Method for enhancing staphylococcus aureus phagocytosis function of macrophages

Abstract

The invention discloses a method for enhancing a staphylococcus aureus phagocytosis function of macrophages. Belongs to the field of immunometabolism. The method comprises the following specific steps: (1) unfreezing RAW264.7 macrophage cryopreservation liquid, transferring the cells into a centrifugal tube after the cells are completely melted, adding a DMEM culture medium into the centrifugal tube, centrifuging, and discarding supernatant after the centrifugation is finished; adding 1mL of a complete medium to resuspend the cells, transferring the cells into a cell culture bottle, uniformly distributing the cells, and starting passage when the confluence degree of the cells reaches about 80%; (2) inoculating the cell culture solution obtained in the step (1) into a 96-well plate, and culturing for 24 hours; removing the original culture medium, cleaning the cells for 3 times by using PBS, and carrying out starvation treatment on the macrophages for 1 hour by replacing a DMEM culture medium; the method comprises the following steps: pretreating macrophages for 6 hours by using 10 mu L of manganese gluconate with the final concentration of 0.05-0.25 mM; the staphylococcus aureus phagocytosis function of macrophages can be enhanced through manganese gluconate.

Description

Technical Field

[0001] The present invention belongs to the field of immunometabolism, and particularly relates to a method for enhancing the phagocytic function of macrophages. Background Art

[0002] Staphylococcus aureus (S. aureus ) is a common Gram-positive bacterium and a zoonotic pathogen, which can cause various diseases ranging from mild skin infections to life-threatening septicemia, pneumonia, endocarditis, etc. Its pathogenic process is quite complex, and it can escape host immunity by secreting a large number of virulence factors. Currently, the treatment of Staphylococcus aureus infection mainly relies on antibiotics, but the abuse of antibiotics has led to an increasingly serious problem of drug resistance in Staphylococcus aureus, with the continuous emergence of drug-resistant strains and little efficacy. Therefore, developing new treatment strategies is of great significance.

[0003] Macrophages belong to innate immune cells and play a crucial role in resisting Staphylococcus aureus infection as an important part of the innate immune system. They can recognize, phagocytose, and kill invading pathogens, and recruit other immune cells to participate in the immune response by secreting cytokines and chemokines. However, Staphylococcus aureus has evolved various strategies to escape macrophage immune surveillance, such as secreting toxins to damage macrophage cell membranes, inhibiting phagosome maturation, and resisting the killing of reactive oxygen and reactive nitrogen species. These strategies enable Staphylococcus aureus to survive and even multiply within macrophages, leading to the persistence and spread of infection. Therefore, developing a method that can enhance the phagocytic function of macrophages against Staphylococcus aureus is of great significance for improving the host immune defense ability and controlling Staphylococcus aureus infection. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for enhancing the phagocytic function of macrophages against Staphylococcus aureus, so as to provide a new strategy for the treatment of Staphylococcus aureus infection.

[0005] The specific steps of the method for enhancing the phagocytic function of macrophages against Staphylococcus aureus of the present invention are as follows: (1) Take out the RAW264.7 macrophage cryopreservation solution from liquid nitrogen, immediately put it into a water bath to thaw, transfer the cells to a 15 mL centrifuge tube when the cells are completely melted, add 9 mL of DMEM medium to the 15 mL centrifuge tube, centrifuge at 500 g for 5 min at room temperature, and discard the supernatant after completion; resuspend the cells with 1 mL of complete medium and transfer them into a T25 cell culture flask, shake for 2 min by the cross method to evenly distribute the cells in the culture flask, transfer the cells to an incubator for culturing for use, and start subculturing when the cell confluence reaches about 80%; (2) Inoculate the cell culture solution obtained in step (1) into a 96-well plate, with the number of cells per well being 1×104 One cell was taken, and the cells were gently mixed to make them evenly distributed in the cell well plate. The cells were cultured for 24 h; the original culture medium was discarded, and the cells were washed 3 times with PBS, and then the macrophages were starved by replacing with DMEM medium for 1 h; the macrophages were pretreated with 10 μL of manganese gluconate with a final concentration of 0.05 - 0.25 mM for 6 h.

[0006] In the present invention, RAW264.7 macrophages were cultured, treated with 250 μM manganese gluconate for 6 h, and then S. aureus stimulated the macrophages, collected samples for metabolomics sequencing, screened the metabolites regulated by manganese gluconate for macrophage phagocytosis, and found that when manganese gluconate was added to macrophages, the contents of glucose-6-phosphate, glyceraldehyde-3-phosphate, 3-phosphoglycerate, lactic acid and other sugar metabolism-related products increased significantly. qPCR verification of the metabolomics results showed that the sugar metabolism-related genes Glut1, Hk1, Pkm, and Ldha were significantly up-regulated, which was consistent with the metabolomics results. This result indicates that manganese gluconate can enhance macrophage sugar metabolism.

