Application of clofampol in preparation of medicine for preventing and / or treating non-alcoholic fatty liver disease

Abstract

The invention discloses an application of clofampol in preparation of a medicine for preventing and / or treating non-alcoholic fatty liver disease, and belongs to the technical field of medicines. It is found that the clofamprol can reduce the liver index, effectively improve liver lipid metabolism disorder, inhibit hepatic cell fatty degeneration, inflammatory response and fibrosis processes, reduce the level of alanine transaminase, aspartic transaminase, total bilirubin, triglyceride and low-density lipoprotein, improve the level of albumin and high-density lipoprotein, and inhibit liver cell fatty degeneration, inflammatory response and fibrosis processes. Therefore, effective treatment on the non-alcoholic fatty liver is realized.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of clofocol in preparing medicines for preventing and / or treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver injury closely associated with insulin resistance and genetic susceptibility. It is characterized by diffuse hepatocyte steatosis, accompanied by chronic hepatocyte inflammation and apoptosis. The development of NAFLD involves multiple risk factors, including obesity, insulin resistance, hyperglycemia, and hypertension.

[0003] Currently, intervention and treatment for NAFLD primarily rely on lifestyle changes (such as a healthy diet and exercise), but patient compliance is poor, necessitating the use of surgical and medication-based treatments. Surgical treatment carries a significant economic burden, high postoperative recurrence rates, and a high risk of cardiovascular complications. Treatments for NAFLD include insulin sensitizers, metformin, antioxidants, lipid-lowering drugs, and anti-inflammatory drugs, but these have limited applicability and are associated with certain side effects and uncertainties. Therefore, new therapeutic agents are needed. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes the use of clofoctol in the preparation of a medicament for preventing and / or treating fatty liver.

[0005] The present invention also proposes the use of clofocol or α-estradiol.

[0006] The present invention also provides a method for increasing the expression level of CSDE1 protein for non-diagnostic and therapeutic purposes.

[0007] Use of clofocol according to the first embodiment of the present invention in the preparation of a medicament for preventing and / or treating fatty liver.

[0008] The present invention has at least the following beneficial effects:

[0009] Chlorpheniramine can effectively treat non-alcoholic fatty liver disease, reduce liver index, effectively improve liver lipid metabolism disorders, inhibit liver cell fatty degeneration, inflammatory response and fibrosis process, reduce the levels of alanine aminotransferase, total bilirubin, aspartate aminotransferase, and triglycerides, and increase the levels of total protein, albumin, total bile acid, and high-density lipoprotein.

[0010] According to some embodiments of the present invention, the use of the medicament includes at least one of the following A1) to A7):

[0011] A1) Reduce liver index;

[0012] A2) inhibits the accumulation of fat in the liver;

[0013] A3) Improve liver fibrosis;

[0014] A4) Reduce liver inflammation;

[0015] A5) inhibits hepatocyte fatty degeneration;

[0016] A6) reducing the level of at least one of the following: serum triglyceride (TG), aspartate aminotransferase (AST), total bilirubin (TBIL), alanine aminotransferase (ALT), and low-density lipoprotein (LDL);

[0017] A7) Increased serum albumin (ALB) levels.

[0018] According to some embodiments of the present invention, the fatty liver is non-alcoholic fatty liver.

[0019] According to some embodiments of the present invention, the fatty liver is fatty liver caused by a high-fat diet.

[0020] According to some embodiments of the present invention, the drug is in the form of an oral dosage form, an injection dosage form, or a transdermal dosage form. For example, the oral dosage form may be a tablet, capsule, or oral solution; the injection dosage form may be an injection or powder injection; and the transdermal dosage form may be an adhesive transdermal device.

[0021] According to some embodiments of the present invention, the medicament comprises a pharmaceutically acceptable excipient.

[0022] According to some embodiments of the present invention, the excipient includes at least one of a diluent, a binder, an antioxidant, a pH adjuster, a preservative, a lubricant, and a disintegrant.

