SiRNA targeting cd177 and use thereof in the preparation of a medicament for treating diseases associated with nlrp3 gene mutations

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to siRNA targeting CD177 and application thereof in preparation of a medicine for treating NLRP3 gene mutation related diseases, the siRNA targeting CD177 can significantly reduce peripheral blood CD177 expression and significantly reduce CD177 mRNA expression in skin, liver and spleen; can significantly reduce the number of peripheral blood neutrophils and IL-6 and IL-1 beta positive cell proportion; can significantly reduce IL-6, IL-1 beta and TNF-alpha expression in tissues and serum. The siRNA targeting CD177 can effectively relieve NLRP3 mutation related multi-organ inflammation, and the effect is better than that of traditional anti-inflammatory treatment (such as IL-6 blocking or IL-1 beta neutralizing antibody), and is a very promising treatment strategy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to siRNA targeting CD177 and its application in the preparation of drugs for treating diseases related to NLRP3 gene mutations. Background Technology

[0002] The NLRP3 inflammasome is a key regulator of the innate immune system, and its abnormal activation is closely related to various autoinflammatory diseases (such as CAPS, gout, and atherosclerosis). Studies have shown that gain-of-function mutations in the NLRP3 gene lead to overactivation of the inflammasome, which in turn promotes the release of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α, triggering a systemic inflammatory response. Currently, treatments for NLRP3 mutation-related diseases (such as the IL-1 receptor antagonist Anakinra) have limitations in efficacy, significant side effects, and high costs. Summary of the Invention

[0003] In order to overcome the shortcomings and deficiencies of the existing technology, the purpose of this invention is to provide siRNA targeting CD177 and its application in the preparation of drugs for treating diseases related to NLRP3 gene mutation.

[0004] The objective of this invention is achieved through the following technical solution: siRNA targeting CD177, wherein the sequences of the siRNA targeting CD177 are: si-CD177-F: 5'-GGAUCAUCUCUGAUCUGAATT-3', si-CD177-R: 5'-UUCAGAUCAGAGAUGAUCCTT-3'.

[0005] Another objective of this invention is achieved through the following technical solution: the application of the CD177-targeting siRNA described above in the preparation of drugs for treating diseases related to NLRP3 gene mutations.

[0006] Preferably, the CD177-targeting siRNA can significantly reduce CD177 expression in peripheral blood and significantly reduce CD177 mRNA expression in skin, liver and spleen.

[0007] Preferably, the siRNA targeting CD177 can significantly reduce the number of neutrophils in peripheral blood and the proportion of IL-6 and IL-1β positive cells.

[0008] Preferably, the CD177-targeting siRNA can significantly reduce the expression of IL-6, IL-1β and TNF-α in tissues and serum, and promote significant recovery of damaged structures in the skin, liver and spleen, and reduce inflammatory infiltration.

[0009] Another object of the present invention is achieved by the following technical solution: a drug for treating diseases related to NLRP3 gene mutation, comprising the above-mentioned siRNA targeting CD177 and a pharmacologically acceptable vector.

[0010] Preferably, the CD177-targeting siRNA can significantly reduce CD177 expression in peripheral blood and significantly reduce CD177 mRNA expression in skin, liver and spleen.

[0011] Preferably, the siRNA targeting CD177 can significantly reduce the number of neutrophils in peripheral blood and the proportion of IL-6 and IL-1β positive cells.

[0012] Preferably, the CD177-targeting siRNA can significantly reduce the expression of IL-6, IL-1β and TNF-α in tissues and serum, and promote significant recovery of damaged structures in the skin, liver and spleen, and reduce inflammatory infiltration.

[0013] Another objective of the present invention is achieved by the following technical solution: the application of CD177 in the preparation of a kit for diagnosing diseases related to NLRP3 gene mutations.

