Application of pyruvate dehydrogenase kinase 2 inhibitor in preparation of medicine for treating autoimmune hepatitis

Abstract

The invention belongs to the technical field of medicines and disease treatment, and particularly relates to application of a pyruvate dehydrogenase kinase 2 inhibitor in preparation of a medicine for treating autoimmune hepatitis. The amino acid sequence of the pyruvate dehydrogenase kinase 2 is as shown in SEQ ID NO. 1. Research finds that in an in-vivo experiment, compared with a Control group, PDK2 expression in liver tissue of a Con A group mouse is obviously increased; after the PDK2 inhibitor is injected into the intraperitoneal cavity of a mouse to inhibit the activity of the PDK2, the death rate of the mouse is obviously reduced, liver tissue damage is relieved, and the proportion of myeloid-derived suppressor cells (MDSCs) is obviously increased. In-vitro experiment results show that inhibition of PDK2 activity can up-regulate the proportion of MDSCs and promote expression of related immunosuppressive molecules iNOS and Arg1. The research further shows that the PDK2 aggravates the Con A-induced autoimmune hepatitis by inhibiting the aggregation of the MDSCs and the expression of the immunosuppressive factor of the MDSCs, and the aggregation of the MDSCs in the liver tissue and the expression of the related suppressive factor of the MDSCs can be obviously promoted by using the PDK2 inhibitor, so that the Con A-induced autoimmune hepatitis is relieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine and disease treatment, and particularly relates to the use of a pyruvate dehydrogenase kinase 2 inhibitor in the preparation of a medicine for treating autoimmune hepatitis. Background Art

[0002] Autoimmune hepatitis (AIH) is a severe, progressive disease mediated by an abnormal autoimmune response, with unclear etiology and pathogenesis. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of cells derived from the bone marrow that exert potent immunosuppressive effects by expressing and secreting immunosuppressive factors. Studies have shown that MDSCs are key cells in maintaining liver immune homeostasis. Pyruvate dehydrogenase kinase 2 (PDK2) is a key rate-limiting enzyme in oxidative phosphorylation of glucose metabolism.

[0003] Currently, standard clinical treatments for autoimmune hepatitis include glucocorticoids (such as prednisone), immunosuppressants (such as azathioprine and tacrolimus), and ursodeoxycholic acid. However, since these drugs generally relieve symptoms by nonspecifically suppressing the overall function of the immune system, long-term use can easily lead to a decline in immune function. Summary of the Invention

[0004] To address the problem that existing immunosuppressants generally alleviate symptoms by nonspecifically suppressing the overall function of the immune system, and that long-term use can easily lead to a decline in immune function, the present invention provides the use of a pyruvate dehydrogenase kinase 2 inhibitor in the preparation of a medicament for treating autoimmune hepatitis. To achieve this objective, the present invention employs the following technical solutions.

[0005] The present invention provides the use of an inhibitor of pyruvate dehydrogenase kinase 2 in the preparation of a medicament for treating autoimmune hepatitis. The amino acid sequence of the pyruvate dehydrogenase kinase 2 is shown in SEQ ID NO. 1: MRWVWALLKNASLAGAPKYIEHFSKFSPSPLSMKQFLDFGSSNACEKTSFTFLRQELPVRLANIMKEINLLPDRVLSTPSVQLVQSWYVQSLLDIMEFLDKD PEDHRTLSQFTDALVTIRNRHNDVVPTMAQGVLEYKDTYGDDPVSNQNIQYFLDRFYLSRISIRMLINQHTLIFDGSTNPAHPKHIGSIDPNCNVSEVVKDA YDMAKLLCDKYYMASPDLEIQEINAANSKQPIHMVYVPSHLYHMLFELFKNAMRATVESHESSLILPPIKVMVALGEEDLSIKMSDRGGGVPLRKIERLFSYMYSTAPTPQPGTGGTPLAGFGYGLPISRLYAKYFQGDLQLFSMEGFGTDAVIYLKALSTDSVERLPVYNKSAWRHYQTIQEAGDWCVPSTEPKNTSTYRVS.

[0006] Among them, the English abbreviation of pyruvate dehydrogenase kinase 2 is PDK2.

[0007] The gene version number of the nucleotide encoding the pyruvate dehydrogenase kinase 2 is NC_000017.11:50094737-50112152.

[0008] Preferably, the drug contains the pyruvate dehydrogenase kinase 2 inhibitor as an active ingredient and is supplemented with a pharmaceutically acceptable excipient or carrier.

[0009] Preferably, the medicament is used to upregulate the proportion of myeloid-derived suppressor cells in the liver.

[0010] Preferably, the pyruvate dehydrogenase kinase 2 inhibitor comprises at least one of dichloroacetic acid, dichloroacetate, dibromoacetic acid, difluoroacetic acid, myricetin, quercetin, rapamycin and AZD7545.

[0011] Preferably, the excipients include any one or more of a diluent, a binder, a disintegrant, a lubricant and a glidant.

[0012] Preferably, the carrier comprises any one or more of microcrystalline cellulose, a solvent, a matrix, a buffer salt and a surfactant.

[0013] Preferably, the drug is a solution.

[0014] The solution is prepared by mixing the pyruvate dehydrogenase kinase 2 inhibitor with a solvent.

[0015] The solvent includes any one of water, ethanol and glycerol.

[0016] Preferably, the concentration of the pyruvate dehydrogenase kinase 2 inhibitor in the solution is 1.25 μg / g to 5 μg / g.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides the use of a pyruvate dehydrogenase kinase 2 inhibitor in the preparation of a medicament for treating autoimmune hepatitis, providing a new method for treating autoimmune hepatitis. This pyruvate dehydrogenase kinase 2 inhibitor has significant immunomodulatory effects, effectively suppressing overactive immune responses by altering cellular metabolic reprogramming, reducing liver inflammation, and ameliorating liver cell damage. This addresses the problem that prior art immunosuppressants generally alleviate symptoms by nonspecifically suppressing the overall function of the immune system, and long-term use can easily lead to decreased immune function.