[0023] According to some embodiments of the present invention, the drug contains clofoquinol as a main active ingredient.

[0024] According to some embodiments of the present invention, the content of clofoquinol in the medicament is a therapeutically effective amount.

[0025] According to some embodiments of the present invention, the content of clofocol in the drug can be 0.1%-100% by mass, for example, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%.

[0026] According to some embodiments of the present invention, the subject of the drug is a mammal.

[0027] According to some embodiments of the present invention, the mammal is at least one of a human, a mouse, a rabbit, and a dog.

[0028] According to the second aspect of the present invention, the use of clofocol or α-estradiol in any one of B1) to B2):

[0029] B1) increasing the expression level of CSDE1 protein;

[0030] B2) preparing a product for increasing the expression level of CSDE1 protein.

[0031] The present invention has at least the following beneficial effects:

[0032] Chloroform and α-estradiol can effectively increase the expression level of CSDE1 protein.

[0033] According to some embodiments of the present invention, the product is selected from a drug, a reagent or a kit.

[0034] According to the third aspect of the present invention, a method for increasing the expression level of CSDE1 protein for non-diagnostic therapeutic purposes comprises the following steps: contacting the clofocol or α-estradiol with target cells.

[0035] According to some embodiments of the present invention, the target cells include but are not limited to liver cancer cells.

[0036] According to some embodiments of the present invention, the liver cancer cells include but are not limited to Hepg2 cells.

[0037] According to some embodiments of the present invention, the effective concentration of clofoquinol is above 10 μM.

[0038] According to some embodiments of the present invention, the effective concentration of clofocol is 10 μM-17 μM, for example, 10 μM, 10.5 μM, 11 μM, 11.5 μM, 12 μM, 12.5 μM, 13 μM, 13.5 μM, 14 μM, 14.5 μM, 15 μM, 15.5 μM, 16 μM, 16.5 μM or 17 μM.

[0039] According to some embodiments of the present invention, the effective concentration of α-estradiol is above 10 μM.

[0040] According to some embodiments of the present invention, the effective concentration of α-estradiol is 10 μM-17 μM, for example, 10 μM, 10.5 μM, 11 μM, 11.5 μM, 12 μM, 12.5 μM, 13 μM, 13.5 μM, 14 μM, 14.5 μM, 15 μM, 15.5 μM, 16 μM, 16.5 μM or 17 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The results of affinity chromatography purification of the full-length CSDE1 protein are shown; A: CSDE1 full-length prokaryotic expression plasmid map, B: affinity chromatography purification results;

[0042] Figure 2 The results of ion exchange chromatography purification of the full-length CSDE1 protein; A: Coomassie Brilliant Blue staining verification, B: ion exchange chromatography purification elution curve;

[0043] Figure 3 The results of gel exclusion chromatography purification of the full-length CSDE1 protein; A: Coomassie Brilliant Blue staining verification, B: gel exclusion chromatography purification elution curve;

[0044] Figure 4 The docking results of clofocol and CSDE1 molecules; A: Schematic diagram of ligand-protein molecular docking results, B: Specific interaction between ligand and protein;

[0045] Figure 5 Surface plasmon resonance (SPR) results for the analysis of clofocol-CSDE1 binding affinity;

[0046] Figure 6 This is the virtual screening result based on the structure of chloroform;

[0047] Figure 7 Effects of clofolate and α-estradiol on CSDE1 protein expression levels; A: Western blotting results; B: Gray value analysis results; *** indicates significant differences (p<0.001);

[0048] Figure 8 This is a schematic diagram of the experimental process of Example 2;

[0049] Figure 9 The improvement of the liver of NAFLD mice by different drugs; A: Representative photos of liver morphology, B: Liver index; * indicates significant difference (p < 0.05);

[0050] Figure 10The histological evaluation results of NAFLD mice before and after model establishment and after treatment with different drugs; A: Oil Red O, H&E, and Sirius Red staining results of liver tissue; B: Histological evaluation of the brain, heart, spleen, kidney, testis, and pancreas;