[0014] The beneficial effects of this invention are as follows: the CD177-targeting siRNA of this invention can significantly reduce CD177 expression in peripheral blood and significantly reduce CD177 mRNA expression in skin, liver, and spleen; it can significantly reduce the number of neutrophils in peripheral blood and the proportion of IL-6 and IL-1β positive cells; and it can significantly reduce the expression of IL-6, IL-1β, and TNF-α in tissues and serum. This CD177-targeting siRNA can effectively alleviate NLRP3 mutation-related multi-organ inflammation, with effects superior to traditional anti-inflammatory treatments (such as IL-6 blockade or IL-1β neutralizing antibodies), representing a highly promising therapeutic strategy. Attached Figure Description

[0015] Figure 1 This study revealed the abnormal transcriptional regulation of the NLRP3 L573W mutation in human neutrophils. A shows the peripheral blood transcriptome analysis (RNA-seq) of patients with NLRP3 L573W mutation-related autoinflammatory disease (PT) and healthy controls (H1 / H2), with Veen plots displaying differentially expressed genes (DEGs). B shows a heatmap analysis of significantly upregulated inflammatory pathways. CD shows qPCR validation of NLRP3 and CD177 mRNA levels in neutrophils (**P < 0.01, ***P < 0.001). EJ shows the effects of NLRP3 agonist and inhibitor treatments on the expression of CD177 and inflammatory factors such as IL-1β, IL-6, and TNF-α in neutrophils.

[0016] Figure 2Phenotypic heterogeneity of the NLRP3 L573W mutant mouse model is shown; where A represents the appearance of wild-type (WT), mild, and severe inflammatory mice; B represents weight changes (days 12–22); C represents peripheral blood neutrophils detected by flow cytometry; DF represents H&E staining of skin, liver, and spleen showing inflammatory infiltration; G represents serum inflammatory factor levels detected by microarray method; HI represents IL-6 and IL-1β expression in neutrophils detected by flow cytometry; and JL represents tissue mRNA levels of IL-6, IL-1β, and TNF-α.

[0017] Figure 3 The study showed upregulated CD177 expression in NLRP3 L573W mutant mice. A represents flow cytometry analysis of peripheral blood CD177+ neutrophils; B represents tissue CD177 mRNA levels (qPCR); CE represents Western blot analysis of CD177 protein in skin, liver, and spleen; and FG represents the effect of NLRP3 agonist-treated MCF-7 cells on inflammatory cytokine mRNA and CD177 protein levels.

[0018] Figure 4 The therapeutic effect of IL-6 antibody on NLRP3-AID mice was shown; AB represents CD177 expression in peripheral blood (A) and tissue (B) after treatment; CE represents the differences in CD177+ (C), IL-6+ (D), and IL-1β+ (E) neutrophils after treatment detected by flow cytometry; FH represents the mRNA level of inflammatory factors in tissues; IK represents H&E staining of tissues after treatment; and LN represents immunohistochemistry of Ly6G+ neutrophils.

[0019] Figure 5 The study showed that targeting CD177 significantly improved the pathological phenotype of NLRP3-AID mice; where A represents the change in body weight after treatment with anti-IL-1β and siCD177; BC represents CD177 expression in peripheral blood (B) and tissue (C); and DF represents H&E staining for organ inflammation.

[0020] Figure 6 The study showed that CD177 blockade reversed the inflammatory phenotype in NLRP3-AID mice; where C represents neutrophil CD177 (A), IL-6+ (B), and IL-1β+ (C) cells detected by flow cytometry; DE represents serum IL-6 and IL-1β levels (ELISA); FH represents tissue inflammatory factor mRNA levels; and IK represents Ly6G immunohistochemistry. Detailed Implementation

[0021] To facilitate understanding by those skilled in the art, the following description is provided in conjunction with embodiments and appendices. Figure 1-4 The present invention will be further described below, and the content mentioned in the embodiments is not intended to limit the present invention.

[0022] Example 1

[0023] The siRNA targeting CD177 has the following sequences: si-CD177-F: 5'-GGAUCAUCUCUGAUCUGAATT-3', si-CD177-R: 5'-UUCAGAUCAGAGAUGAUCCTT-3'.

[0024] The aforementioned CD177-targeting siRNA is used in the preparation of drugs for treating NLRP3 gene mutation-related diseases. The CD177-targeting siRNA can significantly reduce CD177 expression in peripheral blood and CD177 mRNA expression in the skin, liver, and spleen; significantly reduce the number of neutrophils and the proportion of IL-6 and IL-1β-positive cells in peripheral blood; significantly reduce the expression of IL-6, IL-1β, and TNF-α in tissues and serum; and promote significant recovery of damaged structures in the skin, liver, and spleen, reducing inflammatory infiltration.

