Application of East Asian scorpion toxin polypeptide in the preparation of drugs for treating and / or preventing liver diseases caused by intrahepatic lipid accumulation

Abstract

The present invention discloses the use of the East Asian scorpion toxin polypeptide Makatoxin‑3 or a pharmaceutical composition containing the same in the preparation of a drug for treating and / or preventing liver diseases caused by lipid accumulation in the liver, in particular, in the preparation of a drug for treating and / or preventing non-alcoholic fatty liver disease. The present invention finds that Makatoxin‑3, as a PPARα agonist, significantly activates the transcription of genes related to fatty acid β-oxidation, activates the transcription of genes related to fatty acid β-oxidation, promotes lipid oxidation and decomposition to inhibit lipid accumulation in the liver, and reduces inflammation and liver fibrosis. Therefore, Makatoxin‑3 can be used to prepare a drug for treating non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically to the use of the East Asian scorpion toxin polypeptide Makatoxin-3 in the preparation of medicines for treating and / or preventing liver diseases caused by intrahepatic lipid accumulation, and particularly to the use of the Makatoxin-3 in non-alcoholic fatty liver disease. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is a fatty liver disease associated with genetic susceptibility and metabolic dysfunction. Its disease spectrum includes simple fatty liver, non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). However, there is currently no effective drug approved for the treatment of NASH.

[0003] Peroxisome proliferator-activated receptor α (PPARα) is a ligand-regulated transcription factor that plays a key role in the transcriptional regulation of glucose, lipid metabolism and inflammatory response. Studies have shown that the pathological mechanism of NASH is closely related to PPARα dysfunction, and the liver PPARα level is negatively correlated with the severity of NASH in patients with NAFLD. PPARα activation causes the transcription of enzymes and genes related to lipid metabolism, promotes liver fatty acid β-oxidation, and reduces liver lipid deposition; it can also help reduce plasma triglyceride and cholesterol levels by regulating apolipoproteins. In terms of regulating inflammatory responses, PPARα ligand activation inhibits NF-kB activation in lymphocytes, and PPARα reduces the expression of inflammatory factors such as IFN-γ and TNF-α by trans-inhibiting the transcription of pro-inflammatory genes. Therefore, PPARα is an important target for studying NASH therapeutic drugs.

[0004] The whole scorpion is the dried body of the East Asian scorpion of the family Pincers, which has very high medicinal value. The venom of the East Asian scorpion contains a large number of active peptides, which may be one of the important ways for it to exert its medicinal effects. New drugs for the treatment of NASH based on the venom of the East Asian scorpion have not yet been effectively developed. Makatoxin-3 is a sodium channel toxin polypeptide isolated from scorpion venom. The inventor disclosed in the previous Chinese patent application CN112210569B the preparation method of Makatoxin-3 and its related mutants, and that such polypeptides have anti-inflammatory and analgesic effects and can be used to treat pain. Summary of the invention

[0005] The purpose of the present invention is to provide a new medical use of the East Asian scorpion toxin polypeptide Makatoxin-3, that is, its use in the preparation of a drug for treating and / or preventing liver diseases caused by intrahepatic lipid accumulation, wherein the East Asian scorpion toxin polypeptide is an East Asian scorpion toxin polypeptide, and its amino acid sequence is shown in SEQ ID NO.1 (GRDAYIAKKENCTYFCALNPYCNDLCTKNGAKSGYCQWAGRYGNACWCIDLPD KVPIRIPGPCIG). The detailed information of the East Asian scorpion toxin polypeptide Makatoxin-3 is disclosed in Chinese patent application CN112210569B.

[0006] Specifically, the liver disease caused by intrahepatic lipid accumulation includes any one or a combination of non-alcoholic fatty liver disease, alcoholic fatty liver disease, viral hepatitis, and drug-induced liver disease.

[0007] Wherein, the non-alcoholic fatty liver disease includes any one or a combination of liver steatosis, non-alcoholic fatty hepatitis, liver fibrosis and liver cirrhosis.

[0008] The present application discovered that the East Asian scorpion toxin polypeptide or a composition comprising the same acts as a PPARα agonist and / or a transcription activator of genes related to fatty acid β-oxidation; more specifically, the East Asian scorpion toxin polypeptide Makatoxin-3 or a pharmaceutical composition comprising the same activates PPARα transcriptional activity, promotes fatty acid β-oxidation in hepatocytes, reduces lipid deposition in hepatocytes, and reduces inflammation and liver fibrosis.

[0009] Specifically, the fatty acid β-oxidation related gene includes at least one of PPARα, ACOX1, CPT1A and PGC1α.

