantly increased, while the proportion of M-MDSCs was decreased. Figure 7 As shown in Figure A-7F), the effect of lactate on the differentiation of mouse bone marrow MDSCs in vitro was also examined. The results showed that the ability of lactate-treated bone marrow MDSCs to differentiate into G-MDSCs was significantly higher than that of the WT control group. Figure 7 As shown in section G-7I), the effect of lactate on MDSC proliferation was also examined. The results showed that, compared with the WT control group, the proliferation of MDSCs in the lactate-treated group was significantly increased. Figure 7 (As shown in the J-7K section).

[0047] (8) Lactic acid affects the expression of immune cell-related functions in mouse liver: Transcriptomic analysis revealed that, compared with WT mice, 369 genes were highly expressed and 343 genes were lowly expressed in the liver of lactate-treated mice. Figure 8 Gene set enrichment analysis (GSEA) of A-8B showed that the most significantly enriched KEGG pathways included the cell cycle and NF-κB signaling pathways. Figure 8 As shown in Part C), there was a significant difference in the enrichment of immune cell-related gene sets (such as NK cell lectin-like receptor binding-related gene sets) between the two groups. Figure 8 (D-8F).

[0048] (9) Lactic acid reduces the activation of STAT1 and STAT3 signaling pathways in mouse liver under ConA stimulation: The activation of STATs can regulate and control the development and progression of liver disease. Therefore, liver tissues of WT mice and lactated mice before and after ConA stimulation were isolated, liver tissue proteins were extracted, and the phosphorylation levels of STAT1 and STAT3 were detected by Western Blot. The results showed that there was no significant change in the phosphorylation levels of STAT1 and STAT3 in the livers of the two groups of mice without ConA stimulation. Figure 9 A), however, when stimulated with ConA, the phosphorylation levels of STAT1 and STAT3 in the liver of the lactate-treated group were significantly lower than those in the control group ( Figure 9 (B) indicates that the attenuation of the IFN-γ signaling pathway in lactate-treated mice after ConA treatment was closely related to STAT1 and STAT3. For details on the activation of STAT1 and STAT3 after lactate treatment, please refer to [link to relevant documentation]. Figure 9 , Figure 9 In this study, mice were divided into four groups: a lactate-only treatment group, a ConA-only treatment group, a combined lactate and ConA treatment group, and a control group receiving no treatment. After the corresponding treatments, Western blotting was used to detect and analyze the expression levels of target genes in the four groups of mice. Figure 9 (AB) is a band diagram showing the phosphorylation levels of STAT1 and STAT3 after lactate treatment.

[0049] (10) Lactic acid upregulates CXCR2 expression in mouse liver: Previous experimental results showed that lactic acid significantly increased the proportion of MDSCs in mouse liver regardless of whether the mice received ConA stimulation. To investigate the mechanism by which lactic acid promotes MDSC migration to the liver, the expression levels of chemokine receptors and their corresponding ligands in mouse liver before and after ConA stimulation were detected by RT-PCR. The results showed that compared with the WT control group, the expression levels of CXCR4, CXCR2 and their ligands CXCL2, CXCL5 and CXCL10 in the liver of mice treated with lactic acid were significantly upregulated ( Figure 10 (A-10F) To further verify the changes in the expression of CXCR2 and CXCR4 in MDSCs before and after lactate treatment, flow cytometry was used to detect the expression of CXCR2 and CXCR4 in mouse liver MDSCs after ConA stimulation. The results showed that, compared with the WT group, there was no significant difference in the expression of CXCR4 in the liver MDSCs of mice in the lactate-treated group. Figure 10 G-10H), while CXCR2 expression was significantly increased ( Figure 10 (I-10J) The above results suggest that lactate may effectively resist ConA-induced liver injury by specifically recruiting MDSCs via CXCR2 through chemokines. For information on lactate upregulating CXCR2 expression in mouse liver, please refer to [link to relevant documentation]. Figure 10 , Figure 10 In this study, RNA was extracted from the liver tissues of two groups of mice. RT-PCR was used to detect the expression levels of chemokines in the lactate group and the control group after ConA stimulation. Flow cytometry analysis was then used to further verify changes in gene content. Figure 10 The middle (AF) section represents the expression levels of CXCR4, CXCR2 and their ligands CXCL2, CXCL5 and CXCL10 in the WT and WT-LA groups after lactate treatment for ConA-induced liver injury. Figure 10 (HJ) represents the expression of CXCR2 and CXCR4 in mouse liver MDSCs after ConA stimulation; Figure 10 The value of GI indicates the expression levels of CXCR2 and CXCR4 in MDSC cells in the liver as detected by flow cytometry.

