Pharmaceutical application of oxidative phosphorylation inhibitor

Abstract

The invention belongs to the technical field of biological medicine, and relates to pharmaceutical application of an oxidative phosphorylation inhibitor, in particular to application of the oxidative phosphorylation inhibitor in preparation of an immunomodulatory drug, and the immunomodulatory drug has the advantages that the function damage of newborn myeloid-derived suppressor cells caused by antagonism BCG (bacillus calmette guerin) vaccination is inhibited, and the immunomodulatory effect is improved. The compound is used for preventing or treating neonatal immune-related diseases caused by bacillus calmette guerin vaccine inoculation. The invention finds that the BCG can cause damage to the immunosuppression function of the MDSC of the suckling mouse through up-regulation oxidative phosphorylation, and the negative effect caused by inoculation of the BCG can be reversed by using the OXPHOS inhibitor, so that the immunosuppression function of the MDSC of the suckling mouse is successfully recovered in an in-vivo and in-vitro model, and the immune-related adverse reaction caused by inoculation of the BCG is expected to be improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the pharmaceutical use of oxidative phosphorylation inhibitors. Background Technology

[0002] BCG (Bacillus Calmette-Guérin) is currently the only approved vaccine for the prevention of infectious tuberculosis and meningeal tuberculosis. This drug is routinely administered to full-term newborns within the first week of life. Notably, epidemiological studies have linked BCG vaccination with a reduction in childhood all-cause mortality, an effect attributed to its non-specific mechanism of action, effectively protecting the body from non-tuberculous pathogens. This broader immunomodulatory function is thought to involve the formation of innate immune memory through induced training immunity, involving metabolic reprogramming and epigenetic rewiring.

[0003] Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population. Immature myeloid cells proliferate abnormally under various pathological conditions, such as cancer and chronic infections, effectively suppressing immune responses. MDSCs are generally divided into two main subsets: polymorphonuclear MDSCs (PMN-MDSCs) and mononuclear MDSCs (M-MDSCs). PMN-MDSCs are the dominant subset, primarily mediating immunosuppression through the production of reactive oxygen species (ROS), peroxynitrite, and prostaglandin E2. In contrast, M-MDSCs mainly suppress T cell function through the expression of arginase-1 and its product nitric oxide. Besides pathological effects, myeloid-derived suppressor cells can also undergo transient proliferation under specific physiological conditions such as pregnancy, neonatal development, aging, and post-vaccination responses. In newborns, their transient accumulation is thought to help regulate inflammatory responses associated with initial microbiota colonization. Given that the neonatal period is a critical window for vaccination to establish durable immune protection, it is necessary to investigate whether and how early BCG vaccination affects the function and characteristics of neonatal MDSCs. Summary of the Invention

[0004] The inventors discovered that BCG vaccination leads to immunosuppressive damage in neonatal rat MDSCs by upregulating oxidative phosphorylation (OXPHOS), while the negative effects induced by BCG vaccination can be reversed using an OXPHOS inhibitor (oligomycin), successfully restoring the immunosuppressive function of neonatal rat MDSCs in in vitro and in vivo models. Based on these findings, this invention provides pharmaceutical applications for oxidative phosphorylation inhibitors.

[0005] The technical solution provided by this invention is as follows: This invention provides the use of oxidative phosphorylation inhibitors in the preparation of immunomodulatory drugs, which are used to prevent or treat BCG-induced neonatal immune-related diseases by antagonizing functional damage to neonatal myeloid-derived suppressor cells caused by BCG vaccination.

[0006] In some embodiments of the present invention, the oxidative phosphorylation inhibitor is oligomycin or a pharmaceutically acceptable salt thereof.

[0007] In some embodiments of the present invention, the neonatal immune-related disease is neonatal necrotizing enterocolitis.

[0008] In some embodiments of the present invention, the newborn is a premature infant or a low birth weight infant.

[0009] In some embodiments of the present invention, the immunomodulatory drug is an injectable preparation.

[0010] In some embodiments of the present invention, the myeloid-derived suppressor cells are PMN-MDSCs or M-MDSCs.

[0011] In some embodiments of the present invention, the functional impairment is manifested as a significant decrease in the ability of myeloid-derived suppressor cells to inhibit T cell proliferation.