[0025] Drugs for treating NLRP3 gene mutation-related diseases include the aforementioned CD177-targeting siRNA and pharmacologically acceptable vectors. The CD177-targeting siRNA significantly reduces CD177 expression in peripheral blood and CD177 mRNA expression in the skin, liver, and spleen; significantly reduces the number of neutrophils and the proportion of IL-6 and IL-1β-positive cells in peripheral blood; significantly reduces the expression of IL-6, IL-1β, and TNF-α in tissues and serum; and promotes significant recovery of damaged structures in the skin, liver, and spleen, reducing inflammatory infiltration.

[0026] Application of CD177 in the preparation of kits for diagnosing diseases related to NLRP3 gene mutations.

[0027] To test the beneficial effects of the present invention, the inventors conducted the following experiments on the CD177-targeting siRNA obtained in Example 1:

[0028] I. Experimental Materials

[0029] Sample source: Patients with NLRP3 L573W mutation were admitted to the Eighth People's Hospital of Dongguan City; healthy controls were laboratory student volunteers. Venous blood samples from both groups of subjects were collected by professional nurses using a vacuum blood collection system and stored at low temperature in vacuum blood collection tubes containing EDTA anticoagulant for later use.

[0030] Cells and Animals: MCF-7 cells (purchased from GeneMedi, China); NLRP3 L573W mutant mice were constructed by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. An NLRP3 point mutation mouse model was constructed using CRISPR / Cas9 gene editing technology. sgRNA was designed targeting exon 3 of the NLRP3 gene (NCBI Gene ID: 216799, chromosomal location: Chr11: 59,345,672-59,370,981). Through homologous recombination, codon 573 CTG (leucine) was site-directed to TGG (tryptophan) (c.1718T>G, p.Leu573Trp). The obtained F0 generation C57BL / 6J mice were amplified by PCR (primer sequences: F: 5'-GGAAGTGGACTGCGAGAGATTCTAC-3', R: 5'-GGGAAAGTGGTCCATGGCACT-3') and verified by Sanger sequencing. These mice were then crossed with wild-type mice to establish a stable heterozygous strain. All experimental animals were housed in an SPF-grade barrier environment. This experimental protocol and any modifications thereto were reviewed and approved by the IACUC committee.

[0031] All animal experimental procedures in this study were approved by the Laboratory Animal Ethics Committee of Guangdong Medical University (GDY2502138). This study was also approved by the Ethics Committee of Dongguan Eighth People's Hospital (LL2023060903), and informed consent forms were obtained from the participants before the study.

[0032] II. Experimental Methods:

[0033] 2.1 Transcriptome Analysis

[0034] In this study, peripheral venous blood samples were collected from subjects, and peripheral blood mononuclear cells (PBMCs) were extracted using a human peripheral blood mononuclear cell isolation kit (LZS11131, TBD, China). Cells were then lysed using TRIZOL reagent (15596018CN, Invitrogen, USA). All samples were transported to the Shenzhen BGI Genomics Center on dry ice for whole transcriptome sequencing analysis. Samples were stored at -80℃ before analysis. Bioinformatics analysis showed that 1590 differentially expressed genes (DEGs) were identified between the patient group and healthy control group 1 (screening criteria: |Log2FC|>1 and P<0.05), and 768 DEGs were obtained compared with healthy control group 2. VENN plot analysis revealed 352 differentially expressed genes shared by both groups. Further hierarchical clustering heatmap analysis was performed on the top 20 significantly upregulated genes (screening criteria: Log2FC>1 and sorted by expression level change). Figure 1B). CD177 was ultimately selected as the key candidate gene, which not only showed significant expression changes but is also a neutrophil membrane protein.

[0035] 2.2 LPS Induction Experiment

[0036] In this study, neutrophils were isolated using density gradient centrifugation with a kit (LZS11131, TBD, China), and the cell concentration was adjusted to 1×10⁶. 6 After being seeded at a concentration of cells / mL, the cells were cultured in 24-well cell culture plates and cultured in complete peripheral blood neutrophil medium (Pricella, CM-H197, China). The entire experiment was conducted in a 37℃, 5% CO2 incubator.