[0007] In the present invention, RAW264.7 macrophages were cultured, treated with 250 μM manganese gluconate for 6 h, and then S. aureus stimulated the macrophages. After culturing for 24 h, RIPA cell lysate was added to lyse the cells. 20 μL of the lysate was spread on TSA medium, and the culture plate was placed in an incubator at 37 °C overnight. In addition, an IFN-γ detection kit was used to detect the content of IFN-γ in the cell supernatant. The results showed that when manganese gluconate was added to macrophages, the macrophages S. aureus phagocytosis rate was significantly enhanced, and the content of IFN-γ increased significantly. Specifically, when the concentration of manganese gluconate was 50 μM, the macrophages S. aureus phagocytosis rate was 15%; when the concentration of manganese gluconate was 100 μM, the macrophages S. aureus phagocytosis rate was 25%; when the concentration of manganese gluconate was 150 μM, the macrophages S. aureus phagocytosis rate was 60%; when the concentration of manganese gluconate was 200 μM, the macrophages S. aureus phagocytosis rate was 96%; when the concentration of manganese gluconate was 250 μM, the macrophages S. aureus phagocytosis rate was as high as 133%.

[0008] In the present invention, by adding the sugar metabolism inhibitor 2-DG to macrophages, it was detected whether the phagocytosis S. aureus ability and the levels of IL-6, IFN-β, and IFN-γ produced changed when macrophage sugar metabolism was inhibited. It was found that when sugar metabolism was inhibited, macrophage phagocytosis S. aureusThere was a significant decrease in ability, and the levels of IL-6, IFN-β, and IFN-γ were significantly reduced; to detect how manganese gluconate affects macrophage glucose metabolism and then regulates its phagocytosis S. aureus ability, Western blot was used to detect the phosphorylation levels of the key proteins P38, JNK, and ERK in the MAPK signaling pathway, and the MAPK signaling pathway was inhibited with the P38 inhibitor SB203580, the JNK inhibitor SP600125, and the ERK inhibitor FR180204 to detect macrophage phagocytosis S. aureus ability. The results showed that manganese gluconate could up-regulate the phosphorylation levels of p38 and JNK proteins in macrophages and down-regulate the phosphorylation level of ERK protein in cells. When the MAPK signaling pathway was inhibited, macrophage phagocytosis S. aureus ability decreased significantly. The above experimental data indicate that manganese gluconate affects macrophage metabolism by regulating the p38, JNK, and ERK signaling pathways.

[0009] The method provided by the present invention for improving macrophage function by adding manganese gluconate to macrophages to change the metabolic pathway can clarify the mechanism by which manganese gluconate enhances macrophage phagocytosis S. aureus mechanism, and macrophages with strong phagocytosis ability can be obtained through its mechanism. Brief Description of the Drawings

[0010] Figure 1 Shows the results of the effect of manganese gluconate at different concentrations on macrophage toxicity after pretreatment; Figure 2 Shows the results of the volcano plot analysis of differential metabolites affected by manganese gluconate in LC-MS / MS liquid phase secondary mass spectrometry technology; Figure 3 Shows the results of the heat map analysis of the effects of manganese gluconate on genes and metabolites related to macrophage glucose metabolism; Figure 4 Shows the results of the changes in the mRNA expression levels of genes related to macrophage glucose metabolism under the stimulation of manganese gluconate; Figure 5 Shows the results of the changes in the protein expression levels of genes related to macrophage glucose metabolism under the stimulation of manganese gluconate; Figure 6 Shows macrophage phagocytosis under the stimulation of manganese gluconate S. aureus experimental results; Figure 7 Shows macrophage phagocytosis under the stimulation of manganese gluconate S. aureus phagocytosis rate detection results; Figure 8 Shows the detection results of IFN-γ secreted by macrophages under the stimulation of manganese gluconate; Figure 9Detection results of the phagocytic ability of macrophages after inhibiting metabolism-related genes; Figure 10 Detection results of the secretion of IL-6, IFN-β, and IFN-γ by macrophages after inhibiting metabolism-related genes; Figure 11 Results of detecting the changes in the phosphorylation levels of key proteins in the MAPK signaling pathway under the stimulation of manganese gluconate; Figure 12 Detection results of the phagocytic ability of macrophages after inhibiting the MAPK signaling pathway. Detailed implementation methods Detailed implementation method 1

