CN117562904A - Application of vilazodone in preparing medicament for preventing or treating NLRP3 inflammatory small body related diseases - Google Patents

Abstract

The invention belongs to the field of medicines, specifically relates to the field of new uses of medicines, and specifically relates to the application of vilazodone in the preparation of medicines for preventing or treating NLRP3 inflammasome-related diseases. The present invention takes vilazodone as the research object and first examines the effect of vilazodone on the activation of NLRP3 inflammasome in macrophages. The results show that the addition of vilazodone can effectively inhibit the maturation and activation of caspase-1 and IL-1β. secretion, and this inhibitory effect is dose-dependent. Finally, the mechanism of action and binding site of vilazodone in inhibiting NLRP3 was clarified, providing an NLRP3 inhibitor with clear clinical pharmacological effects and high clinical safety.

Description

Verazodone in the preparation of drugs for preventing or treating NLRP3 inflammasome-related diseases Applications in

Technical field

The invention belongs to the field of new uses of drugs, and specifically relates to the application of vilazodone in preparing drugs for preventing or treating NLRP3 inflammasome-related diseases.

Background technique

The NOD-like receptor (NLR) family NLRP3 inflammasome is an intracellular protein complex composed of NLRP3 protein, connexin ASC and cysteine protease caspase-1. NLRP3 serves as an innate immune sensor that can detect a variety of stimuli such as viral RNA, microbial toxins, uric acid crystals, and environmental and host factors. Upon activation, NLRP3 recruits ASC and pro-caspase-1 to assemble the NLRP3 inflammasome, leading to autoproteolysis of pro-caspase-1. The main function of caspase-1 is to induce the maturation and production of pro-inflammatory cytokines IL-1β and IL-18 and mediate pyroptosis.

Studies have shown that abnormal activation of the NLRP3 inflammasome is related to the pathogenesis of a variety of human inflammatory diseases, including sepsis, peritonitis, cryogen-related periodic syndrome, opioid analgesia tolerance, gout, atherosclerosis, Type 2 diabetes, etc. In particular, neuroinflammatory diseases such as multiple sclerosis, Alzheimer's disease, stroke, and depression have also been linked to NLRP3 inflammasome dysregulation. Therefore, targeting NLRP3 represents a promising strategy for treating these inflammation-driven diseases.

In recent years, some drugs targeting the NLRP3 inflammasome have been proven to be effective in reducing inflammatory diseases through animal experiments. These drugs include MCC950, CY-09, tranilast, OLT177, oridonin, etc. However, its clinical pharmacological effects and specificity are still unclear, and its clinical safety needs further evaluation. Many drugs, such as MNS, CY-09, oridonin, have not yet entered the clinical trial stage. Although research on NLRP3 inflammasome targets is rapidly evolving, the utility of these drugs in treating NLRP3-related diseases in humans remains uncertain. Therefore, we still need NLRP3 inhibitors that are closer to clinical application and have good safety profile.

Contents of the invention

(1) Technical problems solved

The technical problem to be solved by the present invention is to find an NLRP3 inhibitor that is close to clinical application and has good safety, and can be used for the prevention and treatment of NLRP3 inflammasome-related diseases.

(2) Technical solutions

In order to achieve the above objects, the present invention provides in one aspect the use of vilazodone or its pharmaceutically acceptable salts, crystal forms, and solvates in the preparation of drugs for preventing or treating NLRP3 inflammasome-related diseases; The NLRP3 inflammasome-related diseases described above do not include depression.

In one embodiment, the NLRP3 inflammasome-related diseases include gout, peritonitis, Alzheimer's disease, enteritis, amyotrophic lateral sclerosis, obesity, type II diabetes, metabolic syndrome, sepsis, and sepsis. , fatty liver, hepatitis, arthritis, atherosclerosis, Parkinson's disease, myocardial infarction, asbestosis, silicosis and silicosis, asthma or acute respiratory distress syndrome, acute and chronic tissue damage caused by infection, ultraviolet-induced skin damage Sunburn, contact hypersensitivity or Muhl-Weil syndrome or cold porphyrin-associated periodic syndrome (CAPS), opioid analgesic tolerance, stroke, multiple sclerosis, stroke.

In one embodiment, the NLRP3 inflammasome-related disease is multiple sclerosis.

In one embodiment, the NLRP3 inflammasome-related disease is stroke.

In a second aspect, the present invention provides the use of a pharmaceutical composition containing vilazodone or a pharmaceutically acceptable salt, crystal form, or solvate thereof in the preparation of a drug for preventing or treating NLRP3 inflammasome-related diseases; The NLRP3 inflammasome-related diseases do not include depression.

