Application of allicin in medicine for preventing and treating Kawasaki disease

Abstract

The invention provides an application of allicin in a medicine for preventing and treating Kawasaki disease. The invention proves the inhibition effect of allicin on coronary artery injury and endothelial pyroptosis for the first time. Experiments further prove that allicin relieves Kawasaki disease coronary artery injury or endothelial cell pyroptosis by activating a TRPV1 / A1 channel and down-regulating an EGFR / Sp1 / ASM axis, new evidence is provided, and it is powerfully proved that allicin is a very effective component for treating Kawasaki disease coronary artery endothelial cell injury.

Description

Technical Field

[0001] The invention relates to the field of biopharmaceuticals, and in particular to application of allicin in medicines for preventing and treating coronary artery damage caused by Kawasaki disease. Background Art

[0002] Kawasaki disease (KD) is an acute, self-limited vasculitis of unknown etiology that occurs primarily in children younger than 5 years of age and is currently considered the most common cause of acquired heart disease in children in developed countries. It is well known that the main features of KD are coronary artery structural damage accompanied by inflammatory infiltration, endothelial damage, and dedifferentiation of vascular smooth muscle cells. Among them, endothelial dysfunction plays a key role in the development of coronary artery abnormalities. Damaged endothelial cells can both disrupt endothelial barrier function and enhance inflammatory responses by releasing proinflammatory cytokines, ultimately destroying the coronary artery structure. The main serious complication of KD is coronary artery damage, ranging from self-limited coronary artery dilatation to giant coronary artery aneurysms.

[0003] The standard treatment for KD is IVIG plus aspirin, which is effective for most patients with KD. However, some patients are "hard bones" who are resistant to this treatment strategy, and these patients face a much higher risk of developing coronary artery damage and may develop coronary artery aneurysms. Therefore, elucidating the molecular mechanisms of coronary artery abnormalities in KD and developing potentially effective therapeutic drugs are very important for the clinical diagnosis, treatment, and management of KD patients.

[0004] So far, a variety of phytochemicals and natural products have shown therapeutic effects on KD, including berberine, melatonin, Xijiao Dihuang decoction, forsythiaside B, etc. Allicin, as the main component of garlic, can be easily absorbed through the cell membrane without causing any damage to the phospholipid bilayer, and then rapidly metabolized to exert its pharmacological effects. Currently, allicin has been used in clinical practice as an alternative to antibiotics for the treatment of deep fungal and bacterial infections. In addition to its antibacterial function, allicin also has a wide range of other pharmacological activities, such as antidiabetic, antihypertensive, and cardiovascular protective activities. However, it is still unclear whether allicin can play a protective role in the treatment of KD. Summary of the invention

[0005] In view of the defects existing in the prior art, the purpose of the present invention is to provide the use of allicin in drugs for preventing and / or treating coronary artery damage in Kawasaki disease. The present invention studies have confirmed the protective effect of allicin on coronary artery damage in the KD mouse model induced by Candida albicans cell wall extract (CAWS). Mechanism studies have shown that allicin activates TRP channels, inhibits JNK / c-Jun activation and EGFR / Sp1 / ASM axis, and ultimately blocks CTSB-mediated NLRP3 inflammasome activation, endothelial cell pyroptosis and coronary artery damage.

[0006] In one aspect, the present application provides a use of allicin in a drug for preventing and / or treating Kawasaki disease.

[0007] On the other hand, the present application provides a use of allicin in a drug for preventing and / or treating coronary artery damage caused by Kawasaki disease.

[0008] On the other hand, the present application provides a use of allicin in a drug for preventing and / or treating endothelial cell pyroptosis in Kawasaki disease.

[0009] The present invention first confirmed in vivo the inhibitory effect of allicin on cardiovascular inflammation characterized by aortitis and coronary arteritis, endothelial cell activation and inflammatory factors in the CAWS-induced KD vasculitis mouse model. NLRP3 inflammasome-mediated endothelial cell pyroptosis is a key cellular mechanism in the pathophysiology of KD. Therefore, the present application observed and verified the inhibitory effect of allicin on endothelial cell pyroptosis.

[0010] On the other hand, the present application provides a use of allicin in regulating the levels of lysosomal ASM and ceramide, characterized in that allicin reduces the levels of lysosomal ASM and ceramide.

[0011] The present study found that the levels of total ASM, lysosomal ASM and ceramide increased in endothelial cells treated with KD serum, while allicin significantly reduced these levels. In vitro and in vivo experiments confirmed that the inhibitory effects of allicin-mediated endothelial cell pyroptosis and cardiovascular inflammation were reversed by ASM overexpression. The present application first discovered that allicin inhibits endothelial damage by regulating ASM expression.

[0012] On the other hand, the present application provides a use of allicin in regulating the binding of Sp1 to the SMPD1 promoter. Preferably, allicin reduces the binding of Sp1 to the SMPD1 promoter.

[0013] The research data of this application confirmed that KD serum promoted the binding of Sp1 to the SMPD1 promoter, while allicin inhibited this binding. The data showed that SP1 overexpression significantly reversed the allicin-mediated downregulation of ASM and pyroptosis effector proteins GSDMD and GSDMD p30. These results revealed that allicin inhibits endothelial cell pyroptosis by regulating the Sp1-ASM axis.

[0014] On the other hand, the present application provides a use of allicin in regulating the level of EGFR in the cell nucleus. Preferably, allicin reduces the level of EGFR in the cell nucleus.

[0015] This study found that allicin can prevent the increase in nuclear EGFR distribution caused by KD serum due to increased total EGFR levels and enhanced nuclear translocation. Further experiments showed that the three ligands of β-Cellulin, TGF-α and HB-EGF contribute to the nuclear transport of EGFR, the upregulation of ASM and the pyroptosis of endothelial cells under KD conditions.

[0016] On the other hand, the present application provides a use of an inhibitor of the EGFR / Spl / ASM pathway in a drug for preventing and / or treating Kawasaki disease.

[0017] The present application found that the nuclear interaction between EGFR and Sp1 promoted the expression of ASM; and through a series of experiments, it was confirmed that the expression changes of EGFR and Sp1 mediated by allicin depended on TRPV1 and TRPA1 channels, which was achieved through the JNK / c-Jun axis.

[0018] In another aspect, the present application provides a biomarker combination for evaluating coronary artery damage in patients with Kawasaki disease, wherein the biomarkers include EGFR, Sp1 and / or ASM.

[0019] On the other hand, the present application provides a detection kit comprising the marker combination.

[0020] In another aspect, the present application provides the use of the marker combination or the kit in detecting coronary artery damage in patients with Kawasaki disease.