[0018] 2. This study investigates the regulation of pyruvate dehydrogenase kinase 2 (PDK2) on the aggregation and immunosuppressive function of MDSCs and examines its effects on a mouse model of concanavalin A (Con A)-induced autoimmune hepatitis (AIH). The results show that intraperitoneal administration of a PDK2 inhibitor significantly increases the proportion of MDSCs in the spleen. In vitro experiments revealed that inhibition of PDK2 activity also increased the proportion of MDSCs and the expression of iNOS and Arg1. In vivo experiments revealed a significant increase in PDK2 expression in the liver tissue of Con A-treated mice compared to the control group. Inhibition of PDK2 activity also reduced mouse mortality, alleviated liver tissue damage, and significantly increased the proportion of MDSCs. PDK2 may aggravate Con A-induced autoimmune hepatitis by inhibiting MDSC aggregation and the expression of immunosuppressive-related factors.

[0019] 3. This study focuses on the regulatory mechanisms of pyruvate dehydrogenase kinase 2 (PDK2), systematically revealing its key role in the pathogenesis of autoimmune hepatitis (AIH). By targeting the PDK2-mediated metabolic reprogramming pathway, it elucidates the molecular mechanisms underlying MDSC function. This innovative exploration of immune intervention strategies based on metabolic regulation aims to elucidate the dynamic regulatory network of the MDSCs metabolic-functional axis in the pathological progression of AIH. This provides multi-dimensional theoretical support for the development of novel therapeutic modalities targeting the immune metabolic microenvironment and offers an interdisciplinary research paradigm for the mechanistic study of other autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The expression of pyruvate dehydrogenase kinase 2 in the liver of the present invention; wherein, ( Figure 1A) The mRNA expression levels of pyruvate dehydrogenase kinase 2 in the livers of patients with autoimmune hepatitis (AIH) and healthy controls (normal individuals) were obtained from the GSE206364 dataset in the Gene Expression Omnibus (GEO). An autoimmune hepatitis mouse model was established. Concanavalin A (Con A, 20 mg / kg) was administered to the mice 12 hours later, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed ( Figure 1 B), Western blotting ( Figure 1 C and D) and immunofluorescence staining ( Figure 1 E and F) Detection of pyruvate dehydrogenase kinase 2 expression in mouse liver tissue.

[0021] Figure 2 This is a heat map of genes positively correlated (red) and negatively correlated (blue) with pyruvate dehydrogenase kinase 2 (PDK2) in AIH patients obtained by analyzing the autoimmune hepatitis (AIH) dataset in the present invention.

[0022] Figure 3 is the biological information obtained by analyzing the autoimmune hepatitis (AIH) dataset in the present invention. Figure 3 A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of genes positively correlated with PDK2; ( Figure 3 B) Gene Ontology (GO)-biological process analysis of genes positively correlated with PDK2; ( Figure 3 C) GO-cellular component analysis.

[0023] Figure 4 is the biological information obtained by analyzing the autoimmune hepatitis (AIH) dataset in the present invention. Figure 4 A) GO-molecular function analysis; ( Figure 4 B) Protein-protein interaction network analysis of mitogen-activated protein kinase (MAPK) pathway-related proteins that interact with PDK2.

[0024] Figure 5 The PDK2 inhibitor (PDK-IN) of the present invention can alleviate the pathological liver damage in mice with autoimmune hepatitis; wherein, ( Figure 5 A) Survival of mice after administration of concanavalin A (Con A, 25 mg / kg) (10 mice per group). Overall survival was estimated using the Kaplan-Meier method, and differences in survival were analyzed using the Log-rank test; ( Figure 5 B~ Figure 5E) Mice were injected with Con A (20 mg / kg) 2 hours after PDK-IN administration, and serum and liver tissues were collected 12 hours later; ( Figure 5 B and Figure 5 C) Analysis of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels; ( Figure 5 D and Figure 5 E) Hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining of liver tissue; green indicates PDK2, and blue indicates 4′,6-diamidino-2-phenylindole (DAPI).

[0025] Figure 6 The PDK2 inhibitor (PDK-IN) of the present invention can reduce the liver oxidative stress induced by concanavalin A (Con A); mice were first given PDK-IN for 2 hours, and then injected with Con A (20 mg / kg), and liver tissue was collected for detection 12 hours later; wherein, ( Figure 6 A) Glutathione (GSH) content in liver tissue; ( Figure 6 B) Superoxide dismutase (SOD) activity; ( Figure 6 C) Malondialdehyde (MDA) levels; ( Figure 6 D) Determination of myeloperoxidase (MPO) activity.

[0026] Figure 7 The PDK2 inhibitor (PDK-IN) of the present invention reduces the production of inflammatory cytokines in mice with autoimmune hepatitis; the mice were first given PDK-IN for 2 hours, followed by an injection of concanavalin A (Con A, 20 mg / kg), and serum and liver tissue were collected 12 hours later; enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin (IL)-6 ( Figure 7 A in), IL-12 / p40 ( Figure 7 B in), tumor necrosis factor (TNF)-α ( Figure 7 C) and IL-1β ( Figure 7 Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression of Il-6 ( Figure 7 E in), Il-12 / p40 ( Figure 7 F in), TNF-α ( Figure 7 G) and Il-β ( Figure 7 H) mRNA expression levels in .

[0027] Figure 8The PDK2 inhibitor (PDK-IN) of the present invention can inhibit the mitogen-activated protein kinase (MAPK) / nuclear factor-κB (NF-κB) signaling pathway; mice were first given the PDK2 inhibitor for 2 hours, followed by injection of concanavalin A (Con A, 20 mg / kg), and liver tissue was collected 12 hours later; wherein, ( Figure 8 A) Western blot analysis of p38 ( Figure 8 B), extracellular regulated protein kinase (ERK) ( Figure 8 C in), c-Jun N-terminal kinase (JNK) ( Figure 8 D in), and p65 protein ( Figure 8 E) Expression levels.

