Application of miR159 and its composition in the preparation of drugs for preventing or treating NLRP3 inflammasome-related diseases

Abstract

The present invention discloses the use of miR159, miR159 mimics, or miR159 promoters, and compositions thereof, in the preparation of medicaments for preventing or treating NLRP3 inflammasome-associated diseases. The nucleotide sequence of miR159 is shown in SEQ ID NO. 1. The present invention, for the first time, discovers that miR159 can inhibit the activation of the NLRP3 inflammasome by inhibiting the interaction between NEK7 and NLRP3, thereby treating or preventing NLRP3 inflammasome-associated diseases. The present invention provides novel pharmaceutical uses of miR159 and compositions thereof.

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 host immune responses. It consists 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 best-characterized pattern recognition receptor, NLRP3 can detect microbial invasion, endogenous danger, and stress signals. Upon stimulation, NLRP3, ASC, and pro-caspase-1 proteins assemble to form the NLRP3 inflammasome, which induces the autocleavage of pro-caspase-1 to form the 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 pyroptosis. This releases the mature inflammatory cytokines IL-1β, IL-18, and lactate dehydrogenase into the extracellular space, promoting immune and inflammatory responses. However, abnormal activation of the NLRP3 inflammasome may lead to the amplification of inflammatory effects, thereby triggering 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 the NLRP3 inflammasome is an effective way to treat the above diseases.

[0003] However, currently marketed monoclonal antibodies (anakinra, canakinumab, and rilonacept) cannot address all the 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] MiRNAs are a class of endogenous, non-coding, single-stranded small RNAs with a size of approximately 18-25 base pairs and widely found 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, which encodes the Wnt signaling transcription factor (Andrew R Chin et al., Cell Res. 2016, 26(2): 217-228). However, no research has yet found that miR 159 can prevent or treat NLRP3 inflammasome-related diseases. Summary of the Invention

[0005] Purpose of the Invention: To address the problems of the prior art, the present invention provides the use of miR159 and compositions thereof in the preparation of drugs for preventing or treating diseases associated with the NLRP3 inflammasome. The downstream pathway of the NLRP3 inflammasome involves multiple inflammatory factors, including IL-1β, IL-18, LDH, and Gasdermin D. This invention, for the first time, discovers that miR159 can inhibit NLRP3 inflammasome activation by inhibiting the interaction between NEK7 and NLRP3. This method can effectively inhibit the production and release of multiple inflammatory factors, thereby addressing the inflammatory storm in various major diseases, such as inflammatory bowel disease and bacterial infections, and has excellent prospects for clinical translation.

[0006] Technical solution: To achieve the above-mentioned 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 (miRNA mimics) are chemically synthesized to mimic endogenous miRNAs in living organisms and can enhance the function of endogenous miRNAs. Specifically, the miR159 mimic is a small double-stranded miRNA designed and synthesized to target the mature form of miR159. It acts similarly to the naturally occurring mature miR159 in plants, inhibiting the interaction between NEK7 and NLRP3, thereby suppressing the abnormal activation of the NLRP3 inflammasome.

[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 genetic tool that increases the expression or activity of miR159 can be a substance or genetic tool that further increases the expression or activity of miRNA-1293 when miR159 is normally expressed; it can also be a substance or genetic tool that relieves the inhibition of miR159 when miR159 is inhibited; it can also be a substance or genetic tool that repairs the normal expression of miR159 when genetic material mutations cause miR159 to be unable to express normally. In short, those substances or genetic tools that can increase the expression of miR159 or enhance the activity of miR159, whether through direct or indirect action, can be referred to as 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, a miR159 mimetic, and / or a miR159 promoter, wherein the nucleotide sequence of miR159 is shown in SEQ ID NO. 1. The miR159, miR159 mimetic, and miR159 promoter are as described above.

[0021] As an optional embodiment, the composition consists of miR159, miR159 mimics and / or miR159 promoters 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 juice, 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 a lyophilized powder injection, injection, tablet, capsule, or patch. The composition can be delivered by direct naked RNA injection, liposome-encapsulated RNA direct injection, bacteria-borne plasmid-expressed RNA or virus-encapsulated RNA expression, nanomaterial assembly, and micromaterial assembly.

