阅读说明：本技术 β-羟基丙酮酸在制备人胰岛淀粉样多肽聚集抑制剂中的应用 (Application of beta-hydroxy pyruvic acid in preparing human islet amyloid polypeptide aggregation inhibitor ) 是由 郝海平 郑秋凌 夏丹丹 杨蕾 徐小为 于 2021-01-29 设计创作，主要内容包括：本发明公开了一种内源性代谢物β-羟基丙酮酸(HPA)在制备人胰岛淀粉样蛋白(IAPP)聚集抑制剂中的应用。本发明以内源性代谢物为目标,筛选出具有抑制IAPP纤维化潜力的差异代谢物HPA,经ThT法检测表明HPA通过作用于IAPP寡聚体形成阶段,显著延缓IAPP寡聚体生成速度,降低β折叠结构含量,达到抑制纤维化的作用。经TEM表征,与HPA作用后所形成的纤维形貌发生显著变化,无典型IAPP纤维生成,进一步表明HPA可有效抑制IAPP纤维化,对发现和设计抑制IAPP聚集及由其引起的生理毒性的药物具有非常重要的意义。(The invention discloses application of an endogenous metabolite beta-Hydroxy Pyruvic Acid (HPA) in preparation of an inhibitor for human islet amyloid protein (IAPP) aggregation. The invention takes endogenous metabolites as targets, screens out differential metabolites HPA with the potential of inhibiting IAPP fibrosis, and the ThT method detection shows that the HPA remarkably delays the generation speed of IAPP oligomer by acting on the formation stage of IAPP oligomer, reduces the beta-folding structure content and achieves the effect of inhibiting the fibrosis. The appearance of the fiber formed after the fiber is acted by HPA is obviously changed by TEM representation, no typical IAPP fiber is generated, further, the HPA can effectively inhibit IAPP fibrosis, and the method has very important significance for discovering and designing the medicines for inhibiting IAPP aggregation and physiological toxicity caused by the IAPP.)

1. Application of beta-hydroxy pyruvic acid in preparing human islet amyloid polypeptide aggregation inhibitor.

2. Application of beta-hydroxy pyruvic acid in preparing medicine for preventing and treating type II diabetes.

3. The use of claim 1 or 2, wherein the beta-hydroxypyruvate delays or prevents human amylin aggregation.

4. The use of claim 1 or 2, wherein the β -hydroxy pyruvate inhibits human amylin fibrosis.

5. The use according to claim 4, wherein the beta-hydroxy pyruvic acid acts on the formation stage of the human amylin oligomer to delay the formation speed of the human amylin oligomer and reduce the content of beta-sheet structure to inhibit fibrosis.

Technical Field

The invention belongs to the biomedical technology, and particularly relates to application of beta-hydroxy pyruvic acid in preparation of a human islet amyloid polypeptide aggregation inhibitor.

Background

Aggregation of Human islet amyloid polypeptide (IAPP) is thought to be closely related to the development of type II diabetes (T2 DM). Studies have shown that metabolic disorders caused by T2DM lead to misfolding and aggregation of IAPP and that the resulting fiber deposition further leads to islet beta cell dysfunction, causing cytotoxicity by inducing intracellular oxidative stress, endoplasmic reticulum stress and mitochondrial damage. Therefore, inhibition of IAPP aggregation is of great importance for the prevention and treatment of T2 DM. Currently, research on therapeutic methods for delaying or preventing IAPP aggregation and fibrogenesis is progressing, including amyloid mimetic peptide fragments, amyloid antibodies, nanoparticles, and the like. In addition, the development of small molecule inhibitors has attracted considerable attention and many have been demonstrated to be effective in inhibiting fibrogenesis and reducing cytotoxicity caused by protein aggregation in vitro experiments. However, many of the currently available small molecule inhibitors are derived from chemical synthesis or extraction from natural products, and have low bioavailability and gastrointestinal absorption rate, thereby limiting their effect in vivo.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the technical problems in the prior art, the application takes an endogenous metabolite as a target, finds that the endogenous metabolite beta-hydroxypyruvate has the effects of inhibiting IAPP aggregation and cytotoxicity caused by aggregation, and can be used for preparing a human islet amyloid polypeptide aggregation inhibitor.

