Application of zinc pyrithione in preparation of NLRP3 inflammasome inhibitor

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of zinc pyrithione in inhibition of NLRP3 inflammasome activation. The technical problem to be solved by the invention is to provide a new choice for treatment of NLRP3 inflammasome related diseases. The technical scheme of the invention is application of zinc pyrithione in preparation of an NLRP3 inflammasome inhibitor. The invention finds that the zinc pyrithione can inhibit the activation of NLRP3 inflammasomes, and shows a good inhibition effect in mouse and human cells; zinc pyrithione is expected to become a candidate drug for treating NLRP3 inflammasome related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of zinc pyrithione in inhibiting the activation of NLRP3 inflammasome. Background Art

[0002] Current studies have shown that NLRP3 inflammasomes can be activated by a variety of pathogens and sterile inflammatory signals, including PAMPs from fungi, bacteria, and viruses, as well as host-derived molecules such as reactive oxygen species (ROS), extracellular ATP, and crystalline and particulate matter; in addition, interference with intracellular homeostasis can also activate NLRP3 inflammasomes. After the NLRP3 inflammasome is activated, it can recruit the adaptor protein ASC and caspase-1 precursor to form the NLRP3 inflammasome. After the NLRP3 inflammasome is assembled, the caspase-1 precursor will self-cleave to form caspase-1 with cleavage activity. Caspase-1 then cleaves IL-1β precursor and IL-18 precursor, inducing the maturation and secretion of IL-1β and IL-18, promoting immune response and inflammatory response, and maintaining the body's immune homeostasis.

[0003] Some small molecule compounds have been found to directly bind to NLRP3 and inhibit its activity. These compounds may become therapeutic drugs targeting NLRP3 inflammasomes and are currently a hot topic of research.

[0004] MCC950 is a highly selective NLRP3 inhibitor that can directly bind to NLRP3 and inhibit its activity. Studies have shown that MCC950 may inhibit the ATPase activity of NLRP3 by binding to the NACHT domain of NLRP3, thereby preventing NLRP3 aggregation and NLRP3 inflammasome activation.

[0005] Oridonin is a polycyclic diterpene natural product. Due to its unique chemical structure and potential biological activity, it can induce apoptosis of various cancer cells and has multiple biological activities such as anti-tumor and anti-inflammatory. Studies have found that Oridonin specifically covalently binds to NLRP3, forms a covalent bond with cysteine ​​279 in the NACHT domain, blocks the interaction between NLRP3 and NEK7, and thus inhibits the assembly and activation of the NLRP3 inflammasome.

[0006] Tranilast is an analog of tryptophan metabolites. Studies have shown that Tranilast directly binds to the NACHT domain of NLRP3, blocks NLRP3 oligomerization and NLRP3 inflammasome assembly, and inhibits NLRP3 inflammasome activation.

[0007] CY-09 is a novel NLRP3 inhibitor. It may inhibit NLRP3-ATPase activity by directly binding to the ATP binding motif of the NACHT domain of NLRP3, thereby preventing the assembly and activation of the NLRP3 inflammasome. CY-09 is characterized by high selectivity and specificity, but is less effective in inhibiting other inflammasomes such as AIM2 and NLRC4. CY-09 has shown anti-inflammatory effects in a variety of disease models, such as osteoarthritis and diabetic nephropathy.

[0008] Although these compounds are expected to become drugs for the treatment of NLRP3 inflammasome-related diseases, they still need to undergo lengthy efficacy and safety evaluations as well as clinical trials. Therefore, it is very important to find a clinical drug that can inhibit NLRP3 inflammasomes to treat related diseases. This will not only greatly shorten the drug development time, but also ensure its safety.

[0009] Zinc pyrithione (ZnPT) is a coordination compound formed by zinc (Zn) and pyrithione ligands. It is widely used in hair care products and skin care products and is an FDA-approved clinical drug for the treatment of dandruff and seborrheic dermatitis. Its chemical structure, especially the coordination mode of zinc atom with sulfur and oxygen atoms in pyrithione ligands, significantly affects its physical and chemical properties, giving it certain chemical stability and reactivity. Low solubility in water is a key feature of ZnPT in antiseptic and antifungal applications, and this low solubility ensures its durability in products. Its excellent chemical and thermal stability allows it to remain effective in product formulations under various pH and temperature conditions. The most critical feature of ZnPT is its antibacterial and antifungal activity, and it is widely used to treat dandruff and seborrheic dermatitis (SD), which are usually manifested by skin scaling, erythema and itching. These diseases are closely related to the NLRP3 inflammasome, but it is still unclear whether ZnPT exerts its anti-inflammatory effects by affecting NLRP3 inflammasome activation.

