Use of dicarbonyl / l-xylulose reductase as a target in screening and / or preparing a drug for treating kawasaki disease

Abstract

The application provides application of dicarbonyl / L-xylulose reductase as a target in screening and / or preparation of a medicine for treating Kawasaki disease, in particular, Kawasaki disease without response to immunoglobulin G and / or Kawasaki disease with coronary artery lesions; further, the medicine has at least one of the following functions: increasing expression level of dicarbonyl / L-xylulose reductase, enhancing activity of dicarbonyl / L-xylulose reductase, promoting L-xylulose metabolism or improving function of glucuronate metabolism pathway. The application firstly finds that dicarbonyl / L-xylulose reductase is closely related to occurrence of Kawasaki disease without response to immunoglobulin G, provides a brand-new metabolic treatment target for Kawasaki disease, provides a new use of dicarbonyl / L-xylulose reductase as a treatment target for Kawasaki disease, and provides a new treatment scheme for Kawasaki disease without response to immunoglobulin G.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and molecular medicine, and relates to the metabolic regulation mechanism and targeted treatment strategy of Kawasaki disease (KD). In particular, it relates to the application of a dicarbonyl / L-xylitol reductase as a target in the screening and / or preparation of drugs for the treatment of Kawasaki disease. Background Technology

[0002] Kawasaki disease (KD) is an acute febrile illness that primarily affects children under 5 years old, characterized by systemic inflammation of small and medium-sized blood vessels; the coronary arteries are the most commonly affected target organ. In recent years, KD has become one of the leading causes of acquired cardiovascular disease in children. If left untreated, it can further develop into coronary artery dilation, coronary aneurysm, thrombosis, myocardial ischemia, and even sudden death. Therefore, controlling the inflammatory response and preventing coronary artery damage as early as possible has always been a key focus of clinical treatment.

[0003] Currently, high-dose intravenous immunoglobulin (mainly composed of IgG) is the first-line treatment for Kassym-Jowls (KD). The vast majority of KD children experience rapid fever reduction and a significant decrease in the incidence of coronary artery disease after their first IgG treatment. However, approximately 15%–25% of children still experience persistent fever or recurrence of fever within 36 hours after the completion of standard IgG treatment; these are defined as IgG-unresponsive KD. In recent years, epidemiological surveys have consistently indicated a gradually increasing incidence of IgG-unresponsive KD. These children have a significantly higher risk of developing coronary artery aneurysms, giant coronary artery aneurysms, and long-term cardiovascular complications compared to IgG-sensitive children, making this one of the most pressing issues to be addressed in the field of precision medicine for KD.

[0004] Current treatments for IgG-unresponsive Karnofsky Performance Status (KD) mainly include re-infusion of IgG, glucocorticoids, TNF-α inhibitors, immunosuppressants, and plasma exchange. While some patients achieve some efficacy, the overall efficacy is poor. Furthermore, repeated IgG therapy increases the incidence of hemolytic reactions and medical costs; long-term use of glucocorticoids can lead to immunosuppression, abnormal growth and development, and metabolic disorders; TNF-α inhibitors and immunosuppressants are expensive and have limited applicability; and plasma exchange is highly invasive and not suitable for widespread use.

[0005] Studies have shown that the occurrence of KD and IgG non-responsive KD is closely related not only to host genetic background, immune regulatory imbalance, and oxidative stress, but also to host metabolic disorders. Furthermore, metabolomics studies suggest that phenylalanine metabolism, tryptophan metabolism, arginine metabolism, and the pentose phosphate pathway may all be closely related to the occurrence of KD. Phenylalanine metabolism, in particular, may be closely related to the development of gamma globulin non-responsive KD. However, the mechanism of gamma globulin non-responsive KD remains unclear, and no key metabolites have been identified that can be directly used as therapeutic targets.

[0006] Dicarbonyl / L-xylulose reductase (DCXR) is an important oxidoreductase involved in the glucose-pentose phosphate pathway. It is highly expressed primarily in the liver and can catalyze the reduction of L-xylulose to xylitol in a NADPH-dependent manner, thereby maintaining L-xylulose homeostasis and linking glucuronide metabolism with pentose phosphate metabolism. In addition to its role in glucose metabolism, DCXR also has biological functions such as scavenging α-dicarbonyl compounds, reducing the formation of advanced glycation end products (AGEs), and mitigating oxidative stress damage. Therefore, it is considered a protective metabolic enzyme.

[0007] Current research on DCXR mainly focuses on its biological functions in diseases such as diabetes, liver fibrosis, kidney injury, reproductive system disorders, and tumors. It is generally believed that decreased DCXR expression can lead to increased oxidative stress, metabolic homeostasis imbalance, and aggravated inflammatory response; conversely, increasing DCXR expression can help improve metabolic abnormalities and reduce tissue damage. Summary of the Invention

[0008] The purpose of this invention is to solve the technical problems of existing intensified treatments for Kawasaki disease that lacks a clear therapeutic target and has limited efficacy and significant side effects.