[0028] Figure 9 The PDK2 inhibitor (PDK-IN) of the present invention protects the liver by regulating the accumulation of myeloid-derived suppressor cells (MDSCs); mice pretreated with the PDK2 inhibitor (PDK2-IN) were injected with concanavalin A (Con A, 20 mg / kg) 12 hours later, and then hepatic mononuclear cells (HMNCs) were prepared; wherein, ( Figure 9 A~ Figure 9 E) Detection of CD11b in HMNCs by flow cytometry + Gr-1 + MDSCs ( Figure 9 C in the figure), CD11b + Ly6G + G-MDSCs ( Figure 9 D) and CD11b + Ly6C + M-MDSCs ( Figure 9 The ratio of E in ( Figure 9 F~ Figure 9 H) Western blot was used to detect the expression of arginase-1 (Arg-1) in liver tissue ( Figure 9 G) and inducible nitric oxide synthase (iNOS) ( Figure 9 The expression of H in Figure 9 (I and J) Its mRNA level was further verified by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR).

[0029] Figure 10 The PDK2 inhibitor (PDK-IN) of the present invention can enhance the immunosuppressive activity of myeloid-derived suppressor cells (MDSC) in vitro; wherein, ( Figure 10 A~ Figure 10E) Mouse bone marrow cells were cultured with different concentrations of PDK2 inhibitor (PDK2-IN, 10 μM, 20 μM, and 40 μM) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF, 40 ng / mL) and interleukin-6 (IL-6, 40 ng / mL) for 4 days. CD11b was detected by flow cytometry. + Gr-1 + MDSCs ( Figure 10 C in the figure), CD11b + Ly6G + G-MDSCs ( Figure 10 D) and CD11b + Ly6C + M-MDSCs ( Figure 10 The ratio of E in ( Figure 10 F~ Figure 10 H) Western blotting was used to detect the expression of arginase-1 (Arg-1) in MDSCs after treatment with PDK2-IN (10 μM, 20 μM, and 40 μM). Figure 10 G) and inducible nitric oxide synthase (iNOS) ( Figure 10 The expression level of H in ( Figure 10 Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was also used to further verify its mRNA expression level.

[0030] Figure 11 The CellTiter-Lumirn luminescence cell viability detection kit of the present invention was used to detect the viability of MDSCs after in vitro administration of PDK-IN to determine the in vitro drug concentration of PDK-IN. The optimal in vitro drug concentrations of PDK2-IN were determined to be 10 μM, 20 μM, and 40 μM. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0032] The mice used in the examples of this invention were all C57BL / 6 strains and housed in an SPF, specific pathogen-free animal facility. Laboratory Animal Use Permit Number: (No. JNMC-2023-DW-131).

[0033] Among them, the English name of specific pathogens is specific pathogen free, SPF.

[0034] All mice used in the experiment were 8-week-old male mice, and all mouse experiments complied with the "Regulations on the Use of Experimental Animals" formulated by the national and Jiangxi University of Traditional Chinese Medicine Ethics Committee.

[0035] Example 1 The present invention aims to verify the specific mechanism by which pyruvate dehydrogenase kinase 2 (PDK2) affects liver injury in AIH mice by regulating MDSCs and to explore the potential therapeutic effects of drug intervention. The following studies were conducted.

[0036] 1. Expression of PDK2 in liver tissue of autoimmune hepatitis (AIH) (1) Expression of PDK2 in liver tissue of patients with autoimmune hepatitis The expression of PDK2 in liver tissues of healthy individuals and patients with autoimmune hepatitis was analyzed using GSE206364 data obtained from the GEO database. Figure 1 As shown in Figure A, PDK2 expression was significantly increased in liver tissues of AIH patients compared with liver tissues of healthy individuals.

[0037] (2) Expression of PDK2 in Con A-induced AIH mouse model: According to the experimental design, C57BL / 6 mice were divided into a control group (control group) and a Con A group. The Con A group received a tail vein injection of Con A (20 mg / kg) to establish a liver injury model. The control group received a tail vein injection of the same volume of PBS. Twelve hours later, liver tissues were harvested and partially paraffin-embedded in 4% paraformaldehyde.

[0038] 1) Detection of PDK2 mRNA expression in liver tissues of the above two groups of mice by qRT-PCR ① Extraction of tissue RNA Take an appropriate amount of frozen liver tissue and add 1 mL of TRlzol to each tube, thoroughly grinding. Add 200 mL of chloroform, shake vigorously, invert and mix for 30 seconds, and let it rest for 5 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes. Carefully collect the upper aqueous phase into a 15 mL EP tube without enzyme, add an equal amount of isopropanol, gently invert and shake, and let it rest for 10 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes. Discard the supernatant, remove water with absorbent paper, add 700 mL of 75% by volume DEPC water and ethanol, and gently invert to suspend the precipitate. Centrifuge at 7,500 rpm at 4°C for 5 minutes. Discard the supernatant, remove excess water with absorbent paper, and bake in a 37°C oven for 10 minutes. Add DEPC water according to the amount of RNA, and place in a 56°C thermostat for 10 minutes to dissolve the RNA sample. Take 2 μL of the RNA sample and measure the OD value.

[0039] ②Reverse transcription synthesis of cDNA Reaction system: 5× reaction buffer - 2 mL.

[0040] 10 mmol / L dNTP mixture - 1 μL.

[0041] RNase inhibitor - 0.5 μL.

[0042] Reverse transcriptase - 0.5 μL.

[0043] Oligo (dT) 18 primer - 0.5 μL.

[0044] Sample - calculated based on sample concentration.

[0045] DEPC water - 5.5 μL - sample volume.

[0046] Reaction program: 42°C, 60 min, 70°C, 5 min, 4°C.

[0047] ③Real-time fluorescence quantitative PCR: PCR primer sequences are shown in Table 1: Table 1 PCR primers Reaction system: cDNA (dilute the above reverse transcription product 20-fold) - 4 μL.

[0048] Upstream primer - 0.5 μL.

[0049] Downstream primer - 0.5 μL.

[0050] SYBR Green q-PCR Supermix-5μL.

