Application of glucosylceramide synthase inhibitor in preparation of product for preventing and / or treating metabolic stress liver injury related diseases

Abstract

The invention provides application of a glucosylceramide synthase inhibitor in preparation of a product for preventing and / or treating metabolic stress liver injury related diseases, and relates to the technical field of biological medicines. It is found for the first time that the glucosylceramide synthase inhibitor (GCS enzyme inhibition) can inhibit bacteroides polymorpha sphingolipid monoglycosylation, and the glucosylceramide synthase inhibitor has the effects of improving hyperlipidemia and relieving hepatotoxicity of non-alcoholic steatohepatitis. Experiments prove that the GCS enzyme inhibitor induces a peripheral cell compartment to transfer to an immunosuppressive ecological niche, and mediates immune cells to inhibit inflammation of non-alcoholic steatohepatitis. The invention provides important treatment strategies and theoretical basis for clinical medicine.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a glucosylceramide synthase inhibitor in the preparation of a product for preventing and / or treating metabolic stress-induced liver damage-related diseases. Background Art

[0002] The lipid-rich cell membrane is not only a simple mechanical barrier, but also an important component of the cell. It also communicates with the external environment. Sphingomyelin is an important class of phospholipids in the membrane, which has multiple functions. In recent years, the interest in the lipid body of sphingomyelin, a key component of the cell membrane, has become the focus of intensive research. Through further research, the functions of sphingomyelin and its derivatives in diseases can be gradually elucidated. Sphingomyelin is composed of ceramide and its derivatives, including ceramide-1-phosphate glucosylceramide and sphingosine-1-phosphate. Glucosylceramide synthase is the key enzyme for ceramide glycosylation. The activity of glucosylceramide synthase directly affects the level of glucosylceramide in cells, which in turn affects the function of cells. In recent years, the relationship between glucosylceramide and its metabolic enzymes and diseases has become a relatively new research field.

[0003] Metabolic diseases are a general term for diseases that occur due to metabolic disorders in the body, including hyperglycemia, hyperlipidemia, non-alcoholic fatty liver disease, etc. With the dramatic increase in obesity and type 2 diabetes, non-alcoholic fatty liver disease is increasingly recognized as a major healthcare problem. Given that persistent lipid accumulation in the liver can in some cases progress to more severe non-alcoholic steatohepatitis and cirrhosis, effective prevention and interventional treatments are greatly needed. Although to date, glycosphingolipids (GSLs) have not been considered to play a role in the occurrence of hepatic steatosis.

[0004] Bacteroides is an important type of Gram-positive bacteria in the intestine, and some studies have shown that its content in the intestine is reduced in patients with diabetes, systemic inflammation, and corresponding disease animal models. However, no study has found that Bacteroides has a relieving effect on fatty liver hepatitis, and there is no report on whether its glucosylceramide synthase (GCS) can relieve metabolic diseases such as non-alcoholic fatty liver. Summary of the invention

[0005] In view of this, the object of the present invention is to provide the use of a glucosylceramide synthase inhibitor in the preparation of a product for preventing and / or treating metabolic stress-induced liver damage-related diseases.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of a glucosylceramide synthase inhibitor in the preparation of a product for preventing and / or treating metabolic stress-induced liver damage-related diseases.

[0008] Preferably, the metabolic stress liver damage-related diseases include one or more of hyperglycemia, hyperlipidemia, non-alcoholic fatty liver disease, decompensated cirrhosis, hepatocellular carcinoma and liver transplant recurrence.

[0009] Preferably, the glucosylceramide synthase inhibitor treats metabolic stress-induced liver damage-related diseases by inhibiting glucosylceramide synthase of Bacteroides.

[0010] More preferably, the Bacteroides is Bacteroides thetaiotaomicron.

[0011] Preferably, the glucosylceramide synthase inhibitor is Miglustat.

[0012] Preferably, the product comprises one or more of medicine, food, and feed.

[0013] The present invention provides a medicine for preventing and / or treating metabolic stress liver damage-related diseases, which comprises an effective dose of a glucosylceramide synthase inhibitor and a pharmaceutically acceptable adjuvant.

[0014] Preferably, the auxiliary materials include one or more of carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and flavors.

