Application of rosaxostat in preparation of medicine for treating steatohepatitis related to metabolic dysfunction

Abstract

The invention discloses an application of rosaxostat in preparation of a medicine for treating metabolic dysfunction related steatohepatitis, in-vivo and in-vitro experiments prove that the rosaxostat improves liver cell lipid accumulation, inflammatory response and fibrosis in an MASH model through a thyroid hormone receptor beta, enlarges the indications of the rosaxostat, and improves the metabolic dysfunction related steatohepatitis. The compound is expected to become the first drug for treating MASH in China.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to an application of rosuvastatin in preparing a drug for treating metabolic dysfunction-related fatty hepatitis. Background Art

[0002] Metabolic dysfunction-associated steatohepatitis (MASH), formerly known as nonalcoholic steatohepatitis (NASH), is a progressive metabolic dysfunction-associated steatotic liver disease (MASLD) or nonalcoholic fatty liver disease (NAFLD) that usually follows simple fatty liver and includes liver steatosis, hepatitis, and liver fibrosis with or without liver fibrosis. MASLD has become the most common liver disease in the world, and the latest adult prevalence of MASLD in China is as high as 40.3%. MASH is a metabolic dysfunction-associated fatty liver disease in a transitional and unstable state. If not effectively controlled, it can develop into cirrhosis, liver cancer, or death.

[0003] At present, the pathogenesis of MASH is still unclear, and the development and exploration of related drugs are relatively slow. Roxadustat, as an innovative hypoxia-inducible factor proline hydroxylase inhibitor (HIF-PHI), was first approved for marketing in China in December 2018 and is suitable for anemia caused by chronic kidney disease (CKD). Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide an application of rosuvastatin in the preparation of a drug for treating fatty liver disease associated with metabolic dysfunction.

[0005] Technical solution: The use of rosuvastatin described in the present invention in the preparation of a drug for treating fatty liver disease associated with metabolic dysfunction.

[0006] Preferably, the disease symptoms of metabolic dysfunction-associated steatohepatitis are the coexistence of liver steatosis, liver inflammation and liver fibrosis.

[0007] Preferably, rosuvastatin is used to treat metabolic dysfunction-related fatty liver disease via thyroid hormone receptor β.

[0008] Preferably, hepatocyte lipid accumulation in the MASH model is improved.

[0009] Preferably, the inflammatory response in the MASH model is improved.

[0010] Preferably, fibrosis in the MASH model is improved.

[0011] Preferably, rosuvastatin is used in combination with other drugs to prepare a drug for treating metabolic dysfunction-related fatty hepatitis.

[0012] Preferably, rosuvastatin is prepared into an oral preparation, an injection or an external preparation.

[0013] The drug for treating metabolic dysfunction-related fatty hepatitis of the present invention includes rosuvastatin or a pharmaceutically acceptable salt of rosuvastatin.

[0014] Preferably, it also includes a pharmaceutically acceptable carrier.

[0015] Principle of the invention: The traditional clinical treatment of metabolic dysfunction-related fatty liver disease is anti-inflammatory symptomatic treatment, and the treatment outcome is often poor, and there is a lack of targeted drugs for etiological treatment. Rosuvastatin was originally a drug for the treatment of renal anemia that has been marketed. The present invention found through molecular docking and pharmacological studies that rosuvastatin treats metabolic dysfunction-related fatty liver disease through thyroid hormone receptor β, which not only expands the clinical indications of rosuvastatin, but also provides a potential drug for the treatment of metabolic dysfunction-related fatty liver disease.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. Thyroid hormone receptor β is the target of the pathogenesis of MASH, and roxadustat is a THRβ receptor agonist, that is, roxadustat is an etiological treatment drug for MASH, which is superior to the current clinical symptomatic treatment for liver protection; 2. Roxadustat is an old drug for new uses, which reduces the time cost and economic cost of roxadustat anti-MASH clinical trials; 3. There are currently no drugs for the treatment of MASH on the market in China, and roxadustat is expected to become the first drug for the treatment of MASH in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a diagram showing the docking of Roxadustat and THRβ protein;

[0018] Figure 2 This is a diagram showing the effect of roxadustat on lipid metabolism induced by FFA in L02 hepatocytes; Figure 2 A: Oil red staining, Figure 2 B: Oil red staining semi-quantitative, Figure 2 C and Figure 2 D: Contents of triglyceride and cholesterol in hepatocytes; Ctr is the normal control group, Mdl is the MASH model group, R is the roxadustat group, and B is the positive drug bezafibrate group;