[0051] Amplify using appropriate reaction conditions according to the primer instructions. -△△Ct Methods Analytical data.

[0052] The results are as follows Figure 1 As shown in Figure B, the expression of PDK2 in the liver tissue of AIH mice was significantly higher than that in the control mice.

[0053] 2) Detection of PDK2 protein expression in liver tissues of the above two groups of mice by Western blotting ① Preparation of protein samples: Excise liver tissue, add protein lysis buffer, grind thoroughly, and shake on a shaker at 4°C for 10 minutes to ensure complete cell lysis. Centrifuge at 12,000 rpm for 5 minutes at 4°C. Transfer the supernatant to a new set of EP tubes. Add 5x loading buffer to the protein sample to a final concentration of 1x. Boil the protein at 99°C for 8 minutes. Chill on ice, then aliquot and freeze for later use.

[0054] ②Prepare SDS-PAGE gel and electrophoresis: Wash the glass plates and air-dry them for later use. Determine the concentration of the separating gel based on the molecular weight of the target protein. Prepare the separating gel and fill it three-quarters of the way up the glass plates. Flatten it with 75% ethanol by volume. Once a clear separation is visible, discard the ethanol and remove as much liquid as possible by aspirating. Prepare the stacking gel and fill it between the glass plates, carefully inserting a comb to prevent air bubbles from forming around the comb. Allow the separating gel to solidify after approximately 40 minutes. Place the glass plates in the electrophoresis tank and add the electrophoresis solution (starting between the glass plates) until the comb is submerged, then remove the comb vertically. Slowly add the sample using a 10µm range gun and level each well with a 1x loading buter. Run the electrophoresis at room temperature for approximately 40 minutes at a constant voltage of 80V. Once a red band of the protein marker appears, increase the voltage to 120V. Determine the run time based on the location of the target protein.

[0055] ③ Film transfer and exposure: After electrophoresis, remove the glass plate and retain a specific area of ​​gel according to the protein marker's instructions. Place the gel in a container filled with transfer buffer until ready for use. Based on the area of ​​gel retained, cut a PVDF membrane of appropriate size (the pore size of the membrane should be determined by the size of the target protein) and immerse it in methanol for approximately 1 minute. Prepare the transfer "sandwich": Arrange the transfer template in the following order: black side of the transfer template, sponge, filter paper, gel, PVDF membrane, filter paper, sponge, and transparent side of the transfer template. Be careful not to trap air bubbles between the gel and PVDF membrane. Place the transfer "sandwich" in an electroporation tank, fill it with pre-chilled transfer buffer, and place it in zero-degree water to prevent overheating during transfer. Maintain a constant voltage of 100V and adjust the transfer time based on the molecular weight of the target protein. Disconnect the power supply to stop the transfer, separate the front and back of the PVDF membrane, and block it with 3% BSA on a shaker at low speed for 2 hours. Dilute the primary antibody in TBST and incubate the PVDF membrane overnight at 4°C. The next day, wash the membrane five times with TBST (10 minutes each). Dilute the secondary antibody in TBST and incubate the PVDF membrane at room temperature for 2 hours. Wash the membrane six times with TBST (10 minutes each). Mix equal amounts of ECL Reagent Solution A and Solution B. Place the PVDF membrane flat, front-side up, on a dedicated tray. Add the prepared ECL substrate to the membrane and carefully place it in a gel imager for automatic exposure. Analyze and save the imaging results.

[0056] The results are as follows Figure 1 C and Figure 1 As shown in D: Compared with the Control group, the expression level of PDK2 protein in the liver tissue of mice in the Con A group was significantly increased, and the difference was statistically significant ( P <0.01).

[0057] (3) The expression and localization of PDK2 in the liver tissues of the two groups of mice were detected by immunofluorescence histochemistry staining.

[0058] Paraffin-embedded liver tissue sections were routinely dewaxed and hydrated. Antigen retrieval was performed on the sections using microwave-assisted citrate buffer. The sections were then left to air at room temperature and washed three times with PBS for 5 minutes each. Blocking serum was added dropwise and incubated at room temperature for 20 minutes. The serum was then removed. Rabbit anti-PDK2 polyclonal antibody (1:200) was added dropwise and incubated overnight at 37°C. The sections were then washed three times with PBS for 5 minutes each. AlexaFluor® 647-labeled goat anti-rabbit antibody (1:500) was added dropwise and incubated at room temperature for 20 minutes. The sections were then washed five times with PBS for 5 minutes each. 5 μg / mL DAPI was added dropwise and incubated at room temperature for 30 minutes. The sections were then washed three times with PBS, mounted with an anti-fluorescence quencher, and observed under a fluorescence microscope.

[0059] The results are as follows Figure 1 As shown in Figures E and F, the expression of PDK2 in the liver tissues of mice in the Con A group was significantly higher than that in the control group. Western and immunofluorescence histochemical staining results showed that PDK2 is involved in the development of autoimmune hepatitis.

[0060] 2. Bioinformatics analysis of autoimmune hepatitis (1) Data preprocessing and identification of differentially expressed genes In R language, the getGEO function was used to download the relevant data set from the GEO database. The GEOquery package was used to process the data and generate an expression matrix. Subsequently, differential expression analysis was performed, and the screening criteria for differentially expressed genes were set as follows: P The value was less than 0.05 and the |logFC| was greater than 1. Next, the dplyr package was used to filter the PDK2 expression profile in the normal and autoimmune hepatitis groups from the overall data set. The Pearson correlation coefficient between PDK2 and other genes in the dataset was calculated. Based on the resulting correlation coefficient, genes were categorized as positively correlated (correlation coefficient > 0.5) or negatively correlated (correlation coefficient < -0.5) with PDK2. Finally, heatmaps were created in R using the heatmap package to visualize the expression patterns of genes positively and negatively correlated with PDK2.