[0012] (1) Culturing RAW264.7 macrophages: Take out the RAW264.7 macrophage cryopreservation solution from liquid nitrogen, immediately place it in a water bath to thaw. When the cells are completely melted, transfer the cells to a 15 mL centrifuge tube. Add 9 mL of DMEM medium to the 15 mL centrifuge tube, centrifuge at 500 g for 5 min at room temperature, and discard the supernatant after centrifugation; add 1 mL of complete medium (90% DMEM medium + 10% fetal bovine serum) to resuspend the cells and transfer them into a T25 cell culture flask. Shake the flask crosswise for 2 min to evenly distribute the cells in the culture flask, and then transfer the cells to an incubator for culturing for later use. When the cell confluence reaches about 80%, start subculturing.

[0013] (2) Detecting the effects of different concentrations of manganese gluconate on the toxicity of macrophages by CCK-8 staining method: Count RAW264.7 macrophages using a hemocytometer and inoculate them into a 96-well plate. The number of cells per well is 1×10 4 cells. Gently mix the cells to evenly distribute them in the cell wells, and culture the cells for 24 h. Discard the original medium, wash the cells 3 times with PBS, and replace the DMEM medium to starve the macrophages for 1 h. Pretreat the macrophages with 10 μL of manganese gluconate at concentrations of 0 μM, 50 μM, 100 μM, 150 μM, 200 μM, and 250 μM for 6 h respectively. Set 3 replicates for each gradient, and separately set a control group and a zero adjustment group. After a certain period of time, add 10 μL of CCK8 solution, and then add 90 μL of serum-free medium. Culture them in an incubator at 37 °C and 5% CO 2 for 3 h. Use a multi-functional microplate reader to detect the absorbance value of macrophages at 450 nm, and repeat 3 times. Analyze the cytotoxicity detection of macrophages after treatment with different concentrations of manganese gluconate according to the calculation formula.

[0014] Cell survival rate = (OD 450nm experimental group - OD4 50nm blank group) / (OD450nm Control group - OD 450nm × 100% (blank group) To examine the effect of manganese gluconate on the toxicity of macrophages and to determine the safe dose of manganese gluconate for treating macrophages, the survival rate of macrophages treated with different concentrations of manganese gluconate was detected by CCK-8 staining method. As Figure 1 shown, the survival rate of macrophages varied with the dose of manganese gluconate, but the change was not significant. The results showed that treating macrophages with 0 μM, 50 μM, 100 μM, 150 μM, 200 μM, and 250 μM of manganese gluconate had no toxic or side effects on macrophages.

[0015] (III) Preparation and detection of metabolomics samples: Seed RAW264.7 macrophages at 2.5 × 10 6 cells / well in a 6-well plate and incubate overnight in a cell culture incubator until the cells are completely adherent. After that, discard the cell supernatant, wash the cells 3 times with PBS, add DMEM medium for starvation treatment for 3 h, add 250 μmol / L manganese gluconate for treatment for 6 h, and then S. aureus infect the cells with Newman strain at a multiplicity of infection of 1:20 as the experimental group, and use untreated cells as the control group. Set 3 replicates for each group. After 3 h, add gentamicin to remove extracellular S. aureus Newman. After 2 h, wash away gentamicin with PBS, place the treated macrophages in a cell culture incubator for 24 h, aspirate the complete medium (90% DMEM medium + 10% fetal bovine serum), wash the cells with cold PBS, and then transfer the cells to a 15 mL centrifuge tube, centrifuge at 500 g for 5 min, discard the supernatant, resuspend and count with 1 mL PBS, and take 1 × 10 7Transfer the cells at a density of cells / mL to a 1.5 mL centrifuge tube, centrifuge at 1000 g for 5 min, discard the supernatant, wash the cells 3 times with PBS, centrifuge at 1000 g for 5 min, discard the supernatant, and collect the obtained cell samples for metabolome analysis. Add 4 volumes of 80% methanol to the cells to precipitate proteins, and sonicate for 6 min; repeat the above steps once, vortex, let stand on ice for 5 min, centrifuge at 4 °C and 8000 g for 10 min, take an appropriate amount of the supernatant, dilute the methanol with mass spectrometry-grade ultrapure water to make its content 53%, and place it in a centrifuge tube, centrifuge at 4 °C and 5000 g for 10 min, collect the supernatant, take an equal volume of samples from each experimental sample and mix them as a quality control sample, use a Hypersil Goldcocumn chromatographic column to process metabolites, the column temperature is 40 °C, the flow rate is 0.2 mL / min, in the positive mode, mobile phase A (0.1% formic acid) and mobile phase B (methanol), in the negative mode, mobile phase A (5 mM ammonium acetate formic acid) and mobile phase B (methanol). The mass spectrometry scanning range is selected as 100 - 1500 m / z, the spray voltage is 3.5 kv, the sheath gas flow rate is 35 psi, the ion transfer tube temperature is 320 °C, and start LC-MS detection.