[0009] To address the aforementioned technical problems, embodiments of the present invention disclose the application of dicarbonyl / L-xylitol reductase as a target in screening and / or preparing drugs for treating Kawasaki disease.

[0010] According to another specific embodiment of the present invention, the application disclosed in the embodiments of the present invention includes Kawasaki disease that is unresponsive to gamma globulin and / or Kawasaki disease with coronary artery lesions.

[0011] According to another specific embodiment of the present invention, the application disclosed in the embodiments of the present invention includes a drug comprising a first formulation having at least one of the following functions: increasing the expression level of dicarbonyl / L-xylulose reductase, enhancing the activity of dicarbonyl / L-xylulose reductase, promoting L-xylulose metabolism, or improving the function of the glucuronic acid metabolism pathway.

[0012] According to another specific embodiment of the present invention, the application disclosed in the embodiments of the present invention, a first formulation having the function of improving the expression level of dicarbonyl / L-xylitol reductase includes at least one of: dicarbonyl / L-xylitol reductase protein, a recombinant expression vector encoding dicarbonyl / L-xylitol reductase, an mRNA formulation, a DNA expression vector, an adeno-associated virus vector, an adenovirus vector, a lentiviral vector, and a lipid nanoparticle delivery system.

[0013] According to another specific embodiment of the present invention, the application disclosed in the embodiments of the present invention includes at least one of the following: natural dicarbonyl / L-xylulose reductase protein, recombinant dicarbonyl / L-xylulose reductase protein, and protein having a functional fragment with dicarbonyl / L-xylulose reductase activity.

[0014] According to another specific embodiment of the present invention, the application disclosed in the embodiments of the present invention further includes a second formulation, the second formulation including at least one of gamma immunoglobulin, glucocorticoids, and aspirin; the first formulation and the second formulation are used in combination.

[0015] According to another specific embodiment of the present invention, the application disclosed in the embodiments of the present invention further includes at least one of a pharmaceutically acceptable carrier, excipient, diluent, stabilizer, and buffer.

[0016] According to another specific embodiment of the present invention, the dosage form of the drug disclosed in the embodiments of the present invention is at least one of injection, tablet, capsule, granule, powder, oral liquid, and inhaler.

[0017] According to another specific embodiment of the present invention, the application disclosed in the embodiments of the present invention includes at least one of injection administration, oral administration, and nebulized administration of the drug.

[0018] Beneficial technical effects of the present invention:

[0019] This invention is the first to discover a close correlation between dicarbonyl / L-xylitol reductase and the occurrence of gamma globulin-unresponsive Kawasaki disease. Furthermore, inhibiting dicarbonyl / L-xylitol reductase directly affects the therapeutic effect of gamma globulin, providing a novel metabolic therapeutic target for Kawasaki disease and offering a new application for dicarbonyl / L-xylitol reductase as a therapeutic target, particularly for gamma globulin-unresponsive Kawasaki disease. This invention not only expands the novel biological functions of dicarbonyl / L-xylitol reductase and provides a new targeted therapeutic strategy for Kawasaki disease, but also possesses significant scientific value, clinical application value, and industrial transformation prospects. Attached Figure Description

[0020] Figure 1 This is a bar chart showing the quantitative analysis results of L-xylulose concentration in the serum of IgG-sensitive and IgG-unresponsive children with KD in Example 1 of the present invention.

[0021] Figure 2 This is a bar chart showing the concentration of L-xylulose in the serum of normal mice, KD mice, IgG-sensitive KD mice, and IgG-unresponsive KD mice in Example 1 of the present invention.

[0022] Figure 3 This is a diagram showing the results of Western blot analysis of DCXR protein expression in the livers of normal mice, KD mice, IgG-sensitive KD mice, and IgG-unresponsive KD mice in Example 2 of this invention (where β-ACTIN (β-actin) was used as an internal reference protein for calibration).

[0023] Figure 4 Image J is used in Embodiment 2 of this invention. Figure 3 Grayscale values ​​of DCXR protein expression in the livers of normal mice, KD mice, IgG-sensitive KD mice, and IgG-unresponsive KD mice are shown in the figure.

[0024] Figure 5 This is a diagram showing the results of Western blot analysis of DCXR protein expression in the hearts of normal mice, KD mice, IgG-sensitive KD mice, and IgG-unresponsive KD mice in Example 2 of this invention (GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal reference protein for calibration).

[0025] Figure 6 Image J is used in Embodiment 2 of this invention. Figure 5 Grayscale values ​​of DCXR protein expression in the hearts of normal mice, KD mice, IgG-sensitive KD mice, and IgG-unresponsive KD mice are shown in the figure.

[0026] Figure 7This is a correlation analysis diagram of clinical liver function indicators ALT, AST, ALB and TBil with IgG responsiveness in children with KD who are IgG sensitive and IgG unresponsive according to Example 2 of the present invention.

[0027] Figure 8 This is a graph showing the results of Western blot analysis of DCXR protein expression in the livers of normal mice, KD mice, IgG-sensitive KD mice, DCXR-supplemented KD mice, and DCXR-supplemented IgG-sensitive KD mice in Example 3 of this invention (where β-ACTIN was used as an internal reference protein for calibration).