[0010] The present application also found that the East Asian scorpion toxin polypeptide Makatoxin-3 or a pharmaceutical composition comprising the same can reduce the content of TG and TC in the liver.

[0011] The East Asian scorpion toxin polypeptide Makatoxin-3 or a pharmaceutical composition comprising the same reduces serum ALT and AST levels.

[0012] Wherein, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition is in the form of an injection.

[0013] Beneficial effects: The present application unexpectedly discovered that Makatoxin-3 can act as a PPARα agonist and / or a transcriptional activator of fatty acid β-oxidation-related genes (ACOX1, CPT1A, and PGC1α), significantly activating the transcription of genes related to fatty acid β-oxidation, and promoting lipid oxidation and decomposition to inhibit lipid accumulation in the liver. Therefore, Makatoxin-3 and a composition containing the same can be used to prepare a drug for treating non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0015] Figure 1 This is a graph showing the effects of Makatoxin-3 on lipid accumulation, inflammation and fibrosis in liver tissue of NAFLD model mice;

[0016] Figure 2 This is the result diagram of the effect of Makatoxin-3 on the transcriptional activity of PPARα in hepatocytes;

[0017] Figure 3 This is the result diagram of the effect of Makatoxin-3 on fatty acid β-oxidation mRNA in hepatocytes; DETAILED DESCRIPTION

[0018] The present application unexpectedly discovered for the first time that Makatoxin-3 can activate PPARα transcriptional activity and promote fatty acid β-oxidation in liver cells. Further experiments show that Makatoxin-3 can reduce lipid deposition, inflammatory response and fibrosis in liver cells, and has the potential to treat non-alcoholic fatty liver disease. On this basis, the present invention is completed. In order to better understand the technical solution of the present invention, the present invention is further described below in conjunction with the embodiments.

[0019] Example 1: Preparation and purification of the functional polypeptide of the present invention.

[0020] 1 Experimental Materials and Methods

[0021] (1) Vector construction: The Makatoxin-3 gene and the MBP protein treated with double enzyme digestion were fused to the periplasmic expression vector using double enzyme digestion technology to construct a recombinant Makatoxin-3 or its mutant expression vector. In the constructed vector, the N-terminus of the MBP protein carries a His6 tag, and the Makatoxin-3 gene is located at the C-terminus of the MBP, and is connected to the Makatoxin-3 through the His-MBP at the N-terminus.

[0022] (2) Expression: The prepared recombinant Makatoxin-3 expression vector is transferred into the expression host, and high-copy transformants are obtained by screening. Then, the transformants are cultured and induced to express soluble His6-MBP fused Makatoxin-3 using an inducer. These fusion proteins are the recombinant toxin polypeptide Makatoxin-3. The periplasmic expression vector fused with the MBP protein is pLicC-MBP. The expression host is Escherichia coli. The inducer is any one of lactose, galactose or isopropyl galactoside (IPTG), the induction temperature is between 15-37°C, and the induction time is 2-24 hours.

[0023] (3) Isolation and purification: The cells were lysed using a homogenizer to release soluble recombinant MBP-Makatoxin-3, which was then used The MBP-Makatoxin-3 fusion protein was purified by avant 150 system. The MBP-Makatoxin-3 fusion protein was purified by nickel ion affinity chromatography, and then digested by TEV-protease specific protease, which can recognize and cut the MBP fragment in the fusion protein to release MBP. Subsequently, nickel ion affinity chromatography was used to separate the MBP protein containing the His6 tag and the recombinant Makatoxin-3 without the His6 tag. The amino acid sequence of the recombinant Makatoxin-3 is shown in SEQ ID NO.1.

[0024] For the detailed preparation method, please refer to the contents disclosed in Example 1 of Chinese Patent CN112210569B.

[0025] Example 2: Makatoxin-3 improves lipid accumulation, inflammation and fibrosis in liver tissue of NAFLD model mice.

[0026] 1 Experimental Materials and Methods

[0027] (1) Animal source: Experimental animals were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. (License No.: SCXK(Su)2016-0010). SPF grade C57BL / 6 male mice, 8-10 weeks old, weighing 20-22 g. Room temperature 21-25°C, light 12 h / day.

[0028] (2) Animal grouping and drug administration: After one week of adaptive feeding, the mice were sorted according to their weight from small to large, and randomly grouped using Excel into four groups: normal chow diet (NCD), high fat diet (HFD), Makatoxin-3 low-dose group (HFD-MkTx3-L), and Makatoxin-3 high-dose group (HFD-MkTx3-H). The mice were fed a high-fat diet for modeling, Research Diet (D09100301). Drug administration began 16 weeks after modeling, with intraperitoneal injection of Makatoxin-3 (75 or 150 nmol / kg) for 8 weeks.

