CN119700741A - Application of allicin in medicine for preventing and treating Kawasaki disease - Google Patents

Abstract

The present invention provides an application of allicin in a drug for preventing and treating Kawasaki disease. The present invention confirms for the first time the inhibitory effect of allicin on coronary artery damage and endothelial cell pyroptosis. The present invention further confirms through experiments that allicin relieves Kawasaki disease coronary artery damage or endothelial cell pyroptosis 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 Kawasaki disease coronary endothelial cell damage.

Description

Application of allicin in medicine for preventing and treating Kawasaki disease

Technical Field

The invention relates to the field of biological pharmacy, in particular to application of allicin in a medicine for preventing and treating Kawasaki disease coronary artery injury.

Background

Kawasaki Disease (KD) is an acute self-limiting vasculitis of unknown etiology, occurring mainly in children older than 5 years, and is currently considered to be the most common cause of acquired heart disease in children in developed countries. It is well known that KD is mainly characterized by structural damage to the coronary arteries, accompanied by inflammatory infiltrates, endothelial damage and vascular smooth muscle cell dedifferentiation. Among them, endothelial dysfunction plays a key role in the development of coronary abnormalities. Damaged endothelial cells can disrupt both endothelial barrier function and inflammatory response by releasing pro-inflammatory cytokines, ultimately destroying coronary artery structure. The main serious complication of KD is coronary artery injury, ranging from self-limiting coronary artery dilation to giant coronary aneurysms.

The standard treatment for kawasaki disease is IVIG plus aspirin, which is effective in most kawasaki patients. However, some patients are "hard bones" that are resistant to this therapeutic strategy, and this part of the patients face a much higher risk of developing coronary lesions, possibly developing coronary aneurysms. Therefore, elucidating the molecular mechanisms of KD coronary abnormalities, the development of potentially effective therapeutic drugs is important for clinical diagnosis, treatment and management of KD patients.

To date, a variety of phytochemicals and natural products have been shown to have therapeutic effects on KD, including berberine, melatonin, luteolin decoction, forsythoside B, and the like. Allicin, which is the main ingredient of garlic, can be easily absorbed through cell membranes without any damage to phospholipid bilayer and then rapidly metabolized to exert pharmacological effects. Currently, allicin has been used clinically as an antibiotic replacement therapy for the treatment of deep fungal and bacterial infections. In addition to antibacterial functions, allicin also has a wide range of other pharmacological activities, such as antidiabetic, antihypertensive and cardiovascular protective activities. However, it is not clear whether allicin can play a protective role in KD therapy.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the application of allicin in medicaments for preventing and/or treating Kawasaki disease coronary artery injury. The study of the invention proves the protection effect of allicin on KD mouse model coronary artery injury induced by candida albicans cell wall extract (CAWS). The mechanism research shows that allicin blocks JNK/c-Jun activation and EGFR/Sp1/ASM axis by activating TRP channel, and finally blocks CTSB-mediated NLRP3 inflammatory body activation, endothelial cell apoptosis and coronary artery injury.

In one aspect, the application provides application of allicin in a medicine for preventing and/or treating Kawasaki disease.

In another aspect, the application provides an application of allicin in a medicine for preventing and/or treating Kawasaki disease coronary artery injury.

In another aspect, the application provides an application of allicin in a medicament for preventing and/or treating kawasaki disease endothelial cell apoptosis.

The application firstly confirms the inhibition of allicin on cardiovascular inflammation characterized by aortic inflammation, coronary artery inflammation, endothelial cell activation and inflammatory factors in CAWS-induced KD vasculitis mouse model in vivo. NLRP3 inflammatory body mediated endothelial cell apoptosis is a key cellular mechanism of KD pathophysiology. Thus, the present application observes and verifies the inhibitory effect of allicin on endothelial cell apoptosis.

In another aspect, the application provides the use of allicin to modulate lysosomal ASM and ceramide levels, wherein the allicin reduces lysosomal ASM and ceramide levels.

The present inventors have found that the levels of total ASM and lysosomal ASM and ceramide are elevated in KD serum treated endothelial cells, while allicin significantly reduces these levels. In vitro and in vivo experiments demonstrate that allicin mediated inhibition of endothelial cell apoptosis and cardiovascular inflammation is reversed by ASM overexpression. The application discovers that allicin inhibits endothelial injury by regulating ASM expression for the first time.

In another aspect, the application provides the use of an allicin to modulate the binding of Sp1 to an SMPD1 promoter, preferably an allicin reduces the binding of Sp1 to an SMPD1 promoter.

The study data of the present application demonstrate that KD serum promotes Sp1 binding to the SMPD1 promoter, while allicin inhibits such binding. The data indicate that SP1 overexpression significantly reversed allicin-mediated down-regulation of ASM and pyrosis effector proteins GSDMD and GSDMD p. These results reveal that allicin inhibits endothelial cell apoptosis by modulating the Sp1-ASM axis.

In another aspect, the application provides a use of an allicin to modulate the level of EGFR in the nucleus, preferably the allicin reduces the level of EGFR in the nucleus.

The study of the application shows that allicin can prevent the increase of nuclear EGFR distribution caused by the increase of total EGFR level and the enhancement of nuclear translocation due to KD serum. Further experiments showed that three ligands of beta-Cellulin, TGF-alpha and HB-EGF contribute to EGFR nuclear transport under KD conditions, ASM upregulation and endothelial cell apoptosis.

In another aspect, the application provides an EGFR/Spl/ASM pathway inhibitor for use in a medicament for preventing and/or treating Kawasaki disease.

The findings of the present application indicate that nuclear interactions of EGFR and Sp1 promote ASM expression, and through a series of experiments, it is confirmed that allicin-mediated changes in EGFR and Sp1 expression are dependent on TRPV1 and TRPA1 channels, which are achieved through the JNK/c-Jun axis.

In another aspect of the application, a biomarker combination is provided for assessing coronary artery injury in a kawasaki patient, the biomarker comprising EGFR, sp1 and/or ASM.

