Application of miR159 and composition thereof in preparation of medicine for preventing or treating NLRP3 inflammasome related diseases

Abstract

The invention discloses miR159, a miR159 simulant or a miR159 accelerant and application of a composition of the miR159, the miR159 simulant or the miR159 accelerant to preparation of a medicine for preventing or treating NLRP3 inflammasome related diseases. The nucleotide sequence of the miR159 is shown as SEQ ID NO. 1. It is found for the first time that miR159 can inhibit activation of NLRP3 inflammasome by inhibiting interaction of NEK7 and NLRP3, so that NLRP3 inflammasome related diseases are treated or prevented, and new medicine application and a composition form of miR159 are provided.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of miR159 and a composition thereof in the preparation of drugs for preventing or treating NLRP3 inflammasome-related diseases. Background Art

[0002] The NLRP3 (nucleotide-binding oligomerization domain-like receptor 3) inflammasome is a multiprotein complex involved in the host immune response, consisting of the sensor molecule NLRP3, the adaptor protein ASC (Apoptosis-associated speck-like protein containing a caspase recruitment domain), and the effector protein pro caspase-1. As the most characterized pattern recognition receptor, NLRP3 can detect microbial invasion, endogenous danger, and stress signals. When stimulated, NLRP3, ASC, and pro caspase-1 proteins assemble to form the NLRP3 inflammasome, inducing pro caspase-1 to self-cleave to generate caspase-1p10 and p20 complexes, which in turn cleave pro IL-1β and pro IL-18 to form IL-1β and IL-18. Finally, Gasdermin D is cleaved and inserted into the cell membrane, forming pores and inducing cell pyroptosis, releasing mature inflammatory factors IL-1β, IL-18, and lactate dehydrogenase to the extracellular space to promote immune and inflammatory responses. However, abnormal activation of NLRP3 inflammasomes may lead to amplified inflammatory effects, thereby inducing or aggravating a series of major diseases, such as Muckle-Wells syndrome, infantile neurocutaneous arthritis, stroke, bacterial infection, ischemic brain injury, inflammatory bowel disease, Huntington's disease, gout, atherosclerosis, Alzheimer's disease, multiple sclerosis, Parkinson's disease, hypertension, heart failure, familial Mediterranean fever, autoimmune encephalomyelitis, rheumatoid arthritis, non-alcoholic fatty liver disease and / or cirrhosis (Ying Chen et al., Cell Mol Biol Lett. 2023, 28(1):51). Therefore, inhibiting abnormal activation of NLRP3 inflammasomes is an effective way to treat the above diseases.

[0003] However, currently marketed monoclonal antibodies (anakinra, canakinumab, and rilonacept) cannot address all consequences of NLRP3 inflammasome activation, such as IL-18 release; small molecule drugs are still in the early stages of exploration (MCC950, DFV-890 / IFM-2427, ZYIL1, OLT-1177 / Dapansutrile, VTX-2735, Somalix, Selnoflasst / RG-6418 / IZD-334 / RO-7486967, Emlenoflast / IZD-174, NT-0249, NT-0796, VTX3232, VENT-01, VENT-02) (Na Li et al., J Med Chem. 2023, 66(21): 14447-14473). Although MCC950 had previously undergone a phase II clinical trial for rheumatoid arthritis, liver toxicity led to the suspension of its program. Therefore, it is still necessary to seek safer and more effective NLRP3 inflammasome inhibitors.

[0004] miRNA is a class of endogenous non-coding single-stranded small RNAs with a size of about 18-25 base pairs and widely present in eukaryotes. They can regulate gene expression at the post-transcriptional level by binding to the 3' non-coding region of the target gene, thereby treating diseases. For example, miR 159 can inhibit breast cancer by targeting TCF7 encoding the Wnt signal transduction transcription factor (Andrew R Chin et al., Cell Res. 2016, 26 (2): 217-228). However, no research has been found to show that miR 159 can prevent or treat NLRP3 inflammasome-related diseases. Summary of the invention

[0005] Purpose of the invention: In order to solve the problems existing in the prior art, the present invention provides the use of miR159 and its composition in the preparation of drugs for preventing or treating NLRP3 inflammasome-related diseases. The downstream of NLRP3 inflammasome involves multiple inflammatory factors such as IL-1β, IL-18, LDH, Gasdermin D, etc. The present invention first discovered that miR159 can inhibit the activation of NLRP3 inflammasome by inhibiting the interaction between NEK 7 and NLRP 3, and can effectively inhibit the production and release of multiple inflammatory factors to cope with the inflammatory storm in various major diseases, such as inflammatory bowel disease and bacterial infection, and has good prospects for clinical transformation.

[0006] Technical solution: To achieve the above purpose, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the use of miR159, a miR159 mimic or a miR159 promoter in the preparation of a drug for preventing or treating NLRP3 inflammasome-related diseases, wherein the nucleotide sequence of miR159 is shown in SEQ ID NO.1.