[0028] Figure 9 In Embodiment 3 of this invention, Image J is used to... Figure 8 Grayscale values ​​of DCXR protein expression in the livers of normal mice, KD mice, IgG-sensitive KD mice, DCXR-supplemented KD mice, and DCXR-supplemented IgG-sensitive KD mice are shown in the figure.

[0029] Figure 10 These are images of the tongue and abdominal aorta of normal mice, KD mice, IgG-sensitive KD mice, DCXR-supplemented KD mice, and DCXR-supplemented IgG-sensitive KD mice in Example 3 of this invention.

[0030] Figure 11 This is a diagram showing the HE and EVG staining results of abdominal aortic tissues from normal mice, KD mice, IgG-sensitive KD mice, DCXR-supplemented KD mice, and DCXR-supplemented IgG-sensitive KD mice in Example 3 of the present invention.

[0031] Figure 12 This is a bar chart analyzing the abdominal aortic diameter / body weight ratio of normal mice, KD mice, IgG-sensitive KD mice, DCXR-supplemented KD mice, and DCXR-supplemented IgG-sensitive KD mice in Example 3 of the present invention.

[0032] Figure 13 This is a diagram showing the HE and EVG staining results of heart tissues from normal mice, KD mice, IgG-sensitive KD mice, DCXR-supplemented KD mice, and DCXR-supplemented IgG-sensitive KD mice in Example 3 of the present invention.

[0033] Figure 14 This is a graph showing the results of Western blot analysis of the expression of inflammatory factors and vascular damage factors in the cardiac tissues of normal mice, KD mice, IgG-sensitive KD mice, KD mice supplemented with DCXR, and IgG-sensitive KD mice supplemented with DCXR (GAPDH was used as an internal reference protein for calibration) in Example 3 of this invention.

[0034] Figure 15 In Embodiment 3 of this invention, Image J is used to... Figure 14 Grayscale values ​​of TNF-α expression in cardiac tissues of normal mice, KD mice, IgG-sensitive KD mice, DCXR-supplemented KD mice, and DCXR-supplemented IgG-sensitive KD mice are shown in the figure.

[0035] Figure 16 In Embodiment 3 of this invention, Image J is used to... Figure 14 Grayscale values ​​of IL-1β expression in cardiac tissues of normal mice, KD mice, IgG-sensitive KD mice, DCXR-supplemented KD mice, and DCXR-supplemented IgG-sensitive KD mice are shown in the figure.

[0036] Figure 17 In Embodiment 3 of this invention, Image J is used to... Figure 14 Grayscale values ​​of the expression of the vascular injury factor THBD in normal mice, KD mice, IgG-sensitive KD mice, KD mice supplemented with DCXR, and IgG-sensitive KD mice supplemented with DCXR are shown in the figure.

[0037] Figure 18 In Embodiment 3 of this invention, Image J is used to... Figure 14 Gray-scale results showing the expression of vascular injury factor vWF in normal mice, KD mice, IgG-sensitive KD mice, KD mice supplemented with DCXR, and IgG-sensitive KD mice supplemented with DCXR.

[0038] Figure 19 This is a diagram showing the results of Western blot analysis of DCXR protein expression in the livers of normal mice, KD mice, IgG-sensitive KD mice, EGCG-supplemented KD mice, and EGCG-supplemented IgG-sensitive KD mice in Example 4 of this invention (where β-ACTIN (as an internal reference protein) is used for calibration).

[0039] Figure 20 In Embodiment 4 of this invention, Image J is used to... Figure 19 Grayscale values ​​of DCXR protein expression in the livers of normal mice, KD mice, IgG-sensitive KD mice, EGCG-supplemented KD mice, and EGCG-supplemented IgG-sensitive KD mice are shown in the figure.

[0040] Figure 21These are images of the tongue and abdominal aorta of normal mice, KD mice, IgG-sensitive KD mice, EGCG-supplemented KD mice, and EGCG-supplemented IgG-sensitive KD mice in Example 4 of this invention.

[0041] Figure 22 This is a diagram showing the HE and EVG staining results of abdominal aortic tissues from normal mice, KD mice, IgG-sensitive KD mice, EGCG-supplemented KD mice, and EGCG-supplemented IgG-sensitive KD mice in Example 4 of this invention.

[0042] Figure 23 This is a bar chart analyzing the abdominal aortic diameter / body weight ratio of normal mice, KD mice, IgG-sensitive KD mice, EGCG-supplemented KD mice, and EGCG-supplemented IgG-sensitive KD mice in Example 4 of the present invention.

[0043] Figure 24 This is a diagram showing the HE and EVG staining results of heart tissues from normal mice, KD mice, IgG-sensitive KD mice, EGCG-supplemented KD mice, and EGCG-supplemented IgG-sensitive KD mice in Example 4 of this invention. Detailed Implementation

[0044] This invention discloses the use of dicarbonyl / L-xylitol reductase (DCXR) as a target in the screening and / or preparation of drugs for treating Kawasaki disease (KD).