Preferably, the NLRP3 inflammasome-related diseases include gout, peritonitis, Alzheimer's disease, enteritis, amyotrophic lateral sclerosis, obesity, type II diabetes, metabolic syndrome, sepsis, sepsis, and fatty liver. , hepatitis, arthritis, atherosclerosis, Parkinson's disease, myocardial infarction, asbestosis, silicosis and silicosis, asthma or acute respiratory distress syndrome, acute and chronic tissue damage caused by infection, ultraviolet-induced skin sunburn, Contact hypersensitivity or Muhlwehr syndrome or cryoporphyrin-associated periodic syndrome (CAPS), opioid analgesia tolerance, stroke, multiple sclerosis, stroke.

In one embodiment, the NLRP3 inflammasome-related disease is multiple sclerosis.

In one embodiment, the NLRP3 inflammasome-related disease is stroke.

In one embodiment, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

In a third aspect, the present invention provides the use of pharmaceutical preparations containing vilazodone or its pharmaceutically acceptable salts, crystal forms, and solvates in the preparation of drugs for preventing or treating NLRP3 inflammasome-related diseases; The NLRP3 inflammasome-related diseases described above do not include depression.

Preferably, the NLRP3 inflammasome-related diseases include gout, peritonitis, Alzheimer's disease, enteritis, amyotrophic lateral sclerosis, obesity, type II diabetes, metabolic syndrome, sepsis, sepsis, and fatty liver. , hepatitis, arthritis, atherosclerosis, Parkinson's disease, myocardial infarction, asbestosis, silicosis and silicosis, asthma or acute respiratory distress syndrome, acute and chronic tissue damage caused by infection, ultraviolet-induced skin sunburn, Contact hypersensitivity or Muhlwehr syndrome or cryoporphyrin-associated periodic syndrome (CAPS), opioid analgesia tolerance, stroke, multiple sclerosis, stroke.

In one embodiment, the NLRP3 inflammasome-related disease is multiple sclerosis or stroke.

In one embodiment, the types of pharmaceutical preparations include solid preparations, semi-solid preparations, liquid preparations, and gas preparations.

In one embodiment, the liquid dosage forms include solutions and injections; the solid dosage forms include tablets, granules, and capsules; the semi-solid dosage forms include ointments and gels; and the gas dosage forms include aerosols. agents, sprays.

(3) Beneficial effects

Verazodone is a serotonin partial agonist-reuptake inhibitor (SPARI) used to treat major depressive disorder. The present invention takes vilazodone as the research object, and first examines the effect of vilazodone on the activation of NLRP3 inflammasome in macrophages. The results show that the addition of vilazodone can effectively inhibit the maturation and secretion of caspase-1 and IL-1β, and this inhibitory effect is dose-dependent. Finally, the mechanism and binding site of vilazodone for inhibiting NLRP3 were clarified, and provided An NLRP3 inhibitor with clear clinical pharmacological effects and high clinical safety. At the same time, the present invention selects EAE, stroke, etc. to confirm that vilazodone improves the symptoms of the above-mentioned animal models, indicating that the application of vilazodone or its pharmaceutically acceptable salts, crystal forms, and solvates can improve the symptoms of the above-mentioned diseases.

Description of the drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting creative efforts.

Figure 1 The effect of velazodone on inhibiting Nigericin-induced NLRP3 inflammasome activation in Example 1 of the present invention; Figure 1A: Structural formula of velazodone; Figure 1B: ELISA results suggest that velazodone inhibits Nigericin-induced IL-1β The maturation and secretion of p20 and IL-1β; Figure 1C: WB results suggest that vilazodone inhibits the maturation and secretion of p20 and IL-1β induced by Nigericin;

Figure 2 In Example 1 of the present invention, vilazodone inhibits the activation of NLRP3 inflammasome induced by multiple agonists; Figure 2A: ELISA results suggest that vilazodone inhibits IL- Maturation and secretion of 1β; Figure 2B: WB results suggest that vilazodone inhibits NLRP3 inflammasome activation induced by multiple NLRP3 inflammasome agonists;

Figure 3 In Example 1 of the present invention, vilazodone inhibits the activation of non-classical NLRP3 inflammasome induced by transcytosed lipopolysaccharide (cLPS); Figure 3A: ELISA results suggest that velazodone can inhibit inflammation induced by transcytosed LPB Activation of corpuscles; Figure 3B: WB results suggest that vilazodone can inhibit inflammasome activation induced by transcytosis of LPB;

Figure 4 Figure 4A: ELISA results indicate that vilazodone cannot inhibit PolyA:T-activated AIM2 inflammasome; Figure 4B: WB results indicate that vilazodone cannot inhibit PolyA:T-activated AIM2 inflammasome; Figure 4C: ELISA The results indicate that vilazodone cannot inhibit the pyrin inflammasome activated by C3 toxin; Figure 4D: WB results indicate that vilazodone cannot inhibit the pyrin inflammasome activated by C3 toxin; Figure 4E: ELISA results indicate that vilazodone cannot inhibit the pyrin inflammasome activated by C3 toxin Salmonella-activated NLRC4 inflammasome; Figure 4F: WB results indicate that vilazodone cannot inhibit Salmonella-activated NLRC4 inflammasome;