[0012] In some embodiments of the present invention, the T cells are CD8 cells. + and CD4 + cell.

[0013] In some embodiments of the present invention, the medicament comprises a therapeutically effective amount of an oxidative phosphorylation inhibitor and a pharmaceutically acceptable carrier.

[0014] In some embodiments of the present invention, the immunomodulatory drug is used to restore the immunosuppressive function of MDSC cells.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides a novel drug target and a clear treatment strategy for the clinical prevention and treatment of immune-related adverse reactions that may be caused by BCG vaccination, and has important clinical translational value. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Transcriptional profiles of BCG-inoculated neonatal rat M-MDSC cells; including: (A) Schematic diagram of single-cell RNA sequencing experimental design and analysis workflow; (BC) Comparison of spleen-derived M-MDSCs after PBS treatment and BCG inoculation; CD3 values ​​of all cells from PBS or BCG inoculation conditions.- CD19 - CD11b + M-MDSCs in cells are displayed by uniform manifold approximation and projection integration, and presented according to experimental conditions; the colors in the figure indicate cell types; (D) Heatmap shows the normalized expression levels of the most significantly changed genes in M-MDSCs from PBS or BCG-inoculated suckling mice; (E) KEGG metabolic pathway enrichment scores in single-cell transcriptomes of M-MDSCs from BCG-inoculated and PBS control mice. Figure 2 Transcriptional map of MDSCs in neonatal mice remodeled by BCG vaccination; (A) Key marker genes of cell type; (B) Projected distribution of differentially expressed genes on UMAP maps of PBS-treated neonatal mouse MDSCs; Scale bar indicates gene expression level; Figure 3 The study aimed to restore the immunosuppressive function of BCG-inoculated suckling mouse M-MDSC cells by oxidative phosphorylation inhibitors. The results included: (A) GO analysis of differentially expressed genes in BCG-inoculated and PBS-treated suckling mouse M-MDSC cells; (B) Heatmap of oxidative phosphorylation-related gene expression in suckling mouse spleen M-MDSC cells on day 3 after BCG inoculation (log2 (fold change) > 1, corrected p < 0.05); (C) qRT-PCR analysis of oxidative phosphorylation-related genes in suckling mouse M-MDSC cells (n = 4); (D) Mitochondrial mass in M-MDSC cells on day 1 after BCG inoculation (MitoTracker Red fluorescence intensity); representative histograms and summed mean fluorescence intensities (n = 3–4); (E) Total ATP levels in M-MDSC cells on day 3 after BCG inoculation (n = 4); (F) Schematic diagram of in vitro oxidative phosphorylation inhibition experiment; (G) Effects of M-MDSC cells on CD8+ under in vitro treatment with PBS, BCG, or oligomycin. + With CD4 + Inhibitory capacity of T cell proliferation (n = 3–4); all data are expressed as mean ± standard error. This means P < 0.05. This means P < 0.01. P < 0.001, ns indicates no statistical significance; Figure 4: BCG vaccination reconstructs the transcriptome of neonatal mouse PMN-MDSC cells; where: (AB) Comparison of spleen-derived PMN-MDSCs after PBS treatment and BCG vaccination; PMN-MDSCs under all treatment conditions are integrated and projected by UMAP and displayed according to experimental conditions; color in the figure indicates cell type determination; (C) Comparison of KEGG metabolic pathway enrichment scores of neonatal mouse PMN-MDSC clusters after BCG vaccination and PBS vaccination based on single-cell RNA sequencing; (D) Heatmap of selected gene expression in PMN-MDSC clusters between PBS treatment and BCG vaccination groups; (E) Comparison of oxidative phosphorylation scores among PMN-MDSC clusters in PBS treatment and BCG vaccination groups; Figure 5 The study aimed to restore immunosuppressive function in BCG-inoculated PMN-MDSC cells from suckling mice by inhibiting oxidative phosphorylation. The results included: (A) KEGG analysis of differentially expressed genes in PMN-MDSC cells using RNA-seq sequencing; (B) a heatmap of oxidative phosphorylation-related gene expression in PMN-MDSC cells treated with PBS and inoculated with BCG, showing the results of RNA-seq sequencing; (C) qRT-PCR detection of mRNA levels in suckling mouse PMN-MDSC cells (n = 6); (D) MitoTracker expression levels in PMN-MDSC cells detected by flow cytometry on day 1 after BCG inoculation; representative histograms and summed mean fluorescence intensity data (n = 3–4); (E) Measurement of total ATP levels in PMN-MDSCs on day 3 after BCG inoculation (n = 6); (F) Measurement of oxygen consumption rate in PMN-MDSC cells on day 3 after BCG inoculation using an oxidative phosphorylation stress test (n = 4); (G) Schematic diagram of in vitro oxidative phosphorylation inhibition; (H) PMN-MDSCs treated with PBS, BCG, or oligomycin inhibited antigen-specific CD8. + T cell proliferation (n = 3); data are expressed as mean ± standard error. This means P < 0.05. This means P < 0.01. This means P < 0.001.