[0010] Therefore, it is of great significance to further strive to find inhibitors that target NLRP3 inflammasome and specifically inhibit NLRP3 inflammasome activation and have therapeutic effects on NLRP3 inflammasome-related diseases. Summary of the invention

[0011] The technical problem to be solved by the present invention is to provide a new option for the treatment of NLRP3 inflammasome-related diseases.

[0012] The technical solution of the present invention is the application of zinc pyrithione in the preparation of NLRP3 inflammasome inhibitors.

[0013] Specifically, the NLRP3 inflammasome is derived from at least one of the following diseases: colitis, psoriasis, septic shock, type II diabetes, metabolic syndrome, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, Behcet's disease, sepsis, gout, arthritis, multiple sclerosis, obesity, viral inflammation such as viral hepatitis and pneumonia, acute and chronic tissue damage caused by infection, atherosclerosis, myocardial infarction, UV-induced skin sunburn, contact hypersensitivity, familial cold autoinflammatory syndrome, chronic infantile neurocutaneous joint syndrome, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, depression, asbestosis, silicosis and silicosis, asthma or acute respiratory distress syndrome, peritonitis, Muller-Weigler syndrome and cryoporphyrin-associated periodic syndrome (CAPS) or cytokine storm caused by tumor treatment (such as CART therapy and immune checkpoint inhibitor therapy).

[0014] In particular, the inhibitor inhibits the activation or assembly of the NLRP3 inflammasome.

[0015] Furthermore, the NLRP3 inflammasome assembly and activation is induced by agonist.

[0016] Specifically, the agonist is nigericin, adenosine triphosphate (ATP), monosodium urate crystals (MSU) and / or intracellular transfection lipopolysaccharide (cLPS).

[0017] The present invention also provides the use of zinc pyrithione in the preparation of a cytokine inhibitor that depends on NLRP3 inflammasome.

[0018] Specifically, the NLRP3 inflammasome is derived from at least one of the following diseases: colitis, psoriasis, septic shock, type II diabetes, metabolic syndrome, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, Behcet's disease, sepsis, gout, arthritis, multiple sclerosis, obesity, viral inflammation such as viral hepatitis and pneumonia, acute and chronic tissue damage caused by infection, atherosclerosis, myocardial infarction, UV-induced skin sunburn, contact hypersensitivity, familial cold autoinflammatory syndrome, chronic infantile neurocutaneous joint syndrome, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, depression, asbestosis, silicosis and silicosis, asthma or acute respiratory distress syndrome, peritonitis, Muller-Weigler syndrome and cryoporphyrin-associated periodic syndrome (CAPS) or cytokine storm caused by tumor treatment (such as CART therapy and immune checkpoint inhibitor therapy).

[0019] Specifically, the cytokine is IL-1β.

[0020] The present invention also provides the use of zinc pyrithione in the preparation of a drug for treating NLRP3 inflammasome-related diseases.

[0021] Specifically, the NLRP3 inflammasome-related disease is colitis, psoriasis, septic shock, type II diabetes, metabolic syndrome, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, Behcet's disease, sepsis, gout, arthritis, multiple sclerosis, obesity, viral inflammation such as viral hepatitis and pneumonia, acute and chronic tissue damage caused by infection, atherosclerosis, myocardial infarction, UV-induced skin sunburn, contact hypersensitivity, familial cold autoinflammatory syndrome, chronic infantile neurocutaneous joint syndrome, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, depression, asbestosis, silicosis and silicosis, asthma or acute respiratory distress syndrome, peritonitis, Muller-Weigler syndrome and cryoporphyrin-associated periodic syndrome (CAPS) or at least one of cytokine storms caused by tumor treatment (such as CART therapy and immune checkpoint inhibitor therapy).

[0022] The present invention also provides a drug for inhibiting NLRP3 inflammasome, wherein the main component of the drug is pyrithione zinc.

[0023] Specifically, the NLRP3 inflammasome is derived from at least one of the following diseases: colitis, psoriasis, septic shock, type II diabetes, metabolic syndrome, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, Behcet's disease, sepsis, gout, arthritis, multiple sclerosis, obesity, viral inflammation such as viral hepatitis and pneumonia, acute and chronic tissue damage caused by infection, atherosclerosis, myocardial infarction, UV-induced skin sunburn, contact hypersensitivity, familial cold autoinflammatory syndrome, chronic infantile neurocutaneous joint syndrome, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, depression, asbestosis, silicosis and silicosis, asthma or acute respiratory distress syndrome, peritonitis, Muller-Weigler syndrome and cryoporphyrin-associated periodic syndrome (CAPS) or cytokine storm caused by tumor treatment (such as CART therapy and immune checkpoint inhibitor therapy).