[0008] SEQ ID NO. 1: 5'-UUUGGAUUGAAGGGAGCUCUA-3'.

[0009] As an optional embodiment, the miR159 is derived from plants or is chemically or biologically synthesized.

[0010] As an optional embodiment, any one or more ribonucleotides of miR159 are modified;

[0011] As an optional embodiment, the miR159 is a mature miRNA, a precursor miRNA or a primary transcript. Preferably, the miR159 is a mature miRNA.

[0012] As an optional embodiment, the miR159 mimic contains the sequence shown in SEQ ID NO.1 and / or its complementary sequence; preferably, the complementary sequence is shown in SEQ ID NO.2.

[0013] SEQ ID NO. 2: 5'-GAGCUCCCUUCAAUCCAAAUU-3'.

[0014] miRNA mimics are miRNAs that mimic endogenous miRNAs in organisms. They are synthesized by chemical synthesis and can enhance the function of endogenous miRNAs. Specifically, the miR159 mimic is a small double-stranded miRNA designed and synthesized for the mature form of miR159. It has the same function as the natural mature miR159 in plants and can inhibit the interaction between NEK7 and NLRP3, thereby inhibiting the abnormal activation of NLRP3 inflammasomes.

[0015] As an optional embodiment, the miR159 promoter is a substance or gene tool that increases the expression level or activity of miR159.

[0016] The substance or gene tool that increases the expression or activity of miR159 can be a substance or gene tool that further increases the expression or activity of miRNA-1293 when miR159 is normally expressed; it can also be a substance or gene tool that relieves the inhibition of miR159 when miR159 is inhibited; it can also be a substance or gene tool that repairs the normal expression of miR159 when the genetic material mutates and causes miR159 to be unable to express normally. In short, those substances or gene tools that can increase the expression of miR159 or improve the activity of miR159, whether by direct action or indirect action, can be called miR159 promoters in the present invention.

[0017] Preferably, the miR159 promoter is a vector containing a miR159 nucleic acid fragment;

[0018] More preferably, the vector containing the miR159 nucleic acid fragment is a plasmid containing the miR159 nucleic acid fragment.

[0019] As an optional embodiment, the NLRP3 inflammasome-related disease is Muckle-Wells syndrome, infantile neurocutaneous articular syndrome, stroke, bacterial infection, ischemic brain injury, inflammatory bowel disease, Huntington's disease, gout, atherosclerosis, Alzheimer's disease, multiple sclerosis, Parkinson's disease, hypertension, heart failure, familial Mediterranean fever, autoimmune encephalomyelitis, rheumatoid arthritis, non-alcoholic fatty liver disease and / or cirrhosis, etc.

[0020] In a second aspect, the present invention provides a composition for preventing or treating NLRP3 inflammasome-related diseases, the composition comprising miR159, miR159 mimics and / or miR159 promoter, the nucleotide sequence of miR159 being as shown in SEQ ID NO. 1. The miR159, miR159 mimics and miR159 promoter are as described above.

[0021] As an optional embodiment, the composition consists of miR159, miR159 mimics and / or miR159 promoter and pharmaceutically acceptable excipients; or the composition is a plant extract containing miR159.

[0022] As a specific embodiment, the plant extract is mainly obtained by squeezing the plant, collecting the juice, and then centrifuging and purifying it to obtain plant vesicle-like nanoparticles.

[0023] As a specific embodiment, the plant extract is selected from vesicle-like nanoparticles of plants such as black plum and dandelion.

[0024] More specifically, the composition can be in the form of freeze-dried powder, injection, tablet, capsule or patch. The delivery methods of the composition include: direct naked RNA injection, liposome-encapsulated RNA direct injection, bacteria carrying plasmid expression RNA or virus packaging expression RNA, nanomaterial assembly method, micron material assembly method.

[0025] In a third aspect, the present invention provides a use of the composition in the preparation of a drug for preventing or treating NLRP3 inflammasome-related diseases. The NLRP3 inflammasome-related diseases are as described above.

[0026] Beneficial effects: Compared with the prior art, the present invention discovers for the first time that miR159 can inhibit the activation of NLRP3 inflammasome by inhibiting the interaction between NEK7 and NLRP3, thereby treating or preventing NLRP3 inflammasome-related diseases, and provides new drug uses and composition forms of miR159. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Preparation and characterization of PM-EVLPs. (A) Preparation process of PM-EVLPs. (B) Collection of sucrose gradient band 2 (left) for transmission electron microscopy observation of PM-EVLPs morphology (right). (CD) Surface charge (C) and particle size distribution (D) of PM-EVLPs. (EG) RNAomics (E), proteomics (F) and lipidomics (G) results of PM-EVLPs.