[0045] Specifically, when screening drugs for treating KD, a drug screening system is constructed using DCXR as the target to screen for effective drugs and verify their efficacy. Specifically, DCXR promoters that promote DCXR expression or activity, and / or DCXR inhibitors that inhibit DCXR expression or activity can be selected and applied to KD animal models. Based on the effects on the KD animal models, drugs suitable for treating KD are screened and identified. Furthermore, preparations that promote L-xylulose metabolism or improve the function of the glucuronide metabolism pathway can be selected, such as exogenous DCXR or preparations with the same function as DCXR, and / or preparations that promote L-xylulose metabolism or improve the function of the glucuronide metabolism pathway. These preparations are applied to KD animal models, and based on the effects on the KD animal models, drugs suitable for treating KD are screened and identified.

[0046] When used in the preparation of drugs for the treatment of KD, DCXR is used as the target. DCXR promoters or preparations that can promote L-xylulose metabolism or improve the function of the glucuronic acid metabolism pathway are combined with pharmaceutically acceptable carriers, excipients, sustained-release matrices, targeted delivery carriers, etc. to prepare drugs in different dosage forms such as injections and oral preparations for the treatment of KD patients.

[0047] It should be noted that DCXR is mainly involved in glucuronic acid metabolism, L-xylulose metabolism and late AGEs clearance, playing a protective role in maintaining metabolic homeostasis. Furthermore, no evidence has been found that promoting DCXR expression can induce new diseases, nor has it been found to have any biological effects such as carcinogenicity, pro-inflammatory effects or promotion of organ fibrosis. Therefore, DCXR has a good physiological safety basis and is suitable as a potential drug development target.

[0048] In one specific embodiment of the present invention, KD includes KD that is unresponsive to IgG, i.e., DCXR is preferably used as a target for treating IgG-unresponsive KD. Specifically, after a diagnosis of KD is made and treatment with IgG is initiated, if the treatment effect is not good (i.e., IgG-unresponsive KD), a first agent is further applied.

[0049] The lack of clearly defined molecular therapeutic targets in IgG-unresponsive Kashin-D disease (KD) is a significant cause of coronary artery disease and long-term cardiovascular complications in children with KD. Current treatment regimens primarily target the inflammatory response with broad-spectrum immunosuppression, without addressing the core mechanisms underlying IgG non-responsiveness. Consequently, treatment efficacy varies considerably among individuals, and various toxic side effects also occur.

[0050] This invention is the first to discover that DCXR is closely related to the occurrence of IgG-unresponsive KD, and that inhibiting DCXR directly affects the therapeutic effect of IgG, providing a novel metabolic therapeutic target for KD and offering a new use for DCXR as a therapeutic target for KD, especially providing a new treatment option for IgG-unresponsive KD.

[0051] In one specific embodiment of the present invention, KD includes KD with coronary artery disease.

[0052] Coronary artery disease is one of the most serious complications of kidney disease (KD), including coronary artery dilation and aneurysms, which can lead to myocardial infarction or sudden death. This invention demonstrates that DCXR can improve vascular endothelial damage and coronary artery disease, reducing the incidence of coronary artery injury. It should be noted that if a KD patient is assessed as having a high risk of developing coronary artery disease, drugs screened using DCXR as a target can also be used for treatment.

[0053] In one specific embodiment of the present invention, the drug for treating KD screened and / or prepared includes a first formulation having at least one of the following functions: increasing the expression level of DCXR, enhancing the activity of DCXR, promoting L-xylulose metabolism, or improving the function of the glucuronic acid metabolism pathway.

[0054] Specifically, formulations that enhance DCXR expression levels include at least one of the following: formulations that can directionally upregulate DCXR enzyme synthesis, supplements that can assist in upregulating DCXR enzyme expression, gene formulations for enhancing DCXR enzyme expression, and other formulations capable of enhancing DCXR expression levels. These can specifically be small molecule compounds, peptides, natural active substances, expression vectors, etc. Furthermore, this invention confirms that supplements that can assist in upregulating DCXR expression include recombinant DCXR proteins. Further, gene formulations for enhancing DCXR expression include recombinant expression vectors encoding DCXR, mRNA formulations encoding DCXR, DNA expression vectors encoding DCXR, adeno-associated virus (AAV) vectors encoding DCXR, adenovirus vectors, lentiviral vectors, and lipid nanoparticle delivery systems. Further, gene formulations for enhancing DCXR enzyme expression can be commercially available products, such as the human DCXR complete ORF expression clone with catalog number RC200023 manufactured by OriGene, which uses the pCMV6-Entry vector with a Myc-DDK tag and can be used for mammalian cell expression; gene formulations can also be prepared using existing technologies.

[0055] Formulations that enhance the activity of DCXR include: DCXR enzyme inducers, formulations that improve the stability of DCXR proteins, agonists that enhance the catalytic activity of DCXR, formulations that restore the activity of DCXR, and at least one of other pharmaceutical compositions that can enhance the activity of DCXR.