[0018] Figure 6 In vivo oxidative phosphorylation inhibitors restore immunosuppressive function in BCG-inoculated neonatal mouse M-MDSCs and PMN-MDSCs; where: (A) Schematic diagram of in vivo oxidative phosphorylation inhibition; (B) Effect of PBS, BCG or oligomycin treatment on CD8 in neonatal mouse-derived M-MDSCs. + With CD4 + Inhibitory capacity of T cell proliferation (n = 3-4); (C) Inhibition of antigen-specific CD8 by PMN-MDSCs from suckling mice treated with PBS, BCG, or oligomycin.+ T cell proliferation (n = 3–4); (D) ROS expression level in neonatal mouse PMN-MDSCs detected by flow cytometry; representative histograms and summative mean fluorescence intensity data (n = 3–4); data are expressed as mean ± standard error. This means P < 0.05. This means P < 0.01. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0020] The technical solution provided by the present invention will be described in detail below with reference to the embodiments.

[0021] Unless otherwise specified, the methods used in the embodiments are as follows: 1) BCG vaccination method for newborn mice: Freeze-dried BCG vaccine was purchased from Chengdu Institute of Biological Products and stored at 4°C until injection. Following previous methods, newborn mice were subcutaneously injected with 5 × 10⁵ BCG vaccine on days 3-4 after birth. 5 BCG colony-forming units of CFU.

[0022] 2) Flow cytometry analysis and sorting: First, single-cell suspensions were stained with surface antibodies for 30 minutes at 4°C using cold FACS buffer containing 1% FBS and 2mM EDTA. After surface staining, dead cells were excluded using LIVE / DEAD staining. Flow cytometry analysis was performed using specifically fluorescently labeled mouse antibodies and a CytoFLEX flow cytometer. Cell sorting was performed using a MoFlo Astrios EQs flow cytometer, and data were analyzed using FlowJo V10 software. The flow cytometry analysis and sorting strategy was as follows: Mouse M-MDSCs were identified as CD11b. + Ly6C hi Ly6G - PMN-MDSC is CD11b + Ly6G + Ly6C lo MDSC is CD11b + Gr1 + All anti-mouse antibodies used in this study were purchased from Biolegend.

[0023] 3) MDSC cell immunosuppressive function assay: Newborn mice were inoculated with BCG on days 3-4 after birth. Splenic M-MDSC or PMN-MDSC cells were isolated from BCG-inoculated mice and cultured in vitro. After treatment with DMSO or 1 μM Ligomycin for 24 hours, subsequent experiments were performed. M-MDSC cell immunosuppressive function assay: M-MDSC cells were collected from the spleen of BCG-inoculated mice 3 days after birth. CD3+ cells were collected from the spleen of adult mice. + T cells, labeled with CFSE (2 μM), were cultured alone or co-cultured with M-MDSC cells at an 8:1 ratio for 3 days on anti-CD3 (5 μg / ml) coated plates and soluble anti-CD28 (1 μg / ml) antibody. T cell proliferation was analyzed by CFSE fluorescence intensity. PMN-MDSC cell immunosuppressive function assay: PMN-MDSC cells were collected from the spleens of suckling mice 3 days after BCG inoculation. OT-I mouse spleen cells were then cultured with WT mouse CD8... + T cells were mixed at a 1:4 ratio, labeled with CFSE (2 μM), and then administered via OVA with a specific peptide. 257-264 In the presence of the virus, T cells were co-incubated with PMN-MDSC cells at a 2:1 ratio for 60 hours, and T cell proliferation was analyzed by CFSE fluorescence intensity.