Figure 5 In Example 2 of the present invention, vilazodone inhibits the interaction between NLRP3 and serine/threonine protein kinase NEK7 and inhibits the assembly of NLPR3 inflammasome; Figure 5A: During the process of vilazodone inhibiting the activation of NLRP3 inflammasome Oligomerization of ASC; Figure 5B: Verazodone inhibits the interaction between endogenous NLRP3 and NEK7; Figure 5C: Verazodone inhibits the interaction between endogenous NLRP3 and ASC;

Figure 6: The effect of vilazodone directly binding to NLPR3 protein in Example 3 of the present invention; Figure 6A: Verazodone does not inhibit the degradation of NEK7, ASC, pro-caspase-1 and β-actin, but inhibits the degradation of NLRP3; Figure 6B: Verazodone does not inhibit the degradation of overexpressed NLRP1b protein; Figure 6C: Verazodone inhibits the degradation of overexpressed NLRP3 protein; Figure 6D: Verazodone does not inhibit the degradation of overexpressed NLRC4 protein; Figure 6E : Verazodone does not inhibit the degradation of overexpressed AIM2 protein;

Figure 7 In Example 3 of the present invention, vilazodone specifically binds to the NACHT domain of NLRP3; Figure 7A: Verazodone does not inhibit protein degradation of overexpressed LRR, Figure 7B: Verazodone does not inhibit overexpressed PYD. Protein degradation, Figure 7C: Verazodone inhibits protein degradation of overexpressed NACHT;

Figure 8 Figure 8A: Verazodone can significantly inhibit the occurrence and development of EAE; Figure 8B: Verazodone improves the weight loss of EAE mice; Figure 8C: Representative flow cytometry results suggest that vilazodone inhibits EAE mice Figure 8D: Histogram suggests that vilazodone can inhibit central infiltration in EAE mice; Figure 8E: QPCR results indicate that vilazodone can inhibit central inflammation levels in EAE mice.

Figure 9 Figure 9A: Schematic diagram of vilazodone administration after stroke modeling; Figure 9B: Slice staining shows that vilazodone reduces stroke infarct volume; Figure 9C: Verazodone improves neurological function scores after stroke; Figure 9 9D: Verazodone improves body weight of mice after stroke; Figure 9E: Verazodone improves rotarod grasping time after stroke; Figure 9F: QPCR results suggest that vilazodone can inhibit central inflammation in mice with stroke level.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments These are some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

Detailed description is provided below through specific embodiments.

The instruments, reagents, and materials used in the present invention are all well known to those skilled in the art and can be purchased through commercial institutions.

Example 1: In vitro study on the inhibitory effect of vilazodone on NLRP3 inflammasome

1. Verazodone inhibits Nigericin-induced P20 and IL-1β secretion and maturation

1. Differentiation of mouse bone marrow-derived macrophages (BMDM): Take the bone marrow of C57BL/6 mice about 8 weeks old, take the bone marrow cells, lyse the red blood cells, and use DMEM culture medium (containing 10% fetal bovine serum and Differentiation with 25ng/ml M-CSF (macrophage colony-stimulating molecule) for 4-5 days.

2. Distribute the differentiated BMDM cells into a 12-well plate, with 5×10 ⁵ cells in each well. The next day, cells were pretreated with opti-MEM medium (containing 1% fetal calf serum and 50ng/ml LPS) for 3 hours. Then, different concentrations (0 μM, 0.5 μM, 1 μM, 2 μM) of vilazodone were added for half an hour, and then 5 μM nigericin was added to stimulate the cells for 15 minutes.

3. Collect the cell supernatant and cell lysate, and detect the secretion of IL-1β and p20 in the supernatant by WB and ELISA respectively.

As shown in Figures 1A, 1B, and 1C, in Figure 1B, the ELISA results suggest that vilazodone inhibits the maturation and secretion of IL-1β induced by Nigericin; in Figure 1C, the WB results suggest that vilazodone inhibits Nigericin-induced p20 and the maturation and secretion of IL-1β. The above results indicate that vilazodone dose-dependently inhibits the maturation and secretion of p20 and IL-1β induced by Nigericin.