[0056] Furthermore, DCXR proteins include at least one of the following: native DCXR protein, recombinant DCXR protein, and a protein containing a functional fragment with DCXR activity. Even further, the recombinant DCXR protein can be a commercially available product, such as the DCXR recombinant protein with catalog number HY-P70082 manufactured by MedChemExpress (MCE).

[0057] The recombinant DCXR protein can also be prepared in-house using methods including: prokaryotic expression in *E. coli* followed by His tagging, Ni-NTA affinity purification, and the HEK293 / HEK293T eukaryotic expression system. It should be noted that the gene encoding DCXR has Entrez ID 51181 and HGNC ID 18985; the DCXR protein sequence number is NP_057370.1, consisting of 244 amino acids with a molecular weight of approximately 27-28 kDa.

[0058] The first formulation can be used alone or in combination with other drugs for treating KD, or other anti-inflammatory and immunomodulatory drugs. In one specific embodiment of the invention, the drug for treating KD further includes a second formulation, which includes at least one of IgG, glucocorticoids, and aspirin; and the first and second formulations are used in combination, further wherein the first and second formulations can be used sequentially or simultaneously.

[0059] In one specific embodiment of the present invention, the drug further includes at least one of a pharmaceutically acceptable carrier, excipient, diluent, stabilizer, and buffer.

[0060] Specifically, all of the above-mentioned excipients are conventional pharmaceutical excipients in the field of pharmaceutical preparations. They have good biocompatibility, safety and physicochemical stability, no toxic side effects, and will not adversely affect the pharmacological activity, chemical structure, dissolution performance and in vivo absorption of the active ingredients.

[0061] In one specific embodiment of the present invention, the dosage form of the drug is at least one of injection, tablet, capsule, granule, powder, oral liquid, and inhaler.

[0062] In one specific embodiment of the present invention, the route of administration of the drug includes at least one of injection, oral administration, and nebulization. The dosage form is preferably an injection, and the route of administration is preferably injection, specifically intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, etc.

[0063] To make the objectives, technical solutions, and effects of the present invention clearer, the embodiments of the present invention will be further described in detail below by way of specific examples.

[0064] Example 1

[0065] This embodiment collected serum samples from 15 children with KD who were sensitive to gamma immunoglobulin (IgG) and 3 children with KD who were unresponsive to IgG. Untargeted metabolomics detection was performed using ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry. The specific method included: transferring 100 μL of sample to a 1.5 mL centrifuge tube, adding 400 μL of extraction buffer (methanol:acetonitrile = 1:1 (v:v)) containing 0.02 mg / mL internal standard (L-2-chlorophenylalanine), and extracting at 1500 rpm for 15 min at 10 °C; incubating the sample at -20 °C for 20 min, then centrifuging at 18000 g for 20 min at 4 °C; collecting the supernatant, drying it under nitrogen, and then reconstituted it with 100 μL of reconstitution solution (acetonitrile:water = 1:1 (v:v)); mixing at 1500 rpm for 5 min at 10 °C, centrifuging for 10 min, and then transferring the supernatant to a sample vial. The concentration of metabolites was detected using an ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry (UHPLC-Q Exactive HF-X) system. The liquid chromatography detection conditions included: an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm id, 1.8 μm; Waters, Milford, The column temperature was 40℃, and the sample manager temperature was 10℃. Mobile phase A consisted of 95% water + 5% acetonitrile (containing 0.1% formic acid), and mobile phase B consisted of 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). The flow rate was 0.4 mL / min, and the sample volume was 3 μL. Gradient elution was performed. The mass spectrometry detection conditions included: ESI ion source, ion source voltage of 3.5 kV, capillary temperature of 326℃, heating temperature of 425℃, sheath gas flow rate of 50 arb, auxiliary gas flow rate of 13 arb, scan range of 70-1050 Da, FulI MS resolution of 60,000, and MS2 resolution of 7500.

[0066] The results of serum L-xylulose concentration are as follows: Figure 1 As shown, the average L-xylulose concentration in the serum of children with IgG non-responsive KD was about 13 µmol / L, which was significantly higher than the average L-xylulose concentration in the serum of children sensitive to IgG treatment (about 5 µmol / L), and the difference was statistically significant.

[0067] To further validate clinical results, this embodiment used a single injection of 1 mg / mL Lactobacillus casei wall extractive (LCWE) to establish a KD mouse model. On day 5 after LCWE injection, a single injection of 20 mg / kg IgG was administered as an IgG-treated KD model. On day 14 after LCWE injection, changes in the mouse tongue were observed and recorded. The mice were then euthanized by cervical dislocation, and the abdominal aorta was dissected and recorded. Simultaneously, the heart was removed and observed. Mice exhibiting tongue congestion and swelling, abdominal aortic dilation, cardiac inflammatory infiltration, and elevated levels of inflammatory factors and vascular damage factors in the cardiac tissue were identified as IgG non-responsive KD mice. Conversely, mice without these characteristics were identified as IgG-sensitive KD mice. The serum L-xylulose concentration was measured using the same method described above in the normal mouse group, KD mouse group, IgG-sensitive KD mouse group, and IgG non-responsive KD mouse group. The results are as follows: Figure 2 As shown, the average concentration of L-xylulose in the serum of IgG-unresponsive KD mice was about 33 µmol / L, which was significantly higher than that of mice in other groups, consistent with clinical samples, further demonstrating that elevated L-xylulose is closely related to IgG treatment non-response.