[0021] The beneficial effects of the present invention include but are not limited to:

[0022] 1. The present invention confirms for the first time the inhibitory effect of allicin on coronary artery damage and endothelial cell pyroptosis.

[0023] 2. The present invention further confirms through experiments that allicin relieves coronary artery damage or endothelial cell pyroptosis in Kawasaki disease by activating TRPV1 / A1 channels and downregulating EGFR / Sp1 / ASM axis. The present invention provides new evidence, which strongly proves that allicin is a very effective ingredient for treating coronary endothelial cell damage in Kawasaki disease.

[0024] 3. The experimental analysis of the present invention shows that EGFR, Sp1 and ASM are valuable biomarkers for evaluating coronary artery damage in KD patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of intraperitoneal injection of allicin to prevent or treat CAWS-induced KD mouse model;

[0026] Figure 2 The results of H&E staining of endothelial cells in the KD mouse model;

[0027] Figure 3 The results of immunohistochemical staining of the coronary artery intima in the KD mouse model;

[0028] Figure 4 is the relative expression level of mRNA of inflammation-related factors in the KD mouse model;

[0029] Figure 5 The effect of different concentrations of allicin on LDH release;

[0030] Figure 6 The effect of different concentrations of allicin on the survival rate of CCK8 cells;

[0031] Figure 7 The effect of allicin on endothelial cell pyroptosis-related proteins;

[0032] Figure 8 The results of immunofluorescence staining of Caspase-1 in endothelial cells;

[0033] Fig. 9 The results of immunofluorescence staining of TUNEL in endothelial cells;

[0034] Fig.10 The results of immunofluorescence staining of PI in endothelial cells;

[0035] Fig.11 is the relative mRNA expression level of GSDMD-N overexpressing endothelial cells;

[0036] Fig.12 is the effect of allicin on the expression level of GSDMD-N in GSDMD-N overexpressing endothelial cells;

[0037] Fig.13 The results of immunofluorescence staining of TUNEL in GSDMD-N overexpressing endothelial cells;

[0038] Fig.14 The results of immunofluorescence staining of PI in GSDMD-N overexpressing endothelial cells;

[0039] Fig.15 Effects of allicin on the levels of inflammatory factors in GSDMD-N overexpressing endothelial cells;

[0040] Fig.16 This is a principal component analysis diagram for endothelial cell RNA sequencing;

[0041] Fig.17 Venn diagram of differentially expressed genes for endothelial cell RNA sequencing;

[0042] Fig.18Heat map of lysosome-related DEGs in endothelial cells;

[0043] Fig.19 The effect of allicin on the expression level of SMPD1 in endothelial cells;

[0044] Fig. 20 is the expression level of ASM and ceramide in KD-treated endothelial cells;

[0045] Fig.21 is the effect of allicin on the expression levels of ASM and ceramide in KD-treated endothelial cells;

[0046] Fig. 22 The effect of allicin on the expression levels of ASM and ceramide in KD mouse model;

[0047] Fig.23 The immunofluorescence staining results of ASM and ceramide in lysosomes;

[0048] Fig.24 is the mRNA level of SMPD1 in SMPD1-overexpressing endothelial cells;

[0049] Fig.25 is the effect of allicin on the expression level of SMPD1 in SMPD1-overexpressing endothelial cells;

[0050] Fig.26 The effect of allicin on the expression of pyroptosis-related proteins in SMPD1-overexpressing endothelial cells;

[0051] Fig. 27 The results of immunofluorescence staining of Caspase-1, TUNEL and PI in SMPD1-overexpressing endothelial cells;

[0052] Fig.28 The results of immunohistochemical staining in endothelial cells overexpressing SMPD1 are shown;

[0053] Fig.29 The results of immunofluorescence staining of TUNEL in SMPD1-overexpressing endothelial cells;

[0054] Fig.30 The results of immunofluorescence staining of PI in SMPD1-overexpressing endothelial cells;

[0055] Fig.31 is the effect of allicin on SMPD1 promoter activity;

[0056] Fig.32 To predict possible binding sites of Sp1 and SMPD1 promoters using JASPAR database;

[0057] Fig.33Luciferase activity of endothelial cells transfected with Sp1 overexpression plasmid;

[0058] Fig.34 Chromatin immunoprecipitation (ChIP) assay of endothelial cells transfected with Sp1 overexpression plasmid;

[0059] Fig.35 is the Sp1 expression level in endothelial cells transfected with Sp1 overexpression plasmid;

[0060] Fig.36 The effect of allicin on the expression of related proteins in endothelial cells transfected with Sp1 overexpression plasmid;

[0061] Fig.37 Schematic diagram of possible interactions between Spl and five proteins;

[0062] Fig.38 is the nuclear distribution of EGFR in treated endothelial cells;

[0063] Fig.39 The effect of allicin on EGFR expression in endothelial cells transfected with EGFR overexpression plasmid;

[0064] Fig.40 The effect of allicin on the distribution of EGFR in endothelial cells transfected with EGFR overexpression plasmid;

[0065] Fig.41 Effects of KD treatment on the contents of β-Cellulin, TGF-a and HB-EGF in THP1 cells;

[0066] Fig.42 The effect of KD treatment on EGF expression in THP1 cells;

[0067] Fig.43 The effect of neutralization of EGF ligand on total EGFR levels;

[0068] Fig.44 The effect of neutralization of EGF ligand on the expression of related pyroptotic effector proteins;

[0069] Fig.45 This is the staining result of co-localization of EGFR and Spl in endothelial cells;

[0070] Fig.46 The effect of allicin on the interaction between EGFR and Spl in endothelial cells;

[0071] Fig.47 The effect of Sp1 and EGFR on SMPD1 promoter activity;

[0072] Fig.48The results of in vitro kinase assays of purified EGFR and Sp 1 are shown;

[0073] Fig.49 Western blot results of EGFR and Spl in endothelial cells;

[0074] Fig.50 Western blot results of EGFR and Sp1 in endothelial cells after inhibition of TRPA1 or TRPV1;

[0075] Fig.51 is the promoter activity of EGFR and SP1 in endothelial cells after treatment;

[0076] Fig.52 Western blot results of phosphorylation of JNK and c-Jun in endothelial cells after inhibition of TRPA1 or TRPV1;

[0077] Fig.53 The results of hematoxylin-eosin (H&E) staining of Trpvl or Trpal knockout mice;

[0078] Fig.54 The results of immunofluorescence staining of endothelial cells in Trpvl or Trpal knockout mice;

[0079] Fig.55 The results of immunofluorescence staining of phosphorylation of JNK and c-Jun in endothelial cells of Trpvl or Trpal knockout mice;

[0080] Fig.56 The results of immunofluorescence staining of EGFR, Sp1 and ASM in endothelial cells of Trpvl or Trpal knockout mice;

[0081] Fig.57 is the content of EGFR ligand in the serum of CAWS-induced KD mice;

[0082] Fig.58 The results of hematoxylin-eosin (H&E) staining of mouse hearts after treatment;

[0083] Fig.59 The results of immunohistochemical staining of mouse hearts after treatment;

[0084] Fig.60 for the effect of pyroptotic effector protein expression after treatment;

[0085] Fig.61 The results of immunofluorescence staining of Caspase-1 and TUNEL in endothelial cells after treatment;

[0086] Fig.62Figure 5. Serum levels of EGFR, Sp1, and ASM in HCs and KD patients with or without coronary artery lesions (CAL). DETAILED DESCRIPTION

[0087] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments are only for the convenience of those skilled in the art to understand the technical solution of the present invention, to implement or use the present invention, and are not intended to limit the protection scope of the present invention.