[0050] (11) Inhibition of CXCR2 can restore liver injury in lactate-protected mice: To verify the role of CXCR2 in liver injury in lactate-protected mice, mice were injected with the CXCR2-specific inhibitor SB225002, followed by lactate and ConA. The levels of MDSCs in the liver and bone marrow of mice after CXCR2 signaling inhibition were detected by flow cytometry. The results showed that after CXCR2 inhibition, the proportion of MDSCs in the liver and bone marrow of mice in the lactate-treated group was also significantly reduced. Figure 11(A-11D), and simultaneously, after CXCR2 inhibition, the serum ALT level in mice significantly recovered, indicating that SB225002 can reverse the protective effect of lactate and restore ConA-induced liver damage in mice. Figure 11 (E) Furthermore, by examining the changes in hepatic immune cells in mice after CXCR2 signaling inhibition, the results showed that after CXCR2 inhibition using SB225002, the proportion of CD4+ T cells and NK cells secreting IFN-γ in the liver of lactate-treated mice significantly increased (Figures 11F-11I). These results indicate that CXCR2 signaling plays a crucial role in lactate recruitment of MDSCs to the liver. CXCR2-mediated MDSC infiltration directly leads to IFN-γ inhibition and reduced liver damage. For further information on how CXCR2 inhibition can restore lactate-protected liver damage in mice, please refer to [reference needed]. Figure 11 , Figure 11 Specifically, after intraperitoneal injection of SB225002 to inhibit CXCR2 signaling, lymphocytes were extracted from mouse liver and bone marrow tissues, and flow cytometry staining was used to analyze changes in MDSC count, immune cell activation function, and ALT levels. Figure 11 The middle (AD) indicates the changes in the number of MDSC cells in the liver and bone marrow of mice; Figure 11 Part (E) shows the change in ALT levels 2 hours after ConA1 injection following lactate treatment; Figure 11 The secretion of IFN-γ by CD4+ T cells and NKT cells in the liver of mice (FI).

[0051] This invention utilizes a novel mechanism to exert hepatoprotective effects by specifically recruiting granulocyte-like myeloid-derived suppressor cells (G-MDSCs) through the chemokine-receptor axis. Pre-treatment with lactate significantly reduces liver damage, particularly by decreasing hepatocyte necrosis and inflammatory factor infiltration. This is achieved through remodeling the liver's immune microenvironment, specifically manifested in anti-inflammatory and repair mechanisms. Phase 1: Lactic acid provides anti-inflammatory defense, targeting CD4+ T cells and NK cells to significantly inhibit the production of the key secretory factor IFN-γ, while inhibiting the signal transduction of the upstream pro-inflammatory factor IL-12 and promoting the significant upregulation of the anti-inflammatory factor IL-10, thereby rapidly establishing an immune buffer zone mainly based on anti-inflammation to prevent excessive liver damage. The second stage: Lactic acid promotes tissue repair and enhances the expression of Arg-1, TGF-β and iNOS. These are not only core cytokines for tissue regeneration and repair, but also key effector molecules for immunosuppression. Therefore, it was verified that lactic acid can reshape the liver's immune cytokine network, shifting it from a pro-inflammatory state to an anti-inflammatory state. It is speculated that lactic acid achieves its protective effect against liver damage by regulating the metabolic and functional state of MDSCs.