[0068] Since L-xylulose is an important intermediate metabolite in the glucuronic acid and pentose phosphate metabolism pathways, a sustained increase in its concentration usually indicates a decrease in the expression or activity of the corresponding metabolic enzyme.

[0069] Example 2

[0070] To determine whether the abnormal accumulation of L-xylulose is caused by a decrease in the expression of its key metabolic enzyme DCXR, this example further examines the expression of DCXR protein in the liver and heart tissues of IgG-unresponsive KD mice constructed in Example 1. Total protein was extracted from the liver and heart tissues of normal mice, KD mice, IgG-sensitive KD mice, and IgG-unresponsive KD mice, respectively, and DCXR protein expression was detected by Western blot.

[0071] Experimental results of mouse liver tissue are as follows Figure 3 and Figure 4 As shown, compared with IgG-sensitive KD mice, IgG-unresponsive KD mice showed significantly reduced DCXR protein expression in liver tissue. However, experimental results for mouse heart tissue were as follows... Figure 5 and Figure 6 As shown, there was no significant difference in DCXR protein expression among the groups in cardiac tissue.

[0072] Since liver damage is common in clinically diagnosed children with KD, liver function indicators such as ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALB (albumin), and TBil (total bilirubin) are key indicators to assist clinicians in diagnosing IgG-unresponsive KD. To rule out the possibility that L-xylulose metabolism abnormalities are secondary to liver damage, a correlation analysis was performed between the above-mentioned liver function indicators in clinically diagnosed children with IgG-unresponsive KD and those in IgG-sensitive KD. The results are as follows: Figure 7 As shown, the above indicators did not show a significant correlation with IgG non-responsive KD patients, and the correlation coefficients were all low, indicating that the metabolic disorders exhibited by IgG non-responsive KD patients are less likely to be secondary to liver function damage.

[0073] The experimental results of this embodiment show that the decrease in DCXR expression in children with IgG non-responsive KD mainly occurs in the liver, rather than in target organs such as the heart. At the same time, this change is not significantly correlated with traditional liver injury indicators. Therefore, it is more likely to be a metabolic regulation abnormality in the process of IgG non-responsive KD, rather than a non-specific metabolic disorder caused by hepatocyte damage. Therefore, DCXR can be used as a potential therapeutic target for IgG non-responsive KD.

[0074] Example 3

[0075] To further verify whether reduced DCXR expression is involved in the development of KD and to clarify whether restoring DCXR levels can improve the disease phenotype of KD mice, this embodiment uses exogenous supplementation of DCXR protein to intervene in KD mice.

[0076] This embodiment uses recombinant human DCXR protein as an exogenous supplement for DCXR. The commercially available recombinant human DCXR protein from MedChemExpress (MCE), catalog number HY-P70082, was selected. The protein was prepared according to the product instructions, and the working concentration of the prepared DCXR protein was 1 μg / mL. It should be noted that the recombinant DCXR protein used in this embodiment is only for verifying whether restoring DCXR levels can improve Klebsiella pneumoniae pathological changes and does not constitute a limitation on the form of DCXR administration.

[0077] Based on the KD mouse and IgG-treated KD mouse models constructed in Example 1, starting on day 5 after LCWE injection, some mice were treated with recombinant DCXR protein via intraperitoneal injection at a dose of 100 μL the day after IgG injection. Total protein was extracted from the livers of mice in the normal group (normal group), KD mouse group (KD group), IgG-treated KD mouse group (KD+IgG group), DCXR-supplemented KD mouse group (KD+DCXR group), and DCXR-supplemented IgG-treated KD mouse group (KD+IgG+DCXR group), and DCXR-supplemented IgG-treated KD mouse group (KD+IgG+DCXR group), and DCXR-supplemented KD mouse group (KD+IgG+DCXR group), and DCXR protein expression was detected.

[0078] Experimental results are as follows Figures 8-9 As shown, the expression of DCXR in the liver of mice supplemented with DCXR (KD+IgG+DCXR group and KD+DCXR group in the figure) was significantly higher than that in the liver of mice not supplemented with DCXR (KD+IgG group and KD group in the figure), and was close to that in the liver of normal mice, indicating that DCXR supplementation can effectively restore DCXR expression in the liver of KD mice.