[0088] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the embodiments, unless otherwise specified, are all conventional methods in the art.

[0089] The materials and methods used in the following examples are as follows:

[0090] This application randomly selected voluntary sampling patients diagnosed with KD in the Second Affiliated Hospital of Wenzhou Medical University and Yuying Children's Hospital. All recruited KD patients met the criteria defined by the 2017 American Heart Association KD guidelines and received intravenous immunoglobulin (IVIG, 2g / kg) and oral aspirin (30-50mg / kg / day) in the acute phase. Age- and sex-matched healthy controls were children undergoing routine physical examinations. The obtained serum samples were routinely stored at -80°C within 4 hours of collection for later use. All participants gave written consent for their clinical information and blood samples to be used for academic research. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University and Yuying Children's Hospital (2023-K-41-01).

[0091] The Candida albicans cell wall extract (CAWS) of the present application is obtained from Candida albicans. In short, CAWS is cultured in CAWS restricted medium at 27°C for 2 days. Next, an equal volume of ethanol is added and placed in a 4°C refrigerator overnight. Then, the culture is collected, dissolved in water and stirred for 2 hours. The soluble portion is then harvested, mixed with an equal volume of ethanol at 4°C overnight, and centrifuged again to obtain a precipitate. Finally, the precipitate is resuspended with acetone and left to stand overnight. The obtained CAWS is dissolved in 1× phosphate buffered saline (PBS) buffer, the concentration of the CAWS solution is 20 mg / ml, and it is autoclaved before use.

[0092] Male C57 BL / 6 mice (3-4 weeks old) were purchased from Wenzhou Medical University, license number SCXK[ZJ]2023-0034, and were housed under standardized experimental conditions. The mice were randomly divided into PBS group, CAWS group, CAWS+Allicin group, CAWS+Allicin+AAV9-Smpd1 group, CAWS+AAV9-ICAM2-sh-Smpd 1 group, CAWS+plicamycin group, and CAWS+lapatinib group. Mice in the PBS group were intraperitoneally injected with 1× phosphate-buffered saline (PBS) buffer 200 μl / mouse once a day for 5 consecutive days; mice in the CAWS group were intraperitoneally injected with CAWS 4 mg / mouse (i.e., CAWS solution 200 μl / mouse) once a day for 5 consecutive days; the CAWS+Allicin group was treated as Figure 1 The CAWS+allicin+AAV9-Smpd 1 group was treated with AAV9-Smpd 1 injected into the tail vein 3 weeks before CAWS injection, and then Figure 1 The CAWS+AAV9-ICAM2-sh-Smpd1 group was treated with AAV9-ICAM2-sh-Smpd1 injected into the tail vein 3 weeks before CAWS injection, and then Figure 1 The mice were injected with CAWS as shown; the CAWS+plicamycin group was treated with intraperitoneal injection of plicamycin starting 6 days before CAWS injection, and then injected every other day until the end of the experiment; the CAWS+lapatinib group was treated with intraperitoneal injection of lapatinib starting 7 days before CAWS injection, and then injected every three days until the end of the experiment.

[0093] On day 28 after the last CAWS injection, mice were anesthetized and sacrificed to harvest heart tissue for subsequent examination in the CAWS+AAV 9-ICAM2-sh-Smpd 1 group. All animal experiments were authorized by the Animal Care and Use Committee of Wenzhou Medical University (wenchw 2022-0722) and performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Chinese Academy of Health Research.

[0094] Hematoxylin-eosin (H&E) staining and immunostaining: The hearts of mice from different groups were fixed in 4% paraformaldehyde and embedded in paraffin. The 5 μm sections were dewaxed, hydrated, and then applied for H&E staining. Briefly, the heart sections were immersed in xylene and ethanol, then stained with hematoxylin for 3 minutes, followed by eosin for 3 minutes. Immunohistochemical (IHC) staining of F4 / 80, neutrophils, and VCAM-1 was performed as follows. The heart sections were dewaxed, hydrated, and then treated with 3% H2O2 for 15 minutes. Next, the sections were blocked with 5% bovine serum albumin (BSA) at 37°C for 30 minutes and then incubated with the primary antibody overnight at 4°C. HRP-conjugated secondary antibodies were incubated for 2 hours. Diaminobenzidine was used as a chromogenic substrate. All slides were counterstained with hematoxylin. These stained sections were observed under an optical microscope. For caspase-1 immunofluorescence staining, the slides were dewaxed, hydrated, and blocked for 30 minutes at 37°C in PBS containing 5% BSA and 0.1% Triton X-100, and then stained with antibodies to CD 31 and caspase-1 at 4°C overnight. After incubation with these primary antibodies, the slides were washed with PBS and incubated with Alexa Fluor 594-conjugated IgG secondary antibodies and Alexa Fluor 488-conjugated IgG secondary antibodies at 37°C for 1 hour. DAPI staining was performed to visualize the nuclei. For in situ TUNEL staining, similar to caspase-1 / CD31 co-staining, the slides were first incubated with CD 31 primary antibodies and then incubated with a mixture of AlexaFluor 594-conjugated IgG secondary antibodies and TUNEL solution. These immunofluorescence images were examined using a LeicaSTELLARIS 5 confocal microscope (Leica). The fluorescence intensity was analyzed blindly using Image J, and the mean fluorescence intensity (MFI) was the average of the fluorescence intensities of different sections in the same group.

[0095] Heart tissue or cells were collected. Total RNA was extracted using TRIzol reagent (Invitrogen) and expressed using PrimeScript TM RT kit (Takara) to obtain cDNA. Next, qPCR was performed using Power TB Green PCR Master Mix (Takara) on an Applied Biosystems QuantStudio 3 Real-Time PCR System (ThermoFisher) with 40 cycles. GAPDH was used as an internal control, and the values ​​of all target genes were normalized to GAPDH. -ΔΔCT The relative quantification method was used to calculate the mRNA levels. The primers used in this study are listed in Table 1.