[0015] Preferably, the dosage form of the drug is one or more of tablets, powders, oral solutions, suspensions and emulsions.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The present invention first discovered that glucosylceramide synthase inhibitors (GCS enzyme inhibition) can inhibit the monosaccharidation of Bacteroides thetaiotaomicron sphingolipids, improve hyperlipidemia and reduce the hepatotoxicity of non-alcoholic steatohepatitis. The present invention provides new insights into intestinal microorganisms and circulating immune factors, which may serve as prognostic markers for GCS enzyme inhibitors in the treatment of metabolic dysfunction-associated steatohepatitis (MASH), as well as new targets or strategies for the treatment of MASH based on intestinal microbiota. It provides important treatment strategies and theoretical basis for clinical medicine.

[0018] (2) It has been experimentally verified that GCS enzyme inhibitors induce the shift of peripheral cell compartments to immunosuppressive niches, mediating immune cell suppression of inflammation in non-alcoholic fatty liver disease. GCS enzyme inhibition mainly changed the myeloid cell clusters in the GCS enzyme high expression group, while B cells and endothelial cell (EC) clusters dominated in the GCS enzyme low expression samples. Among all bone marrow cell subclusters, dendritic cells (DCs) and macrophages (Mφ) were the main cell types in terms of the number of filtered cells. Through unsupervised clustering, the present invention first focused on the intrinsic properties and potential functions of the DCs population, which were identified as CD8α + In the GCS enzyme high expression group, IL-12Rβ2 of Tregs can inhibit CD8α + DCs-derived IL-35, thereby inhibiting the inflammatory process. GCS enzyme inhibitors in the PBMCs of the GCS enzyme high expression group CD4 + Foxp3 + The number of Tregs increased. In addition, the present invention noted that the distribution of DC clusters was associated with the expression of gamma-glutamyl transferase (GGT), a well-recognized serum biomarker for MASH progression. GCS enzyme inhibitors showed that among all DCs in the group with high GCS enzyme expression, GGT + CD1C in samples + The proportion of DCs (cDC2) increased significantly, while DEC205 + The proportion of DCs (cDC1) decreased. The present invention's observations indicate that cDC1 and cDC2 cells have opposite functions in response to GCS enzyme inhibitor treatment.

[0019] (3) For the macrophage compartment, unsupervised clustering generated six clusters with different gene signatures. Two non-classical monocyte clusters enriched in PBMCs: MINCLE + Mφ and S1PR2 + In these macrophage clusters, whether GCS enzyme inhibitors are in the GCS enzyme high expression group (GH-Miglustat) or GCS enzyme inhibitors are in the GCS enzyme low expression group (GL-Miglustat) MINCLE + Mφ was significantly downregulated, S1PR2 + Mφ tended to be expressed in GH-Miglustat. Cell-cell interaction analysis of GCS enzyme inhibitors in GCS enzyme high expression group samples also confirmed that anti-inflammatory macrophages actively participated in the recruitment of T cells through CCL3 / 15-CCR5, CXCL10 / 11-CXCR5 and CXCL12 / 14-CXCR4 signals. In the GCS enzyme high expression group, the dominant Mφ of the CCL22 cluster was represented by MINCLE +Mφ is converted from Mφ. MINCLE of Mφ is a sensor of pathogen-associated molecular patterns and damage-associated molecular patterns, which is associated with the development of steatohepatitis and the pro-inflammatory properties of myofibroblasts. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The workflow diagram for the deletion of GCS genes in Bacteroides thetaiotaomicron is shown below.

[0021] Figure 2 Diagram of the de novo synthesis pathway of azide-labeled hexosylceramide;

[0022] Figure 3 Schematic diagram of the transwell co-culture system;

[0023] Figure 4 Azide-labeled lipids were detected with AlexaFluor 647 and stained with DAPI;

[0024] Figure 5 GCS enzyme inhibitor treatment GF BTWT and GF BT△GCS Schematic diagram of mouse;

[0025] Figure 6 For GF BTWT and GF BT△GCS Representative H&E (upper), Oil Red O (middle), and Sirius Red (lower) staining images of liver sections of mice after treatment with saline and GCS enzyme inhibitors;

[0026] Figure 7 Experimental flow chart of the scRNA-seq landscape;

[0027] Figure 8 Uniform Manifold Approximation and Projection (UMAP) maps of major cell types;

[0028] Fig. 9 This is the pseudo-time trajectory of macrophage branches in the GH-Miglustat group;

[0029] Fig.10 fecal GCS activity, fecal Bacteroides thetaiotaomicron relative abundance, serum CD8α + Dendritic cells, serum MINCLE + Heat map of the correlation between macrophages and liver disease indicators. DETAILED DESCRIPTION

[0030] The present invention provides the use of a glucosylceramide synthase inhibitor in the preparation of a product for preventing and / or treating metabolic stress liver damage-related diseases. In the present invention, the metabolic stress liver damage-related diseases preferably include one or more of hyperglycemia, hyperlipidemia, non-alcoholic fatty liver disease, decompensated cirrhosis, hepatocellular carcinoma and liver transplant recurrence.