[0016] Sequence and perform bioinformatics analysis on the differentially expressed metabolites in macrophages regulated by manganese gluconate, screen out the differentially expressed metabolites and analyze their functions, and explore the regulatory effect of manganese gluconate on macrophages. The results of the differentially expressed metabolites are presented in the form of a volcano plot, and the results are as Figure 2 shown. A total of 1549 differentially expressed metabolites were screened out, including 268 significantly up-regulated differentially expressed metabolites and 22 significantly down-regulated differentially expressed metabolites. Screen out the metabolites related to glucose metabolism from the differentially expressed metabolites, and it was found that the contents of glucose-6-phosphate, glyceraldehyde-3-phosphate, 3-phosphoglycerate, lactic acid, etc. increased significantly, and the results are as Figure 3 shown.

[0017] (4) qPCR detection of differentially expressed genes: Total RNA of macrophages treated with 250 μmol / L manganese gluconate was extracted using an RNA extraction kit, and the operation was carried out according to the kit instructions. The quality concentration of the extracted sample RNA was detected with a spectrophotometer. The quality concentration was greater than or equal to 250 ng / μL, and the OD260nm / OD280nm between 1.8 - 2.0 was considered a qualified sample. The qualified sample RNA (1 μg was taken from each sample) was synthesized into cDNA using a reverse transcription kit. Quantitative detection was performed using a fluorescence quantitative PCR kit. The qPCR reaction system was 20 μl: 1 μl of cDNA template, 0.5 μl of each upstream and downstream primer, 10 μl of 2×ChamQ TM Universal® SYBR qPCR Master Mix, and 8 μl of Rnase-free water. The qPCR running conditions were: 95°C for 1 min, 95°C for 5 s, 58°C for 10 s, 72°C for 15 s, for 40 cycles. The internal reference gene was β-Actin, and each sample had three replicates. The 2 -△△Ct algorithm.

[0018] To detect the accuracy of LC-MS / MS sequencing results, genes related to glucose metabolism were verified by qPCR method, and the results were as Figure 4 shown. The qPCR results of differentially expressed genes were consistent with the LC-MS / MS sequencing results.

[0019] (V) Western blot detection of differentially expressed proteins Total cell proteins were collected, and the protein concentration was measured using a BCA protein concentration assay kit. After quantification, a stacking gel and a separating gel were prepared using an SDS-PAGE gel kit, and the comb was quickly inserted. After 20 - 30 min, when the stacking gel was completely solidified, the comb was removed, and it was installed into the electrophoresis tank. An appropriate amount of electrophoresis buffer was added to the electrophoresis tank, and loading was carried out according to 4 μL / well of Marker and 10 μL / well of protein sample. It was run at 80 V for 20 - 30 min. After observing that the protein sample reached the stacking gel position, the voltage was adjusted to 120 V to complete electrophoresis. After electrophoresis, membrane transfer was started. The membrane transfer clip was assembled and placed in the order of black gel and white membrane. After completion, it was blocked with skim milk for 2 h. After 2 h, the membrane was washed, and finally the corresponding antibody was incubated, and ECL chemiluminescent color developing solution was added for exposure.

[0020] According to the LC-MS / MS sequencing results, manganese gluconate mainly affected macrophage glucose metabolism. Western blot detection was performed on the key proteins of glucose metabolism, glucose transporter Glut1, hypoxia-inducible factor HIF1α, and GAPDH. It was found that under the stimulation of manganese gluconate, the expression levels of the key proteins of glucose metabolism, glucose transporter Glut1, hypoxia-inducible factor HIF1α, and GAPDH were significantly increased. The results were as Figure 5As shown, it indicates that manganese gluconate may enhance macrophage glucose metabolism by regulating glucose transporter Glut1, hypoxia-inducible factor HIF1α, and GAPDH.