2. Verazodone inhibits the inflammasome activated by multiple agonists

1. Distribute the differentiated BMDM cells into a 12-well plate, with 5 × 10 ⁵ cells in each well.

2. The next day, pretreat the cells with opti-MEM medium (containing 1% fetal calf serum and 50ng/ml LPS) for 3 hours. Then they were divided into two groups. The first group added 2 μM vilazodone, and the second group added an equal volume of DMSO for half an hour. The first group was further divided into four groups, and each group added 5 μM Nigericin to stimulate the cells for 15 seconds. minutes, 5mM ATP to stimulate cells for 30 minutes, 700ug/ml urate crystals (MSU) to stimulate cells for 4 hours, and the last group is a negative control. The second largest group was also divided into four groups. Each group added 5 μM Nigericin to stimulate the cells for 15 minutes, 5 mM ATP to stimulate the cells for 30 minutes, and 700 μg/ml urate crystals (MSU) to stimulate the cells for 4 hours. The last group was a negative control.

3. Collect the cell supernatant and cell lysate, and detect the secretion of IL-1β and p20 in the supernatant by WB and ELISA respectively.

As shown in Figure 2A and 2B, in Figure 2A, the ELISA results indicate that vilazodone inhibits the maturation and secretion of IL-1β induced by multiple NLRP3 inflammasome agonists; in Figure 2B, the WB results indicate that vilazodone inhibits NLRP3 inflammasome activation induced by multiple NLRP3 inflammasome agonists. The above results indicate that vilazodone inhibits NLRP3 inflammasome activation induced by multiple NLRP3 inflammasome agonists.

3. Verazodone inhibits non-classical inflammasome activation

1. Distribute the differentiated BMDM cells into a 12-well plate, with 5 × 10 ⁵ cells in each well.

2. The next day, pretreat the cells with opti-MEM medium (containing 1% fetal calf serum and 200ng/ml Pam3CSK4) for 3 hours. Then, different concentrations of vilazodone (0 μM, 0.5 μM, 1 μM, and 2 μM) were added for half an hour, and then the cells were transfected with Lipo2000 and treated with 1 μg LPS for 16 hours.

3. Collect the cell supernatant and cell lysate, and detect the secretion of IL-1β and p20 in the supernatant by WB and ELISA respectively.

As shown in Figure 3, in Figure 3A, the ELISA results suggest that vilazodone can inhibit the activation of inflammasomes induced by transcytosis of LPB; in Figure 3B, the WB results suggest that vilazodone can inhibit the inflammasome activation induced by transcytosis of LPB. activation. The above results indicate that vilazodone inhibits non-classical inflammasome activation in a dose-dependent manner.

4. Verazodone has no inhibitory effect on the AIM2 inflammasome activated by Poly(dA:dT) and the IPAF inflammasome activated by Salmonella.

1. Distribute the differentiated BMDM cells into a 12-well plate, with 5 × 10 ⁵ cells in each well.

2. The next day, pretreat the cells with opti-MEM medium (containing 1% fetal calf serum and 50ng/ml LPS) for 3 hours. Then it was divided into two large groups. The first large group added 2 μM vilazodone, and the second large group added an equal volume of DMSO for half an hour. The first group was further divided into four small groups, and each group added 5 μM nigericin. ) stimulated cells for 15 minutes, 1 μg of poly(dA:dT) stimulated cells for 2 hours, Salmonella typhimurium (Salmonella) stimulated cells for 4 hours, and the last group was a negative control. The second largest group was also divided into four subgroups. Each group added 5 μM nigericin to stimulate the cells for 15 minutes, 1 μg of poly(dA:dT) to stimulate the cells for 2 hours, and Salmonella typhimurium to stimulate the cells for 4 hours. The last group is a negative control.

3. Collect the cell supernatant and cell lysate, and detect the secretion of IL-1β and p20 in the supernatant by WB and ELISA respectively.

As shown in Figure 4, in Figure 4A, the ELISA results indicate that vilazodone cannot inhibit the PolyA:T-activated AIM2 inflammasome; in Figure 4B, the WB results indicate that vilazodone cannot inhibit the PolyA:T-activated AIM2 inflammasome. In Figure 4C, the ELISA results indicate that vilazodone cannot inhibit the pyrin inflammasome activated by C3 toxin; in Figure 4D, the WB results indicate that vilazodone cannot inhibit the pyrin inflammasome activated by C3 toxin; in Figure 4E, The ELISA results indicate that vilazodone cannot inhibit the NLRC4 inflammasome activated by Salmonella; in Figure 4F, the WB results indicate that vilazodone cannot inhibit the NLRC4 inflammasome activated by Salmonella.

The above results show that vilazodone specifically inhibits the activation of NLRP3 inflammasome and does not affect the activation of AIM2 inflammasome and IPAF inflammasome.