[0031] In the present invention, the glucosylceramide synthase inhibitor is preferably a glucosylceramide synthase inhibitor of Bacteroides thetaiotaomicron; more preferably, it is a glucosylceramide synthase inhibitor of Bacteroides thetaiotaomicron. The glucosylceramide synthase inhibitor of the present invention preferably treats metabolic stress liver damage-related diseases by inhibiting the glucosylceramide synthase of Bacteroides thetaiotaomicron (B. thetaiotaomicron); more preferably, the glucosylceramide synthase inhibitor treats metabolic stress liver damage-related diseases by inhibiting the glucosylceramide synthase of Bacteroides thetaiotaomicron (B. thetaiotaomicron). In vitro experiments and animal experiments, it was found that the GCS enzyme of Bacteroides thetaiotaomicron inhibited the monoglycosylation of ceramide; GCS enzyme inhibitors can mediate immune cells to inhibit the inflammation of non-alcoholic fatty liver disease.

[0032] In the present invention, the glucosylceramide synthase inhibitor is preferably Miglustat. Miglustat is an orally active and reversible ceramide glucosyltransferase inhibitor. In the present invention, the Miglustat can be purchased through conventional channels. In a specific embodiment of the present invention, the Miglustat is purchased from MCE Company, CAS No. 72599-27-0.

[0033] In the present invention, the product includes one or more of medicine, food, and feed. In the present invention, the product targets a wide range of targets, can effectively alleviate the monoglycosylation of sphingolipids and mediate immune cells in the target object, significantly alleviate the symptoms of dyslipidemia and non-alcoholic fatty liver disease, and can be used to prepare lipid-lowering and non-alcoholic fatty liver disease. In the present invention, the target object preferably includes humans and animals.

[0034] The present invention provides a medicine for preventing and / or treating metabolic stress liver damage-related diseases, which comprises an effective dose of a glucosylceramide synthase inhibitor and a pharmaceutically acceptable adjuvant.

[0035] In the present invention, the auxiliary material includes one or more of a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener and a flavor. In the present invention, the auxiliary material can be specifically selected from one or more of lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone (PVP), cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil.

[0036] In the present invention, the dosage form of the drug includes one or more of granules, capsules, tablets, powders, oral liquids, suspensions and emulsions. As an practicable method, the drug of the present invention can be produced in the form of enteric coating preparations for oral use by methods well known to those skilled in the art, so that the active ingredients of the pharmaceutical composition can pass through the stomach smoothly without being destroyed by gastric acid; "enteric coating" includes all coatings allowed for conventional drugs, which are not degraded by gastric acid, but can be fully decomposed in the small intestine and quickly release the active ingredient of the present invention, the glucosylceramide synthase inhibitor. The enteric coating of the present invention can be maintained at 36-38°C for more than 2 hours in synthetic gastric acid such as HCl solution with pH=1.0, and preferably decomposed within 1 hour in synthetic intestinal fluid such as buffer with pH=7.0. The enteric coating of the present invention is coated with about 16-30 mg per tablet, preferably 16-25 mg, and more preferably 16-20 mg. The thickness of the enteric coating in the present invention is 5-100 μm, and the preferred thickness is 20-80 μm. The enteric coating component is a polymer conventionally known to those skilled in the art.