In another aspect, the application provides a detection kit comprising the marker combination.

In another aspect, the application provides the use of the marker combination or the kit for detecting coronary artery lesions in a Kawasaki patient.

The beneficial effects of the invention include, but are not limited to:

1. the invention confirms the inhibition of allicin to coronary artery injury and endothelial cell apoptosis for the first time.

2. Experiments further prove that the garlicin can relieve the coronary artery injury or the endothelial cell apoptosis of Kawasaki disease by activating the TRPV1/A1 channel and down regulating the EGFR/Sp1/ASM axis, and the invention provides novel evidence and strongly proves that the garlicin is a very effective component for treating the coronary endothelial cell injury of the Kawasaki disease.

3. The experimental analysis of the present invention shows that EGFR, sp1 and ASM are valuable biomarkers for assessing coronary artery injury in KD patients.

Drawings

FIG. 1 is a schematic representation of a mouse model for the prevention or treatment of CAWS-induced KD by intraperitoneal injection of allicin;

FIG. 2 shows the H & E staining of endothelial cells in KD mouse model;

FIG. 3 shows immunohistochemical staining results of the endocoronary membranes in KD mouse model;

FIG. 4 shows the relative expression levels of mRNA of inflammatory-related factors in KD mouse models;

FIG. 5 is the effect of different concentrations of allicin on LDH release;

FIG. 6 is a graph showing the effect of different concentrations of allicin on CCK8 cell viability;

FIG. 7 is a graph showing the effect of allicin on endothelial cell apoptosis-related proteins;

FIG. 8 shows the result of immunofluorescent staining of Caspase-1 in endothelial cells;

FIG. 9 shows immunofluorescent staining of TUNEL in endothelial cells;

FIG. 10 shows the immunofluorescent staining of PI in endothelial cells;

FIG. 11 is a graph showing the relative mRNA expression levels of GSDMD-N over-expressing endothelial cells;

FIG. 12 is an effect of allicin on GSDMD-N expression levels in GSDMD-N overexpressing endothelial cells;

FIG. 13 shows immunofluorescent staining of TUNEL in GSDMD-N overexpressing endothelial cells;

FIG. 14 shows the result of immunofluorescent staining of PI in GSDMD-N overexpressing endothelial cells;

FIG. 15 is a graph showing the effect of allicin on inflammatory factor levels in GSDMD-N overexpressing endothelial cells;

FIG. 16 is a principal component analysis of endothelial cell RNA sequencing;

FIG. 17 is a Wen diagram of the differential gene from endothelial cell RNA sequencing;

FIG. 18 is a heat map of lysosomal associated DEG in endothelial cells;

FIG. 19 is a graph showing the effect of allicin on endothelial cell SMPD1 expression levels;

FIG. 20 shows the expression levels of ASM and ceramide in KD-treated endothelial cells;

FIG. 21 shows the expression levels of ASM and ceramide in allicin versus KD treated endothelial cells;

FIG. 22 is the effect of allicin on ASM and ceramide expression levels in KD mouse models;

FIG. 23 shows the result of immunofluorescence staining of ASM and ceramide in lysosomes;

FIG. 24 shows the mRNA levels of SMPD1 in SMPD1 overexpressing endothelial cells;

FIG. 25 is a graph showing the effect of allicin on SMPD1 expression levels in SMPD1 overexpressing endothelial cells;

FIG. 26 is a graph showing the effect of allicin on the expression of a scorch-related protein in SMPD1 overexpressing endothelial cells;

FIG. 27 shows immunofluorescence staining of Caspase-1, TUNEL and PI in SMPD1 overexpressing endothelial cells;

FIG. 28 is a graph showing immunohistochemical staining results in SMPD1 overexpressing endothelial cells;

FIG. 29 shows immunofluorescent staining of TUNEL in SMPD1 overexpressing endothelial cells;

FIG. 30 shows the immunofluorescent staining of PI in SMPD1 overexpressing endothelial cells;

FIG. 31 is a graph showing the effect of allicin on SMPD1 promoter activity;

FIG. 32 is a graph showing the prediction of possible binding sites for Sp1 and SMPD1 promoters using JASPAR database;

FIG. 33 shows luciferase activity of Sp1 over-expression plasmid transfected endothelial cells;

FIG. 34 shows a chromatin immunoprecipitation (ChIP) assay of endothelial cells transfected with the Sp1 overexpression plasmid;

FIG. 35 shows the Sp1 expression levels in endothelial cells transfected with the Sp1 overexpression plasmid;

FIG. 36 is an effect of allicin on expression of related proteins in Sp1 overexpressing plasmid transfected endothelial cells;

FIG. 37 is a schematic representation of the possible interactions of Spl with five proteins;

FIG. 38 is a nuclear profile of EGFR in treated endothelial cells;

FIG. 39 is an effect of allicin on EGFR expression in EGFR overexpressing plasmid transfected endothelial cells;

FIG. 40 is an effect of allicin on EGFR distribution in EGFR overexpressing plasmid transfected endothelial cells;

FIG. 41 is a graph showing the effect of KD treatment on beta-Cellulin, TGF-a and HB-EGF levels in THP1 cells;

FIG. 42 is the effect of KD treatment on EGF expression in THP1 cells;

FIG. 43 is the effect of neutralization of EGF ligand on total EGFR levels;

FIG. 44 is the effect of neutralization of EGF ligand on the expression of related focal death effector proteins;

FIG. 45 is a graph showing staining results for EGFR and Spl co-localization in endothelial cells;

FIG. 46 is the effect of allicin on EGFR and Spl interactions in endothelial cells;

FIG. 47 is the effect of Sp1 and EGFR on SMPD1 promoter activity;

FIG. 48 shows the results of in vitro kinase assays for purified EGFR and Sp 1;

FIG. 49 is a Westernblot results of EGFR and Spl in endothelial cells;

FIG. 50 is a Westernblot results of EGFR and Spl in endothelial cells following TRPAl or TRPV1 inhibition;

FIG. 51 shows EGFR and SP1 promoter activity in endothelial cells after treatment;