Example 2: Mechanism of vilazodone inhibiting NLRP3 inflammasome activation

1. Verazodone inhibits ASC oligomerization

1. Distribute differentiated BMDM cells into a 6-well plate, 1×10 ⁶ cells per well.

2. The next day, pretreat the cells with opti-MEM medium (containing 1% fetal calf serum and 50ng/ml LPS) for 3 hours. Then, vilazodone of different concentrations (0 μM, 1 μM, 2 μM) was added for half an hour, and then 5 μM nigericin was added to stimulate the cells for 15 minutes. The culture supernatant from each well was collected, the protein was extracted, and the inhibitory effect of vilazodone on the activation of NLRP3 inflammasome was detected by WB. Add an equal volume of NP-40 containing protease inhibitor to each well, lyse on ice for 30 minutes, collect the lysate, centrifuge to precipitate, remove the supernatant, add cross-linking agent DSS with a final concentration of 2mM, cross-link at room temperature for 30 minutes, pre- Wash the pellet twice with cold PBS, centrifuge the pellet, add protein lysis buffer, and heat at 100°C for 10 minutes.

3. WB detects ASC aggregation.

As shown in Figure 5A, vilazodone dose-dependently inhibited ASC oligomerization during NLRP3 inflammasome activation.

2. Verazodone inhibits the interaction between endogenous NEK7 (NIMArelated kinase 7) and NLRP3 and the interaction between ASC and NLRP3.

1. Distribute differentiated BMDM cells into a 6-well plate, 1×10 ⁶ cells per well.

2. The next day, pretreat the cells with opti-MEM medium (containing 1% fetal calf serum and 50ng/ml LPS) for 3 hours. Then, different concentrations (0 μM, 2 μM) of vilazodone were added for half an hour, and then 5 μM nigericin was added to stimulate the cells for 15 minutes. Remove the supernatant, lyse the cells with NP-40, centrifuge and take the supernatant. A part of it is used as cell lysate. WB detects the expression of NLRP3 and NEK7 or ASC. A part of it is added with Protein G-coated beads and NEK7 or ASC antibodies. Rotate at 4°C. Incubate for 2 hours, centrifuge, wash the beads with NP-40, remove the supernatant, and add protein lysis buffer.

3. WB detects the interaction between NLRP3 and NEK7, NLRP3 and ASC.

As shown in Figures 5B and 5C, in Figure 5B, vilazodone inhibits the interaction between endogenous NLRP3 and NEK7; in Figure 5C, vilazodone inhibits the interaction between endogenous NLRP3 and ASC. The above results indicate that vilazodone inhibits the interaction between endogenous NLRP3 and NEK7, NLRP3 and ASC, thereby inhibiting the assembly of NLRP3 inflammasome.

Example 3: Study on the binding mode and binding site of vilazodone and NLRP3

1. Verazodone can directly bind to NLRP3

1. Distribute differentiated BMDM cells into a 6-well plate, 1×10 ⁶ cells per well.

2. On the next day, BMDM cells were pretreated with opti-MEM medium (containing 1% fetal calf serum and 50ng/ml LPS) for 3 hours, lysed the cells with NP-40 lysis buffer, and mixed the cell lysate with different doses ( 0mM, 0.2mM, 1mM) vilazodone were incubated overnight at 4°C. Incubate with pronase (25ng/μg of protein) at room temperature for 30 minutes to perform target stability experiments of drug affinity reactions (Chen, Y., He, H., Lin, B. et al. RRx-001ameliorates inflammatorydiseases by acting as a potent covalent NLRP3 inhibitor. Cell Mol Immunol 18,1425–1436(2021).).

3. WB detected the expression levels of NLRP3, NEK7, ASC, pro-caspase-1 and β-actin in the cell lysate.

As shown in Figure 6A, vilazodone cannot inhibit the degradation of NEK7, ASC, pro-caspase-1, and β-actin, but can inhibit the degradation of NLRP3. Verazodone was confirmed to bind directly to NLRP3.

2. Verazodone can directly bind to NLRP3

a) Transfer NLRP3, AIM2, NLRC4, and NLRP1b plasmids into 293T cells for 24 hours respectively;

b) Use NP-40 lysis buffer to lyse the obtained 293T cells that overexpress the corresponding plasmids.

The cell lysate was incubated with different doses (0mM, 1mM) of vilazodone at 4°C overnight. Incubate with pronase (25ng/μg of protein) at room temperature for 30 minutes to perform a target stability experiment for drug affinity reaction.

c) WB detects the expression level of the corresponding protein in the cell lysate.

As shown in Figures 6B-6E, it can be seen from Figure 6B that vilazodone does not inhibit the degradation of overexpressed NLRP1b protein; it can be seen from Figure 6C that vilazodone inhibits the degradation of overexpressed NLRP3 protein; Figure As can be seen in 6D, vilazodone does not inhibit the degradation of overexpressed NLRC4 protein; as can be seen in Figure 6E, vilazodone does not inhibit the degradation of overexpressed AIM2 protein. Verazodone cannot inhibit the degradation of overexpressed AIM2, NLRC4, and NLRP1b proteins, but it can inhibit the degradation of NLRP3.