[0096] Table 1 Primers used in this study

[0097]

[0098] Human umbilical vein endothelial cells (HUVEC) and human monocytic leukemia cell line THP1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were identified by Genetic Testing Biotechnology Corporation (Suzhou, China) using short tandem repeat (STR) markers, and no mycoplasma contamination was detected. HUVECs were cultured in ECM supplemented with 5% fetal bovine serum (FBS), THP1 cells were cultured in RPMI-1640 medium containing 10% FBS, and HUVECs were co-cultured with THP1 cells according to previous literature reports. In the co-culture system, HUVECs are cultured in the lower chamber, while THP1 cells are placed in the upper chamber, which allows soluble molecules to diffuse. Serum from healthy controls (HC) or KD patients is then added to the upper chamber, and THP1 cells are treated for 24 hours. At the same time, diffuse molecules also affect HUVECs in the lower chamber.

[0099] Western blot analysis: Total protein from cardiac tissue or endothelial cells was extracted using protein extraction reagent.

[0100] The protein concentration was determined by an assay kit. Next, equal amounts of protein were separated on a 10% or 12% SDS-PAGE gel and electro-transferred to a PVDF membrane. After blocking with 5% skim milk for 2 h, the membranes were incubated with the following primary antibodies overnight at 4°C: (1:1000; ABclonal Technology, A16672), anti-ASM antibody (1:1000; Affinity BioReagents, DF13384), anti-procaspase-1+p10+p12 antibody (1:1000; Abcam, ab 179515), anti-cathepsin B antibody (1:1000; Cell Signaling Technology, 31718), anti-GAPDH antibody (1:1000; Proteintech, 10494-1-AP), anti-GSDMD antibody (1:500; Santa Cruz Biotechnology, sc-393581), anti-IL-1β antibody (1:1000; Affinity BioReagents, AF 5103), anti-IL-18 antibody (1:1000; Affinity BioReagents, DF6252), anti-NLRP 3 antibody (1: 1000; Abcam, ab 263899), anti-SP1 antibody (1: 1000; Cell Signaling Technology, 9389), anti-EGFR antibody (1: 1000; Abcam, ab 52894), anti-PARP antibody (1: 1000; Cell Signaling Technology, 9542), anti-lamin B1 antibody (1: 10000; Bioworlde Technology, AP6001). The membrane was then incubated with HRP-conjugated total protein of cardiac tissue or endothelial cells, and total protein was extracted using a protein extraction reagent. The protein concentration was detected using a BCA protein assay kit. Next, equal amounts of protein were separated on a 10% or 12% SDS-PAGE gel and electrotransferred to a polyvinylidene fluoride (PVDF) membrane.After blocking with 5% skim milk for 2 h, the membranes were incubated with the following primary antibodies overnight at 4°C: (1:1000; ABclonal Technology, A16672), anti-ASM antibody (1:1000; Affinity BioReagents, DF 13384), anti-procaspase-1+p10+p12 antibody (1:1000; Abcam, ab179515), anti-cathepsin B antibody (1:1000; Cell Signaling Technology, 31718), anti-GAPDH antibody (1:1000; Proteintech, 10494-1-AP), anti-GSDMD antibody (1:500; Santa Cruz Biotechnology, sc-393581), anti-IL-1β antibody (1:1000; Affinity BioReagents, AF 5103), anti-IL-18 antibody (1:1000; Affinity BioReagents, DF 6252), anti-NLRP 3 antibody (1: 1000; Abcam, ab 263899), anti-SP1 antibody (1: 1000; Cell Signaling Technology, 9389), anti-EGFR antibody (1: 1000; Abcam, ab 52894), anti-PARP antibody (1: 1000; Cell Signaling Technology, 9542), anti-lamin B1 antibody (1: 10000; Bioworlde Technology, AP 6001). The membrane was then incubated with HRP-conjugated secondary antibody (1: 10,000) for 2 hours. Western blot bands were analyzed using the ChemiDocTM XRS+ imaging system (Bio-Rad Laboratories, Hercules, CA, USA), and band density was quantified using ImageJ's Multi Gauge software (National Institutes of Health, Bethesda, USA) bundled with Java 1.8.0_112.

[0101] Immunofluorescence staining of endothelial cells: The collected endothelial cells were fixed in 4% paraformaldehyde for 30 min and incubated at 37°C for 30 min.

[0102] C for 30 minutes with 5% BSA. Then the cells were incubated with the designated primary antibodies at 4°C overnight. Afterwards, the cells were washed in PBS and incubated with IgG secondary antibodies coupled to Alexa Fluor 488 or 594 (ThermoFisher Scientific) at 37°C for 1 hour. Finally, DAPI staining was performed to mark the cell nucleus. Images were observed under an immunofluorescence confocal microscope (LeicaSTELLARIS 5).

[0103] Enzyme-linked immunosorbent assay (ELISA): Human ASM ELISA kit (JL 12600) and human ceramide

[0104] ELISA kit (JL 19781) detects the content of ASM in HC and KD serum and ASM and ceramide in the supernatant of endothelial cells after treatment. In order to detect the levels of EGF, HB-EGF, TGF-α and β-cellulose in the supernatant of treated THP 1 cells, the experiment was performed as follows. Specifically, THP 1 cells were treated with HC or KD serum for 24 hours. Then, the washed THP 1 cells were continued to be incubated for the next 24 hours. The levels of EGF, HB-EGF, TGF-α and β-Cellulin in the culture supernatant of THP 1 cells were detected by human EGF ELISA kit (JL 10101), human HB-EGF ELISA kit (JL 11929), human TGF-α ELISA kit (JL 10974) and human β-Cellulin ELISA kit (JL 10994), respectively. The concentrations of Sp1 and EGFR in the sera of HC and KD patients were detected using human Sp1 ELISA kit (JL 14476) and human EGFR ELISA kit (JL 14169), respectively. All these ELISA kits were purchased from Shanghai Jianglai Industrial Co., Ltd. (Shanghai, China).

[0105] Luciferase reporter gene analysis: HUVEC were seeded in 24-well plates and stained with pGL3-Basic vector, pGL3-

[0106] SMPD1 promoter, PGL3-SMPD1 promoter mSp1, pGL3-SP1 promoter or pGL3-EGFR promoter were transfected. After the transfected cells were treated accordingly, the dual luciferase activity (luminescence ratio of firefly / sea renilla) was measured using the dual luciferase reporter assay system (Promega).

[0107] Chromatin immunoprecipitation (ChIP)-qPCR assay: ChIP assay was performed according to the manufacturer's protocol as described previously.