FIG. 52 is a Westernblot results of phosphorylation of JNK and c-Jun in endothelial cells following inhibition of TRPAl or TRPV 1;

FIG. 53 shows the hematoxylin-eosin (H & E) staining results of Trpvc or Trpal knockout mice;

FIG. 54 shows immunofluorescent staining of endothelial cells of Trpvc or Trpal knockout mice;

FIG. 55 shows immunofluorescent staining results of phosphorylation of JNK and c-Jun in endothelial cells of Trpvc or Trpal knockout mice;

FIG. 56 shows immunofluorescence staining of EGFR, spl and ASM in endothelial cells of Trpvl or Trpal knockout mice;

FIG. 57 shows EGFR ligand content in serum of CAWS-induced KD mice;

FIG. 58 shows hematoxylin-eosin (H & E) staining of the heart of mice after treatment;

FIG. 59 shows immunohistochemical staining of heart of mice after treatment;

FIG. 60 is the effect of focal death effector protein expression following treatment;

FIG. 61 shows the results of immunofluorescent staining of Caspase-1 and TUNEL in endothelial cells after treatment;

FIG. 62 shows EGFR, spl and ASM levels in serum of KD patients with HCs, with or without coronary lesions (CAL).

Detailed Description

The present invention is described in detail below with reference to specific embodiments, which are only for the purpose of facilitating understanding of the technical solutions of the present invention by those skilled in the art, and are not intended to limit the scope of the present invention.

In the present invention, unless specified, the raw materials, equipment and the like used are commercially available or commonly used in the art. The methods of the examples, unless otherwise specified, are all conventional in the art.

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

The application randomly selects voluntary sampling patients diagnosed as KD at a second hospital affiliated with the university of Winzhou medical science and a child-care hospital affiliated with the university of Winzhou. All enrolled KD patients met the criteria defined by the american heart association KD guidelines in 2017 and received intravenous immunoglobulin (IVIG, 2 g/kg) and oral aspirin (30-50 mg/kg/day) during the acute phase. The age and sex matched healthy control group was children receiving routine physical examination. The resulting serum samples were routinely stored at-80 ℃ for later use within 4 hours of collection. All participants were informed by written consent to use their clinical information and blood samples for academic research. The study was approved by the ethical committee of the second hospital affiliated with the university of medical science, wenzhou, child-care hospital (2023-K-41-01).

The candida albicans cell wall extract (CAWS) of the present application is obtained from candida albicans. Briefly, CAWS was cultured in CAWS-limiting medium at 27 ℃ for 2 days. Next, an equal volume of ethanol was added and placed in a 4 ℃ refrigerator overnight. Then, the culture was collected, dissolved in water and stirred for 2 hours. The soluble fraction was then harvested, mixed with an equal volume of ethanol at 4 ℃ overnight, and centrifuged again to give a precipitate. Finally, the pellet was resuspended in acetone and allowed to stand overnight. The resulting CAWS was dissolved in 1 Xphosphate buffered saline (PBS) buffer, the concentration of the CAWS solution was 20mg/ml, and autoclaved before use.

Male C57 BL/6 mice (3-4 weeks old) were purchased from the university of Winzhou medical science, license number SCXK [ ZJ ]2023-0034 and kept under standardized experimental conditions. Mice were randomly divided into PBS group, CAWS group, CAWS+ Allicin (allicin) group, CAWS+allicin+AAV 9-Smpd group, CAWS+AAV9-ICAM2-sh-Smpd 1 group, CAWS+plicamycin group and CAWS+lapatinib group. The treatment of the PBS group mice was injected intraperitoneally with 1 Xphosphate buffered saline (PBS) buffer 200 μl/mouse, 1 day for 1 day, the treatment of the CAWS group mice was injected intraperitoneally with CAWS 4 mg/mouse (i.e., CAWS solution 200 μl/mouse) 1 time a day for 1 day for 5 days, the treatment of the CAWS+allicin group was as shown in FIG. 1, the treatment of the CAWS+allicin+AAV 9-Smpd group was injected with AAV9-Smpd1 by the tail vein 3 weeks before the CAWS injection, the treatment of the CAWS+AAV9-ICAM2-sh-Smpd group was injected with AAV9-ICAM2-sh-Smpd1 by the tail vein 3 weeks before the CAWS injection, the treatment of the CAWS+plicin group was started intraperitoneally with plicin 6 days before the CAWS injection, then every other day until the end of the experiment, the treatment of the CAWS+plicin group was injected three days before the CAWS 7 by the three days after the end of the experiment.

CAWS+AAV 9-ICAM2-sh-Smpd group mice were anesthetized and sacrificed on day 28 after the last CAWS injection to harvest heart tissue and for subsequent examination. All animal experiments were conducted with the national institutes of health guidelines for animal care and use, as authorized by the institutional animal care and use professional committee (wenchw 2022-0722).

Hematoxylin-eosin (H & E) staining and immunostaining different groups of mouse hearts were fixed in 4% paraformaldehyde and embedded in paraffin. The 5 μm sections were dewaxed, hydrated and then applied to H & E staining. Briefly, heart sections were immersed in xylene and ethanol, then stained with hematoxylin for 3 minutes, then with 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%H ₂O₂ min. Next, sections were blocked with 5% Bovine Serum Albumin (BSA) at 37 ℃ for 30min, then incubated with primary antibody overnight at 4 ℃. The HRP-conjugated secondary antibody was incubated for 2h. Diaminobenzidine is used as a chromogenic substrate. All slides were counterstained with hematoxylin. The stained sections were observed under an optical microscope. For caspase-1 immunofluorescent staining, slides were dewaxed, hydrated, and blocked in PBS containing 5% BSA and 0.1% Triton X-100 at 37℃for 30min, and stained with antibodies to CD31 and caspase-1 at 4℃overnight. After incubation with these primary antibodies, the slides were washed with PBS and IgG secondary antibodies coupled to Alexa Fluor 594 and Alexa Fluor 488 were incubated for 1 hour at 37 ℃. DAPI staining was performed to visualize the nuclei. For in situ TUNEL staining, similar to caspase-1/CD 31 co-staining, slides were first incubated with CD31 primary antibody, then with a mixture of AlexaFluor 594 conjugated IgG secondary antibody and TUNEL solution. These immunofluorescence images were examined using LEICA STELLARIS confocal microscopy (Leica). Fluorescence intensities were analyzed using Image J blinding, and the Mean Fluorescence Intensity (MFI) was the mean of the fluorescence intensities of the same group of different sections.