[0024] 4) qRT-PCR detection method: Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions. cDNA was synthesized using the StarScript III cDNA synthesis kit. Real-time quantitative PCR was performed using SYBR qPCR Mix, and gene mRNA levels were determined using the relative standard curve method. β-actin was used as an internal reference gene for standardization. The primer sequences used in this study are as follows: β-actin forward primer: 5'-GACGGCCAGGTCATCACTATTG-3' (SEQ ID NO.1) β-actin reverse primer: 5'-AGGAAGGCTGGAAAAGAGCC-3' (SEQ ID NO.2); Ndufab1 forward primer: 5'-ATGGCGTCTCGTGTCCTCT-3' (SEQ ID NO.3). Ndufab1 reverse primer: 5'- GCGGCACAAATGTGTGACT-3' (SEQ ID NO.4); Ndufs3 forward primer: 5'-TGGCAGCACGTAAGAAGGG-3' (SEQ ID NO.5), Ndufs3 reverse primer: 5'-CTTGGGTAAGATTTCAGCCACAT-3' (SEQ ID NO.6); Sdhb forward primer: 5'-AATTTGCCATTTACCGATGGGA-3' (SEQ ID NO.7). Sdhb reverse primer: 5'-AGCATCCAACACCATAGGTCC-3' (SEQ ID NO.8); Sdhd forward primer: 5'-TGGTCAGACCCGCTTATGTG-3' (SEQ ID NO.9) Sdhd reverse primer: 5'- GGTCCAGTGGAGAGATGCAG-3' (SEQ ID NO.10); Uqcrfs1 forward primer: 5'- GAGCCACCTGTTCTGGATGTG-3' (SEQ ID NO.11), Uqcrfs1 reverse primer: 5'- GCACGACGATAGTCAGAGAAGTC-3' (SEQ ID NO.12); Cox4i forward primer: 5'-ATTGGCAAGAGAGCCATTTCTAC-3' (SEQ ID NO.13). Cox4i reverse primer: 5'-CACGCCGATCAGCGTAAGT-3' (SEQ ID NO.14); Atp5a forward primer: 5'-TCTCCATGCCTCTAACACTCG-3' (SEQ ID NO.15) Atp5a reverse primer: 5'-CCAGGTCAACAGACGTGTCAG-3' (SEQ ID NO.16).

[0025] 5) Single-cell RNA sequencing library construction: Newborn mice were subcutaneously injected with BCG or PBS on days 3-4 after birth. On day 3 after BCG inoculation, CD3+ was isolated from the spleen of the newborn mice using flow cytometry. - CD19 - CD11b +Cells were pooled and loaded at 100,000 cells per channel to generate single cells using a Chromium Controller instrument. The simplified workflow involved partitioning cells in a Gem Code instrument, followed by lysis of individual cells and mixing with magnetic beads carrying unique barcodes within oil droplets. Sequencing libraries were constructed using the Chromium Single Cell 3' kit according to the manufacturer's instructions. Each library was sequenced at 150 bp paired ends on an Illumina NovaSeq platform.

[0026] 6) Single-cell RNA sequencing data processing and analysis: Single-cell RNA sequencing data were processed using the Cell Ranger software suite. For each single-cell RNA sequencing dataset in the BCG and PBS groups, the cell ranger count output was loaded into the R statistical environment using the Read10X function of the Seurat package. Cells with fewer than 500 or more than 7,500 genes were removed, along with cells with a mitochondrial read ratio higher than 5% indicating potential stress signals. After filtering, a total of 21,269 high-quality myeloid cell transcriptomes were obtained. The first 2,000 variable genes identified in each group were batch-corrected using canonical correlation analysis of the Seurat package. Cell clustering was performed using the FindNeighbors and FindClusters functions based on the first 42 principal components estimated by the RunPCA function. Cell clusters were annotated based on published marker genes. The FindAllMarkers function was used to identify cluster-specific expressed genes. Gene characteristics and pathways of each subpopulation were compared using the AddModuleScore function of Seurat. The ggsignif R package was used to calculate the statistical differences between groups for PMN subpopulation significance scores. For the subpopulation analysis of PMN-MDSC, the first 20 principal components were used for cell clustering, and visualization was performed using a uniform manifold approximation and projection.