[0108] qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme). The SMPD 1 forward primer for SP1 was 5'CTTCCACCGACCACATCACA-3', and the reverse primer was 5'GACTCTCCCTGAGGTCACCA-3'. qRT-PCR was performed using the Applied Biosystems QuantStudio 3 Real-Time PCR System (ThermoFisher).

[0109] Nuclear and Cytoplasmic Protein Extraction Kit,

[0110] Beyotime Biotechnology, P0027) instructions for nuclear cytoplasm separation. Specifically, the treated endothelial cells were collected and treated with cytoplasmic protein extraction reagent A supplemented with PMSF, followed by vigorous vortexing for 5 seconds and placed in an ice bath for 10-15 minutes. Next, cytoplasmic protein extraction reagent B was added. After vigorous vortexing for 5 seconds and centrifugation at 16000r for 5 minutes at 4°C, the supernatant was pipetted into a pre-cooled plastic tube for extracted cytosolic protein (do not touch the bottom precipitate). After aspirating the supernatant, a nuclear protein extraction reagent containing PMSF was added to the precipitate, vortexed vigorously for 15s, then ice bathed for 1min, and continued to vortex for 15s. Repeat the vortex and ice bath procedure for 30min. Finally, the supernatant was centrifuged at 16000r for 10min at 4°C to obtain nuclear protein.

[0111] Immunoprecipitation: About 200 μg of nuclear or cytoplasmic proteins of endothelial cells were mixed with 5 μg of anti-Sp1 antibody (ab

[0112] 157123, Abcam), anti-EGFR antibody (ab 52894, Abcam) or IgG control were incubated overnight at 4°C, and then 25 μl of protein A / G agarose beads (Epizyme Biotech) were added and incubated overnight at 4°C. The precipitate was then washed and boiled in SDS sample buffer. The supernatant was immunoblotted with anti-Sp1 antibody (1:1000; Cell Signaling Technology, 9389S) or anti-EGFR antibody (1:5000; Proteintech, 66455-1-IG).

[0113] In vitro kinase assay: A mixture of recombinant human EGFR kinase domain (#81271, Active Motif), recombinant human SP1 protein (#81181, Active Motif), 200 μM ATP (#9804, Cell Signaling Technology), 10× kinase buffer (#9802, Cell Signaling Technology) and γ-32P-ATP was incubated at 30°C for 15 min for phosphorylation reaction. The reaction was terminated by adding loading buffer and incubated at 95°C for 10 min. The proteins in the mixture were immediately separated by SDS-polyacrylamide gel electrophoresis (PAGE) and detected by Coomassie brilliant blue staining and autoradiography.

[0114] Adeno-associated virus (AAV) construction and in vivo delivery: All AAV vectors were purchased from Vigene Biosciences (Shandong, China).

[0115] China). In order to knock down Smpdl in vivo, AAV 9-RGDLRVS-ICAM 2-miR30-sh-Smpdl was designed and constructed using standard methods. Oligonucleotide sequence for gene knockdown: 5'-TGGAGCTGGAATTATTACA-3'. In order to construct an AAV overexpression vector for ASM, the full-length sequence of the mouse Smpd 1 gene was inserted into the pAAV9-CMV-P2A-Flag vector to construct the pAAV9-CMV-P2A-Smpd1-Flag plasmid. The knockout efficiency and overexpression efficiency of each sequence were detected by immunofluorescence. A total of 1012 viral particles were injected into mice via the tail vein.

[0116] Statistics: The above examples were statistically analyzed using GraphPad Prism 8.0 software. All data are expressed as mean ± standard error of the mean (SEM). The Shapiro-Wilk test was used to detect the normality of data distribution. For comparisons between two groups, the F test was applied to determine the homogeneity of variance. If the data variances were equal, the unpaired Student t test was used. Otherwise, the Welch t test was used. When the data were not normally distributed, the Mann-Whitney U test was used for intergroup comparisons. For comparisons between three or more groups, the Brown-Forsythe test was used to assess the homogeneity of variance. If the data passed the equal variance test, a one-way ANOVA analysis was used, followed by a Bonferroni post hoc test; otherwise, a Welch ANOVA test was performed, followed by a report of a post hoc analysis using the Tamhane T2 method. In order to compare the effects of two variables, a two-way ANOVA was used, followed by a Bonferroni post hoc analysis.

[0117] Example 1 Study on the Effect of Allicin on Coronary Artery Damage

[0118] In order to preliminarily analyze the effect of allicin on coronary artery injury in KD, allicin was added as Figure 1 The results showed that allicin was intraperitoneally injected into the KD mouse model induced by Candida albicans cell wall extract (CAWS). HE staining results showed that treatment with allicin significantly reduced coronary artery inflammation ( Figure 2 ). It is well known that vascular cell adhesion molecule 1 (VCAM-1), as a marker of endothelial cell activation, plays an important role in mediating the recruitment of many leukocytes to the damaged vascular endothelium. Therefore, the present application used immunohistochemistry (IHC) analysis to detect the expression changes of VCAM-1. Consistent with previous studies, CAWS significantly increased the expression level of VCAM-1 in the coronary artery intima, accompanied by an increase in the infiltration of inflammatory cells including neutrophils and macrophages ( Figure 3 In addition, the expression levels of inflammation-related factors, including cytokines (IL-1β, TNF-α, and IL-6), adhesion molecules (VCAM-1 and E-selectin), and chemokines (MCP1, CCL3, and CCL5), were significantly increased in the KD mouse model ( Figure 4 ). However, allicin significantly decreased the expression level of endothelial VCAM-1, reduced inflammatory cell infiltration, and downregulated the expression of inflammation-related factors, indicating that allicin plays a protective role in KD coronary artery injury.

[0119] Example 2 Study on the effect of allicin on endothelial cell pyroptosis

[0120] The present application first determined the optimal allicin concentration by CCK8 and LDH release analysis. Figure 5-6 As shown, 10ug / ml allicin showed the best therapeutic effect. In subsequent experiments, this concentration was selected to pretreat endothelial cells. The results showed that KD-mediated upregulation of pyroptosis-related proteins, including CTSB, NLRP3, ASC, Caspase-1, GSDMD, GSDMD p30, IL-1β, and IL-18, was significantly reversed by allicin ( Figure 7 In addition, the increased Caspase-1 fluorescence intensity and the percentage of TUNEL or PI-positive cells in the KD group were significantly reduced by allicin ( Figure 8-10 ). These results suggest that allicin may exert its effects by inhibiting endothelial cell pyroptosis.