[0051] Figure 11 The test results of triglyceride (TG), low-density lipoprotein (LDL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), and total bilirubin (TBIL) in the serum of NAFLD mice after treatment with different drugs; ns indicates no significant difference, * indicates a significant difference (p<0.05), and ** indicates a significant difference (p<0.01). DETAILED DESCRIPTION

[0052] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0053] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0054] Example 1

[0055] 1. The full-length CSDE1 fragment (NCBI reference sequence: NM_001007553.3) was cloned into a modified prokaryotic expression vector (pMAL-p2x vector, with an MBP tag and a PreScission (3C) protease site) to construct a prokaryotic expression plasmid for the full-length CSDE1 protein (L15170 CSDE1 pMAL-p2x vector). This plasmid was transformed into Escherichia coli Tssetta (DE3) for prokaryotic protein expression and purification. After induction at 30°C and 2 mM IPTG for 4 hours, the bacteria were harvested by centrifugation at 4000g and 4°C for 20 minutes. The bacteria were lysed by sonication, and the protein was extracted and purified using different columns depending on the tag carried by the protein. Using MBP tag affinity chromatography, Dextrin Beads were first fixed to a gravity column, the upper and lower end stoppers were removed, and the protective solution in the gravity column was drained. Add 5 column volumes of MBP equilibration solution (20mM Tris HCl, 200mMNaCl, 1mM EDTA, pH=7.4) to the column tube to equilibrate the filler. Add the sample to the balanced gravity column, place it at 4℃ and shake slowly for 1h, and repeat the above operation once. Collect the natural effluent twice respectively. Then wash with 10 column volumes of washing solution (same as MBP equilibration solution) to remove impurities, repeat the above operation once, and collect washing solutions W1 and W2 respectively. Further, use 1 column volume of MBP equilibration solution containing Prescission Protease (20μL / mL) to perform on-column enzymatic digestion on the filler, shake slowly at 4℃ overnight, and collect the eluate E1. Then use 1 column volume of MBP equilibration solution to flow through naturally and collect the effluent E2. All the above collected components were filtered through a 0.22μm filter membrane, and the E1 component was concentrated using a 50KDa Millipore centrifugal ultrafiltration tube. The plasmid map of L15170 CSDE1 pMAL-p2x and the Coomassie Brilliant Blue verification results are as follows Figure 1 shown.

[0056] The obtained protein was further purified. First, the physicochemical properties of the CSDE1 protein were analyzed using an online tool (https: / / web.expasy.org / protparam / ), and the isoelectric point was 5.88. When using a neutral or alkaline buffer (pH 7.4), the protein is negatively charged in the solution. In order to obtain a higher purity protein, anion exchange chromatography was used for further purification, and a high concentration of NaCl solution was used for gradient elution. The peak components were collected and the purity of the obtained protein could reach more than 90%. Coomassie brilliant blue staining verification and elution curve are shown in Figure 2. Figure 2 shown.

[0057] In order to obtain a more homogeneous full-length CSDE1 protein, gel exclusion chromatography (i.e., molecular sieve chromatography) was used for further purification. Proteins were separated according to molecular weight, with proteins with larger molecular weight eluted from the column first and proteins with smaller molecular weight eluted later. The molecular weight of the protein at the corresponding elution volume was determined based on the standard, and the corresponding peak components were collected. Coomassie brilliant blue staining was used for verification and the elution curve was shown in Figure 2. Figure 3 , and finally obtained highly pure CSDE1 full-length protein.

[0058] 2. In MOE software, import the 3D structure of chloroform and minimize the structure. Import the PDB file O75534CSDE1_HUMAN (AF-O75534-F1) and perform QuickPrep. Then, perform Compute-Dock and select the ligand for docking.

[0059] The molecular docking results of clofocol and CSDE1 are as follows Figure 4 shown.