[0052] In summary, this invention reveals the mechanism of action of lactate in ConA-induced liver injury in mice, and verifies that its metabolite lactate can protect WT mice from ConA-induced liver injury, increase the proportion of MDSCs and the expression level of CXCR2 in the mouse liver, thereby inhibiting CXCR2 expression can aggravate liver injury and reduce the proportion of MDSCs in the liver, thus regulating liver injury in mice, providing new ideas and new targets for drug development, clinical diagnosis and treatment.

[0053] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for treating acute liver injury using lactate-enhanced MDSC immunosuppression, characterized in that, Includes the following steps: Step 1: Establishment of ConA-induced acute liver injury model: Dissolve concanavalin A powder in phosphate buffer, randomly group experimental animals according to the principle of balanced body weight and mark individual numbers, and inject ConA into C57 / B6 wild-type mice through the inner canthal vein to establish an acute liver injury model. Step 2, Dynamic ALT Level Measurement: Blood was collected from the orbital region of mice at different time points according to experimental requirements, and the serum ALT level was detected using an ALT kit. Step 3: Cytokine and Liver Injury Detection: Collect mouse blood according to experimental requirements, separate serum, and use ELISA to detect the secretion of various cytokines; collect mouse liver tissue, perform HE staining on paraffin sections, and detect liver damage in wild-type mice and mice supplemented with lactate. Step 4: Extraction of liver lymphocytes from mice: Wild-type mice were harvested, the eyeballs were removed to expel blood, and the liver tissue was taken out and placed in pre-prepared ice-cold PBS for later use. Liver lymphocytes were extracted using the Percoll separation method. Step 5, Flow cytometry: Lymphocytes are isolated from designated organs of mouse liver, spleen and bone marrow, and the cell surface and intracellular cytokine staining is performed. The proportion of these cytokines is then analyzed by flow cytometry. Step 6, Lactic acid content determination: The amount of lactic acid produced in the WT-LA group was determined by lactic acid detection kit II, and the wild-type mouse group without in vitro lactic acid injection was used as a blank control. Step 7: Bone marrow MDSC induction differentiation experiment: Mouse bone marrow progenitor cells were isolated and induced to differentiate into MDSCs using GM-CSF and IL-6. The proportion of G-MDSCs and changes in CXCR2 expression were detected by flow cytometry.

2. The method for treating acute liver injury using lactate-enhanced MDSC immunosuppression according to claim 1, characterized in that: The establishment of the ConA-induced acute liver injury model in mice in step one: S1.

1. Open the biosafety cabinet in advance and irradiate it with ultraviolet light for 25-30 minutes; S1.2 Dissolve ConA in sterile PBS buffer to prepare a ConA solution with a concentration of 10 mg / mL. Mix well by pipetting to ensure complete dissolution. S1.3 Place the prepared ConA solution in a refrigerator or ice bath at 1-4℃ and let it stand for 0.4-0.5 hours. During this period, mix it with a pipette every 10-12 minutes. S1.4 After 25-30 minutes, once the solution is confirmed to be completely dissolved, the same batch of mice are anesthetized using inhalation anesthesia at the same time. S1.5 After anesthesia, liver injury was induced by intravenous injection of ConA solution at a dose of 10 mg / kg based on the mouse's body weight. S1.6 After 10-12 hours, the mice were euthanized using the cervical dislocation method, and their livers were removed for further analysis.

3. The method for treating acute liver injury using lactate-enhanced MDSC immunosuppression according to claim 1, characterized in that: The experimental animals in step one – dosage and treatment methods for each group of mice: (1) WT group: ConA with a content of 10 mg / kg was injected into the inner canthus, and the body was dissected 12 hours later; (2) WT-LA group: 24 hours in advance, lactate with a content of 160 mg / kg was injected intraperitoneally, followed by ConA with a content of 10 mg / kg at the inner canthus, and the body was dissected 12 hours later; (3) WT-LA-Inhibitor group: 24 hours in advance, lactic acid with a content of 160 mg / kg and SB225002 with a content of 5 mg / kg were injected intraperitoneally; 2 hours in advance, ConA with a content of 10 mg / kg was injected into the inner canthus, followed by another intraperitoneal injection of SB225002 with a content of 5 mg / kg; and the body was dissected 12 hours after the ConA injection.