[0024] Wherein, the medicine also includes pharmaceutically acceptable auxiliary ingredients.

[0025] The present invention also provides a drug for treating NLRP3 inflammasome-related diseases, wherein the main component of the drug is pyrithione zinc.

[0026] Specifically, the NLRP3 inflammasome-related disease is colitis, psoriasis, septic shock, type II diabetes, metabolic syndrome, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, Behcet's disease, sepsis, gout, arthritis, multiple sclerosis, obesity, viral inflammation such as viral hepatitis and pneumonia, acute and chronic tissue damage caused by infection, atherosclerosis, myocardial infarction, UV-induced skin sunburn, contact hypersensitivity, familial cold autoinflammatory syndrome, chronic infantile neurocutaneous joint syndrome, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, depression, asbestosis, silicosis and silicosis, asthma or acute respiratory distress syndrome, peritonitis, Muller-Weigler syndrome and cryoporphyrin-associated periodic syndrome (CAPS) or at least one of cytokine storms caused by tumor treatment (such as CART therapy and immune checkpoint inhibitor therapy).

[0027] Wherein, the medicine also includes pharmaceutically acceptable auxiliary ingredients.

[0028] The invention also provides a medicine for treating colitis, the main component of which is pyrithione zinc.

[0029] Wherein, the medicine also includes pharmaceutically acceptable auxiliary ingredients.

[0030] The invention also provides a medicine for treating septic shock, the main component of which is pyrithione zinc.

[0031] Wherein, the medicine also includes pharmaceutically acceptable auxiliary ingredients.

[0032] The invention also provides a medicine for treating colitis and psoriasis, the main component of which is pyrithione zinc.

[0033] Wherein, the medicine also includes pharmaceutically acceptable auxiliary ingredients.

[0034] Beneficial effects of the present invention: The present invention discovered a new application of zinc pyrithione, an FDA-approved drug for the treatment of seborrheic dermatitis, that is, it has a significant effect in inhibiting the activation of NLRP3 inflammasomes. Zinc pyrithione can effectively block the activation of NLRP3 inflammasomes caused by a variety of agonists, and shows good inhibitory effects in both mouse BMDM cells and human THP-1 cells. More importantly, zinc pyrithione significantly alleviates inflammasome-related diseases such as LPS-induced septic shock, DSS-induced colitis in mice, and IMQ-induced psoriasis in mice by inhibiting the activation of NLRP3 inflammasomes. This discovery not only expands the drug application field of zinc pyrithione, but also provides a new option for the treatment of NLRP3 inflammasome-related diseases. As an old drug, zinc pyrithione has great potential to become a candidate drug for the treatment of NLRP3 inflammasome-related diseases due to its safety and wide use. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 In Example 1 of the present invention, zinc pyrithione (ZnPT) inhibits the activation of NLRP3 inflammasome induced by Nigericin (name: Nigericin; stimulates the activation of NLRP3 inflammasome, thereby stimulating the secretion of IL-1β cytokine) in BMDM cells, and has no effect on non-inflammasome-related cytokines; wherein A shows the protein expression of IL-1β and p20 in the cell culture supernatant (SN), B shows the secretion level of inflammatory factor IL-1β in the cell culture supernatant, C and D show that ZnPT has no effect on non-inflammasome-dependent cytokines such as TNF-α and IL-6 in the cell culture supernatant, and E shows the half inhibitory concentration (IC) of ZnPT for inhibiting the activation of NLRP3 inflammasome in BMDM cells 50 ) is 0.2 μM.

[0036] Figure 2 In Example 1 of the present invention, ZnPT inhibits Nigericin-induced NLRP3 inflammasome activation in human THP-1 cells and LPS-induced NLRP3 inflammasome activation in human PBMCs; wherein, A shows the protein expression of p20 in the THP-1 cell culture supernatant (SN), B shows the secretion level of the inflammatory factor IL-1β in the THP-1 cell culture supernatant, C shows that ZnPT has no effect on the non-inflammasome-dependent cytokine TNF-α in the THP-1 cell culture supernatant, D shows that ZnPT inhibits the secretion level of the inflammatory factor IL-1β in the human PBMC culture supernatant, and E shows that ZnPT has no effect on the non-inflammasome-dependent cytokine TNF-α in the human PBMC culture supernatant.

[0037] Figure 3 The effect of zinc pyrithione in Example 1 of the present invention on inhibiting NLRP3 inflammasome activation induced by MSU and ATP; wherein A and C show the protein expression of IL-1β and p20 in the cell culture supernatant (SN), and B and D show the secretion level of the pro-inflammatory cytokine IL-1β in the cell culture supernatant.

