Application of UGCG enzyme inhibitor in preparation of medicine for treating sepsis

Abstract

The invention provides an application of a UGCG enzyme inhibitor in preparation of a medicine for treating sepsis, and particularly relates to an application of a UGCG enzyme activity inhibitor Eliglulukast as a medicine for treating sepsis. The UGCG enzyme is remarkably increased in sepsis patients, and organ injury and systemic inflammatory response related to sepsis are effectively relieved by inhibiting the expression quantity or activity of the UGCG enzyme, influencing MIF release and inhibiting PI3K / AKT / mTOR signal channel activation, so that the survival rate of the sepsis patients is remarkably increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a UGCG enzyme inhibitor in the preparation of a medicament for treating sepsis. Background Art

[0002] UDP-glucose ceramide glucosyltransferase (UGCG) is a biosynthetic enzyme widely present in the Golgi apparatus of neurons (primarily Schwann cells and oligodendrocytes). Glycosphingolipids derived from UGCG have been shown to play a role in myelin synthesis, neural development and growth, and regulating the activity of some transmembrane receptors. Eliglustat, also known as eliglustat, eliglustat, and eliglustat, is a ceramide-like substance and an inhibitor of UGCG enzyme activity. It is indicated for the long-term treatment of adult patients with type 1 Gaucher disease who are poor, intermediate, or extensive metabolizers as determined by cytochrome P450 2D6 (CYP2D6) genotyping.

[0003] Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. According to the Sepsis 3.0 diagnostic criteria, sepsis is diagnosed with a score of ≥2 on the SOFA scale and has a mortality rate exceeding 10%. Septic shock, defined as sepsis combined with severe circulatory, cellular, and metabolic abnormalities, has a mortality rate exceeding 40%. With the rapid aging of the population, the incidence of sepsis is increasing annually, placing a heavy burden on the socioeconomic landscape. Therefore, finding more effective treatments and drugs for sepsis has become an urgent need in this field. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide an inhibitor of UGCG enzyme activity for use in the preparation of a drug for treating sepsis.

[0005] To achieve this object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides the use of a UDP-glucoseceramide glucosyltransferase (UGCG) inhibitor in the preparation of a medicament for treating sepsis.

[0007] In the present invention, it was found in a clinical cohort that UGCG levels were significantly elevated in patients with sepsis. By constructing a mouse model of sepsis induced by LPS or Klebsiella pneumoniae (KP) stimulation, it was further verified that in the sepsis model, UGCG expression was significantly upregulated in monocytes and alveolar macrophages, and apoptosis and autophagy were crosstalked through the UGCG-MIF-PI3K axis. This disruption leads to metabolic disorders and affects systemic and pulmonary immune homeostasis. Therefore, inhibiting the UGCG enzyme will significantly inhibit the occurrence of these conditions.

[0008] As a preferred technical solution of the present invention, the UGCG enzyme inhibitor is a UGCG enzyme expression inhibitor and / or a UGCG enzyme activity inhibitor, preferably a UGCG enzyme activity inhibitor.

[0009] As a preferred technical solution of the present invention, the UGCG enzyme activity inhibitor includes Eliglustat.

[0010] Eliglustat is currently used clinically to treat Gaucher disease. This study found that the use of Eliglustat, an inhibitor of UGCG enzyme activity, can treat sepsis. Eliglustat significantly prolongs survival, effectively alleviates lung damage and systemic inflammatory responses, improves inflammatory infiltration in lung tissue, and mitigates LPS-induced damage to the heart, liver, spleen, and kidneys, providing new insights into the treatment of sepsis.

[0011] As a preferred technical solution of the present invention, the drug for treating sepsis includes a UGCG enzyme activity inhibitor and a pharmaceutically acceptable carrier.

[0012] As a preferred technical solution of the present invention, the dosage form of the drug for treating sepsis is granules, tablets, capsules, pills or oral liquid preparations.

[0013] In a second aspect, the present invention also provides the use of an inhibitor of UGCG enzyme activity in the preparation of a drug for inhibiting the PI3K / AKT / mTOR signaling pathway.

[0014] As a preferred technical solution of the present invention, the drug is used to treat diseases caused by activation of the PI3K / AKT / mTOR signaling pathway.

[0015] The present invention also attempts to elucidate the specific cellular mechanism through a large number of experiments and data analysis. The results show that UGCG may enhance the generation of MIF signals. The UGCG-MIF axis mainly acts through the PI3K / AKT / mTOR signaling pathway, thereby affecting the regulation of cell death signals in the monocyte-macrophage cell lineage and playing an important role in sepsis-related lung injury. Therefore, targeting UGCG (including regulating gene expression and inhibiting enzyme activity with eliglustat) can reverse the crosstalk between apoptosis and autophagy.

[0016] Preferably, the drug is used to inhibit the activation of the PI3K / AKT / mTOR signaling pathway in sepsis.

[0017] In a third aspect, the present invention also provides the use of an inhibitor of UGCG enzyme activity in the preparation of a drug for reducing M1 macrophages.

[0018] In the present invention, the effect of UGCG enzyme activity inhibitor on M1 macrophages is studied under inflammatory conditions. In other conditions such as tumor diseases, UGCG enzyme is also elevated, but its effect on PI3K or M1 macrophages or MIF remains to be studied.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention found that UGCG was significantly elevated in patients with sepsis. UGCG enhanced the generation of MIF signals and exerted its effects by activating the PI3K / AKT / mTOR signaling pathway, thereby affecting the regulation of cell death signals in the monocyte-macrophage cell lineage.