4. The method for treating acute liver injury using lactate-enhanced MDSC immunosuppression according to claim 1, characterized in that: The ALT dynamic level measurement process in step two: S2.

1. Take the mice to be tested and use capillary glass tubes to collect blood from the retro-orbital vein at different time points. Collect 0.2-0.3 mL each time and place it in an EP tube. The specific time interval is every 4 h or 6 h. Continuously monitor for 72 h to obtain the dynamic changes in ALT levels. S2.2 After blood samples are collected, each group of samples is marked with time and group. S2.

3. Place the whole blood sample in a room temperature environment for 1 hour to allow for coagulation. S2.

4. After the blood has been coagulated, the blood sample is basically separated into layers and placed in a constant temperature centrifuge for the first centrifugation process. The speed is set to 3500-3800 rpm × 15-16 min. S2.

5. Use a 200µL pipette to transfer the pale yellow supernatant to a new sterile 1.5mL centrifuge tube; S2.6 Under the same temperature control conditions, the transferred supernatant is centrifuged a second time, with the speed set at 3000-3500 rpm and the centrifugation time at 5-6 min. S2.7 Finally, use a micropipette to aspirate the clear serum; do not aspirate red blood cells. S2.

8. After diluting the collected serum samples 5 times with physiological saline, the changes in ALT and AST levels of the two groups in the serum at different time points were detected using ALT and AST kits. Finally, the OD values ​​were detected using an ELISA reader to plot the changes in the curve.

5. The method for treating acute liver injury using lactate-enhanced MDSC immunosuppression according to claim 1, characterized in that: In step three, mouse liver RNA extraction is performed as follows: (1) After the mice were euthanized in step one, the abdomen was opened, and a piece of liver tissue the size of a mung bean was cut off with tweezers and placed into an enzyme-free EP tube. 200 μL of Trizol was added to the tissue and the mixture was ground completely with a mixer until no tissue was visible to the naked eye. Then, 800 μL of Trizol was added to the EP tube. (2) Add 200 μL of chloroform along the wall, shake vigorously up and down to mix evenly, let stand on ice for 8-10 min, then transfer to a centrifuge at 1-4℃ and centrifuge at 12000 rpm for 15 min; (3) After centrifugation, carefully aspirate 500 μL of the clear supernatant and transfer it into a new enzyme-free EP tube; (4) Add 500 μL of isopropanol to the EP tube, mix until there are no filaments, and let stand in an ice bath for 8-10 minutes; (5) Transfer to a centrifuge at 1-4℃ and centrifuge at 10000-12000rpm for 15-16min; (6) Remove the EP tube, discard the supernatant and add 1 mL of 75% ethanol. Use a vortex mixer to shake for 4-5 min at 1-4℃ and 7000-7500 rpm. (7) Remove the EP tube and use a vacuum pump to remove the supernatant; (8) Add 10-20 μL of RNase-free water according to the amount of precipitate, mix well by pipetting to dissolve RNA, and transfer to a refrigerator at 1-4℃ for 30 min-1 h; (9) Measure the RNA concentration using the instrument, take 1-2 μL of RNA and judge the RNA purity according to the A260 / 280 ratio, and store the rest at -80℃ for short-term use; (10) Estimate the required RNA concentration for reverse transcription based on the measured RNA concentration.