[0037] Four groups were set up for the experiment: normal control group (NC), lipopolysaccharide stimulation group (LPS), LPS+Nigericin (NIG) group and LPS+NIG+CY-09 group. The intervention protocols were as follows: (1) LPS group: 1 μg / mL LPS (L5293, Sigma-Aldrich, Germany) was added for 2 h of stimulation; (2) LPS+NIG group: 10 μM NLRP3 agonist Nigericin (N849347, MACKLIN, China) was added for 1 h of continuous intervention after 2 h of LPS pretreatment; (3) LPS+NIG+CY-09 group: 10 μM NLRP3 inhibitor CY-09 (S5774, Selleck, USA) was added at the same time as NIG treatment. After the intervention of each group, RNA and protein samples were extracted in parallel using TRIzol reagent and RIPA lysis buffer, respectively. All samples were stored at -80℃ for testing.

[0038] 2.3 ELISA Detection

[0039] This study used enzyme-linked immunosorbent assay (ELISA) to quantitatively detect the levels of inflammatory factors. Mice were injected intraperitoneally with the anesthetic azedarach (JX750100, Genxion, China) (300 mg / kg). Whole blood was collected via retroorbital venous plexus puncture and injected into anticoagulant-free EP tubes, incubated at room temperature for 2 h to promote coagulation. The coagulated blood samples were centrifuged at 3000×g for 15 min using a pre-cooled centrifuge at 4°C (Eppendorf, Germany). The supernatant was carefully aspirated and aliquoted as serum, stored at -80°C for later analysis. Mouse-specific detection kits for IL-6 (CSB-E04639m, Cusabio, China) and IL-1β (CSB-E08054m, Cusabio, China) were used according to the manufacturer's instructions. The absorbance was read at 450 nm using a Synergy H1 (BioTek, USA) microplate reader to calculate the concentration.

[0040] 2.4 qPCR detection

[0041] Tissues or cells were added to lysis buffer and lysed using a homogenizer (cells did not need to be homogenized). RNA was extracted using an RNA extraction kit (EZB-RN4, EZB, CHN). cDNA was synthesized using a reverse transcription kit (A0010CGQ, EZB, CHN). Quantitative PCR was performed using SYBR Green qPCR Master Mix (A0012, EZB, CHN) on a Roche cobas Z 480 PCR instrument (Switzerland). Finally, the 2–ΔΔCt value was calculated.

[0042] The primer sequences used in this experiment are shown in the table below:

[0043]

[0044] 2.5 Flow cytometry detection

[0045] This study employed multicolor flow cytometry for immunophenotypic analysis. Human or mouse peripheral blood was lysed using erythrocyte lysis buffer (349202, BD Biosciences, USA), washed with PBS, and incubated with an appropriate amount of antibody at 4°C for 1 h. Flow cytometry (BD FACSAria, USA) was used to analyze cell count and immunophenotype. Flowjo (FLOWJO, LLC, Ashland, USA) was used for population selection and fluorescence intensity analysis. Mouse peripheral blood was labeled with CD177, IL-6, and IL-1β; human peripheral blood was labeled with CD3+ T cells, CD16+ / CD66+ neutrophils, and CD14+ monocytes. The antibodies used in the experiment included CD177 (566599, BD Biosciences, USA), IL-6 (504504, BioLegend, USA), IL-1β (11-7114-82, Invitrogen, USA), CD16 (360703, BioLegend, USA), CD3 (300419, BioLegend, USA), CD14 (301807, BioLegend, USA), CD66 (305111, BioLegend, USA), and CD177 (315804, BioLegend, USA).

[0046] 2.6 Western blot detection

[0047] Tissues or cells were added to protein lysis buffer (R0010, Solarbio, China) and protease inhibitor (A8260, Solarbio, China), and lysed using a homogenizer (cells did not require homogenization). Protein was quantified using a BCA kit (P0010, Beyotime, China), and the amount of protein loaded was calculated. After SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane (ISEQ00010, Millipore, USA), blocked with 5% skim milk powder, and then incubated overnight at 4°C with primary antibody. The membrane was then washed with TBST, incubated with mouse or rabbit secondary antibody, washed again with TBST, and developed using an exposure unit. The antibodies used in this experiment were CD177 (sc-376329, SANTA, USA), GAPDH (sc-137179, SANTA, China), β-actin (sc-58673, SANTA, USA), Mouse antibody (K-20002M, Solarbio, China), and Rabbit antibody (SPA-238, Solarbio, China).