[0028] Figure 2 Figure 3 The therapeutic effect of PM-EVLPs on ulcerative colitis associated with NLRP3 inflammasome activation. (A) Body weight change. (B) DAI score. (C) Quantification of MPO activity in the colon. (D) Colon length. (E) Histopathological damage of the colon. (F) Neutrophil infiltration in the colonic lamina propria. (GH) Cleaved caspase-1 (G) and IL-1β and IL-18 content (H) in colonic macrophages. Data are expressed as mean ± SEM, 6 mice per group. ## P<0.01vs normal group; * P < 0.05 and ** P<0.01vs DSS group.

[0029] Figure 3Effects of PM-EVLPs on NLRP3 inflammasome activation in macrophages. (AB) Protein contents of cleaved caspase-1 and cleaved IL-1β in the supernatant after PM-EVLPs treatment under LPS and ATP stimulation (A) and IL-1β and IL-18 contents (B). (CF) Protein contents of cleaved caspase-1 and cleaved IL-1β in the supernatant after PM-EVLPs treatment under LPS and nigericin (or MSU) stimulation (C, E) and IL-1β and IL-18 contents (D, F). Data are expressed as mean ± SEM, 3 independent in vitro experiments in each group. ## P<0.01vs normal group; * P < 0.05 and ** P<0.01 vs DSS or LPS+

[0030] ATP / nigericin / MUS group.

[0031] Figure 4 Effects of different components of PM-EVLPs on NLRP3 inflammasome activation. (AB) Protein expression of cleaved caspase-1 and cleaved IL-1β in cell culture supernatants after treatment with PM-EVLPs with different treatments (A) and levels of IL-1β and IL-18 (B). (C) The six most abundant miRNAs in PM-EVLPs. (D) Secretion of IL-1β and IL-18 in culture supernatants after treatment with different miRNA mimics. (EF) Expression of cleaved caspase-1 and cleaved IL-1β in supernatants after treatment with miR159 mimics (E) and levels of IL-1β and IL-18 (F). (GH) Phosphorylation and nuclear translocation of p65 in cells after treatment with miR159 mimics. (IJ) Effects of miR159 mimic treatment on the formation of NEK7 / NLRP3 and NLRP3 / ASC / Pro-caspase-1 complexes. Data are presented as mean ± SEM from three independent in vitro experiments per group. ## P<0.01vs normal group; ** P<0.01vs LPS+ATP group; && P<0.01vs PM-EVLPs (untreated) group.

[0032] Figure 5 The therapeutic effect of miR159 on DSS-induced ulcerative colitis. (A) Animal experiment process.

[0033] (B) Body weight change. (C) DAI score. (D) Colon length. (E) Histopathological lesions of colon.

[0034] (F) Quantification of MPO activity in the colon. (G) Proportion of neutrophils in the colonic lamina propria. Data are presented as mean ± SEM, 6 mice per group. ## P<0.01vs normal group; * P < 0.05 and ** P<0.01vs DSS group.

[0035] Figure 6 The therapeutic effect of miR159 on TNBS-induced ulcerative colitis. (A) Colon length.

[0036] (B) Colon photograph. (C) Colon MPO activity. (D-E) Colon histopathological damage. (F) Infiltration of neutrophils in the colonic lamina propria. (G) Colon MPO activity. Data are expressed as mean ± SEM, 6 mice per group. ## P<0.01vs normal group; * P < 0.05 and ** P<0.01vs TNBS group.

[0037] Figure 7 The effect of TH-EVLPs on NLRP3 inflammasome activation induced by S. aureus exotoxin. (AB) IL-1β content (A) and LDH activity (B) in cell supernatants.

[0038] Figure 8 The effects of different components of TH-EVLPs on NLRP3 inflammasome activation induced by S. aureus exotoxin. (AB) Effects of TH-EVLPs knockout or non-knockout proteins on IL-1β content (A) and LDH (B) activity in the supernatant of cells stimulated by S. aureus exotoxin. (CD) Effects of liposomes prepared from TH-EVLPs-derived lipids on IL-1β content (C) and LDH (D) activity in the supernatant of cells stimulated by S. aureus exotoxin. (EF) Effects of TH-EVLPs-derived RNA on IL-1β content (E) and LDH (F) activity in the supernatant of cells stimulated by S. aureus exotoxin.

[0039] Fig. 9 To explore the main active miRNAs that inhibit NLRP3 inflammasome in total RNA of TH-EVLPs.

[0040] (AB) Effects of the top five abundant miRNAs on the IL-1β content (A) and LDH activity (B) in the supernatant of cells stimulated by S. aureus exotoxin. (CD) Effects of miR159 on the IL-1β content (C) and LDH activity (D) in the supernatant of cells stimulated by S. aureus exotoxin.

[0041] Fig.10 Preparation and characterization of miR159 fluorescently labeled liposomes. (AC) Particle size and concentration of miR159 fluorescently labeled liposomes prepared with 20 mg (A), 40 mg (B), and 80 mg (C) TH-EVLPs-derived lipids.

[0042] Fig.11 Evaluation of the cellular uptake efficiency of miR159 liposomes.