The technical scheme is as follows: the application discloses application of beta-hydroxy pyruvic acid in preparation of a human islet amyloid polypeptide aggregation inhibitor.

The application discloses application of beta-hydroxy pyruvic acid in preparation of medicines for preventing and treating type II diabetes.

Wherein said beta-hydroxypyruvate delays or prevents human amylin aggregation.

Further, the beta-hydroxypyruvate inhibits human islet amyloid polypeptide fibrosis. Specifically, the beta-hydroxy pyruvic acid acts on the formation stage of the human amylin oligomer, so that the generation speed of the human amylin oligomer is delayed, the content of a beta folding structure is reduced, and the purpose of inhibiting fibrosis is achieved.

The endogenous metabolite beta-hydroxy pyruvic acid (HPA, beta-hydroxypyruvic acid) is obtained by screening through non-targeted metabonomics analysis and targeting the endogenous metabolite, and the structure is as follows:

experiments prove that the beta-hydroxy pyruvic acid has the function of inhibiting IAPP aggregation and has very important significance for discovering and designing medicines for inhibiting IAPP aggregation and physiological toxicity caused by the IAPP aggregation.

The terminology used in this application is as follows:

the term "HPA" means: beta-hydroxy pyruvic acid, beta-hydroxypyruvic acid.

The term "HFIP" refers to: hexafluoroisopropanol, 1,1,1,3,3, 3-Hexafluoro-2-propanol.

The term "ThT" refers to: thioflavin T, Thioflavin T.

Has the advantages that: the beta-hydroxy pyruvic acid HPA is an endogenous micromolecule, and is expected to overcome the defects of low bioavailability, poor gastrointestinal absorption rate and the like of other micromolecule inhibitors; HPA is a diabetes differential metabolite screened by a non-targeted metabonomics method, is a serine metabolite, can inhibit the aggregation of IAPP from the perspective of metabolic regulation, and has important significance for deeply understanding the relationship between IAPP and the pathogenesis of T2 DM.

Drawings

FIG. 1 is a transmission electron microscope characterization of the modulation of IAPP aggregation-forming fiber morphology by candidate metabolites, wherein (A) is the IAPP fiber morphology; (B) the morphology of the fiber formed when the mixing concentration ratio of HPA to IAPP is 10: 1; (C) the shape (D) of the fiber formed when the mixing concentration ratio of the 3-methyl-L-histidine to the IAPP is 10:1 is the shape of the fiber formed when the mixing concentration ratio of the 3-hydroxybutyric acid to the IAPP is 10: 1;

figure 2 is a dose-dependent representation of HPA characterization by fluorospectrophotometry after ThT staining on the kinetics of controlled IAPP aggregation.

Detailed Description

The present application will be described in detail with reference to specific examples.

Source of beta-hydroxy pyruvate HPA:

entrusted Wuhan Matteville Biotech Co., Ltd based on ABSciex6500⁺LC-MS/MS detection platform non-targeted metabolomics analysis was performed on 27 serum samples (8 type ii diabetic patients, 10 obese patients, 9 normal persons). Based on a self-built target standard database MWDB (metal database), qualitative analysis is carried out on the information and the secondary spectrum data according to the retention time of the detected substances and the primary and secondary ion pairs. And acquiring peak area data of the metabolites in the sample by utilizing a multi-reaction monitoring mode of the triple quadrupole mass spectrometry to obtain the relative content of the metabolites in different samples. And analyzing the data result by using an orthogonal partial least square method, and screening the differential metabolites. The screening criteria were: the fold change is more than or equal to 2 or less than or equal to 0.5, and on the basis, a metabolite of VIP more than or equal to 1 is selected. After obtaining the differential metabolites, the KEGG (Kyoto Encyclopedia of Genes and Genes) and HMDB (human Metabolome database) databases were used to perform functional annotation and related disease analysis on the differential metabolites, and 3 metabolites related to amyloid diseases, including 3-N-methyl-L-histidine, 3-hydroxybutyric acid and HPA of the present application, were screened. And then the HPA is characterized by a TEM electron microscope to find that only the HPA has an inhibiting effect on IAPP aggregation, so that the HPA is selected for subsequent verification.