[0048] 2.7 Animal Experiments

[0049] Grouping: NLRP3 mutant mice were divided into severe and mild types according to their phenotype.

[0050] Treatment: This study used targeted antibodies and siRNA for in vivo intervention. The specific dosing regimen was as follows: Anti-IL-6 (A2118, Selleck, USA) treatment dose 10 mg / kg, intraperitoneal injection once every three days, for a total of four times; Anti-IL-1β (BE0246-1MG, BioXcell, USA) treatment dose 10 mg / kg, intraperitoneal injection once every three days, for a total of four times; siCD77 (GeneMedi, CHN) treatment dose 5 mg / kg, intraperitoneal injection once every three days, for a total of four times.

[0051] Detection indicators: serum ELISA, tissue qPCR / WB.

[0052] 2.8 HE staining and immunohistochemical detection

[0053] Mouse tissue samples were immediately fixed in 4% paraformaldehyde (24197555, Bioshap, China), then dehydrated with alcohol, embedded in paraffin, cut into 2mm sections, dewaxed, stained with hematoxylin and eosin (C0105S, Beyotime, China), dehydrated, mounted, and subjected to pathological analysis. Immunohistochemistry was performed using a kit (SA1020, Boster, China), with sodium citrate for antigen retrieval, 3% hydrogen peroxide to eliminate endogenous active enzymes, blocking with goat serum, incubated with a suitable primary antibody overnight, incubated with a secondary antibody, stained with a DAB kit (AR1027, Boster, China), and mounted with neutral resin after dehydration.

[0054] 2.9 Detection of inflammatory factors

[0055] After anesthetizing mice, blood was collected from the retroorbital region, allowed to stand for 2 hours, and then centrifuged at 3000 rpm for 15 minutes at 4 degrees Celsius. The supernatant was collected and cryopreserved at -80°C. The samples were then sent to Guangzhou Juyan Biotechnology Co., Ltd. for analysis using dry ice. Clustering heatmap analysis was performed using GraphPad Prism 9.0.

[0056] 2.10 Statistical Analysis

[0057] We used GraphPad Prism 8.0 (GraphPad Software, San Diego, CA) for data analysis. T-test and one-way ANOVA analyses were performed; p < 0.05 was considered statistically significant.

[0058] III. Experimental Results

[0059] 1. CD177 is significantly elevated in NLRP3-AID patients.

[0060] To investigate the molecular mechanisms of NLRP3 L573W mutant autoinflammatory disease (AID), we first analyzed the differential gene expression in peripheral blood immune cells from patients with the NLRP3 L573W mutation using transcriptome sequencing. Figure 1 As shown in Figure A, the results indicated that compared to age- and sex-matched healthy controls, the expression of 352 genes in the peripheral blood immune cells of patients was significantly upregulated, among which CD177 was one of the genes with the most significant increase in expression. Figure 1 B). CD177 is mainly expressed on the surface of neutrophils and has important immunomodulatory functions.

[0061] like Figure 1As shown in C-1D, further validation using qPCR revealed significantly increased CD177 expression in neutrophils from patients with the NLRP3 L573W mutation. To clarify whether enhanced NLRP3 function specifically promotes CD177 expression, we treated neutrophils from healthy individuals with an NLRP3 agonist (nigratin) and an inhibitor (CY-09). Figure 1 As shown in E-1J, qPCR results revealed that NLRP3 agonists significantly promoted CD177 expression in neutrophils, and this effect could be significantly reversed by NLRP3 pathway inhibitors. These data indicate that enhanced NLRP3 function can specifically upregulate CD177 expression.

[0062] 2. NLRP3 heterozygous mutant mice exhibit phenotypic heterogeneity.

[0063] To further investigate the molecular mechanism of NLRP3 L573W mutant AID, we constructed an NLRP3 L573W heterozygous mutant mouse model. Figure 2 As shown in A-2B, the study found that heterozygous mutant mice exhibited significant individual differences:

[0064] Mild inflammatory mice: Their size and weight were not significantly different from wild-type mice. They initially showed only mild skin inflammation, and most recovered spontaneously after four weeks.

[0065] Severe inflammatory mice: These mice are significantly smaller and lighter than normal mice, exhibiting severe skin inflammation that is difficult to resolve over a long period.