[0038] Figure 4 In Example 1 of the present invention, zinc pyrithione has no effect on the activation of AIM2 inflammasome induced by Poly (A:T); wherein A shows the protein expression of IL-1β and p20 in the cell culture supernatant (SN), and B shows the secretion level of the inflammatory cytokine IL-1β in the cell culture supernatant.

[0039] Figure 5In Example 2 of the present invention, zinc pyrithione does not affect the LPS-mediated priming stage; wherein, A and B show the secretion levels of proinflammatory cytokines TNF-α and IL-6 in the cell culture supernatant, and C shows the protein expression levels of IL-1β precursor (Pro-IL-1β), NLRP3, ASC, Pro-casp1 and β-actin in the cell lysate.

[0040] Figure 6 In Example 2 of the present invention, zinc pyrithione does not affect nigericin-induced mitochondrial damage, reactive oxygen species production and K + Ion outflow; A shows the confocal microscopy observation of BMDM cell mitochondrial damage and reactive oxygen production, and B shows the inductively coupled plasma emission spectrometer detection of intracellular potassium ion content.

[0041] Figure 7 In Example 3 of the present invention, zinc pyrithione inhibits the binding of endogenous NEK7 to NLRP3, and NLRP3 to ASC; wherein A shows the levels of NLRP3 and NEK7 proteins in cell immunoprecipitation (IP) and cell lysate (Input), and B shows the levels of NLRP3 and ASC proteins in cell immunoprecipitation (IP) and cell lysate (Input).

[0042] Figure 8 In Example 3 of the present invention, zinc pyrithione reversibly binds to the NLRP3 inflammasome.

[0043] Fig. 9 The effect of zinc pyrithione in Example 4 of the present invention on alleviating LPS-induced septic shock in mice; wherein A, B, and C show the secretion levels of inflammatory factors IL-1β, TNF-α, and IL-6 in mouse serum, and D, E, and F show the secretion levels of inflammatory factors IL-1β, TNF-α, and IL-6 in mouse peritoneal fluid; G shows that zinc pyrithione prolongs the survival time of mice with LPS-induced septic shock.

[0044] Fig.10 In Example 4 of the present invention, zinc pyrithione alleviates the disease activity index, weight loss, and spleen and colon tissue lesions induced by DSS in mice; wherein A shows the disease activity index of mice, B shows the change in mouse weight, C shows the spleen weight, D and E show the change in colon length, F shows that zinc pyrithione inhibits the secretion of IL-1β in the colon of mice induced by DSS, and G shows HE staining of colon tissue sections.

[0045] Fig.11The effect of zinc pyrithione on reducing the proportion of Th17 cells and Tc1 cells in the mesenteric lymph nodes and spleen of mice induced by DSS in Example 4 of the present invention; wherein A shows the representative CD4 + IL-17A + Flow cytometry and CD4 + IL-17A in T cells + The percentage of cells is shown in B. The representative CD8 + INF-γ + Flow cytometry and CD8 + INF-γ in T cells + The percentage of cells is shown in Figure 2. C shows the representative CD4 + IL-17A + Flow cytometry and CD4 + IL-17A in T cells + The percentage of cells is shown in Figure 4. D shows the representative CD8 + INF-γ + Flow cytometry and CD8 + INF-γ in T cells + The percentage of cells was counted.

[0046] Fig.12 The effect of pyrithione zinc in Example 4 of the present invention on alleviating the morphology and PASI index of psoriatic dermatitis in mice induced by imiquimod; wherein A shows the morphology of psoriatic dermatitis in mice, and B shows the severity of skin inflammation, including measurement of skin thickening, desquamation, erythema and cumulative score.

[0047] Fig.13 In Example 4 of the present invention, zinc pyrithione inhibits the histological characteristics of psoriatic dermatitis in mice induced by imiquimod and the proliferation and differentiation of epidermal cells; wherein A shows the histological evaluation of HE staining of the back skin of mice, and B shows the immunohistochemical detection of proliferating cell nuclear antigen (PCNA) in each group.

[0048] Fig.14 In Example 4 of the present invention, zinc pyrithione alleviates the expression of NLRP3 inflammasome-related components in the psoriasis skin tissue of mice induced by imiquimod. DETAILED DESCRIPTION

[0049] Through the preliminary screening of multiple drugs in the compound library developed and sold by Taosu (the library brings together clinical drugs approved by the FDA, and there is no disclosure that ZnPT can inhibit NLRP3 inflammasome activation), it was found that copper pyrithione zinc has a significant inhibitory effect on NLRP3 inflammasome activation. Zinc pyrithione (ZnPT) is an important coordination compound that has attracted attention due to its wide application in the treatment of skin inflammation. Its structure is as follows: .