[0021] (2) UGCG inhibitors such as Eliglustat effectively relieve pulmonary edema and reduce inflammatory responses in sepsis. Experimental studies have found that Eliglustat can affect MIF release and inhibit the activation of the PI3K / AKT / mTOR signaling pathway in vivo by inhibiting UGCG enzyme activity, thereby maintaining metabolic homeostasis and reducing M1 macrophages, thereby reducing sepsis-related organ damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Comparison of UGCG mRNA expression levels in peripheral blood leukocytes and plasma UGCG protein levels in patients with pneumonia (n=31), patients with PAS (n=20), and healthy controls (Hc, n=11); Panel A shows UGCG mRNA expression levels, and Panel B shows UGCG plasma protein levels. (Data are presented as medians with 95% CIs).

[0023] Figure 2Figure 2 shows comparative data analysis of mice with LPS-induced sepsis before and after treatment with Eliglustat. Panel A is a schematic diagram of the experimental method for Eliglustat treatment. Panel B shows the survival rate before and after treatment. Panel C shows the weight loss rate of mice. Panel D shows the mRNA expression level of UGCG in peripheral blood. Panel E shows the mRNA expression levels of inflammatory cytokines (IL-1β, IL-6, IL-10, and TNF-α). Panel F shows plasma protein levels. Panel G shows the appearance of lung tissue. Panel H shows the lung wet-to-dry weight ratio. (Data are presented as dotted bar graphs, with error bars representing the median and 95% CI. The PBS group is represented by light green bars, the LPS group by blue bars, and the Eliglustat-treated group by pink bars. Each group was repeated three times. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.)

[0024] Figure 3 Comparative data analysis of mice with KP-induced sepsis before and after treatment with eliglustat in Example 2; Panel A is a schematic diagram of the experimental method for eliglustat treatment, Panel B shows the survival rate before and after treatment, and Panel C shows the weight loss rate of mice. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)

[0025] Figure 4 These are the HE staining results of lung tissue, heart, liver, spleen and kidney of mice with LPS-induced sepsis in Example 2 before and after treatment with Eliglustat.

[0026] Figure 5 Figure 3 is the transcriptome analysis result of PBMCs in septic mice in Example 3; Figure A is the volcano plot of DEGs in KP-induced septic mice and PBS group, Figure B is the GO enrichment analysis diagram of DEGs, and Figure C is the KEGG enrichment analysis diagram of DEGs.

[0027] Figure 6 Figure 3 shows the distribution of Ugcg in mouse PBMCs and lung tissue cell populations; Figure A shows the cell population identification results in mouse PBMCs; Figure B shows the gene expression of Ugcg in the KP group and PBS group; Figure C shows the distribution of Ugcg in the PBMCs cell population; Figure D shows the cell population identification results of mouse lung tissue; Figure E shows the gene expression of Ugcg in the KP group, LPS group and PBS group; Figure F shows the distribution of Ugcg in different cell subpopulations in lung tissue; Figure G shows the KP-Ugcg hi The cell communication signal strength of different cell subpopulations in the group.

[0028] Figure 7 KP-Ugcg in Example 3 hi Figure 2 shows the correlation analysis results between monocytes / alveolar macrophages and cell death signals and MIF signals; Figure A shows the GSEA analysis of mouse PBMCs monocytes; Figure B shows the KP / LPS UGCG of mice hi Figure 3 GSEA analysis of monocytes and alveolar macrophages in the PBMCs group; Figure C shows the incoming signal pattern, outgoing signal pattern and overall signal pattern of different signal pathways in different cell subsets in PBMCs; the bluer the color, the stronger the signal; Figure D shows the communication pattern of the MIF signaling pathway in different cell populations; Figure E shows the KP-UGCG in PBMCs low / UGCG hi The expression of Ugcg and Mif genes in the group.

[0029] Figure 8 Figure 3 is the result of cell communication analysis of mouse lung tissue in Example 3; Figure A shows the incoming signal pattern, outgoing signal pattern and overall signal pattern of different signal pathways in different cell subpopulations of the KP / LPS-UGCGhi group in lung tissue; Figure B shows the UGCG in KP lung tissue low / UGCG hi Figure C shows the expression of Ugcg and Mif genes in LPS lung tissue. low / UGCG hi The expression of Ugcg and Mif genes in the group.

[0030] Figure 9 Figures 1 and 2 show the results of the validation of MIF increase and THP-1 cell death in Example 4. Panel A shows the changes in UGCG protein levels after LPS stimulation in THP-1 cells; Panel B shows the changes in UGCG protein levels after knockdown and overexpression of UGCG; Panels C, D, E, and F show the levels of MIF protein, inflammatory factors, autophagy, and apoptosis-related proteins in THP-1 cells in each group, respectively; Panels G and H show the results of flow cytometry analysis of apoptosis in THP-1 cells in each group after PI / Annexin V double staining. (Data are represented by dotted bars, and error bars represent the mean and standard deviation (SD); each group had three replicates. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.)

[0031] Figure 10Figures 1 and 2 show the results of the validation of MIF increase and MH-S cell death in Example 4; Panel A shows the changes in UGCG protein levels in MH-S cells after LPS stimulation; Panel B shows the changes in UGCG protein levels after UGCG knockdown. Panels C, D, E, and F show the levels of MIF protein, inflammatory factors, autophagy, and apoptosis-related proteins in MH-S cells of each group, respectively; Panels G, H, and I show flow cytometric analysis of apoptosis in MH-S cells of each group following PI / Annexin V double staining. (Data are presented as dotted bars; error bars represent the mean and standard deviation (SD); three replicates were performed for each group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.)