[0121] To further confirm this, endothelial cells were transfected with an empty vector (negative control) or a plasmid containing the GSDMD-N gene. The data showed that the GSDMD-N overexpression plasmid significantly upregulated the expression of GSDMD-N ( Fig.11 ), and abolished the allicin-mediated downregulation of GSDMD-N ( Fig.12 In addition, GSDMD-N overexpression significantly abolished the inhibitory effect of allicin on endothelial cell pyroptosis, such as increased percentages of TUNEL and PI-positive cells, and enhanced release of LDH, IL-1β, and IL-18 ( Figure 13-15 ). These results indicate that allicin can inhibit endothelial cell pyroptosis.

[0122] Example 3 Mechanism of action of allicin in inhibiting endothelial cell pyroptosis

[0123] To explore the potential mechanism, RNA-seq was performed on the treated endothelial cells. This application first used principal component analysis (PCA) to obtain a lower dimensional feature set ( Fig.16 ), and then Venn diagram analysis was performed, which showed that there were 1032 differentially expressed genes (DEGs) in both KD vs HC and KD+allicin vs KD ( Fig.17 Considering that CTSB is released from lysosomes, lysosome-related DEGs are shown in the heat map, including SMPD1 ( Fig.18 ). The acid sphingomyelinase (ASM) encoded by SMPD1 can release ceramide from sphingomyelin. The generated ceramide will trigger lysosomal CTSB activation and cytoplasmic release, ultimately leading to XIAP degradation in natural killer / T lymphoma cell pyroptosis or NLRP3 inflammasome activation in endothelial cell pyroptosis. Therefore, the present application assumes that allicin-mediated KD-induced pyroptosis inhibition is related to SMPD1. The present application first verified the expression changes of SMPD1 at the mRNA and protein levels ( Fig.19). The levels of ASM and ceramide were then evaluated in KD-treated endothelial cells with or without allicin treatment. The results showed that the levels of ASM and ceramide were significantly increased in KD-treated endothelial cells and decreased after allicin treatment ( Fig. 20 ). In the CAWS-induced KD mouse model, endothelial ASM and ceramide expression were also significantly increased compared with the PBS group. However, allicin treatment significantly reduced their expression levels ( Fig.21 ). It is known that ASM acts on lysosomes and the outer leaflet of the plasma membrane after activation. Only the accumulation of lysosomal ceramide can destroy the stability of the lipid bilayer, ultimately leading to increased lysosomal membrane permeability and the release of CTSB into the cytoplasm. Therefore, the present application analyzed the expression of ASM and ceramide in lysosomes. The results showed that compared with the HC group, the levels of lysosomal ASM and ceramide in the KD group increased significantly, and were significantly reduced after allicin treatment ( Figure 22-23 ). These results suggest that allicin reduces lysosomal ASM and ceramide levels.

[0124] To further confirm whether allicin inhibits endothelial cell pyroptosis by regulating ASM, endothelial cells were transfected with empty plasmid (negative control) or SMPD1 overexpression plasmid, which significantly upregulated the mRNA level of SMPD1 ( Fig.24 ), reversing the allicin-mediated downregulation of SMPD1 at both mRNA and protein levels ( Fig.25 ). Moreover, SMPD1 upregulation abolished the allicin-mediated reduction of pyroptosis-related proteins, including CTSB, NLRP3, ASC, caspase-1, GSDMD, GSDMD p30, IL-1β, and IL-18 ( Fig.26 ), significantly reversed the allicin-mediated decrease in Caspase-1 expression, the percentage of TUNEL and PI-positive cells, and LDH release ( Fig. 27 ). These results suggest that allicin inhibits endothelial cell pyroptosis by inhibiting ASM expression. To confirm this in vivo, AAV9-Smpd1 was injected into the allicin-treated KD mouse model via the tail vein. The results showed that Smpd1 overexpression significantly reversed the allicin-mediated inhibition of coronary artery inflammation, ( Fig.28 ). In addition, compared with the CAWS+allicin group, Smpd1 upregulation significantly increased the expression of pyroptosis-related proteins, the immunofluorescence intensity of Casapase-1, and the percentage of TUNEL-positive endothelial cells ( Figure 29-30 ). Taken together, these data suggest that allicin improves KD coronary artery endothelial injury by regulating ASM expression.

[0125] The above data indicate that allicin can reduce the expression of SMPD1 at the mRNA and protein levels. To further confirm whether allicin regulates SMPD1 expression through a transcriptional mechanism, the luciferase reporter system was used to detect the SMPD1 promoter activity. The results showed that the SMPD1 promoter activity increased in the KD group, which decreased after allicin treatment ( Fig.31 ). In order to predict potential transcription factors that can regulate SMPD1 expression, the AnimalTFDB v4.0 database (http: / / guolab.wchscu.cn / AnimalTFDB4 / # / ) was used to screen out the specific protein 1 (Sp1) with the highest score (Supplementary Table 4). Then the JASPAR database (https: / / jaspar.genereg.net / ) was applied to predict the possible binding sites of Sp1 and SMPD1 promoter ( Fig.32 ). Next, endothelial cells were transfected with a pGL3-luciferase construct containing a mutated Sp1 binding site. The data showed that the mutation of the Sp1 binding site in the SMPD1 promoter significantly reduced the luciferase activity in both the HC and KD groups ( Fig.33 ), revealing that the binding of Sp1 is necessary for activating the SMPD1 promoter activity. To demonstrate whether allicin downregulates SMPD1 transcription through Sp1, a chromatin immunoprecipitation (ChIP) assay was performed. ChIP-qPCR results showed that KD serum increased the binding of Sp1 to the SMPD1 promoter, which was reduced after allicin treatment ( Fig.34 ). To further demonstrate the role of Sp1 in allicin-mediated pyroptosis inhibition, Sp1 was overexpressed in endothelial cells. The data showed that the Sp1 overexpression plasmid significantly reversed the allicin-mediated decrease in Sp1 expression at both the mRNA and protein levels ( Figure 35-36 ). In addition, compared with the KD+allicin group, overexpression of Sp1 significantly increased the protein expressions of ASM, pyroptosis effector GSDMD, and GSDMD p30 ( Fig.36 ), indicating that allicin inhibits endothelial cell pyroptosis via Sp1.