The above results confirmed that vilazodone can specifically bind to NLRP3.

3. Verazodone specifically binds to the NACHT domain of NLRP3

1. Transfer the Flag-NACHT, Flag-LRR, and Flag-PYD plasmids into 293T cells for 24 hours respectively (the plasmids were donated by the research group of Professor J. Tschopp (University of Lausanne). For plasmid construction methods, please see Chen Y, He H, Lin B,etal.RRx-001 ameliorates inflammatory diseases by acting as a potent covalentNLRP3 inhibitor[J].Cellular&Molecular Immunology, 2021,18(6):1425-1436.);

2. Lyse the obtained 293T cells that overexpress the corresponding plasmids with NP-40 lysis buffer, and incubate the cell lysate with different doses of vilazodone at 4°C overnight. Incubate with pronase (25ng/μg of protein) at room temperature for 30 minutes to perform a target stability experiment for drug affinity reaction.

3. WB detects the expression level of the corresponding protein in the cell lysate.

As shown in Figures 7A-7C, it can be seen from Figure 7A that vilazodone does not inhibit the protein degradation of overexpressed LRR. It can be seen from Figure 7B that vilazodone does not inhibit the protein degradation of overexpressed PYD. Figure As can be seen in 7C, vilazodone inhibited protein degradation of overexpressed NACHT.

The above results show that vilazodone cannot inhibit the protein degradation of overexpressed LRR and PYD, but can inhibit the degradation of NACHT, confirming that vilazodone can bind to the NACHT domain. Example 4: In vivo study of vilazodone inhibiting NLRP3 inflammasome

1. Verazodone inhibits the occurrence and development of EAE (experimental allergic encephalomyelitis)

1. Twenty 8-week-old male C57BL/6J mice were divided into 2 groups, with 10 mice in each group.

2. Each group is processed as follows:

Group 1: MOG peptide was injected subcutaneously on Day0, PTX was injected intravenously on Day0 and Day2 to induce EAE, and an equal volume of solvent (90% PBS and 10% DMSO) with vilazodone was injected intraperitoneally every day starting from Day0.

Group 2: MOG peptide was injected subcutaneously on Day 0, PTX was injected intravenously on Day 0 and Day 2 to induce EAE, and 10 mg/kg vilazodone (dissolved in 90% PBS and 10% DMSO) was injected intraperitoneally every day starting from Day 0.

3. Score the mice for EAE disease every day. On the 14th day, 5 mice from each group were sacrificed, and the remaining mice were observed throughout the disease process. Mouse spinal cord tissue was taken for section staining, and flow cytometry was used to measure central infiltration in the mouse brain and spinal cord. Analyze the immune cells infiltrating into the spinal cord.

As shown in Figure 8, it can be seen from Figure 8A that vilazodone can significantly inhibit the occurrence and development of EAE; it can be seen from Figure 8B that vilazodone improved the weight loss of EAE mice; in Figure 8C, The representative diagram of the flow cytometry results shows that vilazodone inhibits the central infiltration of EAE mice; in Figure 8D, the bar chart shows that vilazodone can inhibit the central infiltration of EAE mice; in Figure 8E, QPCR (QPCR refers to "2. "Lazodone inhibits the progression of stroke mouse model" section) The results suggest that vilazodone can inhibit the central inflammation level in EAE mice.

The above results show that vilazodone can significantly inhibit the occurrence and development of EAE, inhibit spinal cord lesions and immune cell infiltration in mice.

2. Progress of vilazodone in inhibiting stroke mouse models

1. Ten 8-week-old male C57BL/6J mice were divided into 2 groups, 5 mice in each group.

2. Each group was treated as follows: All mice were subjected to stroke modeling using photochemical methods (Feng Y, Liao S, Wei C, Jia D, Wood K, Liu Q, et al. Infiltration and persistence of lymphocytes during late- stage cerebral ischemia in middle cerebral artery occlusion and photothrombotic stroke models.J Neuroinflammation.2017;14(1):248;Jones KA,Maltby S,Plank MW,Kluge M,Nilsson M,Foster PS,et al.Peripheralimmune cells infiltrate into sites of secondary neurodegeneration afterischemic stroke. Brain Behav Immun. 2018;67:299-307.). Mice undergoing photothrombosis surgery were anesthetized with inhaled 3.5% isoflurane and maintained with 1.0–2.0% inhaled isoflurane in 30% _O . Five minutes before light exposure, mice were intraperitoneally injected with rose Bengal dye at a dose of 150 mg/kg. After receiving rose Bengal dye, mice were placed in a stereoscope. The fontanelle was identified and the end of a 4 mm diameter fiber optic cable was placed on top of the skull, in the center of the fontanel and approximately 2 mm away from the fontanel. Five minutes after the injection of Rose Bengal, the cold light source with a green bandpass filter was turned on and the skull was illuminated for 20 minutes. Remove the mouse from the stereoscope, suture the incision and disinfect it.