[0032] Figure 11 Figure 4 shows the transmission electron microscopy results of THP-1 cells in Example 4, which are divided into THP-1 cells after LPS stimulation, Eliglustat treatment, UGCG knockdown and overexpression, including autophagosomes (black arrows), autolysosomes (red arrows) and apoptotic bodies.

[0033] Figure 12 Figure 4 shows the transmission electron microscopy results of MH-S cells in Example 4, which are divided into MH-S cells after LPS stimulation, Eliglustat treatment, and UGCG knockdown, including transmission electron microscopy analysis of autophagosomes (black arrows), autolysosomes (red arrows), and apoptotic bodies.

[0034] Figure 13 Figures 1 and 2 show the relationship between MIF levels and apoptosis in septic mice in Example 4. Figure A shows changes in autophagy-related and apoptosis-related proteins in mouse lung tissue before and after eliglustat treatment. Figure B shows MIF protein levels in mouse plasma and BALF. Figure C shows changes in autophagosomes (black arrows), autolysosomes (red arrows), and apoptotic bodies in lung tissue of mice in different groups. Figure D shows the effect of eliglustat treatment on apoptosis in mouse lung tissue. TUNEL staining shows red fluorescence, and nuclear staining shows blue fluorescence. Figure E shows the experimental design for ISO-1 treatment of septic mice (green arrow indicates intraperitoneal injection of PBS, black arrow indicates intraperitoneal injection of LPS, blue arrow indicates intraperitoneal injection of blank drug solvent, and orange arrow indicates intraperitoneal injection of ISO-1 (20 mg / kg)). Figure F shows the survival rate of septic mice. Figure G shows the weight loss rate of septic mice. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.)

[0035] Figure 14This is the correlation analysis between the monocyte-macrophage cell line and the inflammation and immune-related signaling pathways in Example 5; Figure A shows the KP / LPS-UGCG in mouse PBMCs and lung tissues hi MIF hi Correlation analysis between cells and 14 inflammatory and immune-related signaling pathways. The redder the color, the stronger the positive correlation, and the greener the color, the stronger the negative correlation. Figure B is a Venn diagram showing KP / LPS-UGCG hi MIF hi Intersection of the top five pathways associated with monocytes and alveolar macrophages.

[0036] Figure 15 Figure 5 shows the analysis results of the validation of the UGCG-MIF-PI3K signaling pathway in inflammatory THP-1 cells. Figure A shows the levels of inflammatory factors; Figure B shows the levels of MIF; Figure C shows the changes in the levels of PI3K, AKT, mTOR, and their phosphorylated proteins in different groups; Figure D shows the changes in apoptosis / autophagy-related proteins in different groups; Figure E shows the effect of adding a PI3K agonist after knocking down UGCG on the apoptosis rate of THP-1 cells; Figure F shows the effect of blocking the PI3K pathway after overexpressing UGCG on the apoptosis rate of THP-1 cells. (Data are represented by dotted bars, and errors are expressed as mean and standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.)

[0037] Figure 16 Figure 5 shows the analysis results of the validation of the UGCG-MIF-PI3K signaling pathway in inflammatory MH-S cells. Figure A shows the levels of inflammatory factors; Figure B shows the levels of MIF; Figure C shows the changes in the levels of PI3K, AKT, mTOR, and their phosphorylated proteins in different groups; Figure D shows the changes in apoptosis / autophagy-related proteins; Figure E shows the effect of adding a PI3K agonist after knockdown of UGCG on the apoptosis rate of MH-S cells; Figure F shows the effect of blocking the PI3K pathway after LPS stimulation on the apoptosis rate of MH-S cells. (Data are represented by dotted bars, and errors are expressed as mean and standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.)

[0038] Figure 17Figures are provided for analysis and validation of the in vivo effects of the UGCG-MIF-PI3K signaling pathway in Example 5. Figure A shows the experimental method for treating septic mice with LY294002 (green arrows indicate intraperitoneal injection of PBS, black arrows indicate intraperitoneal injection of LPS, blue arrows indicate intraperitoneal injection of blank drug solvent, and green arrows indicate intraperitoneal injection of LY294002 (10 mg / kg) for treatment. The dosing time is shown below the x-axis, and the observation endpoint is shown below the horizontal arrow). Figure B shows the therapeutic effect of LY294002 on the survival rate of septic mice. Figure C shows the therapeutic effect of LY294002 on the weight loss rate in sepsis. Figure D shows the changes in the levels of PI3K, AKT, mTOR, and their phosphorylated proteins in lung tissue before and after eliglustat treatment. (Each experiment was repeated three times. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.)

[0039] Figure 18 These are PCA and PLSDA analysis diagrams of the metabolomics of plasma and bronchoalveolar lavage fluid of mice in different groups in Example 6.

[0040] Figure 19 Figures 2 and 3 show differential metabolite enrichment analysis results for mice before and after eliglustat treatment in Example 6. Panel A shows differential metabolite enrichment analysis in mouse plasma; Panel B shows differential metabolite enrichment analysis in mouse bronchoalveolar lavage fluid. (Red represents upregulated pathways, light green represents downregulated pathways; larger |differential abundance (DA)| values ​​indicate stronger correlations.)