6. The method for treating acute liver injury using lactate-enhanced MDSC immunosuppression according to claim 1, characterized in that: The preparation and staining of paraffin sections in step three: S3.1 Preparation of paraffin sections: (1) Sample processing: After the experimental animals were sacrificed, the liver tissue was completely removed and fixed in 4% paraformaldehyde for 24 hours; (2) Gradient dehydration: After the sample is removed from the fixative, it is first rinsed with running water for 3-5 minutes, and then subjected to gradient dehydration treatment according to the standard procedure; (3) Paraffin embedding: Place the tissue block impregnated with paraffin into the embedding box, pour in liquid paraffin, and after cooling and solidification, form a regular paraffin block containing the tissue; (4) Serial sectioning: Fix the embedded sample on the microtome, set the section thickness to 4-8 μm, collect the tissue sections using an anti-roll plate, and quickly transfer them to a constant temperature water bath at 41-42℃ for development; (5) Slide attachment: The flattened tissue sections are attached to pre-labeled glass slides and dried in a slide dryer at 55-60℃; S3.2 HE staining procedure for paraffin sections: (1) Pretreatment of glass slides: Place the glass slides in a constant temperature oven at 60-65℃ and bake for ≥12h to enhance tissue adhesion; (2) Tissue transparency and hydration treatment: Tissue transparency is achieved by soaking the tissue in xylene to completely dissolve the paraffin and make the tissue transparent. Then, hydration treatment is performed by immersing the sections in a gradient of alcohols to gradually absorb water. Finally, the sections are rinsed with distilled water to remove any alcohol residue, thus completing the transition from an organic environment to an aqueous environment. (3) Immerse in hematoxylin stain for 5-8 minutes, rinse with running deionized water, and then absorb the residual liquid with absorbent paper. (4) Counterstain the cytoplasm with eosin solution for 1-2 min, and stop the staining reaction with distilled water; (5) Gradient dehydration and wax impregnation treatment: Immersion in molten paraffin to allow the wax to fully penetrate the interstitial spaces; (6) Add neutral resin to the tissue area, cover with a coverslip, cure at room temperature in the dark for 24 hours, and then seal and store.

7. The method for treating acute liver injury using lactate-enhanced MDSC immunosuppression according to claim 1, characterized in that: The extraction of mouse lymphocytes in step four: S4.1, Liver lymphocyte extraction: (1) Enucleation and bloodletting of the eyes of WT mice; (2) Transfer the extracted liver tissue sample to a sterile culture dish containing pre-cooled sterile phosphate buffer for temporary storage; (3) In a sterile culture dish, assemble a 200-mesh stainless steel cell sieve and spread the liver tissue evenly on the surface of the sieve; (4) Use the piston end of a 5mL syringe to perform mechanical homogenization, apply vertical pressure and grind repeatedly until the tissue is completely dissociated; (5) During homogenization, pre-cooled PBS was continuously added dropwise; S4.2, Cell suspension treatment: (1) Rinse the cell sieve with pre-cooled PBS; (2) Use a pipette to transfer the homogenized mixture to a 50mL centrifuge tube and centrifuge at 1-4℃, 500rpm×1min; (3) Filter the supernatant into a new 50mL centrifuge tube using a stainless steel mesh, centrifuge at 2200rpm×10min, discard the supernatant and keep the precipitate; S4.3, Perform Percoll separation: (1) Prepare a separation solution of 40% Percoll and 70% Percoll; (2) Resuspend the precipitate after centrifugation in 3 mL of 40% Percoll, dispense 70% Percoll into 15 mL centrifuge tubes, 3 mL / tube, and centrifuge briefly and quickly until no solution remains on the wall. (3) Use a sterile Pasteur pipette to add the resuspended 40% Percoll dropwise to the upper layer of 70% Percoll to form a clear boundary layer; (4) Perform density gradient centrifugation in a centrifuge at 1-4℃; S4.4 Cell Collection: (1) After 30 minutes, use a sterile Pasteur pipette to slowly aspirate 3-4 mL of the middle milky white mononuclear cell layer and transfer it to a new 15 mL centrifuge tube; (2) Add sterile PBS pre-cooled at 1-4℃, bring the volume to 14mL, and vortex the cells; (3) Centrifuge again at 300g×10min and discard the supernatant to retain the cell pellet. The obtained cell pellet is mainly enriched with lymphocyte populations derived from liver tissue. S4.5, Extraction of mouse bone marrow lymphocytes: (1) Place the mouse on a dissecting tray and use dissecting scissors to cut open the skin to expose the femur; (2) Use pliers to fix the femur, then use another pair of pliers to break the femur at the joint, remove the skin and tissue to expose the bone, and cut off both ends to expose the bone marrow; (3) Using a 5mL syringe plunger with a 1mL syringe pillow, draw up PBS buffer and gently rinse the bone marrow; (4) Centrifuge at 1-4℃, 1400rpm×7min, discard the supernatant and keep the precipitate; (5) Add 2 mL of red blood cell lysis buffer, mix by pipetting, let stand for 6-7 min, then stop with 10 mL of PBS, and then filter through a 0.22 µm steel mesh into a new 15 mL centrifuge tube. (6) Centrifuge again at 1400 rpm for 7 min, and perform subsequent MDSC flow cytometry staining on the resulting cell pellet; S4.6 Extraction of single lymphocytes from the spleen: (1) Pour PBS into a sterile culture dish, place the spleen on the ground surface of a glass slide, rub the upper and lower ground surfaces together until no clumps of particles are visible to the naked eye, and rinse with PBS. (2) Take the mixture and filter it through a 0.22µm steel mesh into a 15mL centrifuge tube. Centrifuge at 1-4℃ and 1300-1400rpm for 7-9min. (3) Discard the supernatant, add 2 mL of red blood cell lysis buffer, pipette mix well and let stand for 5-7 min; (4) Stop the red lysis with 10 mL PBS and filter it again into a new 15 mL centrifuge tube. Repeat centrifugation at 1400 rpm for 7 min. (5) Discard the supernatant to obtain cell precipitate and perform flow cytometry single-staining.