Heart tissue or cells are collected. Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse transcribed using PRIMESCRIPT ^TM RT kit (Takara) to obtain cDNA. Next, qPCR was performed on Applied Biosystems QuantStudio 3 real-time PCR system (thermo fisher) using Power TB green PCR MASTER Mix (Takara) with 40 cycles. GAPDH was used as an internal control, and the values of all target genes were normalized to GAPDH. mRNA levels were calculated by the relative quantification method of 2 ^-ΔΔCT. The primers used in this study are shown in Table 1.

TABLE 1 primers used in this study

Human Umbilical Vein Endothelial Cells (HUVEC) and human monocytic leukemia cell line THP1 were purchased from american type culture collection (ATCC, manassas, VA, USA). These cells were identified by GENETIC TESTING Biotechnology Corporation (su, 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 for the diffusion of soluble molecules. Serum from Healthy Control (HC) or KD patients was then added to the upper chamber and THP1 cells were treated for 24 hours. At the same time, the diffusing molecules also affect the HUVEC in the lower chamber.

Western blot analysis total protein of heart tissue or endothelial cells was extracted using protein extraction reagents. With BCA proteins

The assay kit detects protein concentration. Then, an equal amount of protein was separated on 10% or 12% SDS-PAGE gel and electrotransferred onto PVDF membrane. After blocking with 5% skim milk for 2 hours, the membranes were incubated overnight at 4 ℃ (1:1000;ABclonal Technology,A16672), anti-ASM antibody (1:1000;Affinity BioReagents,DF 13384), anti-caspase zymogen-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; st. KluyI Biotechnology, sc-393581), anti-IL 1 beta 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-SP 1 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 total protein of heart tissue or endothelial cells, and total protein was extracted using protein extraction reagents. Protein concentration was measured using BCA protein assay kit. Then, an equal amount of protein was separated on 10% or 12% SDS-PAGE gel and electrotransferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk for 2 hours, the membranes were incubated overnight at 4 ℃ (1:1000;ABclonal Technology,A16672), anti-ASM antibody (1:1000;Affinity BioReagents,DF 13384), anti-caspase zymogen-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; st. KluyI Biotechnology, sc-393581), anti-IL 1 beta 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-SP 1 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 ChemiDocTM XRS + imaging system (Bio-Rad Laboratories, hercules, calif., USA) and band densities were quantified using MultiGauge software of ImageJ (National Institutes ofHealth, bethesda, USA) bound to Java 1.8.0_112.

Immunofluorescent staining of endothelial cells collected endothelial cells were fixed in 4% paraformaldehyde for 30 min and at 37 °

Blocking with 5% BSA for 30min at C. The cell-designated primary antibody was then incubated overnight at 4 ℃. Cells were then washed in PBS and incubated with Alexa Fluor 488 or 594 conjugated IgG secondary antibody (ThermoFisher Scientific) for 1 hour at 37 ℃. Finally, DAPI staining was performed to label the nuclei. The images were observed under an immunofluorescence confocal microscope (LEICA STELLARIS).

ELISA (enzyme-linked immunosorbent assay) using human ASM ELISA kit (JL 12600) and human ceramide, respectively

ELISA kit (JL 19781) detects ASM and ceramide content in HC and KD serum and in treated endothelial cell supernatant. To detect the levels of EGF, HB-EGF, TGF-alpha and beta-cellulose in the supernatant of treated THP 1 cells, experiments were performed as follows. Specifically, THP 1 cells were treated with HC or KD serum for 24 hours. Next, the washed THP 1 cells were further incubated for the following 24 hours. Levels of EGF, HB-EGF, TGF-alpha and beta-Cellulin in THP 1 cell culture supernatants were detected using human EGF ELISA kit (JL 10101), human HB-EGF ELISA kit (JL 11929), human TGF-alpha ELISA kit (JL 10974) and human beta-Cellulin ELISA kit (JL 10994), respectively. Sp1 and EGFR concentrations in serum of HC and KD patients were measured using the human Sp1 ELISA kit (JL 14476) and the human EGFR ELISA kit (JL 14169), respectively. All these ELISA kits were purchased from Shanghai Jiang Lai Utility Co., ltd (Shanghai, china).

Luciferase reporter assay HUVEC were inoculated in 24-well plates and used with pGL3-Basic vector, pGL3-

The SMPD1 promoter, PGL3-SMPD1 promoter mSp, pGL3-SP1 promoter or pGL3-EGFR promoter. After the corresponding treatment of the transfected cells, the double luciferase activity (firefly/Renilla luminescence ratio) was measured using a double luciferase reporter assay system (Promega).

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

QPCR was performed using ChamQ Universal SYBR QPCR MASTER Mix (Vazyme). The SMPD 1 forward primer of SP1 was 5 'CTTCCACCGACACACATCACA-3' and the reverse primer was 5 'GACTCTCCCTGAGGTCACA-3'. qRT-PCR was performed using Applied Biosystems QuantStudio real-time PCR system (ThermoFisher).

Nuclear separation experiments based on nuclear separation products (Nuclear and Cytoplasmic Protein Extraction Kit,

Beyotime Biotechnology, P0027) for nuclear mass isolation. Specifically, the treated endothelial cells were collected, treated with cytoplasmic protein extractant A supplemented with PMSF, then vigorously vortexed for 5 seconds, and placed in an ice bath for 10-15 minutes. Then, cytoplasmic protein extractant B was added. After vortexing vigorously for 5 seconds and centrifuging at 16000r at 4 ℃ for 5 minutes, the supernatant was pipetted into a pre-chilled plastic tube for extraction of cytosolic proteins (without contacting the bottom pellet). After the supernatant was aspirated, a PMSF-containing nucleoprotein extraction reagent was added to the pellet, vortexed vigorously for 15s, then ice-bath for 1min, and vortexing continued for 15s. The vortex and ice bath procedure was repeated for 30min. Finally, the supernatant was centrifuged at 16000r for 10min at 4 ℃ to obtain nucleoprotein.