[0037] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0039] Example 1

[0040] Materials and methods: Donors were asked to collect stool into containers and send them to the hospital within 6 hours after morning bowel movement. Before processing, stool samples were stored in a biosafety cabinet at room temperature (20-30°C). Processing of fecal microbiota transplant suspension was performed within 2 hours after collection. 10 g of stool sample was diluted with 50 mL of 0.9% sterile saline and then vortexed for 10 minutes. The mixture was filtered twice with sterile gauze to remove impurities. The filtrate was mixed with glycerol (10%) and then divided into small aliquots and frozen in centrifuge tubes at -80°C for subsequent fecal microbiota transplantation processing. On the day of the experiment, the stool suspension was thawed in a warm water bath (37°C), and the B. thetaiotaomicron strains isolated from the participants' stool within 6 hours after thawing were compared by comparing the 16S rRNA gene sequence with the NCBI reference database. The DNA fragment encoding the full-length GCS gene was cloned into the pET28a vector with an N-terminal His tag using standard molecular cloning methods. GCS was deleted in E. coli BL21 (DE3) and cultured at 37°C with an OD600 of 0.6. An internal fragment (610 bp) of the E. coli GCS gene was cloned into the pGERM suicide vector containing selectable markers for E. coli (bla) and B. thetaiotaomicron. The constructed vector was subsequently transformed into the conjugated E. coli S17 strain. E. coli S17 was used as the donor strain and B. thetaiotaomicron was used as the recipient strain (introduced into the NBU2 att-1 site) and co-cultured under aerobic conditions and transferred to BHI medium containing gentamicin (200 μg / ml) and erythromycin (25 μg / mL). PCR identification was performed to verify the absence of GCS using primers W3274 and W4760 for the junction region of pGERM and GCS genes to select resistant colonies. E. coli and GCS-deficient E. coli were cultured in LB medium at 37°C for 24 h. The bacterial culture medium was centrifuged at 8000 g and 4°C for 10 min, and then all pellets were resuspended in oxygen-free PBS to obtain GCS-deficient B. thetaiotaomicron (BTΔGCS).

[0041] according to Figure 1 The results showed that the GCS gene was missing in B. thetaiotaomicron.

[0042] Example 2

[0043] Materials and Methods

[0044] A co-culture model of L-02 cells and B. thetaiotaomicron strains was established to verify the hypothesis that metabolites were transferred from BTWT to hepatocytes ( Figure 2), L-02 cells induced with 500 μM sodium oleate were incubated with BTWT and BTΔGCS in tissue culture dishes, and the bacterial cells were placed on a 0.4 μM filter, which was located 1 mm above the L-02 cell monolayer. 50 mL of culture medium (OD 600 =0.25) was added with 25 μM CerA mixture and poured into the upper chamber. After 4 h of CerA induction, the contents of the upper BTWT or BTΔGCS insert and the lower L-02 cell-filled plate were collected ( Figure 3 ). L-02 cells containing azide were plated on UV-sterilized No. 1.5 coverglass in 6-well plates and labeled with Alexa Fluor 647 fluorophore using Click iT Cell Reaction Buffer Kit (Thermo Fisher Scientific). A portion of L-02 cells in each group were used for qPCR, and the remaining portion was mounted on a glass slide and imaged under a Leica SP8 confocal microscope to observe the transfer of fluorescent metabolites. Azide-labeled lipids were detected with Alexa Fluor 647, and DNA was stained with DAPI.

[0045] according to Figure 4 The results showed that after the azide-labeled L-02 cells were co-cultured with BTΔGCS cells, the fluorescence intensity increased sharply and reached a plateau at 0.876 h, while the fluorescence intensity of the BTWT co-culture group increased slowly within 2 h, indicating that the GCS enzyme inhibitor reduced the concentrations of azide-containing Hex1Cer (d18:1 / 16:0) and Hex1Cer (d18:1 / 24:1).

[0046] Example 3: Animal experiments to verify the effects of BTWT and BTΔGCS on non-alcoholic fatty liver disease

[0047] Materials and methods:

[0048] All animal operations were performed in accordance with protocols approved by the Experimental Animal Center of Peking University First Hospital (Ethics Number: 2022-184257). Six-week-old C57BL / 6GF or SPF mice were placed in rigid autoclaved cages, 6 mice per cage, with a 12-h light-dark cycle. The MASH model was first constructed by feeding mice with a MASH diet containing 21.1% kcal fat, 1.25% kcal cholesterol, and 41.0% kcal sucrose in a sugar-water solution of 23.1 g / L d-fructose (MilliporeSigma, G8270) and 18.9 g / L d-glucose (MilliporeSigma, F0127) for 16 weeks. Colonization of B. thetaiotaomicron strains was as follows. Figure 5As shown, each group of MASH mice was fed a fat-free diet containing 0% kcal fat, 76% kcal carbohydrates, and 24% kcal protein for 12 weeks, and the diet was supplemented with BTWT or BTΔGCS strains (10 8 CFU / μLPBS, 3 times a week) and 5mg / kg sodium azide (Cat#S2002, Sigma-Aldrich) modified ceramide mixture (Cat#22853, CayManChemical) for 4 weeks. Then GCS enzyme inhibitor was gavaged for 8 weeks to eliminate exogenous lipid interference except for the azide urea-cer (CerA) mixture. Body weight and food intake were monitored weekly, and fecal samples were collected for metagenomic and metabolomics analysis. Finally, all mice were euthanized by bleeding, and their colon, serum and liver samples were collected for intestinal flora colonization ability, serology, standard H&E, Sirius red staining and Oil Red O histological analysis, respectively.