[0043] Fig.12 The therapeutic effect of miR159 on Staphylococcus aureus wound infection. (A) Representative images of mouse wounds at 0, 3, 6, 9, 12, and 15 days. (B) The healing rates of mouse wounds at 3, 6, 9, 12, and 15 days.

[0044] Fig.13 The therapeutic effect of miR159 on systemic infection with Staphylococcus aureus. (AB) Bacterial load in mouse lungs (A) and representative images (B). (CD) IL-1β (C), TNF-α (D), and IL-6 (E) levels in mouse peritoneal fluid. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The experimental methods used in the examples are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available unless otherwise specified.

[0046] Example 1: Preparation and characterization of ebony vesicle-like nanoparticles (PM-EVLPs)

[0047] After squeezing the black plum juice, the juice was first centrifuged in the following order to remove large particles: 200g centrifugation for 10min, 2000g centrifugation for 20min and 10000g centrifugation for 30min. The supernatant was carefully collected, ultracentrifuged at 100000g for 60min, and the precipitate was resuspended in sterile PBS solution. To purify PM-EVLPs, the above mixture was transferred to a gradient sucrose solution and centrifuged at 150000g for 60min. Finally, the fraction containing PM-EVLPs was diluted in sterile PBS and filtered with a 0.45μm membrane to obtain PM-EVLPs. Subsequently, PM-EVLPs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis, Malvern particle size analyzer, proteomics, lipidomics and RNA sequencing.

[0048] The results showed that the PM-EVLPs obtained were mainly concentrated at the 30 / 45% interface of the sucrose gradient ( Figure 1 B left, band 2), showing a unique micro-cup-shaped morphology ( Figure 1 B right), potential about -27mV ( Figure 1 C), particle size about 119nm ( Figure 1 D). RNA deep sequencing showed that the RNA content in PM-EVLPs was mainly composed of miRNA ( Figure 1 E), among which miR159 ranked third in abundance. Proteomic analysis showed that the proteins in PM-EVLPs were mainly involved in single-organism cellular processes (53%), purine ribonucleoside metabolism (14%), and general cellular processes (12%) ( Figure 1 F). PM-EVLP lipidomic analysis showed that the main lipids were diacylglycerol (20%), lysophosphatidylcholine (15%-20%) and N-arachidonoylglycine (15%) ( Figure 1 G).

[0049] Example 2: Therapeutic effect of PM-EVLPs on ulcerative colitis associated with NLRP3 inflammasome activation

[0050] In order to study the therapeutic effect of PM-EVLPs on ulcerative colitis, mice were randomly divided into 5 groups (6 mice / group): normal group, DSS group, PM-EVLPs (0.5×10 10 / mL, 1.5×10 10 / mL) group and 5-ASA (200mg / kg) group. Physical activity, body weight, stool consistency and the presence of gross blood in the stool were monitored daily, and the disease activity index (DAI) score was calculated. At the end of the experiment, the mice were killed and the colon was collected. The distal colon was fixed in 10% formalin at room temperature for 24h or longer, embedded in paraffin, and sliced ​​at a thickness of 8μm, stained with hematoxylin-eosin (H&E), and analyzed for histopathological damage.

[0051] The results showed that PM-EVLPs significantly alleviated DSS-induced weight loss in mice ( Figure 2 A) and DAI score increased ( Figure 2 B). In addition, PM-EVLPs also improved colon shortening and increased myeloperoxidase (MPO) activity in the colon ( Figure 2 C, D) and improved the colonic epithelial damage, mucosal and submucosal edema, extensive neutrophil and macrophage infiltration, and crypt loss caused by DSS ( Figure 2 E). Finally, neutrophils (CD11b + / Ly6G + ) in the colonic lamina propria was significantly reduced after PM-EVLPs treatment ( Figure 2 F). These results indicate that PM-EVLPs can significantly improve DSS-induced colonic inflammation in mice. Abnormal activation of NLRP3 inflammasome in macrophages is closely related to the onset and progression of colitis. The results showed that PM-EVLPs significantly inhibited the expression of cleaved caspase-1 and the production of IL-1β and IL-18 in colonic macrophages of mice with DSS-induced colitis ( Figure 2 G, H). Therefore, PM-EVLPs can treat ulcerative colitis by inhibiting the activation of NLRP3 inflammasome.

[0052] Example 3: Effect of PM-EVLPs on NLRP3 inflammasome activation in macrophages

[0053] BMDM and PMA-stimulated THP-1 cells were seeded in 6-well plates and incubated with 100 ng / mL LPS for 3 h, followed by treatment with PM-EVLPs for 1 h, and finally incubated with 5 mM ATP for 45 min, 4 μM nigericin for 3 h, or 150 μg / mL sodium urate crystals (MSU) for 6 h. Growth medium and cells were harvested to evaluate the expression of cleaved IL-1β and caspase-1, respectively.