[0019] Figure 3 This is a graph showing the effect of roxadustat on FFA-induced inflammatory factors in L02 hepatocytes;

[0020] Figure 4 Effect of roxadustat on liver lipid metabolism in MASH mice - liver HE staining;

[0021] Figure 5 This is a diagram showing the effect of roxadustat on liver lipid metabolism in MASH mice; Figure 5 A: NAS score, Figure 5 B and Figure 5 C is the content of triglyceride (TG) and cholesterol (TC) in the liver, Figure 5 D: Oil red staining, Figure 5 E: Semiquantitative analysis of Oil Red staining;

[0022] Figure 6 This is a diagram showing the effect of roxadustat on liver inflammation in MASH mice; Figure 6 A: Immunohistochemistry of CD68 in liver, Figure 6 B: Semi-quantitative analysis of CD68 positive cells, Figure 6 C. Figure 6 D. Figure 6 E is the mRNA expression of hepatic inflammatory factors;

[0023] Figure 7 This is a diagram showing the effect of roxadustat on liver fibrosis in MASH mice; Figure 7 A: Liver sirius red staining, Figure 7 B: Semi-quantitative analysis of picrosirius red staining, Figure 7 C. Figure 7 D. Figure 7 E is the mRNA expression of liver fibrosis. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0025] Example 1

[0026] The molecular docking and in vivo and in vitro pharmacology experimental study plans of roxadustat are as follows:

[0027] 1. Molecular docking analysis of rosuvastatin and THRβ receptor of the present invention

[0028] First, the protein structure of THRβ receptor was obtained from the PDB database, and the molecular docking of rosuvastatin and THRβ receptor was performed using Schrödinger software. The XP Gscore value was set to be less than -6, indicating that the ligand and protein have stable binding performance. The MM-GBSAdG Bind value was set to be less than -30 kcal / mol, indicating that the binding free energy is low and the ligand and protein are stably bound.

[0029] 2. In vitro study of the effect of roxadustat on MASH model hepatocytes

[0030] ① L02 cell grouping and drug administration

[0031] L02 cells with good growth status were digested and subcultured, and 5×10 5 Cells were seeded at a density of cells / mL in 6-well plates and incubated at 37°C and 5% CO 2 Cultured in an incubator. Experimental groups: normal control group; model group (1mM FFA); low-dose roxadustat group (1mM FFA+10μM); medium-dose roxadustat group (1mM FFA+20μM); high-dose roxadustat group (1mM FFA+20μM). FFA and roxadustat were added to serum-free medium at the same time and cultured for 24 hours, and then the cells were collected.

[0032] ② Oil red O staining of hepatocytes

[0033] Remove the culture medium from the 6-well plate and wash three times with 37°C PBS. Fix with 4% paraformaldehyde at 4°C for 15 minutes, discard the paraformaldehyde, and wash three times with tap water, 5 minutes each time. Add Oil Red O working solution and add 1.0mL to each well for staining for 30 minutes. Discard the Oil Red O staining solution, wash three times with triple distilled water, 5 minutes each time, and take pictures under an inverted microscope. After that, add 100μL of isopropanol to each well and incubate at room temperature for 15 minutes, shake gently, transfer to a 96-well plate, and detect its absorbance value with an enzyme reader at a wavelength of 520nm.

[0034] ③ Determination of triglyceride and cholesterol content in hepatocytes

[0035] The cells in the 6-well plate were collected and the TG and TC contents in the cells were determined according to the instructions of the TG and TC kits. The total protein content in the cells was detected by BCA protein concentration assay.

[0036] ④Gene expression of THRβ and inflammatory factors in hepatocytes.

[0037] The cells in the 6-well plates were collected, and the mRNA expressions of THRβ and inflammatory factors TNFα, IL-1B, and IL-6 in hepatocytes were detected by RT-qPCR.