[0079] Further examination of the phenotype, vascular pathology, and inflammatory expression in different groups of mice yielded the following results: Figures 10-13 As shown, from Figure 10 It can be seen that untreated KD mice all showed obvious tongue congestion, swelling and abdominal aortic dilation. After DCXR supplementation, the tongue of the mice returned to normal and the degree of abdominal aortic dilation was significantly reduced. The improvement effect was more obvious when DCXR was supplemented after combined IgG treatment (KD+IgG+DCXR group in the figure). Figure 11 HE staining and EVG staining of the mid-abdominal aorta showed that the vessel wall in the KD group was significantly thickened, the inner diameter of the abdominal aorta was enlarged, and the elastic fibers were disordered. After DCXR supplementation, the above pathological conditions were significantly improved. Figure 12 Analysis of the mid-abdominal aortic diameter / body weight ratio showed that the ratio was significantly higher in the KD group and significantly lower after DCXR supplementation, further confirming that DCXR can significantly reduce vascular dilation. Simultaneously, cardiac tissue was stained with hematoxylin and eosin (HE) and EVG to observe coronary artery inflammation and changes in elastic fibers. Figure 13 HE staining and EVG staining results of pericoronary tissue showed that after DCXR supplementation, pericoronary inflammatory cell infiltration was significantly reduced and elastic fibers were neatly arranged, indicating that DCXR can improve coronary artery lesions in the KD model.

[0080] The expression of inflammatory factors TNF-α (tumor necrosis factor-α) and IL-1β (interleukin-1β), as well as vascular endothelial injury factors vWF (von Willebrand factor) and THBD (thrombomodulin) in cardiac tissue was further detected by Western blot. The results are as follows: Figures 14-18 As shown, the expression of inflammatory factors and vascular damage factors in the KD group was significantly increased, while the expression of the above proteins was significantly decreased after DCXR supplementation; the decrease was even more significant after combined IgG treatment, with statistical differences.

[0081] This embodiment verifies that exogenous DCXR supplementation can restore DCXR expression in the liver of KD mice and improve pathological damage. These results indicate that restoring DCXR expression in KD patients not only reduces local inflammation but also improves vascular endothelial damage and coronary artery lesions, suggesting that DCXR has potential value as a therapeutic target for IgG-unresponsive KD in drug development.

[0082] It should be noted that this embodiment uses exogenous supplementation of recombinant DCXR protein to restore DCXR levels and achieve a significant therapeutic effect, indicating that increasing the effective level of DCXR protein in vivo is an important way to improve KD vascular inflammation and vascular damage. Therefore, based on the above experimental results, those skilled in the art can reasonably infer that, in addition to directly supplementing recombinant DCXR protein, using an expression vector capable of expressing DCXR in vivo or in target tissues can also promote the metabolism of L-xylitol to xylitol by increasing DCXR protein levels, thereby exerting the same or similar therapeutic effect as in this embodiment. The expression vector for expressing DCXR can be a plasmid vector containing the DCXR coding sequence, a viral vector, or other nucleic acid delivery systems capable of mediating DCXR expression. The specific vector type and delivery method can be selected according to the actual application.

[0083] Furthermore, since the core mechanism of action of this invention lies in restoring DCXR-mediated L-xylulose metabolism, any preparation that can increase DCXR expression levels, enhance DCXR enzyme activity, or promote normal L-xylulose metabolism and improve the function of related glucuronic acid metabolic pathways can theoretically be used as an alternative intervention method of this invention. The aforementioned preparations can exert their effects by increasing DCXR protein supply, promoting DCXR gene expression, or enhancing its enzyme activity, with the ultimate goal of reducing abnormally accumulated L-xylulose levels, thereby alleviating inflammation and vascular damage in KD, especially in IgG-unresponsive KD. The therapeutic results obtained from exogenous DCXR supplementation in this embodiment provide experimental evidence and mechanistic support for the aforementioned different intervention methods.

[0084] Example 4

[0085] To further verify the key role of DCXR in the treatment of KD with IgG, this embodiment uses the DCXR inhibitor epigallocatechin gallate (EGCG) to intervene in the KD mouse model and the IgG-treated KD mouse model constructed in Example 1, and observes whether the inhibition of DCXR can lead to the failure of IgG treatment, thus verifying the important role of DCXR in IgG treatment from the reverse perspective.

[0086] Starting on day 5 after KD modeling, some mice were given EGCG. EGCG at a concentration of 5 mg / ml was administered by gavage at a rate of 200 μL per day starting on day 5 after LCWE injection. The EGCG was purchased from MCE, catalog number HY-13653.

[0087] Total protein was extracted from the livers of mice in the following groups: normal group, KD group, IgG-treated KD group, EGCG-supplemented KD group, and EGCG-supplemented IgG-treated KD group. The expression of DCXR in mouse liver tissue was detected, and the results are as follows: Figures 19-20 As shown, the expression of DCXR protein in liver tissue decreased significantly after EGCG treatment, proving that EGCG can effectively reduce the expression level of DCXR in mouse liver tissue.