8. The method for treating acute liver injury using lactate-enhanced MDSC immunosuppression according to claim 1, characterized in that: In step five, flow cytometry magnetic beads are used to sort MDSC cells. (1) Select WT and WT-LA group mice and extract liver and bone marrow lymphocytes; (2) Add 1×10 cells labeled with CD11b-Biotin and Gr-1-Biotin antibodies. 7 cells / 10μL, gently pipette to mix; (3) Place the mixture in a refrigerator at 1-4℃ and incubate in the dark for 15-30 minutes, stirring and mixing once every 4-5 minutes; (4) After incubation, add 10 mL of pre-cooled PBS to terminate the binding, centrifuge at 1400-1500 rpm for 5-6 min, discard the supernatant and keep the precipitate; (5) Add 1×10 7 / 30μL of Anti-Biotin magnetic beads were used to prepare 1mL of magnetic bead suspension. The magnetic beads were resuspended on ice and bound for 18-20min. (6) Place the magnetic sorting rack in the clean bench, slowly add the labeled cell suspension into the sorting column, let it stand for 8-10 minutes, collect the suspension after the MDSC cells bind to the magnetic beads, and detect them by flow cytometry. The proportion of double-positive cells.

9. The method for treating acute liver injury using lactate-enhanced MDSC immunosuppression according to claim 1, characterized in that: The lactic acid treatment method in step six: S5.

1. Select male C57BL / 6N mice aged 6 to 8 weeks as experimental subjects. Weigh lactic acid and dissolve it in high-pressure deionized water to prepare a lactic acid solution with a concentration of 16 mg / mL, and dispense it for use. S5.

2. 24 hours in advance, calculate the required lactate injection dose based on the weight of the experimental animals, use a 1mL syringe to draw the corresponding volume of lactate solution, inject it into the peritoneum, and continue to observe the condition of the mice. S5.3 24 hours after anesthesia, ConA was injected into the inner canthus to induce liver injury. 12 hours later, blood and tissue samples were collected to observe the performance of lactated mice in ConA-induced liver injury. ALT and liver lymphocytes were analyzed, and the expression of MDSC-related chemokines was detected by RT-PCR.

10. The method for treating acute liver injury using lactate-enhanced MDSC immunosuppression according to claim 1, characterized in that: In step seven, the in vitro bone marrow cell MDSC differentiation induction experiment was performed as follows: After in vitro injection of lactate and ConA, the extracted WT and WT-LA mouse bone marrow lymphocyte suspensions were seeded into 24-well cell culture plates, with 1 mL of cell suspension added to each well; RPMI-1640 containing GM-CSF and IL-6 was added and the cells were cultured completely at 37°C and 5% CO2 for 96 h; finally, the cells were collected for surface staining, and the proportion of MDSC cells and the expression level of CXCR2 were detected by flow cytometry to evaluate the ability of MDSCs to differentiate into G-MDSCs and M-MDSCs.

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