[0066] like Figure 2 As shown in Figure C, flow cytometry analysis revealed that the proportion of neutrophils in severely inflamed mice was significantly higher than that in mildly inflamed mice. Figure 2 As shown in D-2F, further pathological analysis revealed that the stratum corneum, stratum spinosum, and dermis of the skin in the severely affected mice were significantly thickened, while the fat layer and muscles were atrophied, accompanied by extensive inflammatory cell infiltration. Furthermore, the liver and spleen also exhibited significant structural changes.

[0067] Liver: Hepatocytes showed extensive vacuolar degeneration, with a loose reticular structure in the cytoplasm, and inflammatory cell infiltration was observed in the portal areas.

[0068] Spleen: The normal structure of the white pulp is lost, the boundaries are blurred, and some areas are replaced by inflammatory cells or fibrous tissue. The red pulp is diffusely dilated, and the splenic sinuses are highly congested and filled with inflammatory cells.

[0069] like Figure 2 As shown in G, analysis of inflammatory factor microarrays revealed significantly elevated levels of inflammatory factors such as IL-6, IL-1β, MCP-1, and TNF-α in the peripheral blood of severely affected mice, with IL-6 showing the most significant increase; while mildly affected mice only exhibited slight increases in IL-6 and IFN-γ. Figure 2 H-2I flow cytometry and such Figure 2 The qPCR results of J-2L further confirmed that IL-6 and IL-1β were significantly elevated in the peripheral blood and inflamed tissues of severe mice, while the elevation was not obvious in mild mice.

[0070] The above results indicate that NLRP3 heterozygous mutant mice can exhibit inflammatory phenotypes of varying severity due to individual differences, which is consistent with the clinical heterogeneity of NLRP3 mutant patients, suggesting that this mouse model is suitable for studying the pathogenesis of NLRP3-AID.

[0071] 3. CD177 expression is upregulated in NLRP3 mutant mice.

[0072] Previous studies have found significantly elevated CD177 expression in NLRP3 L573W mutant patients and in NLRP3-activated neutrophils. To verify whether CD177 is highly expressed in NLRP3 mutant mice, we examined the expression level of CD177 in mice. Figure 3 Flow cytometry results for A showed that CD177 was significantly elevated in peripheral blood immune cells of severely ill mice compared to wild-type mice, while only slightly elevated in mildly ill mice. Figure 3 B's qPCR and such Figure 3 Western blot results of C-3E further confirmed that the mRNA and protein levels of CD177 were significantly higher in the skin, liver, and spleen of heavy mice than in wild-type mice, while no significant changes were observed in light mice.

[0073] In addition, such as Figure 3 As shown in F-3G, in MCF-7 cells treated with NLRP3 agonist, siRNA knockdown of CD177 significantly inhibited the expression of inflammatory factors such as IL-6, IL-1β, and TNF-α.

[0074] These results suggest that CD177 is not only a potential diagnostic biomarker for NLRP3 L573W mutant AID, but may also be a key factor driving the disease.

[0075] 4. The efficacy of IL-6 antibodies in NLRP3-AID is limited.

[0076] Analysis of NLRP3 L573W mutant patients and mouse models showed that IL-6 was the most significantly elevated inflammatory cytokine, suggesting that targeting IL-6 may be a potential therapeutic strategy. We tested the therapeutic effect of IL-6 monoclonal antibodies on NLRP3 L573W mutant severe mice. Figure 4 A-4B and Figure 4Flow cytometry results for C-4E showed that IL-6 antibody failed to significantly reverse the increased proportion of neutrophils in the peripheral blood of severely ill mice, nor did it significantly reduce the expression levels of IL-6, IL-1β, and CD177. Figure 4 The qPCR results of F-4H showed that the expression of IL-6 and CD177 in the skin, liver and spleen of mice treated with IL-6 antibody did not decrease significantly, but the expression of IL-1β in the skin tissue was significantly reduced, and the expression of TNF-α was inhibited in multiple tissues.

[0077] like Figure 4 As shown in I-4K, pathological examination revealed that skin inflammation in mice treated with the IL-6 antibody improved, but structural damage to the liver and spleen only slightly subsided. Figure 4 As shown in L-4N, immunohistochemical results indicate that a large number of neutrophils still infiltrate the inflamed tissues of the treated mice.