[0038] 3. In vivo study of the effect of roxadustat on the liver of MASH mice

[0039] ① Animal experiment grouping, drug administration and material collection

[0040] 32 C57BL6J mice were housed in a barrier system at room temperature of 20℃~25℃ and 12 hours of light per day. After one week of adaptation, they were randomly divided into 4 groups according to body weight and randomized into 8 groups by randomized block method. The normal group was given ordinary feed, and the model group was fed with choline-methionine-deficient feed (MCD) to establish the MASH model. After 6 weeks of modeling, they were randomly divided into 3 groups and began to be given rosuvastatin intervention until the end of the 10th week (drug intervention for 4 weeks). The administration time was 10:00 am every day. The specific groups are as follows: normal control group: ordinary feed + 0.5% CMC-Na; model group: MCD feed + 0.5% CMC-Na; low-dose rosuvastatin group: MCD feed + rosuvastatin (10 mg / kg); high-dose rosuvastatin group: MCD feed + rosuvastatin (20 mg / kg); two tissue blocks with a volume of approximately 1 cm×1 cm×1 cm were collected, which were used to make paraffin sections and frozen sections respectively. The remaining liver was quickly frozen in liquid nitrogen and then stored at -80°C for testing liver tissue biochemical indicators.

[0041] ②Hematoxylin and eosin (HE) staining and liver histopathological diagnosis

[0042] The collected liver tissues were embedded in wax blocks, sliced, and stained with HE to observe the morphology and pathological phenomena of liver cells. The pathological diagnosis criteria of MASH adopt the guidelines set by the 2005 Pathology Committee of the National Institutes of Health NASH (or MASH) Clinical Research Network, which is recommended by the "Asia-Pacific Consensus on the Diagnosis and Treatment of Non-Alcoholic Fatty Liver Disease", and are evaluated according to the NAFLD activity score (NAS) developed by them (as shown in Table 1). Among them, NAS ≥ 5 points can confirm the diagnosis of NASH, NAS < 3 points can exclude NASH, and those between the two are possible NASH.

[0043] Table 1 NAFLD activity score

[0044]

[0045] ③ Liver Oil Red O staining and determination of TG and TC in liver tissue

[0046] The fresh rat liver tissue blocks were frozen and sliced, and the frozen sections were stained with Oil Red O dye, and the lipid accumulation was observed under a microscope. 100 mg of liver tissue was accurately weighed, 900 μL of methanol was added, and the liver tissue was homogenized with an electric homogenizer; according to the ratio of chloroform / methanol (2:1, v / v), the liver tissue was extracted for 12 hours, centrifuged at 3,500 rpm / min for 10 minutes; all the supernatant was taken as much as possible and placed in a new centrifuge tube, and vortexed for 2 minutes; the content of TG and TC in rat liver was determined according to the method described in the kit instructions.

[0047] ④ Immunohistochemistry to detect the level of liver inflammatory response

[0048] The liver tissue paraffin blocks were sliced ​​and stained with CD68 antibody for immunohistochemistry to detect the expression level of Kupffer cell positive marker CD68 in liver tissue, and imagJ was used for semi-quantification of CD68 positive cells.

[0049] ⑤ Detection of liver fibrosis and gene expression of fibrotic factors by picrosirius red staining and RT-qPCR

[0050] Liver tissue wax blocks were sliced ​​and stained with picrosirius red to detect the expression of liver collagen, and imagJ was used for semi-quantification of collagen. RT-qPCR was used to detect the mRNA expression of liver fibrosis factors.

[0051] The experimental results are:

[0052] 1. Molecular docking analysis of rosuvastatin and THRβ receptor of the present invention

[0053] Based on the XP docking and MM-GBSA analysis results, the docking score of Roxadustat with THRβ is -17.033, and the MM-GBSA analysis result is -69.63 kcal / mol. Both the binding free energy and the docking score are very low, indicating that Roxadustat binds very stably to THRβ. The docking score of Roxadustat with THRα is -4.367, and the MM-GBSA analysis result is -20.48 kcal / mol. Both the binding free energy and the docking score are low, indicating that Roxadustat binds relatively stably to THRα (as shown in Table 2). Roxadustat penetrates deep into the active pocket of the THRβ protein, forming a hydrogen bond with protein residue ASN331, two hydrogen bonds with residue MET313, a hydrogen bond and a salt bridge with residue ARG282, and a π-π bond with residue PHE269 (as shown in Table 2). Figure 1 As shown in the figure, ## and ### represent the significance compared with the normal control group at P<0.05, P<0.01; *, *, ** represent the significance compared with the model group at P<0.05, P<0.01, P<0.001).