[0049] according to Figure 6 The experimental results showed that H&E and Oil Red O (ORO) staining showed that BTWT Compared with mice treated with salt, GF BT△GCS Hepatocyte ballooning and lobular inflammation were reduced in mice, whereas treatment with a GCS enzyme inhibitor only reduced GF BTWT The degree of fatty degeneration in mice, while GF BT△GCS Mice showed no further changes.

[0050] Example 4 Identification of cell types that inhibit nonalcoholic steatohepatitis with GCS enzyme inhibitors

[0051] Fresh peripheral blood was collected from 18 cases in EDTA anticoagulation tubes, and 5 mL of peripheral blood was layered in HISTOPAQUE-1077 (MilliporeSigma) solution to isolate PBMCs. After centrifugation, immune cells remaining at the plasma-histopaque-1077 interface were transferred to a new tube, washed twice with PBS, and resuspended in sorting buffer. The number of viable cells was determined in 10 μL of each single-cell suspension using an automated cell counter (Luna-II, Logos Biosystems). Cell concentration and viability were subsequently assessed using a RigelS3 fluorescent cell analyzer. To avoid cell composition bias caused by any enrichment step, the original unsorted cell suspension was used for library construction. The cell suspension was loaded onto the 10x Genomics single-cell platform to generate gel beads (GEMs), and the library was sequenced with 150 base end pairs by the Illumina NovaSeq 6000 sequencer. Cell Ranger (version 3.1) was used to align and quantify the scRNA-seq data with the human reference genome GRCh38. The unique molecular identifier (UMI) counts for each gene in each cell were summarized. The raw UMI count matrix was filtered using the R package Seurat (version 3.2.3), and the cell quality was assessed according to the following criteria: (1) the total number of UMI counts per cell (library size) was less than 30,000; (2) the number of genes detected per cell was more than 500 and less than 6,500 (neutrophils have a low number of transcripts, ranging from 100 to 6,000); (3) the percentage of UMIs from mitochondrial genes was less than 20%. Subsequently, a cluster-level approach was used to remove bimodal cells. The raw counts and log2 (normalized counts) of each cell were calculated. As immune cells and stromal cells were well mixed across samples, no obvious batch effects were observed. The Python package scanpy (version 1.6)13 was used to identify the major cell types. A total of 2,000 HVGs were selected by highly variable gene functions, and the top 50 principal components were calculated for the first round of clustering and annotation. The nearest neighbor graph was constructed using the neighbor function and clustering was performed using the community algorithm using the Louvain function with a resolution of 1.0. The expression of typical marker genes was used to confirm the annotation of each cell cluster. Based on each major cell type, clusters within the major cell types were identified using the same clustering protocol mentioned in BMKCloud (www.biocloud.net).

[0052] according to Figures 7-8 The results showed that myeloid cell clusters constituted the main altered cellular components in GH-Miglustat, while B cells and EC clusters dominated in GL-Miglustat samples.

[0053] Example 5 Trajectory Analysis

[0054] The R package Monocle (version 2.12) was used to construct the cell differentiation trajectories. A CellDataSet object was created using the UMI count matrix and negative binomial. The size parameter was set in the default settings using the new CellDataSet function. Dimensionality reduction was performed using the DDR Tree algorithm, and then the cells were sorted along the trajectory. The likelihood ratio test was used to identify the altered genes along the trajectory using the differential gene test function in the monocle package. The minimum spanning tree of the cells was plotted by the plot-complex-cell-trajectory or plot-cell-trajectory functions. The BEAM test with all default parameters was performed on the first branch point of the cell lineage.