[0054] The results show that if Figure 3 As shown in A, PM-EVLP (1×10 7 , 3×10 7 / mL) significantly inhibited the protein expression of cleaved caspase-1 and cleaved IL-1β in cells induced by ATP, and inhibited the secretion of IL-1β and IL-18 ( Figure 3 B). Similarly, PM-EVLPs (1×10 7 , 3×10 7 / mL) treatment also inhibited the protein expression of cleaved caspase-1 and cleaved IL-1β and the secretion of IL-1β and IL-18 stimulated by nigericin or MSU in BMDM and PMA-stimulated THP-1 cells ( Figure 3 CF), indicating that PM-EVLPs act as a broad-spectrum inhibitor of NLRP3 inflammasome.

[0055] Example 4: Effects of different components of PM-EVLPs on NLRP3 inflammasome activation

[0056] PM-EVLPs were untreated or digested with proteases at 37°C for 24 h to digest proteins, or DNase I / RNase A to remove most RNA, or chloroform / methanol solution to extract lipids. BMDM and PMA-stimulated THP-1 cells were cultured with LPS (100 ng / mL) for 3 h, then treated with PM-EVLPs that had undergone the above treatment for 1 h, followed by the addition of ATP (5 mM) for 45 min. Growth medium and cells were harvested to evaluate the expression of cleaved caspase-1, cleaved IL-1β, and the secretion of IL-1β and IL-18 in the supernatant, respectively.

[0057] The results showed that PM-EVLPs treated with proteinase K still strongly inhibited the protein expression of cleaved caspase-1 and cleaved IL-1β and the secretion of IL-1β and IL-18 in the supernatant, suggesting that the proteins in PM-EVLPs are not necessary for inhibiting NLRP3 inflammasome activation ( Figure 4 A, B). Similarly, the extracted lipids did not attenuate the activation of the NLRP3 inflammasome. In contrast, depletion of RNA in PM-EVLPs using DNase I / RNase I abolished the inhibitory effect of PM-EVLPs, as evidenced by the intracellular expression of cleaved caspase-1 and cleaved IL-1β and the extracellular secretion of IL-1β and IL-18 ( Figure 4 A, B). Therefore, RNA is the key active substance of PM-EVLPs in inhibiting NLRP3 inflammasome.

[0058] Example 5: Exploration of the main active miRNAs that inhibit NLRP3 inflammasome in total RNA of PM-EVLPs

[0059] In order to identify the key miRNAs responsible for inhibiting NLRP 3 inflammasome activation in PM-EVLPs, miRNAs were extracted and deeply sequenced, and the top six miRNAs in abundance were synthesized for activity exploration. Specifically, after LPS (100 ng / mL) acted on BMDM cells for 3 h, the synthesized miR159, miR482, miR6300, miR1222, miR396 and miR166 mimics were added for 1 h, and then ATP (5 mM) was added for 45 min, and then the secretion of IL-1β and IL-18 in the culture supernatant was detected by ELISA.

[0060] The results showed that after quality control and read filtering, 94 unique miRNAs were identified from the total RNA of PM-EVLPs, among which miR 482, miR 6300, miR 159, miR 1222, miR 396, and miR 166 were the most abundant ( Figure 4 C). Interestingly, only miR159 mimic (40 nmol) could effectively inhibit the secretion of IL-1β and IL-18 in LPS+ATP-treated BMDM ( Figure 4 D). Further dose-response experiments confirmed that miR159 mimic (20, 40, 80 nmol) inhibited the autocleavage of caspase-1 and the production of IL-1β and IL-18 in a dose-dependent manner, similar to the effects of intact PM-EVLPs ( Figure 4 E, F). In addition, miR159 mimic showed no significant inhibitory activity on the phosphorylation modification or nuclear translocation of p65 in LPS-stimulated BMDM ( Figure 4 G, H). However, NEK7-NLRP3 interaction and NLRP3 inflammasome assembly were significantly inhibited by miR159 mimic at the indicated doses ( Figure 4 I, J). Therefore, miR159 is the main active component of PM-EVLPs in inhibiting NLRP 3 inflammasome.

[0061] Example 6: Therapeutic effect of miR159 on ulcerative colitis

[0062] C57 BL / 6 and BALB / c mice were used to induce acute colitis models with DSS and TNBS, respectively. MiR159 agomir (400 pmol and 800 pmol) and 5-ASA (200 mg / kg) were orally administered for 10 consecutive days, and body weight and DAI scores were recorded. Piglets were killed after 10 days, and colon length, MPO activity, and tissue damage were calculated.