[0054] Table 2. Results of molecular docking of rosuvastatin and THRβ

[0055]

[0056] 2. In vitro study of the effect of roxadustat on MASH model hepatocytes

[0057] In vitro, the FFA-induced MASH HepG2 cell model was used to detect the effect of rosuvastatin on lipid accumulation in hepatocytes. Rosuvastatin dose-dependently reduced lipid accumulation in hepatocytes and significantly reduced the content of TG and TC in hepatocytes ( Figure 2 , ## and ### in the figure represent the significance compared with the normal control group at P<0.05, P<0.01; *, *, ** represent the significance compared with the model group at P<0.05, P<0.01, P<0.001). In addition, rosuvastatin can dose-dependently reduce the gene expression of inflammatory factors (Tnfa, Il6, Mcp1) in FFA-induced MASH hepatocytes ( Figure 3 In the figure, ## and ### represent the significance compared with the normal control group, P<0.05, P<0.01; *, *, ** represent the significance compared with the model group, P<0.05, P<0.01, P<0.001).

[0058] 3. In vivo study of the effect of roxadustat on the liver of MASH mice

[0059] In vivo, a high-fat, high-sugar diet was used to induce a MASH mouse model, and the effects of roxadustat on lipid metabolism and inflammatory response in the liver of MASH mice were examined. Roxadustat dose-dependently reduced hepatocyte lipid accumulation and significantly reduced the content of TG and TC in the liver. In addition, roxadustat dose-dependently reduced NAS Score, indicating that roxadustat can significantly improve or block the progression of MASH ( Figure 4 and Figure 5 In the figure, ## and ### represent the significance compared with the normal control group, P<0.05, P<0.01; *, *, ** represent the significance compared with the model group, P<0.05, P<0.01, P<0.001).

[0060] In the experimental liver lipid metabolism disorder model, long-term chronic inflammation can activate the most important resident macrophage in the liver, Kupffer cells, and their positive marker CD68 protein expression is upregulated. The results of CD68 immunohistochemistry in the livers of MASH mice in each group showed that rosuvastatin dose-dependently reduced the number of CD68-positive cells and the expression of inflammatory factors (Tnfa, Il6, Mcp1) in the livers of MASH mice. Figure 6 In the figure, ## and ### represent the significance compared with the normal control group, P<0.05, P<0.01; *, *, ** represent the significance compared with the model group, P<0.05, P<0.01, P<0.001).

[0061] Liver fibrosis can be observed under ordinary light microscopy by staining the liver with Sirius red. In the normal control group, the liver collagen fibers are red and the cell nuclei are blue. They are mainly distributed in the portal area and around the central vein of the liver, and their content is relatively low. In the model group, the collagen fibers in the portal area and around the central vein of the liver increased significantly, the area increased, and appeared bright red. At the same time, Sirius red staining also indirectly reflects the activity of astrocytes in the liver. Figure 7 As shown in the figure (## and ### represent the significance compared with the normal control group at P<0.05, P<0.01; *, *, ** represent the significance compared with the model group at P<0.05, P<0.01, P<0.001), rosuvastatin dose-dependently reduced the content of liver collagen fibers and the expression of fibrosis factors (Tgfb, Asma, Mmp13) in MASH mice.

Claims

1. The use of roxadustat in the preparation of drugs for the treatment of fatty liver disease associated with metabolic dysfunction.

2. The application according to claim 1, characterized in that: The disease symptoms of metabolic dysfunction-associated steatohepatitis are the coexistence of liver steatosis, liver inflammation and liver fibrosis.

3. The application according to claim 1, characterized in that: Roxadustat treats metabolic dysfunction-related steatohepatitis via thyroid hormone receptor β.

4. The use according to claim 1, characterized in that: Improves hepatocyte lipid accumulation in the MASH model.

5. The use according to claim 1, characterized in that: Improves inflammatory response in the MASH model.

6. The use according to claim 1, characterized in that: Improves fibrosis in the MASH model.

7. The use according to claim 1, characterized in that: Rosuvastatin is used in combination with other drugs to prepare drugs for treating fatty liver disease associated with metabolic dysfunction.

8. The use according to claim 1, characterized in that: Rosuvastatin is prepared into oral preparations, injections or external preparations.

9. A drug for treating metabolic dysfunction-related fatty liver disease, characterized in that: Including roxadustat or a pharmaceutically acceptable salt of roxadustat.

10. A drug for treating metabolic dysfunction-related fatty hepatitis according to claim 9, characterized in that: Also included are pharmaceutically acceptable carriers.

Images