Immunoprecipitation by combining about 200. Mu.g of endothelial cell nuclear or cytoplasmic protein with 5. Mu.g of anti-Sp 1 antibody (ab

157123, Abcam), anti-EGFR antibody (ab 52894, abcam) or IgG control was incubated overnight at 4℃and then 25 μl protein A/G agarose beads (Epizyme Biotech) were added and incubated overnight at 4 ℃. The precipitate was then washed and boiled in SDS sample buffer. The supernatants were immunoblotted with anti-Sp 1 antibodies (1:1000;Cell Signaling Technology,9389S) or anti-EGFR antibodies (1:5000; proteintech, 66455-1-IG).

In vitro kinase assay A mixture of recombinant human EGFR kinase domain (# 81271,Active Motif), recombinant human SP1 protein (# 81181,Active Motif), 200 μMATP (# 9804,Cell Signaling Technology), 10 Xkinase buffer (# 9802,Cell Signaling Technology) and gamma-32P-ATP was incubated at 30℃for 15min for phosphorylation. The reaction was stopped by adding loading buffer and incubated at 95 ℃ for 10min. Proteins in the mixture were immediately separated using SDS-polyacrylamide gel electrophoresis (PAGE) and detected by Coomassie blue staining and autoradiography.

Construction and in vivo delivery of adeno-associated virus (AAV) all AAV vectors are composed of Vigene Biosciences (Shandong,

China) construction. For in vivo knockdown Smpdl, AAV 9-RGDLRVS-ICAM 2-miR30-sh-Smpdl was designed and constructed using standard methods. The oligonucleotide sequences were used for gene knockdown 5'-TGGAGCTGGAATTATTACA-3'. To construct an AAV overexpression vector for ASM, the full-length sequence of the mouse Smpd1 gene was inserted into the pAAV9-CMV-P2A-Flag vector to construct the pAAV 9-CMV-P2A-Smpd-Flag plasmid. Immunofluorescence was used to detect the knockdown and overexpression efficiency of each sequence. A total of 1012 viral particles were injected into mice via the tail vein.

Statistics the above examples were analyzed statistically using GRAPHPAD PRISM 8.0.0 software. All data are expressed as mean ± Standard Error of Mean (SEM). The data distribution normalization was checked using the Shapiro-Wilk test. For comparison between the two groups, the F test was applied to determine the variance alignment. If the data variances are equal, the unpaired Student t test is used. Otherwise, a Welch t-test was used. When the data is not normally distributed, the inter-group comparison is verified with Mann-Whitney U. For comparisons between three or more groups, the variance alignment was assessed using the Brown-Forsythe test. If the data passes the isovariational test, a one-way ANOVA analysis is used followed by Bonferroni post-hoc test, otherwise, welchANOVA tests are performed followed by post-hoc reporting using the Tamhane T2 method. To compare the effect of the two variables, two-way ANOVA was used, followed by Bonferroni post hoc analysis.

EXAMPLE 1 investigation of the Effect of allicin on coronary artery injury

To preliminary analyze the effect of allicin on KD coronary artery injury, allicin was intraperitoneally injected into candida albicans cell wall extract (CAWS) -induced KD mouse models as shown in fig. 1. HE staining results showed that treatment with allicin significantly reduced coronary inflammation (figure 2). It is well known that vascular cell adhesion molecule 1 (VCAM-1) acts as a marker of endothelial cell activation and plays an important role in mediating the recruitment of many leukocytes to damaged vascular endothelium. Thus, the present application utilizes Immunohistochemical (IHC) analysis to detect changes in the expression of VCAM-1. Consistent with previous studies, CAWS significantly increased expression levels of VCAM-1 in the coronary intima, with an increase in inflammatory cell infiltration including neutrophils and macrophages (fig. 3). In addition, the expression levels of inflammatory-related factors, including cytokines (IL-1β, TNF- α, and IL-6), adhesion molecules (VCAM-1 and E-selectin), and chemokines (MCP 1, CCL3, and CCL 5) were significantly elevated in the KD mouse model (FIG. 4). However, allicin significantly reduced endothelial VCAM-1 expression levels, decreased inflammatory cell infiltration, down-regulated expression of inflammatory-related factors, indicating that allicin plays a protective role in KD coronary artery injury.

EXAMPLE 2 investigation of the Effect of allicin on endothelial cell apoptosis

The application first determines the optimal allicin concentration by CCK8 and LDH release analysis. As shown in FIGS. 5-6, 10ug/ml allicin showed the best therapeutic effect. In subsequent experiments, this concentration was chosen to pretreat endothelial cells. The results showed that KD-mediated upregulation of focal death-related proteins, including CTSB, NLRP3, ASC, caspase-1, GSDMD, GSDMD p, IL-1β and IL-18, was significantly reversed by garlicin (FIG. 7). In addition, the increased Caspase-1 fluorescence intensity and percentage of TUNEL or PI positive cells in KD groups was significantly reduced by allicin (fig. 8-10). These results indicate that allicin may act by inhibiting endothelial cell apoptosis.

To further confirm this, endothelial cells were transfected with empty vector (negative control) or plasmid containing GSDMD-N gene. The data shows that GSDMD-N over-expression plasmid significantly up-regulated GSDMD-N expression (fig. 11) and eliminated allicin-mediated downregulation of GSDMD-N (fig. 12). In addition, GSDMD-N overexpression significantly abrogated the inhibition of endothelial apoptosis by allicin, such as increased percentage of TUNEL and PI positive cells, increased LDH, IL-1 β and IL-18 release (fig. 13-15). These results indicate that allicin is able to inhibit endothelial cell apoptosis.