[0088] Further examination of the phenotype, vascular pathology, and inflammatory expression in different groups of mice yielded the following results: Figures 21-24 As shown, from Figure 21 It can be seen that after IgG treatment combined with EGCG treatment, the mice in the KD mouse group again showed obvious tongue congestion, edema and abdominal aortic dilation. Figure 22 Pathological staining results of the abdominal aorta in different groups of mice showed that the abdominal aortic wall of mice treated with EGCG was significantly thickened, inflammatory cells re-accumulated in large quantities, and elastic fibers re-brooded and rearranged. Further analysis of the abdominal aortic diameter / body weight ratio yielded the following results: Figure 23 As shown, the ratio in the EGCG treatment group was significantly higher than that in the IgG treatment group; Figure 24 HE and EVG staining results of heart tissues from different groups showed that after EGCG treatment, the coronary arteries again showed extensive inflammatory cell infiltration and disordered elastic fiber arrangement. These results suggest that inhibiting DCXR can re-induce vasodilation and vascular damage in mice sensitive to IgG treatment.

[0089] The test results in this embodiment all indicate that the performance of KD mice after DCXR inhibition is highly consistent with the IgG unresponsive KD model, suggesting that DCXR inhibition can significantly weaken the therapeutic effect of IgG. The DCXR inhibitor EGCG in this embodiment can reverse the therapeutic effect of IgG, further confirming that DCXR is an important therapeutic target for IgG unresponsive KD.

[0090] The above results indicate that downregulation of DCXR expression is an important metabolic mechanism in the development of IgG-unresponsive KD, and clarify that DCXR, as a key enzyme in liver metabolism, affects the therapeutic effect of IgG by regulating L-xylulose metabolism.

[0091] Based on the above results, this invention proposes DCXR as a novel therapeutic target for KD, realizing the transformation from a metabolic marker to a therapeutic target. This invention proposes a novel precision treatment strategy that restores normal L-xylulose metabolism by promoting DCXR expression and enhancing DCXR enzyme activity, providing a new direction for drug development in KD, especially IgG-unresponsive KD.

[0092] Furthermore, the treatment strategy targeting DCXR established in this invention does not simply suppress inflammation, but rather treats children with KD, especially those with IgG-unresponsive KD, by restoring normal metabolic homeostasis, thus providing precise intervention at the disease pathogenesis level. This invention not only expands the new biological functions of DCXR and provides a new targeted treatment strategy for KD, but also has significant scientific value, clinical application value, and industrial transformation prospects.

[0093] It should be noted that, in addition to the specific embodiments described above, those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Although the description of the present invention is presented in conjunction with preferred embodiments, this does not mean that the features of the invention are limited to these embodiments. On the contrary, the purpose of describing the invention in conjunction with the embodiments is to cover other options or modifications that may be derived based on the claims of the present invention. To provide a deep understanding of the invention, many specific details are included in the above description, and the invention may also be implemented without using these details. Furthermore, to avoid confusion or obscuring the focus of the invention, some specific details will be omitted in the description. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0094] While the present invention has been described and illustrated with reference to certain preferred embodiments, those skilled in the art should understand that the above description is a further detailed explanation of the invention in conjunction with specific embodiments, and should not be construed as limiting the specific implementation of the invention to these descriptions. Various changes in form and detail can be made by those skilled in the art, including several simple deductions or substitutions, without departing from the spirit and scope of the invention.

Claims

1. Application of dicarbonyl / L-xylitol reductase as a target in screening and / or preparation of drugs for treating Kawasaki disease.

2. The application as described in claim 1, characterized in that, The Kawasaki disease mentioned includes Kawasaki disease that is unresponsive to gamma immunoglobulin and / or Kawasaki disease with coronary artery disease.

3. The application as described in claim 1, characterized in that, The drug comprises a first formulation having at least one of the following functions: It can increase the expression level of dicarbonyl / L-xylulose reductase, enhance the activity of dicarbonyl / L-xylulose reductase, promote L-xylulose metabolism, or improve the function of the glucuronic acid metabolism pathway.

4. The application as described in claim 3, characterized in that, The first formulation having the function of increasing the expression level of dicarbonyl / L-xylulose reductase comprises at least one of: dicarbonyl / L-xylulose reductase protein, a recombinant expression vector encoding dicarbonyl / L-xylulose reductase, an mRNA formulation, a DNA expression vector, an adeno-associated virus vector, an adenovirus vector, a lentiviral vector, and a lipid nanoparticle delivery system.

5. The application as described in claim 3, characterized in that, The dicarbonyl / L-xylulose reductase protein includes at least one of the following: natural dicarbonyl / L-xylulose reductase protein, recombinant dicarbonyl / L-xylulose reductase protein, and protein containing a functional fragment with dicarbonyl / L-xylulose reductase activity.

6. The application as described in claim 3, characterized in that, The drug also includes a second formulation, which includes at least one of gamma immunoglobulin, glucocorticoids, and aspirin; The first formulation and the second formulation are used in combination.

7. The application as described in any one of claims 3-6, characterized in that, The drug also includes at least one of a pharmaceutically acceptable carrier, excipient, diluent, stabilizer, and buffer.

8. The application as described in any one of claims 3-6, characterized in that, The dosage form of the drug is at least one of the following: injection, tablet, capsule, granule, powder, oral liquid, and inhaler.

9. The application as described in any one of claims 3-6, characterized in that, The drug can be administered via at least one of the following routes: injection, oral administration, or nebulization.

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