[0021] RAW264.7 macrophages in this embodiment were purchased from Wuhan Punosai Life Science Co., Ltd. S. aureus Newman was preserved in the Bioengineering Laboratory of Heilongjiang Bayi Agricultural University. DMEM medium was purchased from HyClone, USA. Fetal bovine serum was purchased from ExCell Bio. Manganese gluconate was purchased from Macklin. RNA extraction kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. Reverse transcription kit was purchased from Lamboid. Fluorescent quantitative PCR kit was purchased from Nanjing Novoprotein Scientific Co., Ltd. SDS-PAGE gel kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. BCA protein concentration assay kit was purchased from Beijing Coolaber Co., Ltd. Specific Embodiment 2 (I) Detection of the effect of manganese gluconate on macrophage phagocytosis of S. aureus Newman strain by the plate coating method: Inoculate 2.5×10 5 RAW264.7 macrophages into a 24-well plate, gently mix them left, right, front, and back to evenly distribute the cells in the well plate. After standing, add DMEM for starvation treatment for 2 h, and add 0 μM, 50 μM, 100 μM, 150 μM, 200 μM, and 250 μM of manganese gluconate for pretreatment for 6 h. Use the cells treated with Ca 2+ as the control group, with three replicates in each group. Then, infect S. aureus Newman strain. After 3 h, add gentamicin to remove extracellular S. aureuss Newman. After 1 h, wash off gentamicin with PBS. Add complete medium (90% DMEM medium + 10% fetal bovine serum) and culture the cells for 24 h. Then, wash the macrophages 5 times with PBS solution, add 200 μL of RIPA cell lysate to lyse the cells. To ensure sufficient cell lysis, gently shake the cell culture plate every 5 min. Collect the solution into a 2 mL centrifuge tube, take 20 μL of the lysate and spread it on TSA medium. Place the culture plate in an incubator at 37°C overnight. The next day, count the S. aureus Newman colonies and calculate the number of phagocytosed S. aureus Newman. The results are as Figure 6As shown, when the concentration of manganese gluconate was 50 μM, the number of bacteria phagocytized by macrophages was 207 CFU; when the concentration of manganese gluconate was 100 μM, the number of bacteria phagocytized by macrophages was 225 CFU; when the concentration of manganese gluconate was 150 μM, the number of bacteria phagocytized by macrophages was 288 CFU; when the concentration of manganese gluconate was 200 μM, the number of bacteria phagocytized by macrophages was 352 CFU; when the concentration of manganese gluconate was 250 μM, the number of bacteria phagocytized by macrophages was 420 CFU. As the concentration of manganese gluconate increased, the phagocytosis S. aureus ability of macrophages gradually increased.

[0023] (2) Detection of the effect of manganese gluconate on macrophage phagocytosis of S. aureus RN4220 strain by fluorescence method: 2.5×10 5 RAW264.7 macrophages were seeded in 24-well plates, gently mixed left and right, front and back, so that the cells were evenly distributed in the 24-well plates. After standing for 2 min, DMEM was added for starvation treatment for 1 h. Then, manganese gluconate at 0 μM, 50 μM, 100 μM, 150 μM, 200 μM, and 250 μM was added for pretreatment for 6 h. Each group had three replicates. The cells were washed 3 times with PBS, and then infected with S. aureus RN4220 strain expressing green fluorescent protein (multiplicity of infection was 1:20). After 3 h, gentamicin was added to remove extracellular S. aureus RN4220. After 1 h, gentamicin was washed away with PBS, and complete medium (90% DMEM medium + 10% fetal bovine serum) was added. After culturing the cells for 24 h, the macrophages were washed 5 times with PBS solution. The phagocytosis of S. aureus RN4220 by macrophages after pretreatment with different concentrations of manganese gluconate was observed under a fluorescence microscope. The results were as Figure 6 shown. As the concentration of manganese gluconate increased, the ability of macrophages to phagocytize S. aureus RN4220 became stronger and stronger.

[0024] The detection results of the phagocytosis rate of macrophages phagocytizing S. aureus were as Figure 7 shown. When the concentration of manganese gluconate was 50 μM, the phagocytosis rate of macrophages was 15%; when the concentration of manganese gluconate was 100 μM, the phagocytosis rate of macrophages was 25%; when the concentration of manganese gluconate was 150 μM, the phagocytosis rate of macrophages was 60%; when the concentration of manganese gluconate was 200 μM, the phagocytosis rate of macrophages was 96%; when the concentration of manganese gluconate was 250 μM, the phagocytosis rate of macrophages was as high as 133%. The trend of the fluorescence method was consistent with that of the plate coating method, indicating that manganese gluconate could enhance the phagocytosis S. aureus ability after acting on macrophages.