Example 3 mechanism of action of allicin to inhibit endothelial cell apoptosis

To explore the underlying mechanism, RNA-seq was performed on treated endothelial cells. The present application first uses Principal Component Analysis (PCA) to obtain a lower dimensional feature set (fig. 16), followed by wien diagram analysis, which shows 1032 Differentially Expressed Genes (DEGs) in both KD vs HC and kd+allicin vs KD (fig. 17). Considering the release of CTSB from lysosomes, lysosomal related DEG is shown in the heat map, which includes SMPD1 (fig. 18). SMPD1 encoded Acid Sphingomyelinase (ASM) is capable of liberating ceramide from sphingomyelin. The resulting ceramide will trigger lysosomal CTSB activation and cytoplasmic release, ultimately leading to XIAP degradation in natural killer/T lymphoma cell pyrosis or NLRP3 inflammatory platelet activation in endothelial cell pyrosis. Thus, the present application assumes that allicin-mediated KD-induced inhibition of pyrosis is associated with SMPD 1. The present application first validated the changes in SMPD1 expression at the mRNA and protein levels (fig. 19). ASM and ceramide levels were then assessed in KD-treated endothelial cells with or without allicin treatment. The results showed that the levels of ASM and ceramide increased significantly in KD-treated endothelial cells and decreased after allicin treatment (fig. 20). Endothelial ASM and ceramide expression were also significantly increased over PBS groups in the CAWS-induced KD mouse model. However, allicin treatment significantly reduced their expression levels (fig. 21). ASM is known to act upon lysosomes and outer leaflets of plasma membranes after activation. Only accumulation of lysosomal ceramides breaks down the stabilization of lipid bilayer, ultimately leading to enhancement of lysosomal membrane permeability and release of CTSB into the cytosol. Thus, the present application analyzes the expression of ASM and ceramide in lysosomes. The results showed that lysosomal ASM and ceramide levels were significantly increased in KD group compared to HC group and significantly decreased after allicin treatment (fig. 22-23). These results indicate that allicin reduced lysosomal ASM and ceramide levels.

To further confirm whether allicin inhibited endothelial cell apoptosis by modulating ASM, endothelial cells were transfected with either empty plasmid (negative control) or SMPD1 overexpression plasmid, which significantly upregulated the mRNA level of SMPD1 (fig. 24), reversing allicin-mediated downregulation of SMPD1 at mRNA and protein levels (fig. 25). Moreover, SMPD1 upregulation abrogated the allicin-mediated reduction of focal death-related proteins, including CTSB, NLRP3, ASC, caspase-1, GSDMD, GSDMD p, IL-1β and IL-18 (fig. 26), significantly reversing the allicin-mediated decrease in Caspase-1 expression, percentage of TUNEL and PI positive cells, and LDH release (fig. 27). These results indicate that allicin inhibits endothelial cell apoptosis by inhibiting ASM expression. To confirm this in vivo, AAV9-Smpd1 was injected into an allicin-treated KD mouse model via the tail vein. The results show that Smpd1 overexpression significantly reversed allicin-mediated inhibition of coronary inflammation (fig. 28). Furthermore, smpd1 upregulation significantly increased the expression of the pyrosis-related protein, the immunofluorescence intensity of Casapase-1 and the percentage of TUNEL positive endothelial cells compared to the caws+allicin group (fig. 29-30). Taken together, these data indicate that allicin ameliorates KD coronary endothelial injury by modulating ASM expression.

The above data indicate that allicin can reduce SMPD1 expression at both mRNA and protein levels. To further confirm whether allicin regulates SMPD1 expression by the transcriptional mechanism, the SMPD1 promoter activity was detected using a luciferase reporter system. The results showed that the SMPD1 promoter activity increased in the KD group, which decreased after allicin treatment (fig. 31). To predict potential transcription factors that could modulate SMPD1 expression, animalTFDB v 4.0.0 database (http:// guolab.wchscu.cn/AnimalTFDB 4/#) was used to screen for specific protein 1 (Sp 1) with the highest score (Supplementary Table 4). The JASPAR database (https:// jaspar. Geneg. Net /) was then used to predict the likely binding sites for Sp1 and SMPD1 promoters (FIG. 32). Next, endothelial cells were transfected with pGL 3-luciferase construct containing the mutated Sp1 binding site. The data show that mutation of the Sp1 binding site in the SMPD1 promoter significantly reduced luciferase activity in the HC and KD groups (fig. 33), revealing that Sp1 binding is necessary to activate SMPD1 promoter activity. To demonstrate whether allicin down-regulates SMPD1 transcription by Sp1, a chromatin immunoprecipitation (ChIP) assay was performed. The ChIP-qPCR results showed that KD serum increased binding of Sp1 to SMPD1 promoter, which was reduced after allicin treatment (fig. 34). To further demonstrate the role of Sp1 in allicin-mediated inhibition of pyrosis, sp1 was overexpressed in endothelial cells. The data show that Sp1 over-expression plasmids significantly reversed allicin-mediated reduction of Sp1 expression at mRNA and protein levels (fig. 35-36). In addition, over-expression of Sp1 significantly increased protein expression of ASM, pyro-apoptosis effectors GSDMD and GSDMD p compared to kd+allicin group (fig. 36), indicating that allicin inhibits endothelial pyro-apoptosis by Sp1.