[0050] ZnPT is known for its excellent chemical and thermal stability, low solubility, and significant antibacterial and antifungal activities. ZnPT is delivered to the skin epithelium as microparticles, and the antifungal activity depends on the skin availability of the bioactive monomer molecule. ZnPT has antifungal activity against the most common Malassezia globosa and Malassezia restrica in scalp skin, with an average in vitro minimum inhibitory concentration of 10-15 ppm. The efficacy of ZnPT depends on its targeted delivery to the skin sites where these yeasts inhabit, including the scalp surface and the infundibulum of hair follicles. These activities are achieved by disrupting the integrity of the microbial cell membrane and interfering with the normal function of intracellular metalloenzymes, thereby inhibiting the growth and reproduction of microorganisms. This broad spectrum of antimicrobial activity makes it an ideal choice for many hair and skin care products. These properties of ZnPT not only make it popular in personal care products, but also occupy an important position in scientific research. However, the pharmacological mechanism of zinc pyrithione in the prevention and treatment of colitis, Alzheimer's disease, alum-induced peritonitis, and gout induced by uric acid crystal accumulation has not been reported.

[0051] The main materials and consumables used in the following examples are shown in Tables 1 to 4.

[0052] Table 1 Cell and mouse information Table 2 Experimental consumables Table 3 Antibody information Table 4 Experimental reagents Example 1 ZnPT inhibits the activation of NLRP3 inflammasome in monocytes and macrophages in vitro 1. ZnPT inhibits Nigericin-induced NLRP3 inflammasome activation in mouse BMDM cells Mouse BMDM cells were pre-stimulated with LPS (lipopolysaccharide) for 3 h, and then treated with different doses of ZnPT (0 μM, 0.2 μM, 0.4 μM, 0.6 μM) for 30 min. Nigericin was then added to induce the activation of NLRP3 inflammasome. The inhibitory effect of ZnPT on Nigericin-induced IL-1β and caspase-1 secretion, as well as the effects on NLRP3 inflammasome-independent cytokines TNF-α and IL-6 were detected by ELISA and Western Blot, respectively. The IC of ZnPT for inhibiting BMDM cells was calculated. 50 value.

[0053] The results are as follows Figure 1 As shown in Figure 2, ZnPT inhibited the activation of NLRP3 inflammasome in BMDM cells in a dose-dependent manner without affecting the secretion of TNF-a and IL-6. ZnPT inhibited the IC 50 The value is 0.2 μM.

[0054] ZnPT inhibits NLRP3 inflammasome activation in human THP-1 cells and human PBMCs The effect of ZnPT on human THP-1 cells was further investigated. The cells were pretreated with LPS for 3 h, then treated with ZnPT (0 μM, 0.2 μM, 0.4 μM, 0.6 μM) for 30 min, and finally Nigericin was added to stimulate and activate NLRP3 inflammasome. The inhibitory effect of ZnPT on Nigericin-induced IL-1β and caspase-1 secretion, as well as the effect on NLRP3 inflammasome-independent cytokine TNF-α were detected by ELISA and Western Blot, respectively.

[0055] There is an alternative NLRP3 inflammasome activation pathway in human monocytes. Human monocytes have constitutively activated caspase-1, and LPS stimulation alone can mediate the release of IL-1β. To further explore the effect of ZnPT on human PBMC, healthy human blood was drawn, and PBMCs were separated using Ficoll separation solution and cultured overnight. First, ZnPT (0 μM, 0.2 μM, 0.4 μM, 0.6 μM) was added to treat the cells for 30 min, and the cells were stimulated with LPS for 16 h to activate NLRP3 inflammasomes. ELISA was used to detect the inhibitory effect of ZnPT on LPS-induced IL-1β, as well as the effect on NLRP3 inflammasome-independent cytokine TNF-α.

[0056] like Figure 2As shown, ZnPT inhibited NLRP3 inflammasome activation in human THP-1 and human PBMC cells in a dose-dependent manner without affecting TNF-α secretion.

[0057] 3. ZnPT inhibits NLRP3 inflammasome activation induced by other agonists In addition, considering that MSU and ATP are also agonists that can effectively activate NLRP3 inflammasomes, ZnPT was added to LPS-pretreated BMDM cells, and then stimulated with MSU and ATP. The inhibitory effect of ZnPT on the secretion of IL-1β and caspase-1 induced by various NLRP3 inflammasome agonists was detected by ELISA and Western Blot, respectively.

[0058] like Figure 3 As shown, ZnPT inhibited MSU- and ATP-induced NLRP3 inflammasome activation.