[0078] The above results suggest that the IL-6 antibody has limited therapeutic effect on severely ill NLRP3 L573W mutant mice and may not be suitable for clinical treatment of NLRP3 mutant patients.

[0079] 5. Targeting CD177 significantly reversed the inflammatory phenotype in NLRP3-AID mice.

[0080] To determine whether CD177 drives the occurrence of NLRP3 L573W mutant AID, such as Figure 5 As shown in Figure A, we tested the effect of inhibiting CD177 expression on the inflammatory phenotype in severely ill mice, using IL-1β monoclonal antibody (NLRP3-AID first-line treatment) as a positive control. Figure 5 B's flow cytometry and such Figure 5 qPCR results showed that CD177 siRNA significantly reduced the expression of CD177 in peripheral blood and inflamed tissues of severely ill mice.

[0081] like Figure 5 As shown in D-5F, pathological analysis revealed that compared to the untreated group, mice treated with CD177 siRNA and IL-1β antibodies showed significant recovery in skin, liver, and spleen structure, and reduced inflammatory infiltration. Furthermore, the degree of skin and spleen recovery in the CD177 siRNA-treated group was superior to that in the IL-1β antibody-treated group. Figure 6 As shown in I-6K, immunohistochemical results revealed a significant reduction in neutrophil infiltration in the inflamed tissues of both groups of treated mice.

[0082] Through such Figure 6 A-6C flow cytometry and such Figure 6ELISA analysis of D-6E showed that the proportion of peripheral blood neutrophils and serum IL-6 and IL-1β levels were significantly reduced in mice treated with CD177siRNA and IL-1β antibodies. Figure 6 qPCR of F-6H further confirmed that CD177 siRNA can significantly inhibit the expression of IL-6, IL-1β and TNF-α in the skin, liver and spleen.

[0083] The above data indicate that targeting CD177 can alleviate NLRP3 mutation-related multi-organ inflammation by inhibiting the expression of IL-1β and IL-6, suggesting that CD177 is a potential therapeutic target for NLRP3 L573W mutant AID.

[0084] The above embodiments are preferred implementations of the present invention. In addition, the present invention can be implemented in other ways. Any obvious substitutions without departing from the concept of the present invention are within the protection scope of the present invention.

Claims

1. The application of CD177-targeting siRNA in the preparation of drugs for treating NLRP3 L573W mutant autoinflammatory disease in mice, characterized by: The sequences of the siRNA targeting CD177 are: si-CD177-F: 5'-GGAUCAUCUCUGAUCUGAATT-3', si-CD177-R: 5'-UUCAGAUCAGAGAUGAUCCTT-3'.

2. The application according to claim 1, characterized in that: The siRNA targeting CD177 can reduce CD177 expression in peripheral blood and CD177 mRNA expression in skin, liver and spleen.

3. The application according to claim 1, characterized in that: The siRNA targeting CD177 can reduce the number of neutrophils in peripheral blood and the proportion of IL-6 and IL-1β positive cells.

4. The application according to claim 1, characterized in that: The siRNA targeting CD177 can reduce the expression of IL-6, IL-1β and TNF-α in tissues and serum, and promote the recovery of damaged structures in the skin, liver and spleen, and reduce inflammatory infiltration.

5. A drug for treating autoinflammatory disease of NLRP3 L573W mutant in mice, characterized in that: Includes siRNA targeting CD177 and a pharmacologically acceptable vector, wherein the sequences of the siRNA targeting CD177 are: si-CD177-F: 5'-GGAUCAUCUCUGAUCUGAATT-3', si-CD177-R: 5'-UUCAGAUCAGAGAUGAUCCTT-3'.

6. The drug according to claim 5, characterized in that: The siRNA targeting CD177 can reduce CD177 expression in peripheral blood and CD177 mRNA expression in skin, liver and spleen.

7. The drug according to claim 5, characterized in that: The siRNA targeting CD177 can reduce the number of neutrophils in peripheral blood and the proportion of IL-6 and IL-1β positive cells.

8. The drug according to claim 5, characterized in that: The siRNA targeting CD177 can reduce the expression of IL-6, IL-1β and TNF-α in tissues and serum, and promote the recovery of damaged structures in the skin, liver and spleen, and reduce inflammatory infiltration.

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