[0060] 3. Analyze the affinity of chlorfenapyr binding to CSDE1 based on SPR technology. Using a CM5 chip, the full-length CSDE1 protein was fixed on the chip surface by amino coupling. Chlorfenapyr was dissolved in HBS-P+ buffer, and samples with different concentration gradients (16μM, 24μM, 36.1μM, 54.1μM, 81.2μM, 121.8μM, 182.7μM, 274μM) were prepared. Set the experimental parameters in the Biacore 8K+ system, select the single-cycle kinetics mode (Single-Cycle Kinetics), and inject samples of different concentrations in sequence. Calculate the affinity constant (KD) of the small molecule and the protein by fitting a 1:1 binding model.

[0061] The results are as follows Figure 5 shown.

[0062] Chloroform has a certain binding ability with CSDE1, KD = 9.56×10 -4 M.

[0063] Example 2

[0064] 1. Pass Using the 2018-1 software, import the 3D structure of clofocalone and select the "Shape Screening" function. For "Screen Structure," select the FDA-approved drug compound library, which has been processed with 3D structures. For "Volume Scoring," select "Pharmacophore Types." Set the Similarity threshold to 0.45. After completing the settings, run the screening program to obtain clofocalone-like compounds, their similarity (Shape Similarity) scores, and rank them.

[0065] The results are as follows Figure 6 shown.

[0066] The top 10 drugs in terms of scores were estradiol, diethylstilbestrol, tolterodine tartrate, tolterodine, estrone, estradiol (cypionate), estrone sulfate piperazine, clioquinol, chloroxine, and α-estradiol.

[0067] 2. Hepg2 cells were inoculated into cell culture plates. When the cell confluence reached 60%-80%, clofoquinol and the top 10 drugs screened were administered at a concentration of 10 μM for 24 h. Cell proteins were extracted and quantified by BCA before Western Blot analysis (with β-tubulin as the internal reference). The relative grayscale values ​​were analyzed to compare the effects of each drug on the expression level of CSDE1 protein at the cellular level.

[0068] The results are as follows Figure 7 shown.

[0069] Chloroform and α-estradiol can promote the expression of CSDE1 protein in Hepg2 cells.

[0070] Example 3

[0071] C57BL / 6 mice (20-25 g, male, purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd., SCXK (Su) 2023-0009) were randomly divided into a control group (4 mice), a model group (4 mice), a clofocalone group (4 mice), and an α-estradiol group (4 mice). The control group was fed a conventional diet. Mice in the model, clofocalone, and α-estradiol groups were fed a high-fat diet (D09100310, 40% fat for energy, 20% fructose for energy, and 2% cholesterol supplemented) for 16 weeks. At weeks 6 and 9, a clofolate intraperitoneal injection solution was prepared (pre-dissolve the drug in DMSO, then add corn oil (filtered through a 0.22 μm filter) until the drug was completely dissolved. The ratio of clofolate, DMSO, and corn oil was 62.5 mg:1 mL:9 mL) and an α-estradiol intraperitoneal injection solution (α-estradiol:DMSO:corn oil:46.3 mg:1 mL:9 mL, prepared in the same manner as for the clofolate intraperitoneal injection) were prepared. A mixture of 10% DMSO and 90% corn oil was used as a solvent control. After preparation, all solutions were sterilized by filtering through a 0.22 μm filter and stored in the dark. The mice were weighed before administration. The mice in the clofocalone group were intraperitoneally injected with clofocalone (62.5 mg / kg) at the 6th and 9th weeks of feeding, respectively. The mice in the α-estradiol group were intraperitoneally injected with α-estradiol (46.3 mg / kg) at the 6th and 9th weeks of feeding, respectively. The mice in the model group were intraperitoneally injected with solvent control (10 mL / kg) at the 6th and 9th weeks of feeding. At the 16th week, the brain, heart, spleen, kidney, testis, pancreas, liver and serum of the mice were collected. The improvement of each drug on NAFLD mice was evaluated based on liver morphology, histology (oil red O staining, H&E staining and Sirius red staining of liver tissue), liver indicators, and serological indicators. The specific animal experiment design process is as follows. Figure 8 shown.