[0126] The above findings confirmed that Spl can bind to the SMPD1 promoter and regulate its expression, which is inhibited by allicin. Therefore, it is speculated that allicin may inhibit this process through certain proteins. To investigate it, this application used the SwissTargetPrediction database (http: / / www.swisstargetprediction.ch / ) to classify the potential targets of allicin and selected 26 targets because their probability >0 (Excel File I in the DataSupplement). Next, the STRING database (https: / / cn.string-db.org) was applied to evaluate the protein-protein interactions (PPIs) between Spl and the 26 targets of allicin. The data showed that Spl may interact with five proteins, including CASP3, CA9, EGFR, KDR and PARPI ( Fig.37 ). Considering that Spl is a nuclear transcription factor, the selected protein should be able to enter the nucleus. Studies have shown that Spl is a substrate protein of CASP3, which can cleave Sp1. However, it has been reported that the expression of CASP is inhibited by allicin, which may affect CASP3-mediated Sp1 cleavage, leading to the upregulation of Spl. This is inconsistent with the results of the present application. Carbonic anhydrase-9 (CA9) is a classic marker of hypoxia, involved in the catalytic hydration of carbon dioxide to carbonic acid, and is mainly located in the serous membrane. The current study showed that CA expression is regulated by Spl and is located downstream of Spl. Therefore, CASP and CA were excluded. EGFR and KDR (VEGFR2) are both membrane receptors that can migrate to the nucleus to regulate gene transcription under certain conditions. Previous studies have shown the interaction between VEGFR2 and Spl. In order to preliminarily analyze which proteins may interact with Spl under KD conditions, immunofluorescence assays were performed to observe the nuclear localization of VEGFR2 and EGFR. Observations showed that the nuclear distribution of EGFR was significantly increased in KD-treated endothelial cells, whereas allicin significantly decreased ( Fig.38 ). However, KD serum barely induced nuclear localization of VEGFR2. Therefore, VEGFR2 was not considered either. PARP1 is a nuclear enzyme whose transcription depends on the regulation of Spl, and PARP1 can also interact with Spl in a DNA-independent manner. To investigate whether EGFR or PARP1 is a potential allicin target affecting ASM expression, EGFR and PARP1 were overexpressed, respectively. The results showed that compared with the KD+allicin group, the EGFR overexpression plasmid significantly increased the expression of EGFR at the mRNA and protein levels and significantly reversed the allicin-mediated downregulation of ASM ( Fig.39). However, PARP1 overexpression did not exclude allicin-mediated reduction of ASM expression. These results suggest that allicin regulates ASM expression through EGFR.

[0127] To further study how EGFR exerts its function, the present application detected the level of EGFR in the nucleus. The data showed that the distribution of EGFR in the nucleus increased in the KD group and decreased after treatment with allicin ( Fig.40 ). In order to exclude the influence of the increased total EGFR expression on the increased nuclear EGFR, the present application detected the percentage of nuclear EGFR to total EGFR. The data showed that the percentage of nuclear EGFR increased in the KD group compared with the HC group, and was significantly reduced after allicin treatment. These results indicate that the increase in nuclear EGFR levels induced by KD is attributed to the increase in total EGFR levels and the enhancement of nuclear translocation, both of which can be inhibited by allicin.

[0128] It has been reported that the internalization and translocation of EGFR to the nucleus depends on ligand binding. Ligands known to mediate its nuclear translocation include β-Cellulin, TGF-a, HB-EGF, and EGF. To explore which ligands play a role in EGFR translocation, the levels of these EGFR ligands in the supernatant of THP1 cells treated with KD serum were examined. The results showed that the levels of β-Cellulin, TGF-a, and HB-EGF in the supernatant of THP1 cells were significantly increased after treatment with KD serum ( Fig.41 However, there was no significant difference in EGF between the KD and HC groups ( Fig.42 ). To further confirm that the nuclear distribution of EGFR is related to these ligands, neutralizing antibodies against β-cellulin, TGF-a, and HB-EGF were used respectively. The results showed that all three neutralizing antibodies significantly reduced nuclear EGFR expression compared with the KD group, while these three antibodies did not change the total EGFR level ( Fig.43 ), suggesting that the nuclear distribution of EGFR depends on the presence of its ligands. Next, the present application observed the effects of the three ligands on ASM expression and pyroptosis. The data showed that neutralization of these ligands significantly reduced the expression level of ASM, as well as the pyroptosis effector proteins GSDMD and GSDMD p30 ( Fig.44 ). These data suggest that EGFR ligands produced by monocytes, including HB-EGF, TGF-a, and β-cellulin, promote the translocation of EGFR to the nucleus and are involved in regulating ASM expression and endothelial cell pyroptosis under KD conditions. All these results suggest that allicin alleviates endothelial cell pyroptosis via EGFR.

[0129] In order to explore how EGFR regulates ASM expression through Sp1, the present application first observed the colocalization of EGFR and Sp1. As expected, EGFR and Sp1 colocalized in the nucleus ( Fig.45 ). Next, nuclear proteins were extracted and co-immunoprecipitation analysis was performed. The data showed that EGFR was immunoprecipitated by anti-Sp1 antibody but not by control IgG, and vice versa. In addition, in the KD group, there was a stronger interaction between EGFR and Sp1, and the interaction was reduced by allicin ( Fig.46 ). To further demonstrate whether the interaction between EGFR and Spl affects SMPD1 transcription, endothelial cells were simultaneously transfected with a pGL3-luciferase construct containing the SMPDI promoter and plasmids containing Spl, EGFR, or both. Next, cells were stimulated with 100ng / ml EGF for 24 hours before the experiment. The data showed that luciferase activity was increased by Spl and further increased by Spl+EGFR. However, EGFR alone had little effect on luciferase activity ( Fig.47 ). These results suggest that the interaction between nuclear EGFR and Sp1 promotes Sp1-mediated SMPD1 expression. It is well known that Sp1 phosphorylation can enhance its mediated transcription. It has been reported that EGFR is able to regulate the phosphorylation of proteins containing tyrosine residues, such as PCNA and FAM129B. Therefore, it is speculated that EGFR exerts its effects by phosphorylating the tyrosine residues of Sp1, ultimately promoting the expression of SMPD1. To confirm this, in vitro kinase assays were performed with purified EGFR and Sp 1. Unexpectedly, EGFR was unable to directly phosphorylate Sp1 ( Fig.48 ). All these results indicate that EGFR enhances ASM expression by binding to Sp1.