[0059] 4. ZnPT does not affect AIM2 inflammasome activation To explore the specificity of ZnPT's inhibitory effect, the study also evaluated its effect on AIM2 inflammasomes. AIM2 inflammasomes can respond to dsDNA and mediate defense responses. In the experiment, BMDM cells pretreated with LPS were treated with ZnPT, and AIM2 inflammasomes were activated by transfection of Poly(A:T). The inhibitory effect of ZnPT on Poly(A:T)-induced IL-1β and caspase-1 secretion was detected by ELISA and Western Blot, respectively.

[0060] like Figure 4 As shown, ZnPT had no effect on AIM2 inflammasome activation.

[0061] Example 2 ZnPT does not affect the upstream signaling stage of NLRP3 inflammasome assembly and activation 1. ZnPT does not affect LPS-mediated priming In order to further understand the mechanism of ZnPT's effect on NLRP3 inflammasome activation, this study first explored whether ZnPT would interfere with the priming stage of NLRP3 inflammasome activation. The activation of NLRP3 inflammasome can be divided into two stages: priming and assembly activation. ZnPT was added before and after LPS stimulation of BMDM cells, and the expression of inflammatory cytokines IL-6 and TNF-α was detected by ELISA, and the expression of key components of NLRP3 inflammasome NLRP3, ASC and Pro-casp1 was detected by Western Blot.

[0062] like Figure 5As shown, ZnPT did not affect the LPS-mediated priming phase.

[0063] 2. ZnPT does not affect mitochondrial damage, reactive oxygen species production, and potassium ion efflux Mitochondrial damage, reactive oxygen species production and potassium ion outflow are upstream signals of NLRP3 inflammasome activation, and play an important role in the activation of NLRP3 inflammasome. After BMDM cells were pretreated with LPS (100 ng / mL) for 3 h, they were treated with ZnPT (0 μM, 0.6 μM) for 30 min, and then Nigericin (10 μM) was added to treat the cells for 30 min. The fluorescent probes MitoTracker and MitoSox were stained and observed by laser confocal microscopy. Alternatively, cells were treated with different concentrations of ZnPT (0 μM, 0.2 μM, 0.4 μM, 0.6 μM) for 30 min, and then Nigericin (10 μM) was added to treat the cells for 30 min. After the cells were lysed with concentrated nitric acid, the intracellular potassium ion content was detected by plasma atomic chromatography.

[0064] like Figure 6 As shown, ZnPT had no effect on mitochondrial damage, reactive oxygen species production, and potassium ion efflux.

[0065] Example 3 Mechanism of ZnPT Inhibiting NLRP3 Inflammasome Activation 1. ZnPT blocks the interaction between endogenous NEK7 and NLRP3, and between NLRP3 and ASC The interactions between NEK7 and NLRP3, and between NLRP3 and ASC, play a key role in the activation and assembly of the NLRP3 inflammasome. These interactions are not only crucial for the structural composition of the NLRP3 inflammasome, but also play a central role in its functional regulation. Specifically, the direct binding of NEK7 to NLRP3 is a key step in regulating NLRP3 oligomerization, ASC spot formation, and activation of caspase-1 under specific conditions (such as potassium ion efflux).

[0066] BMDM cells were pretreated with LPS (100 ng / mL) for 3 h, and treated with ZnPT for 30 min. Nigericin (10 μM) was then added to activate the cells, and the cell culture supernatant and cell lysate were collected. Western blot was used to detect the protein levels of NLRP3, NEK7, NLRP3, and ASC in cell immunoprecipitation (IP) and cell lysate (Input).

[0067] like Figure 7 As shown, ZnPT inhibits the binding of NEK7 to NLRP3 and NLRP3 to ASC.

[0068] 2. ZnPT binds to NLRP3 protein reversibly To further explore the interaction characteristics between ZnPT and NLRP3 inflammasome, elution experiments were performed to evaluate the reversibility of this interaction.

[0069] After 15 minutes of ZnPT treatment, BMDM cells were rinsed with fresh DMEM medium, once every 5 minutes, for a total of three rinses to wash out ZnPT that was not bound to NLRP3. Subsequently, Nigericin was added to stimulate the cells for 30 minutes, and the cell supernatant culture medium was collected, and the secretion of inflammatory factor IL-1β was detected by ELISA.

[0070] like Figure 8 As shown, ZnPT can reversibly bind to the NLRP3 inflammasome.

[0071] Example 4 In vivo study of ZnPT inhibiting NLRP3 inflammasome in mice 1. ZnPT alleviates LPS (lipopolysaccharide)-induced septic shock in mice Septic shock is closely related to the abnormal activation of NLRP3 inflammasome.

[0072] Construction of LPS-induced septic shock model in mice: (1) Animal grouping: Eight-week-old male C57BL / 6J mice with similar body weight were selected and randomly divided into three groups: blank control group (WT group, n=3), septic shock model group (LPS group, n=9), and ZnPT-pretreated septic shock model group (ZnPT+LPS group, n=9).

