NLRP3 small-molecule inhibitor and application thereof

Abstract

The invention discloses an NLRP3 small-molecule inhibitor and an application thereof. The NLRP3 small-molecule inhibitor is asiaticoside or an asiaticoside analogue. The invention provides an application of asiaticoside or an asiaticoside analogue in preparation of a medicine for regulating an NLRP3 inflammasome pathway to relieve neuroinflammation, in particular to an application in preparation of a medicine for preventing Parkinson's disease.

Description

Technical Field

[0001] The present invention belongs to the field of life sciences, and specifically relates to an NLRP3 small molecule inhibitor and an application thereof. Background Art

[0002] NLRP3 inflammasome is a type of cytoplasmic multimeric protein complex composed of sensor protein NLRP3, adapter protein ASC and effector protein Caspase-1. Current studies have shown that NLRP3 inflammasome plays an important role in ALI / ARDS caused by a variety of pathogenic microorganisms such as influenza virus, Pseudomonas aeruginosa, Staphylococcus aureus, etc., and its cleaved mature inflammatory factors IL1β and IL-18 are involved in the occurrence and development of ALI / ARDS. Therefore, inhibiting the excessive activation of inflammasomes, reducing the inflammatory factor storm, and improving organ damage are of great significance for the treatment of ALI / ARDS. In addition, studies have found that NLRP3 inflammasomes also play a key role in the occurrence and development of diseases such as acute pancreatitis, acute myocardial infarction, traumatic spinal injury, ulcerative colitis, Crohn's disease, diabetes, ischemic stroke, Alzheimer's disease (AD), traumatic brain injury, brain tumors, etc., indicating that regulating the function of NLRP3 inflammasomes is expected to become a new target for the treatment of inflammatory diseases.

[0003] The molecular formula of Centella asiatica is C48H78O16, and the molecular weight is 959.12g / mol. Centella asiatica is odorless, bitter, and slightly hygroscopic. It has the effects of clearing away heat and dampness, detoxifying and reducing swelling, and promoting blood circulation and stopping bleeding. It has the effect of promoting wound healing and is used to treat trauma, anti-tumor, and anti-anxiety. Summary of the invention

[0004] The purpose of the present invention is to provide a NLRP3 small molecule inhibitor, which is asiaticoside, and provide its application.

[0005] The present invention is achieved through the following technical solution: a NLRP3 small molecule inhibitor, wherein the NLRP3 small molecule inhibitor is asiaticoside or an asiaticoside analogue.

[0006] Further preferably, the Centella asiatica glycoside alleviates neuroinflammation by regulating the NLRP3 inflammasome pathway.

[0007] Further preferably, the asiaticoside analogue is selected from asiatic acid, madecassic acid, madecassoside or pharmaceutically acceptable salts of asiaticoside, asiatic acid, madecassoside or madecassoside.

[0008] The present invention also provides the use of asiatica glycoside in the preparation of a drug for regulating the NLRP3 inflammasome pathway to alleviate neuroinflammation.

[0009] The present invention also provides the use of asiatica glycoside in preparing medicine for preventing Parkinson's disease.

[0010] The present invention also provides a pharmaceutical composition for regulating the NLRP3 inflammasome pathway to alleviate neuroinflammation, which comprises asiatica glycoside or asiatica glycoside analogs as the only active ingredient, and other pharmaceutically acceptable carriers.

[0011] The present invention also provides a pharmaceutical composition for preventing Parkinson's disease, characterized in that it comprises asiatica glycoside or asiatica glycoside analogs as the only active ingredient, and other pharmaceutically acceptable carriers.

[0012] The present invention finds that asiaticoside or asiaticoside analogs can be used as NLRP3 small molecule inhibitors, and can be used to prepare drugs for regulating the NLRP3 inflammasome pathway to relieve neuroinflammation, especially for preparing drugs for preventing Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The effect of different concentrations of Centella asiatica on the activity of BV2 cells.

[0014] Figure 2 Western blot image showing that Centella asiatica inhibits NLRP3 inflammasome in BV2 microglia cells.

[0015] Figure 3 This is the docking posture of Centella asiatica glycoside and NLRP3 protein (PDB 8ERT).

[0016] Figure 4 The interaction relationship of asiatica glycoside NLRP3 protein (PDB 8ERT).

[0017] Figure 5 Schematic diagram of the experiment on using Centella asiatica to improve movement disorders in mice with Parkinson's disease. DETAILED DESCRIPTION

[0018] The present invention is further explained in detail below with reference to the examples.

[0019] Centella asiatica can alleviate neuroinflammation by regulating the NLRP3 inflammasome pathway

[0020] First, the MTT method was used to evaluate the cytotoxicity of asiatica glycoside on BV2 cells. It was found that 5-200 μM asiatica glycoside had no significant effect on the cell survival rate after acting on BV2 cells, while 400 μM asiatica glycoside caused significant toxicity to BV2 cells ( Figure 1 ), Figure 1BV2 cells were treated with different concentrations of asiaticoside for 24 hours, and then the cell viability was determined using the MTT method. n = 3, *** indicates P < 0.001, AS: asiaticoside. This shows that the toxicity of asiaticoside is very low, and it will only have significant cytotoxicity at high concentrations. Therefore, the concentration below 200 μM was selected for subsequent experiments.