[0061] (2) Bioinformatics analysis To explore the biological function of PDK2 in autoimmune hepatitis, we identified a group of genes positively correlated with PDK2 in AIH patients ( r >0.7; see Figure 2 The KEGG and GO enrichment analyses were performed. KEGG pathway analysis was performed using the clusterAnalyzer toolkit in the clusterProfiler package in R. A list of genes positively correlated with PDK2 was extracted from the previous analysis. Subsequently, the enrichKEGG function in the clusterProfiler package was used to search the KEGG database using these genes as input to identify significantly enriched pathways. P Pathways with a correlation ratio <0.05 were considered to be key biological processes in which genes positively correlated with PDK2 may be involved.

[0062] To further elucidate the functions of these genes, we also performed Gene Ontology (GO) enrichment analysis, covering three components: biological process (BP), cellular component (CC), and molecular function (MF). GO enrichment analysis was performed in the R statistical computing environment using the enrichGO function in the clusterProfiler package. A list of genes positively correlated with PDK2 was compiled from the previous correlation analysis and used for GO enrichment analysis. After the analysis, the enrichment results for MF, CC, and BP were visualized separately. Scatter plots were created for each GO category using the dotplot function in the clusterProfiler package to visually demonstrate the significance and abundance of the enrichment results.

[0063] The results showed that the genes upregulated by PDK2 were mainly enriched in the mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB) and Ras signaling pathways (see Figure 3 In biological process (BP) enrichment analysis, these genes were mainly related to proteasome-mediated ubiquitin-dependent protein degradation, response to oxidative stress, and ubiquitin-dependent protein degradation (see Figure 3 B in Figure 1). Cellular component (CC) enrichment results showed that these genes were mainly enriched in mitochondria, endoplasmic reticulum membrane and mitochondrial matrix (see Figure 3 C in ). Molecular function (MF) enrichment involves RNA binding, ubiquitin protein ligase binding, and oxidoreductase activity (see Figure 4 A in Figure 1). We then integrated genes positively correlated with PDK2, screened for genes enriched in the MAPK pathway, and constructed a protein-protein interaction (PPI) network. The CytoHubba plug-in was used to calculate the node degrees and rank them, and Cytoscape was used to screen out 17 key hub genes (see Figure 4B in the figure), its functions are mainly enriched in processes such as cell proliferation, differentiation, apoptosis and oxidative stress.

[0064] 3. Effects of PDK2-IN inhibition of PDK2 activity on liver damage in AIH mice: (1) Experimental groups and treatments C57BL / 6 mice were divided into control group, PDK2-IN group, Con A group, and Con A+PDK2-IN group. The treatment method of each group was as follows: Control group: 100 μL of PBS was injected into the tail vein 2 h later, and then 100 μL of PBS was injected into the tail vein again to serve as the control group.

[0065] PDK2-IN group: 100 μL of PDK2-IN (5 μg / g) was injected into the tail vein 2 h later, followed by 100 μL of PBS injection into the tail vein.

[0066] Con A group: 100 μL of PBS was injected into the tail vein 2 h later, followed by 100 μL of Con A (20 mg / kg) injected into the tail vein.

[0067] Con A+PDK2-IN group: 100 μL of PDK2-IN (1.25 μg / g, 2.5 μg / g, and 5 μg / g) was injected into the tail vein 2 h before 100 μL of Con A (20 mg / kg) was injected into the tail vein.

[0068] PDK2-IN is AZD7545, which is sourced from MedChemExpress.

[0069] The concentration of PBS was 10 mM, pH 7.3, and the source was Guangzhou Jiecheng Technology Co., Ltd.

[0070] (2) Detect serum ALT and AST levels using a fully automatic biochemical analyzer Twelve hours after Con A was injected into the tail vein of mice, the eyeballs were removed and blood was collected. After centrifugation at 4000 rpm for 10 min, serum was collected and the levels of ALT and AST in serum were measured using Roche cobas8000 automatic biochemical analyzer.

[0071] See the results Figure 5 B and Figure 5 Figure C: There were no significant differences in serum ALT and AST levels between the control and PDK2-IN groups, but these levels were significantly higher in the Con A group. Compared with the Con A group, the Con A + PDK2-IN (1.25 μg / g, 2.5 μg / g, and 5 μg / g) groups showed statistically significant decreases in ALT and AST levels.

[0072] (3) Observation of liver histological changes using HE staining Liver tissue was collected from mice, fixed in 4% paraformaldehyde, embedded in paraffin, cut into 5-μm sections, and stained with hematoxylin-eosin (H&E). Pathological changes in the liver tissue were observed under a light microscope.

[0073] (4) Detection of cell apoptosis in liver tissue using TUNEL Paraffin-embedded pathological sections were placed in a 65°C oven for 2 hours. The sections were then placed in xylene for 10 minutes, twice, 100% alcohol (volume percentage) for 5 minutes, twice, and 95% alcohol (volume percentage), 90% alcohol (volume percentage), 85% alcohol (volume percentage), 80% alcohol (volume percentage), and 75% alcohol (volume percentage) for 3 minutes each. The sections were washed with PBS for 5 minutes, then excess liquid was removed. 200 μg / mL proteinase K working solution was added dropwise and incubated at 37°C for 30 minutes. The sections were washed with PBS for 5 minutes, three times, and excess liquid was removed. TUNEL assay solution was prepared by adding 2 μL of enzyme to 48 μL of labeling solution per sample, adding 50 μL of TUNEL assay solution to each sample and 50 μL of labeling solution to the negative control. The sections were incubated at 37°C in the dark for 60 minutes. The sections were washed with PBS for 5 minutes, three times in the dark. Antiquencher was added, and the sections were covered with a coverslip. The sections were stored in the dark and images were acquired using a fluorescence microscope.

[0074] HE and TUNEL test results are as follows Figure 5 D and Figure 5 As shown in Figure E: Liver tissue was normal in mice in the control and PDK2-IN groups. However, liver tissue architecture was disrupted in mice in the ConA group, with significant hepatocellular necrosis, disorganized hepatic cords, hepatocellular nuclear dissolution, and significant hemorrhage and inflammatory cell infiltration. In contrast, mice treated with PDK2-IN (5 μg / g) showed significant improvement in liver function indicators, with a decrease in hepatocellular apoptosis.