Example 1

This example employs transmission electron microscopy for characterizing the effect of candidate metabolites on IAPP aggregation-generated fiber morphology.

IAPP dispersion method: 4.74mg IAPP was weighed out, 2.456mL HFIP was added and dissolved at a concentration of 0.5mM, and sonicated for 2 minutes.

2. Prepare 2.5 × PBS (containing 50mM NaCl) solution: 0.2g of KCl,1.1688g of NaCl,0.22g of KH were weighed out₂PO₄,2.08g Na₂HPO₄·12H₂O, 250mL of ultrapure water was measured and dissolved completely.

3. Preparing HPA stock solution: 5mg of HPA was weighed and dissolved in 600. mu.L of the above-prepared 2.5 XPBS solution (containing 50mM NaCl) to prepare an 80mM HPA stock solution.

4. Preparing a 3-methyl-L-histidine stock solution: 1mg of 3-methyl-L-histidine was weighed, and 739. mu.L of the 2.5 XPBS solution (containing 50mM NaCl) prepared above was added and dissolved to prepare an 8mM stock solution of 3-methyl-L-histidine.

5. Preparing 3-hydroxybutyric acid stock solution: mu.L of 3-hydroxybutyric acid (1.126g/ml) was measured and dissolved in 1080. mu.L of the 2.5 XPBS solution (containing 50mM NaCl) prepared above to prepare an 80mM stock solution of 3-hydroxybutyric acid.

6. A10. mu.L of IAPP stock solution was measured and added to 990. mu.L of 2.5 XPBS (50mM NaCl) to prepare a control solution. Detection was performed after 6 hours incubation at room temperature.

The preparation method of the IAPP-HPA mixed solution comprises the following steps: 0.625. mu.L of HPA stock solution was measured, added to 989.35. mu.L of 2.5 XPBS (50mM NaCl), and mixed with 10. mu.L of IAPP stock solution to prepare HPA: IAPP is group 10: 1. Detection was performed after 6 hours incubation at room temperature.

The preparation method of the IAPP-3-methyl-L-histidine mixed solution comprises the following steps: 6.25. mu.L of 3-methyl-L-histidine stock was measured, added to 983.75. mu.L of 2.5 XPBS (50mM NaCl), and mixed with 10. mu.L of IAPP stock to prepare 3-methyl-L-histidine: IAPP is group 10: 1. Detection was performed after 6 hours incubation at room temperature.

The preparation method of the IAPP-3-hydroxybutyric acid mixed solution comprises the following steps: 0.625. mu.L of 3-hydroxybutyric acid stock solution was measured, added to 989.35. mu.L of 2.5 XPBS (50mM NaCl), and mixed with 10. mu.L of IAPP stock solution to prepare 3-hydroxybutyric acid: IAPP is group 10: 1. Detection was performed after 6 hours incubation at room temperature.

10. Preparing an electron microscope sample: and (3) dropwise adding 50 mu L of solution to be tested on the surface of the ultrathin carbon-plated copper mesh, standing for 10 minutes, and sucking away the residual liquid. 20 mu L of 1mg/mL ammonium phosphotungstate aqueous solution is dripped on the surface of the copper mesh on which the sample is deposited, the sample is deposited for 5 minutes, and the residual liquid is sucked away. The prepared sample was dried in a desiccator for 30 minutes. Imaging was performed using a biological transmission electron microscope (Hitachi HT7700, japan).