[0041] Figure 20 Figure 6 is a quantitative analysis of differential metabolites associated with M1 macrophage polarization in Example 6; Figure A shows the classification of differential metabolites in the plasma of mice in different groups; Figure B shows the classification of differential metabolites in the alveolar lavage fluid of mice in different groups (the redder the color, the more differential metabolites, and the greener the color, the fewer differential metabolites); Figure C shows the changes in differential metabolites associated with M1 macrophage polarization in mouse plasma before (blue) and after (pink) eliglustat treatment for 24 hours; Figure D shows the changes in differential metabolites associated with M1 macrophage polarization in the alveolar lavage fluid of mice before (blue) and after (pink) eliglustat treatment for 24 hours.

[0042] Figure 21Figure 6 shows the changes in M1 macrophage numbers in different treatment groups of monocyte-macrophage cell lines; Panel A shows the THP-1 cell line; Panel B shows the MH-S cell line. (Statistical data are presented as dotted bar graphs, with errors expressed as mean and standard deviation (SD). Each experiment was repeated three times. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.)

[0043] Figure 22 Figure 6 shows the changes in M2 macrophage numbers in different treatment groups of monocyte-macrophage cell lines; Panel A shows the THP-1 cell line; Panel B shows the MH-S cell line. (Statistical data are presented as dotted bar graphs, with errors expressed as mean and standard deviation (SD). Each experiment was repeated three times. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.)

[0044] Figure 23 Schematic diagram of the action mechanism of UGCG-MIF-PI3K. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further illustrated below with reference to the accompanying drawings and through specific implementation methods. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0046] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0047] Example 1

[0048] The present invention found in a clinical cohort that UDP-glucose ceramide glucosyltransferase (UGCG) was significantly elevated in patients with pneumonia-related sepsis.

[0049] A total of 62 participants were included in the cohort study, including 20 patients with pneumonia-associated sepsis (PAS), 31 patients with pneumonia, and 11 healthy controls.

[0050] The gene expression of peripheral blood leukocytes was detected by RT-qPCR, and the results were as follows Figure 1 As shown in Figure A, compared with the healthy control group, UGCG was significantly increased in the pneumonia group and PAS group (p < 0.05), and the upregulation degree of these genes in the PAS group was significantly higher than that in the pneumonia group (p < 0.05).

[0051] ELISA test results (such as Figure 1(As shown in Figure B) Consistent with the RT-qPCR results, the plasma levels of UGCG in the pneumonia group and PAS group were significantly higher than those in the healthy control group, and the PAS group reached the highest level (p < 0.05).

[0052] Example 2

[0053] In this example, the therapeutic effect of Eliglustat, an inhibitor of UGCG enzyme activity, in lipopolysaccharide (LPS)-induced and Klebsiella pneumoniae (KP)-induced sepsis mouse models was evaluated.

[0054] (1) LPS-induced sepsis mouse model

[0055] Eliglustat treatment experimental methods such as Figure 2 In Figure A, the green arrow indicates intraperitoneal injection of PBS, the black arrow indicates intraperitoneal injection of LPS (10 mg / kg), the blue arrow indicates intragastric administration of corn oil, and the pink arrow indicates intragastric administration of eliglustat (20 mg / kg). The administration time point is indicated below the x-axis, and the observation time is indicated below the horizontal arrow.

[0056] like Figure 2 As shown in Figure B, after intraperitoneal injection of LPS (10 mg / kg, LPS group), the survival rate of mice decreased significantly from 100% to 30% compared with the PBS group (p < 0.01). In contrast, the Eliglustat group (administered orally at a dose of 20 mg / kg every 12 hours, starting 2 hours before LPS stimulation for 3 consecutive days) significantly improved the survival rate of mice.

[0057] like Figure 2 As shown in Figure C, in both the LPS and Eliglustat groups, mice began to lose weight at 24 hours and died at 72 hours. The weight recovery time in the Eliglustat group (72 hours) was significantly earlier than that in the LPS group (96 hours).

[0058] Therefore, we collected peripheral blood and lung tissue from mice at these time points to further evaluate the therapeutic effects in mice with LPS-induced sepsis.

[0059] like Figure 2 As shown in Figure D, the mRNA expression level of UGCG in the LPS group was significantly higher than that in the PBS group, especially at 72 hours. Eliglustat significantly reduced the mRNA expression level of UGCG at 72 hours. In addition, Eliglustat reduced the mRNA and plasma protein levels of peripheral blood inflammatory factors IL-1β, IL-6, and TNF-α, but had no significant effect on IL-10 (see Figure 2 Figures E and F in the middle).

[0060] This example further evaluated the therapeutic effect of Eliglustat on organ damage in septic mice.

[0061] Mice in the LPS group showed significant pulmonary edema and congestion (e.g. Figure 2 The wet-to-dry ratio (W / D) of lung tissue increased 4-fold compared with the PBS group. However, Eliglustat treatment effectively alleviated pulmonary edema and reduced inflammatory response (see Figure 2 Figures G and H).

[0062] (2) KP-induced sepsis model

[0063] Eliglustat treatment experimental methods such as Figure 3 As shown in Figure A, the green arrow indicates the administration of PBS through endotracheal intubation, and the gray arrow indicates the administration of KP (2×10 4 CFU). The blue arrow indicates treatment with corn oil by gavage, and the pink arrow indicates treatment with Eliglustat (20 mg / kg). The red arrow indicates intraperitoneal injection of levofloxacin (15 mg / kg). The administration time point is shown below the x-axis, and the observation time is shown below the horizontal arrow.

[0064] Although all mice eventually died, Eliglustat significantly prolonged survival, especially in the group treated with levofloxacin ( Figure 3 Eliglustat also improved the rate of weight loss in mice with KP-induced sepsis ( Figure 3 (as shown in Figure C).