The above findings demonstrate that Spl can bind to and regulate expression of the SMPD1 promoter, which is inhibited by allicin. Thus, it is speculated that allicin may inhibit this process by certain proteins. To investigate it, the present application uses SwissTargetPrediction database (http:// www.swisstargetprediction.ch /) to sort the potential targets of allicin and selects 26 targets because of their probability >0 (Excel FILE I IN THE DATA support). Next, protein-protein interactions (PPI) between Spl and 26 targets of allicin were evaluated using the STRING database (https:// cn. STRING-db. Org). The data show that Spl may interact with five proteins, including CASP3, CA9, EGFR, KDR and PARPI (fig. 37). Considering Spl as a nuclear transcription factor, the protein of choice should be able to enter the nucleus. Studies have shown that Spl is a substrate protein for CASP3, and that CASP3 can cleave Sp1. However, it was reported that expression of CASP was inhibited by allicin, which may affect CASP 3-mediated Sp1 cleavage, resulting in upregulation of Spl. This is not consistent with the results of the present application. Carbonic anhydrase-9 (CA 9) is a classical marker of hypoxia, involved in the catalytic hydration of carbon dioxide to carbonic acid, mainly located in serosa. Current studies indicate that CA expression is regulated by Spl, downstream of Spl. Thus, CASP and CA are excluded. Both EGFR and KDR (VEGFR 2) are membrane receptors that, under certain conditions, can migrate to the nucleus to regulate gene transcription. Previous studies have shown interactions between VEGFR2 and Spl. To initially analyze which proteins might interact with Spl under KD conditions, immunofluorescence assays were performed to observe nuclear localization of VEGFR2 and EGFR. Observations showed that the nuclear distribution of EGFR was significantly increased, while allicin was significantly decreased in KD-treated endothelial cells (fig. 38). However, KD serum hardly induced nuclear localization of VEGFR 2. Therefore, VEGFR2 is also not considered. PARP1 is a ribozyme whose transcription is dependent 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 over-expressed, respectively. The results indicate that EGFR over-expressed plasmids significantly increased EGFR expression at mRNA and protein levels and significantly reversed allicin-mediated ASM down-regulation compared to kd+allicin group (fig. 39). However, PARP1 overexpression does not exclude allicin-mediated reduction of ASM expression. These results indicate that allicin regulates ASM expression through EGFR.

To further investigate how EGFR functions, the present application examined the level of EGFR in the nucleus. The data indicate that the distribution of EGFR in the nucleus increases in KD groups and decreases after treatment with allicin (fig. 40). To rule out the effect of increased total EGFR expression on increased intra-nuclear EGFR, the present application detects the percentage of nuclear EGFR to total EGFR. The data show that the percentage of nuclear EGFR in KD group is increased compared to HC group, while allicin treatment is significantly reduced. These results indicate that KD-induced increases in nuclear EGFR levels are due to an increase in total EGFR levels and an increase in nuclear metastasis, both of which can be inhibited by allicin.

Internalization and translocation of EGFR to the nucleus is reported to be dependent on ligand binding. Ligands known to mediate nuclear translocation thereof include β -Cellulin, TGF-a, HB-EGF and EGF. To explore which ligands play a role in EGFR translocation, levels of these EGFR ligands in THP1 supernatants after KD serum treatment were examined. The results showed that the content of β -Cellulin, TGF-a and HB-EGF in THP1 cell supernatant was significantly increased after treatment with KD serum (FIG. 41). However, there was no significant difference in EGF between KD and HC groups (fig. 42). To further confirm that EGFR nuclear distribution is associated with these ligands, neutralizing antibodies to beta-Cellulin, TGF-a and HB-EGF were used, respectively. The results show that all three neutralizing antibodies significantly reduced nuclear EGFR expression compared to KD set, while these three antibodies did not alter total EGFR levels (fig. 43), suggesting that the nuclear distribution of EGFR is dependent on the presence of its ligand. Next, the present application observes the effect of three ligands on ASM expression and pyrosis. The data show that neutralization of these ligands significantly reduced the expression levels of ASM, as well as the focal death effector proteins GSDMD and GSDMD p (fig. 44). These data indicate that monocyte-produced EGFR ligands, including HB-EGF, TGF-a and beta-Cellulin, promote EGFR translocation to the nucleus and are involved in regulating ASM expression and endothelial cell apoptosis under KD conditions. All these results indicate that allicin relieves endothelial cell apoptosis through EGFR.

To explore how EGFR regulates ASM expression by Spl, the present application first observed co-localization of EGFR and Sp 1. As expected, EGFR and Spl were co-localized in the nucleus (fig. 45). Next, nucleoprotein was extracted and subjected to co-immunoprecipitation analysis. The data show that EGFR was immunoprecipitated with anti-Sp 1 antibodies, but not control IgG, and vice versa. Furthermore, there was a stronger interaction between EGFR and Spl in KD groups, which interaction could be reduced by allicin (fig. 46). To further demonstrate whether interaction of EGFR with Spl would affect SMPD1 transcription, endothelial cells were transfected simultaneously with pGL3-luciferase construct containing SMPDI promoter and plasmid containing Spl, EGFR or both. Next, cells were stimulated with 100ng/ml EGF for 24 hours prior to the experiment. The data show that luciferase activity is increased by Spl, and further increased by spl+egfr. However, EGFR alone had little effect on photochemical enzyme activity (fig. 47). These results indicate that interaction of nuclear EGFR and Spl promotes Spl-mediated SMPD1 expression. It is well known that Spl phosphorylation can enhance its mediated transcription. EGFR has been reported to be capable of modulating protein phosphorylation containing tyrosine residues, such as PCNA and FAM129B. Thus, EGFR is presumed to act by phosphorylating the tyrosine residue of Spl, ultimately promoting the expression of SMPD 1. 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 Spl.