[0025] (3) Effect of Manganese Gluconate on IFN-γ Secretion by Macrophages Perform relevant operations according to the instructions provided by the ELISA kit. Mix 10 μL of cell supernatant with 40 μL of sample diluent. Add PBS to the blank well for control. Except for the blank well, add 100 μL of the detection antibody labeled with horseradish peroxidase (HRP) to each standard well and sample well, and incubate in an incubator at a constant temperature for 60 min. Quickly discard the liquid. Use absorbent paper to drain the remaining liquid as much as possible, and repeat the plate washing 5 times with the washing solution. Add 50 μL of substrate A and 50 μL of substrate B to each well, pretreat at 37 °C for 15 min, and finally add 50 μL of the termination solution to each well. Measure the OD value of each well at a wavelength of 450 nm using a multifunctional microplate reader.

[0026] To explore the effect of manganese gluconate on IFN-γ secretion by macrophages, a cytokine detection kit was used to detect the effect of different concentrations of manganese gluconate on IFN-γ secretion by macrophages. The results are as Figure 8 shown. Compared with the control group, the expression level of IFN-γ was significantly increased after treatment with manganese gluconate, indicating that manganese gluconate can promote IFN-γ secretion by macrophages.

[0027] The RAW264.7 macrophages in this embodiment were purchased from Wuhan Punosai Life Science Co., Ltd.; S. aureus Newman was preserved in the Bioengineering Laboratory of Heilongjiang Bayi Agricultural University; S. aureus The RN4220 strain was preserved in the Bioengineering Laboratory of Heilongjiang Bayi Agricultural University; The DMEM medium was purchased from HyClone, USA; Fetal bovine serum was purchased from ExCell Bio; Manganese gluconate was purchased from Macklin; The cytokine IFN-γ detection kit was purchased from Shenzhen Dakewei Biotechnology Co., Ltd.; Specific Embodiment 3

[0028] (1) Detection of the effect of the sugar metabolism inhibitor 2-DG on the phagocytic ability of macrophages by the plate coating method: Inoculate 2.5×10 5 RAW264.7 macrophages into a 24-well plate, gently mix left, right, front, and back to evenly distribute the cells in the well plate. After standing, add DMEM medium for starvation treatment for 2 h, add 2-DG for treatment for 6 h, with three replicates in each group. Then infect with S. aureus Newman strain respectively. After 3 h, add gentamicin to remove extracellular S. aureusNewman. After 1 h, gentamicin was washed away with PBS. After culturing the cells in complete medium (90% DMEM medium + 10% fetal bovine serum) for 24 h, the macrophages were washed 5 times with PBS solution, and 200 μL of RIPA cell lysate was added to lyse the cells. 20 μL of the lysate was spread on a TSA plate, and the culture plate was placed in an incubator at 37 °C overnight. The next day, for S. aureus the Newman colonies were counted, and the number of phagocytosed S. aureus was calculated.

[0029] (2) Detection of the effect of the glycolysis inhibitor 2-DG on the phagocytic ability of macrophages by fluorescence method: 2.5×10 5 RAW264.7 macrophages were seeded in a 24-well plate, gently mixed left, right, front and back to evenly distribute the cells in the 24-well plate. After standing for 2 min, DMEM medium was added for starvation treatment for 2 h, and 2-DG was added for pretreatment for 6 h. Each group had three replicates. The cells were washed 3 times with PBS and then medium was added. Then, the cells were infected with S. aureus RN4220 strain expressing green fluorescent protein (multiplicity of infection was 1:20). After 3 h, gentamicin was added at a final concentration of 100 μg / mL to remove extracellular S. aureus RN4220. After 1 h, gentamicin was washed away with PBS. After culturing the cells in complete medium (90% DMEM medium + 10% fetal bovine serum) for 24 h, the macrophages were washed 5 times with PBS solution, and the phagocytosis of macrophages after pretreatment with the glycolysis inhibitor 2-DG was observed using a fluorescence microscope. S. aureus situation.

[0030] To further confirm that manganese gluconate enhances the phagocytic ability of macrophages by regulating glycolysis, 2-DG, a glycolysis inhibitor, was added before adding manganese gluconate to pretreat macrophages, and the phagocytic ability of macrophages was verified bidirectionally by immunofluorescence method and plate coating method. The results were as Figure 9 shown. After adding 2-DG, the phagocytic ability of macrophages decreased significantly. The above experiments indicate that manganese gluconate enhances the phagocytosis of macrophages by promoting the activation of the glycolysis pathway.