[0063] The results showed that oral administration of miR159 agomir (400, 800 pmol) for 10 consecutive days significantly alleviated DSS-induced colitis, as shown by less weight loss, lower DAI scores, and less shortening of colon length ( Figure 5 AD). In addition, MPO activity in the colon was also significantly inhibited by the designated dose of miR159 agomir ( Figure 5 F). Finally, miR159 agomir (400, 800 pmol) significantly reduced neutrophil infiltration in the colonic lamina propria and improved intestinal epithelial damage ( Figure 5 E, G). It is noteworthy that miR159 agomir also has a protective effect on TNBS-induced colitis mice ( Figure 6 AG). These results suggest that miR159 is a key component of PM-EVLPs-mediated colitis alleviation, and PM-EVLPs treat ulcerative colitis by inhibiting NLRP3 inflammasome.

[0064] Example 7: Effect of dandelion vesicle-like nanoparticles (TH-EVLPs) on Staphylococcus aureus exotoxin-induced NLRP3 inflammasome activation

[0065] Fresh dandelions were washed 2-3 times with deionized water at room temperature (25°C) to remove dirt from the surface and then squeezed using a juicer. The obtained juice was centrifuged at 700×g for 20 min, 4000×g for 60 min, and 10000×g for 60 min to remove large particles and fibers. The supernatant containing TH-EVLPs was passed through 100 kDa and 3 kDa ultrafiltration membranes in sequence, and TH-EVLPs present in the 3 kDa retentate were collected.

[0066] The groups were Blank, Staphylococcus aureus exotoxin, 0.5×10 9 TH-EVLPs+Staphylococcus aureus exotoxin, 1.0×10 9 TH-EVLPs+Staphylococcus aureus exotoxin, 2.0×10 9 After incubation of iBMDM cells with TH-EVLPs of different concentrations for 4 h, 5x10 6 CFU / mL Staphylococcus aureus exotoxin was stimulated for 2 h, and then LDH and IL-1β in the cell supernatant were detected.

[0067] The results showed that TH-EVLPs inhibited the release of LDH and IL-1β induced by Staphylococcus aureus exotoxin in a dose-dependent manner (7A, B). LDH and IL-1β are key active factors in the NLRP3 inflammasome pathway, so TH-EVLPs have the effect of inhibiting NLRP3 inflammasome.

[0068] Example 8: Effects of different components of TH-EVLPs on NLRP3 inflammasome activation induced by Staphylococcus aureus exotoxin

[0069] TH-EVLPs were treated as follows: ① heat treatment at 95℃ for 5min to inactivate the proteins; ② lipids in TH-EVLPs were extracted by chloroform / methanol method and prepared into liposomes by thin film dispersion method; ③ RNA in TH-EVLPs was extracted by Trizol method. 9 After incubation with iBMDM cells for 4 h, 5x10 6 CFU / mL Staphylococcus aureus exotoxin was stimulated for 2 h, and then LDH and IL-1β in the cell supernatant were detected. For lipids, TH-EVLPs-derived lipids (0.5, 1, 2×10 9 After incubation with iBMDM cells for 4 h, 5x10 6 CFU / mL Staphylococcus aureus exotoxin was stimulated for 2 h, and then LDH and IL-1β in the cell supernatant were detected. For RNA, TH-EVLPs-derived RNA (100, 200, 400, 800 ng / mL) was transfected into iBMDM cells using a transfection reagent for 1 h, and then 5x10 6 CFU / mL Staphylococcus aureus exotoxin was stimulated for 2 h, and then LDH and IL-1β in the cell supernatant were detected.

[0070] The results showed that there was no significant difference in the levels of LDH and IL-1β in the cell supernatant before and after protein inactivation ( Figure 8 A, B), indicating that protein is not the main active ingredient of TH-EVLPs. Similarly, liposomes prepared with the same concentration of TH-EVLPs-derived lipids did not reduce the levels of LDH and IL-1β in the cell supernatant, that is, lipids were not the main active ingredients ( Figure 8 C, D). However, different concentrations of TH-EVLPs-derived RNA inhibited the release of LDH and IL1β induced by S. aureus exotoxin in a dose-dependent manner ( Figure 8 E, F), indicating that RNA is the main active substance of TH-EVLPs in inhibiting S. aureus exotoxin-induced NLPR3 inflammasome activation.

[0071] Example 9: Exploration of the main active miRNAs that inhibit NLRP3 inflammasome in total RNA of TH-EVLPs

[0072] Studies have shown that the RNA in plant EVLPs is mainly miRNA, so we first performed deep sequencing of miRNA in TH-EVLPs, and after excluding confirmed inactive miRNAs, synthesized the top 5 miRNAs for activity testing. Specifically, 800 nmol / mL of miR166-3p, miR6118-3p, miR159-3p, miR396-5p, and miR166b were transfected into iBMDM cells for 1 h using a transfection reagent, and then 5×10 6 CFU / mL Staphylococcus aureus exotoxin was stimulated for 2 hours, and finally LDH and IL1β in the cell supernatant were detected.