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

[0026] Beneficial effects: Compared with the prior art, the present invention discovered 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 providing new drug uses and composition forms of miR159. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Preparation and characterization of PM-EVLPs. (A) PM-EVLP preparation process. (B) Sucrose gradient lane 2 (left) was collected for transmission electron microscopy observation of PM-EVLP morphology (right). (C-D) Surface charge (C) and particle size distribution (D) of PM-EVLPs. (E-G) RNAomics (E), proteomics (F), and lipidomics (G) results of PM-EVLPs.

[0028] Figure 2 Figure 3. The therapeutic effects 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 lesions of the colon. (F) Neutrophil infiltration in the colonic lamina propria. (G, H, and L) Cleaved caspase-1 (G) and IL-1β and IL-18 levels (H) in colonic macrophages. Data are presented as mean ± SEM, 6 mice per group. ## P<0.01 vs normal group; * P < 0.05 and ** P<0.01vs DSS group.

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

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

[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<0.01 vs 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) Colonic MPO activity. (D-E) Colonic histopathological lesions. (F) Neutrophil infiltration in the colonic lamina propria. (G) Colonic MPO activity. Data are presented as mean ± SEM, 6 mice per group. ## P<0.01 vs normal group; * P < 0.05 and ** P<0.01vs TNBS group.

[0037] Figure 7 Effects 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 Figure 3. Effects of different TH-EVLP components on NLRP3 inflammasome activation induced by S. aureus exotoxin. (AB) Effects of TH-EVLP protein knockout or non-knockout on IL-1β content (A) and LDH activity (B) in the supernatant of cells stimulated by S. aureus exotoxin. (CD) Effects of liposomes prepared with TH-EVLP-derived lipids on IL-1β content (C) and LDH activity (D) in the supernatant of cells stimulated by S. aureus exotoxin. (EF) Effects of TH-EVLP-derived RNA on IL-1β content (E) and LDH activity (F) in the supernatant of cells stimulated by S. aureus exotoxin.

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

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

[0041] Figure 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) of TH-EVLPs-derived lipids.

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

[0043] Figure 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) Healing rates of mouse wounds at 3, 6, 9, 12, and 15 days.

[0044] Figure 13 The therapeutic effect of miR159 on systemic Staphylococcus aureus infection. (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 will be further described below with reference to 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 10 minutes, 2000g centrifugation for 20 minutes, and 10,000g centrifugation for 30 minutes. The supernatant was carefully collected, ultracentrifuged at 100,000g for 60 minutes, 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 150,000g for 60 minutes. 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), the potential is 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 cell processes (53%), purine ribonucleoside metabolism (14%) and general cell processes (12%) ( Figure 1 F). PM-EVLP lipidomic analysis showed that the main components 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 (200 mg / 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, mice were sacrificed and the colons were harvested. The distal colon was fixed in 10% formalin at room temperature for 24 hours or longer, embedded in paraffin, and sectioned at 8 μm thickness. Hematoxylin and eosin (H&E) staining was performed for analysis of histopathological lesions.

[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), 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 the NLRP3 inflammasome in macrophages is closely associated with 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 assess 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 in BMDM and PMA-stimulated THP-1 cells stimulated by nigericin or MSU ( Figure 3 CF), indicating that PM-EVLPs act as a broad-spectrum inhibitor of the 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 hours to digest proteins, or with DNase I / RNase A to remove most RNA, or with chloroform / methanol solution to extract lipids. BMDM and PMA-stimulated THP-1 cells were cultured with LPS (100 ng / mL) for 3 hours and then treated with PM-EVLPs that had undergone the above treatments for 1 hour, followed by the addition of ATP (5 mM) for 45 minutes. Growth medium and cells were harvested to assess the expression of cleaved caspase-1 and cleaved IL-1β, as well as 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, as well as 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 Similarly, extracted lipids did not attenuate NLRP3 inflammasome activation. In contrast, depletion of RNA from PM-EVLPs using DNase I / RNase I abolished the inhibitory effect of PM-EVLPs, as evidenced by increased intracellular expression of cleaved caspase-1 and cleaved IL-1β, and 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 PM-EVLPs total RNA

[0059] To identify the key miRNAs responsible for inhibiting NLRP 3 inflammasome activation in PM-EVLPs, miRNAs were extracted and deep sequenced, and the top six most abundant miRNAs were synthesized for activity exploration. Specifically, after LPS (100 ng / mL) was applied to BMDM cells for 3 hours, synthetic miR159, miR482, miR6300, miR1222, miR396, and miR166 mimics were added for 1 hour, and then ATP (5 mM) was added for 45 minutes. The secretion of IL-1β and IL-18 in the culture supernatant was then 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 caspase-1 autolysis and IL-1β and IL-18 production in a dose-dependent manner, similar to the effects of intact PM-EVLPs ( Figure 4 E, F). In addition, miR159 mimics did not show 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 NLRP3 inflammasome.