[0031] (3) Detection of the effect of the glycolysis inhibitor 2-DG on the secretion of IL-6, IFN-β, and IFN-γ by macrophages by ELISA method: RAW264.7 macrophages with adjusted concentration were seeded in a 12-well plate, 1 mL of cells per well, and 3 wells were seeded in each group. 2-DG was added to the cells, and then gently tapped and shaken by hand to mix evenly. Then, the 12-well plate was placed in 5% CO 2In the cell culture incubator, culture continuously at 37°C for 6h, then completely aspirate the cell culture medium in the 12-well plate and transfer it to a sterile centrifuge tube, centrifuge the culture medium at 4000g for 20min at 4°C, and then collect the supernatant. Use the cytokine IL-6, IFN-β, and IFN-γ detection kits to detect cytokines IL-6, IFN-β, and IFN-γ. According to the instructions of the kit, set up the standard wells and sample wells. The standard wells are set to 6 groups, and 50μL of S0-S5 are added respectively, corresponding to different concentrations of standards. The sample wells should be divided into groups, and 10μL of the corresponding cell supernatant is added to each well, and then 40μL of sample diluent is added. Then, 100μL of HRP-labeled detection antibody is added to all wells, and the mixture is gently blown with a pipette, and the film is sealed and placed in a 37°C incubator for incubation for 60min. After the incubation, discard the liquid in the wells, pat dry the water on clean absorbent paper, add 200 μL of washing solution to each well, and then place it on a horizontal shaker to wash thoroughly for 1 minute. After the end, discard the washing solution and pat dry on absorbent paper, and repeat the washing steps 5 times. After the last wash, add 50 μL of substrate A and B solution to each well, put it in a 37°C incubator, incubate it in the dark for 15 minutes, add 50 μL of stop solution to each well, and immediately use an enzyme marker to measure the OD value of each well at a wavelength of 450 nm and record it. According to the concentration of the standard and the measured OD value, draw a standard curve, and then calculate the cytokine concentration of the sample well. The final expansion of 5 times is the actual IL-6, IFN-β, and IFN-γ concentration of each group of samples.

[0032] In order to further confirm that manganese gluconate enhances the secretion of IL-6, IFN-β, and IFN-γ by macrophages by regulating glucose metabolism, the glucose metabolism inhibitor 2-DG was added before pre-treatment of macrophages with manganese gluconate, and the effect of glucose metabolism on the secretion of IL-6, IFN-β, and IFN-γ by macrophages was detected by cytokine detection kit. The results are as follows: Figure 10 As shown in the figure, when macrophage glucose metabolism is inhibited, the ability to secrete IL-6, IFN-β, and IFN-γ is significantly reduced. The above experiments show that manganese gluconate promotes the secretion of IL-6, IFN-β, and IFN-γ by regulating macrophage glucose metabolism.

[0033] (IV) Western blot detection of the phosphorylation levels of key proteins P38, JNK, and ERK in the MAPK signaling pathway under manganese gluconate treatment when glucose metabolism was inhibited: Macrophages were counted using a hemocytometer and inoculated into 6-well plates, with 2.5×10 cells in each well. 6 Slowly mix the cells back and forth and left and right to evenly distribute them in the well plate. Place the inoculated cells in a 37°C, 5% CO 2Cultivate in an incubator for 24 h. On the second day, discard the original medium, wash the cells 3 times with PBS, replace with serum-free medium for starvation treatment for 1 h, wash the cells 3 times with PBS, add 2 mL of complete medium, and then add 250 μM manganese gluconate to pretreat macrophages as the experimental group, and pretreat macrophages with the same concentration of calcium gluconate as the control group. Pretreat each group for 6 h and wash the cells 3 times with PBS. Add S. aureus Infect with Newman strain for 3 h. Add 30 μL of cell lysate to each well and place on an ice box for 30 min. Gently shake the cell culture plate every 5 min to better lyse the cells. Scrape the cells in the cell well plate with a cell scraper and collect them in a 2 mL EP tube. Centrifuge the lysed cells at 12,000 rpm at 4 °C for 10 min, discard the precipitate, and aspirate the supernatant and transfer it to a sterile EP tube.

[0034] After detecting the protein concentration using a BCA protein concentration detection kit, start preparing the gel using an SDS-PAGE gel kit. After loading the samples, start transferring the membrane, place them in the order of black membrane and white membrane. After the membrane transfer is completed, block with skim milk for 2 h, then wash the membrane, select appropriate antibodies for incubation, and finally expose.