[0027] 7) Batch RNA sequencing library construction: Newborn mice were subcutaneously injected with BCG on days 3-4 after birth. On day 3 post-inoculation, approximately 5 × 10⁶ RNA samples were isolated from the spleen of the newborn mice. 5 M-MDSC or PMN-MDSC cells were used. Cells were resuspended in 500 μl TRIzol reagent and reverse transcribed using M-MuLV reverse transcriptase. Subsequently, PCR amplification was performed according to the manufacturer's protocol using Phusion high-fidelity DNA polymerase, universal PCR primers, and Index primers. RNA-seq data were obtained by paired-end 150 bp sequencing using the Illumina NovaSeq platform.

[0028] 8) Batch RNA Sequencing Data Processing and Analysis: RNA-seq data were aligned to the mouse reference genome using STAR, and gene expression was quantified using RSEM. The significance of differentially expressed genes between the two groups was assessed using DESeq2, and the Benjamini-Hochberg method was used to correct p-values ​​to control for false detection rate. Genes with a corrected p-value < 0.05 and a |log² change| > 1 were defined as differentially expressed genes. KEGG enrichment analysis was performed on differentially expressed genes using clusterProfiler.

[0029] 9) Seahorse Analysis: Newborn mice were subcutaneously injected with BCG on days 3–4 post-birth. PMN-MDSC cells were isolated from the spleen of the newborn mice on day 3 post-inoculation. Oxygen consumption rate was detected using the Seahorse XFe96 energy metabolism analysis system according to the manufacturer's protocol. Microplates were coated with 22.4 µg / ml Cell-Tak solution in 200 mM sodium bicarbonate solution. Approximately 1 × 10⁻⁶ cells were then placed in each well. 5 PMN-MDSC cells were seeded in OCR detection medium, and oligomycin, FCCP, rotenone, and antimycin A were added sequentially for detection.

[0030] 10) Flow cytometry analysis of mitochondrial quality: Fresh cells were stained with 50 nM MitoTracker at 37°C and 5% CO2 for 30 minutes to assess mitochondrial quality. After washing twice with cold PBS, cells were stained with surface antibodies and analyzed by flow cytometry.

[0031] 11) Establishment of an in vivo experimental model of Oligomycin in neonatal mice: Newborn mice were subcutaneously injected with 5×10⁻⁶ mmol / L on days 3-4 after birth. 5 BCG colony-forming units of CFU were used. On the next day, neonatal mice in the BCG group were injected intraperitoneally with 100ul / mouse or 1ug / mouse of Oligomycin. Three days later, M-MDSC and PMN-MDSC cells of the spleen of neonatal mice were sorted by flow cytometry for functional analysis.

[0032] 12) Statistical Analysis: Statistical analysis was performed using SPSS 20.0 and GraphPad Prismversion 8.0. Statistical values ​​are expressed as mean ± standard error (Mean ± SEM). For normally distributed data, the t-test was used for statistical analysis between two groups; one-way ANOVA was used for statistical analysis of three or more groups. For non-normally distributed data, the Mann-Whitney U test was used. Survival rates were analyzed using the log-rank (Mantel-Cox) test. In all tests, p < 0.05 was considered statistically significant. This means p < 0.05. This means p < 0.01. This indicates p < 0.001, ns: no significant difference.

[0033] The technical solution of the present invention will be described in detail below through specific embodiments, including the cell sources used in the following embodiments: BCG group: Newborn mice on the 3rd to 4th day after birth were subcutaneously injected with 5×10⁵ CFU of BCG vaccine; Control group: Newborn mice on days 3-4 after birth were subcutaneously injected with an equal volume of PBS.

[0034] On day 3 post-inoculation, M-MDSC and PMN-MDSC cells were isolated from the spleen of suckling mice.