[0065] Histological analysis by HE staining showed that Eliglustat significantly improved inflammatory infiltration in lung tissue and alleviated LPS-induced damage to the heart, liver, spleen, and kidneys ( Figure 4 ).

[0066] The above results indicate that Eliglustat effectively alleviates lung injury and systemic inflammatory response in septic mice by inhibiting UGCG enzyme activity, thereby significantly improving the survival rate.

[0067] In addition, given the significant improvement of survival rate and weight loss rate by Eliglustat in the LPS-induced model, this model was selected for subsequent studies in the present invention.

[0068] Example 3

[0069] This example uses single-cell transcriptome analysis to reveal the enrichment of cell death pathways and MIF signaling in the high UGCG expression group.

[0070] In order to explore the mechanism of the effect of UGCG on lung injury, this example performed transcriptome analysis on peripheral blood mononuclear cells of KP-induced septic mice.

[0071] like Figure 5 As shown in Figure A, red dots indicate highly expressed DEGs in KP, blue dots indicate lowly expressed DEGs in KP, and gray dots indicate genes with no statistical significance. The screening criteria were: |logFC| > 2, p < 0.05. In other words, UGCG expression was significantly increased in septic mice (KP group) compared with the PBS group.

[0072] Based on the median expression level of UGCG gene, the KP group was divided into high expression (KP-UGCG hi ) and low expression (KP-UGCG low ) subgroup.

[0073] GO enrichment analysis (see Figure 5 Figure B) and KEGG enrichment analysis (see Figure 5 In the results of Figure C, pathways related to apoptosis and autophagy are highlighted with red boxes, and KP-UGCG hi Apoptosis and autophagy signaling pathways were significantly enriched in the UGCG group, suggesting that UGCG may have a potential correlation with cell death signaling pathways.

[0074] To elucidate the specific cellular mechanisms, this example also analyzed the constructed single-cell transcriptome data of PBMCs from KP-induced septic mice (GSE262512), and collected single-cell transcriptome data of lung tissue from KP- and LPS-induced septic mice (GSE19026217).

[0075] In the PBMCs single-cell transcriptome data, after quality control and deduplication, PBMCs were divided into four cell populations: monocytes, T cells, B cells, and NK cells according to the standard annotation of immune cell marker genes. A small number of granulocytes were also detected in the KP group (see Figure 6 (Figure A in the middle).

[0076] Compared with the PBS group, the expression of UGCG in the KP group was significantly increased and was widely expressed in myeloid and lymphocyte subsets, with the highest expression levels observed in NK cells, T cells, and monocytes (see Figure 6 (Figures B and C in the middle).

[0077] In the lung tissues of PBS-, KP-, and LPS-treated mice, 17 different cell subsets were identified based on standard annotation of multiple cell marker genes, including monocytes, alveolar macrophages, and Fn1 + Macrophages (see Figure 6 (Figure D in the middle).

[0078] Compared with the PBS group, the expression of UGCG in the lung tissue of the KP group was significantly increased, and reached the highest level in the LPS group (see Figure 6 In these three groups, UGCG was widely expressed in all cell subsets (see Figure 6 (Figure F in the middle).

[0079] The results of cell communication of PBMCs showed that KP-UGCG hi The strongest autocrine signaling occurs between monocytes (see Figure 6 Since alveolar macrophages are the main immune cells in lung tissue, monocytes are considered to be a bridge between bone marrow progenitor cells and terminally differentiated tissue macrophages.

[0080] Therefore, this example focused on analyzing the role of monocytes and alveolar macrophages in sepsis-related acute lung injury. GSEA analysis results showed that in KP / LPS-UGCG hi In the group, apoptosis and autophagy signaling pathways of monocytes and alveolar macrophages were significantly enriched ( Figure 7 (as shown in Figures A and B).

[0081] Based on the above results, the following hypothesis is proposed: UGCG participates in the pathological process of sepsis-related lung injury by regulating the cell death signaling pathway in the monocyte-macrophage lineage.

[0082] The results of cell communication analysis between mouse PBMCs and lung tissue showed that (see Figure 7 Figure C and Figure 8 Middle A), KP / LPS-UGCG hi In the group, MIF signal is the strongest communication signal.

[0083] In PBMCs (see Figure 7 Figures C and D), KP-UGCG hi Monocytes interfere with their own communication through autocrine MIF signaling.

[0084] Likewise, Figure 8 As shown in Figure A, in KP / LPS-UGCG hi In lung tissue, monocytes and alveolar macrophages exert significant effects on different cells and themselves through paracrine and autocrine MIF signals. In addition, UGCG is expressed in PBMCs and lung tissues of septic mice. hi The MIF gene expression level of the UGCG group was higher than that of the UGCG group. low Group (see Figure 7 China E map, Figure 8Figures B and C).

[0085] Therefore, UGCG may affect the regulation of cell death signaling in monocyte-macrophage cell lines by enhancing the production of MIF signals.

[0086] Example 4

[0087] This example verifies the relationship between UGCG and MIF under inflammatory conditions. The results show that the increase of MIF can promote the hi Apoptosis and reduced autophagy in monocyte-macrophage cell lines.

[0088] 1. In this example, THP-1 and MH-S cell lines were used to construct LPS-induced cell injury models ( Figure 9 Corresponding to the experimental results of THP-1 cell line, Figure 10 Corresponding experimental results of MH-S cell line).

[0089] (1) Protein expression: Figure 9 A picture and Figure 10 As shown in Figure A, when these two cell lines were cultured under the stimulation of LPS at a concentration of 500 ng / ml for 24 hours, the protein level of UGCG reached a peak.