3. Conduct scoring and rotarod experiments at 6h, 24h, 48h, and 72h respectively. The Longa 5-point scale is used as the scoring standard: 0 points: no neurological deficit; 1 point: the forelimb on the paralyzed side is adducted and unable to fully extend when lifting the tail; 2 points: rotates to the paralyzed side; 3 points: tilts to the paralyzed side when walking. ; 4 points: unable to walk or unconscious. Results: Scores were conducted 6 hours after surgery, and 1-3 were classified as effective models. 4 points and 0 points are invalid. The rotating speed of the rotating rod is 15 rpm/min, the total time is 300s, and the time of falling from the rotating rod is recorded. We injected vilazodone intraperitoneally for the first time 6 hours after surgery, and then intraperitoneally injected 10 mg/kg vilazodone (dissolved in 90% PBS and 10% DMSO) and normal saline at 24h, 48h, and 72h respectively.

The experiment ended at 4.72 hours, and the animals were immediately anesthetized and quickly decapitated. Take the complete brain, mark the group and number, and freeze it in a -20°C refrigerator for 15 to 20 minutes until the brain becomes slightly hard. Remove the olfactory bulb and cerebellum, and then use a stainless steel brain slicing mold to cut the brain tissue into slices every 2 mm, evenly cut into 4 to 5 layers. The brain tissue sections were placed in a 6-well plate containing 2% TTC solution and incubated in a 37°C incubator in the dark for 20 min. During this period, the brain was turned from time to time to evenly contact the staining solution. Take photos after dyeing is complete. Part of the brain tissue was taken for QPCR detection.

5. Fluorescent quantitative PCR (QPCR) detection

(1)RNA extraction

(A) Collect tissues, add 1ml trizol to each sample, fully lyse the cells, and transfer the cell lysate to a 1.5ml EP tube.

(B) Add 200 μl of chloroform to each sample, shake the solution until the mixture turns milky white, put the sample in an ice bath for 2 minutes, and centrifuge at 12,000 rpm for 15 minutes in a 4°C centrifuge.

(C) Transfer the colorless liquid in the upper layer after centrifugation (about 400 μl) to a new 1.5 ml RNAase-free EP tube. Add 400 μl isopropyl alcohol to each sample, mix well, and incubate the sample on ice for 10 min at 4°C. Centrifuge in a centrifuge at 12,000 rpm for 10 min.

(D) Discard the centrifugation supernatant, add 1 ml of pre-cooled 75% ethanol solution to each sample to clean the RNA precipitate, and centrifuge at 7500 rpm for 5 minutes in a 4°C centrifuge. Repeat twice.

(E) Discard the supernatant, dry the RNA pellet at room temperature, and add 50 μl DEPC water to each sample to dissolve the RNA pellet.

(2)Reverse transcription

(A) Add the following reagents to the RNAase-free PCR tube:

Random primer 200ng

mRNA1μg

dNTP Mix(10mM)1μl

Add H2O to 12μl

(B) Mix the above reagents, place the PCR tube in the PCR machine, set to 65°C for 5 minutes, and then keep in ice for 2 minutes.

(C) Continue to add the following reagents to the PCR tube:

DEPC water 1μl

0.1M DTT 2μl

5×First-strand buffer 4μl

(D) Mix the above mixture, place the PCR tube in the PCR machine, set to 37°C for 2 minutes.

(E) Add 1 μl of reverse transcriptase MLV (200 units) to the PCR tube, mix well, place the PCR tube in the PCR machine, and set the program as:

25℃10min

37℃50min

70℃15min

4℃∞

(F) After completion, cDNA can be obtained.

(3).QPCR

(A) Add the following reagents into the QPCR tube

Forward Primer(5μM)1μl

Reverse Primer(5μM)1μl

cDNA1μl

2×SYRB Green 10μl

H2O 7μl

(B) Mix the above reagents, place the QPCR tube in the QPCR instrument, and set the program as:

95℃10min;

95℃10s;

60℃45s;45cycles

(C) Data analysis.