[0073] (2) Model establishment and treatment: The blank control group was injected with an equal volume of PBS. The ZnPT+LPS group was first injected with ZnPT (10 mg / kg, 20 mg / kg), and 1 hour later, LPS (20 mg / kg) was injected together with the LPS group to establish a septic shock model. 4 hours after model establishment, all mice were sampled and killed by cervical dislocation. Alternatively, a survival curve of mice within 72 hours after model establishment was drawn.

[0074] (3) Sample collection and processing: The eyeballs of mice were removed to obtain eyeball blood, and then the mice were killed by cervical dislocation. The blood samples were left at room temperature for 3-4 hours, and then centrifuged at 8500 rpm for 5 minutes at 4°C. The upper layer of serum was transferred to a new EP tube, taking care to avoid touching the cell layer.

[0075] (4) Collect the mouse peritoneal lavage fluid and centrifuge it, then transfer the supernatant to a new EP tube.

[0076] The mouse intraperitoneal injection of LPS model was used to simulate human septic shock, which can cause a significant increase in the levels of proinflammatory cytokines (such as IL-1β, TNF-α and IL-6) in mice.

[0077] (5) ELISA kits were used to detect the levels of inflammatory factors in the serum and peritoneal lavage fluid of mice.

[0078] like Fig. 9 As shown, ZnPT alleviated LPS-induced septic shock in mice.

[0079] 2. ZnPT alleviates DSS (dextran sulfate sodium)-induced colitis in mice Colitis is another inflammatory disease closely related to the overactivation of NLRP3 inflammasome. Construction of DSS (dextran sulfate sodium)-induced colitis model: (1) Animals and groups: Ten-week-old male C57BL / 6J mice with similar body weight were randomly divided into the following three groups, and each mouse was numbered: blank control group (Control group, n=3), DSS-induced colitis model group (DSS group, n=6), and DSS-induced colitis model group after ZnPT treatment (ZnPT + DSS group, n=6).

[0080] (2) Model establishment and treatment: The blank control group was continuously provided with normal drinking water. The DSS-induced group was provided with 3% DSS drinking water for 6 consecutive days. On the 7th day, it was replaced with normal high-pressure tap water. During this period, new 3% DSS drinking water was replaced every two days to ensure drug efficacy. The ZnPT-treated group was intraperitoneally injected with ZnPT (20 mg / kg) for 10 consecutive days, and 3% DSS drinking water was provided for the first 6 days. It was replaced with normal high-pressure tap water on the 7th day. The replacement of drug drinking water was the same as above.

[0081] (3) Observation and recording: Monitor and record the weight of mice at a fixed time every day. Continuously observe the disease activity of mice, and pay attention to the following indicators: loose stools, bloody stools, diarrhea, erect hair, rectal prolapse, etc.

[0082] (4) Experimental results and sample collection: On the 11th day of the experiment, the mice were killed. The spleen of the mice was weighed, the mesenteric lymph nodes and spleen lymphocytes of the mice were separated, and the changes in T cell subsets were analyzed by flow cytometry. The length of the mouse colon was measured and photographed. Part of the colon was selected for HE staining. The colon tissue samples were stored at low temperature, ground, and the colon tissue proteins were extracted. The supernatant cytokine IL-1β was detected by ELISA and the NLRP3 inflammasome-related component proteins were detected by Western blot.

[0083] like Fig.10As shown, ZnPT alleviated DSS-induced weight loss, disease activity index, IL-1β secretion in spleen and colon tissues, and lesion severity. Fig.11 As shown, ZnPT reduces the proportion of Th17 cells and Tc1 cells.

[0084] 3. ZnPT alleviates Imiquimod (IMQ) cream-induced psoriasis in mice Abnormally activated immune response is the key to the development of psoriasis, and the formation of NLRP3 inflammasome contributes to the inflammatory response of psoriasis. This study explored whether ZnPT could alleviate IMQ-induced psoriasis by inhibiting the activation of NLRP3 inflammasome. Construction of IMQ cream-induced psoriasis model in mice: (1) Experimental animals and grouping: Male C57BL / 6J mice of 10 weeks of age and similar body weight were selected and randomly divided into three groups: a blank group (Control), a positive control group (IMQ), and a drug treatment group (IMQ+ZnPT).

[0085] (2) Model establishment and treatment: 62.5 mg of ImQ cream was applied topically to the 2 cm × 3 cm skin area on the back of mice in the positive control group and the drug treatment group once a day for 7 days. At the same time, the blank control group mice were applied with vaseline at the same frequency on the same skin area. The drug treatment group mice were treated with 15 mg / kg of IMQ cream 4 hours after each application of IMQ cream.