[0021] Network pharmacology analysis found that NLRP3 is a potential target for Centella asiatica to prevent Parkinson's disease, and the NOD-like receptor signaling pathway is a potential pathway for Centella asiatica to alleviate Parkinson's disease. Therefore, by constructing a BV2 neuroinflammation model, we verified whether Centella asiatica can regulate NLRP3 inflammasomes and thus alleviate neuroinflammation. The results showed that when BV2 cells were treated with LPS and ATP, it would lead to the activation of NLRP3 inflammasomes, release caspase1 p20 and IL-1βp17, and cause severe inflammatory responses. In the Centella asiatica treatment group, pro-IL-1β expression was significantly downregulated, and the release of caspase1p20 and IL-1βp17 in the cell culture supernatant was significantly reduced ( Figure 2 ), which indicates that Centella asiatica can alleviate neuroinflammation by regulating the NLRP3 inflammasome pathway. Figure 2 BV2 cells were pretreated with different concentrations of asiaticoside (50 μM, 100 μM, 200 μM) for 1 h, then treated with 1 μg / mL LPS for 3 h, and then stimulated with 2.5 mM ATP for 30 min. Western blot analysis of the corresponding proteins in the supernatant (SN) and total cell protein (Input) was performed.

[0022] Binding of asiatica glycoside to NLRP3 protein

[0023] In the MOE 2019 system, the PDB 8ERT crystal structure and the Centella asiatica structure were imported for molecular docking. The results showed that the binding capacity of Centella asiatica to 8ERT was as high as -8.72 kcal / mol, the rmsd value was 1.96, and the docking posture was as follows Figure 3 As shown, it may form hydrogen bonds with LYS9, LYS93, LYS86, ASP31, ARG43, and CYS38 ( Figure 4 ).

[0024] Centella asiatica improves motor coordination in Parkinson's disease model mice

[0025] This experiment used an open field test to conduct behavioral tests on mice, measuring the total movement distance, movement speed and movement trajectory of mice within 5 minutes to reflect the autonomous behavior ability of mice. Preliminary experiments showed that when mice were injected with 30mg / kg MPTP for 30 minutes, they showed tremors, piloerection, convulsions and other phenomena. After 5 consecutive days of injection and then resting for 7 days, the autonomous movement ability of mice decreased significantly, and the expression of TH protein in the striatum and substantia nigra was significantly downregulated. In the formal experiment, mice were placed in the open field for testing 7 days after the last injection of MPTP. The data showed that the movement distance and movement speed of mice in the model group injected with MPTP were significantly lower than those of the normal group, and the number of activities of mice in the central area was significantly reduced. After the mice were pretreated with Centella asiatica glycoside, their movement distance and movement speed rebounded significantly and returned to normal levels, and the activity trajectory of the central area also returned to normal ( Figure 5 ), which indicates that asiatica glycoside can improve MPTP-induced movement disorders in mice. Figure 5 AC mice were pretreated with asiaticoside (10 mg / kg, 60 mg / kg) by gavage for 3 days, then injected with 30 mg / kg MPTP intraperitoneally for 5 days, and then continued to be gavaged with asiaticoside (10 mg / kg, 60 mg / kg) for 7 days. Figure 5 A in the middle is the total distance the mouse moves. Figure 5 B in the middle is the movement speed of the mouse. Figure 5 Middle C is the movement trajectory of mice. n = 6, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. AS: asiaticaside.

[0026] Although the embodiments of the present invention have been shown and described, as detailed above, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A NLRP3 small molecule inhibitor, characterized in that: The NLRP3 small molecule inhibitor is asiatica glycoside or an asiatica glycoside analog.

2. The NLRP3 small molecule inhibitor according to claim 1, characterized in that The Centella asiatica glycoside alleviates neuroinflammation by regulating the NLRP3 inflammasome pathway.

3. The NLRP3 small molecule inhibitor according to claim 1, characterized in that The asiaticoside analogue is selected from asiatic acid, madecassic acid, madecassoside or pharmaceutically acceptable salts of asiaticoside, asiatic acid, madecassoside or madecassoside.

4. Use of Centella asiatica glycoside or Centella asiatica glycoside analogs in the preparation of drugs that regulate the NLRP3 inflammasome pathway to alleviate neuroinflammation.

5. Use of asiatica glycoside or asiatica glycoside analogs in the preparation of drugs for preventing Parkinson's disease.

6. The use according to claim 4 or 5, characterized in that: The asiaticoside analogue is selected from asiatic acid, madecassic acid, madecassoside or pharmaceutically acceptable salts of asiaticoside, asiatic acid, madecassoside or madecassoside.

7. A pharmaceutical composition for regulating the NLRP3 inflammasome pathway to relieve neuroinflammation, characterized in that: The invention comprises asiatica glycoside or asiatica glycoside analogs as the sole active ingredient, and other pharmaceutically acceptable carriers.

8. A pharmaceutical composition for regulating the NLRP3 inflammasome pathway to relieve neuroinflammation according to claim 7, characterized in that: The asiaticoside analogue is selected from asiatic acid, madecassic acid, madecassoside or pharmaceutically acceptable salts of asiaticoside, asiatic acid, madecassoside or madecassoside.

9. A pharmaceutical composition for preventing Parkinson's disease, characterized in that: The invention comprises asiatica glycoside or asiatica glycoside analogs as the sole active ingredient, and other pharmaceutically acceptable carriers.

10. A pharmaceutical composition for preventing Parkinson's disease according to claim 9, characterized in that: The asiaticoside analogue is selected from asiatic acid, madecassic acid, madecassoside or pharmaceutically acceptable salts of asiaticoside, asiatic acid, madecassoside or madecassoside.

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