The above results indicate that allicin improves endothelial cell thermalization by modulating the EGFR/Spl/ASM axis. However, how allicin modulates EGFR and Spl expression remains unclear. Allicin is reported to be capable of activating TRP channels, including TRPV1 and TRPA1. Whether allicin-mediated changes in EGFR and Spl expression are associated with both channels was initially explored, and Ruthenium Red (RR) was used to inhibit both channels. The data show that TRPV1/Al inhibition significantly reversed allicin-mediated reduction in EGFR and Spl expression (fig. 49). To investigate which channel is the TRP acting, TRPA1 and TRPV1 were inhibited by HC030031 and AMG9810, respectively. The results show that TRAl or inhibition of TRPV1 significantly increased protein levels of EGFR and Spl compared to kd+allicin group, suggesting that both TRPA1 and TRPV1 are involved in the mediated down-regulation of EGFR and Spl by allicin (figure 50). To investigate whether changes in EGFR and Spl expression are regulated by transcriptional mechanisms, the present application examined EGFR and SP1 promoter activity. As expected, the EGFR and SP1 promoter activity of KD groups increased and their activity could be reduced by allicin. However, HC030031 and AMG9810 significantly increased their promoter activity (fig. 51). It is well known that the promoters of both EGFR and SPl can be bound and regulated by c-Jun. Furthermore, 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 mediated by the JNK/c-Jun axis. The present application first demonstrates that EGFR and Spl expressions are indeed modulated by the JNK/c-Jun axis under KD conditions. It was then observed by the present application that allicin significantly reduced the levels of phosphorylation of JNK and c-Jun, which in turn could be reversed by TRPV1 or inhibitors of TRPA1 (fig. 52). These results indicate that allicin down-regulates JNK/c-Jun axis mediated EGFR and Spl expression through TRPV1/A1 channels.

To confirm in vivo that allicin plays a protective role through TRPV1/A1 channel, cdh5-Cre mice were infected with AAV-DIO-Trpv-shRNA or AAV-DIO-Trpa-shRNA. The knockout of endothelial Trpvl or Trpal significantly reduced the protective effects of allicin, such as increased inflammatory regions, increased endothelial Caspase-1 expression, and increased TUNEL-positive endothelial cells (FIGS. 53-54). Furthermore, the levels of phosphorylation of JNK and c-Jun were also significantly increased for EGFR, spl and ASM compared to caws+allicin (fig. 55-56). All these results indicate that allicin inhibits EGFR/Spl/ASM pathway mediated endothelial cell thermalization by activating the TRPV1/A1 channel.

Example 4 EGFR/Sp1/ASM pathway was involved in the study of potential diagnostic markers that might be KD coronary abnormalities

The above studies illustrate the molecular mechanism by which allicin plays a therapeutic role in KD coronary lesions. During this process, the above data also indirectly demonstrate that EGFR, spl and ASM are involved in KD endothelial injury. As described above, nuclear translocation and subsequent endothelial apoptosis of EGFR depends on the presence of EGFR ligands. To investigate whether the EGFR/Sp1/ASM axis plays an important role in KD coronary lesions, the present application first examined the amount of EGFR ligand, including EGF, HB-EGF, TGF-a and B-Cellulin, in mouse serum after PBS and CAWS treatment. ELISA results showed that the serum levels of EGF, HB-EGF, TGF-a and B-Cellulin were significantly increased in the KD group mice compared to the PBS group mice (FIG. 57). CAWS-induced KD mice were then intravenously injected with AAV9-ICAM2-sh Smpdl to specifically interfere with endothelial Smpdl expression, either with Spl inhibitors (primycin) or with EGFR inhibitors (lapatinib). The results indicate that interference with ASM, spl or EGFR significantly reduced coronary inflammation, reduced VCAM-1 expression and neutrophil and macrophage infiltration compared to the CAWS group (fig. 58-59). Furthermore, interference with these proteins significantly reduced ASM, GSDMD, and GSDMD p expression, caspase-1 fluorescence intensity, and TUNEL positive cell percentages (fig. 60-61), indicating significant reduction in focal death of coronary endothelial cells. These results demonstrate that EGFR/Spl/ASM pathway mediated endothelial cell apoptosis is involved in KD coronary abnormalities.

To further confirm whether the EGFR/Spl/ASM pathway can serve as a biomarker for clinical KD coronary abnormalities, EGFR, spl and ASM levels in serum of KD patients with or without coronary lesions (CAL) were also tested and the results are shown in FIG. 62. The results indicate that the levels of the three proteins in KD without CAL are significantly increased compared to HC. Furthermore, the levels of the remaining proteins, except ASM, were further elevated in the serum of KD patients with CAL compared to KD patients without CAL. We then plotted the subject characteristics (ROC) curve and calculated the area under the curve (AUC) (auc=1 represents the highest correlation). Sp1, EGFR and ASM levels were able to distinguish between KD patients (NCAL and CAL groups) and HC, spl with an AUC value of 0.902 (95% Confidence Interval (CI), 0.845-0.959), EGFR with a value of 0.878 (95% CI, 0.787-0.969) and ASM with a value of 0.884 (95% CI, 0.816-0.953). The calculated AUCs for Sp1, EGFR and ASM to distinguish CAL and NCAL groups 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 pathology. All these results indicate that Sp1, EGFR and ASM are both biomarkers of the pathology of coronary artery disease and are involved in the coronary pathology.

The application of the allicin in the medicine for preventing and treating Kawasaki disease provided by the invention is described in detail. The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to facilitate an understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several modifications and adaptations of the invention can be made without departing from the principles of the invention, and such modifications and adaptations fall within the scope of the invention as defined in the following claims.

Claims (10)

1. Application of allicin in medicine for preventing and/or treating Kawasaki disease is provided.

2. Application of allicin in preparing medicine for preventing and/or treating Kawasaki disease coronary artery injury is provided.

3. Application of allicin in medicine for preventing and/or treating kawasaki disease endothelial cell apoptosis is provided.

4. Use of allicin for modulating lysosomal ASM and ceramide levels, wherein allicin reduces lysosomal ASM and ceramide levels.

5. Use of allicin to modulate the binding of Sp1 to an SMPD1 promoter, wherein the allicin reduces the binding of Sp1 to the SMPD1 promoter.

6. Use of allicin to modulate the level of EGFR in the nucleus, wherein the allicin reduces the level of EGFR in the nucleus.

Use of an inhibitor of the egfr/Spl/ASM pathway in a medicament for the prevention and/or treatment of kawasaki disease.

8. A biomarker combination for assessing coronary artery injury in a kawasaki patient, the biomarker comprising EGFR, sp1 and/or ASM.

9. A test kit comprising the marker combination of claim 8.

10. Use of the marker combination of claim 8 or the kit of claim 9 for detecting coronary lesions in a patient suffering from kawasaki.