[0090] (2) Construction of UGCG overexpression (OE) and knockdown (si) cell lines (protein expression results as shown in Figure 9 Middle B and Figure 10 (as shown in Figure B).

[0091] (3) To evaluate the effect of UGCG on the production and release of MIF, this example used ELISA to detect the MIF content in the cell supernatant under different conditions.

[0092] The results are as follows Figure 9 Figure C and Figure 10 As shown in Figure C, LPS stimulation significantly elevated MIF levels in the cell supernatant compared to the PBS control group or the empty plasmid overexpression group (OE NC group). Overexpression of UGCG further elevated MIF levels. ISO-1 (7 μM), a MIF inhibitor, served as a positive control and effectively inhibited MIF production.

[0093] Under LPS stimulation, the addition of Eliglustat (24 nM) and ISO-1 reduced MIF levels regardless of UGCG overexpression. Knockdown of UGCG significantly reduced MIF release.

[0094] After knocking down UGCG, adding exogenous MIF (THP-1: 12.71 ng / mL, MH-S: 10 ng / mL) could restore the MIF level to the level of the control group (siNC group).

[0095] (4) The results of inflammatory cytokine level determination are as follows Figure 9 D map and Figure 10 Figure D shows that the addition of eliglustat, ISO-1, or knockdown of UGCG reduced the levels of IL-1β, IL-6, and TNF-α, while overexpression of UGCG or supplementation of exogenous MIF increased the levels of these cytokines. However, the level of IL-10 was not affected.

[0096] (5) WB analysis results are as follows Figure 9 E-map and Figure 10 Figure E shows that neither the addition of ISO-1 nor exogenous MIF changed the level of UGCG protein, indicating that UGCG acts upstream of MIF, thereby promoting the production and release of MIF.

[0097] 2. This example further investigated the relationship between the UGCG-MIF axis and cell death.

[0098] (1) Western blot analysis Figure 9 Figures E and F, Figure 10 Results (Figures E and F) show that compared with the control group, LPS stimulation or UGCG overexpression increased the LC3BII / LC3BI ratio and P62 levels, indicating inhibition of the autophagic degradation pathway. Furthermore, an increase in the cleaved-caspase-3 / caspase-3 ratio suggests enhanced apoptosis.

[0099] (2) Under LPS-stimulated inflammatory conditions, this effect caused by UGCG overexpression was more significant. In contrast, knockdown of UGCG or treatment with Eliglustat and ISO-1 reversed the above results, leading to enhanced autophagic degradation, restored autophagic flux, and reduced apoptosis levels.

[0100] These results indicate that both UGCG and MIF promote apoptosis and inhibit autophagy. Supplementation of exogenous MIF after UGCG knockdown restored the reduced P62 levels and the LC3BII / LC3BI and cleaved-caspase-3 / caspase-3 ratios, further validating the regulatory role of the UGCG-MIF axis in cell death.

[0101] (3) Flow cytometry analysis results ( Figure 9 Figure G and Figure H, Figure 10 Figures G, H, and I further confirmed that the use of Eliglustat, ISO-1, or knockdown of UGCG could reduce the proportion of apoptotic cells induced by LPS, while overexpression of UGCG further increased the apoptotic proportion.

[0102] (4) Transmission electron microscopy results ( Figure 11 and Figure 12 ) showed that LPS stimulation or UGCG overexpression could lead to the accumulation of autolysosomes, autophagosomes, and apoptotic bodies, which were alleviated after the use of Eliglustat and knockdown of UGCG.

[0103] 3. This example further verifies the effect of UGCG inhibitor Eliglustat on MIF release and cell death in vivo.

[0104] (1) Figure 13 Figure A in the middle shows that Eliglustat significantly reduced the LC3BII / LC3BI ratio, cleaved-caspase3 / Caspase 3 ratio, and P62 protein level, indicating that Eliglustat promoted autophagic flux and inhibited apoptosis.

[0105] (2) Figure 13 Figure B in the middle shows that Eliglustat significantly reduced the levels of MIF in the plasma and bronchoalveolar lavage fluid of mice with LPS-induced sepsis, confirming that UGCG also promoted the release of MIF in vivo.

[0106] (3) Transmission electron microscopy analysis of lung tissue ( Figure 13 Middle (C) shows that Eliglustat reduced the accumulation of autolysosomes and autophagosomes in the lung tissue of septic mice, further supporting its role in promoting autophagy.

[0107] (4) TUNEL staining results ( Figure 13 (Center D) shows that red fluorescence intensity in lung tissue increased significantly after LPS stimulation, indicating elevated levels of apoptosis. Eliglustat treatment significantly reduced red fluorescence intensity, particularly after 72 hours of LPS stimulation. These results confirm that Eliglustat reduces apoptosis and promotes autophagy in vivo.

[0108] 4. Based on the verification of the in vitro effect of MIF, this example also evaluated its in vivo effect by intraperitoneal injection of the MIF inhibitor ISO-1 (20 mg / kg, twice a day for three consecutive days). The method of use is as follows: Figure 13 As shown in Figure E.

[0109] The results are as follows Figure 13 Figures F and G in the middle showed that ISO-1 treatment significantly increased the survival rate of septic mice from 20% to 40% (p < 0.05) and alleviated the body weight loss rate.

[0110] These results suggest that the UGCG-MIF axis plays an important role in sepsis-associated lung injury by promoting apoptosis and inhibiting autophagy. Targeting UGCG (including regulating gene expression and inhibiting enzyme activity with eliglustat) can reverse the crosstalk between apoptosis and autophagy.