[0029] (3) Sampling: After the experiment, mice were anesthetized with isoflurane, and blood and liver tissue were collected. After the whole blood was allowed to stand at room temperature for 30 min, it was centrifuged at 4°C, 4000×g, for 30 min, and the supernatant was collected. The liver tissue was stored at -80°C, and the other part of the tissue was fixed in 4% paraformaldehyde for pathological examination.

[0030] (4) H&E staining of liver tissue: The liver tissue was fixed with 4% paraformaldehyde, dehydrated, embedded and sliced, with a slice thickness of 0.4 μm. After dewaxing, the slices were stained with hematoxylin for 2 min, and then blued with tap water. Subsequently, the slices were stained with eosin for 5 min and sealed with neutral resin.

[0031] (5) Oil Red O staining: The fixed liver tissue was placed in a gradient (20% and 30%) sucrose for dehydration, and then embedded in OCT and sliced ​​to a thickness of 8 μm. A 0.5% Oil Red O storage solution was prepared with an isopropanol solution, and the storage solution was diluted with ultrapure water in a ratio of 3:2 to prepare an Oil Red O working solution, which was filtered, packaged, and stored in the dark. First, the liver tissue sections were rinsed with 60% isopropanol for 2 minutes, then stained with Oil Red O working solution for 5 minutes, adjusted with 60% isopropanol, washed with ultrapure water for 2 minutes, then stained with hematoxylin for 1 minute, and blued with a blueing solution, and finally sealed with 20 μL of Kisser sealing solution.

[0032] (6) CD11b staining of liver tissue: The liver tissue was fixed with 4% paraformaldehyde, dehydrated, embedded and sliced, with a thickness of 0.4 μm. The sections were repaired by microwave heating with antigen repair solution for 5 min, incubated with 0.1% TritonX-100 at room temperature for 15 min, blocked with 5% BSA for 1 h, incubated overnight with CD11b antibody (Proteintech, 21851-1-AP, 1:500), and detected after incubation with secondary antibody for 1 h.

[0033] (7) Picrosirius Red (PSR) staining of liver tissue: The liver tissue was fixed with 4% paraformaldehyde, dehydrated, embedded and sliced, with a thickness of 0.4 μm. The slices were stained with Picrosirius Red solution for 1 h, rinsed with running water for 10 s, and treated with 0.5% acetic acid for 20 s. After dehydration and transparency, the slices were sealed with neutral gum.

[0034] (6) Determination of triglyceride (TG) and total cholesterol (TC) content in liver tissue: Lysate was added at a weight / volume ratio of 1:9, homogenized on a tissue homogenizer, centrifuged, 4°C, 10,000 × g, 15 min, and the supernatant was collected for triglyceride and cholesterol detection. The protein concentration of the lysate was detected using a BCA kit. The TG and TC contents were measured using the enzyme reaction method. The detection kit was purchased from Nanjing Jiancheng Bioengineering Institute and the detection method was referred to the kit method.

[0035] (7) Determination of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice: After the mouse serum was equilibrated at room temperature, it was centrifuged at 4°C, 4000×g, for 30 min, and the supernatant was collected. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured according to the enzyme reaction method. The detection kit was purchased from Nanjing Jiancheng Bioengineering Institute and the detection method was referred to the kit method.

[0036] (8) Data processing and statistical analysis: Data were analyzed using GraphPad Prism 7.0 software, and the data results were presented as mean ± standard error (mean ± SEM). One-way analysis of variance was used to analyze the differences between multiple groups of data. When P < 0.05, it was considered to have a statistically significant difference, and when P < 0.01, it was considered to have a statistically significant difference. Compared with the NCD group, #, P < 0.05, ##, P < 0.01. Compared with the HFD group, *, P < 0.05, **, P < 0.01.

[0037] 2 Experimental results

[0038] H&E staining and NAFLD activity score (NAS) results showed that HFD mice exhibited significant hepatocellular damage compared with NCD-fed mice. However, Makatoxin-3 administration significantly reduced hepatic steatosis, ballooning, and inflammatory cell infiltration compared with HFD group mice ( Figure 1 A). Meanwhile, Oil Red O (ORO) staining showed that the liver lipid levels of Makatoxin-3 treated mice were significantly reduced ( Figure 1 B). Immunohistochemistry results showed that Makatoxin-3 administration significantly reduced CD11b-positive macrophage infiltration compared with the HFD group mice ( Figure 1C). PSR results showed that HFD mice exhibited significant liver fibrosis compared with NCD-fed mice. However, Makatoxin-3 administration significantly reduced liver fibrosis compared with HFD group mice ( Figure 1 D). The results of liver tissue TG (triglyceride) and TC (blood lipid index) showed that Makatoxin-3 significantly reduced the content of TG and TC in the liver ( Figure 1 E). The results of liver injury-related indicators ALT (alanine aminotransferase) and AST (aspartate aminotransferase) showed that Makatoxin-3 significantly reduced serum ALT and AST levels ( Figure 1 F).