[0073] The results showed that 136 miRNAs were identified in TH-EVLPs, among which miR166-3p, miR2916, miR6118-3p, miR159-3p, miR396-5p, and miR166b were highly abundant. However, previous efficacy tests showed that miR2916 had no significant inhibitory effect on NLRP3 inflammasome, so we synthesized the remaining 5 miRNAs. Efficacy experiments showed that miR159-3p had a significant inhibitory effect on the release of LDH and IL-1β induced by Staphylococcus aureus exotoxin and was comparable to TH-EVLPs, while the remaining miRNAs did not show a significant inhibitory effect ( Fig. 9 A, B). In addition, further studies have shown that miR159 can inhibit the release of LDH and IL-1β in a dose-dependent manner ( Fig. 9 C, D). Therefore, miR159 is the main active substance of TH-EVLPs to inhibit NLRP3 inflammasome activation.

[0074] Example 10: Preparation and characterization of miR159 fluorescently labeled liposomes

[0075] After extracting TH-EVLPs lipids by chloroform-methanol method, 1 mL of 20 mg / mL, 40 mg / mL, and 80 mg / mL lipid chloroform solutions were placed in round-bottom flasks, spun to dry to form a thin film, and dried with nitrogen for 10-15 min. Then, 1 mL of 2 nmol / mL Cy5 fluorescently labeled miR159 (cy5-miR159) solution was added to the round-bottom flask, sonicated in a light-proof water bath for 10 min, filtered through a 0.45 μm filter membrane, and the filtrate was taken. The filtrate was passed through a 100 kDa ultrafiltration tube to remove free Cy5-miR159, and the retentate was Cy5-miR159 liposome (Cy5-miR159-Liposome). Subsequently, the encapsulation efficiency was calculated using a fluorescence spectrophotometer, and the particle size and concentration of Cy5-miR159-Liposome liposomes were measured using NTA.

[0076] The results showed that the encapsulation efficiency of the three Cy5-miR159-Liposomes was above 95%, and the particle sizes were 113.9±0.4nm, 111.0±1.9nm, and 128.5±1.4nm, respectively. Fig.10 AC), the concentrations are 3.11×10 11 particles / mL, 2.30×10 12 particles / mL、3.91×10 12 particles / mL, indicating that Cy5-miR159-Liposome was successfully prepared.

[0077] Example 11: Evaluation of cellular uptake efficiency of miR159 liposomes

[0078] 1×10 5 The iBMDM cells were seeded in a 12-well plate at a density of 100 cells / well. After culturing at 37°C for 12 hours, 1 mL of pure DMEM medium was replaced. The same volume of PBS, Cy5-miRNA and Cy5-miRNA Liposome (50 nM) prepared from TH-EVLPs-derived lipids of different masses (20, 40, 80 mg) were added. After transfection at 37°C for 1 hour, the medium was removed, 2 mL of PBS was added to wash the wells 3 times, 0.3 mL of trypsin was added to digest and collect the cells, centrifuged at 1200 rpm for 5 minutes, and PBS was discarded. 600 μL of pre-cooled PBS was added to resuspend the cells, filtered through a 300-mesh nylon membrane, and detected by flow cytometry. (n=3)

[0079] As shown in the figure, free miR159 has a low cellular uptake efficiency. On the contrary, the encapsulation of TH-EVLPs with different mass sources of lipids can significantly increase the cellular uptake efficiency of miR159, which is beneficial to the efficacy of miR159 ( Fig.11 ). In addition, the promoting effect of liposomes on miR159 cellular uptake increased with the increase of lipid mass. This innovative discovery provides a useful reference for the development of miR159 clinical formulation design.

[0080] Example 12: Therapeutic effect of miR159 on Staphylococcus aureus wound infection

[0081] The mouse model of Staphylococcus aureus exotoxin infection was established by skin incision and instillation of Staphylococcus aureus exotoxin. Before the experiment, the mice were adaptively fed for 1 week, and the backs of the mice were depilated 1 day in advance. After anesthesia with 4% chloral hydrate, two full-thickness skin defect wounds with a diameter of 6 mm were prepared on the backs of the mice with a puncher, and 30 μL of Staphylococcus aureus exotoxin (OD=3.6) was instilled into the wounds. Then the mice were randomly divided into the following 4 groups: control group (n=4): PBS was subcutaneously injected every 3 days, and 30 μL of sterile PBS was instilled into the wounds; model group (n=4): PBS was subcutaneously injected every 3 days, and 30 μL of Staphylococcus aureus exotoxin was instilled into the wounds; NC agomir group (n=4): 5 nmol NC agomir was subcutaneously injected every 3 days, and 30 μL of Staphylococcus aureus exotoxin was instilled into the wounds; miR159 agomir group (n=4): 5 nmol miR159 agomir was subcutaneously injected every 3 days, and 30 μL of Staphylococcus aureus exotoxin was instilled into the wounds. The wounds were photographed with a camera on days 0, 3, 6, 9, 12, and 15, and the wound area was measured using Image J image analysis software. The wound healing rate was calculated according to the formula: Wound healing rate (%) = (initial wound area - current wound area) / initial wound area × 100.