[0035] To verify whether manganese gluconate enhances macrophage glycolysis and thus phagocytosis by regulating the MAPK signaling pathway S. aureus Pretreat macrophages with manganese gluconate and infect them with S. aureus Use β-actin antibody, P38 antibody, JNK antibody, ERK antibody, P-P38 antibody, P-JNK antibody, and P-ERK antibody to detect the phosphorylation levels of the key proteins P38, JNK, and ERK in the MAPK pathway. The results are as Figure 11 shown. Compared with the control group, after pretreatment of cells with manganese gluconate, it can upregulate the phosphorylation levels of p38 and JNK proteins in macrophages and downregulate the phosphorylation level of ERK protein in cells. The above experimental data indicate that manganese gluconate regulates the p38, JNK, and ERK signaling pathways, thereby affecting macrophage metabolism.

[0036] (5) Detection of the effect of inhibitors of key proteins in the MAPK signaling pathway on macrophage phagocytosis ability by the plate coating method: Seed 2.5×10 5 RAW264.7 macrophages into a 24-well plate, gently mix them left, right, front, and back to evenly distribute the cells in the well plate. After standing, add DMEM for starvation treatment for 2 h, and then add inhibitors of key proteins P38 inhibitor SB203580, JNK inhibitor SP600125, and ERK inhibitor FR180204 in the MAPK signaling pathway for treatment for 6 h, with three replicates in each group. Then infect them respectively withS. aureus The Newman strain, gentamicin was added after 3 h to remove extracellular S. aureus Newman, after 1 h, the gentamicin was washed away with PBS. After culturing the cells for 24 h, the macrophages were washed 5 times with PBS, 200 μL of RIPA cell lysate was added to lyse the cells, 20 μL of the lysate was spread on TSA medium, and it was placed in an incubator at 37 °C overnight. The next day, S. aureus the Newman colonies were counted to calculate the phagocytosis S. aureus number.

[0037] Using fluorescence method and plate coating method to detect the phagocytic ability of macrophages in two-way after the MAPK signaling pathway was inhibited. The results are as Figure 12 shown. After adding the corresponding inhibitor, the phagocytic ability of macrophages decreased significantly. The experimental results showed that manganese gluconate regulated macrophage metabolism through the MAPK signaling pathway and then regulated macrophage phagocytosis S. aureus .

[0038] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention are all within the protection scope of the present invention.

[0039] The RAW264.7 macrophages in this embodiment were purchased from Wuhan Punosai Life Science Co., Ltd.; S. aureus RN4220 was preserved by the Bioengineering Laboratory of Heilongjiang Bayi Agricultural University; DMEM medium was purchased from HyClone Company, USA; Fetal bovine serum was purchased from ExCell Bio Company; Manganese gluconate was purchased from Macklin Company; Cell factor IL-6, IFN-β, IFN-γ detection kits were purchased from Shenzhen Dakewei Biotechnology Co., Ltd.; β-actin antibody, P38 antibody, JNK antibody, ERK antibody, P-P38 antibody, P-JNK antibody, P-ERK antibody were purchased from Cell Signaling Technology Company; SDS-PAGE gel kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; BCA protein concentration assay kit was purchased from Beijing Coolaber Company; P38 inhibitor SB203580, JNK inhibitor SP600125, ERK inhibitor FR180204 were purchased from MCE Company.

Claims

1. A method for enhancing the phagocytic function of macrophages against Staphylococcus aureus, characterized in that The specific steps of this method are as follows: (i) Take out the RAW264.7 macrophage cryopreservation solution from liquid nitrogen and immediately thaw it in a water bath. When the cells are completely melted, transfer the cells to a 15 mL centrifuge tube, add 9 mL of DMEM medium to the 15 mL centrifuge tube, centrifuge at 500 g for 5 min at room temperature, and discard the supernatant; add 1 mL of complete medium to resuspend the cells and transfer them to a T25 cell culture flask, shake for 2 min to evenly distribute the cells in the culture flask, transfer the cells to an incubator for culture, and start subculturing when the cell confluence reaches about 80%; (ii) The cell culture medium obtained in step (i) was inoculated into a 96-well plate, with 1×10 cells per well. 4 cells, gently mix the cells to make them evenly distributed in the cell well plate, and culture the cells for 24 hours; The original culture medium was discarded, the cells were washed three times with PBS, and the DMEM culture medium was replaced to starve the macrophages for 1 hour; 10 μL of manganese gluconate with a final concentration of 0.05-0.25 mM was used to pretreat the macrophages for 6 hours.

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