[0111] Example 5

[0112] This example is used to verify that the UGCG-MIF axis mainly acts through the PI3K / AKT / mTOR signaling pathway.

[0113] 1. First, to clarify the specific mechanism by which the UGCG-MIF axis regulates cell death, this example conducted a correlation analysis on cells that simultaneously overexpress UGCG and MIF (KP / LPS-UGCGhiMIFhi cells) in septic mice to explore their relationship with 14 inflammatory and immune-related pathways.

[0114] like Figure 14 As shown in Figures A and B, this example identified the top five signaling pathways that were positively or negatively correlated with PBMCs and monocytes and alveolar macrophages in lung tissue.

[0115] After intersecting these signaling pathways, it was found that UGCG hi MIF hi Type monocytes and alveolar macrophages were positively correlated with the PI3K signaling pathway, while no signaling pathway was negatively correlated with them.

[0116] Based on these results, it is speculated that the UGCG-MIF axis may exert its pro-inflammatory effects through the PI3K signaling pathway.

[0117] 2. Secondly, to verify the hypothesis that the UGCG-MIF axis regulates inflammatory response through the PI3K / AKT / mTOR signaling pathway, this example used the PI3K pathway inhibitor LY294002 (20 μM) to treat THP-1 and MH-S cells. Figure 15 and Figure 16 shown.

[0118] (1) Figure 15 A picture and Figure 16 As shown in Figure A, LY294002 significantly reduced the release of IL-1β, IL-6, and TNF-α induced by LPS or UGCG overexpression. Conversely, treatment with the PI3K agonist 740YP (30 μM) restored the levels of inflammatory factors reduced by UGCG knockdown. However, neither LY294002 nor 740YP had an effect on IL-10 levels.

[0119] (2) Further experiments showed that neither LY294002 nor 740YP changed the MIF protein level (see Figure 15 Middle B and Figure 16 Figure 3 (middle panel B) confirms that PI3K functions downstream of MIF. Increased phosphorylation of PI3K, AKT, and mTOR is a hallmark of activation of the canonical PI3K signaling pathway.

[0120] (3) 740YP and LY294002 were used as positive controls for PI3K / AKT / mTOR pathway activation and inhibition, respectively.

[0121] The results are as follows Figure 15 Figure C and Figure 16 As shown in Figure C, LPS stimulation or UGCG overexpression significantly increased the phosphorylation levels of PI3K, AKT, and mTOR, which was comparable to that of the positive control group and significantly higher than that of the negative control group.

[0122] In contrast, compared with the LPS group, UGCG knockdown or Eliglustat treatment reduced the phosphorylation levels of PI3K, AKT, and mTOR. In addition, neither LY294002 nor 740YP treatment changed the UGCG protein level, further supporting the upstream role of UGCG in promoting the activation of the PI3K / AKT / mTOR pathway.

[0123] Furthermore, compared with the LPS group, ISO-1 treatment reduced the phosphorylation levels of PI3K, AKT, and mTOR, while the addition of exogenous MIF after UGCG knockdown increased the phosphorylation levels of these proteins. Combined with the results of previous studies, these findings suggest that the UGCG-MIF axis enhances the activation of the PI3K / AKT / mTOR signaling pathway.

[0124] 3. Based on the clarification of the relationship between the UGCG-MIF axis and cell death, this example further explored whether this axis regulates cell death through the PI3K signaling pathway.

[0125] WB experimental results (such as Figure 15 D map and Figure 16 Figure D) shows that the PI3K inhibitor LY294002 significantly reduced the LC3BII / LC3BI ratio, cleaved-caspase 3 / caspase 3 ratio, and P62 protein level induced by LPS stimulation or UGCG overexpression. At the same time, it also reduced the proportion of apoptotic cells (see Figure 15 Figures E, F, and Figure 16 Figures E and F in the middle).

[0126] These findings further confirmed that the UGCG-MIF axis promoted cell apoptosis and inhibited autophagy through the PI3K / AKT / mTOR signaling pathway.

[0127] 4. To verify the role of the UGCG-MIF-PI3K axis in vivo, this example treated septic mice by intraperitoneal injection of LY294002 (10 mg / kg, administered 0.5 hours after LPS stimulation) (the experimental method is as follows Figure 17 (as shown in Figure A).

[0128] The results showed that LY294002 significantly improved the survival rate of mice compared with the LPS group (see Figure 17 Figure B) and reduced the rate of weight loss ( Figure 17 Similarly, eliglustat treatment significantly reduced the phosphorylation levels of PI3K, AKT, and mTOR in the lung tissues of septic mice ( Figure 17 (Figure D in the middle).

[0129] Therefore, the PI3K / AKT / mTOR signaling pathway plays an indispensable role in the pathological process of sepsis. These results show that targeting UGCG inhibits the activation of the PI3K / AKT / mTOR signaling pathway in vitro and in vivo, reducing sepsis-related organ damage.

[0130] Example 6

[0131] Given the role of UGCG as a key enzyme in sphingolipid metabolism, it is speculated that UGCG causes metabolic disorders by regulating the sphingolipid metabolic pathway. Therefore, this example was used to verify that Eliglustat can maintain metabolic homeostasis and reduce M1 macrophages.

[0132] 1. This example performed non-targeted metabolomics analysis on mouse plasma (Serum) and bronchoalveolar lavage fluid (BALF).