[0075] (5) Observation of mouse mortality The mice in the Con A+PDK2-IN group were injected with 100 μL of PDK2-IN (5 μg / g) through the tail vein 2 hours later, and then injected with 100 μL of a lethal dose of Con A (25 mg / kg) through the tail vein. The mice in the Con A group were injected with 100 μL of PBS through the tail vein 2 hours later, and then injected with 100 μL of a lethal dose of Con A (25 mg / kg) through the tail vein. The survival rates of the mice in the two groups were observed.

[0076] See the results Figure 5 A: The survival rate of mice in the Con A+PDK2-IN group was significantly higher than that in the Con A group, and the difference was statistically significant.

[0077] (6) Liver tissue oxidative stress detection The levels of superoxide dismutase (SOD), myeloperoxidase (MPO), malondialdehyde (MDA), and glutathione (GSH) in the liver were measured using corresponding kits according to the instructions, and the level of oxidative stress in liver tissue was comprehensively assessed.

[0078] The results are as follows Figure 6 As shown in Figure 2, after injection of Con A into the tail vein, the oxidative stress level in the liver of mice was significantly increased; while after injection of PDK2-IN, the activities of GSH and SOD in the liver tissue were significantly increased (see Figure 6 A and Figure 6 B in the figure), MDA levels were significantly reduced (see Figure 6 MPO, a marker of neutrophil infiltration and oxidative damage, was significantly decreased in the PDK2-IN pretreatment group compared with the Con A group (see Figure 6 These results indicate that PDK2-IN can significantly inhibit the oxidative stress response induced by autoimmune hepatitis (AIH), thereby protecting the liver.

[0079] (7) Detect the levels of inflammatory cytokines IL-6, IL-12, TNF-α, and IL-1β in serum by ELISA. The specific method is as follows:

[0080] Coat a 96-well plate with 100 μL of antibody and incubate overnight at 4°C. Shake the plate clean, add 200 μL of Washbufer, tap gently, and wash four times. Add 200 μL of Assay dilute, and block at 37°C on a thermostatted shaker for 1 hour. Prepare standards: Dissolve and serially dilute the standards in Assay dilute. Prepare samples: Use Assay dilute to prepare samples to an appropriate concentration so that they are covered by the standard curve. Shake the plate clean, add 200 μL of Washbufer, tap gently, and wash four times. Add 100 μL of sample and standard to each well, in duplicate, and incubate at 37°C on a thermostatted shaker for 2 hours. Prepare detection antibodies in Assay dilute. Shake the plate clean, add 200 μL of Wash buffer, tap gently, and wash four times. Add 100 μL of detection antibody to each well, and incubate at 37°C on a thermostatted shaker for 1 hour. Prepare Assay Dilute with horseradish peroxidase-labeled avidin. Shake the plate clean of detection antibody, add 100 μL of horseradish peroxidase-labeled avidin, and incubate on a constant temperature shaker at 37°C for 30 minutes. Shake the plate clean, add 200 μL of Wash Buffer, tap gently, and wash five times. Add TMB for color development. When the appropriate color develops, add stop buffer and analyze.

[0081] The results are as follows Figure 7 A~ Figure 7 As shown in D: The IL-6, IL-12, TNF-α and IL-1β protein levels in the serum of mice in the Con A group + PDK-IN (1.25μg / , 2.5μg / and 5μg / g) groups were significantly lower than those in the Con A group, especially in the PDK-IN 2.5μg / g and 5μg / g dose groups.

[0082] (8) Detect the mRNA levels of inflammatory cytokines IL-6, IL-12, TNF-α, and IL-1β in the liver tissue of AIH mice by qRT-PCR. The specific method is as follows:

[0083] 1) Extraction of tissue RNA: Take an appropriate amount of frozen liver tissue and add 1 mL of TRlzol to each tube, thoroughly grinding. Add 200 mL of chloroform, shake vigorously, invert and mix for 30 seconds, and let it rest for 5 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes. Carefully collect the upper aqueous phase into a 15 mL EP tube without enzyme, add an equal amount of isopropanol, gently invert and shake, and let it rest for 10 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes. Remove the supernatant, remove moisture with absorbent paper, add 700 mL of 75% by volume DEPC water and ethanol, and gently invert to suspend the precipitate. Centrifuge at 7,500 rpm at 4°C for 5 minutes. Remove the supernatant, remove excess moisture with absorbent paper, and bake in a 37°C oven for 10 minutes. Add DEPC water according to the amount of RNA, and place in a 56°C thermostat for 10 minutes to dissolve the RNA sample. Take 2 μL of the RNA sample and measure the OD value.

[0084] 2) Reverse transcription and cDNA synthesis: Reaction system: 5× reaction buffer - 2 mL.

[0085] 10 mmol / L dNTP mixture - 1 μL.

[0086] RNase inhibitor - 0.5 μL.

[0087] Reverse transcriptase - 0.5 μL.

[0088] Oligo (dT) 18 primer - 0.5 μL.

[0089] Sample - calculated based on sample concentration.

[0090] DEPC water - 5.5 μL - sample volume.

[0091] Reaction program: 42°C, 60 min, 70°C, 5 min, 4°C.

[0092] 3) Real-time fluorescence quantitative PCR: PCR primer sequences are shown in Table 2: Table 2 PCR primers Reaction system: cDNA (dilute the above reverse transcription product 20-fold) - 4 μL.

[0093] Upstream primer - 0.5 μL.

[0094] Downstream primer - 0.5 μL.

[0095] SYBR Green q-PCR Supermix-5μL.

[0096] Amplify using appropriate reaction conditions according to the primer instructions. -△△Ct Methods Analytical data.

[0097] The results are as follows Figure 7 E~ Figure 7 As shown in Figure H, compared with the Con A group, the mRNA levels of IL-6, IL-12, TNF-α, and IL-1β in liver tissue of mice in the Con A + PDK-IN (1.25 μg / g, 2.5 μg / g, and 5 μg / g) groups were significantly decreased. This result was consistent with the trend of IL-6, IL-12, TNF-α, and IL-1β levels in serum. ELISA and qRT-PCR showed that PDK2-IN, after inhibiting PDK2 activity, significantly reduced the levels of inflammatory cytokines IL-6, IL-12, TNF-α, and IL-1β in Con A-induced AIH mice.