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

[0062] C57 BL / 6 and BALB / c mice were induced with DSS and TNBS, respectively, and then orally administered with miR159 agomir (400 pmol and 800 pmol) and 5-ASA (200 mg / kg) for 10 consecutive days. Body weight and DAI scores were recorded. Pigs were sacrificed 10 days later, 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 reduced 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). Notably, miR159 agomir also has a protective effect on TNBS-induced colitis in mice ( Figure 6 These results suggest that miR159 is a key component in PM-EVLPs-mediated colitis remission, and PM-EVLPs treat ulcerative colitis by inhibiting the 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 surface dirt and then squeezed using a juicer. The resulting juice was centrifuged at 700 × g for 20 min, 4000 × g for 60 min, and 10,000 × 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, and the 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 incubating iBMDM cells with TH-EVLPs at different concentrations for 4 h, 5x10 6 The cells were stimulated with S. aureus exotoxin 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 5 min to inactivate the proteins; ② lipids in TH-EVLPs were extracted using chloroform / methanol method and prepared into liposomes using thin film dispersion method; ③ RNA in TH-EVLPs was extracted using Trizol method. 9 After incubation with iBMDM cells for 4 h, 5×10 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, liposomes prepared from TH-EVLPs-derived lipids (0.5, 1, 2×10 9 After incubation with iBMDM cells for 4 h, 5×10 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 The cells were stimulated with S. aureus exotoxin 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 component 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, indicating that lipids are not the main active component ( Figure 8 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 TH-EVLPs total RNA

[0072] Studies have shown that the main RNA in plant EVLPs is miRNA. Therefore, we first performed deep miRNA sequencing on TH-EVLPs, and after excluding confirmed inactive miRNAs, synthesized the top 5 abundant 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 hour using a transfection reagent, and then 5×10 6 The cells were stimulated with CFU / mL Staphylococcus aureus exotoxin for 2 h, and LDH and IL1β in the cell supernatant were detected.

[0073] The results showed that TH-EVLPs identified a total of 136 miRNAs, among which miR166-3p, miR2916, miR6118-3p, miR159-3p, miR396-5p, and miR166b were highly abundant. However, previous efficacy studies showed that miR2916 had no significant inhibitory effect on NLRP3 inflammasome, so we synthesized the remaining five 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 significant inhibitory effects ( Figure 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 ( Figure 9 C, D). Therefore, miR159 is the main active substance of TH-EVLPs in inhibiting NLRP3 inflammasome activation.

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

[0075] After extracting TH-EVLPs lipids using the 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, spin-dried to form a thin film, and blown dry with nitrogen for 10-15 minutes. Then, 1 mL of 2 nmol / mL Cy5 fluorescently labeled miR159 (cy5-miR159) solution was added to the round-bottom flask. The mixture was sonicated in a dark water bath for 10 minutes and filtered through a 0.45 μm filter membrane. The filtrate was collected and passed through a 100 kDa ultrafiltration tube to remove free Cy5-miR159. The retentate was obtained as Cy5-miR159 liposomes (Cy5-miR159-Liposome). The encapsulation efficiency was calculated using a fluorescence spectrophotometer, and the particle size and concentration of the 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 ( Figure 10 AC), the concentrations were 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 iBMDM cells were seeded in 12-well plates at a density of 100 cells / well. After incubation at 37°C for 12 hours, the culture medium was replaced with 1 mL of pure DMEM medium. Equal volumes of PBS, Cy5-miRNA, and Cy5-miRNA liposomes (50 nM) prepared from TH-EVLPs-derived lipids (20, 40, and 80 mg) were added. After transfection at 37°C for 1 hour, the culture medium was removed, the wells were washed three times with 2 mL of PBS, and the cells were digested and harvested with 0.3 mL of trypsin. The cells were centrifuged at 1200 rpm for 5 minutes, and the PBS was discarded. The cells were resuspended in 600 μL of pre-chilled PBS, filtered through a 300-mesh nylon membrane, and analyzed 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 lipids of different masses can significantly increase the cellular uptake efficiency of miR159, which is beneficial to the efficacy of miR159 ( Figure 11 Furthermore, the liposome-mediated promotion of miR159 cellular uptake increased with increasing lipid mass. This innovative finding provides a useful reference for the design and development of clinical miR159 formulations.