[0035] Example 1: Transcriptional map of M-MDSC cells in neonatal rats remodeled by BCG vaccination Previous research has shown that BCG vaccination in neonatal mice leads to a decrease in the immunosuppressive function of myeloid-derived suppressor cells (MDSCs). This study further explores the molecular mechanism by which BCG vaccination regulates MDSCs in neonatal mice. Transcriptome analysis was performed using single-cell RNA sequencing. For this purpose, we isolated myeloid cells (CD3+) from the spleens of control neonatal mice that were BCG-vaccinated and treated with PBS. - CD19 - CD11b + Single-cell RNA sequencing was performed. Figure 1 A). Through unsupervised cluster analysis, based on the specific enrichment of genes, two distinct MDSC subsets were identified: PMN-MDSCs and M-MDSCs. Figure 1 B, C; Figure 2 Subsequent analysis focused on the M-MDSC subsets. The heatmap showing the most significant gene differences revealed substantial transcriptomic differences between M-MDSCs from the PBS and BCG groups. Figure 1D). Furthermore, KEGG pathway analysis confirmed that oxidative phosphorylation (OXPHOS) is a significantly enriched pathway in M-MDSCs after BCG vaccination. Figure 1 E).

[0036] The above data suggest that the high-resolution transcriptome atlas of M-MDSC cells in neonatal rats reveals the BCG-induced metabolic reprogramming phenomenon and promotes the oxidative phosphorylation process of M-MDSCs.

[0037] Example 2: Oxidative phosphorylation inhibitors restore immunosuppressive function in BCG-inoculated neonatal rat M-MDSC cells To further analyze the metabolic effects of BCG vaccination on neonatal rat M-MDSC cells, this invention performed RNA-seq sequencing on M-MDSC cells isolated from the spleens of neonatal rats in the BCG group and control group. GO analysis showed significant enrichment of oxidative phosphorylation-related pathways (…). Figure 3 A). Consistent with this result, the expression of key oxidative phosphorylation genes was significantly upregulated in M-MDSCs derived from BCG-treated suckling mice. Figure 3 B), this result was further validated by qRT-PCR ( Figure 3 C). MitoTracker staining showed a significant increase in mitochondrial mass in M-MDSC cells of BCG-inoculated suckling mice. Figure 3 D), the total ATP level in cells increased synchronously ( Figure 3 E), indicating enhanced oxidative metabolic activity. To establish a causal relationship between enhanced oxidative phosphorylation and loss of immunosuppressive function, this invention uses oligomycin to inhibit the oxidative phosphorylation pathway in BCG-exposed neonatal rat M-MDSC cells (E). Figure 3 F). Notably, oligomycin treatment restored CD8 uptake in neonatal rat M-MDSC cells. + and CD4 + The inhibitory effect of T cell proliferation ( Figure 3 These results demonstrate that BCG-induced enhanced oxidative phosphorylation directly impairs the immunosuppressive function in neonatal rat M-MDSC cells, and that the use of the oxidative phosphorylation inhibitor oligomycin can reverse the immunosuppressive function in BCG-exposed neonatal rat M-MDSC cells.

[0038] Example 3: BCG vaccination reconstructs the transcriptome of neonatal rat PMN-MDSC cells Given that PMN-MDSC cells are the major subpopulation within the MDSC population, this invention focuses on analyzing this cell population. Cluster analysis of single-cell sequencing data identified three subpopulations of PMN-MDSC cells with different transcriptional levels based on specifically enriched gene characteristics. Figure 4 A, B). GO analysis revealed that oxidative phosphorylation was a pathway significantly enriched in all PMN subpopulations after inoculation. Figure 4 C). Consistent with this finding, the expression of oxidative phosphorylation-related genes was significantly upregulated in the PMN1 and PMN2 subgroups of the BCG group. Figure 4 D). Metabolic score analysis further confirmed that the PMN1 subset had the highest baseline oxidative phosphorylation activity, and BCG inoculation significantly improved the oxidative phosphorylation scores of all PMN subsets. Figure 4 E). These data collectively demonstrate that BCG vaccination can reconstruct the transcriptome map in neonatal mouse PMN-MDSC cells and promote its oxidative phosphorylation process.