[0098] (9) Detection of phosphorylation levels of related proteins in the MAPK / NF-κB signaling pathway in liver tissue of AIH mice by Western blot Mouse liver tissue was treated with lysis buffer to extract protein, and protein concentration was subsequently determined using a BCA protein quantification kit. Appropriate amounts of protein samples were separated by SDS-PAGE and blotted onto a 0.45 μm pore size polyvinylidene fluoride membrane. The membrane was blocked with 5% (v / v) skim milk and incubated with specific primary antibodies targeting p38, phosphorylated p38 (p-p38), c-Jun N-terminal kinase (JNK), p-JNK, ERK, p-ERK, p65, and p-p65 (all diluted 1:1000) overnight at 4°C. After washing to remove unbound primary antibody, the membrane was incubated with the corresponding secondary antibody for 2 hours at room temperature. Finally, chemiluminescence detection was performed.

[0099] The MAPK / NF-κB signaling pathway plays an important role in cell apoptosis, oxidative stress, and inflammatory response. To explore the effect of PDK2-IN on the signaling pathway related to autoimmune hepatitis, the present invention analyzed the expression levels of proteins related to this pathway in the liver of autoimmune hepatitis mice. Western blot results are shown in Figure 2. Figure 8 As shown, the phosphorylation levels of p38, ERK, JNK, and p65 were significantly decreased in the PDK2-IN-treated group compared with the untreated control group. However, there was no significant difference in the expression levels of total p38, ERK, JNK, and p65 proteins between the two groups. These results suggest that PDK2 promotes the activation of the MAPK / NF-κB signaling pathway in the livers of mice with autoimmune hepatitis. The PDK2 inhibitor PDK2-IN can inhibit the activation of the MAPK / NF-κB signaling pathway, thereby alleviating the inflammatory response in the liver tissue of AIH mice.

[0100] (10) Flow cytometry detection of MDSCs in the liver 1) Preparation of liver mononuclear cells: After anesthetizing mice, the heart and liver were exposed. Blood was collected from the right atrial appendage, and 20 mL of PBS was injected into the cardiac apex to rinse the liver until it turned grayish white. The liver was then ground onto a 200-mesh copper grid to a single-cell suspension and centrifuged at 3000 rpm for 10 minutes. The supernatant was discarded, and the cells were resuspended in 45 mL of PBS and centrifuged at 400 rpm for 5 minutes. The supernatant was transferred to a fresh 50 mL centrifuge tube and centrifuged at 2500 rpm for 8 minutes. The supernatant was discarded, and the cells were resuspended in 3 mL of 40% by volume Percoll. The resuspended cells were then layered on top of 2 mL of 70% by volume Percoll. Differential centrifugation was performed at 2500 rpm for 30 minutes. The grayish-white layer was aspirated into a 15 mL centrifuge tube, washed twice with PBS, and the supernatant was discarded. The resuspended cells were then used to obtain liver mononuclear cells. The cell concentration was adjusted with PBS for flow cytometry.

[0101] 2) Flow cytometry to detect the number of MDSCs in the liver: The mouse liver mononuclear cell suspension prepared above was transferred to a flow cytometry tube, and the corresponding volume of antibodies (CD11b-FITC, Gr-1-APC, Ly6G-PE, and Ly6C-APC) was added. After mixing, the tube was incubated at 4°C for 30 minutes. After adding PBS, the tube was centrifuged at 1500 rpm for 5 minutes. The supernatant was removed and the cells were suspended. 300 μL of PBS was added and filtered before detection on the flow cytometer.

[0102] The results are as follows Figure 9 A~ Figure 9Figure E shows that during the pathogenesis of AIH, MDSCs migrate to the liver, and the number of MDSCs in the liver tissue of AIH mice is significantly increased. However, the proportion of MDSCs in the liver tissue of the Con A group is significantly higher than that of the Con A+PDK2-IN group. That is, after PDK2 inhibition, the aggregation of MDSCs in the liver of AIH mice is significantly promoted, especially the accumulation of the G-MDSC subpopulation is significantly increased.

[0103] (11) Western blot and qRT-PCR were used to detect the protein and mRNA expression levels of Arg-1 and iNOS in the liver tissue of AIH mice The specific operation method is as described above. The relevant primer sequences are shown in Table 3:

[0104] Table 3 Related primers See the results Figure 9 F~ Figure 9 In the J, the expression levels of MDSCs-associated inhibitory molecules Arg-1 and iNOS in the livers of AIH mice treated with PDK2-IN were significantly increased compared with those in the untreated group. These results suggest that PDK2 may exacerbate the pathological progression of AIH by regulating the recruitment and function of MDSCs, and that PDK2-IN can significantly alleviate the pathological damage of AIH.

[0105] 4. In vitro induction of mouse bone marrow-derived MDSCs. Treatment of in vitro induced MDSCs with different concentrations of PDK2-IN: (1) Induction of mouse bone marrow-derived MDSCs in vitro After killing the mice by cervical dislocation, they were immersed in 75% alcohol by volume for 5 minutes. Under sterile conditions in a clean bench, the femurs and tibias and fibulae of the mice were removed, and the muscle tissue was removed. PBS was aspirated with a 1 mL syringe to flush the bone marrow cavity until it turned white. The washed cells were filtered into a 15 mL tube and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and the tube was mixed. 1 mL of red blood cell lysis buffer was added to remove red blood cells. After 5 minutes, 5 mL of PBS was added to stop the red blood cell lysis. The tube was centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and 1 mL of DMEM complete medium was added to resuspend the tube to obtain fresh mouse bone marrow cell suspension. The bone marrow cell suspension was diluted to 3×10 with DMEM complete medium containing GM-CSF (10 ng / mL) and IL-6 (10 ng / mL). 5 The cells were seeded into 24-well flat-bottom culture plates and cultured in a carbon dioxide cell culture incubator (37°C, 5% CO2) for 4 days to obtain MDSCs.