TEM characterization reflects the morphological features of IAPP aggregation to form fibers. As shown in fig. 1A (with a 500nm scale), IAPP controls self-aggregated to form the typical long linear amyloid fibrils after 6 hours incubation. As shown in fig. 1B, after HPA is mixed and incubated with IAPP, no typical IAPP fibrosis is formed in the visual field, and the morphology is significantly changed, demonstrating that the small molecule inhibitor can significantly inhibit the fibrosis of IAPP. In contrast, after IAPP incubation with 3-methyl-L-histidine and 3-hydroxybutyrate, respectively (FIGS. 1C and D), a typical long linear amyloid fibril similar to that of FIG. 1A was observed, indicating that 3-methyl-L-histidine and 3-hydroxybutyrate had no inhibitory effect on IAPP fibrosis.

Example 2

In this example, regulation and control of IAPP aggregation ability by HPA was characterized by fluorescence spectrophotometry after ThT staining.

1. Prepare 2.5 × PBS (containing 50mM NaCl) solution: 0.2g of KCl,1.1688g of NaCl,0.22g of KH were weighed out₂PO₄,2.08g Na₂HPO₄·12H₂O, 250mL of ultrapure water was measured and dissolved completely.

2. Preparing a ThT stock solution: 1.25mg of ThT was weighed out, and 1.960mL of ultrapure water was measured out and dissolved.

HPA stock method: as described in example 1.

IAPP dispersion method: as described in example 1.

5. 500. mu.L of the dispersed IAPP stock solution prepared above was measured and added to 49.5mL of 2.5 XPBS (containing 50mM NaCl) prepared above to prepare a control group.

6. To a stock of 16. mu.L HPA was added 49.484mL of 2.5 XPBS (50mM NaCl) and 500. mu.L of the above-prepared stock of dispersed IAPP was mixed to prepare HPA: IAPP is group 5: 1.

7. To a stock of 32. mu.L HPA was added 49.468mL of 2.5 XPBS (50mM NaCl) and 500. mu.L of the above-prepared stock of dispersed IAPP was mixed to prepare HPA: IAPP is group 10: 1.

8. 1980 mu L of each group of solution to be detected is respectively placed in a quartz cuvette, 20 mu L of ThT stock solution is added, and the solution is blown and beaten uniformly. The emission signal at 486nm was collected in a Perkin Elmer 6500 fluorescence spectrophotometer with 450nm as the excitation wavelength and fluorescence values were collected every 30 minutes.

The ThT staining experiment is based on the relation between the beta-sheet structure content and the fluorescence intensity change in solution, and can be used for characterizing the IAPP aggregation kinetic process. The results are shown in FIG. 2, where the fluorescence values for the IAPP control group were plotted as an "S" profile over time, consistent with typical fiber growth kinetics. IAPP undergoes a beta sheet structure transition in solution with a lag phase of 3.85 hours, indicating a transition from monomeric IAPP to oligomeric intermediate. The oligomer is then rapidly formed and the accumulation of beta sheet content leads to a sharp increase in fluorescence intensity, saturating and stabilising at 8 hours, with a maximum fluorescence value of 31,000. When HPA was added (ratio HPA: IAPP 5:1), the lag phase was extended to 4.34 hours, indicating that the addition of HPA can retard the IAPP oligomer formation process. At the same time, the maximum fluorescence value after saturation was reduced to 29,000, indicating that the addition of HPA reduced the total amount of beta sheet structure. When the HPA to IAPP ratio was 10:1, the lag phase was further extended to 5.25 hours and the maximum fluorescence was reduced to 26,000. The results show that the HPA can obviously delay the generation speed of IAPP oligomer, reduce the beta-sheet structure content and reduce the generation of IAPP fiber, and the inhibition effect of the HPA has dose dependence.