[0080] Example 12: Therapeutic Effect of miR159 on Staphylococcus aureus Wound Infection

[0081] A mouse model of S. aureus exotoxin infection was established by skin incision and instillation of S. aureus exotoxin. Mice were acclimated for one week before the experiment, and their backs were depilated one day in advance. After anesthesia with 4% chloral hydrate, two 6-mm-diameter full-thickness skin defects were created on the back of the mice using a punch. 30 μL of S. aureus exotoxin (OD = 3.6) was instilled into the wounds. The mice were then randomly divided into four groups: control group (n = 4): PBS was subcutaneously injected every 3 days, followed by 30 μL of sterile PBS; model group (n = 4): PBS was subcutaneously injected every 3 days, followed by 30 μL of S. aureus exotoxin; NC agomir group (n = 4): 5 nmol NC agomir was subcutaneously injected every 3 days, followed by 30 μL of S. aureus exotoxin; and miR159 agomir group (n = 4): 5 nmol miR159 agomir was subcutaneously injected every 3 days, followed by 30 μL of S. aureus exotoxin. The wounds were photographed 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 main reasons for wound healing difficulties. Its exotoxin can activate the NLRP3 inflammasome to produce inflammatory responses and tissue damage. 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 ( Figure 12 A, B). In contrast, NC agomir showed no therapeutic effect, ruling out the possibility of miRNA stress.

[0083] Example 13: Therapeutic Effects of miR159 on Staphylococcus aureus Systemic Infection

[0084] A systemic infection model in mice was established by intraperitoneal injection of Staphylococcus aureus. Prior to the experiment, mice were adaptively housed for one week and randomly divided into the following five groups. Control group (n=6): Intraperitoneal injection of 0.2 mL of sterile PBS was performed for three consecutive days. Thirty minutes after the injection on the third day, 0.1 mL / 10 g of sterile PBS was injected. Model group (n=6): Intraperitoneal injection of 0.2 mL of sterile PBS was performed for three consecutive days. Thirty minutes after the injection on the third day, 0.1 mL / 10 g of the above-prepared bacterial solution was injected for 12 hours. NC agomir group (n=6): Intraperitoneal injection of 20 nmol of NC agomir was performed for three consecutive days. Thirty minutes after the injection on the third day, 0.1 mL / 10 g of the above-prepared bacterial solution was injected for 12 hours. The miR159 agomir group (n=6) received intraperitoneal injections of 20 nmol miR159 agomir for three consecutive days. Thirty minutes after the third day, the mice were injected intraperitoneally with 0.1 mL / 10 g of the above-prepared bacterial solution for 12 hours. The dexamethasone (DEX) group (n=6) received intraperitoneal injections of 5 mg / kg DEX for three consecutive days. Thirty minutes after the third day, the mice were injected intraperitoneally with 0.1 mL / 10 g of the above-prepared bacterial solution for 12 hours. Subsequently, the mice were sacrificed, and their lungs and peritoneal fluids were collected for analysis of lung bacterial loads and inflammatory factors.

[0085] The infection caused by Staphylococcus aureus invading the peritoneal cavity can spread to multiple organs and tissues, which is a common systemic infection model. Its mechanism is related to the activation of the NLRP3 inflammasome by pathogenic factors, resulting in 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 ( Figure 13 A, B), reduce the content of IL-6, TNF-α and IL-1β in peritoneal lavage fluid ( Figure 13 CE), showing great potential for treating Staphylococcus aureus systemic infections.

[0086] The above embodiments are the best implementation methods of the present invention, but the implementation methods 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of miR159 in the preparation of a drug for preventing or treating Staphylococcus aureus infection, wherein the nucleotide sequence of miR159 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The miR159 is a mature miRNA.

3. Use of a composition in the preparation of a medicament for preventing or treating Staphylococcus aureus infection, characterized in that: The composition comprises miR159, and the nucleotide sequence of miR159 is shown as SEQ ID NO.

1.

4. The use according to claim 3, characterized in that The composition consists of miR159 and pharmaceutically acceptable excipients; or the composition is a plant extract containing miR159.

5. The use according to claim 4, 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 plant vesicle-like nanoparticles.

6. The use according to claim 4, characterized in that The plant extract is selected from the vesicle-like nanoparticles of black plum or dandelion.

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