[0106] (2) Determine the in vitro concentration of PDK-IN using the CellTiter-LumiTM luminescence assay ATP is a key indicator of cellular metabolism, and its content closely reflects the number of viable cells. ATP-dependent luciferase-catalyzed luciferin luminescence allows for quantitative analysis of intracellular ATP. This experiment used the CellTiter-Luminescent Cell Viability Assay Kit to assess MDSC viability after in vitro administration of PDK-IN to determine the in vitro PDK-IN concentration. The specific steps are as follows:

[0107] A black opaque 96-well plate was used and 1 × 10 5 For cells in good growth condition, different concentrations of PDK-IN (0μM, 2.5μM, 5μM, 10μM, 20μM, 40μM and 80μM) were added to different culture wells. A culture medium well without cells was set as a negative control, and the cells were cultured according to the conventional method of MDSCs culture. After the culture was completed, the frozen CellTiter-Lumirm luminescence detection reagent was thawed and equilibrated to room temperature. The cell culture plate was taken out and equilibrated at room temperature for 10 minutes. 100μL CellTiter-Lumirm luminescence detection reagent was added to each well and shaken at room temperature for 2 minutes to promote cell lysis. Incubate at room temperature for 10 minutes to stabilize the luminescence signal. Chemiluminescence detection was performed using a multifunctional enzyme reader and the data was recorded. The results are as follows Figure 11 As shown, the in vitro drug concentrations of PDK-IN were determined to be 10 μM, 20 μM, and 40 μM.

[0108] To further explore the effects of PDK2 on MDSCs, MDSCs derived from mouse bone marrow cells were treated with different doses of PDK2-IN. After four days of culture, the percentage of MDSCs was assessed by flow cytometry, and Arg-1 and iNOS levels in MDSCs were detected using qRT-PCR and Western blotting.

[0109] (3) Flow cytometry was used to detect the effects of different concentrations of PDK-IN on MDSCs differentiation The cultured MDSCs were collected into flow cytometry tubes (1×10 6 / tube), 1500 rpm, centrifuge for 5 min, discard the supernatant, suspend the cells, add 100 μL PBS, and perform flow cytometry antibody staining (CD11b-FITC, Gr-1-APC, Ly6G-PE, and Ly6C-APC). Incubate at 4°C for 30 min, add PBS, centrifuge at 1500 rpm for 5 min, remove the supernatant, suspend the cells, add 300 μL PBS, filter, and then detect on the flow cytometer.

[0110] (4) qRT-PCR detection of the effects of different concentrations of PDK-IN on the levels of iNOS and Arg-1 mRNA expressed in MDSCs (5) Western blot was used to detect the effects of different concentrations of PDK-IN on the expression levels of iNOS and Arg-1 proteins in MDSCs The results are as follows Figure 10 As shown in the results, PDK2-IN significantly increased the percentage of total MDSCs, especially G-MDSCs, and increased the expression of Arg-1 and iNOS in MDSCs, indicating that PDK2 inhibited the expansion of MDSCs, while PDK2-IN treatment promoted the expansion of MDSCs.

[0111] Among them, PDK2-IN refers to the PDK2 inhibitor AZD7545, which comes from MedChemExpress.

[0112] These experimental results demonstrate that pyruvate dehydrogenase kinase 2 (PDK2) plays a crucial role in the pathogenesis of autoimmune hepatitis (AIH). Experimental data demonstrate that PDK2-IN treatment significantly improves the prognosis of AIH mice by: 1) improving mouse survival; 2) reducing serum ALT and AST levels; 3) alleviating liver pathological damage and cell apoptosis; 4) inhibiting the production of proinflammatory cytokines in serum and liver tissue; and 5) blocking activation of the MAPK / NF-κB signaling pathway. Furthermore, PDK2-IN effectively alleviates hepatic oxidative stress and promotes the accumulation of MDSCs. Mechanistic studies have demonstrated that PDK2 inhibition ameliorates Con A-induced AIH liver injury through multiple pathways, including enhancing MDSC accumulation, alleviating oxidative stress, inhibiting MAPK / NF-κB signaling activation, and modulating inflammatory responses. Based on these findings, PDK2 is expected to become a new target for AIH treatment, and its inhibitors hold great promise in the development of AIH therapeutics.

[0113] It should be noted that when the present invention involves a numerical range, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes a preferred embodiment.

[0114] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once the basic inventive concepts become known, and all such changes and modifications fall within the scope of the present invention.

Claims

1. Use of an inhibitor of pyruvate dehydrogenase kinase 2 in the preparation of a medicament for treating autoimmune hepatitis, characterized in that: The amino acid sequence of the pyruvate dehydrogenase kinase 2 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The medicine uses the pyruvate dehydrogenase kinase 2 inhibitor as an active ingredient and is supplemented with a pharmaceutically acceptable excipient or carrier.

3. The use according to claim 2, characterized in that The drug is used to upregulate the proportion of myeloid-derived suppressor cells in the liver.

4. The use according to claim 2, characterized in that The pyruvate dehydrogenase kinase 2 inhibitor includes at least one of dichloroacetic acid, dichloroacetate, dibromoacetic acid, difluoroacetic acid, myricetin, quercetin, rapamycin and AZD7545.

5. The use according to claim 2, characterized in that The excipients include any one or more of a diluent, a binder, a disintegrant, a lubricant and a glidant.

6. The use according to claim 2, characterized in that The carrier comprises any one or more of microcrystalline cellulose, a solvent, a matrix, a buffer salt and a surfactant.

7. The use according to claim 2, characterized in that The medicine is a solution; The solution is prepared by mixing the pyruvate dehydrogenase kinase 2 inhibitor and a solvent; The solvent includes any one of water, ethanol and glycerol.

8. The use according to claim 7, characterized in that The concentration of the pyruvate dehydrogenase kinase 2 inhibitor in the solution is 1.25 μg / g to 5 μg / g.

Images