[0055] according to Fig. 9 The results showed that in the GH-Miglustat group, MINCLE + Mφ is mainly converted into CCL22.

[0056] Summary and analysis:

[0057] In order to study the potential mechanism by which GCS enzyme inhibitor-mediated B. thetaiotaomicron monoglycosylated SLs affect the homeostasis of host liver SLs, a mixed nitrogenated amide-modified ceramide (CerA) was added to the culture to evaluate whether human hepatic steatotic cells can process glycosphingolipids produced by B. thetaiotaomicron. The present invention uses B. thetaiotaomicron (BTWT) and its inactivated GCS strain. L-02 cells were co-cultured with BTWT or BTΔGCS cells in a transwell system, and they were incubated with an azide-conjugated fluorophore (AF647-azide), and the fluorescence intensity of each group was continuously quantified by the fluorophore. After the azide-labeled L-02 cells were co-cultured with BT△GCS cells, the fluorescence intensity increased sharply and reached a plateau at 0.876h, while the fluorescence intensity of the BTWT co-culture group increased slowly within 2h. Among them, in BTWT co-cultured L-02 cells, GCS enzyme inhibitors were beneficial to accelerate the transfer of CerA-derived SLs and downregulate the mRNA levels of liver inflammation and fibrosis-related genes, while BT△GCS co-cultured L-02 cells were not. To further elucidate these azide-containing sphingolipids, the above azide-containing sphingolipids were analyzed by tandem mass spectrometry. GCS enzyme inhibitors only reduced the concentrations of azide-containing Hex1Cer (d18:1 / 16:0) and Hex1Cer (d18:1 / 24:1) in BTWT co-cultured L-02 cells.

[0058] In the MASH diet-induced GF mouse model, BTWT / BT containing CerA mixture was administered before oral administration of GCS enzyme inhibitor 3 mg / kg / day. △GCS The cultures were placed on a fat-free diet for one month to ensure that bacterial-derived lipids assimilated by the host were detected. The results showed that treatment with GCS enzyme inhibitors did not affect BT△GCS (GF BT△GCS )-colonized GF mice, while BTWT-colonized GF mice (GF BTWT ) after GCS enzyme inhibitor treatment had a significantly lower weight gain than the control group (p<0.01). BTWT The circulating TG, TC and liver TC of mice were improved, while GF BT△GCS There was no improvement in mice. H&E, Oil Red O (ORO) and Sirius staining showed that BTWT Compared with mice treated with salt, GF BT△GCS Hepatocyte ballooning and lobular inflammation were reduced in mice, whereas treatment with a GCS enzyme inhibitor only reduced GF BTWT The degree of fatty degeneration in mice, while GF BT△GCS Mice do not. BTWT In mice, GCS enzyme inhibitors can reduce SAF scores, overall steatosis / hepatocyte percentage, oil red / % area and mRNA levels of liver fibrosis-related genes Col1a1, Timp1 and Mmp2. In addition, the present invention found that GCS enzyme inhibitors only promoted the transfer of azide-labeled SLs from GF BTWT Intestinal metastasis in mice, but not GF BT△GCS Mouse.

[0059] In order to understand in detail the unique immunological role of intestinal microbial GCS in all immune cell populations during GCS enzyme inhibitor treatment, the present invention used a droplet-based single-cell platform (10x Genomics Chromium) to perform single-cell RNA sequencing (scRNA-seq) and analyze 18 peripheral blood mononuclear cell (PBMC) samples. Including 3 fecal GCS high-activity samples treated with placebo (GH-PL), 3 fecal GCS high-activity samples treated with 160mg Miglustat (GH-Miglustat) and 3 fecal GCS low-activity samples treated with 160mg Miglustat (GL-Miglustat), which were treated on day 0 and day 14, respectively. A total of 57 cell clusters were identified from 128,031 cells screened from the GH-PL, GH-Miglustat, and GL-Miglustat data sets. Marker-based annotation obtained with SingleR v.1.0 defined four major cell types: T cells, NK cells and B cells, myeloid cells, endothelial cells (ECs), and mesenchymal cells. The ratio of observed cell number to expected cell number (R o / e ) to quantify the relative enrichment of the main cell clusters, the present invention found that the myeloid cell cluster constituted the main altered cell component in GH-Miglustat, while the B cell and EC clusters dominated in the GL-Miglustat samples. In view of the above results, it is shown that GCS enzyme inhibitors treat fatty hepatitis through the GCS enzyme of B. thetaiotaomicron, and myeloid cells are focused on in the specific cell compartment analysis between GH-Miglustat and GL-Miglustat.