[0039] Example 3: Makatoxin-3 activates PPARα transcriptional activity.

[0040] 1 Experimental Materials and Methods

[0041] (1) Plasmid transfection: Use 3000 peroxisome proliferator response element (PPRE)-X3-TK-LUC plasmid (0.05 μg) and PPARα expression vector (0.01 μg) were transfected into HepG2 cells.

[0042] (2) Seed plate: 6 hours after transfection, 5×10 4 The cells were plated in 96-well plates at a density of 10 cells / well and cultured in an incubator for 24 h.

[0043] (3) Administration: Different concentrations of Makatoxin-3 (3 μM and 10 μM) were administered, cultured in an incubator, and tested after 24 hours.

[0044] (4) The dual luciferase reporter gene assay system (Promega) was used to first measure the activity of firefly luciferase and then the activity of Renilla luciferase, and the relative expression levels of (firefly / renilla) luciferase were calculated.

[0045] (5) Data processing and statistical analysis: Data were analyzed using GraphPad Prism 7.0 software, and the data results were presented as means ± standard error (mean ± SEM). One-way analysis of variance was used to analyze the differences between multiple groups of data. When P < 0.05, it was considered to be statistically different, and when P < 0.01, it was considered to be statistically significant. Compared with the NCD group, #, P < 0.05, ##, P < 0.01. Compared with the HFD group, *, P < 0.05, **, P < 0.01.

[0046] 2 Experimental results:

[0047] The effect of Makatoxin-3 on the transcriptional activity level of PPARα was detected. The results are shown in ( Figure 2 ). The results showed that Makatoxin-3 significantly increased PPARα transcriptional activity.

[0048] Example 4: Detecting the effect of Makatoxin-3 on genes related to fatty acid β-oxidation in hepatocytes.

[0049] 1 Experimental Materials and Methods

[0050] (1) Isolation and culture of primary mouse hepatocytes: Anesthetize the mouse and soak it in 75% alcohol for 1 minute for disinfection. Open the abdominal cavity and expose the inferior vena cava and portal vein. Insert a catheter into the inferior vena cava and perfuse with A solution (HBSS without Ca2+). 2+ Mg-free 2+ Without phenol red, EDTA 0.25 mM, HEPES 25 mM), and cut the portal vein until there is no blood in the liver, and replace the pump tube with perfusion solution B (collagenase VI concentration: 1 mg / mL, HBSS containing Ca 2+ Mg 2+ and phenol red, HEPES25mM) for digestion. After cutting off the gallbladder, the liver was completely removed, and the liver surface capsule was torn with forceps to release the primary hepatocytes into the solution. Filtered with a 70μm cell strainer and centrifuged at 50×g, 4°C, 2min. Add 10mL of Percoll solution to the precipitate and centrifuged again at 200×g, 4°C, 10min. Aspirate the supernatant, add 20mL of 5% low-glucose DMEM to resuspend, count the hepatocytes and plate them in a 6-well plate coated with collagen, with a cell density of 2×10 5 Pieces / mL.

[0051] (2) Cell administration: 400 μM palmitate (PA) was administered to the cells for cell modeling, and Makatoxin-3 (3 μM) was administered at the same time, and the cells were cultured in an incubator for 24 h.

[0052] (3) Extraction of total RNA from hepatocytes: After washing with PBS, add 1 mL of Trizol to a 6-well plate, lyse at room temperature for 5 min, and transfer the cell lysate to a 1.5 mL EP tube. Add 1 / 5 volume of chloroform, mix by inverting, let stand at room temperature for 5 min, and then centrifuge at 12,000 × g, 4°C, 15 min. Add 1 mL of isopropanol to the supernatant, let stand at room temperature for 10 min, and then centrifuge at 12,000 × g, 4°C, 15 min. Discard the supernatant, add 1 mL of 75% ethanol to wash the precipitate, and then centrifuge at 7,500 × g, 4°C, 15 min. Discard the supernatant, add DEPC water to dissolve the precipitate, and measure the RNA concentration.