[0082] Staphylococcus aureus infection is one of the important reasons why wounds are difficult to heal. Its exotoxin can produce inflammatory response and tissue damage by activating NLRP3 inflammasome. The results showed that on the 15th day, miR159 agomir could significantly improve the wound ulceration and slow healing caused by Staphylococcus aureus exotoxin, showing great potential for treating Staphylococcus aureus wound infection ( Fig.12 A, B). In contrast, NC agomir showed no therapeutic effect, ruling out the possibility of miRNA stress.

[0083] Example 13: Therapeutic effect of miR159 on systemic infection with Staphylococcus aureus

[0084] The systemic infection model of mice was established by intraperitoneal injection of Staphylococcus aureus. Before the experiment, the mice were adaptively fed for 1 week and randomly divided into the following 5 groups. Control group (n=6): 0.2 mL of sterile PBS was injected intraperitoneally for 3 consecutive days. 30 minutes after the injection on the third day, 0.1 mL / 10 g of sterile PBS was continued to be injected. Model group (n=6): 0.2 mL of sterile PBS was injected intraperitoneally for 3 consecutive days. 30 minutes after the injection on the third day, 0.1 mL / 10 g of the above-prepared bacterial solution was continued to be injected for 12 hours. NC agomir group (n=6): 20 nmol NC agomir was injected intraperitoneally for 3 consecutive days. 30 minutes after the injection on the third day, 0.1 mL / 10 g of the above-prepared bacterial solution was continued to be injected intraperitoneally for 12 hours. miR159 agomir group (n=6): 20 nmol miR159 agomir was intraperitoneally injected for 3 consecutive days. 30 minutes after the injection on the third day, 0.1 mL / 10 g of the above-prepared bacterial solution was continued intraperitoneally for 12 hours. Dexamethasone (DEX) group (n=6): DEX 5 mg / kg was intraperitoneally injected for 3 consecutive days. 30 minutes after the injection on the third day, 0.1 mL / 10 g of the above-prepared bacterial solution was continued intraperitoneally for 12 hours. Subsequently, the mice were killed, and the lungs and peritoneal fluids of the mice were obtained to detect the bacterial load in the lungs and inflammatory factors in the peritoneal fluids of the mice.

[0085] The infection caused by Staphylococcus aureus invading the peritoneal cavity can spread to multiple organs and tissues. It is a common systemic infection model. Its principle is related to the activation of the NLRP3 inflammasome by pathogenic factors, which causes systemic inflammatory response and increased tissue load. The results showed that miR159 agomir can significantly reduce the bacterial load in the lung tissue of mice ( Fig.13 A, B), reducing the levels of IL-6, TNF-α and IL-1β in peritoneal lavage fluid ( Fig.13 CE), showing great potential for treating systemic Staphylococcus aureus infections.

[0086] The above embodiments are the best implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of miR159, miR159 mimics or miR159 promoters in the preparation of drugs for preventing or treating NLRP3 inflammasome-related diseases, wherein the nucleotide sequence of miR159 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Any one or more ribonucleotides of the miR159 are modified; and the miR159 is a mature miRNA, a precursor miRNA or a primary transcript.

3. The use according to claim 1, characterized in that: The miR159 mimic contains the sequence shown in SEQ ID NO.1 and / or its complementary sequence; preferably, the complementary sequence is shown in SEQ ID NO.

2.

4. The use according to claim 1, characterized in that: The miR159 promoter is a substance or gene tool that increases the expression level or activity of miR159.

5. The use according to claim 1, characterized in that: The NLRP3 inflammasome-related diseases are Muckle-Wells syndrome, infantile neurocutaneous articular syndrome, stroke, bacterial infection, ischemic brain injury, inflammatory bowel disease, Huntington's disease, gout, atherosclerosis, Alzheimer's disease, multiple sclerosis, Parkinson's disease, hypertension, heart failure, familial Mediterranean fever, autoimmune encephalomyelitis, rheumatoid arthritis, non-alcoholic fatty liver disease and / or cirrhosis.

6. A composition for preventing or treating NLRP3 inflammasome-related diseases, characterized in that: The composition comprises miR159, miR159 mimics and / or miR159 promoter, and the nucleotide sequence of miR159 is shown in SEQ ID NO.

1.

7. The composition according to claim 6, characterized in that The composition consists of miR159, miR159 mimics and / or miR159 promoter and pharmaceutically acceptable excipients; or the composition is a plant extract containing miR159.

8. The composition according to claim 7, characterized in that The plant extract is mainly prepared by squeezing the plant juice, collecting the juice, and then centrifuging and purifying the juice to obtain the plant vesicle-like nanoparticles.

9. The composition according to claim 7, characterized in that The plant extract is selected from vesicle-like nanoparticles of black plum or dandelion.

10. Use of the composition according to any one of claims 6 to 9 in the preparation of a medicament for preventing or treating NLRP3 inflammasome-related diseases.

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