[0086] (3) Recording and evaluation of dermatitis morphology: The morphology and histological characteristics of psoriasis dermatitis in mice were photographed and recorded daily. The severity of skin inflammation was assessed daily using the Psoriasis Area and Severity Index (PASI). Specifically, it included observations of skin erythema, scaling, and thickening, and was scored using a five-point scale from 0 to 4: 0 (none), 1 (mild), 2 (moderate), 3 (significant), and 4 (very significant).

[0087] (4) Sample collection and storage: 24 h after the last ZnPT application, the eyeballs of the mice were removed to obtain eye blood, and then the mice were killed by cervical dislocation. The skin tissue on the back of the mice was removed. The excised skin samples were cleaned and wiped with normal saline, then quickly frozen in liquid nitrogen and stored at -80°C for subsequent experiments. In addition, some skin specimens were fixed with 10% neutral formalin, embedded in paraffin, and then cut into 5-micron thick sections for pathological observation using H&E staining. Immunohistochemistry was used to detect proliferating cell nuclear antigen (PCNA) in the mouse lesion skin. The skin samples were ground, skin proteins were extracted, and then ELISA was used to detect the cytokine IL-1β and Western blot was used to detect NLRP3 inflammasome-related component proteins.

[0088] like Fig.12As shown in the figure, ZnPT alleviated the morphology of psoriatic dermatitis and PASI index in mice. Fig.13 As shown in Figure 2, ZnPT inhibits the histological features of psoriatic dermatitis in mice and the proliferation and differentiation of epidermal cells. Fig.14 As shown, ZnPT alleviated the expression of NLRP3 inflammasome-related components in psoriasis skin tissue of mice.

Claims

1. Application of zinc pyrithione in the preparation of NLRP3 inflammasome inhibitors.

2. The application according to claim 1, characterized in that: The inhibitor inhibits the activation or assembly of the NLRP3 inflammasome.

3. The application according to claim 2, characterized in that: The NLRP3 inflammasome is induced by an agonist; specifically, the agonist is nigericin, intracellular lipopolysaccharide (cLPS), adenosine triphosphate (ATP) and / or monosodium urate crystals (MSU).

4. Use of zinc pyrithione in the preparation of a cytokine inhibitor that depends on NLRP3 inflammasome, characterized in that: The cytokine is IL-1β.

5. Application of zinc pyrithione in the preparation of drugs for the treatment of NLRP3 inflammasome-related diseases.

6. The use according to claim 5, characterized in that: The NLRP3 inflammasome-related disease is at least one of colitis, psoriasis, septic shock, type II diabetes, metabolic syndrome, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, Behcet's disease, sepsis, gout, arthritis, multiple sclerosis, obesity, viral inflammation such as viral hepatitis and pneumonia, acute and chronic tissue damage caused by infection, atherosclerosis, myocardial infarction, UV-induced skin sunburn, contact hypersensitivity, familial cold autoinflammatory syndrome, chronic infantile neurocutaneous joint syndrome, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, depression, asbestosis, silicosis and silicosis, asthma or acute respiratory distress syndrome, peritonitis, Muller-Weigler syndrome and cryoporphyrin-associated periodic syndrome (CAPS) or cytokine storm caused by tumor treatment (such as CART therapy and immune checkpoint inhibitor therapy).

7. A drug for inhibiting NLRP3 inflammasome, characterized in that: Its main ingredient is zinc pyrithione.

8. A drug for treating NLRP3 inflammasome-related diseases, characterized in that: Its main ingredient is zinc pyrithione.

9. The drug according to claim 8, characterized in that: The NLRP3 inflammasome-related disease is at least one of colitis, psoriasis, septic shock, type II diabetes, metabolic syndrome, non-alcoholic fatty liver disease, alcoholic liver disease, kidney disease, Behcet's disease, sepsis, gout, arthritis, multiple sclerosis, obesity, viral inflammation such as viral hepatitis and pneumonia, acute and chronic tissue damage caused by infection, atherosclerosis, myocardial infarction, UV-induced skin sunburn, contact hypersensitivity, familial cold autoinflammatory syndrome, chronic infantile neurocutaneous joint syndrome, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, depression, asbestosis, silicosis and silicosis, asthma or acute respiratory distress syndrome, peritonitis, Muller-Weigler syndrome and cryoporphyrin-associated periodic syndrome (CAPS) or cytokine storm caused by tumor treatment (such as CART therapy and immune checkpoint inhibitor therapy).

10. The drug according to any one of claims 7 to 8, characterized in that: Pharmaceutically acceptable auxiliary ingredients are also included.

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