[0053] (4) Real-time quantitative fluorescence PCR:

[0054] a. Reverse transcribe mRNA from RNA into cDNA using HiScript Q RT SuperMix for qPCR (+gDNAwiper) kit:

[0055] Table 1 Reaction system for removing genomic DNA

[0056]

[0057] Table 2 Reverse transcription reaction system

[0058]

[0059] b. Primer design

[0060] Table 3 Primer list

[0061] GENE Forward Primer Reverse primer ACOX1 GCCATTCGATACAGTGCTGTGAG CCGAGAAAGTGGAAGGCATAGG PPARα ACCACTACGGAGTTCACGCATG ACCACTACGGAGTTCACGCATG CPT1A GGCATAAACGCAGAGCATTCCTG CAGTGTCCATCCTCTGAGTAGC PGC1α GGACATGTGCAGCCAAGACTCT CACTTCAATCCACCCAGAAAGCT β-actin CATTGCTGACAGGATGCAGAAGG TGCTGGAAGGTGGACAGTGAGG

[0062] c. Real-time PCR:

[0063] Table 4 Real-time PCR reaction system

[0064] Reagents volume STBR Master mix 10μL PCR Forward Primer (10 μM) 0.4μL PCR Reserse Primer (10μM) 0.4μL cDNA template 1μL <![CDATA[ddH20 (double distilled water)]]> 8.2μL Total 20μL

[0065] 50℃, 2min; 95℃, 20s; 95℃, 5s, 60℃, 30s, cycle 40 times.

[0066] (5) Data processing and statistical analysis: Data were analyzed using GraphPad Prism 7.0 software, and the data results were presented as mean ± standard error (mean ± SEM). The difference between the two groups of data was analyzed using t-test. When P < 0.05, it was considered to be statistically different, and when P < 0.01, it was considered to be statistically significant. Compared with the NCD group, #, P < 0.05, ##, P < 0.01. Compared with the HFD group, *, P < 0.05, **, P < 0.01.

[0067] 2 Experimental results

[0068] By detecting the mRNA levels of genes related to fatty acid β-oxidation in hepatocytes ( Figure 3 AD), and found that Makatoxin-3 significantly increased the mRNA levels of PPARα, ACOX1, CPT1A, and PGC1α in hepatocytes.

[0069] In general, the present invention found that Makatoxin-3, as a PPARα agonist, significantly activates the transcription of genes related to fatty acid β-oxidation, promotes lipid oxidation and decomposition to inhibit lipid accumulation in the liver, and reduces inflammation and liver fibrosis. Therefore, Makatoxin-3 can be used to prepare drugs for the treatment of non-alcoholic fatty liver disease.

[0070] The present invention provides a new medical use of Makatoxin-3. There are many methods and approaches to implement the technical solution. 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 protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. Use of a Buthus scorpion toxin polypeptide or a pharmaceutical composition comprising the same in the preparation of a drug for treating liver diseases caused by intrahepatic lipid accumulation, wherein: The East Asian scorpion toxin polypeptide is an East Asian scorpion toxin polypeptide, and its amino acid sequence is shown in SEQ ID NO.1; the liver disease caused by intrahepatic lipid accumulation is non-alcoholic fatty liver disease; the non-alcoholic fatty liver disease includes any one or a combination of hepatic fatty degeneration, non-alcoholic fatty hepatitis, liver fibrosis and cirrhosis; the dosage form of the pharmaceutical composition is an injection.

2. The use according to claim 1, characterized in that: When used, the East Asian scorpion toxin polypeptide or a composition comprising the same serves as a PPARα agonist and / or a transcription activator of genes related to fatty acid β-oxidation.

3. The use according to claim 1, characterized in that: The East Asian scorpion toxin polypeptide or the pharmaceutical composition containing the same activates PPARα transcription activity, promotes hepatocyte fatty acid β-oxidation, alleviates hepatocyte lipid deposition, and reduces inflammation and liver fibrosis.

4. The use according to claim 2, characterized in that: The fatty acid β-oxidation related gene comprises at least one of PPARα, ACOX1, CPT1A and PGC1α.

5. The use according to claim 1, characterized in that: The East Asian scorpion toxin polypeptide or the pharmaceutical composition comprising the same reduces the content of TG and TC in the liver.

6. The use according to claim 1, characterized in that: The East Asian scorpion toxin polypeptide or the pharmaceutical composition comprising the same reduces serum ALT and AST levels.

7. The use according to any one of claims 1 to 6, characterized in that: The pharmaceutical composition includes a pharmaceutically acceptable carrier.

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