[0133] like Figure 18 As shown, principal component analysis (PCA) and partial least squares discriminant analysis (PLSDA) effectively distinguished the different groups of mouse plasma samples, while the distinction of bronchoalveolar lavage fluid samples was less obvious.

[0134] like Figure 19As shown in Figure A, enrichment analysis of differential metabolites (DEMs) in mouse plasma revealed that, compared with the PBS control group, 24-hour LPS stimulation led to decreased activity in metabolic pathways such as tryptophan metabolism, protein digestion and absorption, mineral absorption, central carbon metabolism in cancer, and aminoacyl-tRNA biosynthesis. Eliglustat treatment effectively restored the activity of these metabolic pathways.

[0135] However, at 72 hours, eliglustat only reduced the elevation of protein digestion and absorption metabolic pathways induced by LPS stimulation.

[0136] like Figure 19 As shown in Figure B, in the bronchoalveolar lavage fluid of mice, LPS stimulation for 24 hours significantly upregulated taurine and hypotaurine metabolism, cofactor biosynthesis, and arginine and proline metabolism compared to the PBS group, whereas these metabolic pathways were decreased after eliglustat treatment. By 72 hours, eliglustat significantly downregulated pathways related to the regulation of lipolysis in adipocytes, cholinergic synapses, and choline metabolism in cancer, all of which were upregulated by LPS stimulation.

[0137] These data suggest that UGCG affects multiple metabolic pathways, involving various amino acid, lipid, protein, and carbohydrate metabolic pathways. Eliglustat, an inhibitor of GCS synthesis in sphingolipid metabolism, appears to be able to restore these metabolic disorders.

[0138] 2. Since macrophage metabolic homeostasis affects its polarization state, this example further explored the relationship between UGCG and macrophage polarization.

[0139] (1) Previous studies have shown that LPS stimulation, eliglustat treatment, and UGCG knockdown or overexpression all affected the release of M1 macrophage-related inflammatory factors (such as IL-1β, IL-6, and TNF-α), but did not significantly affect the expression of IL-10, an inflammatory factor associated with M2 macrophages. Therefore, UGCG may increase the number of M1 macrophages.

[0140] (2) Metabolomics differential metabolite analysis (e.g. Figure 20 Figures (A and B) show that multiple metabolites were significantly different in the plasma and bronchoalveolar lavage fluid of septic mice compared with the PBS group.

[0141] 3. In this example, differential metabolites related to M1 polarization were retrieved from the PubMed database and compared with the metabolites with significant differences after eliglustat treatment.

[0142] The results are as follows Figure 20 Figures C and D in the middle show that after 24 hours of eliglustat treatment, 10 differential metabolites related to M1 polarization (including trimethylamine, cysteine, kainic acid, L-proline, L-tryptophan, indole, acetylcholine, DL-tryptophan, pyruvate, and ascorbic acid) were significantly changed.

[0143] 4. This example verifies the number of M1 macrophages in cell lines.

[0144] The changes in CD86 positivity in THP-1 and MH-S cell lines after LPS stimulation (blue) with the addition of Eliglustat (pink), ISO-1 (orange), and LY294002 (dark green), and after UGCG overexpression (red) and knockdown of UGCG (brown) followed by the addition of exogenous MIF (yellow) and 740YP (purple) are shown in Figure 2. Figure 21 As shown, Figure A represents the THP-1 cell line and Figure B represents the MH-S cell line.

[0145] Compared with the PBS group, LPS stimulation significantly increased the number of M1 macrophages (CD86 positive rate).

[0146] After treatment with Eliglustat, ISO-1, or LY294002, the number of M1 macrophages decreased to a level comparable to that of the PBS group.

[0147] The addition of exogenous MIF and the PI3K activator 740YP increased the number of M1 macrophages, offsetting the decrease caused by UGCG knockdown in MH-S cells and restoring it to the control level in THP-1 cells.

[0148] Under inflammatory conditions, UGCG overexpression increased the CD86 positivity rate, which was reduced after treatment with Eliglustat, ISO-1, and LY294002.

[0149] However, combined Figure 22 It can be seen that the CD163 positivity rate (M2 macrophage polarization marker) was not affected by LPS stimulation, UGCG knockdown or Eliglustat addition in both THP-1 (Figure A) and MH-S cells (Figure B).

[0150] These findings suggest that the UGCG-MIF-PI3K axis is involved in the crosstalk between apoptosis and autophagy, leading to metabolic disorders in sepsis and regulating systemic and local immune homeostasis in the lungs. Eliglustat can reverse these results. The schematic diagram of its mechanism of action is shown below. Figure 23 shown.

[0151] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. Application of UGCG enzyme inhibitors in the preparation of drugs for treating sepsis.

2. The use according to claim 1, characterized in that UGCG enzyme inhibitors are inhibitors of UGCG enzyme activity.

3. The use according to claim 2, characterized in that The UGCG enzyme activity inhibitor includes eliglilukast.

4. The use according to claim 1, characterized in that The drug for treating sepsis includes a UGCG enzyme activity inhibitor and a pharmaceutically acceptable carrier.

5. The use according to claim 1, characterized in that The dosage form of the drug for treating sepsis is granules, tablets, capsules, pills or oral liquid preparations.

6. Application of UGCG enzyme activity inhibitors in the preparation of drugs that inhibit PI3K / AKT / mTOR signaling pathways.

7. The use according to claim 6, characterized in that The drug is used to treat diseases in which the PI3K / AKT / mTOR signaling pathway is activated.

8. Application of UGCG enzyme activity inhibitors in the preparation of drugs for reducing M1 macrophages.

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