Among them, the QPCR primers: (Mouse IL1b forward,TGCCACCTTTTGACAGTGATG; Mouse IL1breverse,AAGGTCCACGGGAAAGACAC; Mouse IL-6forward,GTC CTTCCTACCCCAATTTCC; MouseIL-6reverse,GCACTA GGTTTGCCGAGTAGA; Mouse Tnf-αforward,CGATGG GTTGTACCTTGTC; Mouse Tnf-αreverse,CGGACTCCG CAAAGTCTAAG; Mouse Gapdh forward,GGTGAAGGTCGGTGTGAACG; Mouse Gapdh reverse,CTCGCTCCTGGA AGATGGTG).

As shown in Figure 9, Figure 9A is a schematic diagram of vilazodone administration after stroke modeling; in Figure 9B, section staining shows that vilazodone reduces stroke infarct volume; as can be seen from Figure 9C, vilazodone Ketone improves neurological function scores after stroke; as can be seen in Figure 9D, vilazodone improves the body weight of mice after stroke; as can be seen in Figure 9E, vilazodone improves rotarod grasping time after stroke; Figure As can be seen in 9F, the QPCR results suggest that vilazodone can inhibit the central inflammation level in stroke mice.

The above results show that vilazodone can significantly inhibit the occurrence and development of stroke and suppress the central inflammation level in mice.

To sum up, the technical solution provided by the present invention has the following beneficial technical effects:

(1) Verazodone is a serotonin partial agonist-reuptake inhibitor (SPARI) used to treat major depressive disorder. The present invention takes vilazodone as the research object, and first examines the effect of vilazodone on the activation of NLRP3 inflammasome in macrophages. The results show that the addition of vilazodone can effectively inhibit the maturation and secretion of caspase-1 and IL-1β, and this inhibitory effect is dose-dependent. Finally, the mechanism and binding site of vilazodone for inhibiting NLRP3 were clarified, and provided An NLRP3 inhibitor with clear clinical pharmacological effects and high clinical safety;

(2) At the same time, the present invention selects EAE, stroke, chronic restraint mouse animal models, etc. to clarify that vilazodone improves the symptoms of the above animal models, indicating that the application of vilazodone or its pharmaceutically acceptable salts and crystal forms , solvates can improve the symptoms of the above diseases and provide new ways and methods for the prevention and treatment of NLRP3 inflammasome-related diseases.

It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that these entities or operations are mutually exclusive. any such actual relationship or sequence exists between them. Furthermore, the terms "comprises," "comprises," or any other variations thereof are intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also those not expressly listed other elements, or elements inherent to the process, method, article or equipment. Without further limitation, an element defined by the statement "comprises a..." does not exclude the presence of additional identical elements in a process, method, article, or apparatus that includes the stated element.

The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the foregoing embodiments. The recorded technical solutions may be modified, or some of the technical features thereof may be equivalently replaced; however, these modifications or substitutions shall not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of each embodiment of the present invention.

Claims (9)

1. Application of vilazodone or its pharmaceutically acceptable salts, crystal forms, and solvates in the preparation of drugs for preventing or treating NLRP3 inflammasome-related diseases; the NLRP3 inflammasome-related diseases do not include depression disease. 2. Application of pharmaceutical compositions containing vilazodone or its pharmaceutically acceptable salts, crystal forms, and solvates in the preparation of drugs for preventing or treating NLRP3 inflammasome-related diseases; the NLRP3 inflammasome Related illnesses do not include depression. 3. The application according to claim 2, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. 4. Application of pharmaceutical preparations containing vilazodone or its pharmaceutically acceptable salts, crystal forms, and solvates in the preparation of drugs for preventing or treating NLRP3 inflammasome-related diseases; the NLRP3 inflammasome-related Illnesses do not include depression. 5. The application according to claim 4, characterized in that the types of pharmaceutical preparations include solid preparations, semi-solid preparations, liquid preparations and gas preparations. 6. The application according to claim 5, wherein the liquid dosage form includes solutions and injections; the solid dosage forms include tablets, granules, and capsules; and the semi-solid dosage forms include ointments and gels. agent; the gas dosage form includes aerosol and spray. 7. The application according to any one of claims 1 to 6, wherein the NLRP3 inflammasome-related diseases include gout, peritonitis, Alzheimer's disease, enteritis, amyotrophic lateral sclerosis, obesity, and type II diabetes. , metabolic syndrome, sepsis, sepsis, fatty liver, hepatitis, arthritis, atherosclerosis, Parkinson's disease, myocardial infarction, asbestosis, silicosis and silicosis, asthma or acute respiratory distress syndrome, infection Acute and chronic tissue damage, ultraviolet-induced skin sunburn, contact hypersensitivity or Muhl-Wehrmacht syndrome or cryoporphyrin-associated periodic syndrome (CAPS), opioid analgesic tolerance, stroke, multiple Sclerosis, stroke. 8. The application of claim 7, wherein the NLRP3 inflammasome-related disease is multiple sclerosis. 9. The application according to claim 7, wherein the NLRP3 inflammasome-related disease is stroke.