[0072] The results are as follows Figure 9 、 Figure 10 、 Figure 11 shown.

[0073] A normal liver has clear edges, is usually dark red with a bright color, and is soft and elastic. However, the livers of mice modeled with NAFLD were enlarged, with a significantly increased liver index, blunt edges, and a color change from the normal dark red to red and yellow, or even yellow or pale. The liver texture became brittle and felt hard. Compared with the control group, the model group had significantly increased levels of serum triglycerides (TG) (p < 0.001), total cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (ALT), and low-density lipoprotein (LDL) (p < 0.0001), indicating that the NAFLD mouse model was successfully established.

[0074] Compared with the model (DMSO) and α-estradiol groups, the liver morphology of mice in the clofocalone group improved slightly, with clearer edges and a dark red color. Furthermore, the liver index of mice in the clofocalone group was significantly lower than that in the model group. This suggests that clofocalone administration normalized liver volume and improved liver morphology in NAFLD mice.

[0075] H&E and Oil Red O staining of mouse liver tissue revealed that the liver structure of mice treated with chloramphenicol returned to normal, with significantly reduced lipid accumulation. Furthermore, Sirius Red staining revealed a reduction in liver fibrosis in mice treated with chloramphenicol. This suggests that chloramphenicol improves liver fibrosis in NAFLD mice. Furthermore, chloramphenicol did not affect the histomorphology of other organs in the mice.

[0076] After treatment with clofocalone, serum ALT and AST levels decreased in model mice, while ALB and TBIL levels improved to a certain extent. This suggests a reduction in liver damage. Furthermore, serum TG levels decreased, indicating a decrease in circulating lipid levels. Serum LDL levels also decreased significantly, suggesting an improvement in lipid metabolism disorders.

[0077] In summary, clofocol can improve the liver morphology and liver lipid accumulation of NAFLD mice, inhibit hepatocyte fatty degeneration, inflammatory response and fibrosis process, improve liver function and lipid metabolism, and can overall improve the pathological changes in NAFLD mice.

[0078] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the scope of knowledge possessed by ordinary technicians in the relevant technical field without departing from the scope of the present invention.

Claims

1. Use of clofocol in the preparation of a medicament for preventing and / or treating fatty liver.

2. The use according to claim 1, characterized in that The use of the drug includes at least one of the following A1) to A7): A1) Reduce liver index; A2) inhibits the accumulation of fat in the liver; A3) Improve liver fibrosis; A4) Reduce liver inflammation; A5) inhibits hepatocyte fatty degeneration; A6) reduction in serum levels of at least one of triglycerides, aspartate aminotransferase, total bilirubin, alanine aminotransferase, and low-density lipoprotein; A7) Increases serum albumin levels.

3. The use according to claim 1, characterized in that The fatty liver is non-alcoholic fatty liver.

4. The use according to claim 1, characterized in that The fatty liver is fatty liver caused by a high-fat diet.

5. The use according to claim 1, characterized in that The dosage form of the drug is an oral dosage form, an injection dosage form or a transdermal dosage form.

6. The use according to claim 1, characterized in that The drug contains pharmaceutically acceptable excipients.

7. The use according to claim 6, characterized in that The auxiliary materials include at least one of a diluent, a binder, an antioxidant, a pH regulator, a preservative, a lubricant and a disintegrant.

8. Use of clofocol or α-estradiol in any one of items B1) to B2): B1) increasing the expression level of CSDE1 protein; B2) preparing a product for increasing the expression level of CSDE1 protein.

9. A method for increasing the expression level of CSDE1 protein for non-diagnostic and therapeutic purposes, characterized in that: The method comprises the following steps: contacting the clofocol or α-estradiol with target cells.

10. The method according to claim 9, characterized in that The effective concentration of the chlorfenapyr is above 10 μM; And / or, the effective concentration of α-estradiol is above 10 μM.

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