[0039] Example 4: Oxidative phosphorylation inhibitors restore immunosuppressive function in BCG-inoculated neonatal mouse PMN-MDSC cells KEGG pathway analysis using RNA-seq sequencing identified oxidative phosphorylation as one of the most significantly enriched pathways in BCG-inoculated neonatal mouse spleen PMN-MDSC cells. Figure 5 A). The expression of key oxidative phosphorylation genes was significantly upregulated ( Figure 5 B), qRT-PCR further verified the increased expression of oxidative phosphorylation genes in BCG-exposed PMN-MDSC cells ( Figure 5 C). MitoTracker staining showed a significant increase in mitochondrial mass in PMN-MDSC cells of BCG-inoculated suckling mice (C). Figure 5 D), the total ATP level in cells increased synchronously ( Figure 5 E). Through Seahorse metabolic flux analysis, this invention observed a significant increase in oxygen consumption rate in PMN-MDSC cells derived from BCG-treated suckling mice (E). Figure 5 F) provides functional evidence for enhanced mitochondrial respiratory function. To clarify the causal relationship between increased oxidative phosphorylation and loss of immunosuppressive function, this invention uses oligomycin to inhibit the oxidative phosphorylation pathway in BCG-exposed PMN-MDSC cells (F). Figure 5 G). Notably, oligomycin treatment restored CD8+ inhibition in PMN-MDSC cells derived from BCG-treated suckling mice. + The inhibitory effect of T cell proliferation ( Figure 5 These results collectively demonstrate that BCG-induced enhanced oxidative phosphorylation directly impairs the immunosuppressive function in neonatal rat PMN-MDSC cells, and that the use of the oxidative phosphorylation inhibitor oligomycin can reverse the immunosuppressive function in BCG-exposed neonatal rat PMN-MDSC cells.

[0040] Example 5: In vivo experiment: Oxidative phosphorylation inhibitors restore the immunosuppressive function of M-MDSCs and PMN-MDSCs in BCG-inoculated suckling mice. To further clarify the causal relationship between increased oxidative phosphorylation and loss of immunosuppressive function, this invention uses an in vivo mouse model experiment. Newborn mice aged 3-4 days were inoculated with PBS or BCG, or treated with the oxidative phosphorylation inhibitor oligomycin. Three days later, M-MDSC and PMN-MDSC cells from the spleen cells of the mice were separated by flow cytometry, and their immunosuppressive function was detected. Figure 6 A). This invention demonstrates that oligomycin treatment restores CD8+ protection in M-MDSC cells derived from BCG-inoculated suckling mice. + T and CD4 + The inhibitory effect of T cell proliferation ( Figure 6 B); Simultaneously, oligomycin treatment reversed the antigen-specific immunosuppressive function of BCG-inoculated neonatal mouse-derived PMN-MDSC cells (B). Figure 6 C). ROS is an important molecule regulating the immunosuppressive function of PMN-MDSC cells. This invention also shows that oligomycin treatment partially restores ROS expression levels in PMN-MDSCs derived from BCG-inoculated suckling mice. Figure 6 D).

[0041] These results collectively demonstrate that BCG-induced enhanced oxidative phosphorylation directly impairs the immunosuppressive function in neonatal rat MDSC cells, and that the use of oligomycin, an inhibitor of oxidative phosphorylation, in in vitro and in vivo experiments can reverse the immunosuppressive function in BCG-exposed neonatal rat MDSC cells.

[0042] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features of the invention herein.

Claims

1. The use of oxidative phosphorylation inhibitors in the preparation of immunomodulatory drugs, characterized in that, The immunomodulatory drug is used to prevent or treat neonatal immune-related diseases caused by BCG vaccination by antagonizing the functional damage of neonatal myeloid-derived suppressor cells induced by BCG vaccination.

2. The use according to claim 1, characterized in that: The oxidative phosphorylation inhibitor is oligomycin or its pharmaceutically acceptable salt.

3. The use according to claim 1, characterized in that: The neonatal immune-related disease mentioned is neonatal necrotizing enterocolitis.

4. The use according to claim 3, characterized in that: The newborns mentioned are premature or low birth weight infants.

5. The use according to claim 1, characterized in that: The immunomodulatory drug is an injectable form.

6. The use according to claim 1, characterized in that: The myeloid-derived suppressor cells are PMN-MDSCs or M-MDSCs.

7. The use according to claim 1, characterized in that: The functional impairment is manifested as a significant decrease in the ability of myeloid-derived suppressor cells to inhibit T cell proliferation.

8. The use according to claim 7, characterized in that: The T cells are CD8. + and CD4 + cell.

9. The use according to claim 1, characterized in that: The drug contains a therapeutically effective amount of an oxidative phosphorylation inhibitor and a pharmaceutically acceptable carrier.

10. The use according to claim 1, characterized in that: The immunomodulatory drug is used to restore the immunosuppressive function of MDSC cells.

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