[0060] Among all myeloid cell subclusters, dendritic cells (DCs) and macrophages (Mφ) are the main cell types in terms of the number of filtered cells. Through unsupervised clustering, the present invention first focused on the intrinsic properties and potential functions of DC populations, which were identified as downregulated CD8α + DCs. Notably, in the GH-Miglustat group, IL-12Rβ2 of Tregs could inhibit CD8α + DCs-derived IL-35, as previously reported, acquires enhanced inhibitory function for inflammatory processes. Strikingly, CD4 + Foxp3 +The number of Tregs increased. The suppressive function of these Tregs retained the effect of the EBI3 subunit of IL-35 on Tregs, resulting in significantly impaired immunogenicity. This may be due to the abnormal activation of STAT1 / STAT3 and the inhibition of the p38 MAPK / NF-κB signaling pathway. In addition, the present invention noted that the distribution of DC clusters was associated with the expression of gamma-glutamyltransferase (GGT), a recognized serum biomarker for MASH progression. In all DCs in the GH-Miglustat group, GGT was significantly higher than that in the GL-Miglustat or GH-PL group. + CD1C in samples + The proportion of DCs (cDC2) increased significantly, while DEC205 + The proportion of DCs (cDC1) decreased. The present invention's observations indicate that cDC1 and cDC2 cells respond to GCS enzyme inhibitor treatment in opposite ways.

[0061] For the macrophage compartment, unsupervised clustering yielded 6 clusters with distinct gene signatures. Two PBMCs-enriched nonclassical monocyte clusters were characterized: MINCLE + Mφ and S1PR2 + In these macrophage clusters, MINCLE + Mφ was the dominant subpopulation in GH-Miglustat and GL-Miglustat samples, while S1PR2 + Mφ tended to be expressed in the GH-Miglustat group. The present invention studied the cell-cell interaction analysis of GH-Miglustat samples and confirmed that anti-inflammatory macrophages actively participated in the recruitment of T cells through CCL3 / 15-CCR5, CXCL10 / 11-CXCR5 and CXCL12 / 14-CXCR4 signals. In the GH-Miglustat group, the surprisingly found MINCLE + Mφ is mainly converted into CCL22, and AGR1 + Mφ upregulated the expression levels of RPS27, CDKN1C, and KLRF1, indicating that fecal-derived GCS treated with GCS enzyme inhibitors + The above metabolic indicators after GCS enzyme inhibitor treatment, the presence of GCS activity, the relative abundance of B. thetaiotaomicron in feces, and the CD8α in serum were combined. + Dendritic cells, MINCLE + Macrophages were significantly positively correlated with the decrease of liver TG, TC and GGT levels ( Fig.10). These results suggest that B. thetaiotaomicron-mediated immunosuppression of GCS activity may predict remission of MASH in individuals treated with GCS enzyme inhibitors.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of glucosylceramide synthase inhibitors in the preparation of products for preventing and / or treating diseases related to metabolic stress liver damage.

2. The use according to claim 1, characterized in that: The metabolic stress liver injury-related diseases include one or more of hyperglycemia, hyperlipidemia, non-alcoholic fatty liver disease, decompensated cirrhosis, hepatocellular carcinoma and liver transplant recurrence.

3. The use according to claim 1, characterized in that: The glucosylceramide synthase inhibitor treats metabolic stress liver damage-related diseases by inhibiting glucosylceramide synthase of Bacteroides.

4. The use according to claim 3, characterized in that: The Bacteroides is Bacteroides thetaiotaomicron.

5. The use according to claim 1, characterized in that: The glucosylceramide synthase inhibitor is Miglustat.

6. The use according to claim 1, characterized in that: The product includes one or more of medicine, food, and feed.

7. A drug for preventing and / or treating metabolic stress-induced liver damage-related diseases, characterized in that: The medicine comprises an effective dose of a glucosylceramide synthase inhibitor and pharmaceutically acceptable adjuvants.

8. The drug according to claim 7, characterized in that The auxiliary materials include one or more of carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and flavors.

9. The drug according to claim 7, characterized in that The dosage form of the drug includes one or more of granules, capsules, tablets, powders, oral solutions, suspensions and emulsions.

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