[0130] The above results indicate that allicin improves endothelial cell thermalization by regulating the EGFR / Spl / ASM axis. However, how allicin regulates EGFR and Spl expression remains unclear. Allicin has been reported to activate TRP channels, including TRPV1 and TRPA1. In order to preliminarily explore whether allicin-mediated changes in EGFR and Spl expression are related to these two channels, this application used ruthenium red (RR) to inhibit these two channels. The data showed that inhibition of TRPV1 / Al significantly reversed the allicin-mediated decrease in EGFR and Spl expression ( Fig.49). To investigate which TRP channel was responsible, TRPA1 and TRPV1 were inhibited by HC030031 and AMG9810, respectively. The results showed that inhibition of TRAl or TRPV1 significantly increased the protein levels of EGFR and Sp1 compared with the KD+allicin group, indicating that both TRPA1 and TRPV1 were involved in the allicin-mediated downregulation of EGFR and Sp1 ( Fig.50 ). To investigate whether the expression changes of EGFR and Sp1 are regulated by transcriptional mechanisms, the present application detected the promoter activities of EGFR and SP1. As expected, the promoter activities of EGFR and SP1 increased in the KD group, and their activities could be reduced by allicin. However, HC030031 and AMG9810 significantly increased their promoter activities ( Fig.51 ). It is well known that the promoters of EGFR and SPl can be bound and regulated by c-Jun. In addition, c-Jun activation can be inhibited by allicin through c-Jun N-terminal kinase (JNK), suggesting that allicin-mediated changes in EGFR and Spl expression may be regulated by the JNK / c-Jun axis. The present application first demonstrated that under KD conditions, EGFR and Spl expression is indeed modulated by the JNK / c-Jun axis. The present application then observed that allicin significantly reduced the phosphorylation levels of JNK and c-Jun, which could be reversed by inhibitors of TRPV1 or TRPA1 ( Fig.52 ). These results suggest that allicin downregulated the JNK / c-Jun axis-mediated EGFR and Spl expressions through the TRPV1 / A1 channel.

[0131] To confirm in vivo that allicin exerts its protective effect through TRPV1 / A1 channels, Cdh5-Cre mice were infected with AAV-DIO-Trpv1-shRNA or AAV-DIO-Trpa1-shRNA. Knockdown of endothelial Trpvl or Trpal significantly reduced the protective effect of allicin, as evidenced by increased inflammatory areas, increased endothelial Caspase-1 expression, and increased TUNEL-positive endothelial cells ( Figure 53-54 In addition, compared with CAWS+allicin, the phosphorylation levels of JNK and c-Jun, and the expressions of EGFR, Spl, and ASM were also significantly increased ( Figure 55-56 ). All these results indicate that allicin inhibits EGFR / Spl / ASM pathway-mediated endothelial cell thermalization by activating TRPV1 / A1 channels.

[0132] Example 4 Study on the involvement of EGFR / Sp1 / ASM pathway as a potential diagnostic marker for coronary artery abnormalities in KD

[0133] The above studies elucidated the molecular mechanism by which allicin exerts a therapeutic effect on KD coronary artery damage. In this process, the above data also indirectly proved that EGFR, Spl and ASM are involved in KD endothelial damage. As mentioned above, the nuclear translocation of EGFR and the subsequent endothelial cell pyroptosis depend on the presence of EGFR ligands. In order to investigate whether the EGFR / Sp1 / ASM axis plays an important role in KD coronary artery damage, the present application first detected the content of EGFR ligands in the serum of mice after PBS and CAWS treatment, including EGF, HB-EGF, TGF-a and B-Cellulin. ELISA results showed that the serum levels of EGF, HB-EGF, TGF-a and B-Cellulin in the KD group mice were significantly increased compared with those in the PBS group ( Fig.57 ). CAWS-induced KD mice were then intravenously injected with AAV9-ICAM2-sh Smpdl to specifically interfere with endothelial Smpdl expression, Spl inhibitor (purimycin) or EGFR inhibitor (lapatinib). The results showed that interference with ASM, Spl or EGFR significantly alleviated coronary artery inflammation, reduced VCAM-1 expression, and infiltration of neutrophils and macrophages compared with the CAWS group ( Figure 58-59 In addition, interference with these proteins significantly reduced the expression of ASM, GSDMD, and GSDMD p30, the fluorescence intensity of Caspase-1, and the percentage of TUNEL-positive cells ( Figure 60-61 ), these data indicate that pyroptosis of coronary endothelial cells was significantly reduced. These results demonstrate that EGFR / Spl / ASM pathway-mediated endothelial cell pyroptosis is involved in KD coronary artery abnormalities.

[0134] To further confirm whether the EGFR / Spl / ASM pathway can be used as a biomarker for coronary artery abnormalities in KD clinically, the levels of EGFR, Spl, and ASM in the serum of HCs and KD patients with or without coronary artery lesions (CAL) were also measured. Fig.62As shown. The results showed that the levels of the three proteins were significantly increased in KD without CAL compared with HC. In addition, the levels of the remaining proteins, except ASM, were further increased in the serum of KD patients with CAL compared with those of KD patients without CAL. We then plotted the receiver operating characteristic (ROC) curve and calculated the area under the curve (AUC) (AUC = 1 indicates the highest correlation). The levels of Sp1, EGFR, and ASM were able to distinguish between KD patients (NCAL and CAL groups) and HC, with an AUC value of 0.902 (95% confidence interval (CI), 0.845-0.959) for Sp1, 0.878 (95% CI, 0.787-0.969) for EGFR, and 0.884 (95% CI, 0.816-0.953) for ASM. The AUCs of Sp1, EGFR, and ASM calculated to distinguish the CAL group from the NCAL group in KD were 0.903 (95% CI, 0.823-0.983), 0.868 (95% CI, 0.771-0.966), and 0.738 (95% CI, 0.597-0.880), respectively. These data indicate that the levels of these proteins are positively correlated with the severity of coronary artery pathology. All these results indicate that Sp1, EGFR, and ASM are both biomarkers of coronary artery disease pathology and are involved in the coronary artery pathological process.

[0135] The above is a detailed introduction to the application of a kind of allicin provided by the present invention in the prevention and treatment of Kawasaki disease. Specific examples are used herein to illustrate the principle and implementation of the present invention, and the description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, the present invention can also be improved and optimized, and these improvements and optimizations also fall within the scope of protection of the claims of the present invention.

Claims

1. Application of allicin in drugs for preventing and / or treating Kawasaki disease.

2. Application of allicin in the prevention and / or treatment of coronary artery damage in Kawasaki disease.

3. Application of allicin in the prevention and / or treatment of endothelial cell pyroptosis in Kawasaki disease.

4. The use of allicin in regulating lysosomal ASM and ceramide levels, characterized in that: Allicin reduces the levels of lysosomal ASM and ceramide.

5. The use of allicin in regulating the binding of Sp1 to SMPD1 promoter, characterized in that: Allicin reduced the binding of Sp1 to the SMPD1 promoter.

6. The use of allicin in regulating the level of EGFR in the cell nucleus, characterized in that: Allicin reduces the level of EGFR in the nucleus.

7. Use of inhibitors of the EGFR / Spl / ASM pathway in the prevention and / or treatment of Kawasaki disease.

8. A biomarker combination for evaluating coronary artery damage in patients with Kawasaki disease, the biomarkers comprising EGFR, Sp1 and / or ASM.

9. A detection kit comprising the marker combination according to claim 8.

10. Use of the marker combination according to claim 8 or the kit according to claim 9 in detecting coronary artery damage in patients with Kawasaki disease.

Images